Charge pair mutations enable correct heavy-light chain pair formation.

Abstract

This application relates to promoting selective binding between modified VH and modified VL domains by introducing amino acids with complementary charges in the two domains at novel positions within the two domains. These modifications are particularly useful in the design and production of multispecific antibodies, such as bispecific antibodies, in which charge pair mutations in the VH and VL domains can promote desirable pair formation between specific heavy and light chain polypeptides. These charge pair mutations can be used alone or in combination with additional strategies to promote precise pair formation of polypeptide chains.

Description

Technical Field

[0001] Reference to Electronically Submitted Sequence Listing This application includes a sequence listing electronically submitted in XML format, which is hereby incorporated by reference in its entirety. A computer-readable format copy of this sequence listing created on June 5, 2024, is named 10572-WO01-SEC_ST26 and has a size of 8.69 kilobytes.

[0002] This disclosure relates to the generation of multispecific antibodies. For example, this disclosure relates to the use of charged pair mutations in the variable regions of heavy chain polypeptide sequences and light chain polypeptide sequences to facilitate accurate pairing between specific desired heavy chain polypeptides and light chain polypeptides.

Background Art

[0003] Multispecific antibodies, such as bispecific antibodies, represent an interesting development for biological agents, enabling simultaneous or sequential targeting of two or more unique epitopes localized on the same or different targets. This dual recognition capability allows for diverse applications, including the recruitment of immune cells to kill tumor cells, crosslinking of distinct cell surface receptors, or enhanced tissue specificity (Labrijn AF et al., Nat. Rev. Drug Discov. 18:585-608 (2019); Lu RM et al., J. Biomed. Sci. 27:1 (2020); Fan G et al., J. Hematol. Oncol. 8:130 (2015)). For example, Amgen's Bispecific T-cell Engager (BiTE®) creates artificial immune synapses between cytotoxic T cells and target tumor cells by simultaneously binding to CD3 epitopes and tumor-associated antigens on the surface of T cells (Wolf E et al., Drug Discov. Today 10:1237-44 (2005); Kantarjian H et al., N.Engl.J.Med. 376:836-47 (2017)). As of 2019, more than 100 bispecificity formats had been reported, more than 85 were under development, and three had received approval from the U.S. Food and Drug Administration (Labrijn AF et al., Nat. Rev. Drug Discov. 18:585-608 (2019); Brinkmann U and Kontermann RE, MAbs 9:182-212 (2017); Wang Q et al., Antibodies (Basel) 8(3):43 (2019)).

[0004] Two main design strategies exist for generating bispecific molecules. The first approach involves encoding two or more unique fragment variable (Fv) sequences on the same polypeptide chain, as in formats such as BiTE®, IgG-scFv, or DVD-Ig (Wang Q et al. Antibodies (Basel) 8(3):43 (2019); Spiess C et al. Mol Immunol 67:95-106 (2015)). While these single-stranded formats avoid the challenges associated with assembling multiple polypeptide chains into a single molecule, they also tend to exhibit insufficient yield and stability that does not reach optimal conditions. Alternative strategies utilize biophysics and engineering to "guide" individual chains in the correct orientation while simultaneously eliminating mispairing scenarios. Examples include techniques such as knob-into-hole (KiH), charge pair mutation (CPM), and strand-exchange gene manipulation domains (SEEDbody), which are used in molecules requiring hetero-Fc pair formation (Davis JH et al., Protein Eng. Des. Sel. 23:195-202 (2010); Dillon M et al., MAbs 9:213-30 (2017); Gunasekaran K et al., J. Biol. Chem. 285:19637-46 (2010); Ridgway JB et al., Protein Eng. 9:617-21 (1996)). This second approach enables the creation of molecules that more closely mimic the native structure of the IgG molecule, leading to increased stability and a wider variety of format designs (Wang Q et al., Antibodies (Basel) 8(3):43 (2019)). However, these operational strategies are often imperfect, and mispairing of chains continues to pose a challenge (Ha JH et al., Front.Immunol.7:394(2016)).

[0005] Mispairing between heavy and light chains is a significant concern in the production of multispecific antibodies. For example, if an incorrect light chain (LC) pairs with an undesirable heavy chain (HC), the resulting mispaired molecule is converted into an impurity that must be removed. Several protein manipulation strategies have been proposed to address HC-LC pairing (Krah, S et al., Nat. Biotechnol., 39(B):167-173 (2017)), but due to the variability of the Fv interface, a solution designed for one molecule may not necessarily be applicable to the next. Therefore, improvements in methods to facilitate pairing between specific desired heavy and light chain polypeptides are needed in this technology. In particular, pairing within the FV interface is required. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Labrijn AF et al.,Nat.Rev.Drug Discov.18:585-608(2019) [Non-Patent Document 2] Lu RM et al.,J.Biomed.Sci.27:1(2020) [Non-Patent Document 3] Fan G et al.,J.Hematol.Oncol.8:130(2015) [Non-Patent Document 4] Wolf E et al.,Drug Discov.Today 10:1237-44(2005) [Non-Patent Document 5] Kantarjian H et al.,N.Engl.J.Med.376:836-47(2017) [Non-Patent Document 6] Brinkmann U and Kontermann RE,MAbs 9:182-212(2017) [Non-Patent Document 7] Wang Q et al., Antibodies (Basel) 8(3):43(2019)

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Summary of the Invention

Means for Solving the Problems

[0007] This application relates to promoting selective binding between modified VH and modified VL domains by introducing amino acids with complementary charges in the two domains at novel positions within the two domains. For example, modifications can be made at positions VH39 and VL85, VH105 and VL105, or VH91 and VL38. These modifications are particularly useful for the design and production of multispecific antibodies, such as bispecific antibodies, in which charge pair mutations in the VH and VL domains can promote desirable pair formation between specific heavy and light chain polypeptides. These charge pair mutations can be used alone or in combination with additional strategies to promote precise pair formation of polypeptide chains.

[0008] In some embodiments, the isolated protein comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), where VH and VL are bound to each other, and VH and VL comprise at least one of the following sets of charged amino acids: (a) VH contains a charged amino acid at position number 39, and VL contains a charged amino acid at position number 85 that is charge-complementary to the amino acid at position 39 of VH; (b) VH contains a charged amino acid at position number 105, and VL contains a charged amino acid at position number 42 that is charge-complementary to the amino acid at position 105 of VH; or (c) VH contains a charged amino acid at position number 91, and VL contains a charged amino acid at position number 38 that is charge-complementary to the amino acid at position 91 of VH. In these embodiments, the position numbers of the charged amino acids within the VH and VL domains refer to positions according to the Kabat numbering scheme.

[0009] In certain embodiments, the isolated protein comprises VH and VL, which comprise a set of charged amino acids such that VH contains a charged amino acid at position 39, and VL contains a charged amino acid at position 85 that is complementary in charge to the amino acid at position 39 of VH. In some of these embodiments, VH contains a positively charged amino acid at position 39, and VL contains a negatively charged amino acid at position 85. In some embodiments in which VH contains a positively charged amino acid at position 39 and VL contains a negatively charged amino acid at position 85, the positions VH39 (VH39) and VL85 (VL85) comprise: (i) lysine at VH39 and aspartic acid at VL85, (ii) lysine at VH39 and glutamic acid at VL85, (iii) arginine at VH39 and aspartic acid at VL85, or (iv) arginine at VH39 and glutamic acid at VL85. In other embodiments, VH contains an amino acid negatively charged at position 39, and VL contains an amino acid positively charged at position 85. In some embodiments in which VH contains an amino acid negatively charged at position 39 and VL contains an amino acid positively charged at position 85, the VH39 position (VH39) and the VL85 position (VL85) contain: (i) aspartic acid at VH39 and lysine at VL85, (ii) glutamic acid at VH39 and lysine at VL85, (iii) aspartic acid at VH39 and arginine at VL85, or (iv) glutamic acid at VH39 and arginine at VL85.

[0010] In certain embodiments, the isolated protein comprises VH and VL, which comprise a set of charged amino acids such that VH contains a charged amino acid at position 105, and VL contains a charged amino acid at position 42 that is complementary in charge to the amino acid at position 105 of VH. In some of these embodiments, VH contains a positively charged amino acid at position 105, and VL contains a negatively charged amino acid at position 42. In some embodiments, VH contains an amino acid positively charged at position 105, and VL contains an amino acid negatively charged at position 42, where VH105 (VH105) and VL42 (VL42) contain: (i) lysine at VH105 and aspartic acid at VL42, (ii) lysine at VH105 and glutamic acid at VL42, (iii) arginine at VH105 and aspartic acid at VL42, or (iv) arginine at VH105 and glutamic acid at VL42. In other embodiments, VH contains an amino acid negatively charged at position 105, and VL contains an amino acid positively charged at position 42. In some embodiments, the VH105 position contains an amino acid that is negatively charged at position 105 and the VL42 position contains an amino acid that is positively charged at position 42, where the VH105 position (VH105) and VL42 position (VL42) contain: (i) aspartic acid at VH105 and lysine at VL42, (ii) glutamic acid at VH105 and lysine at VL42, (iii) aspartic acid at VH105 and arginine at VL42, or (iv) glutamic acid at VH105 and arginine at VL42.

[0011] In certain embodiments, the isolated protein comprises VH and VL, which comprise a set of charged amino acids such that VH contains a charged amino acid at position 91, and VL contains a charged amino acid at position 38 that is complementary in charge to the amino acid at position 91 of VH. In some of these embodiments, VH contains a positively charged amino acid at position 91, and VL contains a negatively charged amino acid at position 38. In some embodiments in which VH contains a positively charged amino acid at position 91 and VL contains a negatively charged amino acid at position 38, position VH91 (VH91) and position VL38 (VL38) comprise: (i) lysine at VH91 and aspartic acid at VL38, (ii) lysine at VH91 and glutamic acid at VL38, (iii) arginine at VH91 and aspartic acid at VL38, or (iv) arginine at VH91 and glutamic acid at VL38. In other embodiments, VH contains an amino acid negatively charged at position 91, and VL contains an amino acid positively charged at position 38. In some embodiments in which VH contains an amino acid negatively charged at position 91 and VL contains an amino acid positively charged at position 38, the positions VH91 (VH91) and VL38 (VL38) contain: (i) aspartic acid at VH91 and lysine at VL38, (ii) glutamic acid at VH91 and lysine at VL38, (iii) aspartic acid at VH91 and arginine at VL38, or (iv) glutamic acid at VH91 and arginine at VL38.

[0012] In some embodiments, the isolated protein comprises an antibody heavy chain containing VH and an antibody light chain containing VL. In some embodiments, the isolated protein is an antibody. In some embodiments, the isolated protein is an IgG antibody. In some embodiments, the isolated protein is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the isolated protein is a bispecific antibody. In some embodiments, the isolated protein is a bispecific antibody comprising two arms, each arm of the antibody comprising a VH-VL pair consisting of one of the following sets of charged amino acids: (a) a set in which each VH contains a charged amino acid at position 39 and each VL contains a charged amino acid at position 85 that is charge-complementary to the amino acid at position 39 of VH; (b) a set in which each VH contains a charged amino acid at position 105 and each VL contains a charged amino acid at position 42 that is charge-complementary to the amino acid at position 105 of VH; or (c) a set in which each VH contains a charged amino acid at position 91 and each VL contains a charged amino acid at position 38 that is charge-complementary to the amino acid at position 91 of VH.

[0013] In some embodiments, the isolated protein comprises an antibody containing a first human IgG CH3 domain (CH3) and a second human IgG CH3 domain (CH3'), wherein the CH3 domain includes amino acid substitutions by negatively charged amino acids at positions 392, 409, and 439, and the CH3' domain includes amino acid substitutions at positions 356 and 399, and the positional numbers of the charged amino acids in the CH3 and CH3' domains refer to positions according to the EU numbering scheme.

[0014] In some embodiments, the isolated protein comprises an antibody comprising a first heavy chain constant domain and light chain constant domain pair (CH1 and CL), and a second heavy chain constant domain and light chain constant domain pair (CH1’ and CL’); CH1 and CH1’ each contain a modification at position 183 numbered according to the EU numbering scheme; CL and CL’ each contain a modification at position 176 numbered according to the EU numbering scheme; position 183 of CH1 and position 176 of CL’ each contain a positively charged amino acid; and position 183 of CH1’ and position 176 of CL each contain a negatively charged amino acid. In some embodiments, (i) position 183 of CH1 and position 176 of CL’ each contain lysine, and position 183 of CH1 and position 176 of CL each contain aspartic acid; (ii) position 183 of CH1 and position 176 of CL’ each contain lysine, and position 183 of CH1’ and position 176 of CL each contain glutamic acid; (iii) position 183 of CH1 and position 176 of CL’ each contain arginine, and position 183 of CH1’ and position 176 of CL each contain aspartic acid; or (iv) position 183 of CH1 and position 176 of CL’ each contain arginine, and position 183 of CH1’ and position 176 of CL each contain glutamic acid.

[0015] In some embodiments, the bispecific antibody comprises a first heavy chain variable domain and light chain variable domain pair (VH and VL), and a second heavy chain variable domain and light chain variable domain pair (VH' and VL'), wherein the VH and VL pair and the VH' and VL' pair are sets of charged amino acids as follows: (a) VH and VH' each contain a charged amino acid at position 39; VL and VL' each contain a charged amino acid at position 85 that is complementary in charge to the amino acid at position 39 of VH and VH'; and VH and VH' are sets containing amino acids that are complementary in charge; (b) VH and VH' each contain a charged amino acid at position number 105; VL and VL' each contain a charged amino acid at position number 42 that is complementary in charge to the amino acid at position 105 of VH and VH', respectively; and VH and VH' are sets containing amino acids with complementary charges; or (c) VH and VH' each contain a charged amino acid at position number 91; VL and VL' each contain a charged amino acid at position number 38 that is complementary in charge to the amino acid at position 91 of VH and VH', respectively; and VH and VH' include at least one set containing amino acids with complementary charges. In these embodiments, the position numbers of the charged amino acids in the VH and VL domains refer to positions according to the Kabat numbering scheme.

[0016] In certain embodiments, the bispecific antibody comprises a first heavy-chain variable domain and light-chain variable domain pair (VH and VL), and a second heavy-chain variable domain and light-chain variable domain pair (VH' and VL'), where VH and VH' each contain a charged amino acid at position 39; VL and VL' each contain a charged amino acid at position 85 that is complementary in charge to the amino acid at position 39 of VH and VH'; and VH and VH' contain the amino acid with complementary charge. In some embodiments, VH and VL' contain a positively charged amino acid, and VH' and VL contain a negatively charged amino acid. In some of these embodiments, the VH and VL pairs and the VH' and VL' pairs include: (i) lysine at VH39, lysine at VL'85, aspartic acid at VH'39, and aspartic acid at VL85; (ii) lysine at VH39, lysine at VL'85, glutamic acid at VH'39, and glutamic acid at VL85; (iii) arginine at VH39, arginine at VL'85, aspartic acid at VH'39, and aspartic acid at VL85; or (iv) arginine at VH39, arginine at VL'85, glutamic acid at VH'39, and glutamic acid at VL85. In some embodiments, VH and VL' include negatively charged amino acids, and VH' and VL include positively charged amino acids. In some of these embodiments, the VH and VL pairs and the VH' and VL' pairs include: (i) aspartic acid at VH39, aspartic acid at VL'85, lysine at VH'39, and lysine at VL85; (ii) glutamic acid at VH39, glutamic acid at VL'85, lysine at VH'39, and lysine at VL85; (iii) aspartic acid at VH39, aspartic acid at VL'85, arginine at VH'39, and arginine at VL85; or (iv) glutamic acid at VH39, glutamic acid at VL'85, arginine at VH'39, and arginine at VL85.

[0017] In certain embodiments, the bispecific antibody comprises a first heavy chain variable domain and light chain variable domain pair (VH and VL), and a second heavy chain variable domain and light chain variable domain pair (VH’ and VL’), wherein VH and VH’ each contain a charged amino acid at position number 105; VL and VL’ each contain a charged amino acid at position number 42 that is complementary in charge to the amino acid at position 105 of VH and VH’ respectively; and VH and VH’ contain amino acids that are complementary in charge. In some embodiments, VH and VL’ contain positively charged amino acids, and VH’ and VL contain negatively charged amino acids. In some of these embodiments, the VH and VL pair and the VH’ and VL’ pair are: (i) lysine at VH105, lysine at VL’42, aspartic acid at VH’105, and aspartic acid at VL42; (ii) lysine at VH105, lysine at VL’42, glutamic acid at VH’105, and glutamic acid at VL42; (iii) arginine at VH105, arginine at VL’42, aspartic acid at VH’105, and aspartic acid at VL42; or (iv) arginine at VH105, arginine at VL’42, glutamic acid at VH’105, and glutamic acid at VL42. In some embodiments, VH and VL’ contain negatively charged amino acids, and VH’ and VL contain positively charged amino acids. In some of these embodiments, the VH and VL pair and the VH’ and VL’ pair are: (i) aspartic acid at VH105, aspartic acid at VL’42, lysine at VH’105, and lysine at VL42; (ii) glutamic acid at VH105, glutamic acid at VL’42, lysine at VH’105, and lysine at VL42; (iii) aspartic acid at VH105, aspartic acid at VL’42, arginine at VH’105, and arginine at VL42; or (iv) glutamic acid at VH105, glutamic acid at VL’42, arginine at VH’105, and arginine at VL42.

[0018] In certain embodiments, the bispecific antibody comprises a first heavy-chain variable domain and light-chain variable domain pair (VH and VL), and a second heavy-chain variable domain and light-chain variable domain pair (VH' and VL'), where VH and VH' each contain a charged amino acid at position 91; VL and VL' each contain a charged amino acid at position 38 that is complementary in charge to the amino acid at position 91 of VH and VH'; and VH and VH' contain the amino acid with complementary charge. In some embodiments, VH and VL' contain a positively charged amino acid, and VH' and VL contain a negatively charged amino acid. In some of these embodiments, the VH and VL pairs and the VH' and VL' pairs include: (i) lysine at VH91, lysine at VL'38, aspartic acid at VH'91, and aspartic acid at VL38; (ii) lysine at VH91, lysine at VL'38, glutamic acid at VH'91, and glutamic acid at VL38; (iii) arginine at VH91, arginine at VL'38, aspartic acid at VH'91, and aspartic acid at VL38; or (iv) arginine at VH91, arginine at VL'38, glutamic acid at VH'91, and glutamic acid at VL38. In some embodiments, VH and VL' include negatively charged amino acids, and VH' and VL include positively charged amino acids. In some of these embodiments, the VH and VL pairs and the VH' and VL' pairs include: (i) aspartic acid at VH91, aspartic acid at VL'38, lysine at VH'91, and lysine at VL38; (ii) glutamic acid at VH91, glutamic acid at VL'38, lysine at VH'91, and lysine at VL38; (iii) aspartic acid at VH91, aspartic acid at VL'38, arginine at VH'91, and arginine at VL38; or (iv) glutamic acid at VH91, glutamic acid at VL'38, arginine at VH'91, and arginine at VL38.

[0019] Some embodiments provide a method for producing a bispecific antibody, comprising expressing one or more polynucleotides encoding a bispecific antibody containing a charge pair mutation described herein in host cells, culturing the host cells in a culture medium under conditions that produce a constituent polypeptide chain, and recovering the bispecific antibody from the cells or culture medium. Similarly, some embodiments provide a method for producing an isolated protein, comprising expressing one or more polynucleotides encoding a protein containing a charge pair mutation described herein in host cells, culturing the host cells in a culture medium under conditions that produce a constituent polypeptide chain, and recovering the protein from the cells or culture medium. In some embodiments, the host cells comprise one or more plasmids containing one or more polynucleotides. In some embodiments, the host cells are Chinese hamster ovary (CHO) cell lines or human embryonic kidney (HEK) cell lines. In some embodiments, the recovered bispecific antibody or protein is purified by protein A chromatography. In some embodiments, the recovered bispecific antibody or protein is further purified by ion exchange chromatography. In some embodiments, the recovered bispecific antibodies or proteins are purified by protein A chromatography and ion exchange chromatography. [Brief explanation of the drawing]

[0020] [Figure 1A]Figure 1A is a simplified diagram of an exemplary bispecific antibody containing one arm capable of binding to a first antigen (antigen 1) and a second arm capable of binding to a second antigen (antigen 2). The two arms are attached via their respective Fc regions. The antibody heavy chain is linked via interactions in the CH2 and CH3 domains within the Fc region, and Figure 1A identifies the CH2-CH2 and CH3-CH3 interfaces where such interactions occur. Similarly, Figure 1A shows how each heavy chain links to its respective light chain via two different interfaces. The first of these involves an interaction between the CH1 domain of the heavy chain and the CL domain of the light chain (CH1-CL interface), and the second involves an interaction between the VH domain of the heavy chain and the VL domain of the light chain (Fv interface). Finally, Figure 1A shows the regions on each arm containing the CDR regions responsible for interactions with antigen 1 and antigen 2. [Figure 1B] Figure 1B shows some undesirable by-products that may form when generating bispecific antibodies. The undesirable by-products shown are those in which an incorrect light chain is paired with one or both of the heavy chains of the bispecific antibody. [Figure 2A] Figures 2A–2C relate to the process used to identify novel charge pair mutations for introduction into the antibody's Fv interface. Figure 2A shows a rendering of the structural interface between the VH and VL domains, with the interaction indicated by a dotted line. Figure 2B shows a diagram of the intended result when a charge pair modification is made at the interaction site in Figure 2A, where the introduced modification results in an unstable high-energy state in an undesirable "mispair formation" state, and as a result, kinetics cause the antibody chain to assemble in the desired pair formation orientation. Figure 2C shows energy level calculations (REU) for exemplary charge pair mutations that were predicted to have higher energy states in each of their mispair formation structures. [Figure 2B]Figures 2A–2C relate to the process used to identify novel charge pair mutations for introduction into the antibody's Fv interface. Figure 2A shows a rendering of the structural interface between the VH and VL domains, with the interaction indicated by a dotted line. Figure 2B shows a diagram of the intended result when a charge pair modification is made at the interaction site in Figure 2A, where the introduced modification results in an unstable high-energy state in an undesirable "mispair formation" state, and as a result, kinetics cause the antibody chain to assemble in the desired pair formation orientation. Figure 2C shows energy level calculations (REU) for exemplary charge pair mutations that were predicted to have higher energy states in each of their mispair formation structures. [Figure 2C] Figures 2A–2C relate to the process used to identify novel charge pair mutations for introduction into the antibody's Fv interface. Figure 2A shows a rendering of the structural interface between the VH and VL domains, with the interaction indicated by a dotted line. Figure 2B shows a diagram of the intended result when a charge pair modification is made at the interaction site in Figure 2A, where the introduced modification results in an unstable high-energy state in an undesirable "mispair formation" state, and as a result, kinetics cause the antibody chain to assemble in the desired pair formation orientation. Figure 2C shows energy level calculations (REU) for exemplary charge pair mutations that were predicted to have higher energy states in each of their mispair formation structures. [Figure 3A] Figures 3A–3E show spectrographs of cation exchange chromatography of bispecific antibodies after purification from HEK 293 cells using a Protein A column. Each of Figures 3A–3E further provides a list of charge pair mutations present in the bispecific antibodies produced. The location of peaks containing specific antibody species is indicated by a diagram of that species and an arrow pointing to the associated peak. Desired bispecific antibodies are highlighted in each of Figures 3A–3E by a box surrounding their peak. [Figure 3B]Figures 3A–3E show spectrographs of cation exchange chromatography of bispecific antibodies after purification from HEK 293 cells using a Protein A column. Each of Figures 3A–3E further provides a list of charge pair mutations present in the bispecific antibodies produced. The location of peaks containing specific antibody species is indicated by a diagram of that species and an arrow pointing to the associated peak. Desired bispecific antibodies are highlighted in each of Figures 3A–3E by a box surrounding their peak. [Figure 3C] Figures 3A–3E show spectrographs of cation exchange chromatography of bispecific antibodies after purification from HEK 293 cells using a Protein A column. Each of Figures 3A–3E further provides a list of charge pair mutations present in the bispecific antibodies produced. The location of peaks containing specific antibody species is indicated by a diagram of that species and an arrow pointing to the associated peak. Desired bispecific antibodies are highlighted in each of Figures 3A–3E by a box surrounding their peak. [Figure 3D] Figures 3A–3E show spectrographs of cation exchange chromatography of bispecific antibodies after purification from HEK 293 cells using a Protein A column. Each of Figures 3A–3E further provides a list of charge pair mutations present in the bispecific antibodies produced. The location of peaks containing specific antibody species is indicated by a diagram of that species and an arrow pointing to the associated peak. Desired bispecific antibodies are highlighted in each of Figures 3A–3E by a box surrounding their peak. [Figure 3E]Figures 3A–3E show spectrographs of cation exchange chromatography of bispecific antibodies after purification from HEK 293 cells using a Protein A column. Each of Figures 3A–3E further provides a list of charge pair mutations present in the bispecific antibodies produced. The location of peaks containing specific antibody species is indicated by a diagram of that species and an arrow pointing to the associated peak. Desired bispecific antibodies are highlighted in each of Figures 3A–3E by a box surrounding their peak. [Figure 4A] Figures 4A–4C also show spectrographs of cation exchange chromatography of bispecific antibodies after purification from HEK 293 cells using a Protein A column. Each of Figures 4A–4C further provides a list of charge pair mutations present in the bispecific antibodies to be produced. The location of the peak containing the desired antibody species is identified by the diagram of that species and the arrow pointing to the associated peak. The desired bispecific antibody is also highlighted in each of Figures 4A–4C by a box surrounding its peak. [Figure 4B] Figures 4A–4C also show spectrographs of cation exchange chromatography of bispecific antibodies after purification from HEK 293 cells using a Protein A column. Each of Figures 4A–4C further provides a list of charge pair mutations present in the bispecific antibodies to be produced. The location of the peak containing the desired antibody species is identified by the diagram of that species and the arrow pointing to the associated peak. The desired bispecific antibody is also highlighted in each of Figures 4A–4C by a box surrounding its peak. [Figure 4C]Figures 4A–4C also show spectrographs of cation exchange chromatography of bispecific antibodies after purification from HEK 293 cells using a Protein A column. Each of Figures 4A–4C further provides a list of charge pair mutations present in the bispecific antibodies to be produced. The location of the peak containing the desired antibody species is identified by the diagram of that species and the arrow pointing to the associated peak. The desired bispecific antibody is also highlighted in each of Figures 4A–4C by a box surrounding its peak. [Figure 5] Figure 5 also shows a spectrograph of cation exchange chromatography of bispecific antibodies after purification from HEK 293 cells using a Protein A column. Figure 5 further provides a list of charge pair mutations present in the bispecific antibodies to be produced. The location of the peak containing the desired antibody species is identified by the diagram of that species and an arrow pointing to the associated peak. The desired bispecific antibody is also highlighted by a box surrounding its peak. [Figure 6] Figure 6 shows plots of the recovery of various monospecific and bispecific antibody species after purification following production in HEK 293 cells. Figure 5 shows how the identified charge pair mutations promote the production of bispecific antibodies at levels exceeding those of the parental antibody. [Figure 7A] Figures 7A and 7B contain exemplary VH and VL sequences, respectively. These sequences are annotated residue by residue using Kabat numbering, and the figures also include box regions indicating amino acids that may be modified according to the charge pair mutations disclosed herein. Pair A corresponds to VH39 and VL85, pair B corresponds to VH105 and VL105, and pair C corresponds to VH91 and VL38. [Figure 7B]Figures 7A and 7B contain exemplary VH and VL sequences, respectively. These sequences are annotated residue by residue using Kabat numbering, and the figures also include box regions indicating amino acids that may be modified according to the charge pair mutations disclosed herein. Pair A corresponds to VH39 and VL85, pair B corresponds to VH105 and VL105, and pair C corresponds to VH91 and VL38. [Modes for carrying out the invention]

[0021] This application arises from the discovery of a novel method by which antibody VH and VL domains can be manipulated for specific pairing so that they assemble with each other, preferably as part of other VH-VL pairings. Structural analysis of a series of different antibody structures revealed the positions between the VH and VL domains where complementary charge-based modifications can consistently drive such preferential assembly across various antibody sequences. The VL / VH interface residues selected for manipulation are embedded and spatially hidden within the VL / VH interface. The target residues are well conserved across various antibody families.

[0022] These manipulated VH and VL domains are useful in a wide range of situations where specific domain interactions are desired. In some embodiments, these VH and VL modifications can be used to facilitate the formation of polypeptide heterodimer pairs, each containing either a manipulated VH domain or a manipulated VL domain. In some embodiments, the VH and VL domains of two distinct antibody heavy chains and two distinct antibody light chains are modified to form a quadruple heterodimer antibody. Electrostatic steering achieved by manipulating the interface residue between the VL and VH domains prevents mispairing of the light chain to a non-homogeneous heavy chain when two different heavy and light chain pairs assemble to form a desired quadruple heterodimer antibody. As described herein, exemplary strategies include introducing one or more negatively charged residues (e.g., Asp or Glu) in the first VL (VL1) and one or more positively charged residues (e.g., Lys, His, or Arg) in the companion VH (VH1) at the VL1 / VH1 interface, while introducing one or more positively charged residues (e.g., Lys, His, or Arg) in the second VL (VL2) and one or more negatively charged residues (e.g., Asp or Glu) in the companion VH (VH2) at the VL2 / VH2 interface. The electrostatic steering effect induces pairing of VL1 with VH1 and VL2 with VH2 because oppositely charged residues (polarity) at the interface attract each other, while same-type charged residues (polarity) at the interface repel each other, resulting in the suppression of undesirable VL / VH pairing. Therefore, in some embodiments, these VH and VL modifications can be used to facilitate the formation of a desirable bispecific antibody structure, thereby minimizing the formation of contamination substitute species.

[0023] In some embodiments, these VH and VL modifications can be combined with other mechanisms to drive the specific formation of a desired bispecific antibody species. For example, VH and VL modifications are combined with known charge pair mutations in the heavy and light chains of the antibody. The combination of VH and VL modifications with other mechanisms to drive specific pair formation increases the production and purity of the expressed bispecific antibody compared to implementing either strategy alone.

[0024] Other methods for manipulating the light and heavy chains to form specific heterodimers include substituting a pair of charged residues at the VL / VH interface with a pair of cysteine ​​residues to form a disulfide bond and stabilize the Fab region, substituting one or more hydrophilic residues (e.g., glycine) at the VL / VH interface with hydrophobic residues (e.g., glutamine), or manipulating pairs of large / small residues at the VL / VH interface to exert a knob-into-hole effect suitable for precise VL / VH pair formation. Using the strategies described herein, full-length heterodimer antibodies can be efficiently generated from two existing antibodies without the use of artificial linkers. The resulting heterodimer antibodies are stable without excessive aggregation or yield loss and are suitable for commercial production. Heterodimer antibodies can simultaneously target two different antigens, or two different epitopes on the same antigen, and therefore have great potential to treat many diseases unparalleled.

[0025] Specific embodiments and further details relating to the present invention are provided below.

[0026] Definitions of general terms and expressions To facilitate understanding of this disclosure, certain terms are defined first. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure relates. Where used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are provided throughout this application.

[0027] Units, prefixes, and symbols are shown in the format recognized by the International System of Units (SI).

[0028] As used in this disclosure and in the claims, the singular forms "a," "an," and "the" include plural forms unless otherwise explicitly indicated by the context. Unless otherwise specified or made clear from the context, the term "or" as used herein is understood to be inclusive. As used herein in phrases such as "A and / or B," the term "and / or" is intended to include "A and B," "A or B," "A," and "B." Similarly, as used in phrases such as "A, B, and / or C," the term "and / or" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0029] Wherever an embodiment is described herein with the word “comprising,” it is understood that other similar embodiments are also provided, which are described with respect to “consisting of” and / or “essentially from.” In this disclosure, “comprises,” “comprising,” “containing,” and “having,” etc., may have the meanings attributed to them under U.S. patent law, and may mean “includes,” and “including,” etc.; similarly, “consisting essentially of” or “consists essentially,” etc., may have the meanings attributed to them under U.S. patent law, and the terms are open-ended, allowing for entities beyond those enumerated, as long as the basic or novel features of those enumerated are not altered by those entities, but excluding embodiments of the prior art.

[0030] The terms “approximately” or “comprising essentially of” refer to a value or composition that falls within an acceptable margin of error for a value or composition as determined by those skilled in the art, and this depends in part on how the value or composition is measured or determined, i.e., on the limits of the measurement system. For example, “approximately” or “comprising essentially of” may mean within or exceeding one standard deviation according to the practice in the art. In addition, “approximately” or “comprising essentially of” may mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the term may mean up to one order of magnitude or up to five times the value. Where a value or composition is provided in this application and claims, unless otherwise specified, the meaning of “approximately” or “comprising essentially of” should be assumed to be within an acceptable margin of error for that particular value or composition.

[0031] The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof. As used herein, the term "antibody" encompasses polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, recombinant antibodies, multispecific antibodies, and bispecific antibodies. Antibodies may be of one of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or of their subclasses (isotypes) based on the identity of the heavy chain constant domains, designated alpha, delta, epsilon, gamma, and mu, respectively (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Different classes of immunoglobulins have different known subunit structures and three-dimensional configurations. For example, a common configuration for an antibody may have two full-length antibody heavy chains and two full-length antibody light chains.

[0032] As used herein, the term “antibody heavy chain” refers to the antibody heavy chain consisting of a variable region and a constant region, as defined for full-length antibodies. A full-length antibody heavy chain is a polypeptide composed of the antibody heavy chain variable domain (VH), antibody constant heavy chain domain 1 (CH1), antibody hinge region (HR), antibody heavy chain constant domain 2 (CH2), and antibody heavy chain constant domain 3 (CH3) in the N-terminal to C-terminal direction, and is abbreviated as VH-CH1-HR-CH2-CH3. When used in reference to antibodies, the term heavy chain can refer to any distinct type based on the amino acid sequence of the constant domain, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which are the basis for the IgA, IgD, IgE, IgG, and IgM classes of antibodies, e.g., subclasses of IgG (e.g., IgG1, IgG2, IgG3, and IgG4) and subclasses of IgA (e.g., IgA1 and IgA2), respectively. The heavy chain amino acid sequence is publicly known in this technology.

[0033] As used herein, the term "antibody light chain" refers to an antibody light chain consisting of a variable region and a constant region, as defined for a full-length antibody. A full-length antibody light chain is a polypeptide located in the N-terminal to C-terminal direction of the antibody light chain variable domain (VL) and antibody light chain constant domain (CL) (abbreviated as VL-CL). When used in reference to antibodies, the term "light chain" can refer to any distinct type based on the amino acid sequence of the constant domain, e.g., kappa (κ) or lambda (λ). Light chain amino acid sequences are known in the art.

[0034] The term "antibody fragment" refers to a portion of an intact antibody. "Antigen-binding fragment," "antigen-binding domain," or "antigen-binding region" refers to a portion of an intact antibody that binds to an antigen. Antigen-binding fragments may contain the antigen-determining region (e.g., the complementarity-determining region (CDR)) of an intact antibody. Examples of antibody antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single-chain antibodies. Antibody antigen-binding fragments may originate from any animal species, such as rodents (e.g., mice, rats, or hamsters) or humans, or they may be artificially produced.

[0035] The term "multispecific antibody" means that an antigen-binding protein can specifically bind to two or more different antigens. A subcategory of multispecific antibodies is a "bispecific antibody" that can specifically bind to two different antigens. As used herein, an antibody "specifically binds" to a target antigen if, under similar binding assay conditions, it has a significantly higher binding affinity to the target antigen compared to its affinity to other unrelated proteins, and as a result can identify the target antigen. Antigen-binding proteins that specifically bind to an antigen have an equilibrium dissociation constant (K). D ) ≤ 1 × 10 -6 It could be M. The antigen-binding protein is K D ≤ 1 × 10 -8 When it is M, it binds specifically to the antigen with "high affinity".

[0036] An "isolated antibody" refers to a population of antibodies containing a single type of antibody. For example, a particular isolated antibody population consists of antibodies having a single heavy-chain amino acid sequence and a single light-chain amino acid sequence that bind to a single epitope. However, isolated antibodies may exhibit cross-reactivity to other antigens, such as related molecules from different species. Furthermore, a population of antibodies can still be an "isolated antibody" even if it is contaminated with small amounts of other antibody species. In particular, an isolated antibody may contain less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of other antibody species, or it may not contain any at all.

[0037] A "monoclonal antibody" refers to a homogeneous population of antibodies involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is typically in contrast to polyclonal antibodies, which contain different antibodies directed towards different antigenic determinants. Furthermore, "monoclonal" antibodies refer to antibodies that can be produced by several methods, including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals.

[0038] The terms “variable region” or “variable domain” are used interchangeably and are common in this technology. A variable region typically refers to a portion of an antibody, generally a portion of the light or heavy chain, typically the approximately 110–120 or 110–125 amino acids at the amino terminus of a mature heavy chain, and approximately 90–115 amino acids within a mature light chain. These regions exhibit wide-ranging sequence differences between antibodies and are used for the binding and specificity of a particular antibody to its specific antigen. Sequence variability is concentrated within regions called complementarity-determining regions (CDRs), while more highly conserved regions within variable domains are called framework regions (FRs). From the N-terminus to the C-terminus, both naturally occurring light and heavy chain variable regions typically correspond to the following sequence of elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. While not wishing to be constrained by any specific mechanism or theory, the CDRs of the light and heavy chains are thought to be primarily responsible for the antibody’s interaction with the antigen and its specificity.

[0039] The terms "VL," "VL domain," and "VH region" are used interchangeably to refer to the variable region of the antibody light chain.

[0040] The terms "VH," "VH domain," and "VH region" are used interchangeably to refer to the variable region of the heavy chain of an antibody.

[0041] The terms "Kabat numbering" and similar terms are recognized in the art and refer to the numbering scheme for amino acid residues in the heavy and light chain variable regions of antibodies, or in the antigen-binding fragments of antibodies. CDR can be determined according to the Kabat numbering scheme (see, for example, Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering scheme, CDRs within the antibody heavy chain molecule are typically located at positions 31–35 (optionally including one or two additional amino acids following 35 (referred to as 35A and 35B in the Kabat numbering scheme)), (CDR1), 50–65 (CDR2), and 95–102 (CDR3). Using the Kabat numbering scheme, CDRs within the antibody light chain molecule are typically located at positions 24–34 (CDR1), 50–56 (CDR2), and 89–97 (CDR3). The Kabat numbering scheme can be used in conjunction with the EU index, which is used to refer to residues within the constant domain of the antibody (see, for example, Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242).

[0042] Instead, Chothia refers to the location of the structural loop (Chothia and Lesk, J.Mol.Biol.196:901-917(1987)). When numbered using the Kabat numbering rules, the end of a Chothia H1 loop will be between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B exists, the loop ends at 32; if only 35A exists, the loop ends at 33; if both 35A and 35B exist, the loop ends at 34).

[0043] The AbM hypervariable region represents a compromise between the Kabat CDR and the Chothia structural loop and is used by Oxford Molecular's AbM antibody labeling software.

[0044] In addition, the CDR region can be determined according to the IMGT numbering scheme (see, for example, Guidicelli et al., Nucl. Acids Res. 34:D781-D784 (2006); Lefranc et al., Dev. Comp. Immunol. 27:55-77 (2003)). This numbering scheme unifies the numbering across the antibody lambda and kappa light and heavy chains, as well as the T cell receptor chain.

[0045] In addition to the above, the positions of specific amino acids within the framework region of the variable domain (described below) can be described using the Aho numbering scheme. Since the length of the antibody CDR amino acid sequence differs for each antibody, numbering residues based on the linear sequence (assuming the first residue is position 1) results in framework residues with different position numbers between antibodies. Annemarie Honegger and Andreas Plueckthun developed a structure-based numbering scheme (Aho). This scheme introduces gaps within the CDR region to minimize deviations from the average structure of the aligned domain (Honegger, A., and Plueckthun, A. (2001). J.Mol.Biol. 309, 657-670). This results in structurally equivalent positions with the same residue numbers when comparing two different antibodies. This makes it possible to compare the effects of substitutions in the variable domain framework region between antibodies.

[0046] The terms "constant region" and "constant domain" are interchangeable and have general meanings in this technology. The constant region is the carboxyl-terminal portion of the light and / or heavy chain of an antibody moiety, for example, an antibody, which is not directly involved in binding to an antigen but can exhibit various effector functions, such as interaction with the Fc receptor. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence compared to the immunoglobulin variable domain.

[0047] As used herein, the terms “Fc region” and “Fc domain” refer to the C-terminal region of the IgG heavy chain; in the case of IgG1 antibodies, the C-terminal region includes -CH2-CH3 (see above).

[0048] The term "interface" refers to the association surface resulting from the interaction of one or more amino acids in a first antibody domain with one or more amino acids in a second antibody domain. Exemplary interfaces include CH1 / CL, VH / VL, CH2-CH2, and CH3 / CH3 interfaces. In some embodiments, the interface includes, for example, hydrogen bonds, electrostatic interactions, or salt bridges between the amino acids forming the interface.

[0049] The term "chimeric antibody" refers to an antibody whose amino acid sequence originates from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of an antibody derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) that possesses the desired specificity, affinity, and capability, while the constant region is homologous to a sequence derived from another species (usually human), thus avoiding the induction of an immune response in that species.

[0050] A "humanized antibody" refers to a chimeric antibody containing amino acid residues derived from non-human CDRs, as well as amino acid residues derived from the human framework region and constant region. A humanized antibody may contain at least one, typically two, substantially all variable domains, where all or substantially all CDRs correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization. Typically, a humanized antibody is a human immunoglobulin in which CDR-derived residues are replaced with CDR-derived residues from a non-human species (e.g., mouse, rat, rabbit, hamster) that possess the desired specificity, affinity, and capabilities. Therefore, humanized antibodies are also called "CDR-grafted" antibodies. Early examples of methods used to generate humanized antibodies are described in U.S. Patent No. 5,225,539; Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994), and Roguska et al., Protein Eng. 9(10):895-904 (1996). Subsequently, many additional examples and methods related to antibody humanization have been published.

[0051] "Human antibody" refers to an antibody in which both the FR and CDR have variable regions derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also originates from a human germline immunoglobulin sequence. The human antibodies of this disclosure may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, is grafted onto a human framework sequence. The terms "human antibody" and "fully human antibody" are used synonymously.

[0052] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, or may be interrupted by non-amino acid groups. The terms also encompass amino acid polymers that are naturally occurring or modified by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation or modification, such as conjugation with labeling components. Furthermore, the definition includes, for example, polypeptides containing one or more analogues of amino acids (e.g., non-natural amino acids), and other modifications known in the art. Since the polypeptides of the present invention are antibody-based, it is understood that in certain embodiments, the polypeptides may exist as single-chain or associated chains.

[0053] A "complimentary amino acid substitution" means that a substitution of a positively charged amino acid in the heavy chain is paired with a positively charged amino acid substitution of an amino acid in the light chain associated with the heavy chain residue. Similarly, a substitution of a positively charged amino acid in the heavy chain is paired with a positively charged amino acid substitution of an amino acid in the light chain associated with the heavy chain residue.

[0054] As used herein, the term “host cell” can refer to any type of cell, such as primary cells, cells in culture, or cells derived from a cell line. In certain embodiments, the term “host cell” refers to cells transfected with nucleic acid molecules, and their progeny or potential progeny. Such progeny may not be identical to the parent cells transfected with nucleic acid molecules, for example, due to mutations or environmental influences that may occur in subsequent generations or in the integration of nucleic acid molecules into the host cell genome.

[0055] Modification of VH domain and VL domain In some embodiments, the disclosed protein includes modifications within the VH and VL domains, which involve introducing charged amino acids that are electrostatically favorable to the modified VH and VL domains interacting with each other, and unfavorable to the modified VH and VL domains interacting with the unmodified VH and VL domains.

[0056] To drive the specific association of the modified VH domain with the modified VL domain, the two domains may contain complementary charge modifications. Therefore, if the VH domain contains a modification to introduce a negatively charged amino acid, this is paired with a modification complementary to a positively charged amino acid in the VL domain. Conversely, if the VL domain contains a modification to introduce a negatively charged amino acid, this is paired with a modification complementary to a positively charged amino acid in the VH domain. Thus, pairs of complementaryly charged residues may be referred to as "charge pair modifications," "charge pair mutations," or "CPMs."

[0057] The modified amino acids in charge pair modification can be any natural or artificial amino acids that are charged at physiological pH. In some embodiments, the negatively charged amino acid is aspartic acid or glutamic acid. In some embodiments, the positively charged amino acid is lysine, histidine, or arginine.

[0058] One type of charge pair modification that can be used to drive a specific VH-VL association involves a modification at position 39 of the VH domain paired with a modification at position 85 (numbered according to the Kabat scheme) of the VL domain. In some embodiments, the VH domain contains a charged amino acid at position number 39, and the VL domain contains a charged amino acid at position number 85 that is charge-complementary to the amino acid at position 39 of the VH domain. In some embodiments, the VH domain contains a positively charged amino acid at position 39 (e.g., arginine, lysine, or histidine), and the VL domain contains a negatively charged amino acid at position 85 (e.g., glutamic acid or aspartic acid). In some embodiments, the VL domain contains a positively charged amino acid at position 85 (e.g., arginine, lysine, or histidine), and the VH domain contains a negatively charged amino acid at position 39 (e.g., glutamic acid or aspartic acid).

[0059] Another charge pair modification that may be used to drive a specific VH-VL association includes a modification at position 105 of the VH domain paired with a modification at position 42 (numbered according to the Kabat scheme) of the VL domain. In some embodiments, the VH domain contains a charged amino acid at position number 105, and the VL domain contains a charged amino acid at position number 42 that is charge-complementary to the amino acid at position 105 of the VH domain. In some embodiments, the VH domain contains a positively charged amino acid at position 105 (e.g., arginine, lysine, or histidine), and the VL domain contains a negatively charged amino acid at position 42 (e.g., glutamic acid or aspartic acid). In some embodiments, the VL domain contains a positively charged amino acid at position 42 (e.g., arginine, lysine, or histidine), and the VH domain contains a negatively charged amino acid at position 105 (e.g., glutamic acid or aspartic acid).

[0060] Another charge pair modification that may be used to drive a specific VH-VL association includes a modification at position 91 of the VH domain paired with a modification at position 38 (numbered according to the Kabat scheme) of the VL domain. In some embodiments, the VH domain contains a charged amino acid at position number 91, and the VL domain contains a charged amino acid at position number 38 that is charge-complementary to the amino acid at position 91 of the VH domain. In some embodiments, the VH domain contains a positively charged amino acid at position 91 (e.g., arginine, lysine, or histidine), and the VL domain contains a negatively charged amino acid at position 38 (e.g., glutamic acid or aspartic acid). In some embodiments, the VL domain contains a positively charged amino acid at position 38 (e.g., arginine, lysine, or histidine), and the VH domain contains a negatively charged amino acid at position 91 (e.g., glutamic acid or aspartic acid).

[0061] Numerous VH and VL domain sequences are known in the art as part of antibody sequences. Modifications of the VH and VL domains disclosed herein can be applied to VH and VL sequences known in the art. In some embodiments, the modifications of the VH and VL domains disclosed herein can be applied to naturally occurring human VH and VL domain sequences. In some embodiments, the modifications of the VH and VL domains disclosed herein can be applied to naturally occurring mouse or rat VH and VL domain sequences. Exemplary VH and VL sequences are provided as SEQ ID NOs: 1-8. In some embodiments, the VH Domain is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the human germline VH Domain. In some embodiments, the VL Domain is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the human germline VL Domain.

[0062] Proteins containing modifications to the VH domain and VL domain The modified VH and VL domains described herein can be used in a variety of situations where the VH and VL domains are part of one or more polypeptides for which a specific VH-VL interaction is desired. For example, the modified VH and VL domains can be integrated into heavy and light chain polypeptides as part of forming an antibody or antibody fragment. In some embodiments, the modified VH and VL domains help to create specific VH-VL pair formation in heterodimer antibodies, such as bispecific antibodies. However, applications are not limited to antibodies or antibody-like molecules, as the VH and VL domains can function as independent domains or be conjugated or fused to other proteins to facilitate specific interactions.

[0063] Bispecific antibodies are one situation in which the modified VH and VL domains described herein may be used. In some forms, bispecific antibodies contain two different heavy chains and two different light chains, requiring a specific arrangement of the antibody such that each heavy chain binds to a particular light chain. Thus, in some embodiments, the modified VH and VL domains described herein are components of a bispecific antibody comprising a first heavy chain variable domain and light chain variable domain pair (VH and VL) and a second heavy chain variable domain and light chain variable domain pair (VH' and VL'). In such a bispecific antibody, each VH-VL pair may contain a different set of charge pair mutations. In some embodiments, VH and VH' each contain a charged amino acid at the same position; VL and VL' each contain a charged amino acid at the same position that is complementary in charge to the modified amino acid of VH and VH', respectively; and VH and VH' contain amino acids with complementary charges. In such embodiments, VH and VL' contain similar charges (i.e., both positively charged amino acids or both negatively charged amino acids), and VH' and VL contain similar charges (i.e., both positively charged amino acids or both negatively charged amino acids). In some embodiments, the VH-VL and VH'-VL' pairs are modified at the same amino acid position. In some embodiments, the VH-VL and VH'-VL' pairs are modified at different positions.

[0064] In some embodiments, the antibody or antibody fragment comprises a VL Domain that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a human germline lambda chain VL Domain.

[0065] In some embodiments, the antibody or antibody fragment comprises a VL Domain that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a human germline kappa chain VL Domain.

[0066] In some embodiments, the modified VH and VL domains described herein are components of an antibody fragment. In some embodiments, the antibody fragment is any antibody fragment containing both the VH domain and the VL domain. In some embodiments, the antibody fragment is a Fab fragment, a Fab' fragment, an F(ab')2 fragment, or an Fv fragment.

[0067] In some embodiments, the antibody or antibody fragment optionally includes further modifications at the VH-VL interface in addition to the modifications described in the preceding "Modification of VH Domain and VL Domain" section. The VH-VL domain interface residues within the VL domain (i.e., amino acid residues that mediate the assembly of the VH and VL domains) are AHo at position 40 (Kabat 32), 42 (Kabat 34), 43 (Kabat 35), 44 (Kabat 36), 46 (Kabat 38), 49 (Kabat 41), 50 (Kabat 42), 51 (Kabat 43), 52 (Kabat 44), 53 (Kabat 45), 54 (Kabat 46), 56 (Kabat 48), 57 (Kabat 49), 58 (Kabat 50), 67 (Kabat 51), 69 (Kabat 53), 70 (Kabat 54), 71 (Kabat 55), 72 (Kabat 56), 73 (Kabat 57), 74 (Kabat This includes 58), 103 (Kabat 85), 105 (Kabat 87), 107 (Kabat 89), 108 (Kabat 90), and 109 (Kabat 91). In some embodiments, one or more interfacial residues in the VL domain are substituted with charged amino acids, which preferably have the opposite charge to those introduced in the congeneral VH domain. In some embodiments, the amino acid at AHo46 (Kabat 38) of the VL domain is substituted with a positively charged amino acid. In some embodiments, for example, if the amino acid at AHo46 (Kabat 39) in the VH domain is substituted with a positively charged amino acid, then the amino acid at AHo46 (Kabat 38) of the VL domain is substituted with an uncharged amino acid. In some embodiments, the amino acids at AHo51 (Kabat 43) and / or AHo141 (Kabat 100) are substituted with positively charged or uncharged amino acids. Such embodiments may further include the substitution of the amino acid at position AHo46 with a positively charged or uncharged amino acid. In some embodiments, the amino acid at position AHo51 is substituted with a positively charged amino acid, such as lysine. In alternative embodiments, the amino acid at position AHo51 is substituted with an uncharged amino acid, such as aspartic acid.In some embodiments, the amino acid at AHo141 is substituted with a positively charged amino acid, such as lysine. In alternative embodiments, the amino acid at AHo141 is substituted with a negatively charged amino acid, such as aspartic acid. In some embodiments, the amino acids at AHo51 and AHo141 are substituted with a positively charged amino acid, such as lysine, or a negatively charged amino acid, such as aspartic acid. Such embodiments may further include substitution with a positively or negatively charged amino acid at AHo46 (Kabat 38).

[0068] In some embodiments, the antibody or antibody fragment containing the modified VH-VL domain contains a kappa light chain. In some embodiments where the light chain is a kappa light chain, one or more amino acids in the CL domain of the antibody or antibody fragment at a position selected from the group consisting of F116, F118, S121, D122, E123, Q124, S131, V133, L135, N137, N138, Q160, S162, T164, S174, and S176 (EU and Kabat numbering in the kappa light chain) are substituted with a charged amino acid. In some embodiments, an exemplary residue for substitution with a negative or positively charged amino acid is the amino acid at position 176 of the CL domain (EU and Kabat numbering scheme). In some embodiments, the amino acid at position 176 of the CL domain is substituted with a positively charged amino acid. In alternative embodiments, the amino acid at position 176 of the CL domain is substituted with a charged amino acid, such as aspartic acid. In some embodiments, the antibody or antibody fragment comprises a kappa chain that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a human germline kappa chain.

[0069] In some embodiments, the antibody or antibody fragment containing the modified VH-VL domain includes a lambda light chain. In some embodiments where the light chain is a lambda light chain, one or more amino acids in the CL domain of the antibody or antibody fragment at a position selected from the group consisting of T116, F118, S121, E123, E124, K129, T131, V133, L135, S137, E160, T162, S165, Q167, A174, S176, and Y178 (Kabat numbering on the lambda chain) are substituted with a charged amino acid. In some embodiments, an exemplary residue for substitution with a negatively or positively charged amino acid is the amino acid at position 176 of the CL domain (EU and Kabat numbering scheme). In some embodiments, the amino acid at position 176 of the CL domain is substituted with a positively charged amino acid. In alternative embodiments, the amino acid at position 176 of the CL domain is substituted with a charged amino acid, such as aspartic acid. In some embodiments, the antibody or antibody fragment comprises a lambda chain that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a human germline lambda chain.

[0070] In some embodiments, the modified VH and VL domains described herein are components of an antibody or antibody fragment containing a CH1 domain. In some embodiments, the antibody or antibody fragment contains a heavy chain CH1 region that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the human germline heavy chain CH1 region.

[0071] Since the CH1 domain complexes with the light chain constant region (CL), the CH1 and CL domains can be manipulated to increase the pairing efficiency of a particular heavy chain with its congener light chain. Therefore, in some embodiments, the modified VH and VL domains disclosed herein can be combined with modifications to the CH1 and CL domains. For example, assembly can be facilitated by introducing cysteine ​​residues into the heavy and light chains at or near the CH1-CL interface to enable the formation of disulfide bonds, modifying amino acids to create a knob-into-hole effect, and performing electrostatic engineering similar to that described herein on the variable region. In some embodiments, assembly can be facilitated by introducing one or more charged amino acids into the CH1 domain of an antibody or antibody fragment at the EU position selected from the group consisting of F126, P127, L128, A141, L145, K147, D148, H168, F170, P171, V173, Q175, S176, S183, V185, and K213. In this regard, S183 (EU numbering scheme) is a particularly preferred residue for substitution with an uncharged or positively charged amino acid. In some embodiments, S183 is substituted with a positively charged amino acid. In alternative embodiments, S183 is substituted with an uncharged amino acid. In some embodiments, the modification at the S183 position of the CH1 domain is paired with a modification at the 176 position of the CL domain to a complementaryly charged amino acid.

[0072] In some embodiments, the modified VH and VL domains described herein are components of an antibody or antibody fragment containing a constant region. The antibodies described herein may contain any constant region. The light chain constant region may be, for example, a kappa-type or lambda-type light chain constant region, e.g., a human kappa-type or human lambda-type light chain constant region. The heavy chain constant region may be, for example, an alpha-type, delta-type, epsilon-type, gamma-type, or mu-type heavy chain constant region, e.g., a human alpha-type, human delta-type, human epsilon-type, human gamma-type, or human mu-type heavy chain constant region. In one embodiment, the light chain constant region or heavy chain constant region is a naturally occurring fragment, derivative, variant, or mutain of a constant region. The antibodies described herein may include any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) of them based on the identity of the heavy chain constant domains, which are referred to as alpha, delta, epsilon, gamma, and mu, respectively (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The antibodies described herein may include a CH2 domain, a CH3 domain, or a constant domain containing both CH2 and CH3 domains.

[0073] An antibody or antibody fragment described herein, comprising a CH3 domain as part of a heavy chain, wherein the antibody or antibody fragment optionally further comprises two CH3 domains, each containing one or more substitutions, at least one of which introduces a non-naturally charged amino acid into the domain. In some embodiments, each CH3 domain contains one or more amino acid substitutions within the CH3 domain that inhibit homodimerization, more preferably preferentially heterodimerization with the corresponding CH3 domain. International Publication No. 2009 / 089004 and U.S. Patent No. 10,233,237 (both incorporated herein by reference in their entirety) describe compositions and methods for manipulating the CH3 domain interface to reduce homodimerization and increase heterodimerization between two CH3 domain-containing molecules. In some embodiments, one or more amino acids at positions selected from the group consisting of 399, 356, and 357 (EU numbering system) of the CH3 domain are substituted with charged amino acids. In some embodiments, one or more amino acids selected from the group consisting of 370, 392, and 409 (EU numbering system) are substituted with positively charged amino acids. In alternative embodiments, one or more amino acids selected from the group consisting of 399, 356, and 357 (EU numbering system) of the CH3 domain are substituted with positively charged amino acids. In further embodiments, one or more amino acids selected from the group consisting of 370, 392, and 409 (EU numbering system) are substituted with uncharged amino acids. In some embodiments, the heterodimer antibody comprises a first heavy chain containing positively charged amino acids (e.g., D399K and E356K) at positions 399 and 356, and a second heavy chain containing uncharged amino acids (e.g., K392D and K409D) at positions 392 and 409. In some embodiments, the heterodimer antibody comprises a first heavy chain containing positively charged amino acids (e.g., 356K and 409K) at positions 356 and 399, and a second heavy chain containing uncharged amino acids (e.g., 392D, 409D, and 439D) at positions 392, 409, and 439.

[0074] Fc modification The heavy chains of antibodies or antibody fragments described herein may further include one or more mutations affecting the binding of the heavy chain-containing antibody to one or more Fc receptors. One function of the Fc portion of an antibody is to communicate with the immune system when the antibody binds to its target. This is commonly referred to as “effector function.” This communication leads to antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement-dependent cell-mediated cytotoxicity (CDC). ADCC and ADCP are mediated through the binding of Fc to Fc receptors on the surface of cells of the immune system. CDC is mediated through the binding of Fc to complement system proteins, such as C1q.

[0075] IgG subclasses exhibit varying abilities in mediating effector function. For example, IgG1 is superior to IgG2 and IgG4 in mediating ADCC and CDC. By introducing one or more mutations into the Fc, the effector function of an antibody can be increased or decreased. Embodiments of the present invention include heterodimer antibodies in which the Fc has been manipulated to increase effector function (U.S. Patent No. 7,317,091 and Strohl, Curr. Opin. Biotech., 20:685-691, 2009 (both incorporated herein by reference in their entirety)).Examples of IgG1 Fc molecules with enhanced effector function include those having one or more of the following substitutions [numbered according to the EU numbering scheme]: S239D / I332E, S239D / A330S / I332E, S239D / A330L / I332E, S298A / D333A / K334A, P247I / A339D, P247I / A339Q, D280H / K290S, D280H / K290S / S298D, D280H / K290S / S2 98V, F243L / R292P / Y300L, F243L / R292P / Y300L / P396L, F243L / R292P / Y300L / V305I / P396L, G236A / S239D / I33 2E, K326A / E333A, K326W / E333S, K290E / S298G / T299A, K290N / S298G / T299A, K290E / S298G / T299A / K326E, K290 N / S298G / T299A / K326E, K334V, L235S+S239D+K334V, Q311M+K334V, S239D+K334V, F243V+K334V, E294L+K334V , S298T+K334V, E233L+Q311M+K334V, L234I+Q311M+K334V, S298T+K334V, A330M+K334V, A330F+K334V, Q311M+ A330M+K334V, Q311M+A330F+K334V, S298T+A330M+K334V, S298T+A330F+K334V, S239D+A330M+K334V, S239D+S298T+K334V, L234Y+K290Y+Y296W, L234Y+F243V+Y296W, L234Y+E294L+Y296W, L234Y+Y296W, and K290Y+Y296W.

[0076] Further embodiments of the present invention include antibodies and antibody fragments in which Fc has been manipulated to reduce effector function. Examples of Fc molecules exhibiting reduced effector function include those having one or more of the following substitutions [numbered according to the EU numbering scheme]: N297A(IgG1), L234A / L235A(IgG1), V234A / G237A(IgG2), L235A / G237A / E318A(IgG4), H268Q / V309L / A330S / A331S(IgG2), C220S / C226S / C229S / P238S(IgG1), C226S / C229S / E233P / L234V / L235A(IgG1), L234F / L235E / P331S(IgG1), S267E / L328F(IgG1).

[0077] Another method to enhance the effector function of IgG Fc-containing proteins involves reducing the fucosylation of Fc. Removing core fucose from branched complex oligosaccharides attached to Fc significantly increased ADCC effector function without altering antigen-binding or CDC effector function. Several methods are known to reduce or eliminate the fucosylation of Fc-containing molecules, such as antibodies. These include recombinant expression in specific mammalian cell lines, such as FUT8 knockout cell lines, variant CHO cell line Lec13, rat hybridoma cell line YB2 / 0, cell lines containing small interfering RNAs specific to the FUT8 gene, and cell lines co-expressing β-1,4-N-acetylglucosaminyltransferase III and Golgi β-mannosidase II. In addition, Fc-containing molecules may be expressed in plant cells, yeast, or prokaryotic cells, such as non-mammalian cells like Escherichia coli (E. coli). Therefore, in certain embodiments, the composition includes an antibody in which fucosylation is reduced or fucosylation is absent as a whole.

[0078] Polynucleotides encoding manipulated heavy or light chains The present invention encompasses nucleic acids encoding polypeptide chains containing VH and VL domains as described herein. Examples of nucleic acid molecules include DNA and RNA in both single-stranded and double-stranded forms, as well as their corresponding complementary sequences. Examples of DNA include cDNA, genomic DNA, chemosynthetic DNA, PCR-amplified DNA, and combinations thereof. Examples of nucleic acid molecules include full-length genes or cDNA molecules and combinations thereof. While the nucleic acids of the present invention are preferably derived from human sources, the invention also includes those derived from non-human species.

[0079] In some embodiments, the nucleic acids of the present invention are isolated nucleic acids. “Isolated nucleic acids” are, in the case of nucleic acids isolated from a naturally occurring source, nucleic acids that are present in the genome of the organism from which the nucleic acid was isolated, separated from adjacent gene sequences. For example, in the case of nucleic acids synthesized enzymatically or chemically from a template, such as PCR products, cDNA molecules, or oligonucleotides, the nucleic acids obtained from such processes are understood to be isolated nucleic acids. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a distinct fragment, or a nucleic acid molecule as a component of a larger nucleic acid construct. In one preferred embodiment, the nucleic acid is substantially free of endogenous contaminants. The nucleic acid molecule is preferably derived from DNA or RNA isolated at least once in a substantially pure form and in an amount or concentration that allows for the identification, manipulation, and recovery of its constituent nucleotide sequences, by standard biochemical methods (such as those outlined in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). Such sequences are preferably provided and / or constructed in the form of an open reading frame that is not interrupted by internal untranslated sequences or introns, typically found within eukaryotic genes. The sequence of untranslated DNA may be located 5' or 3' from the open reading frame, in which case it does not interfere with the manipulation or expression of the coding region.

[0080] Variants are typically prepared by site-directed mutagenesis of nucleotides in polypeptide-encoding DNA, such as generating variant-encoding DNA using cassette or PCR mutagenesis, or other techniques known in the art, and then expressing recombinant DNA in cell culture, as outlined herein. However, antibodies or antigen-binding fragments containing variant CDRs having up to approximately 100–150 residues can be prepared by in vitro synthesis using established techniques. Variants typically exhibit the same qualitative biological activity as naturally occurring analogs, e.g., binding to antigens, but variants with modified characteristics can also be selected, as outlined in more detail below.

[0081] As will be understood by those skilled in the art, due to the degeneracy of the genetic code, a large number of nucleic acids can be produced, all of which encode the polypeptide of the present invention. Therefore, once a specific amino acid sequence is identified, those skilled in the art will be able to produce any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not alter the amino acid sequence of the encoded protein.

[0082] Furthermore, the present invention provides expression systems and constructs in the form of plasmids, expression vectors, transcriptions, or expression cassettes containing at least one of the aforementioned polynucleotides. In addition, the present invention provides host cells containing such expression systems or constructs.

[0083] Typically, an expression vector used in a host cell will contain a sequence for plasmid maintenance, as well as a sequence for cloning and expressing an exogenous nucleotide sequence. Such sequences, collectively referred to as “flanking sequences,” will, in certain embodiments, typically include the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and one or more selection marker elements. Each of these sequences is discussed below.

[0084] Optionally, the vector may contain an oligonucleotide molecule localized at the 5' or 3' end of a sequence encoding a "tag," i.e., the sequence encoding the polypeptide; the oligonucleotide sequence may encode another "tag," such as polyHis (e.g., hexaHis), or FLAG, HA (influenza virus hemagglutinin), or myc, for which commercially available antibodies exist. The tag is typically fused to the polypeptide immediately after polypeptide expression and can serve as a means for affinity purification or detection of the polypeptide from host cells. Affinity purification can be achieved, for example, by column chromatography using an antibody against the tag as an affinity matrix. Optionally, the tag can then be removed from the purified polypeptide by various means, such as using a specific cleavage peptidase.

[0085] Flanking sequences may be of the same species (i.e., derived from the same species and / or strain as the host cell), different species (i.e., derived from a species other than the host cell's species or strain), hybrid (i.e., a combination of flanking sequences from multiple sources), synthetic, or natural. Therefore, the source of the flanking sequence may be any prokaryote or eukaryote, any vertebrate or invertebrate, or any plant, provided that the flanking sequence functions within and can be activated by the host cell mechanism.

[0086] Flanking sequences useful in the vectors of the present invention may be obtained by any of several methods known in the art. Typically, flanking sequences useful herein are identified in advance by mapping and / or restriction endonuclease digestion and can be isolated from a suitable tissue source using an appropriate restriction endonuclease. In some cases, the whole nucleotide sequence of the flanking sequence may be known. In this case, the flanking sequence can be synthesized using the methods described herein for nucleic acid synthesis or cloning.

[0087] Whether all or only some of the flanking sequences are known, this can be obtained by screening a genomic library with appropriate probes, such as oligonucleotides and / or flanking sequence fragments, using polymerase chain reaction (PCR) and / or from the same or a different species. If the flanking sequences are unknown, the DNA fragments containing the flanking sequences can be isolated, for example, from a larger DNA fragment that may contain coding sequences or other genes. Isolation can be achieved by generating appropriate DNA fragments by restriction endonuclease digestion, followed by isolation using agarose gel purification, Qiagen® column chromatography (Chatsworth, Calif.), or other methods known to those skilled in the art. The selection of suitable enzymes for achieving this purpose will be readily apparent to those skilled in the art.

[0088] Origins of replication (ORS) are typically components of commercially available prokaryotic expression vectors, and these origins are useful for vector amplification in host cells. If the selected vector does not contain an ORS site, it may be chemically synthesized based on a known sequence and ligated into the vector. For example, the ORS derived from plasmid pBR322 (New England Biolabs, Beverly, Mass.) is suitable for most Gram-negative bacteria, and various viral origins (e.g., SV40, polyoma, adenovirus, varicella-stomatitis virus (VSV), or papillomavirus, e.g., HPV or BPV) are useful for vector cloning in mammalian cells. In general, ORS components are not required for mammalian expression vectors (for example, the SV40 origin is often used only because it also contains the viral initial promoter).

[0089] Transcription termination sequences are typically localized 3' to the end of the polypeptide coding region and serve to terminate transcription. In prokaryotic cells, transcription termination sequences are usually GC-rich fragments followed by poly-T sequences. These sequences can be readily cloned from libraries or even commercially purchased as part of vectors, while they can also be readily synthesized using nucleic acid synthesis methods such as those described herein.

[0090] Selection marker genes encode proteins essential for the survival and proliferation of host cells grown in a selective medium. Typical selection marker genes encode (a) proteins that confer resistance to antibiotics or other toxins, such as ampicillin, tetracycline, or kanamycin, to prokaryotic host cells; (b) proteins that compensate for deficiencies in the cellular nutritional requirements; or (c) proteins that supply essential nutrients unavailable from combined or limited media. Specific selection markers include kanamycin resistance genes, ampicillin resistance genes, and tetracycline resistance genes. Advantageously, neomycin resistance genes may also be used for selection in both prokaryotic and eukaryotic host cells.

[0091] Other select genes may be used to amplify the genes that will be expressed. Amplification is the process by which genes required for the production of proteins important for growth or cell survival are repeated in tandem within the chromosomes of successive recombinant cells. Examples of select markers suitable for mammalian cells include the dihydrofolate reductase (DHFR) gene and the promoterless thymidine kinase gene. Mammalian cell transformants are placed under selective pressure so that only those transformants are adapted to survive by the select gene present in the vector. Selective pressure is applied by culturing the transformed cells under conditions of continuously increasing selector concentration in the culture medium, thereby amplifying both the select gene and the DNA encoding another gene, such as the light or heavy chain of an antibody. As a result, large amounts of polypeptides are synthesized from the amplified DNA.

[0092] The ribosome binding site is typically essential for mRNA translation initiation and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). This element is typically localized 3' to the promoter and 5' to the coding sequence of the expressed polypeptide. In certain embodiments, one or more coding regions may be operably ligated to an internal ribosome binding site (IRES), enabling translation of two open reading frames from a single RNA transcript.

[0093] In cases where glycosylation is desired in eukaryotic host cell expression systems, various pre-sequences or pro-sequences can be manipulated to improve glycosylation or yield. For example, the peptidase cleavage site of a specific signal peptide may be modified, or a pro-sequence may be added, which can also affect glycosylation. The final protein product may have one or more additional amino acids associated with expression at position -1 (relative to the first amino acid of the mature protein), which do not have to be completely removed. For example, the final protein product may have one or two amino acid residues found within the peptidase cleavage site attached to the amino terminus. In addition, using several other enzymatic cleavage sites, a slightly truncated form of the desired polypeptide may be produced when enzymatically cleaved at such regions within the mature polypeptide.

[0094] The expression vectors and cloning vectors of the present invention typically contain a promoter that is recognized by a host organism and operably ligated to a molecule encoding a polypeptide. The promoter is a non-transcriptional sequence localized upstream (i.e., 5') of the start codon of a structural gene (generally within about 100-1000 bp) and controls the transcription of the structural gene. Conventionally, promoters are grouped into one of two classes: inducible promoters and constitutive promoters. Inducible promoters, under their control, initiate an increase in the transcription level from DNA in response to any change in culture conditions, such as the presence or absence of nutrients or changes in temperature. Constitutive promoters, on the other hand, transcribe the gene to which they operably ligate uniformly, i.e., with little or no control over gene expression. Numerous promoters recognized by various potential host cells are known in this art. By extracting the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence into the vector, the appropriate promoter is operably ligated to, for example, the DNA encoding the heavy chain or light chain.

[0095] Promoters suitable for use in yeast hosts are also known in this art. It is advantageous for yeast enhancers to be used with yeast promoters. Promoters suitable for use in mammalian host cells are known and include, but are not limited to, those obtained from the genomes of viruses such as polyomaviruses, fowlpox virus, adenoviruses (adenovirus type 2, etc.), bovine papillomavirus, aerosarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and most preferably Simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat shock promoters and actin promoters.

[0096] Potential additional promoters include, but are not limited to, the following: the SV40 initial promoter (Benoist and Chambon, 1981, Nature 290:304-310); the CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad. USA 81:659-663); the promoter contained within the long terminal repeat at the 3' end of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:1444-1445); and promoters and regulatory sequences derived from the metallothionein gene (Prinster et al., 1982, Nature 296:39-42); as well as prokaryotic promoters such as beta-lactamase promoters (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731); or tac promoters (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Furthermore, the target areas are the following animal transcriptional regulatory regions that exhibit tissue specificity and have been utilized in transgenic animals: the elastase I gene regulatory region active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); the insulin gene regulatory region active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122); and the immunoglobulin gene regulatory region active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al. al., 1987, Mol. Cell. Biol.7:1436-1444); Active mouse mammary cancer virus regulatory region in testes, mammary glands, lymphocytes, and mast cells (Leder et al., 1986, Cell 45:485-495); Active albumin gene regulatory region in the liver (Pinkert et al., 1987, Genes and Devel. 1:268-276); Active alpha-fetoprotein gene regulatory region in the liver (Krumlauf et al., 1985, Mol.Cell.Biol. 5:1639-1648; Hammer et al., 1987, Science 253:53-58); Active alpha-1 antitrypsin gene regulatory region in the liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); Active beta-globin gene regulatory region in bone marrow cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94); Active myelin basic protein gene regulatory region in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); Active myosin light chain-2 gene regulatory region in skeletal muscle (Sani, 1985, Nature 314:283-286); and Active gonadotropin-releasing hormone gene regulatory region in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

[0097] Enhancer sequences may be inserted into vectors to increase transcription of the light or heavy chain DNA of the present invention by higher eukaryotes. Enhancers are typically cis-acting elements of DNA, usually about 10–300 bp in length, that act on promoters to increase transcription. Enhancers are relatively orientation and position-independent and are found at both 5' and 3' positions relative to the transcription unit. Several enhancer sequences are known and available from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin). However, viral enhancers are typically used. The SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, and adenovirus enhancer, known in this art, are exemplary enhancing elements for eukaryotic promoter activation. Enhancers may be positioned in the vector either 5' or 3' relative to the coding sequence, but are typically localized at a site 5' from the promoter. Sequences encoding appropriate native or heterologous signal sequences (leader sequences or signal peptides) can be incorporated into expression vectors to promote extracellular antibody secretion. The choice of signal peptide or leader depends on the type of host cell from which the antibody will be produced, and heterologous signal sequences can replace native signal sequences. Examples of signal peptides that function in mammalian host cells include: the signal sequence for interleukin-7 (IL-7) described in U.S. Patent No. 4,965,195; the signal sequence for the interleukin-2 receptor described in Cosman et al., 1984, Nature 312:768; the interleukin-4 receptor signal peptide described in European Patent No. 0367 566; the type I interleukin-1 receptor signal peptide described in U.S. Patent No. 4,968,607; and the type II interleukin-1 receptor signal peptide described in European Patent No. 0460 846.

[0098] A vector may contain one or more elements that promote expression when the vector is integrated into the host cell genome. Examples include the EASE element (Aldrich et al. 2003 Biotechnol Prog. 19:1433-38) and the matrix attachment region (MAR). MARs can protect the integrated vector from "location" effects by mediating the structural organization of chromatin. Therefore, MARs are particularly useful when the vector is used to produce a stable transfectant. Several natural and synthetic MAR-containing nucleic acids are known in the art, for example, U.S. Patent Nos. 6,239,328; 7,326,567; 6,177,612; 6,388,066; 6,245,974; 7,259,010; 6,037,525; 7,422,874; and 7,129,062.

[0099] The expression vector of the present invention may be constructed from an initiation vector, such as a commercially available vector. Such a vector may or may not contain all of the desired flanking sequences. If one or more of the flanking sequences described herein are not initially present in the vector, they may be obtained individually and ligated into the vector. Methods used to obtain each of the flanking sequences are known to those skilled in the art.

[0100] After the vector is constructed and nucleic acid molecules encoding light chains, heavy chains, or light and heavy chain sequences are inserted into appropriate sites within the vector, the completed vector can be inserted into host cells suitable for amplification and / or polypeptide expression. Transformation of the expression vector into selected host cells may be achieved by known methods, e.g., transfection, infection, calcium phosphate coprecipitation, electroporation, microinjection, lipofection, DEAE-dextran mediated transfection, or other known techniques. The chosen method will depend in part on the type of host cell to be used. These methods and other suitable methods are known to those skilled in the art and are described, for example, in Sambrook et al., 2001.

[0101] Methods for producing proteins When cultured under appropriate conditions, host cells synthesize heterodimeric antibodies, which can then be collected from the culture medium (if the host cells secrete heterodimeric antibodies into the medium) or directly from the heterodimeric host cells (if they do not secrete heterodimeric antibodies). The selection of appropriate host cells depends on various factors, including the desired expression level, the polypeptide modifications desirable or essential for activity (such as glycosylation or phosphorylation), and the ease of folding into biologically active molecules. The host cells may be eukaryotes or prokaryotes.

[0102] Mammalian cell lines available as hosts for expression are known in this art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC). Any cell line used in expression systems known in this art can be used to produce the recombinant polypeptide of the present invention. Generally, the host cell is transformed with a recombinant expression vector containing DNA encoding a desired heterodimeric antibody. Possible host cells include prokaryotes, yeasts, or higher eukaryotic cells. Prokaryotes include Gram-negative or Gram-positive organisms, such as Escherichia coli (E. coli) or bacilli. Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, or their derivatives, such as Veggie CHO and related cell lines that grow in serum-free medium (Rasmussen et al., 1998, Cytotechnology 28:31), HeLa cells, BHK (ATCC CRL 10) cell lines, and CV1 / EBNA cell lines derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) as described by McMahan et al., 1991, EMBO J.10:2821, human embryonic kidney cells, such as 293, 293 EBNA, or MSR. 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from in vitro cultures of primary tissues, Examples include primary grafts, HL-60, U937, HaK, or Jurkat cells. Optionally, when the use of polypeptides in various signal transduction assays or reporter assays is desired, mammalian cell lines, such as HepG2 / 3B, KB, NIH3T3, or S49, may be used for polypeptide expression. Beyond these, polypeptides can also be produced in lower eukaryotes such as yeast, or in prokaryotes such as bacteria. Suitable yeasts include budding yeast (Saccharomyces cerevisiae), fission yeast (Schizosaccharomyces pombe), Kluyveromyces strains, Candida strains, or any yeast strain capable of expressing heterologous polypeptides. Suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous polypeptides.

[0103] If antibodies or antibody fragments are produced in yeast or bacteria, it may be desirable to modify the products produced in yeast or bacteria, for example, by phosphorylation or glycosylation at appropriate sites, to obtain functional products. Such covalent bonding can be achieved using known chemical or enzymatic methods. Polypeptides can also be produced by utilizing an insect expression system, in which the isolated nucleic acids of the present invention are operably linked to a suitable regulatory sequence in one or more insect expression vectors. Materials and methods for baculovirus / insect cell expression systems are commercially available, for example, in kit form (MaxBac® kit) from Invitrogen, San Diego, Calif., USA, and such methods are known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987) and Luckow and Summers, Bio / Technology 6:47 (1988). Cell-free translation systems can also be used to produce polypeptides such as antibodies or antibody fragments using RNA derived from the nucleic acid constructs disclosed herein. Cloning and expression vectors suitable for use in bacterial, fungal, yeast, and mammalian cell hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985). A host cell containing the isolated nucleic acid of the present invention, preferably operably ligated to at least one expression regulatory sequence, is a "recombinant host cell."

[0104] In certain embodiments, cell lines may be selected by determining which cell lines have high expression levels and constitutively produce antigen-binding proteins with desirable binding properties. In another embodiment, cell lines derived from B cell lineages that do not produce their own antibodies but have the ability to produce and secrete heterologous antibodies may be selected.

[0105] Further Embodiments Specific embodiments of the present invention include the following: 1. An isolated protein comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein VH and VL are bound to each other, and VH and VL are the following set of charged amino acids: a. VH is a set containing a charged amino acid at position 39, and VL is a set containing a charged amino acid at position 85 that is complementary in charge to the amino acid at position 39 of VH; b. VH is a set containing a charged amino acid at position 105, and VL is a set containing a charged amino acid at position 42 that is complementary in charge to the amino acid at position 105 of VH; or c.VH is a set containing a charged amino acid at position 91, and VL is a set containing a charged amino acid at position 38 that is complementary in charge to the amino acid at position 91 of VH; Includes at least one of the following: The positional numbers of charged amino acids within the VH and VL domains of isolated proteins refer to positions according to the Kabat numbering scheme. 2. VH and VL are isolated proteins of Embodiment 1, comprising the set of charged amino acids in (a). 3. The isolated protein of Embodiment 2, wherein VH contains a positively charged amino acid at position 39, and VL contains a negatively charged amino acid at position 85. 4. The isolated protein of Embodiment 3, wherein the VH39 (VH39) and VL85 (VL85) positions contain: (i) lysine at VH39 and aspartic acid at VL85, (ii) lysine at VH39 and glutamic acid at VL85, (iii) arginine at VH39 and aspartic acid at VL85, or (iv) arginine at VH39 and glutamic acid at VL85. 5. The isolated protein of Embodiment 2, wherein VH contains a negatively charged amino acid at position 39, and VL contains a positively charged amino acid at position 85. 6. The isolated protein of Embodiment 5, wherein the VH39 (VH39) and VL85 (VL85) positions contain: (i) aspartic acid at VH39 and lysine at VL85, (ii) glutamic acid at VH39 and lysine at VL85, (iii) aspartic acid at VH39 and arginine at VL85, or (iv) glutamic acid at VH39 and arginine at VL85. 7. VH and VL are isolated proteins of any of Embodiments 1 to 6, comprising the set of charged amino acids in (b). 8. The isolated protein of Embodiment 7, wherein VH contains a positively charged amino acid at position 105, and VL contains a negatively charged amino acid at position 42. 9. The isolated protein of Embodiment 8, wherein the VH105 (VH105) and VL42 (VL42) positions contain: (i) lysine at VH105 and aspartic acid at VL42, (ii) lysine at VH105 and glutamic acid at VL42, (iii) arginine at VH105 and aspartic acid at VL42, or (iv) arginine at VH105 and glutamic acid at VL42. 10. The isolated protein of Embodiment 7, wherein VH contains a negatively charged amino acid at position 105, and VL contains a positively charged amino acid at position 42. 11. The isolated protein of Embodiment 10, wherein the VH105 (VH105) and VL42 (VL42) positions contain: (i) aspartic acid at VH105 and lysine at VL42, (ii) glutamic acid at VH105 and lysine at VL42, (iii) aspartic acid at VH105 and arginine at VL42, or (iv) glutamic acid at VH105 and arginine at VL42. 12. VH and VL are isolated proteins of any of Embodiments 1 to 11, comprising the set of charged amino acids in (c). 13. An isolated protein of Embodiment 12, wherein VH contains a positively charged amino acid at position 91, and VL contains a negatively charged amino acid at position 38. 14. The isolated protein of Embodiment 13, wherein the VH91 (VH91) and VL38 (VL38) positions contain: (i) lysine at VH91 and aspartic acid at VL38, (ii) lysine at VH91 and glutamic acid at VL38, (iii) arginine at VH91 and aspartic acid at VL38, or (iv) arginine at VH91 and glutamic acid at VL38. 15. An isolated protein of Embodiment 12, wherein VH contains a negatively charged amino acid at position 91, and VL contains a positively charged amino acid at position 38. 16. The isolated protein of Embodiment 15, wherein the VH91 (VH91) and VL38 (VL38) positions contain: (i) aspartic acid at VH91 and lysine at VL38, (ii) glutamic acid at VH91 and lysine at VL38, (iii) aspartic acid at VH91 and arginine at VL38, or (iv) glutamic acid at VH91 and arginine at VL38. 17. The isolated protein is an isolated protein of any of Embodiments 1 to 16, comprising an antibody heavy chain containing VH and an antibody light chain containing VL. 18. The isolated protein is an antibody, as in the isolated protein of Embodiment 17. 19. The isolated protein of Embodiment 18 is an IgG antibody. 20. The isolated protein of Embodiment 19, wherein the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. 21. The isolated protein is a bispecific antibody, an isolated protein of any of Embodiments 17-20. 22. The bispecific antibody comprises two arms, each arm of the antibody comprising a VH-VL pair having a set of charged amino acids in (a), (b), or (c), the isolated protein of Embodiment 21. 23. An isolated protein according to any of Embodiments 18 to 22, comprising a first human IgG CH3 domain (CH3) and a second human IgG CH3 domain (CH3'), wherein the CH3 domain includes amino acid substitutions by negatively charged amino acids at positions 392, 409, and 439, and the CH3' domain includes amino acid substitutions at positions 356 and 399, and the positional numbers of the charged amino acids in the CH3 and CH3' domains refer to positions according to the EU numbering scheme. 24. The antibody comprises a first heavy chain constant domain and a light chain constant domain pair (CH1 and CL), and a second heavy chain constant domain and a light chain constant domain pair (CH1' and CL'); CH1 and CH1' each include a modification at position 183, numbered according to the EU numbering scheme; CL and CL' include modifications at rank 176, as numbered according to the EU numbering scheme; The CH1 group contains positively charged amino acids at position 183 and the CL' group at position 176; The CH1'183 and CL176 positions each contain negatively charged amino acids. An isolated protein from any of embodiments 18 to 23. 25. An isolated protein of Embodiment 24, wherein (i) the CH1 183 position and CL' 176 position each contain lysine, and the CH1 183 position and CL176 position each contain aspartic acid; (ii) the CH1 183 position and CL' 176 position each contain lysine, and the CH1' 183 position and CL176 position each contain glutamic acid; (iii) the CH1 183 position and CL' 176 position each contain arginine, and the CH1' 183 position and CL176 position each contain aspartic acid; or (iv) the CH1 183 position and CL' 176 position each contain arginine, and the CH1' 183 position and CL176 position each contain glutamic acid. 26. It comprises a first heavy chain variable domain and light chain variable domain pair (VH and VL), and a second heavy chain variable domain and light chain variable domain pair (VH' and VL'), where the VH and VL pair and the VH' and VL' pair are the following sets of charged amino acids: a. VH and VH' each contain a charged amino acid at position 39; VL and VL' each contain a charged amino acid at position 85 that is complementary in charge to the amino acid at position 39 of VH and VH'; and VH and VH' are sets containing amino acids with complementary charges. b. VH and VH' each contain a charged amino acid at position 105; VL and VL' each contain a charged amino acid at position 42 that is complementary in charge to the amino acid at position 105 of VH and VH'; and VH and VH' are sets containing amino acids with complementary charges; or c.VH and VH' each contain a charged amino acid at position 91; VL and VL' each contain a charged amino acid at position 38 that is complementary in charge to the amino acid at position 91 of VH and VH'; and VH and VH' are sets containing amino acids with complementary charges. Includes at least one of the following; This is a bispecific antibody in which the positional numbers of charged amino acids within the VH and VL domains refer to positions according to the Kabat numbering scheme. 27. The bispecific antibody of Embodiment 26, comprising a VH and VL pair and a VH' and VL' pair, each containing the set of charged amino acids in (a). 28. A bispecific antibody of Embodiment 27, wherein VH and VL' contain positively charged amino acids, and VH' and VL also contain negatively charged amino acids. 29. The bispecific antibodies of Embodiment 28, wherein the VH and VL pairs and the VH' and VL' pairs contain: (i) lysine at VH39, lysine at VL'85, aspartic acid at VH'39, and aspartic acid at VL85; (ii) lysine at VH39, lysine at VL'85, glutamic acid at VH'39, and glutamic acid at VL85; (iii) arginine at VH39, arginine at VL'85, aspartic acid at VH'39, and aspartic acid at VL85; or (iv) arginine at VH39, arginine at VL'85, glutamic acid at VH'39, and glutamic acid at VL85. 30. A bispecific antibody of Embodiment 27, wherein VH and VL' contain negatively charged amino acids, and VH' and VL also contain positively charged amino acids. 31. The VH and VL pairs and the VH' and VL' pairs are: (i) aspartic acid at VH39, aspartic acid at VL'85, lysine at VH'39, and lysine at VL85; (ii) glutamic acid at VH39, glutamic acid at VL'85, lysine at VH'39, and lysine at VL85; (iii) aspartic acid at VH39, aspartic acid at VL'85, arginine at VH'39, and arginine at VL85; or (iv) glutamic acid at VH39, glutamic acid at VL'85, arginine at VH'39, and arginine at VL85, the bispecific antibodies of Embodiment 30. 32. A bispecific antibody according to any of Embodiments 25 to 31, comprising a VH and VL pair and a VH' and VL' pair, each containing a set of charged amino acids as in (b). 33. A bispecific antibody of Embodiment 32, wherein VH and VL' contain positively charged amino acids, and VH' and VL also contain negatively charged amino acids. 34. The bispecific antibodies of Embodiment 33, wherein the VH and VL pairs and the VH' and VL' pairs contain (i) lysine at VH105, lysine at VL'42, aspartic acid at VH'105, and aspartic acid at VL42; (ii) lysine at VH105, lysine at VL'42, glutamic acid at VH'105, and glutamic acid at VL42; (iii) arginine at VH105, arginine at VL'42, aspartic acid at VH'105, and aspartic acid at VL42; or (iv) arginine at VH105, arginine at VL'42, glutamic acid at VH'105, and glutamic acid at VL42. 35. A bispecific antibody of Embodiment 32, wherein VH and VL' contain negatively charged amino acids, and VH' and VL also contain positively charged amino acids. 36. The bispecific antibody of Embodiment 35, wherein the VH and VL pairs and the VH' and VL' pairs contain: (i) aspartic acid at VH105, aspartic acid at VL'42, lysine at VH'105, and lysine at VL42; (ii) glutamic acid at VH105, glutamic acid at VL'42, lysine at VH'105, and lysine at VL42; (iii) aspartic acid at VH105, aspartic acid at VL'42, arginine at VH'105, and arginine at VL42; or (iv) glutamic acid at VH105, glutamic acid at VL'42, arginine at VH'105, and arginine at VL42. 37. A bispecific antibody according to any of Embodiments 26 to 36, comprising a VH and VL pair and a VH' and VL' pair, each containing a set of charged amino acids in (c). 38. A bispecific antibody of Embodiment 37, wherein VH and VL' contain positively charged amino acids, and VH' and VL also contain negatively charged amino acids. 39. The VH and VL pairs and the VH' and VL' pairs are: (i) lysine at VH91, lysine at VL'38, aspartic acid at VH'91, and aspartic acid at VL38; (ii) lysine at VH91, lysine at VL'38, glutamic acid at VH'91, and glutamic acid at VL38; (iii) arginine at VH91, arginine at VL'38, aspartic acid at VH'91, and aspartic acid at VL38; or (iv) arginine at VH91, arginine at VL'38, glutamic acid at VH'91, and glutamic acid at VL38, the bispecific antibodies of Embodiment 38. 40. A bispecific antibody of Embodiment 37, wherein VH and VL' contain negatively charged amino acids, and VH' and VL also contain positively charged amino acids. 41. The VH and VL pairs and the VH' and VL' pairs are: (i) aspartic acid at VH91, aspartic acid at VL'38, lysine at VH'91, and lysine at VL38; (ii) glutamic acid at VH91, glutamic acid at VL'38, lysine at VH'91, and lysine at VL38; (iii) aspartic acid at VH91, aspartic acid at VL'38, arginine at VH'91, and arginine at VL38; or (iv) glutamic acid at VH91, glutamic acid at VL'38, arginine at VH'91, and arginine at VL38, the bispecific antibodies of Embodiment 40. 42. A method for generating a bispecific antibody, comprising: expressing one or more polynucleotides encoding a bispecific antibody according to any of Embodiments 26 to 41 in host cells; culturing the host cells in a culture medium under conditions that produce constituent polypeptide chains; and recovering the bispecific antibody from the cells or culture medium. 43. The method of Embodiment 42, wherein the host cell comprises one or more plasmids each containing one or more polynucleotides. 44. The method of Embodiment 42, wherein the host cell is a Chinese hamster ovary (CHO) cell line or a human embryonic kidney (HEK) cell line. 45. The recovered bispecific antibody is purified by protein A chromatography, according to the method of Embodiment 42. 46. ​​The recovered bispecific antibody is further purified by ion exchange chromatography, according to any of the methods of Embodiments 42 to 45. 47. A method for producing an isolated protein, comprising: expressing one or more polynucleotides encoding any of the proteins of Embodiments 1 to 25 in a host cell; culturing the host cell in a culture medium under conditions such that a constituent polypeptide chain is produced; and recovering the protein from the cell or culture medium. 48. The method of Embodiment 47, wherein the host cell comprises one or more plasmids each containing one or more polynucleotides. 49. The isolated protein is purified by ion exchange chromatography, as in the method of Embodiment 47 or Embodiment 48. [Examples]

[0106] Example 1: Analysis of Fv structure for candidate charge pair mutations Since the Fv interface can contain up to 50% of the total heavy-light interactions (Figure 1), we hypothesized that engineering solutions localized at the steady-state CH-CL interface may not be sufficient to attract / repel complete polypeptide chains. Therefore, to develop an improved method for facilitating pairing between specific desired heavy-chain and light-chain polypeptides, we undertook research efforts to identify potential locations within the heavy-chain variable region (VH) and light-chain variable region (VL) where charge pair mutations may be located. Generally, the objective was to identify novel pairing mutations that, when undesirable VH and VL combinations are bound (i.e., when VH and VL are mispaired), contribute to unstable high-energy states. Conversely, when desirable VH and VL combinations are bound, novel pairing mutations should contribute to stable low-energy states.

[0107] Figure 1A shows an exemplary bispecific antibody containing four different protein chains. Each arm of the antibody contains a light chain paired with a complementary heavy chain, and the CDR regions of each heavy chain and light chain pair combine to create a binding site for an antigen (antigen 1 or antigen 2 shown in Figure 1A). In such a bispecific antibody, there are four distinct interaction zones. First, the VH and VL regions interact via the Fv interface in each arm of the antibody. Second, the heavy chain constant domain 1 (CH1) and light chain constant domain (CL) form the CH1-CL interface. Third, the heavy chain constant domain 2 (CH2) in each heavy chain forms the CH2-CH2 interface. Fourth, the heavy chain constant domain 3 (CH3) in each heavy chain forms the CH3-CH3 interface. The CH2-CH2 and CH3-CH3 interfaces are responsible for holding the two arms of the antibody together, but they do not affect the pairing between each heavy chain and its corresponding light chain.

[0108] Figure 1B shows exemplary undesirable byproducts formed when an incorrect light chain is paired with one or both of the heavy chains of a bispecific antibody. These byproducts generally reduce the expression of the desired bispecific antibody by using protein chains that would normally be assembled into the desired product.

[0109] A set of known VH-VL interaction structures were used as a starting point for in silico analysis. An exemplary structure showing the interaction between the VH domain (SEQ ID NO: 1) and the VL domain (SEQ ID NO: 5) is shown in Figure 2A (showing the structure of anti-HIV antibody B12). This exemplary structure includes dotted lines between domains indicating distance-based interaction lines between atoms within the VH and VL domains, as calculated using PyMol. Each of the residues involved in these interactions was a candidate for a potential charge pair mutation.

[0110] In silico analysis was performed to identify novel sites for charge pair mutations. The purpose of this analysis was to identify sites where pairing mutations could occur, resulting in an unstable high-energy state in the undesirable “mispaired” state and a stable lower-energy state in the desired pairing pattern. Figure 2B illustrates this dynamic. In Figure 2B, the undesirable states ("mispaired antibody 1" and "mispaired antibody 2") contain unstable mispairs where similarly charged amino acid residues are forced into proximity, as indicated by paired open circles or paired filled circles. These unstable mispairs result in a high-energy state. In comparison, the desired state (antibody 1 or antibody 2) has a lower-energy state because oppositely charged residues are present at the same site. Therefore, the energy difference causes the antibody chain to assemble in an equilibrium state significantly superior to the desired pairing state. In Figure 2B, this is indicated by larger arrows pointing to antibody 1 and antibody 2.

[0111] Nineteen different amino acid residues within the VH domain were selected based on their presence at the VH-VL interface, and amino acid residues within the VL domain were selected for potential charge pairing mutations with each VH residue based on proximity. This yielded 1 to 9 residues selected to pair with each VH residue, and a total of 69 potential CPMs for in silico analysis.

[0112] As the first step in in silico analysis, each potential charge pair mutation identified at a selected location in Fv was designed and evaluated by Rosetta via XML scripts (RosettaScripts) using the talaris2014 scoring function. Asp, Glu, Lys, and Arg mutations were pairwise and minimized by cyclic coordinate descent of a 5-residue fragment containing two residues upstream and downstream of each mutation site. Fv chain pairs were generated by mutations in both correctly paired (+ / -) combinations and mispaired (+ / + and - / -) combinations. This process was repeated using the structures of 18 different Fv sequences combined in 54 different Fv pairs as input structures. Thus, using this strategy, thousands of pairing designs were screened to identify a subset of promising candidates for experimental validation. The desired mutations were selected based on the consistently large difference in total Rosetta scores between correctly paired and mispaired strands across all input structures, and the fact that the total Rosetta score for correctly paired combinations was as low as possible as a prediction of the Fv vs. energy state. An exemplary output is shown in the chart in Figure 2C. This shows that the energy state for one mispaired antibody was predicted to be much higher than the energy state of any of the correctly paired antibodies, while the predicted energy state for the other mispaired antibody was somewhat higher than that of any of the correctly paired antibodies.

[0113] In silico analysis predicted that several CPM designs would function well across various different Fv frameworks. In addition to the previously reported VH:39-VL:38 design (Lewis, SM, et al., Nat Biotechnol, 32(2):191-198 (2014)), several additional pair formations resulting in stable interfaces were identified. Specifically, the introduction of complementary charge mutations at sites VH:39-VL:85, VH:105-VL:42, and VH:91-VL:38 (numbered according to the Kabat scheme) was predicted to function consistently well, regardless of the Fv framework analyzed. The locations of these newly identified CPMs are shown in the exemplary VH and VL sequences in Figures 7A and 7B, which originate from the anti-HIV antibody B12. In Figures 7A and 7B, the VH and VL sequences are annotated by Kabat numbering at the residue level and at the CDR and framework region locations. Furthermore, Figures 7A and 7B include boxes highlighting the amino acids that would be modified by introducing charge pair mutations at VH:39-VL:85 (CPM vs. A), VH:105-VL:42 (CPM vs. B), or VH:91-VL:38 (CPM vs. C).

[0114] Example 2: Plasmid construction, cell culture, and protein expression Unless otherwise specified, plasmid construction, cell culture, plasmid transfection, protein expression, and antibody purification, as referenced in subsequent examples, were performed as described in this example.

[0115] Plasmid Construction: The genes to be expressed as polypeptides were synthesized using Twist Bioscience. The genes were individually cloned into vectors for mammalian transient expression using the Golden Gate assembly method (see Engler C et al., PLoS One 3:e3647 (2008) (the entire work is incorporated herein by reference)). All strands containing the Fc region were constructed using a human IgG1 scaffold (IgG1-SEFL2) with non-glycosylating mutations and manipulated disulfide bonds (see Estes et al., iScience 24:103447 (2021); and Jacobsen FW et al., J. Biol. Chem. 292:1865-75 (2017) (the entire works are incorporated herein by reference)). All molecules requiring hetero-Fc pair formation contained a charge pair mutation ("CPM") v103 within the CH3 region to promote heterodimerization (see Estes et al., iScience 24:103447 (2021)). Briefly, the v103 mutation pattern includes the 392D, 409D, and 439D mutations in one heavy chain and the 356K and 399K mutations in the other heavy chain (numbered according to the EU antibody numbering scheme). Other CPMs were also included within the heavy and light chain sequences shown below.

[0116] After synthesis and cloning, the plasmid sequence was confirmed by Sanger assay, and transfection-grade DNA was prepared using the Maxi plasmid purification kit (Qiagen).

[0117] Cell culture, plasmid transfection, and protein expression: Proteins were transiently expressed in suspended human embryonic kidney 293-6E cells (NRC-BRI) using PEImax transfection reagent. Prior to transfection, cells were cultured in FreeStyle F-17 medium (Gibco, catalog #A1383502) containing 0.1% Kolliphor P188 (Sigma, catalog #K4894), 25 μg / ml G418 (Gibco, catalog #10131027), and 6 mM L-glutamine (Gibco, catalog #25030149). Transfection yielded 2 × 10⁶ cells per 1 mL. 6 To achieve the optimal viable cell density, cells were passaged 26 hours prior to transfection. The purified plasmid was mixed with the 4-stranded hetero-IgG to be tested in a 1:1:1:1 ratio. 0.5 μg of DNA was added to 1 ml of cell medium after incubation for 10 minutes with 1.5 μl of PEImax reagent (Polysciences, catalog #24765-2) in 100 μl of FreeStyle F-17 medium. 24 hours after transfection, the cell culture was fed with tryptone N1 solution (Organotechnie, catalog #19553) and glucose (Thermo Fisher, catalog #A2494001) to final concentrations of 2.5 g / L and 4.5 g / L, respectively. Seven days after transfection, the conditioned medium was collected for purification.

[0118] Protein A and antibody purification by cation exchange chromatography: After collecting 40 mL of culture, the conditioned medium was purified using a protein A affinity capture system (1 ml HiTrap MabSelect SuRe, GE Life Sciences, catalog #GE11-0034-93) equilibrated with 25 mM Tris, 100 mM NaCl, pH 7.4. The protein was eluted with 100 mM sodium acetate, pH 3.6, and immediately afterward, the buffer was exchanged to 10 mM sodium acetate, 150 mM NaCl, pH 5.2 using a 5 ml HiTrap desalting column (GE Life Sciences, catalog #GE17-1408-01).

[0119] Cation exchange chromatography (CIEX) was performed to analyze and separate the antibody species present in the purified protein A. The protein A eluate was diluted with 20 mL of 20 mM MES, pH 6.2, and loaded onto a 1 mL cation exchange column (HiTrap SP-HP, GE Life Sciences, catalog #GE29-0513-24) at a rate of 1 mL / min. After washing with 8 column volumes of dilution buffer at 1 mL / min, the sample was eluted by a linear gradient of 0–400 mM NaCl over 40 column volumes at 0.4 mL / min.

[0120] To identify the species within the fractions collected during CIEX, or to analyze the accuracy of the samples, the samples were analyzed by non-reducing microcapillary electrophoresis (MCE) and analytical size exclusion chromatography (SEC). For non-reducing MCE, 6 μl of protein was mixed with 21 μl of sample buffer (8.4 mM Tris-HCl pH 7.0, 7.98% glycerol, 2.38 mM EDTA, 2.8% SDS, and 2.4 mM iodoacetamide), heated at 85°C for 10 minutes, and then analyzed using a Caliper LabChip GXII Touch instrument (PerkinElmer). For analytical SEC, protein samples were analyzed using an Acquity HPLC instrument (Waters) with a Zenix-C column (300 Å, 3 microns, 4.6 × 300 mm) (Sepax Technologies) and 100 mM sodium phosphate, pH 6.8, and 250 mM NaCl as running buffer at a flow rate of 0.45 ml / min. Fraction pool concentrations were determined using a Nanodrop 8000 (Thermo Fisher).

[0121] Example 3: Analysis of contaminant species when generating antibodies containing charged pair mutations Antibodies containing complementary charged mutations at sites VH:91-VL:38, VH:39-VL:85, and VH:105-VL:42 were tested in bispecific antibodies, regardless of the presence of other CPMs in the antibody's Fab region. Each set of complementary mutations was tested against a background of IgG1 antibodies containing a known set of charged pair mutations in the heavy chain Fc region. This background allowed for testing of novel complementary mutations for heavy-light chain binding only, with reduced contamination due to heavy chain mispairing. In addition, some test antibodies contained charged pair mutations within the CH1-CL interface. The tested antibodies, along with the CPMs present in each of their domains, are listed in Table 1 below. Therefore, all antibodies listed in Table 1 are IgG1 antibodies, the only differences between them being the CDR region (i.e., altering the specificity to bind to antigen A or antigen C) and the listed charged pair mutations.

[0122] [Table 1]

[0123] [Table 2]

[0124] Constructs for expressing each of the bispecific antibodies in Table 1 were prepared according to the method described in Example 2. Briefly, for the expression of these antibodies, four antibody chains for expressing a given antibody were transfected into HEK293 cells in a ratio of 1:1:1:1 (as also described in Example 2). Seven days after transfection, conditioned medium was collected for the purification of secreted antibodies. Initial purification was achieved using protein A affinity capture. Subsequently, antibody species were separated using cation exchange chromatography. Figures 3A–3E, 4A–4C, and 5 show chromatographs of the separation of the purified antibody samples. As part of the CIEX chromatography process, individual fractions eluted from the CIEX column were sequentially collected and analyzed to determine the antibody species present in each major peak.

[0125] The chromatographs in Figures 3A–3E are from the testing of five different bispecific antibody variants, all of which target two antigens commonly referred to as antigen A and antigen C (see Table 1 above). To distinguish the two arms of these bispecific antibodies, the domain in the peptide chain responsible for binding antigen C is indicated by a prime symbol ('), while the domain responsible for binding antigen A lacks such notation. Each of Figures 3A–3E further provides a list of charge pair mutations present in the bispecific antibodies that will be produced. Except for the differences between the listed CPM modifications, the bispecific antibodies in Figures 3A–3E are sequence-identical. In Figures 3A–3E, the location of the peak containing a particular antibody species is indicated by a diagram of that species and an arrow pointing to the associated peak. Additionally, the desired bispecific antibody in each chromatograph is highlighted by a box surrounding its peak. The bispecific antibody variants tested in Figures 3A to 3E were designed to allow testing of the efficacy of CPM at a newly identified VH:91-VL:38 site that prevents antibody chain mispairing.

[0126] Figure 3A shows a CIEX chromatogram of secreted antibodies from cells expressing the components of "Bispecific Antibody 1". Bispecific Antibody 1 contains CPM only in its Fc region, particularly in residues that are part of the heavy chain CH3-CH3' interface. Specifically, its CH3 domain contains modifications 392D, 409D, and 439D, while its CH3' domain contains modifications 356K and 399K (numbered according to the EU antibody numbering scheme). In addition to the peak corresponding to Bispecific Antibody 1, Figure 3A contains peaks corresponding to at least three contaminant species. The desired bispecific antibody peak is the largest in Figure 3A, while a considerable amount of surrogate contaminant antibody species are also present.

[0127] Figure 3B shows the CIEX chromatogram of secreted antibodies from cells expressing the components of "Bispecific Antibody 2". Bispecific Antibody 2 is identical to Bispecific Antibody 1, except that Bispecific Antibody 2 also contains CPM in the CH1-CL and CH1'-CL' interfaces. The additional CPM consists of modifications of 183K in the CH1 domain and 176K in the CL domain, in parallel with modifications of 183K in the CH1' domain and 176E in the CL' domain. In both Figure 3A and Figure 3B, the most prominent peak is the desired bispecific antibody. Both also contain peaks indicating alternative contaminants. The contamination peak for Bispecific Antibody 2 (Figure 3B) is located between approximately 108 and 115 mL, which is lower than that for Bispecific Antibody 1 (Figure 3A). However, the expression and purification of antibodies containing CPM in both the Fc interface and the CH1 interface still resulted in significant contamination.

[0128] To determine whether adding the newly identified CPM to the Fv interface (see Example 1 above) can reduce the presence of undesirable contaminants during the production of bispecific antibodies, "Bispecific Antibody 3" was constructed. Bispecific Antibody 3 is identical to Bispecific Antibody 2, except that Bispecific Antibody 3 also contains CPM in the VH-VL and VH'-VL' interfaces. Specifically, Bispecific Antibody 3 contains CPM at the VH:39-VL:85 sites of each arm of the antibody. These CPMs include modifications of 39R in the VH' domain and 85E in the VL' domain, as well as modifications of 39E in the VH domain and 85R in the VL domain. Figure 3C shows a CIEX chromatogram of secreted antibodies from cells expressing the components of Bispecific Antibody 3. In Figure 3C, the most prominent peak is the desired bispecific antibody. Between approximately 108-115 mL (where contamination was detected for bispecific antibody 2), little to no contaminating antibodies were detected for bispecific antibody 3. A comparison of Figures 3B and 3C shows that the introduction of CPM at the VH:39-VL:85 site in each arm resulted in the elimination of almost all contaminants observed in the purified antibody sample of bispecific antibody 2. This indicates that CPM at the VH:39-VL:85 site can lead to more effective and complete pair formation of the antibody chain, and can improve pair formation even in the presence of other CPMs in the antibody.

[0129] Furthermore, "Bispecific antibody 4" was constructed by combining the CPM in the VH:39-VL:85 region with the CPM in the CH3-CH3' interface. The heavy chain CH3-CH3' interface in bispecific antibody 4 contains modifications 392D, 409D, and 439D in the CH3 domain, while the CH3' domain contains modifications 356K and 399K (numbered according to the EU antibody numbering scheme). Bispecific antibody 4 is identical to bispecific antibody 3, except that bispecific antibody 4 lacks any CPM in its CH1-CL interface. Bispecific antibody 4 differs from bispecific antibody 2 in that bispecific antibody 4 has a CPM in its VH-VL interface, while bispecific antibody 2 has a CPM in its CH1-CL interface. Figure 3D shows the CIEX chromatogram of secreted antibodies from cells expressing the components of bispecific antibody 4. In Figure 3D, the most prominent peak is the desired bispecific antibody. Figure 3D shows that bispecific antibody 4 contained fewer contaminants at approximately 108-115 mL than the antibody containing only CPM at the CH3-CH3' interface (as shown by comparison with the chromatograph of bispecific antibody 2 in Figure 3A). Furthermore, bispecific antibody 4 showed a different pattern of contaminants than bispecific antibody 2. This suggests that CPM at the VH-VL interface, and particularly at the VH:39-VL:85 site, has a complementary effect to that at the CH1-CL interface.

[0130] To enable comparison of CPMs with the 91-VL:38 format with previously known CPMs with the VH:39-VL:38 format, "Bispecific antibody 5" was constructed and tested. Bispecific antibody 5 was constructed by comparing CPMs in the VH:39-VL:38 region with CPMs in the CH3-CH3' interface. Similar to bispecific antibodies 1-4, the CH3 domain of bispecific antibody 5 contains modifications 392D, 409D, and 439D, while its CH3' domain contains modifications 356K and 399K (numbered according to the EU antibody numbering scheme). Similar to bispecific antibodies 2 and 3, bispecific antibody 5 contains CPMs in the CH1-CL and CH1'-CL' interfaces, including modifications 183E in the CH1 domain and 176K in the CL domain, in parallel with modifications 183K in the CH1' domain and 176E in the CL' domain. Therefore, the comparison between bispecific antibody 3 and bispecific antibody 5 provides evidence of the relative effect of VH:91-VL:38 CPM compared to VH:39-VL:38 CPM. Figure 3E shows the CIEX chromatogram of secreted antibodies from cells expressing the components of "bispecific antibody 5". A comparison of Figure 3E with the results in Figure 3C using bispecific antibody 3 shows that less contaminant is present in Figure 3C, between approximately 108 and 115 mL. This indicates that, in the presence of otherwise identical bispecific antibodies, VH:91-VL:38 CPM can drive selective pairing more effectively than VH:39-VL:38 CPM.

[0131] The chromatographs in Figures 4A–4C are from the testing of three different bispecific antibody variants, all of which target two antigens commonly referred to as antigen B and antigen D (see Table 1 above). To distinguish the two arms of these bispecific antibodies, the domain in the peptide chain responsible for binding antigen D is indicated by a prime symbol ('), while the domain responsible for binding antigen B lacks such notation. Each of Figures 4A–4C further provides a list of charge pair mutations present in the bispecific antibody that will be produced. Except for the differences between the listed CPM modifications, the bispecific antibodies in Figures 4A–4C are sequence-identical. In Figures 4A–4C, the location of the peak containing the desired antibody is indicated by the antibody diagram and an arrow pointing to the associated peak. Additionally, the desired bispecific antibody in each chromatograph is highlighted by a box surrounding its peak. The bispecific antibody variants tested in Figures 4A to 4C were designed to allow testing of the efficacy of CPM at a newly identified VH:105-VL:42 site, which prevents antibody chain mispairing.

[0132] Figure 4A shows a CIEX chromatogram of secreted antibodies from cells expressing the components of "Bispecific Antibody 6". Bispecific Antibody 6 contains CPM only in its Fc region, particularly in residues that are part of the heavy chain CH3-CH3' interface. Specifically, its CH3 domain contains modifications 392D, 409D, and 439D, while its CH3' domain contains modifications 356K and 399K (numbered according to the EU antibody numbering scheme). In addition to the peak corresponding to Bispecific Antibody 6, Figure 4A also contains peaks corresponding to contaminant species at approximately 86-89 mL and on the shoulder of the desired peak at approximately 92-93 mL. The desired bispecific antibody peak is the largest in Figure 4A, while a considerable amount of contaminant surrogate antibody species is also present.

[0133] Figure 4B shows the CIEX chromatogram of secreted antibodies from cells expressing the components of "Bispecificity Antibody 7". Bispecificity Antibody 7 is identical to Bispecificity Antibody 6, except that Bispecificity Antibody 7 also contains CPM in the CH1-CL and CH1'-CL' interfaces. The additional CPM consists of modifications of 183E in the CH1 domain and 176K in the CL domain, in parallel with modifications of 183K in the CH1' domain and 176E in the CL' domain. In both Figure 4A and Figure 4B, the most prominent peak is that of the desired bispecificity antibody. The addition of CPM to the CH1-CL and CH1'-CL' interfaces resulted in the removal of the vicinity of the peak eluted before the desired species in Figure 4B, where the prominent peak was observed in Figure 4A. However, Figure 4B shows a peak indicating a contaminant alternative species above the shoulder of the desired peak at approximately 92-93 mL (also observed in Figure 4A). Therefore, the expression and purification of antibodies containing CPM at both the Fc interface and the CH1-CL interface still resulted in significant contamination.

[0134] To determine whether adding the newly identified CPM to the Fv interface (see Example 1 above) can reduce the presence of undesirable contaminants during the production of bispecific antibodies, "Bispecific Antibody 8" was constructed. Bispecific Antibody 8 is identical to Bispecific Antibody 7, except that Bispecific Antibody 8 also contains CPM in the VH-VL and VH'-VL' interfaces. Specifically, Bispecific Antibody 8 contains CPM at the VH:105-VL:42 sites of each arm of the antibody. These CPMs include modifications of 105R in the VH domain and 42E in the VL domain, in parallel with modifications of 105E in the VH' domain and 42R in the VL' domain. Figure 4C shows a CIEX chromatogram of secreted antibodies from cells expressing the components of Bispecific Antibody 8. In Figure 4C, the most prominent peak is the desired bispecific antibody. As observed for bispecific antibody 7 in Figure 4B, CPM of bispecific antibody 8 resulted in the removal of the vicinity of the peak eluted before the desired species in Figure 4C. A comparison of Figures 4B and 4C shows that the introduction of CPM at the VH:105-VL:42 site in each arm resulted in a reduction in the size of the contaminating peak on the shoulder of the desired peak at approximately 92-93 mL. This indicates that CPM at the VH:105-VL:42 site can lead to more effective and complete pair formation of the antibody chain and can improve pair formation even in the presence of other CPMs in the antibody.

[0135] The chromatograph in Figure 5 is from the testing of a bispecific antibody called "Bispecific Antibody 9," which targets two antigens commonly referred to as antigen E and antigen F (see Table 1 above). To distinguish the two arms of the bispecific antibody, the domain in the peptide chain responsible for binding antigen F is indicated by a prime symbol ('), while the domain responsible for binding antigen E lacks such notation. Figure 5 further provides a list of charge pair mutations present in bispecific antibody 9. In Figure 5, the location of the peak containing bispecific antibody 9 is indicated by the antibody diagram and an arrow pointing to the associated peak. Additionally, the desired bispecific antibody in each chromatograph is highlighted by a box surrounding its peak. The tested bispecific antibody variants in Figure 5 were designed to test the effectiveness of a newly identified CPM at the VH:91-VL:38 site in preventing antibody chain mispairing. Specifically, bispecific antibody 9 contains CPM at the VH:91-VL:38 site on each arm of the antibody. These CPMs include modifications to 91R in the VH domain and 38E in the VL domain, in parallel with modifications to 91E in the VH' domain and 38R in the VL' domain. As shown in Figure 5, the inclusion of CPMs at the VH:91-VL:38 site resulted in highly effective pairing of the antibody chain with a low amount of contaminating mispairing antibody species.

[0136] Example 4: Production level of antibodies containing charged pair mutations Furthermore, experiments were conducted to test whether the inclusion of charge pair mutations in the VH:VL interface could lead to increased production and purification of desirable antibody species. The antibodies tested, along with the CPM present in each of their domains, are listed in Table 2 below. All antibodies listed in Table 2 are IgG1 antibodies, and the only differences between them lie in the CDR region (i.e., altering the specificity for binding to antigen A or antigen C) and the listed charge pair mutations.

[0137] [Table 3]

[0138] [Table 4]

[0139] Plasmids encoding the antibodies shown in Table 2 were constructed as described in Example 2 and expressed in HEK 293-6E cells. For monospecific antibodies, the plasmid was transfected in a 1:1 ratio of heavy-chain plasmid to light-chain plasmid, while four bispecific plasmids were transfected in a 1:1:1:1 ratio. Seven days after transfection, conditioned medium was collected for the purification of secreted antibodies. Initial purification was achieved using protein A affinity capture. Subsequently, antibody species were isolated using cation exchange chromatography. As part of the CIEX chromatography process, individual 1 ml fractions eluted from the CIEX column were sequentially collected and analyzed to determine the antibody species present in each major peak. Fractions containing the desired antibodies were pooled and quantified by A280 measurement.

[0140] Figure 6 shows the amount of pooled antibody recovered per transfection. The anti-A antibody and anti-C antibody tests serve as controls for the amount of antibody recovered when expressing and purifying unmodified IgG1 antibody. After purification, approximately 33 mg / L and 43 mg / L of antibody A and antibody C were recovered, respectively. Expression and purification of bispecific antibody 1 (containing Fc CPM but not Fab CPM) resulted in a recovery of 20 mg / L of antibody. This decrease was likely due to a significant proportion of the expressed antibody chain being used as part of unrecovered contaminants. Next, the effect of adding a single charge pair mutation within the Fab region (parallel to Fc CMP) was tested by expressing either bispecific antibody 2 (CH1:183-CL:176) or bispecific antibody 4 (VH:39-VL:85). Including either of these CPMs increased antibody recovery to levels equivalent to or better than either parent antibody. Bispecific antibody 2 was recovered at approximately 43 mg / L, and bispecific antibody 4 was recovered at approximately 45 mg / L.

[0141] Furthermore, the effect of combining multiple CPMs within the Fab region on antibody production was tested (see Figure 6). Both bispecific antibody 3 (CH1:183-CL:176 plus VH:39-VL:85) and bispecific antibody 4 (CH1:183-CL:176 plus VH:39-VL:38) showed increased recovery compared to all other antibodies tested. Bispecific antibody 3 was recovered at approximately 55 mg / L, and bispecific antibody 4 was recovered at approximately 54 mg / L. This represents a 27% increase in production compared to anti-C antibody and a 65% increase compared to anti-A antibody for these two bispecific antibodies.

[0142] The data in Figure 6 demonstrates that, in addition to reducing the number and amount of contaminant species present during antibody production, the newly discovered CPM at the VH-VL interface promotes an increase in antibody production levels.

Claims

1. An isolated protein comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH and VL are bound to each other, and the VH and VL are the following set of charged amino acids: a. The set VH includes a charged amino acid at position number 39, and VL includes a charged amino acid at position number 85 that is complementary in charge to the amino acid at position 39 of VH; b. The set in which VH contains a charged amino acid at position number 105, and VL contains a charged amino acid at position number 42 that is complementary in charge to the amino acid at position 105 of VH; or c. The set VH includes a charged amino acid at position number 91, and VL includes a charged amino acid at position number 38 that is complementary in charge to the amino acid at position 91 of VH; Includes at least one of the following: The isolated protein wherein the positional numbers of the charged amino acids within the VH and VL domains refer to positions according to the Kabat numbering scheme.

2. The isolated protein according to claim 1, wherein VH and VL include the set of charged amino acids in (a).

3. The isolated protein according to claim 2, wherein VH contains an amino acid positively charged at position 39, and VL contains an amino acid negatively charged at position 85.

4. The isolated protein according to claim 3, wherein the VH39 position (VH39) and VL85 position (VL85) contain: (i) lysine at VH39 and aspartic acid at VL85, (ii) lysine at VH39 and glutamic acid at VL85, (iii) arginine at VH39 and aspartic acid at VL85, or (iv) arginine at VH39 and glutamic acid at VL85.

5. The isolated protein according to claim 2, wherein VH contains an amino acid negatively charged at position 39, and VL contains an amino acid positively charged at position 85.

6. The isolated protein according to claim 5, wherein the VH39 position (VH39) and VL85 position (VL85) contain: (i) aspartic acid at VH39 and lysine at VL85, (ii) glutamic acid at VH39 and lysine at VL85, (iii) aspartic acid at VH39 and arginine at VL85, or (iv) glutamic acid at VH39 and arginine at VL85.

7. The isolated protein according to any one of claims 1 to 6, wherein VH and VL include the set of charged amino acids in (b).

8. The isolated protein according to claim 7, wherein VH contains an amino acid positively charged at position 105, and VL contains an amino acid negatively charged at position 42.

9. The isolated protein according to claim 8, wherein the VH105 position (VH105) and VL42 position (VL42) contain: (i) lysine at VH105 and aspartic acid at VL42, (ii) lysine at VH105 and glutamic acid at VL42, (iii) arginine at VH105 and aspartic acid at VL42, or (iv) arginine at VH105 and glutamic acid at VL42.

10. The isolated protein according to claim 7, wherein VH contains an amino acid negatively charged at position 105, and VL contains an amino acid positively charged at position 42.

11. The isolated protein according to claim 10, wherein the VH105 position (VH105) and VL42 position (VL42) contain: (i) aspartic acid at VH105 and lysine at VL42, (ii) glutamic acid at VH105 and lysine at VL42, (iii) aspartic acid at VH105 and arginine at VL42, or (iv) glutamic acid at VH105 and arginine at VL42.

12. The isolated protein according to any one of claims 1 to 11, wherein VH and VL include the set of charged amino acids in (c).

13. The isolated protein according to claim 12, wherein VH contains an amino acid positively charged at position 91, and VL contains an amino acid negatively charged at position 38.

14. The isolated protein according to claim 13, wherein the VH91 position (VH91) and VL38 position (VL38) contain: (i) lysine at VH91 and aspartic acid at VL38, (ii) lysine at VH91 and glutamic acid at VL38, (iii) arginine at VH91 and aspartic acid at VL38, or (iv) arginine at VH91 and glutamic acid at VL38.

15. The isolated protein according to claim 12, wherein VH contains an amino acid negatively charged at position 91, and VL contains an amino acid positively charged at position 38.

16. The isolated protein according to claim 15, wherein the VH91 position (VH91) and the VL38 position (VL38) contain: (i) aspartic acid at VH91 and lysine at VL38, (ii) glutamic acid at VH91 and lysine at VL38, (iii) aspartic acid at VH91 and arginine at VL38, or (iv) glutamic acid at VH91 and arginine at VL38.

17. The isolated protein according to any one of claims 1 to 16, wherein the isolated protein comprises an antibody heavy chain containing VH and an antibody light chain containing VL.

18. The isolated protein according to claim 17, wherein the isolated protein is an antibody.

19. The isolated protein according to claim 18, wherein the antibody is an IgG antibody.

20. The isolated protein according to claim 19, wherein the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.

21. The isolated protein according to any one of claims 17 to 20, wherein the isolated protein is a bispecific antibody.

22. The isolated protein according to claim 21, wherein the bispecific antibody comprises two arms, and each arm of the antibody comprises a VH-VL pair having the set of charged amino acids in (a), (b), or (c).

23. The isolated protein according to any one of claims 18 to 22, wherein the antibody comprises a first human IgG CH3 domain (CH3) and a second human IgG CH3 domain (CH3'), the CH3 domain comprising amino acid substitutions by negatively charged amino acids at positions 392, 409, and 439, the CH3' domain comprising amino acid substitutions at positions 356 and 399, and the position numbers of the charged amino acids in the CH3 and CH3' domains refer to positions according to the EU numbering scheme.

24. The antibody comprises a first heavy chain constant domain and a light chain constant domain pair (CH1 and CL), and a second heavy chain constant domain and a light chain constant domain pair (CH1' and CL'); CH1 and CH1' each include a modification at position 183, which is assigned according to the EU numbering scheme; The aforementioned CL and CL' include modifications at position 176, which are numbered according to the EU numbering scheme; The CH1 group contains positively charged amino acids at positions 183 and CL' at position 176; The isolated protein according to any one of claims 18 to 23, wherein the CH1'183 position and the CL176 position each contain a negatively charged amino acid.

25. (i) the CH1 183 position and the CL' 176 position each contain lysine, and the CH1 183 position and the CL' 176 position each contain aspartic acid; (ii) the CH1 183 position and the CL' 176 position each contain lysine, and the CH1' 183 position and the CL' 176 position each contain glutamic acid; (iii) the CH1 183 position and the CL' 176 position each contain arginine, and the CH1' 183 position and the CL' 176 position each contain aspartic acid; or (iv) the CH1 183 position and the CL' 176 position each contain arginine, and the CH1' 183 position and the CL' 176 position each contain glutamic acid, according to claim 24.

26. It comprises a first heavy chain variable domain and light chain variable domain pair (VH and VL), and a second heavy chain variable domain and light chain variable domain pair (VH' and VL'), wherein the VH and VL pair and the VH' and VL' pair are the following set of charged amino acids: a. VH and VH' each contain a charged amino acid at position number 39; VL and VL' each contain a charged amino acid at position number 85 that is complementary in charge to the amino acid at position 39 of VH and VH'; and VH and VH' are sets containing amino acids with complementary charges; b. VH and VH' each contain a charged amino acid at position number 105; VL and VL' each contain a charged amino acid at position number 42 that is complementary in charge to the amino acid at position 105 of VH and VH'; and VH and VH' are sets containing amino acids with complementary charges; or c. VH and VH' each contain a charged amino acid at position number 91; VL and VL' each contain a charged amino acid at position number 38 that is complementary in charge to the amino acid at position 91 of VH and VH'; and VH and VH' are sets containing amino acids with complementary charges. Including at least one of the following: A bispecific antibody in which the positional numbers of the charged amino acids within the VH and VL domains refer to positions according to the Kabat numbering scheme.

27. The bispecific antibody according to claim 26, comprising the VH and VL pairs and the VH' and VL' pairs in (a), wherein the set of charged amino acids in (a).

28. The bispecific antibody according to claim 27, wherein VH and VL' contain positively charged amino acids, and VH' and VL contain negatively charged amino acids.

29. The bispecific antibody according to claim 28, wherein the VH and VL pair and the VH' and VL' pair comprise: (i) lysine at VH39, lysine at VL'85, aspartic acid at VH'39, and aspartic acid at VL85; (ii) lysine at VH39, lysine at VL'85, glutamic acid at VH'39, and glutamic acid at VL85; (iii) arginine at VH39, arginine at VL'85, aspartic acid at VH'39, and aspartic acid at VL85; or (iv) arginine at VH39, arginine at VL'85, glutamic acid at VH'39, and glutamic acid at VL85.

30. The bispecific antibody according to claim 27, wherein VH and VL' contain negatively charged amino acids, and VH' and VL contain positively charged amino acids.

31. The bispecific antibody according to claim 30, wherein the VH and VL pair and the VH' and VL' pair comprise: (i) aspartic acid at VH39, aspartic acid at VL'85, lysine at VH'39, and lysine at VL85; (ii) glutamic acid at VH39, glutamic acid at VL'85, lysine at VH'39, and lysine at VL85; (iii) aspartic acid at VH39, aspartic acid at VL'85, arginine at VH'39, and arginine at VL85; or (iv) glutamic acid at VH39, glutamic acid at VL'85, arginine at VH'39, and arginine at VL85.

32. The bispecific antibody according to any one of claims 25 to 31, comprising the VH and VL pair and the VH' and VL' pair in (b), wherein the set of charged amino acids in (b).

33. The bispecific antibody according to claim 32, wherein VH and VL' contain positively charged amino acids, and VH' and VL contain negatively charged amino acids.

34. The bispecific antibody according to claim 33, wherein the VH and VL pair and the VH' and VL' pair each contain (i) lysine at VH105, lysine at VL'42, aspartic acid at VH'105, and aspartic acid at VL42; (ii) lysine at VH105, lysine at VL'42, glutamic acid at VH'105, and glutamic acid at VL42; (iii) arginine at VH105, arginine at VL'42, aspartic acid at VH'105, and aspartic acid at VL42; or (iv) arginine at VH105, arginine at VL'42, glutamic acid at VH'105, and glutamic acid at VL42.

35. The bispecific antibody according to claim 32, wherein VH and VL' contain negatively charged amino acids, and VH' and VL contain positively charged amino acids.

36. The bispecific antibody according to claim 35, wherein the VH and VL pair and the VH' and VL' pair comprise: (i) aspartic acid at VH105, aspartic acid at VL'42, lysine at VH'105, and lysine at VL42; (ii) glutamic acid at VH105, glutamic acid at VL'42, lysine at VH'105, and lysine at VL42; (iii) aspartic acid at VH105, aspartic acid at VL'42, arginine at VH'105, and arginine at VL42; or (iv) glutamic acid at VH105, glutamic acid at VL'42, arginine at VH'105, and arginine at VL42.

37. The bispecific antibody according to any one of claims 26 to 36, comprising the VH and VL pair and the VH' and VL' pair in (c) the set of charged amino acids.

38. The bispecific antibody according to claim 37, wherein VH and VL' contain positively charged amino acids, and VH' and VL contain negatively charged amino acids.

39. The bispecific antibody according to claim 38, wherein the VH and VL pair and the VH' and VL' pair comprise: (i) lysine at VH91, lysine at VL'38, aspartic acid at VH'91, and aspartic acid at VL38; (ii) lysine at VH91, lysine at VL'38, glutamic acid at VH'91, and glutamic acid at VL38; (iii) arginine at VH91, arginine at VL'38, aspartic acid at VH'91, and aspartic acid at VL38; or (iv) arginine at VH91, arginine at VL'38, glutamic acid at VH'91, and glutamic acid at VL38.

40. The bispecific antibody according to claim 37, wherein VH and VL' contain negatively charged amino acids, and VH' and VL contain positively charged amino acids.

41. The bispecific antibody according to claim 40, wherein the VH and VL pair and the VH' and VL' pair comprise: (i) aspartic acid at VH91, aspartic acid at VL'38, lysine at VH'91, and lysine at VL38; (ii) glutamic acid at VH91, glutamic acid at VL'38, lysine at VH'91, and lysine at VL38; (iii) aspartic acid at VH91, aspartic acid at VL'38, arginine at VH'91, and arginine at VL38; or (iv) glutamic acid at VH91, glutamic acid at VL'38, arginine at VH'91, and arginine at VL38.

42. A method for producing a bispecific antibody, comprising: expressing one or more polynucleotides encoding the bispecific antibody according to any one of claims 26 to 41 in a host cell; culturing the host cell in a culture medium under conditions that produce constituent polypeptide chains; and recovering the bispecific antibody from the cell or the culture medium.

43. The method according to claim 42, wherein the host cell comprises one or more plasmids each containing one or more polynucleotides.

44. The method according to claim 42, wherein the host cells are a Chinese hamster ovary (CHO) cell line or a human embryonic kidney (HEK) cell line.

45. The method according to claim 42, wherein the recovered bispecific antibody is purified by protein A chromatography.

46. The method according to any one of claims 42 to 45, wherein the recovered bispecific antibody is further purified by ion exchange chromatography.

47. A method for producing an isolated protein, comprising: expressing one or more polynucleotides encoding the protein according to any one of claims 1 to 25 in a host cell; culturing the host cell in a culture medium under conditions such that a constituent polypeptide chain is produced; and recovering the protein from the cell or the culture medium.

48. The method according to claim 47, wherein the host cell comprises one or more plasmids each containing one or more polynucleotides.

49. The method according to claim 47 or 48, wherein the isolated protein is purified by ion exchange chromatography.

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