Protein containing heterodimer antibody FC and preparation method thereof

Abstract

Disclosed herein are novel heterodimeric antibody Fc-containing proteins, such as bispecific antibodies, and novel methods for making such proteins.

Description

[0001] This application is a divisional application of the invention application filed on April 20, 2011, with Chinese national application number 201180030540.9, entitled "Protein containing heterodimer antibody FC and preparation method thereof" (application number 201910248855.9). Invention Field

[0002] This invention relates to novel proteins containing heterodimeric antibody Fc, such as bispecific antibodies, and novel methods for preparing such proteins. Background of the Invention In recent years, monoclonal antibodies have become successful therapeutic molecules, particularly for treating cancer. However, unfortunately, when used as a monotherapy, monoclonal antibodies often fail to cure the disease. Bispecific antibodies have the potential to overcome some limitations of monoclonal antibody therapy, such as acting as mediators to target drugs or toxic compounds to target cells, acting as mediators to retarget effector mechanisms to disease-related sites, or acting as mediators to increase specificity for tumor cells, for example, by combining with target molecules found only on tumor cells.

[0003] A recent review of the different forms and uses of bispecific antibodies can be found in Chames and Baty (2009) Curr Opin Drug Disc Dev 12: 276. One of the major obstacles to the development of bispecific antibodies is the difficulty in preparing sufficient quality and quantity of material using conventional techniques such as hybridomas and chemical conjugation methods (Marvin and Zhu (2005) Acta Pharmacol Sin 26:649). Co-expression of two antibodies composed of different heavy and light chains in host cells, in addition to producing the desired bispecific antibody, can also produce a mixture of possible antibody products.

[0004] Several strategies have been described to facilitate the formation of heterodimers (i.e. bispecific products) after co-expression in different antibody constructs.

[0005] Lindhofer et al. (1995 J Immunol 155:219) have described how the fusion of rat and mouse hybridomas producing different antibodies leads to the enrichment of functional bispecific antibodies due to preferred species-restricted heavy / light chain pairing. Another strategy that promotes heterodimer formation over homodimer formation is the “knob-into-hole” strategy, in which a bulge is introduced at the interface of the first heavy chain polypeptide and a corresponding cavity is introduced at the interface of the second heavy chain polypeptide, allowing the bulge to reside within the cavity to promote heterodimer formation and inhibit homodimer formation. The “bulge” is constructed by replacing a small amino acid side chain at the interface of the first polypeptide with a larger side chain. A complementary “cavity” of the same or similar size as the bulge is established at the interface of the second polypeptide by replacing a large amino acid side chain with a smaller amino acid side chain (US Patent 5,731,168). EP1870459 (Chugai) and WO 2009089004 (Amgen) describe other strategies that facilitate the formation of heterodimers after co-expression of different antibody domains in host cells. In these methods, one or more residues at the CH3-CH3 interface of the two CH3 domains are replaced with charged amino acids, making electrostatically unfavorable for homodimer formation and electrostatically favorable for heterodimerization. WO2007110205 (Merck) describes yet another strategy in which the difference between the IgA and IgG CH3 domains is used to promote heterodimerization.

[0006] Dall'acqua et al. (1998 Biochemistry 37:9266) have identified five energy-critical amino acid residues (366, 368, 405, 407 and 409) involved in the CH3-CH3 contact at the CH3 homodimer interface.

[0007] WO 2008119353 (Genmab) describes an in vitro method for preparing bispecific antibodies, wherein, after incubation under reducing conditions, bispecific antibodies are formed between two monospecific IgG4 antibodies or IgG4-like antibodies via “Fab arm” or “half-molecule” exchange (exchange of heavy chain with linked light chain). This Fab arm exchange reaction is the result of disulfide bond isomerization and CH3 domain dissociation-association, wherein the heavy chain disulfide bond in the hinge region of the parental (originally monospecific) antibody is reduced, and the resulting free cysteine ​​forms an inter-heavy chain disulfide bond with a cysteine ​​residue of another parental antibody molecule (originally with different specificity), while the CH3 domain of the parental antibody is released and reformed through dissociation-association. The resulting product is a bispecific antibody with two Fab arms, which may contain different sequences. However, it should be noted that the process is random, and Fab arm exchange can also occur between two molecules with the same sequence, or two bispecific molecules can participate in Fab arm exchange to regenerate an antibody containing the specificity of the original monospecific parental antibody.

[0008] It has now been surprisingly discovered that by introducing asymmetric mutations into the CH3 region of two single-specific initiation proteins, the Fab arm interchange reaction can be forced to become directional, resulting in highly stable heterodimeric proteins. Invention Overview Therefore, on the one hand, the present invention provides an efficient in vitro method for preparing highly stable proteins containing heterodimeric Fc based on stable homodimeric Fc-containing raw materials. For example, highly stable bispecific antibodies with high yield and purity can be formed based on two stable monospecific antibodies as raw materials.

[0009] Therefore, in one respect, the present invention relates to an in vitro method for generating heterodimeric proteins, the method comprising the following steps: a) Provide a first homodimeric protein comprising an immunoglobulin Fc region, wherein the Fc region comprises a first CH3 region. b) Provide a second homodimeric protein comprising an immunoglobulin Fc region, wherein the Fc region comprises a second CH3 region. 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 stronger than the homodimer interaction between the first CH3 region and the second CH3 region.

[0010] c) Incubate the first protein and the second protein together under reducing conditions sufficient to allow disulfide isomerization of cysteine ​​residues in the hinge region. d) Obtain the heterodimeric protein.

[0011] The method can be used, for example, to prepare heterodimeric proteins in vitro, such as bispecific antibodies, for various applications, including therapeutic or diagnostic uses. An advantage of this in vitro method is that the heavy / light chain pairing remains intact during the reaction, so undesirable combinations of heavy and light chains are not obtained in the product. This contrasts with some prior art co-expression methods (see above), where, due to random heavy / light chain pairing in cells, a common light chain capable of forming functional antibodies with both heavy chains must be identified to avoid the formation of non-functional heavy / light chain products. Furthermore, the in vitro method can be performed in the laboratory, allowing for greater control, flexibility, and yield of heterodimeric proteins compared to co-expression.

[0012] The in vitro method of the present invention can also be used, for example, in screening methods to build larger-sized compound libraries to identify advantageous specific combinations. For example, for some combinations of antibody targets, not every bispecific antibody will be functional, i.e., capable of binding to both targets simultaneously and mediating the desired functional effect. In such cases, bispecific antibodies possessing the desired properties (e.g., optimal target binding or cell killing) can be identified through the following steps: a) Provide a first set of homodimeric antibodies with different variable regions, wherein the antibodies in the first set contain a first CH3 region. b) Provide a second group of homodimeric antibodies with different variable regions, wherein the antibodies in the second group contain a second CH3 region. 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 stronger than the homodimer interaction between the first CH3 region and the second CH3 region. c) Incubate the combination of the first group of antibodies and the second group of antibodies under reducing conditions sufficient to allow disulfide isomerization of cysteine ​​in the hinge region, thereby producing a set of bispecific antibodies. d) Select any recovery condition until non-reducible. e) The bispecific antibody group obtained by measuring the given desired properties, and f) Select bispecific antibodies with the desired properties.

[0013] In another aspect, the present invention relates to heterodimeric proteins obtained or obtainable by the method of the present invention and to a method for preparing heterodimeric proteins of the present invention by co-expression in suitable host cells. Brief description of the attached diagram Figure 1Bispecific antibodies were generated via interspecies Fab arm exchange. The generation of bispecific antibodies following GSH-induced in vitro Fab arm exchange between the EGFR (2F8) and CD20 (7D8) IgG4 antibodies was determined by ELISA. Concentration series (total antibody) ranging from 0–1 μg / mL were analyzed in ELISA. Bispecific binding following Fab arm exchange between rhesus monkey (Rh) and human (Hu) IgG4 antibodies was higher than that following Fab arm exchange between two antibodies from the same species.

[0014] Figure 2 Comparison of the core hinge (i.e., the CPPC sequence of human IgG1, which includes two cysteine ​​residues that may form disulfide bonds between heavy chains, and corresponding residues of other human isotypes or other species) and the amino acid sequence of the CH3-CH3 interface of human and rhesus monkey antibody isotypes.

[0015] Figure 3 Bispecific antibodies were generated using mutant human IgG1 that participated in Fab arm exchange. The generation of bispecific antibodies following 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 presented graph shows the mean of three independent Fab arm exchange assays, with a total antibody concentration of 1 µg / mL used for ELISA. Bispecific binding after Fab arm exchange between IgG1-2F8-CPSC-ITL and IgG4-7D8 was higher than that after Fab arm exchange between the two IgG4 antibodies. Combinations of IgG4-7D8 with IgG1-2F8-CPSC or IgG1-2F8-ITL did not generate bispecific antibodies under the conditions used.

[0016] Figure 4 Bispecific antibodies were generated in vivo via Fab arm exchange between human IgG4 and mutant IgG1 antibodies. The generation of bispecific antibodies following Fab arm exchange between human CD20 (7D8) IgG4 and the indicated human EGFR (2F8) IgG1 and IgG4 mutant antibodies in immunodeficient mice was determined by ELISA. The presented graphs show the mean (n=4). Bispecific reactivity is expressed as a percentage of bispecific antibody concentration relative to total IgG concentration. Human IgG4 with a stable hinge (CPPC) or R409K mutation in the CH3 domain did not participate in Fab arm exchange. IgG1 with both a CPSC sequence in the hinge and a K409R mutation in the CH3 domain participated in Fab arm exchange. (*) Bispecificity for mixtures containing IgG1-2F8, IgG4-2F8-CPPC, or IgG4-2F8-R409K was below the limit of detection and was therefore arbitrarily set to 0.

[0017] Figure 5 Bispecific antibodies were generated by 2-mercapto-ethylamine•HCl- (2-MEA-)-induced Fab arm exchange between human IgG1 and IgG4 antibodies. The generation of bispecific antibodies following 2-MEA-induced in vitro Fab arm exchange between the indicated human EGFR (2F8) and CD20 (7D8) antibodies was determined by ELISA. A series of 0–40 mM 2-MEA concentrations were tested. The presented graph shows the ELISA results, using a total antibody concentration of 20 µg / mL. 2-MEA also effectively induced Fab arm exchange between antibodies containing a stable hinge (CPPC). Regarding the CH3 domain, the combination of human IgG4 x human IgG1 with the triple mutant T350I-K370T-F405L resulted in a higher level of bispecific reactivity compared to the two wild-type IgG4 antibodies.

[0018] Figure 6 Bispecific antibodies were generated by using 2-MEA-induced Fab arm swapping between human IgG1 and IgG4 antibodies.

[0019] For all samples in the 0–40 mM 2-MEA concentration series, mass spectrometry was used to determine the production of bispecific antibodies following 2-MEA-induced in vitro Fab arm exchange between the indicated human EGFR (2F8) and CD20 (7D8) antibodies. (A) Representative mass spectrometric examples of samples from Fab arm exchange reactions between IgG1-2F8-ITL x IgG4-7D8-CPPC using 0 mM, 7 mM, and 40 mM 2-MEA are shown. (B) After quantification of the mass spectrometry data, the percentage of bispecific antibodies was calculated and plotted against the 2-MEA concentration in the Fab arm exchange reaction. IgG4-2F8 x IgG4-7D8 produced approximately 50% bispecific antibodies. IgG1-2F8-ITL x IgG4-7D8-CPPC produced approximately 95% bispecific antibodies.

[0020] Figure 7Stability analysis of heterodimeric bispecific antibodies obtained by 2-MEA-induced Fab arm exchange. Following a GSH-induced Fab arm exchange reaction in the presence of the indicated concentration of irrelevant IgG4, the stability of bispecific samples generated by 2-MEA-induced Fab arm exchange of either IgG1-2F8-ITL x IgG4-7D8-CPPC (A) or IgG4-2F8 xIgG4-7D8 (B) was tested by measuring EGFR / CD20 bispecific binding via ELISA. Bispecific binding is expressed relative to the bispecific binding of the raw material (control) (set to 100%). (A) Bispecific binding of the 2-MEA-induced bispecific product derived from IgG1-2F8-ITL x IgG4-7D8-CPPC was preserved, indicating a stable product that does not participate in Fab arm exchange under GSH conditions. (B) Reduced bispecific EGFR / CD20 binding of the 2-MEA-induced bispecific product derived from IgG4-2F8 x IgG4-7D8 suggests that the product is involved in Fab arm exchange with unrelated IgG4 under GSH conditions.

[0021] Figure 8 Plasma clearance of heterodimeric bispecific antibodies generated by 2-MEA-induced Fab arm exchange. Three groups of mice (n=3 per group) were injected with the antibodies shown: (1) 100 µg of bispecific antibody generated by in vitro 2-MEA-induced Fab arm exchange between IgG1-2F8-ITL and 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 total antibody plasma concentration curves were identical for all antibodies. (B) Bispecific antibody concentration as determined by ELISA. The bispecificity of the injected antibodies was the same with and without the addition of excess irrelevant IgG4. (*) The bispecific binding of the IgG1-2F8-ITL + IgG4-7D8-CPPC mixture was below the detection limit, therefore the corresponding symbol could not be plotted in this figure. Show the mean of the two ELISA experiments.

[0022] Figure 9: Purity of bispecific antibodies generated by Fab arm interchange between human IgG1-2F8 and IgG4-7D8-CPPC. (A) Reducing SDS-PAGE (a) shows the heavy and light chain bands of both the bispecific sample and the IgG1 control sample. Non-reducing SDS-PAGE (b). (B) Peak results from HP-SEC analysis show that >98% of the bispecific sample is homogeneous, with almost no detectable antibody aggregates. (C) Mass spectrometry shows that Fab arm interchange produces approximately 100% bispecific product.

[0023] Figure 10 Comparison of bispecific antibody production between triple mutant (ITL), dual mutant (IT, IL, TL), and single mutant (L) human IgG1-2F8 via Fab arm exchange with human IgG4-7D8. Bispecific antibody production was determined by ELISA between human IgG1-2F8 triple mutants and dual mutants and wild-type IgG4-7D8 with a CPSC hinge (A) or mutant IgG4-7D8-CPPC with a stable hinge (B), or single mutant IgG1-2F8-F405L and IgG4-7D8 with a wild-type CPSC or stable CPPC hinge (C), following 2-MEA-induced in vitro Fab arm exchange. The concentration series (total antibody) analyzed by ELISA in experiments including dual mutants and single mutants ranged from 0–20 μg / mL or 0–10 μg / mL. Combining with the dual mutants IgG1-2F8-IL and IgG1-2F8-TL results in bispecific EGFR / CD20 binding similar to that of the triple mutant IgG1-ITL. Combining with IgG1-2F8-IT does not produce a bispecific product. Combining with the single mutant IgG1-2F8-F405L results in bispecific EGFR / CD20 binding.

[0024] Figure 11 Bispecific antibodies were generated at different temperatures using 2-MEA-induced Fab arm interchange. Bispecific antibodies were generated by combining human EGFR (2F8) and CD20 (7D8) antibodies in an in vitro Fab arm interchange reaction induced by 2-MEA at 0°C, 20°C, and 37°C, followed by immediate ELISA. Bispecific binding was most effective at 37°C and slower at 20°C. At 0°C, no bispecific binding was measured.

[0025] Figure 12Bispecific antibodies were generated by in vitro Fab arm interchange induced with different reducing agents. The concentration series of the reducing agents shown was measured by ELISA in the reduction reaction by bispecific antibodies combining human IgG1-2F8-ITL and IgG4-7D8-CPPC. Bispecific binding was measured after reaction with DTT (maximum value obtained at 2.5 mM DTT) and 2-MEA (maximum value obtained at 25 mM 2-MEA), but not with GSH. (*) Data with GSH concentrations >10 mM were excluded due to antibody aggregation.

[0026] Figure 13 : 2-MEA-induced Fab arm exchange between IgG1-2F8-ITL and IgG1-7D8-K409X mutants. The production of bispecific antibodies following 2-MEA-induced in vitro Fab arm exchange between IgG1-2F8-ITL and the indicated IgG1-7D8-K409X mutant was determined by ELISA. (A) Analysis of concentration series (total antibody) from 0–20 μg / mL. The positive control was a purified batch of bispecific antibody derived from IgG1-2F8-ITL x IgG4-7D8-CPPC. (B) The exchange is represented by bispecific binding at 20 µg / mL relative to the positive control (black bar). Dark gray bars represent IgG4 control (IgG4-7D8 x IgG4-2F8), negative control (IgG1-2F8 x IgG1-7D8-K409R), and bispecific binding between IgG1-2F8-ITL and IgG4-7D8-CPPC. The light gray bars represent the results of the simultaneous Fab arm exchange reaction between the IgG1-7D8-K409X mutant and IgG1-2F8-ITL.

[0027] Figure 14 Antibody deglycosylation does not affect the generation of bispecific antibodies via 2-MEA-induced Fab arm exchange. The generation of bispecific antibodies following 2-MEA-induced in vitro Fab arm exchange between EGFR (2F8) and CD20 (7D8) antibodies was determined by ELISA. Exchanges with 7D8 antibody and its enzymatically deglycosylated variant were compared. A series of concentrations (total antibody) from 0–20 μg / mL were analyzed by ELISA. Fab arm exchange reactions involving deglycosylated (deglyc) antibodies showed the same bispecific binding profiles as their derived glycosylated variants.

[0028] Figure 15The ability to participate in Fab arm exchange is correlated with the strength of CH3-CH3 interactions. (A), (B), and (C) represent the generation of bispecific antibodies by GSH-induced Fab arm exchange between IgG1-2F8 with the mutation shown and IgG1-7D8 (A) or IgG4-2F8 with IgG4-7D8 (B and C) constructs, which are expressed as bispecific binding over time in ELISA. Bispecificity at 24 hours is expressed as a comparison with the IgG4-2F8 x IgG4-7D8 control. (D) and (E) represent the apparent binding at 24 hours for IgG1-based (D) or IgG4-based (E) molecules. K D (Table 2) Relationship with the production of bispecific antibodies Figure 15 (A / B / C).

[0029] Figure 16 Sequence alignment of anti-EGFr antibody 2F8 in the backbones of IgG1, IgG4, and (partially) IgG3. Description based on amino acid numbers according to Kabat and the 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)).

[0030] Figure 17 Bispecific antibodies were generated by in vitro Fab arm exchange induced by different reducing agents. The bispecific antibodies generated by combining human IgG1-2F8-F405L and IgG1-7D8-K409R were measured by ELISA in a series of reduction reactions using the indicated reducing agents. The measured OD values ​​were normalized to the signal of the bispecific control sample derived from Fab arm exchange induced by 2-MEA between IgG1-2F8-ITL and IgG4-7D8-CPPC, set to 100%. Maximum bispecific binding was measured after reaction with DTT at concentrations ranging from 0.5–50 mM, 2-MEA at concentrations ranging from 25–50 mM, and tris(2-carboxyethyl)phosphine (TCEP) at concentrations ranging from 0.5–5.0 mM, but not with GSH. (*) Data for GSH concentrations ≥ 25 mM were excluded due to antibody aggregation.

[0031] Figure 18 Bispecific antibodies were generated by 2-MEA-induced Fab arm swapping between human IgG1-2F8-F405L and IgG1-7D8-K409R.

[0032] (A) The production of bispecific antibodies following 2-MEA-induced in vitro Fab arm exchange was determined by ELISA. The presented graph shows the ELISA results using a total antibody concentration of 20 µg / mL. 2-MEA effectively induced Fab arm exchange. (B) The production of bispecific antibodies following 2-MEA-induced in vitro Fab arm exchange was determined by mass spectrometry for all samples in the 0-40 mM 2-MEA concentration series. After quantification of the mass spectrometry data, the percentage of bispecific antibodies was calculated and plotted against the concentration of 2-MEA in the Fab arm exchange reaction. IgG1-2F8-F405L x IgG1-7D8-K409R produced approximately 100% bispecific antibodies, confirming the ELISA data.

[0033] Figure 19 Purity of bispecific antibodies generated by Fab arm exchange between human IgG1-2F8-F405L and IgG1-7D8-K409R. Mass spectrometry showed that the Fab arm exchange produced approximately 100% bispecific product.

[0034] Figure 20 Plasma clearance of bispecific antibodies generated by 2-MEA-induced Fab arm exchange. Two groups of mice (n=3 per group) were injected with the antibodies shown: (1) 100 µg of bispecific antibody generated by in vitro 2-MEA-induced Fab arm exchange between IgG1-2F8-F405L x 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 total antibody plasma concentration curves were identical for all antibodies. (B) Bispecific antibody concentration as determined by ELISA. The bispecificity of the antibodies was the same with and without the addition of excess irrelevant IgG4.

[0035] Figure 21 Bispecific antibodies derived from Fab arm swaps induced by 2-MEA between IgG1-2F8-F405L and IgG1-7D8-K409R were used to investigate CDC-mediated cell killing in CD20-expressing cells. The antibody concentration series shown were used to test their ability to induce CDC in Daudi (A) and Raji (B) cells. Both cell lines express CD20 but not EGFR. The introduction of K409R into IgG1-7D8 did not affect its ability to induce CDC. Bispecific antibodies derived from Fab arm swaps induced by 2-MEA between IgG1-2F8-F405L and IgG1-7D8-K409R were still able to induce CDC.

[0036] Figure 22Bispecific antibodies generated via Fab arm interchange induced by 2-MEA between IgG1-2F8-F405L and IgG1-7D8-K409R were used to investigate ADCC-mediated cell killing in EGFR-expressing cells. The antibody concentration series shown was used to test their ability to induce ADCC in A431 cells. IgG1-7D8 cannot bind to CD20-negative A431 cells and therefore does not induce ADCC. ADCC was induced by the EGFR antibody IgG1-2F8, and also induced after the introduction of the F405L mutation into the CH3 domain. The ADCC effector function of IgG1-2F8-F405L is preserved in the bispecific form obtained via Fab arm interchange between IgG1-2F8-F405L and IgG1-7D8-K409R.

[0037] Figure 23 : 2-MEA-induced Fab arm exchange between IgG1-2F8-F405X mutant and IgG1-7D8-K409R. The production of bispecific antibodies following 2-MEA-induced in vitro Fab arm exchange between the IgG1-2F8-F405X mutant and IgG1-7D8-K409R was determined by ELISA. (A) A series of concentrations (total antibody) from 0–20 μg / mL were analyzed in ELISA. The positive control was a purified batch of bispecific antibody derived from IgG1-2F8-F405L x IgG1-7D8-K409R. (B) The exchange was expressed as bispecific binding at an antibody concentration of 20 µg / mL relative to the positive control (black bar). Dark gray bars represent bispecific binding between the IgG4 control (IgG4-7D8 x IgG4-2F8) and the negative control (IgG1-2F8 x IgG1-7D8-K409R). The light gray bars represent the results of simultaneous Fab arm exchange reactions between the indicated IgG1-2F8-K405X mutant and IgG1-7D8-K409R or control.

[0038] Figure 24: 2-MEA-induced Fab arm exchange between IgG1-2F8-Y407X mutant and IgG1-7D8-K409R. The production of bispecific antibodies following the 2-MEA-induced in vitro Fab arm exchange between the IgG1-2F8-Y407X mutant and IgG1-7D8-K409R was determined by ELISA. (A) ELISA analysis of concentration series (total antibody) from 0–20 μg / mL. The positive control is a purified batch of bispecific antibodies derived from IgG1-2F8-F405L x IgG1-7D8-K409R. (B) The exchange is expressed as bispecific binding at an antibody concentration of 20 µg / mL relative to the positive control (black bar). Dark gray bars indicate bispecific binding between the IgG4 control (IgG4-7D8 x IgG4-2F8) and the negative control (IgG1-2F8 x IgG1-7D8-K409R). The light gray bars represent the results of simultaneous Fab arm exchange reactions between the indicated IgG1-2F8-Y407X mutant and IgG1-7D8-K409R or the control.

[0039] Figure 25 In non-reduction ( Figure 25 (A)) and reduction ( Figure 25 (B) Under the condition of SDS-PAGE analysis, bispecific antibodies generated by 2-MEA-induced Fab arm interchange were analyzed.

[0040] Figure 26 Homodimeric raw material IgG1-2F8-F405L ( Figure 26 (B)), Homodimeric raw material IgG1-7D8-K409R ( Figure 26 (A) A mixture of two homodimers (1:1) Figure 26 (C)) and the bispecific product generated by 2-MEA-induced Fab arm exchange between IgG1-2F8-F405L x IgG1-7D8-K409R ( Figure 26 HP-SEC curve (D)

[0041] Figure 27 Homodimeric raw material IgG1-2F8-F405L ( Figure 27 (B)), Homodimeric raw material IgG1-7D8-K409R ( Figure 27 (A) A mixture of two homodimers (1:1) Figure 27 (C)) and the bispecific product generated by 2-MEA-induced Fab arm exchange between IgG1-2F8-F405L x IgG1-7D8-K409R ( Figure 27 (D)) Mass spectrometry (ESI-MS).

[0042] Figure 28 Homodimeric raw material IgG1-2F8-F405L ( Figure 28 (A)), Homodimeric raw material IgG1-7D8-K409R ( Figure 28 (B) A mixture of two homodimers (1:1) Figure 28 (C)) and the bispecific product generated by 2-MEA-induced Fab arm exchange between IgG1-2F8-F405L x IgG1-7D8-K409R ( Figure 28 (D) Capillary isoelectric focusing (cIEF) curve.

[0043] Figure 29 Homodimeric raw material IgG1-2F8-F405L ( Figure 29 (A)), Homodimeric raw material IgG1-7D8-K409R ( Figure 29 (B) A mixture of two homodimers (1:1) Figure 29 (C)) and the bispecific product generated by 2-MEA-induced Fab arm exchange between IgG1-2F8-F405L x IgG1-7D8-K409R ( Figure 29 (D) HPLC-CIEX curve.

[0044] Figure 30 Electrospray ionization mass spectrometry analysis of IgG obtained by co-transfection of expression vectors encoding the heavy and light chains of IgG1-7D8-K409R or IgG1-2F8-F405. Heterodimer peaks are indicated by *. Homodimer peaks are indicated by †.

[0045] Figure 31 The exchange reaction of homodimers IgG1-2F8-F405L and IgG1-7D8-K409R was monitored by high-performance liquid chromatography-cation exchange (HPLC-CIEX) after injection at different intervals.

[0046] Figure 32 : Residual homodimers from the exchange reaction, such as in Figure 32 The results were detected using the CIEX method (indicated by arrows).

[0047] Figure 33 : For example, the production of bispecific antibodies as determined by ELISA at various IgG concentrations, 2-MEA concentrations, incubation temperatures, and times.

[0048] Figure 34 : For example, the production of bispecific antibodies at various IgG concentrations, 2-MEA concentrations, incubation temperatures, and times, as determined by ELISA and compared with an arbitrary control set at 100%.

[0049] Figure 35 For example, the production of bispecific antibodies under various IgG concentrations, 2-MEA concentrations, incubation temperatures, and times can be analyzed by HPLC-CIEX.

[0050] Figure 36 The production of bispecific antibodies following 2-MEA-induced in vitro Fab arm exchange between the IgG1-2F8-L368X mutant and IgG1-7D8-K409R was determined by ELISA using a series of total antibody concentrations ranging from 0-20 μg / mL. Figure 37 (A)). The positive control is a purified batch of bispecific antibody derived from IgG1-2F8-F405L x IgG1-7D8-K409R. Figure 37 (B) Shows bispecific binding at 20 µg / mL compared to the positive control (black bar). Dark gray bars indicate bispecific binding between the IgG4 control (IgG4-7D8 x IgG4-2F8) and the negative control (IgG1-2F8 x IgG1-7D8-K409R). Light gray bars indicate the results of the simultaneous Fab arm exchange reaction between the indicated IgG1-2F8-L368X mutant and IgG1-7D8-K409R.

[0051] Figure 37 The production of bispecific antibodies following 2-MEA-induced in vitro Fab arm exchange between the IgG1-2F8-K370X mutant and IgG1-7D8-K409R was determined by ELISA using a series of total antibody concentrations ranging from 0 to 20 μg / mL. Figure 37 (A)). The positive control is a purified batch of bispecific antibody derived from IgG1-2F8-F405L x IgG1-7D8-K409R. Figure 37 (B) Shows bispecific binding at 20 µg / mL compared to the positive control (black bar). Dark gray bars indicate bispecific binding between the IgG4 control (IgG4-7D8 x IgG4-2F8) and the negative control (IgG1-2F8 x IgG1-7D8-K409R). Light gray bars indicate the results of the simultaneous Fab arm exchange reaction between the indicated IgG1-2F8-D370X mutant and IgG1-7D8-K409R.

[0052] Figure 38 The production of bispecific antibodies following 2-MEA-induced in vitro Fab arm exchange between the IgG1-2F8-D399X mutant and IgG1-7D8-K409R was determined by ELISA using a series of total antibody concentrations ranging from 0-20 μg / mL. Figure 38 (A)). Figure 37 (B) Shows bispecific binding at an antibody concentration of 20 µg / mL compared to the positive control (black bar). Dark gray bars indicate bispecific binding between the IgG4 control (IgG4-7D8 x IgG4-2F8) and the negative control (IgG1-2F8 x IgG1-7D8-K409R). Light gray bars indicate the results of a simultaneous Fab arm exchange reaction between the indicated IgG1-2F8-D399X mutant and IgG1-7D8-K409R.

[0053] Figure 39 Examples include 2-MEA-induced Fab arm interchanges among four different IgG1 mutant combinations, as determined by sandwich ELISA after incubation at 15°C for 0, 30, 60, 105, and 200 minutes.

[0054] Figure 40 For example, the 2-MEA-induced Fab arm interchange between different IgG1 mutant combinations, as determined by sandwich ELISA after incubating the antibody at 15°C for 90 minutes.

[0055] Figure 41 c-Met was phosphorylated using a c-Met-specific antibody. A549 cells were incubated with HGF or a different set of antibodies for 15 minutes. Proteins were separated by SDS-PAGE and transferred to a membrane by Western blotting. Phosphorylated c-Met, total c-Met, and β-actin were detected using antibodies against phosphorylated c-Met, total c-Met, or β-actin.

[0056] Figure 42 Proliferation assay using NCI-H441 cells. NCI-H441 cells were incubated with monovalent bispecific IgG1069 / b12 for 7 days, with no control antibodies (IgG1-069, UniBody-069, IgG1-b12). Cell count was determined and plotted as a percentage of the untreated sample (set as 100%).

[0057] Figure 43Bispecific antibodies derived from Fab arm swaps induced by 2-MEA between IgG1-7D8-F405L or IgG1-2F8-F405L and IgG1-7D8-K409R were used to induce CDC-mediated cell killing in CD20-expressing cells. Their ability to induce CDC in Daudi (A) and Raji (B) cells was tested serially using the antibody concentrations shown. Both cell lines expressed CD20 but not EGFR. Bispecific antibodies derived from Fab arm swaps induced by 2-MEA between IgG1-7D8-F405L and IgG1-7D8-K409R induced CDC-mediated cell killing as effectively as IgG1-7D8. Bispecific antibodies derived from Fab arm swaps induced by 2-MEA between IgG2-2F8-F405L and IgG1-7D8-K409R produced monovalent CD20-binding bispecific antibodies, which slightly affected the induction of CDC-mediated cell killing.

[0058] Figure 44 A431 cell killing induced by pre-incubation with anti-κ-ETA'-conjugated HER2 x HER2 bispecific antibody. Viability of A431 cells after 3 days of incubation with HER2 antibody pre-incubated with anti-κ-ETA'. Cell viability was quantified using Alamarblue. Data shown are fluorescence intensities (FI) of A431 cells treated with anti-κ-ETA'-conjugated HER2 antibody and HER2 x HER2 bispecific antibody in one experiment. Astrocytocin was used as a positive control, while an isotype control antibody was used as a negative control.

[0059] Figure 45 HER2 x HER2 bispecific molecule-induced downregulation of the HER2 receptor. Relative percentage of HER2 expression levels in AU565 cell lysates after 3 days of incubation with 10 μg / mL mAb. HER2 levels were quantified using a HER2-specific capture ELISA as a percentage of inhibition compared to untreated cells. Data shown are the mean plus standard deviation of both experiments.

[0060] Figure 46 Co-localization analysis of HER2 x HER2 bispecific antibody (FITC) and lysosomal marker LAMP1 (Cy5). Intensity of FITC pixels overlapping with Cy5 for various monospecific HER2 antibodies and HER2 x HER2 bispecific antibodies. Figure 46 (B) The FITC pixel intensity in the LAMP1 / Cy5 positive pixels of three different images for each tested antibody was plotted. Monospecific antibodies showed lower FITC pixel intensity in the LAMP1 / Cy5 positive pixels than bispecific antibodies. Figure 46(B) represents the average FITC pixel intensity for each LAMP1 / Cy5 positive pixel calculated from three different images. These results together suggest that, after internalization, higher levels of bispecific antibodies localize Lamp1 / Cy5 positive vesicles compared to monospecific antibodies.

[0061] Figure 47 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 HER2 antibody or HER2 x HER2 bispecific antibody. After 3 days, the number of viable cells was quantified using Alamarblue, and cell viability was expressed as a percentage compared to untreated cells. An allotype control antibody was used as a negative control. Data shown are the percentage of viable AU565 cells compared to untreated cells, measured at 5 ± standard deviation. * indicates only one data point is described.

[0062] Figure 48 Binding of monospecific and bispecific IgG1 antibodies and hinge-deficient IgG1 antibodies to human and mouse FcRn at different pH values. Plates containing human and mouse FcRn were incubated with different monospecific and bispecific IgG1 antibodies or hinge-deficient IgG1 molecules. Binding to FcRn was analyzed by ELISA at 405 nm. (A) Binding of monospecific and bispecific IgG1 antibodies and hinge-deficient IgG1 (Uni-G1) molecules to human FcRn at pH 7.4 and 6.0. Binding to human FcRn was very low at neutral pH. At pH 6.0, the (bispecific) antibodies bound human FcRn efficiently unless they contained the H435A mutation. Hinge-deficient IgG1 (Uni-G1) molecules bound human FcRn with low efficiency. (B) Binding of monospecific and bispecific IgG1 antibodies and hinge-deficient 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 (bispecific), antibodies bind to mouse FcRn very efficiently unless they contain the H435A mutation in both Fab arms. Bispecific molecules with the H435A mutation in only one Fab arm can still bind to mouse FcRn. Hinge-deficient IgG1 (Uni-G1) molecules bind to mouse FcRn with moderate efficiency, while hinge-deficient IgG1 (Uni-G1) bispecific molecules with the H435A mutation in only one Fab arm are slightly less efficient.

[0063] Figure 49 T-cell-mediated cytotoxicity of AU565 cells by Her2 x CD3 bispecific antibody and its N297Q mutant. Invention Details definition The term "immunoglobulin" refers to a class of structurally related glycoproteins composed of two pairs of polypeptide chains: a pair of light (L) low molecular weight chains and a pair of heavy (H) chains, all four chains being linked by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)). In short, each heavy chain typically consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region typically contains three domains: CH1, CH2, and CH3. The heavy chains are linked by disulfide bonds in so-called "hinge regions." Each light chain typically consists of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region typically consists of a domain CL. Typically, amino acid residues in constant regions are numbered according to the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991). Figure 16 Overview of the EU and Kabat numbers (WO 02 / 100348) for different isotypes of antibody 2F8. The VH and VL regions can be further subdivided into highly variable regions (or structurally defined loop sequences and / or formally highly variable regions), also known as complementarity-determining regions (CDRs), which are scattered within more conserved regions called framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901 917 (1987)).

[0064] When used in this paper, the term "Fab arm" refers to a heavy-light chain pair.

[0065] When used in this article, the term "Fc region" refers to an antibody region that contains at least a hinge region, a CH2 domain, and a CH3 domain.

[0066] In the context of this invention, the term "antibody" (Ab) refers to an immunoglobulin molecule, an immunoglobulin molecule fragment, or a derivative thereof, which has the ability to specifically bind to an antigen under typical physiological conditions, has a half-life of a significant time period, such as 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, about 24 hours or longer, about 48 hours or longer, about 3, 4, 5, 6, 7 or more days, or any other relevant functionally defined time period (e.g., sufficient time to induce, promote, enhance, and / or regulate physiological responses associated with antibody-bound antigens and / or sufficient time for antibody to recruit effector activity). The variable regions of the heavy and light chains of an immunoglobulin molecule contain binding domains that interact with the antigen. The constant regions of an antibody (Ab) mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system such as C1q, the first component of the classical complement activation pathway. Antibodies can also be bispecific antibodies, diabody, or similar molecules. The term "bispecific antibody" refers to an antibody that is specific to at least two different epitopes, typically non-overlapping epitopes. As indicated above, unless otherwise stated or explicitly contradicted by the context, the term antibody as used herein includes antibody fragments that retain the ability to bind specifically to antigens. Such fragments can be provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant expression. The antigen-binding function of antibodies has been demonstrated to be exercised by fragments of full-length antibodies, such as the F(ab')2 fragment. It should also be understood that, unless otherwise specified, the term antibody also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like peptides, such as chimeric antibodies and humanized antibodies. Antibodies thus generated can possess any isotype.

[0067] When used in this article, the term "full-length antibody" refers to an antibody containing all the constant and variable domains of the heavy and light chains that are normally found in antibodies of the same type.

[0068] As used in this article, “isotype” refers to the class of immunoglobulins encoded by heavy chain constant region genes (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM).

[0069] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutations). However, the term "human antibody" as used herein is not intended to include antibodies in which a CDR sequence derived from another mammalian species, such as a mouse, has been grafted onto a human frame sequence.

[0070] When used in this context, the term "heavy chain antibody" refers to an antibody consisting of only two heavy chains and lacking the two light chains typically found in antibodies. Heavy chain antibodies, naturally occurring in animals such as camels, can bind antigens, although they possess only the VH domain.

[0071] The term "epitope" refers to a protein determinant that can specifically bind to an antibody. Epitopes are typically composed of surface aggregates of molecules (e.g., amino acids or sugar side chains), usually possessing specific three-dimensional structural features and specific charge properties. The difference between conformational and non-conformational epitopes lies in the fact that, in the presence of denaturing solvents, they bind to the former rather than losing binding to the latter. Epitopes can contain amino acid residues that directly participate in binding (also known as the immunodominant component of the epitope) and other amino acid residues that do not directly participate in binding, such as amino acid residues that are effectively blocked by the specific antigen-binding peptide (in other words, the amino acid residues are located within the footprint of the specific antigen-binding peptide).

[0072] In the case of antibody binding to a predetermined antigen, the term "binding" as used herein generally refers to a binding such that, when the antigen is used as a ligand and the antibody as an analyte, and the binding is measured in a BIAcore 3000 instrument using, for example, surface plasmon resonance (SPR) technology, the affinity is equivalent to approximately 10. -6 M or smaller, such as 10 -7 M or smaller, such as about 10 -8 M or smaller, such as about 10 -9 M or smaller, approximately 10 -10 M or smaller, or about 10 -11 M or even smaller K D And the affinity for binding to the predetermined antigen is equivalent to the following K D It is the K affinity of the antibody for binding to non-specific antigens (e.g., BSA, casein) that are not the intended antigen or closely related antigens. D At most 1 / 10, for example, at most 1 / 100, for example, at most 1 / 1,000, for example, at most 1 / 10,000, for example, at most 1 / 100,000. The amount at lower affinity depends on the antibody's K. D This makes it possible if the antibody's K D If the affinity is very low (i.e., the antibody is highly specific), then the amount of affinity for the antigen can be at most 1 / 10,000 of the amount of affinity for the nonspecific antigen. The term "K" as used in this article... D "(M) refers to the dissociation equilibrium constant of a specific antibody-antigen interaction."

[0073] When used in this article, the term "heterodimeric interaction between the first CH3 region and the second CH3 region" refers to the interaction between the first CH3 region and the second CH3 region in the first CH3 / second CH3 heterodimeric protein.

[0074] When used in this article, the term “homodimal interaction between the first CH3 region and the second CH3 region” refers to the interaction between the first CH3 region and another first CH3 region in the first CH3 / first CH3 homodimer protein and the interaction between the second CH3 region and another second CH3 region in the second CH3 / second CH3 homodimer protein.

[0075] As used herein, "isolated antibody" means that the material has been removed from its original environment (e.g., the natural environment if it is naturally occurring, or the host cell if it is recombinantly expressed). It is also advantageous that the antibody is in a purified form. The term "purified" does not require absolute purity; rather, it is intended as a relative definition, indicating an increase in antibody concentration relative to the concentration of contaminants in the composition compared to the raw material.

[0076] As used herein, the term "host cell" is intended to refer to a cell in which an expression vector, such as an expression vector encoding the antibody of the present invention, has been introduced. Recombinant host cells include, for example, transfected tumors, such as CHO cells, HEK293 cells, NS / O cells, and lymphocytes.

[0077] When used in this article, the term “co-expression” of two or more nucleic acid constructs refers to the expression of two constructs in a single host cell.

[0078] The term "tumor cell protein" refers to proteins located on the surface of tumor cells.

[0079] As used herein, the term "effective cell" refers to immune cells that participate in the effector phase of an immune response, which is distinct from the recognition and activation phases of the immune response. Exemplary immune cells include cells of bone marrow or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, and polymorphonuclear cells such as 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 antibody-dependent cytotoxicity (ADCC), such as natural killer cells, which can induce ADCC. In some embodiments, effector cells can phagocytose target antigens or target cells.

[0080] The term "reduction conditions" or "reduction environment" refers to the conditions or environment in which the substrate (in this case, the cysteine ​​residues in the antibody hinge region) is more likely to be reduced than to be oxidized.

[0081] The term "disulfide isomerization" refers to the interchange of disulfide bonds between different cysteine ​​residues, i.e., the reorganization of disulfide bonds.

[0082] Other aspects and embodiments of the invention As described above, in a first aspect, the present invention relates to an in vitro method for generating heterodimeric proteins, the method comprising the steps of: a) Provide a first homodimeric protein comprising an immunoglobulin Fc region, wherein the Fc region comprises a first CH3 region. b) Provide a second homodimeric protein comprising an immunoglobulin Fc region, wherein the Fc region comprises a second CH3 region. 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 stronger than the homodimer interaction between the first CH3 region and the second CH3 region. c) Incubate the first protein and the second protein together under reducing conditions sufficient to allow cysteine ​​residues in the hinge region to undergo disulfide isomerization. d) Obtain the heterodimeric protein.

[0083] Bispecific antibodies can be used in many ways to generate desired combinations of bispecific antibodies. Besides being able to combine antibodies targeting different antigens in a highly selective manner, they can be used to modify desired properties, such as increasing CDC, by combining two different antibodies targeting the same antigen. Additionally, they can be used to remove some of the agonistic activity of antagonistic antibodies or to convert agonistic antibodies into antagonistic antibodies by preparing their bispecific antibodies from irrelevant (inactive) antibodies.

[0084] In one embodiment, the homodimeric protein is selected from (i) an Fc region, (ii) an antibody, (iii) a fusion protein containing an Fc region, such as an Fc region fused with a receptor, cytokine, or hormone, and (iv) an Fc region conjugated with a prodrug, peptide, drug, or toxin.

[0085] In some embodiments, the first and / or second homodimeric protein, in addition to the Fc region, includes one or more or all of the other regions of the antibody, namely the CH1 region, VH region, CL region, and / or VL region. Therefore, in one embodiment, the first homodimeric protein is a full-length antibody. In another embodiment, the second homodimeric protein is a full-length antibody.

[0086] In key embodiments, both the first and second homodimeric proteins are antibodies, preferably full-length antibodies, and bind to different epitopes. In such embodiments, the resulting heterodimeric protein is a bispecific antibody. The epitopes may be located on different antigens or the same antigen.

[0087] However, in other embodiments, only one homodimeric protein is a full-length antibody, while the other homodimeric protein is not, such as an Fc region lacking a variable region, which is expressed together with another protein or peptide sequence, such as a receptor, cytokine, or hormone, or conjugated to a prodrug, peptide, drug, or toxin. In yet another embodiment, neither homodimeric protein is a full-length antibody. For example, both homodimeric proteins may be Fc regions fused to another protein or peptide sequence, such as a receptor, cytokine, or hormone, or conjugated to a prodrug, peptide, drug, or toxin.

[0088] In one embodiment, the Fc region of the first homodimeric protein is an isotype selected from IgG1, IgG2, IgG3, and IgG4, and the Fc region of the second homodimeric protein is an isotype selected from IgG1, IgG2, IgG3, and IgG4. In a preferred embodiment, the Fc regions of both the first and second homodimeric proteins are IgG1 isotypes. In another preferred embodiment, one of the Fc regions of the homodimeric proteins is an IgG1 isotype, and the other is an IgG4 isotype. In a later embodiment, the resulting heterodimer contains both the Fc regions of IgG1 and IgG4, and therefore may possess intermediate properties of interest in effector function activation. Similar products can be obtained if the first and / or second homodimeric proteins contain a mutation removing the receptor site for Asn-linked glycosylation or are otherwise manipulated to alter the glycosylation properties.

[0089] In another embodiment, the compound is added to the culture medium during antibody production, for example as described in US2009317869 or as described in van Berkel et al. (2010) Biotechnol. Bioeng. 105:350, or, for example as described in Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng 87:614, by using FUT8 knockout cells to glycoengineer one or both of the homodimeric proteins to reduce fucose, thereby improving ADCC. ADCC can alternatively be optimized using the method described in Umaña et al. (1999) Nature Biotech 17:176.

[0090] In yet another implementation, one or both of the homodimeric proteins are modified, for example, as described by Natsume et al. (2009) Cancer Sci. 100:2411, to enhance complement activation.

[0091] In yet another implementation, one or both of the homodimeric proteins have been modified to reduce or increase binding to the neonatal Fc receptor (FcRn) in order to manipulate the serum half-life of the heterodimeric protein.

[0092] In yet another embodiment, one of the homodimer initiating proteins has been modified to not bind protein A, thereby allowing the heterodimer protein to be separated from the homodimer initiating protein by passing the product through a protein A column. This may be particularly useful for embodiments in which an excess of one homodimer protein is used compared to another homodimer protein used as a starting material. In such embodiments, it may be useful to modify the excess homodimer protein to eliminate its ability to bind protein A. After the heterodimerization reaction, the heterodimer protein can be separated from the excess unexchanged homodimer protein by passing through a protein A column.

[0093] In another embodiment, a homodimeric protein is a full-length antibody that recognizes an Fc region or an irrelevant epitope, or a full-length antibody containing a germline-derived sequence that has not undergone high somatic mutations and does not bind to self-antigens. In this type of embodiment, the heterodimeric protein functions as a monovalent antibody. In another embodiment, two homodimeric proteins contain the same heavy chain, but only one homodimeric protein contains a light chain that forms a functional antigen-binding site with said heavy chain, while the other homodimeric protein contains a non-functional light chain that does not bind any antigen in combination with said heavy chain. In this type of embodiment, the heterodimeric protein functions as a monovalent antibody. Such a non-functional light chain can be, for example, a germline-derived sequence that has not undergone high somatic mutations and does not bind to self-antigens.

[0094] The antibodies to be used as the homodimer raw materials of this invention can be prepared, for example, by the hybridoma method first described by Kohler et al., Nature 256, 495 (1975), or by a recombinant DNA method. Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, Clackson et al., Nature 352, 624 628 (1991) and Marks et al., J. Mol. Biol. 222, 581 597 (1991). Monoclonal antibodies can be obtained from any suitable source. Thus, for example, monoclonal antibodies can be obtained from hybridomas prepared from mouse spleen B cells obtained by immunizing mice with a target antigen (which, for example, is in the form of cells expressing the antigen on their surface or nucleic acids encoding the target antigen). Monoclonal antibodies can also be obtained from hybridomas derived from antibody-expressing cells of immunized humans or non-human mammals such as rats, dogs, primates, etc.

[0095] The antibody to be used as the homodimer raw material of this invention may be, for example, a chimeric or humanized antibody. In another embodiment, one or both of the homodimer initiating proteins, except for any specified mutation, are human antibodies. Human monoclonal antibodies may be produced using transgenic or transchromosomal mice such as HuMAb mice (which carry a portion of the human immune system rather than the mouse system). HuMAb mice contain the miniloci of the human immunoglobulin gene, which encodes non-rearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences and targeted mutations that inactivate endogenous μ and κ chain loci (Lonberg, N. et al., Nature 368, 856 859 (1994)). Therefore, mice exhibit reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutations to produce high-affinity human IgG,κ monoclonal antibodies (Lonberg, N. et al. (1994), ibid.; see Lonberg, N. Handbook of Experimental Pharmacology 113, 49 101 (1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 13 65 93 (1995) and Harding, F. and Lonberg, N. Ann. NY Acad. Sci 764 536 546 (1995)). The preparation of HuMAb mice is described in detail in 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).See also US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,789,650, US 5,877,397, US 5,661,016, US 5,814,318, US 5,874,299, US 5,770,429, US 5,545,807, WO 98 / 24884, WO 94 / 25585, WO 93 / 1227, WO 92 / 22645, WO 92 / 03918, and WO 01 / 09187. The spleen cells of these transgenic mice can be used to produce hybridomas that secrete human monoclonal antibodies using well-known techniques.

[0096] Furthermore, the human antibodies of the present invention or antibodies of the present invention derived from other species can be identified using display techniques well known in the art, including but not limited to phage display, retrovirus display, ribosome display, mammalian display and other techniques. The resulting molecules can undergo further maturation, such as affinity maturation, as such techniques are well known in the art.

[0097] In yet another embodiment of the invention, the antibody or a portion thereof, such as one or more CDRs, is of a Camelidae species (see WO2010001251), or of a cartilaginous fish species such as the wrasse, or is a heavy chain antibody or a domain antibody.

[0098] In one embodiment of the method of the present invention, the first and second homodimeric proteins provided in steps a) and b) are purified.

[0099] In one embodiment, the first and / or second homodimeric protein is conjugated to a drug, prodrug, or toxin, or contains a receptor group for the drug, prodrug, or toxin. Such receptor groups may be, for example, non-natural amino acids.

[0100] As described above, the sequences of the first CH3 region and the second CH3 region of the homodimer initiation protein are different, which makes the heterodimer interaction between the first CH3 region and the second CH3 region stronger than the homodimer interaction between the first CH3 region and the second CH3 region.

[0101] In one implementation, the increased strength of heterodimer interactions compared to homodimer interactions is due to CH3 modification, rather than the introduction of covalent bonds, cysteine ​​residues, or charged residues.

[0102] In some embodiments, the product of the present invention is highly stable and does not undergo Fab arm interchange in vivo under mild reducing conditions in vitro or, importantly, after administration to humans in vivo. Therefore, in one embodiment, the heterodimeric interaction between the first and second proteins in the resulting heterodimeric protein prevents Fab arm interchange at 0.5 mM GSH under the conditions described in Example 13.

[0103] In another embodiment, the heterodimeric interaction between the first and second proteins in the resulting heterodimeric protein prevents Fab arm swapping in mice under the conditions described in Example 14.

[0104] In another implementation, for example when determined as described in Example 30, the heterodimeric interaction strength between the first and second proteins in the resulting heterodimeric protein is greater than 2 times, for example, greater than 3 times, or for example, greater than 5 times, the strongest interaction strength among the two homodimeric interactions.

[0105] In yet another embodiment, the sequences of the first CH3 region and the second CH3 region result in a dissociation constant of the heterodimeric interaction between the first and second proteins in the resulting heterodimeric protein being less than 0.05 μmol, as determined as described in Example 30.

[0106] In yet another embodiment, the sequences of the first CH3 region and the second CH3 region are such that, when measured as described in Example 21, the dissociation constant of the interaction between the two homodimers is greater than 0.01 μmol, for example greater than 0.05 μmol, preferably 0.01-10 μmol, for example 0.05-10 μmol, more preferably 0.01-5 μmol, for example 0.05-5 μmol, and even more preferably 0.01-1 μmol, for example 0.05-1 μmol, 0.01-0.5, or 0.01-0.1. Embodiments where the homodimer initiating protein is relatively stable may have the advantages of easier preparation of large quantities of initiating protein and, for example, avoidance of aggregation or misfolding.

[0107] In some implementations, stable heterodimeric proteins can be obtained in high yield using the method of the present invention, based on two homodimeric initiating proteins containing only a few fairly conserved asymmetric mutations in the CH3 region.

[0108] Therefore, in one embodiment, the sequences of the first CH3 region and the second CH3 region contain amino acid substitutions at positions that are not exactly the same.

[0109] The amino acid substituents can be natural or non-natural amino acids. Examples of non-natural amino acids are disclosed, for example, in 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.

[0110] In one implementation, the amino acid is a naturally occurring amino acid.

[0111] In one embodiment, the first homodimeric protein has no more than one amino acid substitution in the CH3 region, and the second homodimeric protein has no more than one amino acid substitution in the CH3 region compared to the wild-type CH3 region.

[0112] In one embodiment, the first homodimeric protein has amino acid substitutions at positions selected from the following: 366, 368, 370, 399, 405, 407, and 409, and the second homodimeric protein has amino acid substitutions at positions selected from the following: 366, 368, 370, 399, 405, 407, and 409, wherein the first homodimeric protein and the second homodimeric protein are not substituted at the same positions.

[0113] In one embodiment, the first homodimeric protein has an amino acid substitution at position 366, and the second homodimeric protein has an amino acid substitution at a position selected from the following: 368, 370, 399, 405, 407, and 409. In one embodiment, the amino acid at position 366 is selected from Arg, Lys, Asn, Gln, Tyr, Glu, and Gly.

[0114] In one embodiment, the first homodimeric protein has an amino acid substitution at position 368, and the second homodimeric protein has an amino acid substitution at a position selected from the following: 366, 370, 399, 405, 407, and 409.

[0115] In one embodiment, the first homodimeric protein has an amino acid substitution at position 370, and the second homodimeric protein has an amino acid substitution at a position selected from the following: 366, 368, 399, 405, 407, and 409.

[0116] In one embodiment, the first homodimeric protein has an amino acid substitution at position 399, and the second homodimeric protein has an amino acid substitution at a position selected from the following: 366, 368, 370, 405, 407, and 409.

[0117] In one embodiment, the first homodimeric protein has an amino acid substitution at position 405, and the second homodimeric protein has an amino acid substitution at a position selected from the following: 366, 368, 370, 399, 407, and 409.

[0118] In one embodiment, the first homodimeric protein has an amino acid substitution at position 407, and the second homodimeric protein has an amino acid substitution at a position selected from the following: 366, 368, 370, 399, 405, and 409.

[0119] In one embodiment, the first homodimeric protein has an amino acid substitution at position 409, and the second homodimeric protein has an amino acid substitution at a position selected from the following: 366, 368, 370, 399, 405, and 407.

[0120] Therefore, in one embodiment, the sequences of the first CH3 region and the second CH3 region contain asymmetric mutations, that is, mutations at different positions in the two CH3 regions, such as a mutation at position 405 in one CH3 region and a mutation at position 409 in the other CH3 region.

[0121] 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 amino acid substitutions at positions selected from the following: 366, 368, 370, 399, 405, and 407.

[0122] 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 yet 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 an amino acid other than Phe, Arg, or Gly at position 405.

[0123] In another embodiment, 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 contains an amino acid other than Phe at position 405 and Lys at position 409. In yet another embodiment herein, 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 contains an amino acid other than Phe, Arg, or Gly at position 405 and Lys at position 409.

[0124] In another embodiment, 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 contains Leu at position 405 and Lys at position 409. In yet another embodiment, the first homodimeric protein contains Phe at position 405 and Arg at position 409, and the second homodimeric protein contains an amino acid other than Phe, Arg, or Gly at position 405 and Lys at position 409.

[0125] 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.

[0126] In yet another embodiment, the first homodimeric protein comprises an amino acid at position 409 that is not Lys, Leu, or Met, and the second homodimeric protein comprises Lys at position 409, Thr at position 370, and Leu at position 405.

[0127] In yet another 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.

[0128] In yet another embodiment, the first homodimeric protein comprises Lys at position 370, Phe at position 405, and Arg at position 409, and the second homodimeric protein comprises Lys at position 409, Thr at position 370, and Leu at position 405.

[0129] In another embodiment, the first homodimeric protein comprises an amino acid at position 409 that is not Lys, Leu, or Met, 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.

[0130] In another embodiment, the first homodimeric protein comprises 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.

[0131] 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.

[0132] 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 Ile at position 350, Thr at position 370, Leu at position 405, and Lys at position 409.

[0133] 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 an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407.

[0134] 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, Met, Asn, Val, or Trp at position 407.

[0135] 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 Gly, Leu, Met, Asn, or Trp at position 407.

[0136] In another embodiment, the first homodimeric protein has a Tyr amino acid at position 407 and an amino acid other than Lys, Leu, or Met at position 409, and the second homodimeric protein has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407 and a Lys amino acid at position 409.

[0137] 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 Ala, Gly, His, Ile, Leu, Met, Asn, Val, or Trp at position 407 and Lys at position 409.

[0138] 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, Met, Asn, or Trp at position 407 and Lys at position 409.

[0139] In another embodiment, the first homodimeric protein has Tyr at position 407 and Arg at position 409, and the second homodimeric protein has an amino acid at position 407 that is not Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr and position 409 that is Lys.

[0140] 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, Asn, Val, or Trp at position 407 and Lys at position 409.

[0141] 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 at position 407 and Lys at position 409.

[0142] 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: (i) an amino acid at position 368 that is not Phe, Leu, or Met, or (ii) In a 370-bit Trp, or (iii) An amino acid at position 399 that is not Asp, Cys, Pro, Glu, or Gln.

[0143] In one embodiment, the first homodimeric protein has Arg, Ala, His, or Gly at position 409, and the second homodimeric protein has: (i) in 368-bit Lys, Gln, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser, Thr, Val, or Trp, or (ii) In a 370-bit Trp, or (iii) In the 399th bit of Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, Trp, Phe, His, Lys, Arg or Tyr.

[0144] In one embodiment, the first homodimeric protein has Arg at position 409, and the second homodimeric protein has: (i) In 368-bit Asp, Glu, Gly, Asn, Arg, Ser, Thr, Val, or Trp, or (ii) In a 370-bit Trp, or (iii) In 399-bit Phe, His, Lys, Arg or Tyr.

[0145] In addition to the amino acid substitutions specified above, the first and second homodimeric proteins may contain other amino acid substitutions, deletions, or insertions relative to the wild-type Fc sequence.

[0146] In yet another embodiment, the first and second CH3 regions, in addition to containing the specified mutation, also contain the sequence elucidated by SEQ ID NO:1 (IgG1m(a)): SEQ ID NO:1: GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK In yet another embodiment, the first and second CH3 regions, in addition to containing the specified mutation, also contain the sequence elucidated by SEQ ID NO:2 (IgG1m(f)): SEQ ID NO:2: GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK In yet another embodiment, the first and second CH3 regions, in addition to containing the specified mutations, also contain the sequence elucidated by SEQ ID NO:3 (IgG1m(ax)): SEQ ID NO:3: GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSLSLSPGK In other embodiments, the provided homodimeric protein may be rat and mouse antibodies exhibiting preferential pairing (as described in Lindhofer et al. (1995) J Immunol 155:219 (see above)), or so-called clonal mutant antibodies that enter the well (as described in U.S. Patent 5,731,168 (see above)). However, sometimes the latter homodimeric initiating protein may be more difficult to prepare because the homodimeric CH3-CH3 interaction is too weak. Therefore, the variants described herein with mutations at positions 350, 370, 405, and 409 are preferred.

[0147] The sequence of the hinge region of the homodimer initiator protein can vary. However, the resulting heterodimer protein may be more stable in some cases, preferably IgG1-like, if the hinge region is not IgG4-like.

[0148] Therefore, in one embodiment, neither the first nor the second homodimeric protein contains the Cys-Pro-Ser-Cys sequence in the (core) hinge region.

[0149] In yet another embodiment, both the first and second homodimeric proteins contain a Cys-Pro-Pro-Cys sequence in the (core) hinge region.

[0150] In many embodiments in which the first and second homodimeric proteins are antibodies, the antibodies also comprise light chains. As explained above, the light chains may be different, i.e., have different sequences and each forms a functional antigen-binding domain with only one heavy chain. However, in another embodiment, the first and second homodimeric proteins are heavy chain antibodies that do not require light chains for antigen binding, see, for example, Hamers-Casterman (1993) Nature 363:446.

[0151] As described above, step c) of the method of the present invention includes incubating the first protein and the second protein together under reducing conditions sufficient to allow cysteine ​​in the hinge region to undergo disulfide isomerization. Examples of suitable conditions are given herein. The minimum requirements for cysteine ​​in the hinge region to undergo disulfide isomerization can vary depending on the homodimer initiating protein, and particularly on the exact sequence in the hinge region. Importantly, the homodimer interactions between the first CH3 region and the second CH3 region are sufficiently weak to allow cysteine ​​in the hinge region to undergo disulfide isomerization under given conditions.

[0152] In one embodiment, the reduction conditions in step c) include the addition of a reducing agent, such as a reducing agent selected from the following: 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dierythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol, preferably a reducing agent selected from the following: 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine.

[0153] In one implementation, the reduction conditions enabling controlled Fab arm interchange are described in terms of the desired redox potential. Glutathione (GSH), a tripeptide, is the dominant low-molecular-weight thiol in cells and controls the thiol-disulfide redox state, essential for normal redox signaling in vivo. Cellular redox homeostasis is achieved by maintaining the thiol-to-disulfide state of both reduced GSH and its oxidized form, GSSG. Numerical values ​​of the reduction potential can be measured, as seen in Rost and Rapoport, Nature 201: 185 (1964) and Aslund et al., J. Biol. Chem. 272:30780-30786 (1997). The redox potential E, taking into account the stoichiometry of the oxidation of two GSHs by each GSSG, is... h To quantitatively measure the redox state, E is calculated using the Nernst equation. h E h = E o + (RT / nF)ln ([GSSG (oxidized form)] / [GSH (reduced form)]) 2 Eo is the standard potential of the redox pair at a defined pH, where 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 estimated value of Eh for the GSH / GSSG pair is -260 to -200 mV (Aw, T., News Physiol. Sci. 18:201-204 (2003)). Therefore, terminally differentiated cells maintain an Eh at the level of -200 mV, while actively proliferating cells maintain a more reducing Eh of approximately -260 mV.

[0154] 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, thus exhibiting a more negative redox potential than DTT. However, precise values ​​have not been reported. Therefore, reduction conditions that allow for controlled Fab arm interchangeability can be described in terms of the desired redox potential Eh, preferably lower than the value obtained in vivo under normal plasma conditions, and higher than the redox potential for reducing antibody disulfide bonds other than those located in the hinge region and involved in heavy chain disulfide bond formation.

[0155] Therefore, in yet another embodiment, step c) is carried out under reduction conditions having a redox potential of less than -50 mV, for example less than -150 mV, preferably -150 to -600 mV, for example -200 to -500 mV, more preferably -250 to -450 mV, for example -250 to -400 mV, and even more preferably -260 to -300 mV.

[0156] In yet another embodiment, step c) comprises incubation at a temperature of at least 20°C for at least 90 minutes in the presence of at least 25 mM 2-mercaptoethylamine or at least 0.5 mM dithiothreitol. Incubation may be performed at a pH of 5 to 8, such as pH 7.0 or pH 7.4.

[0157] In yet another implementation, step d) includes, for example, restoring the conditions to a non-reducing or less reducing state by removing the reducing agent, such as by desalination.

[0158] In some embodiments, the method of the present invention yields 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.

[0159] Post-production is more flexible and easier to control compared to existing co-expression-based approaches.

[0160] The post-production properties of preparing bispecific antibodies via Fab exchange under reducing conditions (e.g., by adding 2-MEA), as disclosed in this paper, make it a highly suitable strategy for (high-throughput) screening of multiple specific combinations for bispecific antibody discovery. Furthermore, the in vitro method can be performed on a library, which allows for greater control, flexibility, and yield over heterodimeric proteins compared to co-expression. Another advantage of this strategy is that screening can be performed on the final therapeutic form, eliminating the need for modification after lead selection.

[0161] As explained above, in another aspect, the method of the present invention can be used for “matrix” screening, i.e., based on two groups of antibodies that produce a large number of different combinations of binding specificity, one group of antibodies having an identical first CH3 region and the other group having an identical second CH3 region, wherein the sequences of the first CH3 region and the second CH3 region are different, and such that the heterodimeric interaction between the first CH3 region and the second CH3 region is stronger than the homodimeric interaction between the first CH3 region and the second CH3 region respectively.

[0162] Therefore, in one embodiment, the present invention relates to a method for selecting heterodimeric proteins having desired properties, the method comprising the following steps: a) Provide a first set of homodimeric proteins containing the Fc region, wherein the homodimeric proteins have identical first CH3 regions. b) Provide a second group of homodimeric proteins containing the Fc region, wherein the homodimeric proteins have an identical second CH3 region. 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 stronger than the homodimer interaction between the first CH3 region and the second CH3 region. c) Incubating the combination of the first group of homodimeric proteins and the second group of homodimeric proteins under reducing conditions sufficient to allow disulfide isomerization of cysteine ​​in the hinge region, thereby generating a set of bispecific antibodies. d) Select any recovery condition until non-reducible. e) The heterodimeric proteome obtained by measuring the given desired properties, and f) Select heterodimeric proteins with desired properties.

[0163] In one embodiment, the present invention relates to a method for selecting bispecific antibodies having desired properties, the method comprising the following steps: a) Provide a first group of homodimeric antibodies comprising antibodies with different variable regions, wherein the antibodies in the first group contain identical first CH3 regions. b) Provide a second group of homodimeric antibodies comprising antibodies with different or identical variable regions, wherein the antibodies in the second group comprise identical second CH3 regions. 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 stronger than the homodimer interaction between the first CH3 region and the second CH3 region. c) Incubate the combination of the first group of antibodies and the second group of antibodies under reducing conditions sufficient to allow disulfide isomerization of cysteine ​​in the hinge region, thereby producing a set of bispecific antibodies. d) Select any recovery condition until non-reducible. e) The bispecific antibody group obtained by measuring the given desired properties, and f) Select bispecific antibodies with the desired properties.

[0164] In one implementation, the second group of homodimeric antibodies has different variable regions.

[0165] In one implementation, the second group of homodimeric antibodies has the exact same variable region, but different amino acid or structural variations outside the antigen-binding region.

[0166] The two groups can be composed in many different ways as desired. Therefore, the two groups can target the same epitope or different epitopes on the same antigen. The two groups can also target different antigens, and each group can contain antibodies that bind to the same or different epitopes on the antigen. Furthermore, one or both groups can each contain antibodies targeting different antigens.

[0167] In another embodiment, the desired properties are cell killing, cell lysis, inhibition of cell proliferation, or binding to cells expressing two antigen targets.

[0168] The screening strategy involves two sets of antibody vectors with a certain range of specificity. One set is cloned into a backbone that can participate in Fab arm interchange with the second backbone under reducing conditions (e.g., by adding 2-MEA). For example, the first set is cloned into the IgG1-F405L backbone, and the second set is cloned into the IgG1-K409R backbone (see Examples 19, 28, 29, 30, 35, 36, 37, 38, and 39 for other possible backbone combinations).

[0169] Each member of the two antibody vector groups was then expressed on a small scale. For example, all antibody vectors were transiently transfected into HEK293 cells and expressed in 2.3 mL of culture in 24-well plates. Alternatively, other suitable (small-scale) preparation systems known in the art can be used.

[0170] The expression antibodies from both groups are then mixed in equimolar pairs in a matrix-like manner. For example, all individual antibodies are purified by small-scale protein A chromatography, with antibody concentrations measured by absorbance at 280 nm. Alternatively, other suitable (small-scale) purification methods or methods known in the art for determining protein concentrations can be used. In another embodiment, the purification step can be omitted if the expression medium does not affect downstream applications. Subsequently, the antibody concentrations are normalized so that a suitable volume contains equimolar amounts of both antibodies. For example, a set of eight antibodies in the F405L backbone is each mixed with eight antibodies in the K409R backbone so that a 100 µl mixture of 64 antibodies contains 80 µg / mL antibody A (F405L) and 80 µg / mL antibody B (K409R). Alternatively, if the strategy includes a downstream bispecific antibody-specific purification step, the step of normalizing antibody amounts can be omitted.

[0171] Add an appropriate amount of reducing agent to the antibody mixture and incubate at a permissible temperature for a suitable period of time. For example, add 25 µl of 125 mM 2-MEA (final concentration 25 mM 2-MEA) to 100 µl of antibody A (F405L) and antibody B (K409R) (80 µg / mL) and incubate overnight at 25°C.

[0172] Therefore, the reducing agent is removed from the mixture (now containing bispecific antibodies) to promote disulfide bond oxidation and avoid interference from the reducing agent during screening assays. For example, 2-MEA is removed by buffer exchange of 64 mixtures using a Zeba Spin 96-well desalting plate (Pierce Biotechnology, #89807). Alternatively, other suitable methods for removing the reducing agent known in the art can be used.

[0173] The bispecific antibodies are then biochemically or functionally characterized to identify lead candidates. For example, the inhibitory effect of 64 bispecific antibodies on the proliferation of suitable cell lines or their binding to suitable cell lines is evaluated. The identified lead candidates are then prepared on a larger scale and characterized in more detail.

[0174] Prepared by co-expression The heterodimeric protein of the present invention can also be obtained by co-expressing constructs encoding the first and second polypeptides in a single cell.

[0175] Therefore, in another aspect, the present invention relates to a method for preparing heterodimeric proteins, the method comprising the following steps: a) Provide a first nucleic acid construct encoding a first polypeptide comprising a first Fc region of an immunoglobulin, wherein the first Fc region comprises a first CH3 region. (b) Provide a second nucleic acid construct encoding a second polypeptide containing a second Fc region of an immunoglobulin, wherein the second Fc region includes a second CH3 region. The sequences of the first CH3 region and the second CH3 region are different, resulting in a stronger heterodimer interaction between the first CH3 region and the second CH3 region than the homodimer interaction between the first CH3 region and the second CH3 region. The first homodimeric protein has an amino acid other than Lys, Leu, or Met at position 409, and the second homodimeric protein has amino acid substitutions at positions selected from the following: 366, 368, 370, 399, 405, and 407.

[0176] and / or The sequences of the first and second CH3 regions are such that, when measured as described in Example 21, the dissociation constant of the homodimer interaction of each CH3 region is 0.01-10 μmol, for example 0.05-10 μmol, more preferably 0.01-5, for example 0.05-5 μmol, even more preferably 0.01-1 μmol, for example 0.05-1 μmol, 0.01-0.5, or 0.01-0.1.

[0177] c) Co-express the first and second nucleic acid constructs in host cells, and d) Obtain the heterodimeric protein from cell culture.

[0178] Suitable expression vectors (including promoters, enhancers, etc.) and suitable host cells for antibody preparation are well known in the art. Examples of host cells include yeast, bacteria, and mammalian cells, such as CHO or HEK cells.

[0179] In one embodiment of the method, the first CH3 region has an amino acid that is not Lys, Leu, or Met at position 409, and the second CH3 region has an amino acid that is not Phe at position 405.

[0180] and / or The sequences of the first and second CH3 regions are such that, when measured as described in Example 21, the dissociation constant of the homodimer interaction of each CH3 region is 0.01-10 μmol, for example 0.05-10 μmol, more preferably 0.01-5, for example 0.05-5 μmol, even more preferably 0.01-1 μmol, for example 0.05-1 μmol, 0.01-0.5, or 0.01-0.1.

[0181] In another implementation of this method: The first CH3 region has an amino acid that is not Lys, Leu, or Met at position 409, and the second CH3 region has an amino acid that is not Phe at position 405, such as an amino acid that is not Phe, Arg, or Gly at position 405. or The first CH3 region has an amino acid that is not Lys, Leu, or Met at position 409, and the second CH3 region has an amino acid that is not Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407.

[0182] In some embodiments, the first and second polypeptides are full-length heavy chains that bind to two antibodies with different epitopes (i.e., the first and second nucleic acid constructs encode full-length heavy chains that bind to two antibodies with different epitopes), thus the heterodimeric protein is a bispecific antibody. This bispecific antibody can be a heavy chain antibody, or the host cell can additionally express one or more nucleic acid constructs encoding a light chain. If only one light chain construct is co-expressed with a heavy chain construct, a functional bispecific antibody is formed only if the light chain sequence allows it to form a functional antigen-binding domain with each heavy chain. If two or more different light chain constructs are co-expressed with a heavy chain, multiple products will be formed.

[0183] In other embodiments, the co-expression method of the present invention includes any other features described in the extra-method section above.

[0184] In another aspect, the present invention relates to expression vectors comprising the first and second nucleic acid constructs specified above. In yet another aspect, the present invention relates to host cells comprising the first and second nucleic acid constructs specified above.

[0185] heterodimeric protein In another aspect, the present invention relates to heterodimeric proteins that are obtained or obtainable by the method of the present invention.

[0186] Furthermore, the method of the present invention can form asymmetric molecules, molecules with different characteristics on each Fab arm or each CH3 domain, or molecules with different modifications throughout the molecule, such as molecules with non-natural amino acid substitutions for conjugation. Such asymmetric molecules can be produced by any suitable combination. This is further illustrated below by some non-limiting examples.

[0187] Bispecific antibodies can be used to pre-target cells, including but not limited to tumor cells. Pre-targeting of target cells can be used for imaging studies or immunotherapy purposes.

[0188] In embodiments of the method of the present invention, the first Fab arm of the bispecific molecule binds to tumor cells, such as tumor cell surface proteins or tumor cell surface carbohydrates, such as one of the tumor cell surface proteins listed herein, and the second Fab arm recognizes radioactive effector molecules, including but not limited to radiolabeled peptides or haptens conjugated or linked (via chelating agents). An example of such radiolabeled peptides is indium-labeled diethylenetriaminepentaacetic acid (anti-DTPA(In) van Schaijk et al. Clin. Cancer Res. 2005; 11: 7230s-7126s). Another example is the use of haptens-labeled particles, such as liposomes, polymeric micelles, or nanoparticles (Jestin et al. Q J Nucl Med Mol Imaging 2007; 51:51-60), carrying a radionuclide such as technetium-99.

[0189] In another implementation, an alternative cell growth inhibitory molecule, such as a toxin, conjugated with a hapten is used.

[0190] In another embodiment of the method of the present invention, the first Fab arm of the bispecific molecule is glycosylated at position N297 (EU number), and the second Fab arm of the bispecific molecule is deglycosylated (aglycosylated, for example by mutating N297 to Q or A or E mutation (Bolt S et al., Eur J Immunol 1993, 23:403-411)). Asymmetric glycosylation in the Fc region affects the interaction with the Fcγ-receptor, the antibody-dependent cytotoxic effect of the antibody (Ha et al., Glycobiology April 5, 2011), and the interaction with other effector functional molecules such as C1q.

[0191] In another embodiment of the method of the present invention, the first Fab arm of the bispecific molecule interacts with FcRn (neonatal Fc receptor) (Roopenian DC et al. Nat. Rev. Immunol. 2007, 7:715-725), and the binding of the second Fab arm to FcRn is impaired by mutating the FcRn interaction site on the molecule, for example by generating the H435A mutation (Shields, RL et al., J Biol Chem, 2001; Firan, M. et al., Int Immunol, 2001).

[0192] In another embodiment of the method of the present invention, the first Fab arm of the bispecific molecule interacts with staphylococcal protein A (Protein A, Deisenhofer et al., Biochemistry 20, 2361-2370 (1981) and streptococcal protein G (Protein G, Derrick et al., Nature 359, 752-754 (1992), which are commonly used for antibody purification, while the interaction between the second Fab arm of the bispecific molecule and protein A or G is impaired. As a result, by purifying the bispecific molecule with protein A or G, it is easy to remove the residual homodimer with impaired protein A or G binding after the exchange into heterodimers.

[0193] In another embodiment, binding to Fcγ-receptors or FcRn is improved or reduced on one of the two Fab arms of the bispecific molecule.

[0194] In another implementation, the binding to C1q is improved or reduced on one of the two Fab arms of the bispecific molecule.

[0195] In another implementation, the protein has been modified to enhance complement activation on one or both of the two Fab arms of the molecule.

[0196] In another implementation, each Fab arm present in the bispecific molecule is derived from a different IgG subclass.

[0197] In another implementation, each Fab arm present in the bispecific molecule carries a different heterogeneous mutation (Jefferis & Lefranc, 2009, MABs 1:332-8).

[0198] In another embodiment, another class of asymmetric immunotherapy molecules is generated by replacing one Fab arm of a bispecific molecule with an immunologically active cytokine, an immunostimulatory cytokine, or an immunosuppressive 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, and IL-13. Alternatively, the molecule may include (growth) factors or hormone stimulants or inhibitors.

[0199] In another embodiment, the Fab of one Fab arm is replaced by a cleavage peptide, which is a peptide capable of cleaving tumor cells, bacteria, fungi, etc., including but not limited to antimicrobial peptides such as Xenopus antimicrobial peptide, melitin, silkworm bactericidal peptide, KLAKKLAK and its variants (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 cationic cleavage peptides (CLYP technology, US2009 / 0269341).

[0200] In another embodiment, one or both of the Fabs on the Fab arm are replaced with receptors for cytokines and / or growth factors, creating so-called decoy receptors, of which Enbrel® (etanercept) targeting TNF-α and VEGF-trap targeting VEGF are well-known examples. Combining these two decoy receptors into a single molecule has shown superior activity compared to single decoy receptors (Jung, J. Biol. Chem. 2011; 286:14410-14418).

[0201] In another implementation, another class of asymmetric immunotherapy molecules is generated by fusing an immunologically active cytokine, an immunostimulatory cytokine, or an immunosuppressive cytokine to one or both N-termini or C-termini of the Fab arms present in the bispecific molecule. This can positively influence the antitumor activity of the bispecific molecule. Examples of such molecules are (but are not limited to) 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), and TNF-α. Alternatively, N-terminal or C-terminal fusions of cytokines such as G-CSF, GM-CSF, IL-10, IL-4, IL-6, or IL-13 can positively influence the effector function of bispecific antibody molecules. Alternatively, (growth) factors or hormone stimulants or inhibitors can be included at the N-terminus or C-terminus of the molecule.

[0202] In another embodiment, the fusion of cleavage peptides, such as antimicrobial peptides like Xenopus antimicrobial peptides, bee venom peptides, silkworm bactericidal peptides, KLAKKLAK and its variants (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 cationic cleavage peptides (CLYP technology, US2009 / 0269341) at the N-terminus or C-terminus of one or both Fab arms, can enhance the activity of the molecule.

[0203] In another implementation, another class of asymmetric immunotherapy molecules is a monovalent antibody that interacts with a selected target via a single Fab arm. In such molecules, one Fab arm present in a bispecific molecule targets the selected target molecule, while the second Fab arm of the molecule either does not carry a Fab or has a non-binding / non-functional Fab, as described with MetMab (Genentech; WO 96 / 38557). Alternatively, monomeric Fc-fusion proteins can be produced, such as those described with factors VIII and IX (Peters et al., Blood 2010; 115: 2057-2064).

[0204] Alternatively, any combination of the above-described asymmetric molecules can be generated by the method of the present invention.

[0205] In another aspect, the present invention relates to heterodimeric proteins comprising a first polypeptide containing a first Fc region of an immunoglobulin, the first Fc region comprising a first CH3 region; and a second polypeptide containing a second Fc region of an immunoglobulin, the second Fc region comprising a second CH3 region, wherein the sequences of the first CH3 region and the second CH3 region are different, such that the heterodimeric interaction between the first CH3 region and the second CH3 region is stronger than the homodimeric interaction between the first CH3 region and the second CH3 region respectively. The first homodimeric protein has an amino acid other than Lys, Leu, or Met at position 409, and the second homodimeric protein has amino acid substitutions at positions selected from the following: 366, 368, 370, 399, 405, and 407. and / or The sequences of the first and second CH3 regions are such that, when measured as described in Example 21, the dissociation constant of the homodimer interaction of each CH3 region is 0.01-10 μmol, for example 0.05-10 μmol, more preferably 0.01-5, for example 0.05-5 μmol, even more preferably 0.01-1 μmol, for example 0.05-1 μmol, 0.01-0.5, or 0.01-0.1.

[0206] In one embodiment, the first CH3 region has a non-Lys, Leu, or Met amino acid at position 409 and the second CH3 region has a non-Phe amino acid at position 405. and / or The sequences of the first and second CH3 regions are such that, when measured as described in Example 21, the dissociation constant of the homodimer interaction of each CH3 region is 0.01-10 μmol, for example 0.05-10 μmol, more preferably 0.01-5, for example 0.05-5 μmol, even more preferably 0.01-1 μmol, for example 0.05-1 μmol, 0.01-0.5, or 0.01-0.1.

[0207] In yet another implementation scheme of the heterodimeric protein The first CH3 region has an amino acid at position 409 that is not Lys, Leu, or Met, and the second CH3 region has an amino acid at position 405 that is not Phe, such as an amino acid at position 405 that is not Phe, Arg, or Gly. or The first CH3 region has an amino acid at position 409 that is not Lys, Leu, or Met, and the second CH3 region has an amino acid at position 407 that is not Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr.

[0208] In other embodiments, the heterodimeric protein of the present invention includes any other features described above regarding the preparation method.

[0209] Therefore, in another embodiment of the heterodimeric protein of the present invention, the first polypeptide is the full-length heavy chain of an antibody (preferably a human antibody).

[0210] In another embodiment of the heterodimeric protein of the present invention, the second polypeptide is the full-length heavy chain of an antibody (preferably a human antibody).

[0211] In another embodiment of the heterodimeric protein of the present invention, the first and second polypeptides are both full-length heavy chains of two antibodies (preferably two human antibodies binding to different epitopes), thus the resulting heterodimeric protein is a bispecific antibody. This bispecific antibody can be a heavy chain antibody, or an antibody that, in addition to the heavy chain, contains two full-length light chains (which may be the same or different).

[0212] In another embodiment of the heterodimeric protein of the present invention, the Fc region of the first polypeptide is an isotype selected from IgG1, IgG2, IgG3 and IgG4 (except for a specified mutation), and the Fc region of the second polypeptide is an isotype selected from IgG1, IgG2, IgG3 and IgG4 (except for a specified mutation).

[0213] In another embodiment of the heterodimeric protein of the present invention, the Fc regions of both the first polypeptide and the second polypeptide are of the IgG1 isotype.

[0214] In another embodiment of the heterodimeric protein of the present invention, one Fc region of the polypeptide is of the IgG1 isotype and the other is of the IgG4 isotype.

[0215] In another embodiment of the heterodimeric protein of the present invention, the increased strength of the heterodimeric interaction compared to the individual homodimeric interactions is attributed to CH3 modification, rather than to the introduction of covalent bonds, cysteine ​​residues, or charged residues.

[0216] In another embodiment of the heterodimeric protein of the present invention, the heterodimeric interaction between the first and second polypeptides in the heterodimeric protein prevents Fab arm swapping from occurring at 0.5 mM GSH under the conditions described in Example 13.

[0217] In another embodiment of the heterodimeric protein of the present invention, the heterodimeric interaction between the first and second polypeptides in the resulting heterodimeric protein prevents Fab arm interchange in mice under the conditions described in Example 14.

[0218] In another embodiment of the heterodimeric protein of the present invention, the first CH3 region contains a Phe amino acid at position 405 and a non-Lys, Leu, or Met amino acid at position 409, and the second CH3 region contains a non-Phe amino acid at position 405 and a Lys amino acid at position 409.

[0219] In another embodiment of the heterodimeric protein of the present invention, the first CH3 region contains Phe at position 405 and an amino acid other than Lys, Leu, or Met at position 409, and the second CH3 region contains Leu at position 405 and Lys at position 409.

[0220] In another embodiment of the heterodimeric protein of the present invention, the first CH3 region contains Phe at position 405 and Arg at position 409, and the second CH3 region contains Leu at position 405 and Lys at position 409.

[0221] In another embodiment of the heterodimeric protein of the present invention, the first CH3 region contains an amino acid at position 409 that is not Lys, Leu, or Met, and the second CH3 region contains 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.

[0222] In another embodiment of the heterodimeric protein of the present invention, the first CH3 region contains Arg at position 409, and the second CH3 region contains 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.

[0223] In another embodiment of the heterodimeric protein of the present 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 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.

[0224] In another embodiment of the heterodimeric protein of the present 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 comprises Ile at position 350, Thr at position 370, Leu at position 405, and Lys at position 409.

[0225] In another embodiment of the heterodimeric protein of the present invention, neither the first polypeptide nor the second polypeptide contains the Cys-Pro-Ser-Cys sequence in the hinge region.

[0226] In another embodiment of the heterodimeric protein of the present invention, both the first polypeptide and the second polypeptide contain a Cys-Pro-Pro-Cys sequence in their hinge regions.

[0227] In another embodiment of the heterodimeric protein of the present invention, the first polypeptide and / or the second polypeptide contains a mutation that removes the receptor site for Asn-linked glycosylation.

[0228] target antigen As explained above, in an important embodiment of the present invention, the heterodimeric protein is a bispecific antibody containing two variable regions with different binding specificities (i.e., binding to different epitopes).

[0229] In principle, any specific combination is possible. As mentioned above, bispecific antibodies have the potential to overcome some limitations of monospecific antibodies. One potential limitation of monospecific antibodies is their lack of specificity for the desired target cells, because the target antigen is expressed on other cell types to which the antibody does not intend to bind. For example, a target antigen overexpressed on tumor cells may also be expressed in healthy tissue, which can lead to undesirable side effects after treatment with an antibody targeting that antigen. Bispecific antibodies, which have further specificity for proteins expressed only on the target cell type, have the potential to improve specific binding to tumor cells.

[0230] Therefore, in one embodiment of the invention, the first and second epitopes are located on the same cell, such as a tumor cell. Suitable targets on tumor cells include, but are not limited to, the following targets: erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD19, CD20, CD4, CD38, CD138, CXCR5, c-Met, HERV-capsule protein, periosteal protein, Biggs3, SPARC, BCR, CD79, CD37, EGFrvIII, L1-CAM, AXL, tissue factor (TF), CD74, EpCAM, and MRP3. Possible combinations of tumor cell targets include, but are not limited to: 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.

[0231] In yet another embodiment, the first and second epitopes may be located on the same target antigen, wherein the positions of the two epitopes on the target antigen are such that binding of an antibody to one epitope does not interfere with binding of the antibody to the other epitope. In yet another embodiment, the first and second homodimeric proteins are antibodies that bind to two different epitopes located on the same target antigen but with different target cell, such as tumor cell, killing modes of action. For example, in one embodiment, the target antigen is erbB2 (HER2), and the bispecific antibody combination is a combination of pertuzumab and trastuzumab antigen-binding sites. In another embodiment, the target antigen is erbB1 (EGFr), and the bispecific antibody combination is a combination of zalcimenumab and nimotuzumab antigen-binding sites.

[0232] Bispecific antibodies can also serve as mediators to retarget effector mechanisms onto disease-related tissues such as tumors. Therefore, in another embodiment, the first or second epitope is located on tumor cells, such as tumor cell proteins or tumor cell carbohydrates, while the other epitope is located on effector cells.

[0233] In one implementation, the effector cells are T cells.

[0234] Potential targets on effector cells include the following: FcγRI (CD64): expressed on monocytes, macrophages, and activated neutrophils; FcγRIII (CD16): expressed on natural killer cells and macrophages; CD3: expressed on circulating T cells; CD89: expressed on PMNs (polymorphonuclear neutrophils), eosinophils, monocytes, and macrophages; CD32a: expressed on macrophages, neutrophils, and eosinophils; and FcεRI expressed on basophils and mast cells. In one embodiment, the epitope is located on CD3 expressed on T cells.

[0235] In another embodiment, the first antibody has binding specificity to the pathogenic microorganism, and the second antibody has binding specificity to effector cell proteins (such as CD3, CD4, CD8, CD40, CD25, CD28, CD16, CD89, CD32, CD64, FcεRI, or CD1).

[0236] Additionally, bispecific antibodies can be used to make chemotherapeutic agents more specifically target the cells to which the chemotherapeutic agent is effective. Therefore, in one embodiment, one of the homodimeric proteins is an antibody that recognizes small molecules or peptides, or is capable of forming covalent bonds with such molecules, for example, according to the principles described in Rader et al., (2003) PNAS 100:5396. In yet another embodiment of the method of the invention, the first antibody has binding specificity (i.e., binding to epitopes on said tumor cells or tumor cell surface proteins, such as erbB1, erbB2, erbB3, erbB4, EGFR3vIII, CEA, MUC-1, CD19, CD20, CD4, CD38, EPCAM, c-Met, AXL, L1-CAM, tissue factor, CD74, or CXCR5) to tumor cells or tumor cell surface proteins, and the second antibody has binding specificity to chemotherapeutic agents such as toxins (including radiolabeled peptides), drugs, or prodrugs.

[0237] Bispecific antibodies can also be used to target vesicles, such as electron-dense vesicles, or microcells containing toxins, drugs, or prodrugs targeting tumors. See, for example, MacDiarmid et al. (2009) Nature Biotech 27:643. Microcells are nonchromosomal cells, which are abnormal cell division products that do not contain chromosomal DNA. Therefore, in another embodiment, the first or second epitope is located on tumor cells, such as tumor cell proteins or tumor cell carbohydrates, and the other epitope is located on electron-dense vesicles or microcells.

[0238] Furthermore, the serum half-life of the antibody can be altered by including the binding specificity to serum proteins in the bispecific antibody. For example, the serum half-life can be prolonged by including the binding specificity to serum albumin in the bispecific antibody. Therefore, in yet another embodiment of the method of the present invention, the first antibody has binding specificity to tumor cells or tumor cell proteins such as erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD19, CD20, CD4, CD38, CD138, CXCR5, c-Met, HERV-enveloping protein, periosteum protein, Biggs3, SPARC, BCR, CD79, CD37, EGFrvIII, L1-CAM, AXL, tissue factor (TF), CD74, EpCAM or MRP3, CEA, and the second antibody has binding specificity to blood proteins such as serum albumin. The second binding specificity can also be used to target the antibody to specific tissues, such as the central nervous system or the brain (crossing the blood-brain barrier). Therefore, in another embodiment of the method of the present invention, the first antibody has binding specificity to brain-specific targets, such as amyloid-β (e.g., for the treatment of Alzheimer's disease), Her-2 (e.g., for the treatment of brain metastases from breast cancer), EGFr (e.g., for the treatment of primary brain cancer), Nogo A (e.g., for the treatment of brain injury), TRAIL (e.g., for the treatment of HIV), α-synuclein (e.g., for the treatment of Parkinson's disease), Htt (e.g., for the treatment of Huntington's disease), prions (e.g., for the treatment of mad cow disease), and West Nile virus protein; the second antibody has binding specificity to blood-brain barrier proteins, such as transferrin receptor (TfR), insulin receptor, melanin transferrin receptor (MTfR), lactoferrin receptor (LfR), apolipoprotein E receptor 2 (ApoER2), LDL receptor-associated proteins 1 and 2 (LRP1 and LRP2), receptor for advanced glycation end products (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).

[0239] The binding specificity to blood-brain barrier proteins can also be used to target another non-antibody molecule to specific tissues, such as the central nervous system or the brain (which crosses the blood-brain barrier). Therefore, in yet another embodiment, one homodimer is a full-length antibody with binding specificity to blood-brain barrier proteins (such as TfR, insulin receptor, MTfR, LfR, ApoER2, LRP1, LRP2, RAGE, DTR (= HB-EGF), or gp190), and the other homodimer is an Fc region of another protein linked to its N- or C-terminus, such as cytokines, soluble receptors, or other proteins, such as VIP (vasoactive intestinal peptide), BDNF (brain-derived neurotrophic factor), FGF (fibroblast growth factor), various FGFs, EGF (epidermal growth factor), PNA (peptide nucleic acid), NGF, etc. (Nerve growth factor), neurotrophic protein (NT)-3, NT-4 / 5, glial cell-derived neurotrophic factor, ciliary neurotrophic factor, neurotrophic protein, neuromodulatory protein, interleukin, transforming growth factor (TGF)-α, TGF-β, erythropoietin, hepatocyte growth factor, platelet-derived growth factor, artemin, persephin, cytokinin, cardiotrophin-1, stem cell factor, mid-term factor, pleiotropic factor, bone morphogenetic protein, brain signaling protein, brain signaling protein, leukocyte inhibitory factor, α-L-iduronidase, iduronate-2-sulfatase, N-acetyl-galactosamine-6-sulfatase, arylsulfatase B, acid α-glucosidase or sphingomyelinase (Pardridge, Bioparmaceutical drug targeting the brain, Journal of Drug Targeting 2010, 1-11; Pardridge, Re-engineering Biopharmaceuticals) for delivery to the brain with molecular Trojan horses (a re-engineered biological drug for delivery to the brain using molecular Trojan horses). Bioconjugate Chemistry 2008, 19: 1327-1338.

[0240] Furthermore, the second binding specificity can be used to target coagulation factors to a particularly desired site of action. For example, a bispecific antibody having a first binding specificity to tumor cells and a second binding specificity to coagulation factors can direct blood clotting towards the tumor, thereby stopping tumor growth. Therefore, in yet another embodiment of the method of the present invention, the first antibody has binding specificity to tumor cells or tumor cell proteins such as erbB1, erbB2, erbB3, erbB4, MUC-1, CD19, CD20, CD4, or CXCR5, and the second antibody has binding specificity to proteins involved in coagulation, such as tissue factor.

[0241] Other binding-specific combinations of particular interest include: CD3 + HER2, CD3 + CD20, IL-12 + IL18, IL-1a + IL-1b, 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, and 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-Met, c-Met + non-binding arm, and combinations of G-protein-coupled receptors.

[0242] In yet another embodiment, the bispecific antibody of the present invention can be used to eliminate pathogens, pathogenic autoantibodies, or harmful compounds such as circulating venom and toxins by targeting erythrocytes, essentially as described in Taylor et al., J. Immunol. 158:842-850 (1997) and Taylor and Ferguson, J. Hematother. 4:357-362, 1995. The first epitope is located on erythrocyte (erythrocyte) proteins (including but not limited to erythrocyte complement receptor 1), and the second epitope is located on the compound or organism to be targeted for elimination.

[0243] In another embodiment, the second Fab arm contains a fusion protein representing an autoantigen or a conjugation site such as dsDNA that links an autoantigen. Therefore, the bispecific antibody of this invention, targeting pathogens, autoantibodies, or harmful compounds, followed by erythrocyte-mediated clearance, can have therapeutic efficacy in the treatment of various diseases and syndromes.

[0244] Adhesion In other embodiments of the invention, the first and / or second homodimeric protein is linked to a compound selected from toxins (including radioisotopes), prodrugs, or drugs. Such compounds can, for example, more effectively kill target cells in cancer therapy. The resulting heterodimeric protein is thus an immunoconjugate. Alternatively, a compound may be coupled to the resulting heterodimeric protein, i.e., after Fab arm exchange has occurred.

[0245] Suitable compounds for forming the immunoconjugates of this invention include paclitaxel, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, and dihydroxy anthracin. Dione), mitoxantrone, styromycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabin, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (e.g., nitrogen mustard, 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 (e.g., actinomycin D). (Formerly known as actinomycin), bleomycin, daunorubicin (formerly known as doxorubicin), doxorubicin, idarubicin, styromycin, mitomycin, mitoxantrone, procainamide, atrazomycin (AMC), diphtheria toxin and related molecules (e.g., diphtheria A chain and its active fragments and hybrid molecules), ricin toxin (e.g., ricin A or deglycosylated ricin A chain toxin), cholera toxin, shiga-like toxins (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alarin, soapwort protein, lotus root toxin II, gelanin, acacia root toxin A chain, lotus root toxin II A chain, α-broomycin, tung oil (Aleurites fordii) protein, carnation toxin, American pokeweed ( Phytolacca americana Proteins (PAPI, PAPII, and PAP-S), bitter melon ( momordica Charantia Inhibitors, jatropha toxin, croton toxin, soapwort ( sapaonaria officinalisInhibitors, white azadirachtin, mitogellin, localized aspergillin, phenolmycin, and enoxacin toxin. Other suitable conjugated molecules include ribonuclease (RNase), deoxyribonuclease I, staphylococcal enterotoxin-A, pokeweed antiviral protein, diphtheria toxin, Pseudomonas endotoxin, maytansine alkaloids, auristatins (MMAE, MMAF), cucurbitacin and Duocarmycin analogues (Ducry and Stump, Bioconjugate Chem. 2010, 21: 5-13), Dolostatin-10, Dolostatin-15, irinotecan or its active metabolite SN38, and pyrrolobenzodiazepines (PBDs).

[0246] In yet another embodiment of the invention, the first and / or second homodimeric protein is linked to an α emitter, including but not limited to thorium-227, radium-223, bismuth-212, and actinium-225.

[0247] In yet another embodiment of the invention, the first and / or second homodimeric protein is linked to a β-emitting radionuclide, including but not limited to iodine-313, yttrium-90, fluorine-18, rhenium-186, gallium-68, technetium-99, indium-111, and lutetium-177.

[0248] In another embodiment, the compound to be conjugated comprises a nucleic acid or a nucleic acid-related molecule. In one aspect of the invention, the conjugated nucleic acid is a cytotoxic ribonuclease, an antisense nucleic acid, an inhibitory RNA molecule (such as siRNA), or an immunostimulatory nucleic acid (such as an immunostimulatory DNA molecule containing a CpG motif).

[0249] Any method known in the art for conjugation may be employed, including those described in 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. and Cytochem. 30, 407 (1982). Conjugates can be prepared by chemically conjugating other moieties to the N-terminal or C-terminal side of the protein (see, for example, Antibody Engineering Handbook, edited by Osamu Kanemitsu, published by Chijin Shokan (1994)). Where appropriate, such conjugated antibody derivatives can also be generated by conjugation at internal residues or sugars. The agent may be conjugated directly or indirectly to the protein of the invention. One example of indirect conjugation of a second agent is conjugation via a spacer base. The linking techniques for drug conjugates have recently been summarized in Ducry and Stump (2010) Bioconjugate Chem. 21: 5.

[0250] Composition and Use In another key aspect, the present invention relates to pharmaceutical compositions comprising the heterodimeric protein of the present invention as described herein and a pharmaceutically acceptable carrier.

[0251] Pharmaceutical compositions can be formulated using conventional techniques, such as those disclosed in the following literature: Remington: The Science and Practice of Pharmacy, 19th edition, edited by Gennaro, Mack Publishing Co., Easton, PA, 1995. The pharmaceutical compositions of the present invention may contain, for example, diluents, fillers, salts, buffers, detergents (e.g., nonionic detergents, such as Tween-20 or Tween-80), stabilizers (e.g., amino acids without sugars or proteins), preservatives, tissue fixatives, solubilizers, and / or other substances suitable for inclusion in the pharmaceutical composition.

[0252] Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coating materials, antibacterial and antifungal agents, isotonic agents, antioxidants, and absorption retardants that are physiologically compatible with the compounds of the present invention. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, saline, phosphate buffered solutions, ethanol, glucose, and polyols (e.g., glycerol, propylene glycol, polyethylene glycol). Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersants and sterile powders for the temporary formulation of sterile injectable solutions or dispersants. Appropriate flowability can be maintained, for example, by using coating materials (e.g., lecithin), by maintaining the desired particle size in the case of dispersants, and by using surfactants.

[0253] The pharmaceutical compositions of the present invention may further comprise pharmaceutically acceptable antioxidants, such as (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0254] The pharmaceutical compositions of the present invention may also contain isotonic agents, such as sugars, polyols, such as mannitol, sorbitol, glycerol, or sodium chloride.

[0255] The pharmaceutical compositions of the present invention may also contain one or more adjuvants, such as preservatives, wetting agents, emulsifiers, dispersants, or buffers, suitable for the chosen route of administration and capable of improving the shelf life or effectiveness of the pharmaceutical composition. The compounds of the present invention can be prepared together with a carrier that prevents rapid release of the compound, such as controlled-release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable biocompatible polymers such as ethyl ethylene glycol, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid alone or with waxes, or other substances well known in the art. Methods for preparing such formulations are generally known to those skilled in the art.

[0256] As needed, a sterile injectable solution can be prepared by incorporating the required amount of the active compound, along with one or a combination of the ingredients listed above, into a suitable solvent, followed by sterile microfiltration.

[0257] The actual dose level of the active ingredient in a pharmaceutical composition can be varied to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response for a specific patient, composition, and route of administration while remaining non-toxic to the patient. The selected dose level will depend on several pharmacokinetic factors, including the activity of the specific composition of the invention used, the route of administration, the time of administration, the excretion rate of the specific compound used, the duration of treatment, other drugs, compounds, and / or substances used in combination with the specific composition used, and factors well-known in the medical field such as the age, sex, weight, condition, general health status, and medical history of the patient to be treated.

[0258] The pharmaceutical composition can be administered via any suitable route and method. In one embodiment, the pharmaceutical composition of the invention is administered parenterally. As used herein, "parenteral administration" means a non-enteral and non-local administration method, typically administered by injection, including intradermal, intravenous, intramuscular, intra-arterial, intrasheath, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural, and intrasternal injections and infusions.

[0259] In one embodiment, the pharmaceutical composition is administered via intravenous or subcutaneous injection or infusion.

[0260] In one key aspect, the present invention relates to heterodimeric proteins of the present invention used as pharmaceuticals, such as the bispecific antibodies of the present invention. The heterodimeric proteins of the present invention can be used for a variety of purposes. Specifically, as explained above, the heterodimeric proteins of the present invention can be used to treat various forms of cancer, including metastatic and refractory cancers.

[0261] Therefore, in one respect, the present invention relates to a method for inhibiting the growth and / or proliferation of tumor cells and / or killing tumor cells, comprising administering the heterodimeric protein of the present invention as described herein to an individual in need.

[0262] In another embodiment, the heterodimeric protein of the present invention is used to treat immune diseases and autoimmune diseases, inflammatory diseases, infectious diseases, cardiovascular diseases, CNS and musculoskeletal diseases.

[0263] Adjust the dosage regimen in the above treatment methods and uses to provide the best desired response (e.g., therapeutic response). For example, a single bolus injection may be given, several fractions may be given over time, or the dose may be reduced or increased proportionally according to the severity of the treatment situation.

[0264] The effective dose and dosage regimen of the heterodimeric protein depend on the disease or condition to be treated and can be determined by those skilled in the art. Exemplary, non-limiting ranges for the therapeutically effective amount of the bispecific antibody of this invention are about 0.1-100 mg / kg, such as about 0.1-50 mg / kg, such as about 0.1-20 mg / kg, such as about 0.1-10 mg / kg, such as about 0.5, such as 0.3, about 1, about 3, about 5, or about 8 mg / kg.

[0265] A physician or veterinarian with ordinary skills in the art can readily determine and prescribe an effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian may begin administering a dose of the heterodimeric protein in the pharmaceutical composition below the level required to achieve the desired therapeutic effect, gradually increasing the dose until the desired effect is achieved. Generally, the appropriate daily dose of the compositions of the present invention is the amount of the lowest dose of compound that effectively produces a therapeutic effect. Administration may be, for example, parenteral, intravenous, intramuscular, or subcutaneous.

[0266] The heterodimeric protein of the present invention can also be administered prophylactically to reduce the risk of developing diseases such as cancer, delay the onset of events during disease progression, and / or reduce the risk of relapse when a disease such as cancer is in remission.

[0267] The heterodimeric protein of the present invention, such as the bispecific antibody, can also be administered in combination therapy, i.e., in combination with other therapeutic agents related to the disease or condition to be treated. Thus, in one embodiment, a drug containing a heterodimeric protein is combined with one or more other therapeutic agents, such as cytotoxic agents, chemotherapeutic agents, or anti-angiogenic agents. Such combination administration can be simultaneous, single, or sequential. In yet another embodiment, the present invention provides a method of treating or preventing a disease, such as cancer, comprising administering to a subject in need a therapeutically effective amount of a heterodimeric protein, such as the bispecific antibody of the present invention, in combination with radiotherapy and / or surgery.

[0268] The heterodimeric proteins of the present invention, such as bispecific antibodies, can also be used for diagnostic purposes. Example

[0269] Example 1: Expression vector for expressing human IgG1-2F8 and IgG1-7D8 The VH and VL coding regions of HuMab 2F8 (WO 02 / 100348) and HuMab 7D8 (WO 04 / 035607) were cloned into the expression vector pConG1f (containing the genomic sequence of the human IgG1f isotype constant region (Lonza Biologics)) for the preparation of the human IgG1 heavy chain, and cloned into pConKappa (containing the human κ light chain constant region (Lonza Biologics)) for the preparation of the κ light chain. For the IgG4 antibody, the VH region was inserted into the pTomG4 vector (containing the genomic sequence of the human IgG4 constant region in the pEE12.4 vector (Lonza Biologics)). Alternatively, in subsequent constructs, a vector containing the complete codon-optimized coding region of the heavy chain (IgG1 or IgG4) in the pEE12.4 vector or the human κ light chain of HuMab 2F8 or HuMab 7D8 in the pEE6.4 vector (Lonza Biologics) was used.

[0270] Example 2: Expression vectors for expressing hinge-deficient IgG1-2F8 and CH2-CH3 fragments of human IgG1 and IgG4 containing specific mutations. To introduce mutations in the hinge and CH3 region of the antibody heavy chain, use the Quickchange Directed Mutagenesis Kit (Stratagene, La Jolla, CA) as recommended by the manufacturer. Alternatively, synthesize the complete construct or clone the VH region into a vector that already contains the specific amino acid encoding the substitution.

[0271] Constructs encoding the CH2 and CH3 fragments with fully codon-optimized designs were constructed by PCR or synthesis. These constructs possess an N-terminal signal peptide and a 6-amino acid His tag, containing amino acids 341-447 of the human IgG1 / 4 constant region. The constructs were cloned into pEE12.4.

[0272] To construct a hinge-deficient IgG1 (Uni-G1) molecule, a synthetic DNA construct encoding the Uni-G1 form of an EGFR-specific human IgG1 isotype was prepared. The native hinge region (e.g., defined by the hinge exon) was deleted in this construct. An additional Ser-to-Cys mutation was created at position 158 in the IgG1 construct to salvage the Cys bond between the HC and LC chains in this isotype. The protein sequence is shown below. The construct was inserted into the pEE6.4 vector and named pHG1-2F8.

[0273] QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWDDGSYKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGITMVRGVMKD YFDYWGQGTLVTVSSASTKGPSVFPLAPCSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KRVAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Example 3: Expression vector for expressing rhesus monkey IgG4-2F8 and IgG4-7D8 Vectors containing the coding regions of the Chinese rhesus monkey IgG4 heavy and κ light chains and the VH and VL regions of Humab 2F8 and 7D8 were synthesized, fully codon-optimized, and inserted into pEE12.4 (heavy chain) and pEE6.4 (light chain). The heavy chain constant region sequence used (based on the sequence described by Scinicariello et al., Immunology 111: 66-74, 2004) is as follows (aligned with human sequence): The rhesus monkey light chain constant region (CL) sequence used is: AVAAPSVFIFPPSEDQVKSGTVSVVCLLNNFYPREASVKWKVDGVLKTGNSQESVTEQDSKDNTYSLSSTLTLSSTDYQSHNVYACEVTHQGLSSPVTKSFNRGEC Example 4: Preparation of antibodies by transient expression in HEK-293F cells Antibodies were prepared under serum-free conditions using 293fectin (Invitrogen) by co-transfection of relevant heavy and light chain expression vectors in HEK-293F cells (Invitrogen) according to the manufacturer's instructions.

[0274] Example 5: Purification of IgG1 and IgG4 antibodies IgG1 and IgG4 antibodies were purified by protein A affinity chromatography. Cell culture supernatant was filtered through a 0.20 µM dead-end filter and then loaded onto a 5 mL protein A column (rProtein A FF, GE Healthcare, Uppsala, Sweden). IgG was eluted with 0.1 M lemon-NaOH, pH 3. The eluent 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 aseptically filtered through a 0.20 µM dead-end filter. The concentration of purified IgG was determined by turbidimetry and absorbance at 280 nm. The purified protein was analyzed by SDS-PAGE, IEF, mass spectrometry, and glycoanalysis.

[0275] Example 6: Purification of the CH2-CH3 fragment His-labeled CH2-CH3 protein was subjected to immobilization of metal ions (Ni). 2+ Purified by affinity chromatography (Macherey-Nagel GmbH, Düren, Germany), desalted using a PBS-equilibrated PD-10 column (GE Healthcare), and aseptically filtered through a 0.2 µM dead-end filter. The concentration of the purified protein was determined by absorbance at 280 nm. The quality of the purified protein was analyzed by SDS-PAGE.

[0276] Example 7: Generation of bispecific antibodies via GSH-induced Fab arm exchange between human and rhesus monkey IgG4 antibodies As described above, WO 2008119353 (Genmab) describes an in vitro method for preparing bispecific antibodies, wherein the bispecific antibodies are formed by "Fab arm" or "half-molecule" exchange (exchange of heavy chain and linked light chain) between two monospecific IgG4 antibodies or IgG4-like antibodies after incubation under reducing conditions. This Fab arm exchange reaction is the result of a disulfide bond isomerization reaction, in which the inter-heavy chain disulfide bonds in the hinge region of the monospecific antibody are reduced, and the resulting free cysteine ​​residues form new inter-heavy chain disulfide bonds with cysteine ​​residues of another antibody molecule with different specificities. The resulting product is a bispecific antibody with two Fab arms having different sequences.

[0277] To test Fab arm interchangeability between human and rhesus monkey IgG4 antibodies, all possible combinations of the two antibodies were prepared using human IgG4-2F8 (anti-EGFR), human IgG4-7D8 (anti-CD20), rhesus monkey IgG4-2F8, and rhesus monkey IgG4-7D8. For in vitro Fab arm interchangeability, the antibody mixture (containing each antibody at a final concentration of 4 µg / mL in 0.5 mL PBS) was incubated with 0.5 mM reduced glutathione (GSH) at 37°C for 24 hours. To terminate the reduction reaction, 0.5 mL of PBS / 0.05% Tween 20 (PBST) was added to the reaction mixture.

[0278] The presence of bispecific antibodies was determined by sandwich enzyme-linked immunosorbent assay (ELISA) to assess bispecific binding. An ELISA plate (Greiner bio-one, Frickenhausen, Germany) was coated overnight at 4°C with 2 μg / mL (100 μL / well) of recombinant EGFR extracellular domain in PBS. The plate was washed once with PBST. Serially diluted antibody samples (0–1 μg / mL, in 3-fold dilutions) were transferred to the coated ELISA plate (100 μL / well) in PBST / 0.2% BSA (PBSTB) and incubated at room temperature (RT) on a plate shaker (300 rpm) for 60 minutes. The samples were discarded, and the plate was washed once with PBS / 0.05% Tween 20 (PBST). Next, the plates were incubated for 60 minutes on a plate shaker (300 rpm) with 100 μL / well of PBTB containing 2 μg / mL mouse anti-idiotype monoclonal antibody 2F2 SAB1.1 (against 7D8; Genmab). The plates were then washed once with PBS / 0.05% Tween 20 (PBST). Next, the plates were incubated for 60 minutes at room temperature on a plate shaker (300 rpm) with 100 μL / well of PBSTB containing HRP-conjugated goat anti-mouse IgG (15G; Jackson ImmunoResearch Laboratories, Westgrove, PA, USA; 1:5.000). The plates were washed once with PBS / 0.05% Tween 20 (PBST). ABTS (50 mg / mL; Roche Diagnostics GmbH, Mannheim, Germany) (100 µL / well) was added, and the plates were incubated at room temperature in the dark for 30 minutes. The reaction was terminated with 2% oxalic acid (100 μL / well; Riedel de Haen Seelze, Germany). After 10 minutes at room temperature, the absorbance was measured at 405 nm using an ELISA reader.

[0279] Figure 1 The results show that the combination of human and rhesus monkey IgG4 produces a greater bispecific binding (higher OD 405 nm) compared to individual combinations of IgG4 molecules from the same species. These data indicate that Fab arm exchange occurs between human and rhesus monkey IgG4. Furthermore, the higher bispecific binding suggests that the human IgG4 hemimolecule preferentially dimers (heterodimerizes) with the rhesus monkey IgG4 hemimolecule, causing the equilibrium of the Fab arm exchange reaction to shift towards the bispecific heterodimer rather than towards a random exchange of 50% heterodimer and 50% homodimer.

[0280] Example 8: Sequence analysis of human and rhesus monkey IgG4 The ability of antibodies to participate in Fab arm interchange has been described as involving a third constant domain (CH3) and a so-called permissive (e.g., CPSC-containing) hinge region that requires only a reducing environment for activation (Van der Neut Kolfschoten, 2007, Science). For human antibodies, Fab arm interchange has been found to be an intrinsic feature of IgG4, characterized by an arginine (R) residue at position 409 of the CH3 domain and a permissive hinge (226-CPSC-229) (see WO 2008145142 (Genmab)). In contrast, human IgG1, which does not participate in Fab arm interchange, has a lysine (K) residue at position 409 and a stable (i.e., non-permissive) hinge (226-CPPC-229) (EU number, see also...). Figure 16 ).

[0281] To attempt to elucidate the increased Fab arm interchange between human and rhesus monkey IgG4 compared to Fab arm interchange between IgG4 molecules of the same species, the core hinge and CH3-CH3 interface amino acids of human and rhesus monkey antibodies were analyzed (for a review of human CH3-CH3 interface residues, see, for example, Dall'Acqua et al. (1998) Biochemistry 37: 9266). Figure 2 The core hinge sequence of Chinese rhesus monkey IgG4 is 226-CPAC-229, and the CH3 domain contains a lysine (K) at position 409. Furthermore, sequence alignment shows that rhesus monkey IgG4 is characterized by three additional amino acid substitutions at the CH3-CH3 interface compared to human IgG4: isoleucine (I) at position 350 in rhesus monkeys compared to threonine (T) in humans; threonine (T) at position 370 in rhesus monkeys compared to lysine (K) in humans; and leucine (L) at position 405 in rhesus monkeys compared to phenylalanine (F) in humans.

[0282] Example 9: Generation of bispecific antibodies by GSH-induced Fab arm swapping between human IgG4 and human IgG1 containing the rhesus monkey IgG4 CH3 sequence. As has been described for human antibodies, in order to allow Fab arm interchange to occur in the IgG1 molecule, substitution of the IgG1 core hinge sequence (CPPC) with the human IgG4 sequence (CPSC) via P228S has no effect, but the CH3 mutation to an IgG4-like sequence is necessary for Fab arm interchange activity (Van der Neut Kolfschoten, 2007, Science).

[0283] Based on the Fab arm exchange between human and rhesus monkey IgG4 described in Example 7, the possibility of the CH3 sequence of Chinese rhesus monkey IgG4 participating in the Fab arm exchange of human IgG1 was analyzed. Therefore, in addition to generating the P228S mutation of the hinge sequence CPSC, a triple mutation T350I-K370T-F405L (hereinafter referred to as ITL) was introduced into human IgG1-2F8. The human IgG1-2F8 mutant was combined with human IgG4-7D8 for in vitro GSH-induced Fab arm exchange. An antibody mixture containing various antibodies and 0.5 mM GSH in 0.5 mL PBS at a final concentration of 4 µg / mL was incubated at 37°C for 0–3–6–24 hours. To terminate the reduction reaction, 0.5 mL PBS / 0.05% Tween 20 (PBST) was added to the reaction mixture. Bispecific binding was measured in an ELISA as described in Example 7.

[0284] Figure 3 It was confirmed that the introduction of the CPSC hinge alone did not induce human IgG1-2F8 to participate in GSH-induced Fab arm exchange when combined with human IgG4-7D8. Furthermore, the introduction of rhesus monkey IgG4-specific CH3 interface amino acids (ITL) into human IgG1-2F8 while retaining the wild-type IgG1 hinge also did not lead to Fab arm exchange participation when combined with human IgG4-7D8 under these conditions. Conversely, the variant human IgG1-2F8 backbone sequence containing the CPSC sequence in the hinge and the rhesus monkey IgG4-specific CH3 interface amino acid (ITL) showed increased bispecific binding after GSH-induced Fab arm exchange with human IgG4-7D8 compared to the two human IgG4 antibodies. These data indicate that the combination of the CPSC-containing hinge with CH3 domains containing I, T, and L at positions 350, 370, and 405, respectively, is sufficient to induce human IgG1 to participate in GSH-induced Fab arm exchange, and that the equilibrium of the exchange reaction shifts towards the exchanged bispecific product when combined with human IgG4.

[0285] Example 10: Generation of bispecific antibodies through in vivo Fab arm exchange between human IgG4 and IgG1 or IgG4 mutants. To further identify the characteristics required for Fab arm exchange, human IgG4 and IgG1 variants were analyzed in vivo. Four female SCID mice (Charles River, Maastricht, The Netherlands) were intravenously injected with an antibody mixture containing 600 µg antibody (500 µg 7D8 + 100 µg 2F8) in a total volume of 300 µL. Blood samples were collected from the saphenous vein at 3, 24, 48, and 72 hours post-injection. Blood was collected in heparinized vials and centrifuged at 10,000 g for 5 min to separate cells from plasma. Bispecific antibodies were generated, and the CD20 and EGFR bispecific reactivity of plasma samples, serially diluted in PBSTB as described in Example 7, was assessed in ELISA. The bispecific antibodies in the plasma samples were quantified by nonlinear regression curve fitting (GraphPad Software, San Diego, CA) using the in vitro exchanged antibody mixture as a reference.

[0286] Figure 4 The data shows that when the hinge or CH3 sequence is converted into the corresponding human IgG1 sequence (CPPC or R409K, respectively), human IgG4-2F8 no longer participates in in vivo Fab arm swapping. Conversely, when both the hinge region and the CH3 interface sequence are converted into the corresponding human IgG4 sequences (CPSC and K409R), human IgG1 can participate in in vivo Fab arm swapping. These data indicate that the combination of the CPSC-containing hinge (S at position 228) and the CH3 domain containing arginine (R) at position 409 is sufficient to enable in vivo Fab arm swapping via human IgG1.

[0287] Example 11: Generation of bispecific antibodies via 2-MEA-induced Fab arm interchange: Bypass / disruption of stable hinges 2-Mercaptoethylamine·HCl (2-MEA) is a mild reducing agent and has been described as selectively cleaving disulfide bonds in the hinge region of antibodies while preserving disulfide bonds between the heavy and light chains. Therefore, the ability of a series of concentrations of 2-MEA to induce bispecific antibody production via Fab arm interchange between two antibodies containing CPSC or CPPC hinge regions was tested. Antibody mixtures (containing various 0.5 mg / mL final concentrations of antibody) were incubated with 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 mM) at a total TE volume of 100 µL at 37°C for 90 min. To terminate the reduction reaction, the reducing agent 2-MEA was removed by desalting the sample using a centrifugal column (Microcon centrifugal filter, 30k, Millipore) as recommended by the manufacturer. Bispecific binding was measured in an ELISA as described in Example 7.

[0288] The combination IgG4-2F8 x IgG4-7D8 (which contains the CPSC hinge region and is known to participate in GSH-induced Fab arm interchange) and the combination IgG1-2F8-ITL x IgG4-7D8-CPPC (which does not participate in GSH-induced Fab arm interchange due to its stable hinge region) were described in Example 9. Figure 3 The experiment tested 2-MEA-induced Fab arm interchange. Surprisingly, as determined by non-reducing SDS-PAGE (data not shown), 2-MEA-induced separation of the light and heavy chains was found. However, as... Figure 5 The results showed the generation of functional bispecific antibodies. The maximum level of bispecific binding following the Fab arm interchange between wild-type human IgG4-2F8 and IgG4-7D8 was achieved at a concentration of 2.0 mM 2-MEA, which is comparable to the level achieved with 0.5 mM GSH as described in Example 9. Figure 3 Comparable to IgG1-2F8-ITL and IgG4-7D8-CPPC (with a stable hinge region), 2-MEA can induce Fab arm swapping between the two human antibodies in a dose-dependent manner. While little or no bispecific antibody formation occurs at low 2-MEA concentrations (likely due to the presence of the CPPC sequence in the hinge region of both antibodies), bispecific antibodies are generated very efficiently at higher concentrations of 2-MEA. Maximum bispecific binding is achieved at 25 mM 2-MEA, exceeding the maximum binding after Fab arm swapping between the two wild-type IgG4 antibodies. These maximum binding levels are comparable to those in Example 9 (…). Figure 3 The maximum binding levels described for GSH treatment of the corresponding antibody (IgG1-2F8-CPSC-ITL) containing a CPSC hinge are comparable. Since both IgG1-2F8-ITL and IgG4-7D8-CPPC contain a CPSC hinge, these data suggest that 2-MEA can bypass the need for an in vitro Fab arm interchangeable with a CPSC hinge.

[0289] Example 12: Mass spectrometry analysis after generating bispecific antibodies via 2-MEA-induced Fab arm interchange. The generation of bispecific antibodies by 2-MEA-induced Fab arm interchange is described in Example 11, wherein bispecific binding is demonstrated by ELISA. Figure 5To confirm the formation of bispecific antibodies, the molecular weight of the sample was determined by electrospray ionization mass spectrometry (ESI-MS). First, the sample was deglycosylated by incubating 200 µg of antibody with 0.005 U N-glycanase (catalog number GKE-5006D; Prozyme) in 180 µL PBS overnight at 37°C. The sample was then desalted on an Aquity UPLC™ (Waters, Milford, USA) column using a BEH300 C18, 1.7 µm, 2.1 x 50 mm column at 60°C, followed by gradient elution with a mixture of MQ water (eluent A) containing 0.05% formic acid (Fluka Riedel-de Haën, Buchs, Germany) and LC-MS grade acetonitrile (eluent B) (Biosolve, Valkenswaard, The Netherlands). Time-of-flight electrospray ionization mass spectra were recorded online on a micriOTOF™ mass spectrometer (Bruker, Bremen, Germany) operating in positive ion mode. Prior to analysis, the 500–4000 m / z scale was calibrated using an ES tuning mix (Agilent Technologies, Santa Clara, USA). The mass spectra were deconvoluted using maximum entropy provided by DataAnalysis™ software version 3.4 (Bruker, Bremen, Germany). Based on the antibody molecular weight used for Fab arm interchange in this experiment, the bispecific antibody could be distinguished from the original antibody (also described in Example 15, regarding IgG1-2F8-ITLxIgG4-7D8-CPPC). Figure 9C For the peak of bispecific antibodies, determine the area under the curve and divide it by the total area under the curve to calculate the percentage of bispecific antibodies in each sample.

[0290] Figure 6 A shows three representative mass spectra of the Fab arm interchange reaction between IgG1-2F8-ITL and IgG4-7D8-CPPC using 0 mM 2-MEA (two peaks corresponding to the parental antibody), 7 mM 2-MEA (three peaks corresponding to the parental and bispecific antibodies), and 40 mM 2-MEA (one peak corresponding to the bispecific antibody). The uniform peaks of the bispecific product suggest the absence of light chain mismatch, which would otherwise lead to fragmented peaks. Quantitative data are presented in... Figure 6As shown in Figure B, the Fab arm exchange between IgG1-2F8-ITL and IgG4-7D8-CPPC produces nearly 100% bispecific antibody. Conversely, the Fab arm exchange between wild-type IgG4 antibodies produces less than 50% bispecific product. These data confirm the results of the bispecific binding ELISA described in Example 11. Figure 5 ).

[0291] Example 13: Stability of bispecific antibodies generated by 2-MEA-induced Fab arm interchange The stability of bispecific antibodies generated via 2-MEA-induced in vitro Fab arm exchange was tested. Therefore, 2 µg of bispecific samples (as described in Example 11) generated from IgG1-2F8-ITL and IgG4-7D8-CPPC were prepared using 7.0 mM 2-MEA. Figure 5 A series of concentrations (0, 2, 20, 100 µg) of irrelevant IgG4 (anti-acetylcholine receptor IgG4-MG) were used for GSH-induced Fab arm exchange reactions, where each concentration represented an excess of 0, 1, 10, 50x IgG4-MG compared to 2 µg of bispecific test sample. Fab arm exchange in this reaction resulted in the loss of bispecific EGFR / CD20 binding. The GSH reduction reaction was performed under the same conditions as described in Example 7 (24 hours at 37°C in 0.5 mL PBS / 0.5 mM GSH). To terminate the reduction reaction, 0.5 mL of PBSTB was added to the reaction mixture. Bispecific binding was measured in an ELISA as described in Example 7. Bispecific binding after GSH reduction was expressed as a comparison with bispecific binding measured in the raw material (control) (set as 100%).

[0292] Figure 7 A shows that for the bispecific sample derived from IgG1-2F8-ITL x IgG4-7D8-CPPC, the EGFR / CD20 bispecific binding after GSH-induced Fab arm swapping was not significantly altered in the presence of irrelevant IgG4. This suggests that the bispecific product is stable and does not participate in GSH-induced Fab arm swapping. As a control, Figure 7 Samples derived from IgG4-2F8 x IgG4-7D8 showed reduced EGFR / CD20 bispecific binding following GSH-induced Fab arm interchange in the presence of irrelevant IgG4, suggesting instability of the product. These data indicate that the heterodimer composed of a human IgG1 heavy chain containing a triple mutation T350I-K370T-F405L in the CH3 domain and a human IgG4 heavy chain containing an S228P substitution that produces a stable hinge (CPPC) is stable.

[0293] Example 14: In vivo analysis of the pharmacokinetics and stability of bispecific antibodies generated by 2-MEA-induced Fab arm exchange. Bispecific antibodies generated by in vitro 2-MEA-induced Fab arm exchange between IgG1-2F8-ITL and IgG4-7D8-CPPC were injected into SCID mice to analyze their stability (in vivo Fab arm exchange) and pharmacokinetic properties (plasma clearance) compared to the parental antibodies IgG1-2F8-ITL and IgG4-7D8-CPPC. Three groups of mice (n=3 per group) were intravenously injected with 200 µL of purified antibodies via the tail vein: (1) 100 µg bispecific antibody; (2) 100 µg bispecific antibody + 1,000 µg irrelevant IgG4 (natalizumab, anti-α4-integrin); (3) 50 µg IgG1-2F8-ITL + 50 µg IgG4-7D8-CPPC. Blood samples (50–100 µL) were collected via buccal puncture at predetermined time intervals (10 min, 3 h, 1, 2, 7, 14, 21 days) following antibody administration. The blood was collected in heparinized vials and centrifuged at 14,000 g for 10 min. Plasma was stored at -20°C prior to further analysis.

[0294] The total IgG concentration in plasma samples was determined by ELISA. Subsequent assay conditions were the same as those described in Example 7 for the ELISA. Specific compounds used for the total IgG measurement were as follows: coated with 2 μg / mL mouse anti-human IgG (clone MH16-1; CLB; catalog number M1268); serum sample diluents (1:500 and 1:2,500 for groups 1 and 3, and 1:2,500 and 1:10,000 for group 2); conjugate: HRP-conjugated goat anti-human IgG (clone 11H; Jackson; catalog number 109-035-098; 1:10,000). The presence of bispecific antibodies in plasma samples was determined by quantifying the CD20 and EGFR bispecific reactivity in the ELISA as described in Example 10.

[0295] Figure 8 A shows the total antibody plasma concentration. The plasma clearance curves were identical in shape for all groups, suggesting that plasma clearance of the bispecific antibodies was similar to that of the parental antibodies IgG1-2F8-ITL and IgG4-7D8-CPPC during the analysis interval. Figure 8B shows the plasma concentration of the bispecific antibody over time. Adding a 10-fold excess of irrelevant IgG4 to the bispecific antibody did not affect its concentration, indicating that no Fab arm exchange occurred in vivo. After injection of the parental antibody (IgG1-2F8-ITL + IgG4-7D8-CPPC), no bispecific antibody was detected in plasma, confirming that these antibodies do not participate in in vivo Fab arm exchange. These data suggest that the bispecific antibody product generated by in vitro 2-MEA-induced Fab arm exchange between IgG1-2F8-ITL and IgG4-7D8-CPPC is stable in vivo (without Fab arm exchange) and exhibits pharmacokinetic properties (plasma clearance) comparable to that of the parental monovalent antibody.

[0296] Example 15: Purity of bispecific antibodies generated by 2-MEA-induced Fab arm exchange between two antibodies Bispecific antibody batches generated via 2-MEA-induced Fab arm exchange between human IgG1-2F8-ITL x IgG4-7D8-CPPC were purified on a PD-10 desalted column (catalog number 17-0851-01; GE Healthcare). The purity of the bispecific product was then 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 antibody was confirmed by bispecific binding in ELISA (data not shown).

[0297] SDS-PAGE was performed on 4–12% NuPAGEBis-Tris gels (Invitrogen, Breda, The Netherlands) under reducing and non-reducing conditions using a modified Laemli method (Laemli 1970 Nature 227(5259): 680-5), with samples run at neutral pH. The SDS-PAGE gels were stained with Coomassie stain and digitally imaged using GeneGenius (Synoptics, Cambridge, UK). Figure 9A The antibody sample after Fab arm swapping consisted of intact IgG and trace amounts of half-molecules (H1L1) detectable on the non-reducing gel. Figure 9A -b).

[0298] Use the TSK HP-SEC column (G3000SW) for connection xlHP-SEC fractionation was performed using a Waters Alliance 2695 separation system (Waters, Etten-Leur, The Netherlands) with a Waters 2487 dual λ absorbance detector (Waters) and a Waters 2487 dual λ absorbance detector (Waters). The sample was run at 1 mL / min. Results were processed using Empower software version 2002, and each peak was expressed as a percentage of the total peak height. (Toso Biosciences, via Omnilabo, Breda, The Netherlands) Figure 9B The results showed that >98% of the samples consisted of intact IgG, with almost no aggregates forming.

[0299] Mass spectrometry measurements were performed as described in Example 12. Figure 9C The mass spectra of the raw materials IgG1-2F8-ITL and IgG4-7D8-CPPC, as well as the bispecific product generated by Fab arm interchange between IgG1-2F8-ITL and IgG4-7D8-CPPC, are shown. The product in the Fab arm interchanged sample is 145,901 kDa, which is perfectly matched with the bispecific product derived from IgG1-2F8-ITL (146,259.5 / 2=73,130) + IgG4-7D8-CPPC (145,542.0 / 2=72,771). Moreover, the bispecific antibody product shows a uniform peak, suggesting that no light chain mismatch occurred that would have resulted in a refraction of peaks. These data indicate that Fab arm interchange produces 100% bispecific antibody. The small peaks detected in addition to the main peak (K0) of IgG4-7D8-CPPC and the bispecific sample can be attributed to the presence of one (K1) or two (K2) C-terminal lysine residues.

[0300] These data show that approximately 100% of the functional bispecific antibody samples were generated through 2-MEA-induced Fab arm swapping between IgG1-2F8-ITL and IgG4-7D8-CPPC.

[0301] Example 16: Elucidating the need for Fab arm swapping of human IgG1 to replace T350I, K370T, and F405L To further identify the determinant in the IgG1 CH3 domain required for IgG1 to participate in Fab arm exchange, IgG1 containing the triple mutant T350I-K370T-F405L (ITL) was compared with the double mutants T350I-K370T (IT), T350I-F405L (IL), and K370T-F405L (TL). The single mutant F405L (L) was also tested. In vitro Fab arm exchange was induced using 2-MEA as a reducing agent (50 µg of each antibody in 100 µL PBS / 25 mM 2-MEA at 37°C for 90 min). For the single mutant F405L antibody, unpurified antibody from the transient transfection supernatant was used after exchanging the buffer for PBS using an Amicon Ultra centrifuge (30k, Millipore, catalog number UFC803096). To terminate the reduction reaction, the reducing agent 2-MEA was removed by desalting the sample using a centrifugal column as described in Example 11. The production of bispecific antibodies was determined by measuring bispecific binding in an ELISA as described in Example 7.

[0302] Triple mutations (ITL), double mutations (IT, IL, and TL), and single mutations (L) were introduced into IgG1-2F8. These mutants were then combined with IgG4-7D8 containing a CPSC hinge (wild-type) or a stable hinge (IgG4-7D8-CPPC) and subjected to Fab arm swapping at 37°C for 90 minutes with 25 mM 2-MEA. Figure 10 AB analysis showed that the IgG1-2F8-IL and IgG1-2F8-TL mutants exhibited the same level of Fab arm interchange as the triple mutant ITL, independent of the combined IgG4-7D8 (CPSC or CPPC hinge). In contrast, no bispecific binding was found for the combination with the IgG1-2F8-IT mutant. Figure 10 C shows that the IgG1-2F8-F405L mutant also exhibits Fab arm interchange, independent of the combined IgG4-7D8 (CPSC or CPPC hinge). These data suggest that under the above conditions, the F405L mutation is sufficient to enable human IgG1 to participate in Fab arm interchange.

[0303] Example 17: Generation of bispecific antibodies by 2-MEA-induced Fab arm interchange at different temperatures The ability of 2-MEA to induce bispecific antibodies via Fab arm exchange between two different antibodies was tested at different temperatures. The Fab arm exchange reaction was initiated by incubating 160 µg of human IgG1-2F8-ITL with 160 µg of IgG4-7D8-CPPC in 320 µl PBS / 25 mM 2-MEA (final antibody concentration 0.5 mg / mL) at 0 °C, 20 °C (RT), or 37 °C. From these reactions, 20 µL samples were collected at different time points (0, 2.5, 5, 10, 15, 30, 45, 60, 75, 90, 120, 150, 180, and 240 min). 20 µL of PBS was added to each sample, and the reducing agent 2-MEA was removed by desalting the samples using a Zeba 96-well centrifugation desalting plate (7k, catalog number 89808 Thermo Fisher Scientific) as recommended by the manufacturer. 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 in an ELISA using serially diluted antibody samples (total antibody concentration 0-20 μg / mL, in 25-fold dilutions) as described in Example 7.

[0304] Figure 11 The study found that bispecific antibody production via 2-MEA-induced Fab arm swapping between human IgG1-2F8-ITL and IgG4-7D8-CPPC was most effective at 37°C, reaching maximum bispecific binding after 45 minutes. At room temperature, bispecific antibody production was slower, reaching maximum bispecific binding after 240 minutes. At 0°C, no bispecific binding was observed during the analysis time.

[0305] Example 18: Analysis of the ability of different reducing agents to induce the generation of bispecific antibodies via in vitro Fab arm exchange As shown above, 0.5 mM GSH can induce Fab arm interchange between human IgG4 and IgG1-CPSC-ITL in vitro, but not between human IgG4 and IgG1-ITL containing a stable hinge. Figure 3 Furthermore, it was found that 2-MEA can induce Fab arm interchange between antibodies with stable hinge regions, such as IgG1-ITL x IgG4-CPPC. Figure 5To test whether other concentrations of GSH, 2-MEA, or other reducing agents could induce Fab arm interchange between two different antibodies in vitro, a series of concentrations of 2-MEA, GSH, and DTT (dithiothreitol) were tested. Therefore, a combination of 10 µg human IgG1-2F8-ITL and 10 µg IgG4-7D8-CPPC in 20 µl PBS (final antibody concentration 0.5 mg / mL) was incubated with a series of different concentrations of reducing agents (0.0, 0.04, 0.1, 0.2, 0.5, 1.0, 2.5, 5.0, 12.5, 25.0, and 50.0 mM) at 37°C. After 90 minutes, 20 µL PBS was added to each sample, and the reducing agent was removed by desalting the samples using a centrifugal desalting plate as described in Example 17. The total antibody concentration was determined as described in Example 17. Bispecific binding was measured in ELISA using serial dilutions of antibody samples (total antibody concentration 0-20 μg / mL, in 3-fold dilutions) as described in Example 7.

[0306] Figure 12 2-MEA was confirmed to induce maximum bispecific binding at a concentration of 25 mM. DTT was found to generate bispecific antibodies very efficiently, reaching maximum bispecific binding at 2.5 mM DTT. GSH concentrations in the 0–5 mM range failed to induce bispecific antibody generation via Fab arm exchange between IgG1-ITL and IgG4-CPPC antibodies (both containing stable hinge regions). Higher GSH concentrations (12.5–50 mM) resulted in antibody aggregate formation, as determined by non-reducing SDS-PAGE (data not shown). Therefore, these samples were excluded from the analysis. These data indicate that different reducing agents can induce bispecific antibody generation via Fab arm exchange between two different antibodies.

[0307] Example 19: Determinant cluster at position 409 of IgG1 for use in combination with IgG1-ITL to participate in 2-MEA-induced Fab arm interchange 2-MEA can induce Fab arm interchange between human IgG1-ITL and IgG4-CPPC, as described in Example 11 ( Figure 5 The only difference between human IgG1 and IgG4 at the CH3 interface is position 409: IgG1 has lysine (K) and IgG4 has arginine (R) (described in Example 8). Figure 2Therefore, it was tested whether replacing lysine at position 409 with arginine or any other amino acid (K409X) would enable IgG1 to participate in the 2-MEA-induced Fab arm exchange with IgG1-ITL. A combination of 10 µg human IgG1-2F8-ITL and 10 µg IgG1-7D8-K409X in 20 µl PBS / 25 mM 2-MEA (final antibody concentration 0.5 mg / mL) was incubated at 37°C for 90 min. After replacing the buffer with PBS using an Amicon Ultra centrifuge (30k, Millipore, catalog number UFC803096), unpurified antibodies from the transient transfection supernatant were used. Following the Fab arm exchange reaction, 20 µL PBS was added to each sample, and the reducing agent was removed by desalting the samples using a centrifugal desalting plate as described in Example 17. Bispecific binding was measured in ELISA using serial dilutions of antibody samples (total antibody concentration 0-20 μg / mL, in 3-fold dilutions) as described in Example 7.

[0308] Figure 13 A shows the results of bispecific binding after 2-MEA-induced Fab arm interchange between IgG1-2F8-ITL and IgG1-7D8-K409X. Figure 13 In section B, interchange is represented as bispecific binding relative to a purified batch of bispecific antibody (set to 100%) derived from a 2-MEA-induced Fab arm interchange between IgG1-2F8-ITL and IgG4-7D8-CPPC. As shown in Table 1, these data are also scored as (-) no Fab arm interchange, (+ / -) low, (+) moderate, or (++) high Fab arm interchange. No Fab arm interchange (-) is found when position 409 of IgG1-7D8 is K (=wild-type IgG1), L, or M. Fab arm interchange was found to be moderate (+) when position 409 of IgG1-7D8 is F, I, N, or Y, and high (++) when position 409 of IgG1-7D8 is A, D, E, G, H, Q, R, S, T, V, or W.

[0309] Table 1: Fab arm interchange between IgG1-2F8-ITL and IgG1-7D8-K409X mutants induced by 2-MEA. Bispecific antibody production following in vitro Fab arm interchange between IgG1-2F8-ITL and IgG1-7D8-K409X mutants induced by 2-MEA was determined by sandwich ELISA. (-) None, (+ / -) Low degree, (+) Moderate degree, (++) High degree of Fab arm interchange.

[0310] Example 20: Antibody deglycosylation does not affect the generation of bispecific antibodies via 2-MEA-induced Fab arm interchange. IgG4-7D8 and IgG4-7D8-CPPC samples were deglycosylated by incubating 200 µg of antibody with 0.005 U N-glycanase (catalog number GKE-5006D; Prozyme) in 180 µL PBS overnight at 37°C. These samples were then used directly for the Fab arm exchange reaction. Fab arm exchange was performed by incubating 50 µg of each antibody in 100 µL PBS / 25 mM 2-MEA (final antibody concentration 0.5 mg / mL) at 37°C for 90 minutes. The reducing agent 2-MEA was removed by desalting the samples using a centrifuge column as described in Example 11. Bispecific binding was measured in a sandwich ELISA using serial dilutions of the antibody samples (total antibody concentration 0-20 μg / mL, in 3-fold dilutions) as described in Example 7.

[0311] Mass spectrometry analysis showed that the deglycosylation reaction produced 100% deglycosylated antibody products (data not shown). Figure 14 The data showed no difference between Fab arm swaps involving deglycosylated antibodies and those involving corresponding glycosylated antibodies (IgG4-2F8 x IgG4-7D8-deglycosylated vs. IgG4-2F8 x IgG4-7D8 and IgG1-2F8-ITL x IgG4-7D8-CPPC-deglycosylated vs. IgG1-2F8-ITL x IgG4-7D8-CPPC). These data suggest that deglycosylation does not affect the generation of bispecific antibodies via 2-MEA-induced Fab arm swaps.

[0312] Example 21: Quantification of non-covalent CH3-CH3 interactions The strength of the interaction at the CH3 interface should be such that both heavy chains of the parent antibody are likely to dissociate during the Fab arm exchange reaction and subsequently associate in the heterodimerization reaction. Therefore, the ability to participate in Fab arm exchange and the strength of the non-covalent CH3-CH3 interaction (dissociation constant, K) were analyzed. D The correlation between them. For the following human antibody combinations, GSH-induced Fab arm swapping was performed as described in Example 9 (at 37°C and 0.5 mM GSH): IgG1-2F8 x IgG1-7D8 IgG1-2F8-CPSC x IgG1-7D8-CPSC IgG1-2F8-CPSC-T350I x IgG1-CPSC-7D8-T350I IgG1-2F8-CPSC-K370T x IgG1-7D8-CPSC-K370T IgG1-2F8-CPSC-ITL x IgG1-7D8-CPSC-ITL IgG1-2F8-CPSC-K409R x IgG1-7D8-CPSC-K409R IgG4-2F8 x IgG4-7D8 IgG4-2F8-R409K x IgG4-7D8-R409K IgG4-2F8-R409A x IgG4-7D8-R409A IgG4-2F8-R409L x IgG4-7D8-R409L IgG4-2F8-R409M x IgG4-7D8-R409M IgG4-2F8-R409T x IgG4-7D8-R409T IgG4-2F8-R409W x IgG4-7D8-R409W IgG4-2F8-F405A x IgG4-7D8-F405A IgG4-2F8-F405L x IgG4-7D8-F405L IgG4-2F8-Y349D x IgG4-7D8-Y349D IgG4-2F8-L351K x IgG4-7D8-L351K IgG4-2F8-E357T x IgG4-7D8-E357T IgG4-2F8-S364D x IgG4-7D8-S364D IgG4-2F8-K370Q x IgG4-7D8-K370Q IgG4-2F8-K370E x IgG4-7D8-K370E The production of bispecific antibodies was measured by determining bispecific binding in a sandwich ELISA as described in Example 7. Figure 15 A / B / C show the results of bispecific binding after the Fab arm interchange reaction.

[0313] To measure the effect of the aforementioned CH3 mutation on the strength of CH3-CH3 interactions, fragments consisting only of CH2-CH3 domains were prepared. These fragments lacked hinge regions to prevent covalent heavy-chain disulfide bonds. The fragments were analyzed by natural mass spectrometry. The sample buffer was replaced with 100 mM ammonium acetate at pH 7 using a 10 kDa MWCO centrifugal filter column. Aliquots (approximately 1 μL) of serially diluted samples (20 μM–25 nM; monomer equivalent) were loaded into gold-plated borosilicate capillaries for analysis on a Waters LCT mass spectrometer. The monomer signal M... s Defined as the fraction of the area of ​​a single peak to the total area of ​​all peaks in the spectrum (M s / (M s +D s ), where D s = Dimer signal). The monomer concentration [M] at equilibrium. eq Defined as M s [M]0, where [M]0 is the total protein concentration in terms of monomers. The equilibrium dimer concentration [D] is then considered. eq Defined as ([M]0-[M]) eq ) / 2. Then from [D] eq Comparison [M] eq 2 Calculate K from the slope of the curve. D Table 2 presents the K values ​​for non-covalent CH3-CH3 interactions. D .

[0314] The relationship between the ability to participate in Fab arm interchange and the strength of non-covalent CH3-CH3 interactions was analyzed. Figure 15 D / E display shows the measurement K for the corresponding CH2-CH3 fragment. D The percentage of bispecific bindings after Fab arm swapping was plotted. Figure 15 D regarding IgG1; Figure 15 E regarding IgG4). These data suggest the presence of an apparent K group under the test conditions that allows for efficient Fab arm interchange of CH3-CH3 interactions. D The specific range of values.

[0315] Table 2: K values ​​for non-covalent CH3-CH3 interactions D Example 22: Analysis of the ability of different reducing agents to induce bispecific antibodies generated through in vitro Fab arm exchange between IgG1-2F8-F405L and IgG1-7D8-K409R. It was found that 2-MEA and DTT can induce in vitro Fab arm exchange between human IgG1-ITL and IgG4-CPPC. Figure 12 The ability of these reducing agents to induce in vitro Fab arm interchange between human IgG1-2F8-F405L and IgG1-7D8-K409R was tested. A series of concentrations of 2-MEA, DTT, GSH, and TCEP (tris(2-carboxyethyl)phosphine) were tested. Fab arm interchange was performed as described in Example 18. The series of test concentrations for different reducing agents are as follows: 0.0, 0.04, 0.1, 0.2, 0.5, 1.0, 5.0, 25.0, and 50.0 mM 2-MEA, GSH, DTT, or TCEP.

[0316] Figure 17 2-MEA was confirmed to induce maximum Fab arm exchange at a concentration of 25 mM, which persisted at higher concentrations of 50.0 mM. DTT was found to generate bispecific antibodies very efficiently, reaching maximum Fab arm exchange at 0.5 mM DTT, and persisting at higher concentrations of DTT (1.0–50.0 mM). TCEP was also found to generate bispecific antibodies very efficiently, reaching maximum Fab arm exchange at 0.5 mM. Fab arm exchange via TCEP was interfered with at concentrations ≥25.0 mM. GSH concentrations in the 0.0–5.0 mM range did not induce bispecific antibody production via Fab arm exchange. Higher GSH concentrations (25.0–50.0 mM) led to antibody aggregate formation (data not shown). Therefore, these samples were excluded from the analysis. These data indicate that different reducing agents can induce bispecific antibody production via Fab arm exchange between two different antibodies.

[0317] Example 23: Generation of bispecific antibodies via 2-MEA-induced Fab arm exchange between IgG1-2F8-F405L and IgG1-7D8-K409R To confirm the formation of bispecific antibodies via Fab arm exchange induced by 2-MEA between human IgG1-2F8-F405L and IgG1-7D8-K409R, the molecular weights of samples from Fab arm exchange reactions performed with a series of concentrations of 2-MEA were determined by ESI-MS. 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.

[0318] Figure 18A shows that 2-MEA induces Fab arm swapping between IgG1-2F8-F405L and IgG1-7D8-K409R in a dose-dependent manner, effectively leading to the production of bispecific antibodies, with maximum bispecific binding at a concentration of 15.0 mM 2-MEA. Figure 18 B presents quantitative ESI-MS data showing that the Fab arm swap between IgG1-2F8-F405L and IgG1-7D8-K409R produces nearly 100% bispecific antibodies, confirming the results of the bispecific binding ELISA.

[0319] Example 24: Purity of bispecific antibodies generated by 2-MEA-induced Fab arm exchange between human IgG1-2F8-F405L and IgG1-7D8-K409R The batch of bispecific antibodies generated by 2-MEA-induced Fab arm swapping between human IgG1-2F8-F405L x IgG1-7D8-K409R was purified using a PD-10 desalting column (catalog number 17-0851-01; GE Healthcare). The purity of the bispecific product was then analyzed by mass spectrometry as described in Example 12.

[0320] Figure 19 The mass spectra of the bispecific products generated from the raw materials IgG1-2F8-F405L and IgG1-7D8-K409R, and those generated via Fab arm interchange between IgG1-2F8-F405L and IgG1-7D8-K409R, are shown. The product in the Fab arm interchanged sample is 146,160.7 kDa, which matches the bispecific product derived from IgG1-2F8-F405L (146,606.8 / 2=73,303.3) x IgG1-7D8-K409R (146,312.2 / 2=73,156.1) = 146,459.4 kDa. Furthermore, the bispecific antibody products show uniform peaks, suggesting the absence of light chain mismatches that would lead to refraction. These data indicate that Fab arm interchange produces approximately 100% bispecific antibodies.

[0321] Example 25: In vivo analysis of the stability and pharmacokinetics of bispecific antibodies generated from IgG1-2F8-F405L x IgG1-7D8-K409R via 2-MEA-induced Fab arm exchange. Bispecific antibodies generated via in vitro 2-MEA-induced Fab arm exchange between IgG1-2F8-F405L and IgG1-7D8-K409R were injected into SCID mice as described in Example 14 to analyze their stability (in vivo Fab arm exchange) and pharmacokinetic properties. Two groups of mice (n=3 per group) were analyzed: (1) 100 µg bispecific antibody; (2) 100 µg bispecific antibody + 1,000 µg irrelevant IgG4 (IgG4-637, described in WO2007068255). The total IgG concentration in plasma samples was determined by ELLISA as described in Example 14, but in this example, HRP-conjugated goat anti-human IgG (Jackson, catalog number 109-035-098, 1 / 10,000) was used as the conjugate for detection. The presence of bispecific antibodies in plasma samples was determined and quantified by the CD20 and EGFR bispecific reactivity in a sandwich ELISA as described in Example 14.

[0322] Figure 20 A shows the total antibody plasma concentration over time. The plasma clearance curves of the two groups have the same shape. Figure 20 B shows the plasma concentration of bispecific antibodies over time. Adding a 10-fold excess of irrelevant IgG4 to the bispecific antibody did not affect the bispecific antibody concentration, suggesting that Fab arm exchange does not occur in vivo. These data suggest that the bispecific antibody product generated by in vitro 2-MEA-induced Fab arm exchange between IgG1-2F8-F405Lx and IgG1-7D8-K409R is stable in vivo (without Fab arm exchange).

[0323] Example 26: CDC-mediated cell killing by bispecific antibodies generated via 2-MEA-induced Fab arm exchange between human IgG1-2F8-F405L and IgG1-7D8-K409R. The CD20 antibody IgG1-7D8 effectively kills CD20-expressing cells via complement-dependent cytotoxicity (CDC). Conversely, the EGFR antibody IgG1-2F8 does not mediate CDC on EGFR-expressing target cells. The study tested whether the bispecific antibody generated from the mutant IgG1-7D8-K409R and the Fab arm interchange induced by 2-MEA between IgG1-2F8-F405L x IgG1-7D8-K409R could still induce CDC on CD20-expressing cells. 10 5Daudi or Raji cells were pre-incubated with a series of antibody concentrations in 80 µL of RPMI medium supplemented with 0.1% BSA on a shaker at room temperature for 15 min. 20 µL of normal human serum (NHS) was added as a complement source (20% NHS final concentration), and the cells were incubated at 37°C for 45 min. The CDC reaction was terminated by adding 30 µL of ice-cold RPMI medium supplemented with 0.1% BSA. Dead and live cells were distinguished by adding 10 µL of 10 µg / mL propidium iodide (PI) (1 µg / mL final concentration) and FACS analysis.

[0324] Figure 21 The results showed that IgG1-7D8 had an effect on Daudi (expressing CD20). Figure 21 A) and Raji ( Figure 21 B) CDC-mediated cell killing in cells was unaffected by the introduction of the K409R mutation. Neither Daudi nor Raji cells expressed EGFR, resulting in monovalent binding of bispecific antibodies generated via 2-MEA-induced Fab arm exchange between IgG1-2F8-F405L and IgG1-7D8-K409R. However, the bispecific antibodies still induced CDC-mediated cell killing in CD20-expressing cells. These data suggest that the CDC capacity of the parental antibody is preserved in the bispecific form.

[0325] Example 27: ADCC-mediated cell killing by bispecific antibodies generated via 2-MEA-induced Fab arm exchange between human IgG1-2F8-F405L and IgG1-7D8-K409R. The EGFR antibody IgG1-2F8 can kill EGFR-expressing cells, such as A431 cells, through antibody-dependent cytotoxicity (ADCC). A431 cells do not express CD20, therefore the CD20 antibody IgG1-7D8 does not induce ADCC in these cells. The study tested whether the bispecific antibody generated from the mutant IgG1-2F8-F405L and the Fab arm interchange induced by 2-MEA between IgG1-2F8-F405L and IgG1-7D8-K409R could still induce ADCC in A431 cells. For effector cell isolation, Leucosep was used as recommended by the manufacturer. ® Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy donors using tubes (Greiner Bio-one, catalog number 227290). 100 µCi of PBMCs were cultured in 1 mL of RPMI medium supplemented with 0.1% BSA. 51 Add Cr to 5 x 10 6Target cells were labeled by incubating A431 cells at 37°C with shaking for 60 minutes. The labeled cells were washed and resuspended in RPMI supplemented with 0.1% BSA. 5 x 10⁶ cells were then placed in RPMI supplemented with 0.1% BSA. 4 100 µL of labeled target cells were pre-incubated with a series of antibody concentrations (final concentration range of 0-10 µg / mL in ADCC assay, in 3-fold dilution) at room temperature for 15 minutes. 50 µL of effector cells (5 x 10T) were added at an E:T ratio of 100:1. 6 ADCC assays were initiated with 10 cells. After 4 hours at 37°C, triplicate copies of the experiment were measured in a scintillation counter at counts per minute (cpm). 51 Cr release. The percentage of cytotoxicity was calculated using the following formula: Percentage of specific lysis = (Experimental cpm – Baseline cpm) / (Maximum cpm – Baseline cpm) x 100. This was determined by adding 50 µL of 5% Triton X-100 to 50 µL of target cells (5 x 10⁻⁶ cells per ... 4 (cells) determine the maximum 51 Cr release was measured in the absence of sensitized antibodies and effector cells, with basal release being measured.

[0326] Figure 22 The CD20-specific antibody IgG1-7D8 did not induce ADCC in CD20-negative A431 cells. Both IgG1-2F8 and the mutant IgG1-2F8-F405L induced ADCC in A431 cells, suggesting that introducing the F405L mutation into IgG1-2F8 does not affect its ADCC effector function. A bispecific antibody derived from IgG1-2F8-F405L x IgG1-7D8-K409R also induced ADCC in A431 cells in a dose-dependent manner, suggesting that ADCC effector function is preserved in the bispecific form.

[0327] Example 28: Determinant cluster at position 405 of IgG1 for use in combination with IgG1-K409R to participate in 2-MEA-induced Fab arm interchange Example 16 describes how the F405L mutation, when combined with IgG4-7D8, is sufficient to enable human IgG1 to participate in Fab arm swapping. To further test the determinant cluster at position 405 of IgG1 participating in 2-MEA-induced Fab arm swapping when combined with human IgG1-K409R, all possible IgG1-2F8-F405X mutants (except C and P) were combined with IgG1-7D8-K409R. The procedure was performed using purified antibody as described in Example 19.

[0328] Figure 23The results show the bispecific binding following 2-MEA-induced Fab arm exchange between IgG1-2F8-F405X and IgG1-7D8-K409R. As presented in Table 3, these data were also graded as (-) no Fab arm exchange, (+ / -) low, (+) moderate, or (++) high Fab arm exchange. No Fab arm exchange was found (-) when position 405 of IgG1-2F8 was F (= wild-type IgG1). Low-grade Fab arm exchange was found (+ / -) when position 405 of IgG1-2F8 was G or R. High-grade Fab arm exchange was found (++) when position 405 of IgG1-2F8 was A, D, E, H, I, K, L, M, N, Q, S, T, V, W, or Y. These data suggest that a specific mutation at position 405 of IgG1 allows IgG1 to participate in 2-MEA-induced Fab arm exchange when combined with IgG1-K409R.

[0329] Table 3: 2-MEA-induced Fab arm interchange between IgG1-2F8-F405X mutant and IgG1-7D8-K409R The production of bispecific antibodies following 2-MEA-induced in vitro Fab arm exchange between IgG1-2F8-F405X mutant and IgG1-7D8-K409R was determined by sandwich ELISA. (-) None, (+ / -) Low, (+) Moderate, (++) High Fab arm exchange.

[0330] Example 29: Determinant cluster at position 407 of IgG1 for use in combination with IgG1-K409R to participate in 2-MEA-induced Fab arm interchange In Example 28, it was described that certain single mutations at position F405, when combined with IgG1-K409R, were sufficient to enable human IgG1 to participate in Fab arm exchange. To test whether other determinants involving the Fc:Fc interface position in the CH3 domain could also mediate Fab arm exchange, mutagenesis was performed at position 407 of IgG1, and mutants were tested for participation in 2-MEA-induced Fab arm exchange when combined with human IgG1-K409R. All possible IgG1-2F8-Y407X mutants (except C and P) were combined with IgG1-7D8-K409R. The procedure was performed using purified antibodies as described in Example 19.

[0331] Figure 24The results show the bispecific binding following 2-MEA-induced Fab arm interchange between IgG1-2F8-Y407X and IgG1-7D8-K409R. As presented in Table 4, these data were also scored as (-) no Fab arm interchange, (+ / -) low, (+) moderate, or (++) high Fab arm interchange. No Fab arm interchange (-) was found when position 407 of IgG1-2F8 was Y (=wild-type IgG1), E, ​​K, Q, or R. Fab arm interchange was found to be low (+ / -) when position 407 of IgG1-2F8 was D, F, I, S, or T, moderate (+) when position 407 of IgG1-2F8 was A, H, N, or V, and high (++) when position 407 of IgG1-2F8 was G, L, M, or W. These data suggest that a specific single mutation at position 407 of IgG1, when combined with IgG1-K409R, allows IgG1 to participate in 2-MEA-induced Fab arm swapping.

[0332] Table 4: 2-MEA-induced Fab arm interchange between IgG1-2F8-Y407X mutant and IgG1-7D8-K409R The production of bispecific antibodies following 2-MEA-induced in vitro Fab arm exchange between IgG1-2F8-Y407X mutant and IgG1-7D8-K409R was determined by sandwich ELISA. (-) None, (+ / -) Low, (+) Moderate, (++) High Fab arm exchange.

[0333] Example 30: Quantification of non-covalent CH3-CH3 interactions in IgG1 heterodimers Example 21 describes a specific range of CH3-CH3 homodimer interaction strengths that allow for efficient Fab arm interchange. The strength of the interaction at the CH3 interface should be such that both heavy chains in the parent antibody (homidimer) are likely to dissociate in the Fab arm interchange reaction and subsequently associate in the heterodimer reaction. To produce a stable heterodimer, the strength of the heterodimer interaction should be greater than that of the homodimer interaction, thus favoring heterodimerization relative to homodimerization. To confirm this, the strength of the CH3-CH3 interaction in the heterodimer is measured and compared with the strength of the homodimer. The K2 of the CH2-CH3 fragments derived from the IgG1-K409R, IgG1-F405L, and IgG1-ITL homodimers is measured as described in Example 21. D For K in heterodimers DTo determine the presence of the CH2-CH3 domain fragments (G1-F405L and G1-ITL) and the IgG1Δ hinge fragment containing all antibody domains except the hinge, a mixture was prepared. Both fragments lacked hinge regions to prevent covalent heavy-chain disulfide bonds. The fragments were mixed and analyzed by natural mass spectrometry as described in Example 21 after 24 hours. Table 5 presents the K values ​​for non-covalent CH3-CH3 interactions in the CH2-CH3 fragments or the mixture of CH2-CH3 fragments and the IgG1Δ hinge fragment. D These data suggest that, under the test conditions, the strength of heterodimer interactions is greater than (lower than K). D The corresponding homodimer interactions.

[0334] Table 5 Example 31: Biochemical analysis of bispecific antibodies generated by 2-MEA-induced Fab arm interchange Bispecific antibody batches generated via 2-MEA-induced Fab arm exchange between human IgG1-2F8-F405L x IgG1-7D8-K409R were purified on a PD-10 desalting column (catalog number 17-0851-01; GE Healthcare). The purity of the bispecific products was then analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), high-performance size exclusion chromatography (HP-SEC), mass spectrometry, HPLC cation exchange chromatography (HPLC-CIEX), and capillary isoelectric focusing (cIEF).

[0335] As described in Example 15, in non-reduction ( Figure 25 A) and reduction ( Figure 25 SDS-PAGE was performed under condition B). Figure 25 A shows that the antibody sample after 2-MEA-induced Fab arm swapping consists of intact IgG and trace amounts of half-molecules (H1L1) detectable on the non-reducing gel.

[0336] HP-SEC was performed as described in Example 15. Figure 26 (B) and Figure 26 (A) The HP-SEC curves of raw materials IgG1-2F8-F405L and IgG1-7D8-K409R are shown respectively. Figure 26 C and Figure 26 D shows a mixture of two antibodies (1:1) and a bispecific product generated by 2-MEA-induced Fab arm exchange between IgG1-2F8-F405L and IgG1-7D8-K409R, respectively. Furthermore, Figure 26 D showed that >99% of the samples consisted of intact IgG, with almost no aggregates forming.

[0337] Mass spectrometry (ESI-MS) was performed as described in Example 12. Figure 27 (B) and Figure 27 (A) Mass spectrometry curves of raw materials IgG1-2F8-F405L and IgG1-7D8-K409R are shown respectively. Figure 27 C and Figure 27 Figures D show the mixture of the two antibodies (1:1) and the bispecific product generated by 2-MEA-induced Fab arm interchange between IgG1-2F8-F405L and IgG1-7D8-K409R, respectively. The product in the 2-MEA-induced Fab arm interchange sample was 146,159.7 kDa, perfectly matched with the bispecific product derived from IgG1-2F8-F405L (146,289.0 / 2=73,145) x IgG1-7D8-K409R (146,028.0 / 2=73,014). Furthermore, the bispecific antibody product showed a uniform peak, suggesting the absence of light chain mismatches that would lead to further peak division. These data indicate that 2-MEA-induced Fab arm interchange generates bispecific IgG. Small peaks indicated by (*) are from incomplete deglycosylation prior to analysis. These data show that bispecific antibody samples were generated by 2-MEA-induced Fab arm swapping between IgG1-2F8-F405L and IgG1-7D8-K409R.

[0338] Capillary isoelectric focusing (cIEF) was performed using an iCE280 analyzer (Convergent Biosciences). Figure 28 A and Figure 28 B shows the cIEF curves of raw materials IgG1-2F8-F405L and IgG1-7D8-K409R, respectively. Figure 28 C and Figure 28D shows a mixture of two antibodies (1:1) and a bispecific product generated by Fab arm exchange between IgG1-2F8-F405L and IgG1-7D8-K409R. All samples were desalted before use. The final concentration of the mixture was determined to be 0.3 mg / mL IgG (0.35% methylcellulose; 2% carrier amphoteric electrolyte 3-10; 6% carrier amphoteric electrolyte 8-10.5; 0.5% pI label 7.65 and 0.5% pI label 10.10). Whole-capillary absorption images were acquired using a charge-coupled device camera at 3000 V for 7 minutes. Data were analyzed using EZChrom software after peak correction. pI labels are indicated by (*). These data show that bispecific antibody samples were generated by 2-MEA-induced Fab arm exchange between IgG1-2F8-F405L and IgG1-7D8-K409R.

[0339] Another technique for studying charged isoforms of monoclonal antibodies is high-performance liquid chromatography with cation exchange (HPLC-CIEX). Figure 29 A and Figure 29 B shows the HPLC-CIEX curves of raw materials IgG1-2F8-F405L and IgG1-7D8-K409R, respectively. Figure 29 C and Figure 29 Figures D show a mixture of two antibodies (1:1) and a bispecific product generated by 2-MEA-induced Fab arm exchange between IgG1-2F8-F405L and IgG1-7D8-K409R. Samples were diluted to 1 mg / mL in mobile phase A (10 mM NaPO4, pH 7.0) for injection onto HPLC. Differently charged IgG molecules were separated using a ProPac® WCX-10, 4 mm x 250 mm analytical column at a flow rate of 1 mL / min. Elution was performed using a gradient from mobile phase A to mobile phase B (10 mM NaPO4, pH 7.0, 0.25 M NaCl), and detection was performed at 280 nm. These data demonstrate the generation of bispecific antibody samples via 2-MEA-induced Fab arm exchange between IgG1-2F8-F405L and IgG1-7D8-K409R. Cation exchange is also shown to be a powerful tool for separating residual homodimers from heterodimers. Therefore, another application of cation exchange chromatography is to refine bispecific heterodimers, that is, to purify any residual homodimers after the exchange.

[0340] Example 32: Recombinant expression of heterodimers by simultaneously co-expressing two homodimers To demonstrate that heterodimer formation also occurs when two homodimers are recombinantly co-expressed, HEK-293F cells were co-transfected with four expression vectors (see Example 1) encoding the heavy and light chains of IgG1-7D8-K409R and IgG1-2F8-F405 at a ratio of 1:1:1:1. Antibodies were transiently prepared under serum-free conditions as described in Example 4. Next, IgG was 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.

[0341] Table 6 shows the theoretical masses of the heavy and light chains of IgG1-7D8-K409R and IgG1-2F8-F405.

[0342] Table 6: Theoretical mass of heavy and light chains of IgG1-7D8-K409R and IgG1-2F8-F405 IgG1-2F8-F405 23252.8 49894.6 IgG1-7D8-K409R 23438.1 49579.0 Based on these masses, the following IgG molecules can theoretically be detected (Table 7). The measured masses are shown in the last column. Figure 30 ).

[0343] Table 7: Theoretical detection values ​​of heavy and light chains of IgG1-7D8-K409R and IgG1-2F8-F40 The two most abundant peaks at 146345 and 146159 Da represent heterodimers incorporating a single light chain (from IgG1-7D8-K409R) or two light chains, respectively. Homodimers of the heavy chains of IgG1-7D8-K409R or IgG1-2F8-F405 were detected, but in small amounts. These data indicate that heterodimerization also occurs when both homodimers are co-expressed.

[0344] Example 33: Monitoring the kinetics of 2-MEA-induced Fab arm swapping and quantifying the residual homodimer after swapping using HPLC-CIEX Bispecific antibodies were generated by 2-MEA-induced Fab arm exchange as described in Example 11. In this example, the exchange reaction was monitored by high-performance liquid chromatography-cation exchange (HPLC-CIEX; as described in Example 31) at various time points during the exchange reaction.

[0345] Homodimers IgG1-2F8-F405L and IgG1-7D8-K409R, each at a concentration of 1 mg / mL, were mixed in a 1:1 molar ratio. After adding 25 mM 2-MEA, the sample was placed in an automated HPLC sampler and preheated at 25°C. Figure 31A through 31H represent eight consecutive injections at different time intervals, obtained by HPLC-CIEX from t = 0 min to t = 450 min after the addition of 2-MEA. The data show fairly rapid formation of bispecific IgG, with most homodimers exchanging after 135 min. The heterodimer peak appearing after 45 min resolved into a more homogeneous peak near 180 min, suggesting that the exchange occurred in different phases. Furthermore, Figure 32 A shows that approximately 3% residual homodimer was detected using the CIEX method (indicated by the arrow). As shown, this method is suitable for quantifying the residual homodimer content (homogeneity elution is shown in the image). Figure 32 B), when the exchange reaction is almost complete.

[0346] Example 34: Generation of bispecific antibodies via 2-MEA-induced Fab arm interchange at high antibody concentrations under various 2-MEA concentrations, temperatures, and incubation times. 2-MEA-induced Fab arm swapping was performed at high IgG concentrations. The effects of 2-MEA concentration, incubation temperature, and time on the swapping amount were investigated.

[0347] The exchange process was performed using a combination of IgG1-7D8-K409R and IgG1-2F8-F405L. Both materials were purified by protein A affinity chromatography. After the material concentration reached >20 mg / mL, a series of anion exchange steps (flow-through) were performed using HiPrep Q FF 16 / 10 (GE Health Care, #28-9365-43). The final purified material buffer was exchanged for PBS.

[0348] Bispecific exchange was studied in PBS at final IgG concentrations of 20 mg / mL (final concentration of each homodimer was 10 mg / mL) and 10 mg / mL (final concentration of each homodimer was 5 mg / mL). Separate mixtures of 2-MEA with final concentrations of 10, 25, 50, and 100 mM were prepared for both IgG concentrations. The mixtures were aliquoted into 100 μL eppendorf tubes and stored at 15, 25, and 37 °C. Individual tubes were used for incubation at various temperatures for 90 min, 5 h, and 24 h.

[0349] For both IgG concentrations, a 2-MEA-free mixture was also prepared and stored at 4°C as an untreated control. After appropriate incubation times, 90-minute and 5-hour samples were collected for desalting to remove 2-MEA (the 90-minute sample was initially placed on ice to terminate the exchange reaction). Samples were desalted using a Zeba 96-well desalting plate (7k, catalog number 89808, Thermo Fisher Scientific). The 24-hour sample was desalted separately after 24 hours of incubation.

[0350] Bispecific binding was measured in a sandwich ELISA using serial dilutions of antibody samples as described in Example 7 (total antibody concentration of 10⁻⁶–0.123 μg / mL in 3-fold dilutions for 90-minute and 5-hour samples; 10⁻⁶–0.041 μg / mL in 3-fold dilutions for 24-hour samples). For each plate, controls were included in a purified batch of bispecific antibody derived from a 2-MEA-induced Fab arm interchange between IgG1-2F8-ITL and IgG4-7D8-CPPC (as described in Example 15). Figure 34 (A)-(F) show the bispecific binding results measured in their respective ELISA plates. The highest OD405 value (as determined in the ELISA for a concentration of 10 μg / mL) was used to calculate the bispecific binding compared to the control (which was arbitrarily set to 100%). This yields the percentage (% cFAE) of controlled Fab arm interchange compared to the control, as shown for each 2-MEA concentration. Figure 34 As shown in (A)-(D).

[0351] Data showed that bispecific binding (89-109% compared to control) was achieved at 100 mM 2-MEA for both IgG concentrations under all temperature-time conditions. At 50 mM 2-MEA, maximum binding (88-107%) was achieved at 25°C and 37°C, and also after 24 hours of incubation at 15°C. For the lower concentrations of 25 mM and 10 mM 2-MEA, exchange was more efficient at higher temperatures, increasing with longer incubation times, culminating in maximum exchange at 37°C after 24 hours of incubation with 25 mM 2-MEA. Conditions tested at 10 mM 2-MEA did not produce 100% bispecific product. The exchange process was slightly faster at 10 mg / mL IgG concentration compared to 20 mg / mL total IgG.

[0352] To confirm the formation of bispecific antibodies and to study the bispecific products in more detail, the samples were analyzed using cation exchange chromatography (HPLC-CIEX). Samples incubated for 5 hours and 24 hours with 20 mg / mL IgG and all 2-MEA concentrations were analyzed by HPLC-CIEX as described in Example 31.

[0353] Figure 35 CIEX chromatograms (A)-(D) show the highest yields of the bispecific product obtained at 50 and 100 mM 2-MEA, confirming the results of the bispecific ELISA. However, small amounts of residual homodimers were still detected at 50 and 100 mM 2-MEA (2-3.5% of various homodimers for samples incubated at 25°C and 37°C). Interchanging higher temperatures, longer (24 hours) incubation times, and gradually increasing 2-MEA concentrations resulted in additional peaks at 22-24 minutes in the CIEX curves.

[0354] Minimal additional peaks were obtained when the exchange was completed within 5 hours. To characterize these peaks, SDS-PAGE and HP-SEC analyses were performed. HP-SEC showed amounts less than 1% for all conditions, suggesting the additional peaks were not representative of the aggregates. However, non-reducing SDS-PAGE showed the additional peaks could represent heterodimers lacking one or two light chains. Small amounts of half-molecules were also detected.

[0355] Experiments showed that the exchange reaction occurred at high homodimer concentrations (which makes the method attractive for commercial scale), and that the yield of bispecific antibodies depended on the 2-MEA concentration, temperature, and incubation time.

[0356] Example 35: Determinant cluster at position 368 of IgG1 for use in combination with IgG1-K409R to participate in 2-MEA-induced Fab arm interchange Examples 28 and 29 show that certain single mutations at positions F405 and Y407, when combined with IgG1-K409R, are sufficient to enable human IgG1 to participate in Fab arm exchange. As illustrated in this example, other determinants involving the Fc:Fc interface position of the CH3 domain can also mediate Fab arm exchange mechanisms. For this purpose, mutagenesis was performed at position 368 of IgG1, and mutants participating in 2-MEA-induced Fab arm exchange when combined with human IgG1-K409R were tested. All possible IgG1-2F8-L368X mutants (except C and P) were combined with IgG1-7D8-K409R. The procedure was performed using purified antibody as described in Example 19.

[0357] Figure 36The results show the bispecific binding following 2-MEA-induced Fab arm exchange between IgG1-2F8-L368X and IgG1-7D8-K409R. As presented in Table 8, these data were also graded as (-) no Fab arm exchange, (+ / -) low, (+) moderate, or (++) high Fab arm exchange. No Fab arm exchange (-) was found when position 368 of IgG1-2F8 was L (=wild-type IgG1), F, or M. Low Fab arm exchange (+ / -) was found when position 368 of IgG1-2F8 was Y. Moderate Fab arm exchange (+) was found when position 368 of IgG1-2F8 was K, and high (++) was found when position 368 of IgG1-2F8 was A, D, E, G, H, I, N, Q, R, S, T, V, or W. These data suggest that a specific mutation at position 368 of IgG1, when combined with IgG1-K409R, allows IgG1 to participate in 2-MEA-induced Fab arm swapping.

[0358] Table 8: 2-MEA-induced Fab arm interchange between IgG1-2F8-L368X mutant and IgG1-7D8-K409R The production of bispecific antibodies following 2-MEA-induced in vitro Fab arm exchange between IgG1-2F8-L368X mutant and IgG1-7D8-K409R was determined by sandwich ELISA. (-) No, (+ / -) Low, (+) Moderate, or (++) High Fab arm exchange.

[0359] Example 36: Determinant cluster at position 370 of IgG1 for use in combination with IgG1-K409R to participate in 2-MEA-induced Fab arm interchange Examples 28, 29, and 35 show that certain single mutations at positions F405, Y407, or L368, when combined with IgG1-K409R, are sufficient to enable human IgG1 to participate in Fab arm exchange. As illustrated in this example, other determinants involving the Fc:Fc interface position of the CH3 domain can also mediate Fab arm exchange mechanisms. For this purpose, mutagenesis was performed at position 370 of IgG1, and mutants participating in 2-MEA-induced Fab arm exchange when combined with human IgG1-K409R were tested. All possible IgG1-2F8-K370X mutants (except C and P) were combined with IgG1-7D8-K409R. The procedure was performed using purified antibodies as described in Example 19.

[0360] Figure 37The results show the bispecific binding following 2-MEA-induced Fab arm exchange between IgG1-2F8-K370X and IgG1-7D8-K409R. As presented in Table 9, these data were also graded as (-) no Fab arm exchange, (+ / -) low, (+) moderate, or (++) high Fab arm exchange. No Fab arm exchange (-) was found when position 370 of IgG1-2F8 was K (=wild-type IgG1), A, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, or Y. Only substitution of K370 with W resulted in moderate Fab arm exchange (+). These data suggest that only one mutation at position 370 of IgG1 (K370W) allows IgG1 to participate in 2-MEA-induced Fab arm exchange when combined with IgG1-K409R.

[0361] Table 9: 2-MEA-induced Fab arm interchange between IgG1-2F8-K370X mutant and IgG1-7D8-K409R The production of bispecific antibodies following 2-MEA-induced in vitro Fab arm exchange between IgG1-2F8-K370X mutant and IgG1-7D8-K409R was determined by sandwich ELISA. (-) No, (+ / -) Low, (+) Moderate, or (++) High Fab arm exchange.

[0362] Example 37: Determinant cluster at position 399 of IgG1 for use in combination with IgG1-K409R to participate in 2-MEA-induced Fab arm interchange Examples 28, 29, 35, and 36 show that certain single mutations at positions F405, Y407, L368, or K370, when combined with IgG1-K409R, are sufficient to enable human IgG1 to participate in Fab arm exchange. As illustrated in this example, other determinants involving the Fc:Fc interface position of the CH3 domain can also mediate Fab arm exchange mechanisms. For this purpose, mutagenesis was performed at position 399 of IgG1, and mutants participating in 2-MEA-induced Fab arm exchange when combined with human IgG1-K409R were tested. All possible IgG1-2F8-D399X mutants (except C and P) were combined with IgG1-7D8-K409R. The procedure was performed using purified antibodies as described in Example 19.

[0363] Figure 38The results show the bispecific binding following 2-MEA-induced Fab arm exchange between IgG1-2F8-D399X and IgG1-7D8-K409R. As presented in Table 10, these data were also graded as (-) none, (+ / -) low, (+) moderate, or (++) high Fab arm exchange. No Fab arm exchange (-) was found when position 399 of IgG1-2F8 was D (=wild-type IgG1), E, ​​or Q. Fab arm exchange was found to be low (+ / -) when position 399 of IgG1-2F8 was V, and moderate (+) when position 399 of IgG1-2F8 was G, I, L, M, N, S, T, or W. Fab arm exchange was found to be high (++) when position 399 of IgG1-2F8 was A, F, H, K, R, or Y. These data suggest that a specific mutation at position 399 of IgG1, when combined with IgG1-K409R, allows IgG1 to participate in 2-MEA-induced Fab arm swapping.

[0364] Table 10: 2-MEA-induced Fab arm interchange between IgG1-2F8-D399X mutant and IgG1-7D8-K409R The production of bispecific antibodies following 2-MEA-induced in vitro Fab arm exchange between IgG1-2F8-D399X mutant and IgG1-7D8-K409R was determined by sandwich ELISA. (-) No, (+ / -) Low, (+) Moderate, or (++) High Fab arm exchange.

[0365] Example 38: Determining the range of conditions under which 2-MEA-induced Fab arm interchange occurs suboptimally to distinguish highly effective IgG1 mutants. When 25 mM 2-MEA is used, the 2-MEA-induced Fab arm interchange process occurs efficiently at 37°C. Under these conditions, most permissible IgG1 mutants (such as IgG1 with certain single mutations at positions 368, 370, 399, 405, and 407 and / or 409 as described in Examples 19, 28, 29, and 35-37) exhibit high levels of 2-MEA-induced Fab arm interchange (80%-100%). To determine the experimental conditions that allow for the most efficient differentiation of IgG1 mutants, 2-MEA-induced Fab arms of four different mutant combinations (IgG1-2F8-F405S x IgG1-7D8-K409A, IgG1-2F8-D399R x IgG1-7D8-K409G, IgG1-2F8-L368R x IgG1-7D8-K409H, and IgG1-2F8-F405L x IgG1-7D8-K409R) were studied over time at 15°C and 20°C. The procedure was performed as described in Example 19, except for variations in temperature, time period, and antibody dilution (20, 2, 0.2, and 0.02 µg / mL).

[0366] Compared to the maximum exchange (positive control), the four mutant combinations underwent 2-MEA-induced Fab arm exchange at different rates at 20°C. The maximum level of exchange was reached in IgG1-2F8-L368R x IgG1-7D8-K409H after 105 minutes of incubation, while the maximum values ​​of IgG1-2F8-F405S x IgG1-7D8-K409A, IgG1-2F8-D399R x IgG1-7D8-K409G, and IgG1-2F8-F405L x IgG1-7D8-K409R were reached at 90%, 85%, and 85%, respectively, after 200 minutes of incubation.

[0367] The most significant difference in exchange rate was observed when different combinations of IgG1 mutants were incubated at 15°C (shown in...). Figure 39 The differences in 2-MEA-induced Fab arm exchange among the four mutant combinations were most extreme after 60 and 105 minutes of incubation. Compared with the positive control, the Fab arm exchange efficiency after 200 minutes of incubation was 100% (IgG1-2F8-L368R x IgG1-7D8-K409H), 85% (IgG1-2F8-F405L x IgG1-7D8-K409R and IgG1-2F8-D399R x IgG1-7D8-K409G), or 65% (IgG1-2F8-F405S x IgG1-7D8-K409A).

[0368] Example 39: Analysis of 2-MEA-induced Fab arm interchange efficiency in mutants under suboptimal conditions When using 25 mM 2-MEA, 2-MEA-induced Fab arm swapping occurs efficiently at 37°C. Under these conditions, most permissible IgG1 mutants (such as IgG1 with certain single mutations at positions 368, 370, 399, 405, and 407 and / or 409 as described in Examples 19, 28, 29, and 35-37) exhibit high levels of 2-MEA-induced Fab arm swapping (80-100%). The most significant difference in 2-MEA-induced Fab arm swapping efficiency was described in Example 38 after incubation under so-called suboptimal conditions, i.e., incubation at 15°C for 60 to 105 minutes. A total of 24 IgG1-2F8 mutants in L368, D399, F405, and Y407 exhibiting >90% 2-MEA-induced Fab arm interchanges with IgG1-7D8-K409R (Examples 28, 29, and 35-37) were selected (see Table 11) and compared with IgG1-7D8-K409A, G, H, or R (Fab arm interchange analysis based on results reported in Example 19). To classify these mutant combinations according to their efficiency in generating bispecific antibodies, 2-MEA-induced Fab arm interchanges were performed at 15°C for 90 minutes (suboptimal conditions). Two IgG1-2F8 mutants (Y407Q and D399Q) (Examples 29 and 37) that exhibited weak 2-MEA-induced Fab arm interchange after incubation with IgG1-7D-K409R were used together as additional negative controls to investigate whether incubation with another amino acid (G, H, or W) at the K409 position led to different results. Procedure was performed as described in Example 19, except that the temperature and antibody dilution were varied (20, 2, 0.2, and 0.02 ug / mL).

[0369] All different combinations of IgG1 mutants (as is clear from Table 11) were incubated at 15°C for 90 min to show a range of different 2-MEA-induced Fab arm exchange efficiencies. Bispecific binding results at an antibody concentration of 20 µg / mL are shown in Table 11. The results were categorized into four classes: no (-), low (+ / -), moderate (+), and high (++) bispecific binding efficiencies, as specified in the notes below Table 11. It is clear from these results that, under suboptimal conditions, combinations of certain amino acid mutations in the IgG1 molecule will favor 2-MEA-induced Fab arm exchange.

[0370] Table 11: Bispecific binding (%) between allowed IgG1 mutants (20 µg / mL) at 15°C for 90 minutes Six mutant IgG1-2F8 molecules were selected from those tested (Table 11) for a second analysis to confirm the previously obtained results (Table 11). Several mutants were selected for their high (IgG1-2F8-L368R) and moderate (IgG1-2F8-L368W, IgG1-2F8-F405I, IgG1-2F8-F405L, and IgG1-2F8-Y407W) efficiency of 2-MEA-induced Fab arm exchange. IgG1-2F8-Y407Q was also analyzed a second time because it showed an unexpectedly positive 2-MEA-induced Fab arm exchange reaction with IgG1-7D8-K409H. Overall, these results (in...) Figure 40 The results (presented in Table 11) confirmed the initial analysis, showing that the 2-MEA-induced Fab arm exchange reaction between the mutant IgG1-2F8 molecule and IgG1-7D8-K409H had the highest efficiency. Furthermore, the 2-MEA-induced Fab arm exchange reaction between the mutant IgG1-2F8 molecule and IgG1-7D8-K409R, which was reported as negative in Examples 28, 29, and 35-37, remains of interest as it has the potential to promote IgG1 2-MEA-induced Fab arm exchange.

[0371] Example 40: Using a bispecific form to remove the adverse agonistic activity of antagonistic c-Met antibodies, thereby converting them into a monovalent, bispecific form. Several bivalent antibodies developed for monoclonal antibody therapy exhibit poor agonistic activity upon binding to their targets. This is also true for most IgG1-based receptors that target the receptor tyrosine kinase c-Met. These agonistic antibodies induce receptor dimerization, which then activates several downstream signaling pathways. This results in the induction of (tumor) cell growth and differentiation. Using monovalent antibody forms prevents the induction of receptor dimerization. Combining the Fab arm of an anti-c-Met antibody with the Fab arm of an irrelevant antibody produces a functionally monovalent and therefore completely antagonistic bispecific antibody. Here we combine a partially-(IgG1-069) or fully-(IgG1-058) agonist antibody with IgG1-b12 (first described in Burton DR et al., “Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody”, Science. November 1994;266(5187):1024-1027) in a bispecific antibody. IgG1-b12 is considered an irrelevant non-binding antibody because it is produced against the viral protein (HIV-gp120). The anti-c-Met antibody used in this example is a fully-human monoclonal antibody produced in transgenic mice. IgG1-058 and IgG1-069 bind to different epitopes on c-Met.

[0372] The two anti-c-Met antibodies used are IgG1,κ antibodies with modifications in their Fc region, as further disclosed. They have the following variable heavy and light chain sequences. 058: VH 058 EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYYMYWVRQTPEKRLEWVATISDDGSYTYYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYCAREGLYYYGSGSYYNQDYWGQGTLVTVSS VL 058 AIQLTQSPSSSLSASVGDRVTITCRASQGLSSALAWYRQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFTSYPQITFGQGTRLEIK 069: VH 069 QVQLVQSGAEVKKPGASVKVSCETSGYTFTSYGISWVRQAPGHGLEWMGWISAYNGYTNYAQKLQGRVTMTTDTSSTAYMELRSLRSDDTAVYYCARDLRGTNYFDYWGQGTLVTVSS VL 069 DIQMTQSPSSVSASVGDRVTITCRASQGISNWLAWFQHKPGKAPKLLIYAASSLLSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPITFGQGTRLEIK Receptor phosphorylation Monovalent bispecific c-Met antibodies were generated using 25 mM 2-MEA via a Fab arm interchange reaction of IgG1-058-F405L or IgG1-069-F405L with IgG1-b12-K409R as described in Example 23. The effect of the bispecific antibody on c-Met phosphorylation was evaluated. Following dimerization of two adjacent c-Met receptors via the natural ligand HGF or via an agonistic bivalent antibody, three tyrosine residues (positions 1230, 1234, and 1235) in the c-Met intracellular domain were cross-phosphorylated. This resulted in phosphorylation of several other amino acids in the c-Met intracellular domain and activation of multiple signal transduction cascades. The dimerization and activation of c-Met could be monitored using antibodies specific to the phosphorylated receptors at these positions, which served as a readout of the potential agonistic activity of the anti-c-Met antibody.

[0374] A549 cells (CCL-185 obtained from ATCC) were grown in serum-containing DMEM medium until 70% confluence was achieved. Cells were digested with trypsin, washed, and cultured at 1×10⁻⁶. 6 Cells were seeded per well in serum-containing medium in 6-well plates. After overnight incubation, cells were treated with HGF (R&D system; catalog 294-HG) (50 ng / mL) or an antibody group (30 µg / mL) and incubated at 37°C for 15 min. Cells were washed twice with ice-cold PBS and lysed with lysis buffer (Cell Signaling; catalog 9803) supplemented with a protease inhibitor mixture (Roche; catalog 11836170001). Cell lysate samples were stored at -80°C. Receptor activation was determined by detecting c-Met phosphorylation using a phosphorylated c-Met-specific antibody via Western blotting. Proteins present in the cell lysates were separated on a 4–12% SDS-PAGE gel, transferred to a nitrocellulose membrane, and then stained with an antibody specific to phosphorylated c-Met (Y1234 / 1235) (Cell Signaling, catalog 3129). As controls for gel loading, total β-actin and c-Met levels were determined using anti-c-Met (CellSignaling, catalog number 3127) and anti-β-actin (Cell Signaling, catalog number 4967) antibodies. Western blot results are shown in... Figure 41 .

[0375] Cells treated with tissue culture medium controls and the monovalent form UniBody® (Genmab, WO2007059782 and WO2010063785) of antibody 5D5 (Genentech; WO 96 / 38557) showed no c-Met receptor phosphorylation. The monovalent UniBody form used in this study was IgG4, in which the hinge region was missing and the CH3 region was mutated at positions 405 and 407. In contrast, Western blot analysis of cells treated with the positive control HGF or the agonist antibody IgG1-058 showed a clear band at the expected high level of c-Met phosphorylation. The partially agonist antibody IgG1-069 showed smaller, but detectable, receptor phosphorylation, suggesting some cross-linking of the receptor. However, neither the bispecific IgG1 058 / b12 nor the bispecific 069 / b12 antibodies induced any c-Met phosphorylation, showing a complete absence of agonist activity associated with the parental antibodies. Figure 41 ).

[0376] Effect of c-Met antibody on the in vitro proliferation of NCI-H441 In the lung adenocarcinoma cell line NCI-H441 (ATCC, HTB-174) TM The potential proliferative agonist activity of the c-Met antibody was tested in vitro. 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) in 96-well tissue culture plates (Greiner bio-one, Frickenhausen, Germany) (5,000 cells / well). The anti-c-Met antibody was diluted to 66.7 nM in serum-free RPMI medium and added to the cells. After incubation at 37°C / 5% CO2 for 7 days, the number of viable cells was quantified using Alamarblue (BioSource International, San Francisco, US) according to the manufacturer's instructions. Fluorescence was monitored using an EnVision 2101 Multilabel reader (PerkinElmer, Turku, Finland) with standard Alamarblue settings.

[0377] Unlike IgG1-069, incubation of NCI-H441 cells with the bispecific IgG1-069 / b12 did not induce proliferation. Figure 42 As shown. The UniBody-069 control also did not induce proliferation, comparable to untreated or IgG1-b12 treated controls.

[0378] Example 41: CDC-mediated cell killing of bispecific antibodies generated by Fab arm exchange induced by 2-MEA between human IgG1-2F8-F405L or IgG1-7D8-F405L and IgG1-7D8-K409R. The CD20 antibody IgG1-7D8 effectively kills CD20-expressing cells via complement-dependent cytotoxicity (CDC). Conversely, the EGFR antibody IgG1-2F8 does not mediate CDC on EGFR-expressing target cells. Both the bispecific antibody IgG1-7D8-K409R and the bispecific antibody generated by a 2-MEA-induced Fab arm exchange between IgG1-2F8-F405L and IgG1-7D8-K409R can induce CDC on CD20-expressing cells (as described in Example 26). The bispecific antibody generated by a 2-MEA-induced Fab arm exchange between IgG1-7D8-F405L and IgG1-7D8-K409R was tested to determine whether it could also induce CDC on CD20-expressing cells. 10 5 Daudi or Raji cells were pre-incubated with a series of antibody concentrations in 100 µL of RPMI medium supplemented with 0.1% BSA on a shaker at room temperature for 15 minutes. 25 µL of normal human serum (NHS) was added as a complement source (20% NHS final concentration), and the cells were incubated at 37°C for 45 minutes. After incubation, the plate was placed on ice to terminate the CDC reaction. Dead and live cells were distinguished by adding 10 µL of 10 µg / mL propidium iodide (PI) (0.6 µg / mL final concentration) and FACS analysis.

[0379] Figure 43 The product exhibits bispecificity for IgG1-7D8 and Fab arm interchange induced by 2-MEA between IgG1-7D8-F405L and IgG1-7D8-K409R, and has the same induced CD20 expression in Daudi (…). Figure 43 A) and Raji ( Figure 43B) Efficacy of CDC-mediated cell killing. Neither Daudi nor Raji cells express EGFR, resulting in monovalent binding of bispecific antibodies generated via 2-MEA-induced Fab arm exchange between IgG2-2F8-F405L x IgG1-7D8-K409R. This bispecific product also induced CDC-mediated cell killing, although with slightly lower efficacy. These data suggest that the CDC capacity of the parental antibodies is retained in their bispecific form. CDC-mediated cell killing induced via the bispecific product (IgG1-7D8-F405L x IgG1-7D8-K409R) was slightly more effective than that induced by the monovalent bispecific product (IgG2-2F8-F405L x IgG1-7D8-K409R). The 11B8 antibody targeting CD20 failed to induce CDC-mediated cell killing and served as a negative control.

[0380] Example 42: HER2 x HER2 bispecific antibody tested in an in vitro κ-directed ETA' killing assay This example demonstrates that, using a κ-oriented Pseudomonas exotoxin A (anti-κ-ETA'), a HER2 x HER2 bispecific antibody can deliver a cytotoxic agent into tumor cells after internalization in a standard in vitro cell-based killing assay. This assay utilizes a high-affinity anti-κ domain antibody conjugated to a truncated form of Pseudomonas exotoxin A. Similar antibody-binding proteins (from the IgG-binding motif of streptococcal protein A or protein G) and fusion proteins of diphtheria toxin or Pseudomonas exotoxin A have been previously reported (Mazor Y. et al., J. Immunol. Methods 2007; 321:41-59; Kuo SR. et al., 2009 Bioconjugate Chem. 2009; 20: 1975-1982). Unlike anti-κ-ETA', these molecules bind to the Fc portion of the intact antibody. Following internalization and endocytosis sorting, the anti-κ-ETA' domain antibody undergoes proteolysis and disulfide bond reduction, separating the catalytic and binding domains. The catalytic domain is then transported from the Golgi apparatus to the endoplasmic reticulum via the KDEL-retained motif, and subsequently translocates to the cytoplasm, where it inhibits protein synthesis and induces apoptosis (Kreitman RJ. et al., BioDrugs 2009; 23:1-13).

[0381] The anti-HER2 antibodies used in this embodiment and in Examples 43-45 below are fully human monoclonal antibodies produced in transgenic mice. They bind to different epitopes on HER2.

[0382] They are all IgG1,κ antibodies with modifications in their Fc region, as further disclosed. They possess the following variable sequences for both heavy and light chains. 005: VH005 EVQLVQSGAEVKKPGESLKISCKASGYSFHFYWIGWVRQMPGKGLEWMGSIYPGDSDTRYRPSFQGQVTISADKSISTAYLQWTSLKASDTAIYYCARQRGDYYYFYGMDVWGQGTTVTVSS VL005 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQVPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSS-LTFGGGTKVEIK 025: VH025 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEIHHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGYYDSGVYYFDYWAQGTLVTVSS VL025 DIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAASSLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPITFGQGTRLEIK 153: VH153 QVQLVESGGGVVQPGRSLRLSCAASGFTFSDYVIHWVRQAPGKGLEWVTVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLSAEDTAMYYCARGGITGTTGVFDYWGQGTLVTVSS VL153 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYDASSLQSGVPSRFSGSGYGTDFSLTISSLQPEDFAIYYCQQYKSYPITFGQGTRLEIK 169: VH169 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGISWVRQAPGQGLEWMGWLSAYSGNTIYAQKLQGRVTMTTDTSTTTAYMELRSLRSDDTAVYYCARDRIVVRPDYFDYWGQGTLVTVSS VL169 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPRTFGQGTKVEIK Prior to incubation with A431 cells, the HER2 x HER2 bispecific antibody was pre-incubated with anti-κ-ETA. A431 cells expressed ~15,000 HER2 antibody / cell (as determined by Qifi analysis) and were insensitive to treatment with “naked” HER2-antibody.

[0384] First, the optimal concentration of anti-κ-ETA' for each cell line was determined, i.e., the maximum tolerated dose that does not induce nonspecific cell death. A431 cells (2500 cells / well) were seeded in normal cell culture medium in 96-well tissue culture plates (Greiner bio-one) and allowed to attach for at least 4 hours. These cells were then incubated in normal cell culture medium with a series of anti-κ-ETA' dilutions (100, 10, 1, 0.1, 0.01, 0.001, and 0 µg / mL). After 3 days, the number of viable cells was quantified using Alamarblue (BioSource International, San Francisco, US) according to the manufacturer's instructions. Fluorescence was monitored using an EnVision 2101 Multilabel reader (PerkinElmer, Turku, Finland) with standard Alamarblue settings. The highest self-toxic concentration of anti-κ-ETA' (1 µg / mL for A431 cells) was used in the following experiments.

[0385] Next, the ability of these antibodies to induce cell killing was tested by examining the effects of HER2 x HER2 bispecific and HER2 monospecific antibodies pre-incubated with anti-κ-ETA. A431 cells were seeded as described above. Serial dilutions of HER2-specific antibodies (monospecific and bispecific antibodies) were prepared, pre-incubated with predetermined concentrations of anti-κ-ETA for 30 minutes, and then added to the cells. After incubation at 37 °C for 3 days, the number of viable cells was quantified as described above. The Alamarblue signal of cells treated with antibody-pre-incubated anti-κ-ETA was plotted against untreated cells. EC5 was calculated using GraphPad Prism 5 software. 50Values ​​and maximum cell death. Astrosporin (23.4 µg / mL) was used as a positive control for cell killing. Isotype control antibodies (IgG1 / κ; IgG1-3G8-QITL) were used as negative controls.

[0386] Figure 44 The results showed that all HER2 bispecific antibodies pre-incubated with anti-κ-ETA' were able to kill A431 cells in a dose-dependent manner. These results demonstrate that most of the tested HER2 bispecific antibodies were more effective in this anti-κ-ETA' assay than the monospecific antibodies present in the combination. Furthermore, the efficacy of bispecific antibodies 005X169, 025X169, and 153X169 showed that the efficacy of a monospecific antibody (HER2-specific antibody 169) lacking activity in this in vitro κ-directed ETA' killing can be increased by bispecific combination with another HER2-specific antibody.

[0387] Example 43: Downregulation of HER2 receptor by incubation with bispecific antibodies targeting different HER2 epitopes HER2 x HER2 bispecific antibodies can bind to two different epitopes on two spatially dissimilar HER2 receptors. This allows other HER2 x HER2 bispecific antibodies to bind to the remaining epitopes on these receptors. This can lead to multivalent receptor cross-linking (compared to dimerization induced by monovalent antibodies) and thus enhance receptor downregulation. To investigate whether HER2 x HER2 bispecific antibodies induce enhanced HER2 downregulation, AU565 cells were incubated with both the antibody and the bispecific antibody for 3 days. Total HER2 levels and antibody-bound HER2 levels were determined.

[0388] AU565 cells were seeded in normal cell culture medium in 24-well tissue culture plates (100,000 cells / well) and cultured at 37°C for 3 days in the presence of 10 μg / mL HER2 antibody or HER2 x HER2 bispecific antibody. After washing with PBS, cells were lysed by incubating with 25 μL Surefire lysis buffer (Perkin Elmer, Turku, Finland) at room temperature for 30 min. Total protein levels were quantified using the bicinchoninic acid (BCA) protein assay reagent (Pierce) according to the manufacturer's protocol. HER2 protein levels in the lysates were analyzed using a HER2-specific sandwich ELISA. Rabbit anti-human HER2 intracellular domain antibody (Cell Signaling) was used to capture HER2, and biotinylated goat anti-human HER2 polyclonal antibody (R&D system, Minneapolis, USA) was used to detect bound HER2. The reaction was visualized using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS: one ABTS tablet diluted in 50 mL ABTS buffer [Roche Diagnostics, Almere, Netherlands]) and terminated with oxalic acid (Sigma-Aldrich, Zwijndrecht, The Netherlands). Fluorescence at 405 nm was measured on a microtiter plate reader (Biotek Instruments, Winooski, USA), and the amount of HER2 was expressed as a percentage compared to untreated cells.

[0389] The results are shown in Figure 45 This demonstrates that all tested HER2 x HER2 bispecific antibodies induced ≥40% HER2 downregulation. Interestingly, all HER2 x HER2 bispecific antibodies showed increased HER2 downregulation compared to their monospecific counterparts.

[0390] Example 44: Co-localization of HER2 x HER2 bispecific antibody and lysosomal marker LAMP1 analyzed by confocal microscopy The HER2 downregulation assay described in Example 43 showed that the HER2 x HER2 bispecific antibody increased lysosomal degradation of HER2. To confirm these findings, confocal microscopy was used. AU565 cells were grown on coverslips (1.5 μm thick, Thermo Fisher Scientific, Braunschweig, Germany) in standard tissue medium at 37°C for 3 days. Cells were pre-incubated with leucopeptide (Sigma) for 1 hour to block lysosomal activity, followed by the addition of 10 μg / mL HER2 monospecific antibody or HER2 x HER2 bispecific antibody. Cells were then incubated at 37°C for another 3 or 18 hours. Afterward, they were washed with PBS and incubated at room temperature with 4% formaldehyde (Klinipath) for 30 minutes. The slides were washed with blocking buffer (PBS supplemented with 0.1% saponin [Roche] and 2% BSA [Roche]) and incubated with blocking buffer containing 20 mM NH4Cl for 20 minutes to quench the formaldehyde. Wash the slides again with blocking buffer and incubate them at room temperature with mouse anti-human CD107a (LAMP1) (BDPharmingen) for 45 minutes to stain lysosomes. After washing with blocking buffer, incubate the slides at room temperature with a mixture of secondary antibodies: goat anti-mouse IgG-Cy5 (Jackson) and goat anti-human IgG-FITC (Jackson) for 30 minutes. Wash the slides again with blocking buffer and fix them on microscope slides overnight with 20 μL of fixation medium (6 g glycerol [Sigma] and 2.4 g Mowiol 4-88 [Omnilabo] dissolved in 6 mL distilled water, 12 mL 0.2 M Tris [Sigma] pH 8.5 added, and incubated at 50–60 °C for 10 minutes. Divide the fixation medium into equal portions and store at -20 °C). The slides were imaged using a Leica SPE-II confocal microscope (Leica Microsystems) equipped with a 63x 1.32–0.6 oil immersion objective and LAS-AF software. To allow for quantification of overlapping pixel intensities, pixel saturation should be avoided. 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%. With these settings, bispecific antibodies were clearly presented without pixel saturation, but monospecific antibodies were sometimes difficult to detect. To compare lysosomal colocalization between monospecific and bispecific antibodies, the same settings were maintained for all analyzed confocal slides.

[0391] Using MetaMorph ®Software (Meta Series 6.1, Molecular Devices Inc, Sunnyvale, California, USA) analyzed colocalization in 12-bit images. FITC and Cy5 images were input in an overlay, with the background subtracted. The exact same threshold settings (manually set) were used for all FITC and all Cy5 images. Colocalization was described as the pixel intensity of overlapping region (ROI) d FITC, where the ROI consists of all Cy5-positive regions. To compare different slides stained with several HER2 antibodies or HER2 x HER2 bispecific antibodies, the images were normalized to Cy5 pixel intensity. Goat anti-mouse IgG-Cy5 was used to stain the lysosomal marker LAMP1 (CD107a). The pixel intensity of LAMP1 should not differ between the various HER2 antibodies or HER2 x HER2 bispecific antibodies tested (one cell has approximately 200,000 Cy5 pixel intensities).

[0392] The normalized value of co-location between FITC and Cy5 = [(TPI-FITC x Percentage FITC-Cy5 Co-location) / 100] x [200.000 / TPI-Cy5] In this formula, TPI represents the total pixel intensity.

[0393] The percentage of live cells for various monospecific HER2 antibodies and HER2 x HER2 bispecific antibodies was presented, as measured by the intensity of FITC pixels overlapping with Cy5. For each antibody or bispecific molecule described, three different images were analyzed from a slide containing ~1, 3, or >5 cells. Significant differences were observed between the different images within each slide. However, when compared with their monospecific counterparts, all HER2 x HER2 bispecific antibodies clearly showed increased co-localization with the lysosomal marker LAMP1. These results suggest that once internalized, HER2 x HER2 bispecific antibodies are efficiently sorted into the lysosomal lumen, making them suitable for bispecific antibody-drug conjugation methods.

[0394] Example 45: Inhibition of AU565 cell proliferation after incubation with HER2 monospecific antibody or HER2 x HER2 bispecific antibody The ability of the HER2 bispecific antibody to inhibit the proliferation of AU565 cells in vitro was tested. Due to the high HER2 expression levels in AU565 cells (e.g., ~1,000,000 copies per cell as determined by Qifi-kit), HER2 is constitutively active in these cells and therefore independent of ligand-induced heterodimerization. In 96-well tissue culture plates (Greiner bio-one, Frickenhausen, Germany), 9,000 AU565 cells per well were seeded in serum-free medium in the presence of 10 µg / mL HER2 antibody or HER2 xHER2 bispecific antibody. As a control, cells were seeded in serum-free medium without either antibody or bispecific antibody. After 3 days, the number of viable cells was quantified using Alamarblue (BioSource International, San Francisco, US) according to the manufacturer's instructions. Fluorescence was monitored using an EnVision 2101 Multilabel reader (PerkinElmer, Turku, Finland) with standard Alamarblue settings. The Alamarblue signal of antibody-treated cells was plotted as a percentage compared to untreated cells.

[0395] Figure 47 The Alamarblue fluorescence intensity of AU565 cells after incubation with HER2 antibody and HER2xHER2 bispecific antibody was described. Herceptin® (trastuzumab) was included as a positive control, and as described by Juntilla TT. et al., CancerCell 2009; 15: 429-440, Herceptin® has been shown to inhibit proliferation. All HER2 x HER2 bispecific antibodies were able to inhibit the proliferation of AU565 cells. In this assay, the bispecific antibodies IgG1-005-ITL x IgG1-169-K409R and IgG1-025-ITL x IgG1-005-K409R were both more effective than their monospecific antibody counterparts.

[0396] Example 46: In vitro and in vivo analysis of FcRn binding of bispecific IgG1 antibodies containing one or two FcRn binding sites in the Fc region and hinge-deficient bispecific IgG1 antibodies. This embodiment illustrates the preparation of asymmetric bispecific molecules (i.e., molecules of the present invention that have different characteristics in each Fab arm).

[0397] The neonatal Fc receptor (FcRn) is responsible for the long plasma half-life of IgG by protecting it from degradation. After antibody internalization, FcRn binds to the antibody's Fc region in endosomes, where the interaction is stable in a mildly acidic environment (pH 6.0). Upon recirculation to the neutral plasma membrane (pH 7.4), the interaction is lost, and the antibody is released back into circulation. The antibody's Fc region contains two FcRn binding sites, one on each heavy chain at the CH2-CH3 interface. The H435A mutation in the antibody's Fc region cancels binding to FcRn (Shields, RL et al., J Biol Chem, 2001; Firan, M. et al., Int Immunol, 2001), and the hinge region is also thought to affect FcRn binding (Kim, JK et al., Mol Immunol., 1995). In addition, it has been proposed that the binding of bivalent antibodies to FcRn plays a greater role than that of monovalent antibodies in efficient recycling (Kim, JK, et al., Scand J Immunol., 1994).

[0398] In this embodiment, the effect of FcRn binding valence was evaluated using asymmetric bispecific IgG1 molecules containing a single FcRn binding site. The additional role of the hinge region was evaluated using IgG1 molecules lacking the asymmetric bispecific hinge (Uni-G1).

[0399] The FcRn binding of bispecific IgG1 or hinge-deficient IgG1 (Uni-G1) molecules with or without 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 were prepared according to Examples 2, 3, 4, and 5. Hinge-deficient IgG1 molecules Uni-G1-2F8-ITL, Uni-G1-7D8-K409R, and Uni-G1-7D8-K409R-H435A were prepared according to Example 11. Bispecific IgG1 molecules were generated by 2-MEA-induced Fab arm interchange between IgG1-2F8-ITL and IgG1-7D8-K409R or IgG1-7D8-K409R-H435A molecules. Bispecific hinge-deficient IgG1 molecules were prepared by incubating Uni-G1-2F8-ITL with Uni-G1-7D8-K409R or Uni-G1-7D8-K409R-H435A. A 3-fold dilution series of monospecific and bispecific IgG1 molecules and hinge-deficient IgG1 molecules were added to biotinylated human or mouse FcRn trapped on streptoacid-coated ELISA plates, followed by incubation at pH 6.0 and 7.4 for 1 h. Using horseradish peroxidase-labeled goat anti-human (Fab')2 as a conjugate and ABTS as a substrate, bound antibodies and hinge-deficient IgG1 molecules were observed. The results were measured using an EL808 ELISA reader at a wavelength of 405 nm.

[0400] Figure 48 The results show the binding of monovalent or bivalent IgG1 antibodies and hinge-deficient IgG1 molecules to human FcRn (A) and mouse FcRn (B) at pH 6.0 and pH 7.4. As expected, all tested antibodies (both bispecific IgG1 and hinge-deficient IgG1 molecules) did not bind FcRn efficiently at pH 7.4 (both human and mouse). Under slightly acidic conditions (pH 6.0), monospecific IgG1-2F8-ITL and bispecific IgG1 derived from IgG1-2F8-ITL and IgG1-7D8-K409R showed bivalent binding efficiency to FcRn, although mouse FcRn was 3 times that of the human, mimicking the positive control (IgG1-2F8) for FcRn binding. This suggests that ITL mutation and K409R do not impede binding to FcRn.

[0401] When comparing the binding of IgG1 molecules to human and mouse FcRn at pH 6.0, a significant effect was observed between 2 and 1 compared to 0 FcRn interaction sites. Figure XXA and B, pH 6 (left panel). IgG1-2F8-ITL, IgG1-7D8-K409R, and IgG1-2F8-ITL / IgG1-7D8-K409R (2 FcRn binding sites) bound comparably to the control (IgG1-2F8). The molecule IgG1-7D8-K409R-H435A, with 0 FcRn binding sites, showed no binding at all. The molecule IgG1-2F8-ITL / IgG1-7D8-K409R-H435A, with 1 FcRn binding site, showed moderate binding compared to the molecule with 2 FcRn binding sites.

[0402] Figure 48 (A), pH 6.0, the right figure shows the binding of hinge-deficient IgG1 molecules (Uni-G1) to human FcRn. Compared to the control IgG1 molecule (IgG1-2F8), the interaction of all hinge-deficient molecules with human FcRn was impaired, suggesting that the hinge does indeed affect the interaction with FcRn when evaluated in FcRn binding ELISA. When comparing the binding of these hinge-deficient molecules to human FcRn at pH 6.0, no significant effect was observed at the 2 vs. 1 vs. 0 FcRn interaction sites.

[0403] However, because human IgG binds more strongly to mouse FcRn, when comparing the binding of these hinge-deficient IgG molecules to mouse FcRn at pH 6.0, a significant difference was observed between 2 vs. 1 vs. 0 FcRn interaction sites. Figure 48 (B), pH 6.0 (Right Figure). When compared with the binding of Uni-G1-2F8-ITL, Uni-G1-7D8-409R, and Uni-G1-2F8-ITL / Uni-G1-7D8-K409R (2 FcRn binding sites) and Uni-G1-2F8-ITL-H435A (0 FcRn binding sites, no binding), the binding of Uni-G1-7D8-K409R-H435A / Uni-G1-2F8-ITL (1 FcRn binding site) was moderate.

[0404] Example 47: Her2 x CD3 bispecific antibody tested in in vitro cytotoxicity assay CD3 is a co-receptor of the T-cell receptor complex expressed on mature T cells. In bispecific antibodies, the combination of the Fab arm of a CD3-specific antibody and the Fab arm of a tumor antigen-specific antibody leads to specific targeting of tumor cells by T cells, causing T-cell-mediated tumor cell lysis. Similarly, CD3-positive T cells can target other abnormal cells in the body, infected cells, or directly pathogens.

[0405] Her2 x CD3 bispecific antibodies were generated. The heavy and light chain variable region sequences of the Her2-specific Fab arm are shown in antibodies 153 and 169 in relation to Example 42. The CD3-specific Fab arm uses the following heavy and light chain variable region sequences: YTH12.5 (sequence as described by Routledge et al., Eur J Immunol. 1991, 21(11):2717-25) VHYTH12.5 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFPMAWVRQAPGKGLEWVSTISTSGGRTYYRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKFRQYSGGFDYWGQGTLVTVSS VLYTH12.5 DIQLTQPNSVSTSLGSTVKLSCTLSSGNIENNYVHWYQLYEGRSPTTMIYDDDKRPDGVPDRFSGSIDRSSNSAFLTIHNVAIEDEAIYFCHSYVSSFNVFGGGTKLTVL huCLB-T3 / 4 (Sequence Parren et al., Res Immunol. 1991, 142(9):749-63. Minoramino acid substitutions were introduced to make the sequence resemble the closest human germline as described) VH huCLB-T3 / 4 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMFWVRQAPGKGLEWVATISRYSRYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRPLYGSSPDYWGQGTLVTVSS VL huCLB-T3 / 4 EIVLTQSPATLSLSPGERATLSCSASSSVTYVHWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCFQGSGYPLTFGSGTKLEMR All antibodies were expressed as follows, with their Fc regions modified as follows: IgG1,κ: 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.

[0406] Bispecific antibodies were generated from these Her2 and CD3 specific antibodies as described in Example 11 and tested in an in vitro cytotoxicity assay using AU565 cells.

[0407] AU565 cells were cultured until nearly confluenced. Cells were washed twice with PBS and digested with trypsin at 37°C for 5 minutes. 12 mL of culture medium was added to inactivate the trypsin, and the cells were centrifuged at 800 rpm for 5 minutes. The cells were resuspended in 10 mL of culture medium, and a single-cell suspension was prepared by passing the cells through a cell strainer. 100 μL of 5x10⁻¹⁰ cells was then added to the culture medium. 5 Add 10 cells / mL suspension to each well of a 96-well culture plate and incubate the cells at 37°C and 5% CO2 for at least 3 hours to allow them to adhere to the plate.

[0408] Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy volunteers using Leucosep 30 mL tubes, according to the manufacturer's protocol (Greiner Bio-one). T cells were isolated from the PBMC preparation using the Untouched Human T-cells Dynabead kit (Dynal) with negative selection. The isolated cells were then resuspended in culture medium to 7 x 10⁻⁶. 6 Final concentration of cells / mL.

[0409] Remove the culture medium from the attached AU565 cells and dilute with 50 μl / well 2x concentrated antibody dilution buffer and 50 μl / well 7x10. 6 Replace with 1 T cell / mL (effector:target ratio = 7:1). Incubate the plate at 37°C and 5% CO2 for 3 days. Discard the supernatant and wash the plate twice with PBS. Add 150 μL of culture medium and 15 μL of Alamar blue to each well. Incubate the plate at 37°C and 5% CO2 for 4 hours and measure the absorbance (Envision, Perkin Elmer).

[0410] Figure 49 While control antibodies (Her2 monospecific IgG1-Herceptin, CD3 monospecific IgG1-YTH12.5, and monospecific IgG1-huCLB-T3 / 4, irrelevant antigen monospecific IgG1-b12, and CD3 x b12 bispecific antibody) did not induce T cell-mediated cytotoxicity, the bispecific (Duo) Her2 x CD3 antibodies huCLB / Her2-153, huCLB / Her2-169, YTH12.5 / Her2-153, and YTH12.5 / Her2-169 induced dose-dependent T cell-mediated cytotoxicity in AU565 cells. Bispecific antibodies containing Her2-169 were more effective than those containing Her2-153.

[0411] Mutants of IgG1-hu-CLB-T3 / 4, IgG1-YTH12.5, and Her2-153 were prepared, containing the N297Q mutation to remove the glycosylation site; glycosylation at this site is crucial for IgG-Fcγ receptor interaction (Bolt S et al., Eur J Immunol 1993, 23:403-411). Figure 49 The N297Q mutation and consequent lack of Fc glycosylation in Her2 x CD3 bispecific anti-YTH12.5 / Her2-153 and huCLB / Her2-153 did not affect the potential to induce dose-dependent T cell-mediated cytotoxicity in AU565 cells.

Claims

1. An in vitro method for generating bispecific antibodies, the method comprising the following steps: a) Provides a first homodimeric monospecific antibody comprising an immunoglobulin Fc region, the Fc region comprising a first CH3 region, wherein the first monospecific antibody comprises Phe at position 405 and Arg at position 409 according to EU numbering, and a Cys-Pro-Pro-Cys sequence in the core hinge region. b) Provide a second homodimeric monospecific antibody comprising the Fc region of an immunoglobulin, the Fc region comprising a second CH3 region, wherein the second monospecific antibody comprises Leu at position 405 and Lys at position 409 according to EU numbering, and a Cys-Pro-Pro-Cys sequence in the core hinge region. c) Incubate the first monospecific antibody and the second monospecific antibody together under reducing conditions sufficient to allow cysteine ​​residues in the Cys-Pro-Pro-Cys sequence of the core hinge region to undergo disulfide isomerization. d) Obtain the bispecific antibody; 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 stronger than the homodimer interaction between the first CH3 region and the second CH3 region, and wherein the first monospecific antibody binds to the first epitope and the second monospecific antibody binds to the second epitope, wherein the first epitope and the second epitope are different.

2. The in vitro method of claim 1, wherein the Fc region of the first monospecific antibody belongs to an isotype selected from IgG1, IgG2, IgG3 and IgG4, and wherein the Fc region of the second monospecific antibody belongs to an isotype selected from IgG1, IgG2, IgG3 and IgG4.

3. The in vitro method of any of the preceding claims, wherein the Fc regions of both the first and second monospecific antibodies belong to the IgG1 isotype.

4. The in vitro method of any of the preceding claims, wherein heterodimer interactions occur in the resulting bispecific antibodies. a) Ensures that Fab arm interchange does not occur under the conditions described in Example 13 at 0.5 mM glutathione, and / or b) Ensures that Fab arm interchange does not occur in mice under the conditions described in Example 14, and / or c) The strength is greater than 2 times, for example greater than 3 times, for example greater than 5 times, the strength of the strongest interaction among the two homodimer interactions, for example when measured as described in Example 30.

5. The in vitro method of any of the preceding claims, wherein the sequences of the first and second CH3 regions are such that... a) When measured according to Example 30, the dissociation constant of the heterodimer of the obtained bispecific antibody is less than 0.05 μmol, and / or b) When measured as described in Example 21, the dissociation constants of the two homodimers of the first and second monospecific antibodies are both greater than 0.01 μmol, for example greater than 0.05 μmol, preferably 0.01-10 μmol, for example 0.05-10 μmol, more preferably 0.01-5 μmol, for example 0.05-5 μmol, even more preferably 0.01-1 μmol, for example 0.05-1 μmol, 0.01-0.5 or 0.01-0.1 μmol.

6. The in vitro method of any of the preceding claims, wherein the first homodimeric monospecific antibody has no more than one amino acid substitution in the CH3 region compared to the wild-type CH3 region, and the second homodimeric monospecific antibody has no more than one amino acid substitution in the CH3 region.

7. An in vitro method of any of the preceding claims, wherein the first and second CH3 regions are contained in the sequence described in SEQ ID NO:1, except for the specified mutation.

8. The in vitro method of any of the preceding claims, wherein the first homodimeric monospecific antibody and the second homodimeric monospecific antibody are human or humanized antibodies, except for any specified mutation.

9. The in vitro method of any of the preceding claims, wherein both the first homodimeric monospecific antibody and the second homodimeric monospecific antibody further comprise a light chain.

10. An in vitro method according to any of the preceding claims, wherein the first homodimeric monospecific antibody and / or the second homodimeric monospecific antibody comprises a mutation removing the receptor site for glycosylation of Asn linkage.

11. The in vitro method of any of the preceding claims, wherein the first and second isodimeric monospecific antibodies provided in steps a) and b) are purified.

12. An in vitro method according to any of the preceding claims, wherein the first homodimeric monospecific antibody and / or the second homodimeric monospecific antibody are conjugated with a drug, prodrug, or toxin or contain their receptor groups.

13. The in vitro method of any of the preceding claims, Wherein the first epitope and / or the second epitope are located on tumor cells; The first epitope or the second epitope is located on tumor cells, and the other epitope is located on effector cells; The first or second epitope is located on T cells, for example on CD3 expressed on T cells; The first and second antibodies bind to different epitopes on the same tumor cells; or The first antibody binds to an epitope on tumor cells, and the other antibody is an irrelevant or inactive antibody that has no relevant in vivo binding activity for the intended application.

14. An in vitro method according to any of the preceding claims, wherein the reduction conditions in step c) include the addition of a reducing agent, such as a reducing agent selected from the following: 2-mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine or chemical derivatives thereof, dithioerythritol, glutathione, L-cysteine ​​and β-mercaptoethanol.

15. An in vitro method of any of the preceding claims, wherein step c) is performed under reducing conditions having a redox potential of -150 to -600 mV, for example -250 to -400 mV.

16. An in vitro method according to any of the preceding claims, wherein step c) comprises incubating at a temperature of at least 20°C for at least 90 minutes in the presence of at least 25 mM 2-mercaptoethylamine or in the presence of at least 0.5 mM dithiothreitol.

17. An in vitro method of any of the preceding claims, wherein step d) comprises removing the reducing agent, such as by desalting.

18. A method for selecting bispecific antibodies having desired properties, the method comprising the following steps: a) Provide a first group of homodimeric antibodies comprising antibodies with different variable regions and Cys-Pro-Pro-Cys sequences in the core hinge region, wherein the antibodies in the first group comprise an identical first CH3 region, wherein the CH3 region comprises Phe at position 405 and Arg at position 409 according to EU numbering. b) Provide a second group of homodimeric antibodies comprising a variable region having different or identical variable regions and a Cys-Pro-Pro-Cys sequence in the core hinge region, wherein the antibodies in the second group comprise an identical second CH3 region, wherein the CH3 region comprises Leu at position 405 and Lys at position 409 according to the EU number. c) Incubate the combination of the first group of antibodies and the second group of antibodies under reducing conditions sufficient to allow disulfide isomerization of cysteine ​​in the hinge region, thereby producing a set of bispecific antibodies. d) Select any recovery condition until non-reducible. e) The bispecific antibody group obtained by measuring the given desired properties, and f) Select bispecific antibodies with the desired properties. 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 stronger than the homodimer interaction between the first CH3 region and the second CH3 region, and the first homodimer antibody and the second homodimer antibody bind to different epitopes.

19. A method for preparing bispecific antibodies, the method comprising the following steps: a) Provide a first nucleic acid construct encoding a first polypeptide comprising a first full-length heavy chain of an antibody, said antibody comprising a first CH3 region comprising Phe at position 405 and Arg at position 409 according to EU numbering, and further comprising a Cys-Pro-Pro-Cys sequence in a core hinge region. b) Provide a second nucleic acid construct encoding a second polypeptide comprising a second full-length heavy chain of an antibody, the antibody comprising a second CH3 region comprising Leu at position 405 and Lys at position 409 according to EU numbering, and further comprising a Cys-Pro-Pro-Cys sequence in the core hinge region. c) Co-express the first and second nucleic acid constructs in host cells, and d) Obtain the bispecific antibody from the cell culture; 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 stronger than the homodimer interaction between the first CH3 region and the second CH3 region, and wherein the first full-length heavy chain binds to the first epitope and the second full-length heavy chain binds to the second epitope, wherein the first epitope and the second epitope are different.

20. The method of claim 19, further comprising the features of any one of claims 2 to 13.

21. An expression vector comprising the nucleic acid construct specified in any one of claims 19 to 20.

22. A host cell comprising the nucleic acid construct specified in any one of claims 19 to 20.

23. A bispecific antibody, which is obtained or obtainable by the method of any one of claims 1 to 17, 19 or 20.

24. A bispecific antibody comprising: a first polypeptide containing a first full-length antibody heavy chain, the first full-length antibody heavy chain including a first Fc region, the first Fc region including a first CH3 region; and a second polypeptide containing a second full-length antibody heavy chain, the second full-length antibody heavy chain including a second Fc region, the second Fc region including a second CH3 region. The first heavy chain binds to a first epitope, and the second heavy chain binds to a second epitope, wherein the first epitope and the second epitope are different, and wherein the sequences of the first CH3 region and the second CH3 region are different, such that the heterodimer interaction between the first CH3 region and the second CH3 region is stronger than the homodimer interaction between the first CH3 region and the second CH3 region, and a) wherein the first CH3 region comprises Phe at position 405 and Arg at position 409 according to the EU numbering, and the Cys-Pro-Pro-Cys sequence in the core hinge region, and b) wherein the second CH3 region contains Leu at position 405 and Lys at position 409 according to the EU number, and the Cys-Pro-Pro-Cys sequence in the core hinge region.

25. The bispecific antibody of claim 23 or 24, wherein the first Fc region belongs to an isotype selected from IgG1, IgG2, IgG3 and IgG4, and wherein the second Fc region belongs to an isotype selected from IgG1, IgG2, IgG3 and IgG4.

26. The bispecific antibody of any one of claims 23 to 25, wherein both the first and second Fc regions belong to the IgG1 isotype.

27. The bispecific antibody of any one of claims 23 to 26, wherein the first CH3 region has no more than one amino acid substitution compared to the wild-type CH3 region, and the second CH3 region has no more than one amino acid substitution.

28. The bispecific antibody of any one of claims 23 to 27, wherein the first and second CH3 regions are contained in the sequence described in SEQ ID NO:1, except for the specified mutation.

29. The bispecific antibody of any one of claims 23 to 28, wherein the first full-length heavy chain and the second full-length heavy chain are human or humanized antibodies except for any specified mutation.

30. The bispecific antibody of any one of claims 23 to 29, further comprising two full-length light chains.

31. The bispecific antibody of any one of claims 23 to 30, wherein the first polypeptide and / or the second polypeptide comprises a mutation removing the receptor site for glycosylation of Asn linkage.

32. The bispecific antibody of any one of claims 23 to 31, wherein the first polypeptide and / or the second polypeptide is conjugated to a drug, prodrug, or toxin or contains a receptor group thereof.

33. The bispecific antibody according to any one of claims 23 to 32, Wherein the first epitope and / or the second epitope are located on tumor cells; The first epitope or the second epitope is located on tumor cells, and the other epitope is located on effector cells; The first or second epitope is located on T cells, for example on CD3 expressed on T cells; The first and second heavy chains bind to different epitopes on the same tumor cells; or The first heavy chain binds to an epitope on tumor cells, while the other heavy chain is an irrelevant or inactive antibody that has no relevant in vivo binding activity for the intended application.

34. The bispecific antibody of any one of claims 23 to 33, wherein the first epitope and the second epitope are located on tumor cells, and wherein the bispecific antibody has binding specificity to a target selected from: erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD19, CD20, CD4, CD38, CD138, CXCR5, c-Met, HERV-capsule protein, periosteal protein, Biggs3, SPARC, BCR, CD79, CD37, EGFrvIII, L1-CAM, AXL, tissue factor (TF), CD74, EpCAM, and MRP3.

35. A bispecific antibody according to any one of claims 23 to 33, wherein the first epitope and the second epitope are located on tumor cells, and wherein the bispecific antibody has binding specificity to a combination of targets selected from: 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.

36. The bispecific antibody of any one of claims 23 to 33, wherein the first epitope or the second epitope is located on tumor cells and the other epitope is located on effector cells, wherein the bispecific antibody has binding specificity to a target selected from: FcγRI (CD64), FcγRIII (CD16), CD3, CD89, CD32a, FcεRI.

37. The bispecific antibody of any one of claims 23 to 33, having binding specificity to a combination of targets selected from: CD3 + HER2, CD3 + CD20, IL-12 + IL18, IL-1a + IL-1b, 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 + Combinations of 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-Met, c-Met + non-binding arm, and G-protein-coupled receptors.

38. The bispecific antibody of any one of claims 23 to 37, used as a medicine.

39. A pharmaceutical composition comprising a bispecific antibody of any one of claims 23 to 37 and a pharmaceutically acceptable carrier.

Images