LOAD PAIR MUTALIZATIONS TO ENABLE CORRECT HEAVY-LIGHT CHAIN ​​MATCHING

Abstract

This application relates to facilitating the selective binding between modified VH and VL domains by introducing amino acids with complementary charges into the two domains at novel positions within these domains. These modifications are particularly useful in the design and generation of multispecific antibodies, such as bispecific antibodies, where charge-pair mutations in VH and VL domains can facilitate desired pairing between specific heavy- and light-chain polypeptides. These charge-pair mutations can be used alone or in combination with additional strategies to promote correct pairing of polypeptide chains.

Description

REFERENCE TO THE ELECTRONICALLY SUBMISSIVED SEQUENCE LIST

[0001] The present application contains a sequence list, which was submitted electronically in XML format and is hereby incorporated in its entirety by reference. The copy of the sequence list in computer-readable format, created on June 5, 2024, is named 10572-WO01-SEC_ST26 and is 8.69 kilobytes in size. TECHNICAL AREA

[0002] The disclosure relates to the production of multispecific antibodies. For example, the disclosure relates to the use of charge-pair mutations in the variable regions of heavy and light chain polypeptide sequences to facilitate correct pairing between certain desirable heavy and light chain polypeptides. BACKGROUND

[0003] Multispecific antibodies, such as bispecific antibodies, are an exciting generation of biotherapeutics that enable simultaneous or sequential targeting of two or more unique epitopes located on the same or different targets. This dual recognition capability allows for various applications, such as recruiting immune cells to kill tumor cells, cross-linking different cell surface receptors, or improving 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 ®) an artificial immune synapse between cytotoxic T cells and target tumor cells by simultaneously binding a CD3 epitope on the surface of T cells and a tumor-associated antigen. (Wolf E et al., Drug Discov. Today 10:1237-44 (2005); Kantarjian H et al., N. Engl. J. Med. 376:836-47 (2017)) Since 2019, over 100 bispecific formats have been described, with over 85 in development and three approved by the US 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] There are two important design strategies for generating bispecific molecules. The first approach is to encode two or more unique fragment-variable (Fv) sequences on the same polypeptide chain(s), as in formats such as BiTEs. ®, 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)) These single-chain formats circumvent the problems associated with assembling multiple polypeptide chains into a single molecule, but also tend to exhibit poor yields and suboptimal stability. Alternative strategies utilize biophysics and engineering to "guide" individual chains into the correct orientation while simultaneously discriminating against mismatch scenarios. Examples include technologies such as knob-in-holes (KiH), charge-pair mutations (CPMs), and strand-exchange engineered domains (SEEDbody), which have been used in molecules requiring hetero-Fc pairing. (Davis JH et al., Protein Eng Des. Sci. 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 allows the production of molecules that more closely mimic the native structures of IgG molecules, enabling increased stability and more diverse format design. (Wang Q et al., Antibodies (Basel) 8(3):43 (2019)) However, these technical strategies are often imperfect, and chain mismatch remains a problem. (Ha JH et al., Front. Immunol. 7:394 (2016))

[0005] Heavy- and light-chain mismatch is a significant challenge in the generation of multispecific antibodies. For example, if the wrong light chain (LC) pairs with the undesired heavy chain (HC), the resulting mismatched molecule translates into an impurity that must be removed. Several protein engineering 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 is not necessarily applicable to the next. Therefore, there is a need in the field for improved methods to facilitate pairing between particularly desirable heavy- and light-chain polypeptides. Furthermore, there is a particular need for pairings within the Fv interface. SUMMARY

[0006] This application relates to facilitating the selective binding between modified VH and VL domains by introducing amino acids with complementary charges into the two domains at new positions within these domains. For example, modifications can be made at VH position 39 and VL position 85, at VH position 105 and VL position 105, or at VH position 91 and VL position 38. These modifications are particularly useful in the design and generation of multispecific antibodies, such as bispecific antibodies, where charge-pair mutations in VH and VL domains can facilitate desired pairing between specific heavy- and light-chain polypeptides. These charge-pair mutations can be used alone or in combination with additional strategies to promote correct pairing of polypeptide chains.

[0007] In some embodiments, an isolated protein comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein VH and VL are linked to each other, and wherein VH and VL comprise at least one of the following sets of charged amino acids: (a) VH comprises a charged amino acid at position 39 and VL comprises a charged amino acid at position 85 that is charge complementary to the amino acid at position 39 of VH; (b) VH comprises a charged amino acid at position 105 and VL comprises a charged amino acid at position 42 that is charge complementary to the amino acid at position 105 of VH; or (c) VH comprises a charged amino acid at position 91 and VL comprises a charged amino acid at position 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 in the VH and VL domains refer to their positions according to the Kabat numbering scheme.

[0008] In certain embodiments, the isolated protein comprises a VH and VL, which include a set of charged amino acids, such that the VH includes a charged amino acid at position 39 and the VL includes a charged amino acid at position 85, which is charge complementary to the amino acid at position 39 of the VH. In some of these embodiments, VH comprises a positively charged amino acid at position 39 and VL comprises a negatively charged amino acid at position 85. In some embodiments where VH comprises a positively charged amino acid at position 39 and VL comprises a negatively charged amino acid at position 85, VH position 39 (VH39) and VL position 85 (VL85) comprise: (i) a lysine at VH39 and an aspartic acid at VL85, (ii) a lysine at VH39 and a glutamic acid at VL85, (iii) an arginine at VH39 and an aspartic acid at VL85, or (iv) an arginine at VH39 and a glutamic acid at VL85.In other embodiments, VH comprises a negatively charged amino acid at position 39 and VL comprises a positively charged amino acid at position 85. In some embodiments, where VH comprises a negatively charged amino acid at position 39 and VL comprises a positively charged amino acid at position 85, VH position 39 (VH39) and VL position 85 (VL85) comprise: (i) an aspartic acid at VH39 and a lysine at VL85, (ii) a glutamic acid at VH39 and a lysine at VL85, (iii) an aspartic acid at VH39 and an arginine at VL85, or (iv) a glutamic acid at VH39 and an arginine at VL85.

[0009] In certain embodiments, the isolated protein comprises a VH and VL, which include a set of charged amino acids, such that the VH includes a charged amino acid at position 105 and the VL includes a charged amino acid at position 42, which is charge complementary to the amino acid at position 105 of the VH. In some of these embodiments, VH comprises a positively charged amino acid at position 105 and VL comprises a negatively charged amino acid at position 42. In some embodiments where VH comprises a positively charged amino acid at position 105 and VL comprises a negatively charged amino acid at position 42, VH position 105 (VH105) and VL position 42 (VL42) comprise: (i) a lysine at VH105 and an aspartic acid at VL42, (ii) a lysine at VH105 and a glutamic acid at VL42, (iii) an arginine at VH105 and an aspartic acid at VL42, or (iv) an arginine at VH105 and a glutamic acid at VL42.In other embodiments, VH comprises a negatively charged amino acid at position 105 and VL comprises a positively charged amino acid at position 42. In some embodiments, where VH comprises a negatively charged amino acid at position 105 and VL comprises a positively charged amino acid at position 42, VH position 105 (VH105) and VL position 42 (VL42) comprise: (i) an aspartic acid at VH105 and a lysine at VL42, (ii) a glutamic acid at VH105 and a lysine at VL42, (iii) an aspartic acid at VH105 and an arginine at VL42, or (iv) a glutamic acid at VH105 and an arginine at VL42.

[0010] In certain embodiments, the isolated protein comprises a VH and VL, which include a set of charged amino acids, such that the VH includes a charged amino acid at position 91 and the VL includes a charged amino acid at position 38, which is charge complementary to the amino acid at position 91 of the VH. In some of these embodiments, VH comprises a positively charged amino acid at position 91 and VL comprises a negatively charged amino acid at position 38. In some embodiments where VH comprises a positively charged amino acid at position 91 and VL comprises a negatively charged amino acid at position 38, VH position 91 (VH91) and VL position 38 (VL38) comprise: (i) a lysine at VH91 and an aspartic acid at VL38, (ii) a lysine at VH91 and a glutamic acid at VL38, (iii) an arginine at VH91 and an aspartic acid at VL38, or (iv) an arginine at VH91 and a glutamic acid at VL38.In other embodiments, VH comprises a negatively charged amino acid at position 91 and VL comprises a positively charged amino acid at position 38. In some embodiments, where VH comprises a negatively charged amino acid at position 91 and VL comprises a positively charged amino acid at position 38, VH position 91 (VH91) and VL position 38 (VL38) comprise: (i) an aspartic acid at VH91 and a lysine at VL38, (ii) a glutamic acid at VH91 and a lysine at VL38, (iii) an aspartic acid at VH91 and an arginine at VL38, or (iv) a glutamic acid at VH91 and an arginine at VL38.

[0011] In some embodiments, the isolated protein comprises an antibody heavy chain including the VH and an antibody light chain including the 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, and each arm of the antibody comprises a VH-VL pair with one of the following sets of charged amino acids: (a) each VH comprises a charged amino acid at position 39 and each VL comprises a charged amino acid at position 85 that is charge-complementary to the amino acid at position 39 of a VH; (b) each VH comprises a charged amino acid at position 105 and each VL comprises a charged amino acid at position 42 that is charge-complementary to the amino acid at position 105 of a VH; or (c) each VH comprises a charged amino acid at position 91 and each VL comprises a charged amino acid at position 38 that is charge-complementary to the amino acid at position 91 of a VH.

[0012] In some embodiments, the isolated protein comprises an antibody comprising a first human IgG-CH3 domain (CH3) and a second human IgG-CH3 domain (CH3'), wherein the CH3 domain comprises the substitution of the amino acids at positions 392, 409 and 439 by a negatively charged amino acid, wherein the CH3' domain comprises the substitution of the amino acids at positions 356 and 399, and wherein the position numbers of the charged amino acids in the CH3 and CH3' domains refer to their positions according to the EU numbering scheme.

[0013] In some embodiments, the isolated protein comprises an antibody comprising a first pair of heavy chain constant domain and light chain constant domain (CH1 and CL) and a second pair of heavy chain constant domain and light chain constant domain (CH1' and CL'); wherein CH1 and CH1' each comprise a modification at position 183, as numbered according to the EU numbering scheme; wherein CL and CL' each comprise a modification at position 176, as numbered according to the EU numbering scheme; wherein CH1 position 183 and CL' position 176 each comprise a positively charged amino acid; and wherein CH1' position 183 and CL position 176 each comprise a negatively charged amino acid.In some embodiments, (i) CH1 position 183 and CL' position 176 each comprise lysine, and CH1' position 183 and CL' position 176 each comprise aspartic acid; (ii) CH1 position 183 and CL' position 176 each comprise lysine, and CH1' position 183 and CL' position 176 each comprise glutamic acid; (iii) CH1 position 183 and CL' position 176 each comprise arginine and CH1' position 183 and CL' position 176 each comprise aspartic acid; or (iv) CH1 position 183 and CL' position 176 each comprise arginine and CH1' position 183 and CL' position 176 each comprise glutamic acid.

[0014] In some embodiments, a bispecific antibody comprises a first pair of heavy chain variable domain and light chain variable domain (VH and VL) and a second pair of heavy chain variable domain and light chain variable domain (VH' and VL'), wherein the VH and VL pair and the VH' and VL' pair comprise at least one of the following sets of charged amino acids: (a) VH and VH' each comprise a charged amino acid at position 39; VL and VL' each comprise a charged amino acid at position 85 that is charge-complementary to the amino acid at position 39 of VH and VH', respectively; and VH and VH' comprise amino acids with complementary charges; (b) VH and VH' each comprise a charged amino acid at position 105; VL and VL' each comprise a charged amino acid at position 42 that is charge-complementary to the amino acid at position 105 of VH and VH', respectively.VH' is charge-complementary; and VH and VH' comprise amino acids with complementary charges; or (c) VH and VH' each comprise a charged amino acid at position 91; VL and VL' each comprise a charged amino acid at position 38 that is charge-complementary to the amino acid at position 91 of VH and VH', respectively; and VH and VH' comprise amino acids with complementary charges. In these embodiments, the position numbers of the charged amino acids in the VH and VL domains refer to their positions according to the Kabat numbering scheme.

[0015] In certain embodiments, a bispecific antibody comprises a first pair of heavy chain variable domains and light chain variable domains (VH and VL) and a second pair of heavy chain variable domains and light chain variable domains (VH' and VL'), wherein VH and VH' each comprise a charged amino acid at position 39; VL and VL' each comprise a charged amino acid at position 85 that is charge complementary to the amino acid at position 39 of VH and VH', respectively; and VH and VH' comprise amino acids with complementary charges. In some embodiments, VH and VL' comprise a positively charged amino acid, and VH' and VL comprise a negatively charged amino acid.In some of these embodiments, the VH and VL pair and the VH' and VL' pair comprise: (i) a lysine at VH39, a lysine at VL'85, an aspartic acid at VH'39 and an aspartic acid at VL85; (ii) a lysine at VH39, a lysine at VL'85, a glutamic acid at VH'39 and a glutamic acid at VL85; (iii) an arginine at VH39, an arginine at VL'85, an aspartic acid at VH'39 and an aspartic acid at VL85; or (iv) an arginine at VH39, an arginine at VL'85, a glutamic acid at VH'39 and a glutamic acid at VL85. In some embodiments, VH and VL' comprise a negatively charged amino acid, and wherein VH' and VL comprise a positively charged amino acid.In some of these embodiments, the VH and VL pair and the VH' and VL' pair comprise: (i) an aspartic acid at VH39, an aspartic acid at VL'85, a lysine at VH'39 and a lysine at VL85; (ii) a glutamic acid at VH39, a glutamic acid at VL'85, a lysine at VH'39 and a lysine at VL85; (iii) an aspartic acid at VH39, an aspartic acid at VL'85, an arginine at VH'39 and an arginine at VL85; or (iv) a glutamic acid at VH39, a glutamic acid at VL'85, an arginine at VH'39, and an arginine at VL85.

[0016] In certain embodiments, a bispecific antibody comprises a first pair of heavy chain variable domain and light chain variable domain (VH and VL) and a second pair of heavy chain variable domain and light chain variable domain (VH' and VL'), wherein VH and VH' each comprise a charged amino acid at position 105; VL and VL' each comprise a charged amino acid at position 42 that is charge complementary to the amino acid at position 105 of VH and VH', respectively; and VH and VH' comprise amino acids with complementary charges. In some embodiments, VH and VL' comprise a positively charged amino acid, and VH' and VL comprise a negatively charged amino acid.In some of these embodiments, the VH and VL pair and the VH' and VL' pair comprise: (i) a lysine at VH105, a lysine at VL'42, an aspartic acid at VH'105 and an aspartic acid at VL42; (ii) a lysine at VH105, a lysine at VL'42, a glutamic acid at VH'105 and a glutamic acid at VL42; (iii) an arginine at VH105, an arginine at VL'42, an aspartic acid at VH'105 and an aspartic acid at VL42; or (iv) an arginine at VH105, an arginine at VL'42, a glutamic acid at VH'105 and a glutamic acid at VL42. In some embodiments, VH and VL' comprise a negatively charged amino acid, and wherein VH' and VL comprise a positively charged amino acid.In some of these embodiments, the VH and VL pair and the VH' and VL' pair comprise: (i) an aspartic acid at VH105, an aspartic acid at VL'42, a lysine at VH'105, and a lysine at VL42; (ii) a glutamic acid at VH105, a glutamic acid at VL'42, a lysine at VH'105, and a lysine at VL42; (iii) an aspartic acid at VH105, an aspartic acid at VL42, an arginine at VH'105, and an arginine at VL42; or (iv) a glutamic acid at VH105, a glutamic acid at VL'42, an arginine at VH'105, and an arginine at VL42.

[0017] In certain embodiments, a bispecific antibody comprises a first pair of variable heavy chain domain and variable light chain domain (VH and VL) and a second pair of variable heavy chain domain and variable light chain domain (VH' and VL'), wherein VH and VH' each comprise a charged amino acid at position 91; VL and VL' each comprise a charged amino acid at position 38 that is charge complementary to the amino acid at position 91 of VH and VH', respectively; and VH and VH' comprise amino acids with complementary charges. In some embodiments, VH and VL' comprise a positively charged amino acid, and VH' and VL comprise a negatively charged amino acid.In some of these embodiments, the VH and VL pair and the VH' and VL' pair comprise: (i) a lysine at VH91, a lysine at VL'38, an aspartic acid at VH'91 and an aspartic acid at VL38; (ii) a lysine at VH91, a lysine at VL'38, a glutamic acid at VH'91 and a glutamic acid at VL38; (iii) an arginine at VH91, an arginine at VL'38, an aspartic acid at VH'91 and an aspartic acid at VL38; or (iv) an arginine at VH91, an arginine at VL'38, a glutamic acid at VH'91 and a glutamic acid at VL38. In some embodiments, VH and VL' comprise a negatively charged amino acid, and wherein VH' and VL comprise a positively charged amino acid.In some of these embodiments, the VH and VL pair and the VH' and VL' pair comprise: (i) an aspartic acid at VH91, an aspartic acid at VL'38, a lysine at VH'91 and a lysine at VL38; (ii) a glutamic acid at VH91, a glutamic acid at VL'38, a lysine at VH'91 and a lysine at VL38; (iii) an aspartic acid at VH91, an aspartic acid at VL'38, an arginine at VH'91 and an arginine at VL38; or (iv) a glutamic acid at VH91, a glutamic acid at VL'38, an arginine at VH'91 and an arginine at VL38.

[0018] Some embodiments are a method for producing a bispecific antibody, comprising expressing one or more polynucleotides encoding a bispecific antibody containing charge-pair mutations described herein in a host cell, cultivating the host cells in a culture medium under conditions that generate the component polypeptide chains, and recovering the bispecific antibody from the cell or the culture medium. Similarly, some embodiments are a method for producing isolated proteins, comprising expressing one or more polynucleotides encoding a protein described herein with charge-pair mutations in a host cell, cultivating the host cells in a culture medium under conditions that generate the component polypeptide chains, and recovering the protein from the cell or the culture medium.In some embodiments, the host cells comprise one or more plasmids containing the one or more polynucleotides. In some embodiments, the host cells are a Chinese hamster ovary (CHO) cell line or a human embryonic kidney (HEK) cell line. In some embodiments, the obtained bispecific antibody or protein is purified by protein A chromatography. In some embodiments, the obtained bispecific antibody or protein is further purified by ion-exchange chromatography. In some embodiments, the obtained bispecific antibody or protein is purified by protein A chromatography and ion-exchange chromatography. BRIEF DESCRIPTION OF THE FIGURES Fig. Figure 1A is a simplified diagram of an exemplary bispecific antibody, comprising one arm capable of binding a first antigen (antigen 1) and a second arm capable of binding a second antigen (antigen 2). The two arms are linked by their corresponding Fc regions. The antibody's heavy chains are linked by interactions in the CH2 and CH3 domains within the Fc region, and Fig. Figure 1A shows the CH2-CH2 interface and the CH3-CH3 interface where such interactions occur. Similarly, Figure 1A shows... Fig. 1A, how each heavy chain is connected to its respective light chain via two different interfaces. The first of these contains interactions between the CH1 domain of the heavy chain and the CL domain of the light chain (the CH1-CL interface), and the second contains interactions between the VH domain of the heavy chain and the VL domain of the light chain (the Fv interface). Finally, shows Fig. 1A the regions on each arm that contain the CDR regions responsible for the interaction with antigen 1 and antigen 2. Fig. Figure 1B shows some of the unwanted byproducts that can form during the production of a bispecific antibody. The unwanted byproducts shown are those in which the wrong light chain is paired with one or both of the heavy chains of the bispecific antibody. Fig. 2A-2C refer to the procedure used to identify novel charge-pair mutations for introduction into the Fv interface of antibodies. Fig. Figure 2A shows a rendering of the structural interface between a VH domain and a VL domain, with interactions shown as dotted lines. Fig. Figure 2B shows a diagram of the intended result when charge pair changes occur at the interaction sites of Fig. 2A is performed, whereby the introduced changes lead to an unstable, high-energy state in undesired "mismatched" states, so that kinetics drive the antibody chains to assemble in the desired pairing orientation. Fig. Figure 2C shows the energy level calculation (REU) for an exemplary charge-pair mutation that was predicted to have a higher energy state in each of its mispaired structures. Fig. Figures 3A-3E show cation-exchange chromatography spectrograms of bispecific antibodies after purification from HEK-293 cells using a protein A column. Each of Fig. 3A-3E further provides a list of charge-pair mutations present in the generated bispecific antibody. The locations of peaks containing specific antibody species are indicated with diagrams of the species and arrows pointing to the corresponding peak. The desired bispecific antibody is also highlighted by a box showing its peak in each of Fig. surrounds 3A-3E. Fig. Figures 4A-4C also show cation-exchange chromatography spectrograms of bispecific antibodies after purification from HEK-293 cells using a protein A column. Each of Fig. 4A-4C further provides a list of charge-pair mutations present in the generated bispecific antibody. The location of the peak containing the desired antibody species is indicated with a species diagram and arrows pointing to the corresponding peak. The desired bispecific antibody is also highlighted by a box showing its peak in each of Fig. surrounds 4A-4C. Fig. Figure 5 also shows cation exchange chromatography spectrograms of a bispecific antibody after purification from HEK-293 cells using a protein A column. Fig. Section 5 further provides a list of charge-pair mutations present in the generated bispecific antibody. The location of the peak containing the desired antibody species is indicated by a species diagram and arrows pointing to the corresponding peak. The desired bispecific antibody is also highlighted by a box surrounding its peak. Fig. Figure 6 shows a representation of the recovery of various monospecific and bispecific antibody species when purified after production in HEK-293 cells. Fig. Figure 5 illustrates how the identified charge-pair mutations facilitate the production of bispecific antibodies at levels above parental antibodies. Fig. 7A and Fig. Figure 7B contains an exemplary VH sequence and VL sequence, respectively. These sequences are labelled with the Kabat numbering for each residue, and the figures also include regions in boxes showing the amino acids that could be altered according to charge-pair mutations disclosed herein. Pair A corresponds to VH position 39 and VL position 85, pair B corresponds to VH position 105 and VL position 105, and pair C corresponds to VH position 91 and VL position 38. DETAILED DESCRIPTION

[0019] The present application arises from the discovery of novel methods by which antibody VH and VL domains can be engineered for specific pairing such that they preferentially assemble with each other and not as part of other VH-VL pairings. A structural analysis of a range of different antibody structures revealed positions between VH and VL domains where modifications based on complementary charges can be made to drive such preferential assembly consistently across different antibody sequences. The VL / VH interface residues selected for engineering are buried within the VL / VH interface and are spatially close. The target residues are well conserved across different antibody families.

[0020] These engineered VH and VL domains are useful in a range of contexts where specific domain interactions are desired. In some manifestations, these VH and VL modifications can be used to facilitate the formation of a heterodimeric pair of polypeptides, each containing either the engineered VH domain or the engineered VL domain. In other manifestations, the VH and VL domains of two different antibody heavy chains and two different antibody light chains are engineered to form a four-chain heterodimeric antibody. Electrostatic steering, achieved by manipulating interface residues between the VL and VH domains, prevents mispairing of light chains with the non-cognate heavy chains when two different heavy-chain and light-chain pairs assemble to form a desired four-chain heterodimeric antibody.As described herein, an exemplary strategy includes introducing one or more negatively charged residues (e.g., Asp or Glu) into a first VL (VL1) and one or more positively charged residues (e.g., Lys, His, or Arg) into the accompanying VH (VH1) at the VL1 / VH1 interface, and introducing one or more positively charged residues (e.g., Lys, His, or Arg) into a second VL (VL2) and one or more negatively charged residues (e.g., Asp or Glu) into the accompanying VH (VH2) at the VL2 / VH2 interface. The electrostatic steering effect directs VL1 to pair with VH1 and VL2 to pair with VH2, since the oppositely charged residues (polarity) attract each other at the interface, while the same type of charged residues (polarity) causes repulsion at an interface, resulting in the suppression of the unwanted VL / VH pairings.Therefore, in some manifestations, these VH and VL changes can be used to facilitate the formation of the desired structure of a bispecific antibody and to minimize the formation of contaminating alternative species.

[0021] In some manifestations, these VH and VL alterations can be combined with other mechanisms to drive the specific formation of a desired bispecific antibody species. For example, VH and VL alterations are combined with known charge-pair mutations in the heavy and light chains of antibodies. Combining VH and VL alterations with other mechanisms for driving specific pairing increases the production and purity of an expressed bispecific antibody compared to implementing either strategy alone.

[0022] Other approaches to engineering the light and heavy chains to form specific heterodimers include replacing 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; replacing one or more hydrophilic residues (e.g., glycine) at the VL / VH interface with a hydrophobic residue (e.g., glutamine); or constructing a pair of bulky / small residues at the VL / VH interface to exert a button-in-a-hole effect to achieve the correct VL / VH pairing. The strategy described here can be used to efficiently generate a full-length heterodimeric antibody from two pre-existing antibodies without the use of artificial linkers. The resulting heterodimeric antibodies are stable and suitable for commercial production without excessive aggregation or yield loss.Since heterodimeric antibodies can simultaneously target two different antigens or two different epitopes on the same antigen, they may have significant potential for the specific treatment of numerous diseases.

[0023] Specific embodiments and further details of the invention are provided below. Definitions of general terms and expressions

[0024] To facilitate understanding of this disclosure, certain terms are first defined. Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by a person skilled in the field of disclosure. As used in this application, each of the following terms, unless expressly stated otherwise herein, shall have the meanings given below. Further definitions are set forth throughout the application.

[0025] Units, prefixes and symbols are given in their form recognized by the International System of Units (SI).

[0026] As used in the present disclosure and the claims, the singular forms "a", "a", "an", and "the" include the plural forms unless the context clearly indicates otherwise. Unless expressly stated or evident from the context, the term "or", as used herein, is understood to be inclusive. The term "and / or", as used in an expression such as "A and / or B", is intended to include "A and B", "A or B", "A" and "B". Likewise, the term "and / or", as used in an expression such as "A, B and / or C", 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).

[0027] It is understood that wherever embodiments are described herein by the phrase "comprising," otherwise analogous embodiments described by the terms "consisting of" and / or "consisting substantially of" are also provided. In this disclosure, "comprising," "encompassing," "containing," and "having," and the like, may have the meanings attributed to them in U.S. patent law and may mean "includes," "contain," and the like; "consisting substantially of" or "consisting substantially of" also has the meaning described in U.S. patent law, and the term is open, permitting the presence of more than what is stated, provided that the presence of more than what is stated does not alter fundamental or novel characteristics of the said product, while excluding embodiments in the prior art.

[0028] The terms "approximately" or "substantially encompassing" refer to a value or composition that lies within an acceptable range of error for the value or composition, as determined by a person skilled in the art. This range will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measuring system. For example, "approximately" or "substantially encompassing" may mean within 1 or more than 1 standard deviation according to established technical practice. Alternatively, "approximately" or "substantially encompassing" may mean a range of up to 20%. Furthermore, particularly in biological systems or processes, the terms may mean up to an order of magnitude or up to 5 times a value.Unless otherwise stated, where values ​​or compositions are provided in the application and in the claims, the meaning of "approximately" or "substantially comprising" shall be within an acceptable margin of error for that value or composition.

[0029] The term "antibody" means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combination thereof. As used herein, the term "antibody" includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, recombinant antibodies, multispecific antibodies, and bispecific antibodies. An antibody may belong to any of the five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or to subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy chain constant domains, designated alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins exhibit different and known subunit structures and three-dimensional configurations.For example, a common configuration of an antibody has two full-length antibody heavy chains and two full-length antibody light chains.

[0030] As used herein, the term "antibody heavy chain" refers to an antibody heavy chain consisting of a variable region and a constant region, as defined for a full-length antibody. A full-length antibody heavy chain is a polypeptide consisting, in the N-terminal to C-terminal direction, of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody joint region (HR), an antibody heavy chain constant domain 2 (CH2), and an antibody heavy chain constant domain 3 (CH3), abbreviated as VH-CH1-HR-CH2-CH3. The term “heavy chain”, when used in reference to an antibody, can refer to any particular type, e.g. alpha (δ), delta (α), epsilon (ε), gamma (γ) and mu (µ), based on the amino acid sequence of the constant domain to form IgA, IgD, IgE, IgG and μ, respectively.to produce IgM classes of antibodies, including subclasses of IgG (e.g., IgG1, IgG2, IgG3, and IgG4) and subclasses of IgA (e.g., IgA1 and IgA2). Heavy chain amino acid sequences are known in the art.

[0031] 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 consisting, in the N-terminal to C-terminal direction, of an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), abbreviated as VL-CL. The term "light chain," when used in relation to an antibody, may refer to any particular type, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domains. Light chain amino acid sequences are known in the art.

[0032] The term "antibody fragment" refers to a part of an intact antibody. An "antigen-binding fragment," "antigen-binding domain," or "antigen-binding region" refers to a part of an intact antibody that binds to an antigen. An antigen-binding fragment may contain the antigen-determining regions of an intact antibody (e.g., the complementarity-determining regions (CDRs)). Examples of antigen-binding fragments of antibodies include Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single-chain antibodies. An antigen-binding fragment of an antibody may be derived from any animal species, such as rodents (e.g., mouse, rat, or hamster) or humans, or it may be artificially produced.

[0033] The term “multispecific antibody” means that an antigen-binding protein is capable of binding specifically to two or more different antigens. A subcategory of multispecific antibodies are “bispecific antibodies,” which are capable of binding specifically to two different antigens. As used here, an antibody “binds specifically” to a target antigen if it has a significantly higher binding affinity for that antigen and is consequently able to distinguish that antigen from other, unrelated proteins under similar binding assay conditions based on its affinity. Antigen-binding proteins that bind specifically to one antigen may have an equilibrium dissociation constant (Ke). D ) ≤ 1 × 10 -6 M exhibit. The antigen-binding protein binds an antigen specifically with "high affinity" when K D ≤ 1 × 10 -8 M is.

[0034] "Isolated antibody" refers to an antibody population that comprises a single antibody species. For example, a particular isolated antibody consists of an antibody population with a single heavy chain amino acid sequence and a single light chain amino acid sequence binding to a single epitope. However, an isolated antibody may exhibit cross-reactivity with other antigens, such as related molecules from different species. Furthermore, an antibody population can still be considered an "isolated antibody" even if it is contaminated by small amounts of other antibody species. Specifically, an isolated antibody may contain less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or no other antibody species.

[0035] “Monoclonal antibody” refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This contrasts with polyclonal antibodies, which typically contain different antibodies against different antigenic determinants. Furthermore, “monoclonal” antibody refers to antibodies produced by any number of methods, including but not limited to hybridoma, phage selection, recombinant expression, and transgenic animals.

[0036] The terms "variable region" and "variable domain" are used interchangeably and are common in the field. A variable region typically refers to a part of an antibody, generally a portion of a light or heavy chain, typically around the amino-terminal 110 to 120 amino acids or 110 to 125 amino acids in the mature heavy chain and around 90 to 115 amino acids in the mature light chain. These regions vary considerably in sequence among antibodies and are used to determine the binding and specificity of a given antibody for its corresponding antigen. Sequence variability is concentrated in those regions called complementarity-determining regions (CDRs), while the more conserved regions in the variable domain are called scaffold regions (FRs).From the N-terminus to the C-terminus, naturally occurring light and heavy chain variable regions (CDRs) generally correspond to the following sequence of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Without regard to any particular mechanism or theory, it is assumed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen.

[0037] The terms “VL”, “VL domain”, and “VL region” are used interchangeably and refer to the light chain variable region of an antibody.

[0038] The terms “VH”, “VH domain”, and “VH region” are used interchangeably and refer to the variable region of the heavy chain of an antibody.

[0039] The term "Kabat numbering" and similar terms are recognized in the prior art and refer to a system for numbering amino acid residues in the heavy- and light-chain variable regions of an antibody or an antigen-binding fragment of an antibody. CDRs can be determined according to the Kabat numbering system (see, e.g., 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, US Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs in a heavy chain of an antibody molecule are typically located at amino acid positions 31 to 35, which may optionally include one or two additional amino acids after 35 (designated as 35A and 35B in the Kabat numbering scheme) (CDR1), amino acid positions 50 to 65 (CDR2) and amino acid positions 95 to 102 (CDR3).Using the Kabat numbering system, CDRs in an antibody light chain are typically located at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). The Kabat numbering scheme can be used in conjunction with the EU index, which is used to refer to residues in the constant domains of an antibody (see, e.g., Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242).

[0040] Chothia refers instead to the location of the structure loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR H1 loop, when numbered using the Kabat numbering convention, varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).

[0041] The hypervariable AbM regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody labeling software.

[0042] Alternatively, the CDR regions can be determined according to the IMGT numbering system (see, e.g., Guidicelli et al., Nucl. Acids Res. 34:D781-D784 (2006); Lefranc et al., Dev. Comp. Immunol. 27:55-77 (2003)). This numbering scheme standardizes the numbering of antibody lambda and kappa light chains, heavy chains, and T-cell receptor chains.

[0043] Alternatively, the position of specific amino acids within the scaffold regions of the variable domains (described below) can be described using the Aho numbering system. Since the length of the antibody CDR amino acid sequence varies from antibody to antibody, numbering residues based on the linear sequence (assuming the first residue is position 1) results in scaffold residues with different position numbers between antibodies. Annemarie Honegger and Andreas Plückthun developed a structure-based (Aho) numbering scheme that introduces gaps in the CDR regions to minimize deviations from the mean structure of the aligned domains. (Honegger, A., and Plückthun, A. (2001). J. Mol. Biol. 309, 657-670) This results in structurally equivalent positions with the same residue number when comparing two different antibodies.This allows for comparisons of the effect of substitutions in the scaffold region of the variable domain between antibodies.

[0044] The terms "constant region" and "constant domain" are interchangeable and have their established meanings in the field. A constant region is a part of an antibody, for example, a carboxyl-terminal portion of a light and / or heavy chain, that is not directly involved in antibody 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 better conserved amino acid sequence compared to a variable immunoglobulin domain.

[0045] As used herein, the terms ‘Fc region’ and ‘Fc domain’ refer to a C-terminal region of an IgG heavy chain; in the case of an IgG1 antibody, the C-terminal region comprises -CH2-CH3 (see above).

[0046] The term "interface" refers to the association surface that arises 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. Examples of interfaces include the CH1 / CL, VH / VL, CH2-CH2, and CH3 / CH3 interfaces. In some embodiments, the interface may contain, for example, hydrogen bonds, electrostatic interactions, or salt bridges between the amino acids forming the interface.

[0047] The term "chimeric antibody" refers to an antibody whose amino acid sequence is derived from two or more species. Typically, the variable region of both the light and heavy chains corresponds to the variable region of antibodies derived from one mammalian species (e.g., mouse, rat, rabbit, etc.), with the desired specificity, affinity, and ability, while the constant regions are homologous to sequences derived from another (usually human) species to avoid triggering an immune response in that species.

[0048] “Humanized antibody” refers to a chimeric antibody that includes amino acid residues from non-human CDRs and amino acid residues from human scaffold regions and constant regions. A humanized antibody may include substantially all of at least one and typically two variable domains, with all or substantially all of the CDRs corresponding to those of a non-human antibody and all or substantially all of the FRs corresponding to those of a human antibody. A humanized antibody may optionally include at least part of an antibody's constant region derived from a human antibody. “Humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. Typically, humanized antibodies are human immunoglobulins in which CDR residues are replaced by CDR residues from a non-human species (e.g.,The antibodies are replaced with those from mice, rats, rabbits, and hamsters, which exhibit the desired specificity, affinity, and ability. Accordingly, humanized antibodies are also referred to as "CDR-grafted" antibodies. Early examples of methods used to generate humanized antibodies are described in US Pat. 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). Many more examples and methods for humanizing antibodies have subsequently been published.

[0049] “Human antibody” refers to an antibody with variable regions in which both the FRS and CDRs are derived from human germline immunoglobulin sequences. If the antibody further contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies disclosed may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody,” as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human scaffold sequences. The terms “human antibody” and “fully human antibody” are used interchangeably.

[0050] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The terms also include an amino acid polymer that has been naturally or artificially modified; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included in the definition are, for example, polypeptides containing one or more analogues of an amino acid (including, for example, non-natural amino acids, etc.), as well as other modifications known in the prior art.It is understood that, since the polypeptides of the present invention are based on antibodies, in certain embodiments the polypeptides may occur as single chains or associated chains.

[0051] "Complementary amino acid substitutions" refers to the pairing of a substitution of a positively charged amino acid in the heavy chain with a substitution of a negatively charged amino acid in the light chain, which then aggregates with the remaining amino acid in the heavy chain. Similarly, a substitution of a negatively charged amino acid in the heavy chain is paired with a substitution of a positively charged amino acid in the light chain, which then aggregates with the remaining amino acid in the heavy chain.

[0052] As used here, the term "host cell" can be any type of cell, e.g., a primary cell, a cell in culture, or a cell from a cell line. In specific embodiments, the term "host cell" refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the stem cell transfected with the nucleic acid molecule, e.g., due to mutations or environmental influences that may occur in subsequent generations, or the integration of the nucleic acid molecule into the host cell genome. VH domain and VL domain modifications

[0053] In some embodiments, the disclosed proteins include modifications within VH and VL domains that introduce charged amino acids which 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 unmodified VH and VL domains.

[0054] To control the specific association of a modified VH domain with a modified VL domain, the two domains can include complementary charge changes. Thus, if a VH domain includes a change to introduce a negatively charged amino acid, it will pair with a complementary change in a VL domain to introduce a positively charged amino acid. Conversely, if a VL domain includes a change to introduce a negatively charged amino acid, it will pair with a complementary change in a VH domain to introduce a positively charged amino acid. Accordingly, the pair of complementary charged residues can be referred to as a "charge-pair change," a "charge-pair mutation," or a "CPM."

[0055] The modified amino acids in a charge-pair modification can be any natural or synthetic amino acid that is charged at a physiological pH. In some embodiments, the negatively charged amino acid is aspartic acid or glutamic acid. In other embodiments, the positively charged amino acid is lysine, histidine, or arginine.

[0056] One of the charge-pair modifications that can be used to target a specific VH-VL association involves a modification at position 39 of the VH domain paired with a modification at position 85 of the VL domain (as numbered according to the Kabat scheme). In some embodiments, the VH domain includes a charged amino acid at position 39, and the VL domain includes a charged amino acid at position 85 that is complementary to the amino acid at position 39 of the VH domain. In other embodiments, the VH domain includes a positively charged amino acid at position 39 (e.g., arginine, lysine, or histidine), and the VL domain includes a negatively charged amino acid at position 85 (e.g., glutamic acid or aspartic acid). In some embodiments, the VL domain comprises a positively charged amino acid at position 85 (e.g., arginine, lysine, or histidine), and the VH domain comprises a negatively charged amino acid at position 39 (e.g.,Glutamic acid or aspartic acid).

[0057] Another charge-pair modification that can be used to target a specific VH-VL association involves a modification at position 105 of the VH domain paired with a modification at position 42 of the VL domain (as numbered according to the Kabat scheme). In some embodiments, the VH domain includes a charged amino acid at position 105, and the VL domain includes a charged amino acid at position 42 that is complementary to the amino acid at position 105 of the VH domain. In some embodiments, the VH domain includes a positively charged amino acid at position 105 (e.g., arginine, lysine, or histidine), and the VL domain includes a negatively charged amino acid at position 42 (e.g., glutamic acid or aspartic acid). In some embodiments, the VL domain comprises a positively charged amino acid at position 42 (e.g., arginine, lysine, or histidine), and the VH domain comprises a negatively charged amino acid at position 105 (e.g.,Glutamic acid or aspartic acid).

[0058] Another charge-pair modification that can be used to target a specific VH-VL association involves a modification at position 91 of the VH domain paired with a modification at position 38 of the VL domain (as numbered according to the Kabat scheme). In some embodiments, the VH domain includes a charged amino acid at position 91, and the VL domain includes a charged amino acid at position 38 that is complementary to the amino acid at position 91 of the VH domain. In some embodiments, the VH domain includes a positively charged amino acid at position 91 (e.g., arginine, lysine, or histidine), and the VL domain includes a negatively charged amino acid at position 38 (e.g., glutamic acid or aspartic acid). In some embodiments, the VL domain comprises a positively charged amino acid at position 38 (e.g., arginine, lysine, or histidine), and the VH domain comprises a negatively charged amino acid at position 91 (e.g.,Glutamic acid or aspartic acid).

[0059] Numerous VH and VL domain sequences are known in the prior art as part of antibody sequences. The VH and VL domain modifications disclosed herein can be applied to the VH and VL sequences known in the prior art. In some embodiments, the VH and VL domain modifications disclosed herein can be applied to naturally occurring human VH and VL domain sequences. In some embodiments, the VH and VL domain modifications 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 approximately 70%, at least approximately 71%, at least approximately 72%, at least approximately 73%, at least approximately 74%, at least approximately 75%, at least approximately 76%, at least approximately 77%, at least approximately 78%, at least approximately 79%, at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98% or at least approximately 99% identical to a human germline VH domain.In some embodiments, the VL domain is at least approximately 70%, at least approximately 71%, at least approximately 72%, at least approximately 73%, at least approximately 74%, at least approximately 75%, at least approximately 76%, at least approximately 77%, at least approximately 78%, at least approximately 79%, at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98% or at least approximately 99% identical to a human germline VL domain. Proteins containing VH domain and VL domain modifications

[0060] The modified VH and VL domains described herein can be used in various situations where the VH and VL domains are part of one or more polypeptides and specific VH-VL interactions are desirable. For example, the modified VH and VL domains can be incorporated into heavy- and light-chain polypeptides as part of the generation of an antibody or antibody fragment. In some embodiments, the modified VH and VL domains facilitate the generation of specific VH-VL pairings in heterodimeric 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 with other proteins to facilitate specific interactions.

[0061] Bispecific antibodies are a context in which the modified VH and VL domains described herein can be used. In some forms, bispecific antibodies comprise two different heavy chains and two different light chains and require a specific arrangement of the antibody such that each heavy chain binds to a specific light chain. Accordingly, in some embodiments, the modified VH and VL domains described herein are components of a bispecific antibody comprising a first pair of a heavy chain variable domain and a light chain variable domain (VH and VL) and a second pair of a heavy chain variable domain and a light chain variable domain (VH' and VL'). In such a bispecific antibody, each VH-VL pair can include its own set of charge-pair mutations.In some embodiments, VH and VH' each comprise a charged amino acid at the same position; VL and VL' each comprise a charged amino acid at the same position that is complementary to the modified amino acid of VH and VH', respectively; and VH and VH' comprise amino acids with complementary charges. In such embodiments, VH and VL' comprise the same charges (i.e., both positively charged or both negatively charged amino acids), and VH' and VL comprise the same charges (i.e., both positively charged or both negatively charged amino acids). In some embodiments, the VH-VL and VH'-VL' pairs are modified at the same amino acid positions. In some embodiments, the VH-VL and VH'-VL' pairs are modified at different positions.

[0062] In some embodiments, the antibody or antibody fragment comprises a VL domain that is at least approximately 70%, at least approximately 71%, at least approximately 72%, at least approximately 73%, at least approximately 74%, at least approximately 75%, at least approximately 76%, at least approximately 77%, at least approximately 78%, at least approximately 79%, at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% identical to a Human germline lambda chain VL domain is.

[0063] In some embodiments, the antibody or antibody fragment comprises a VL domain that is at least approximately 70%, at least approximately 71%, at least approximately 72%, at least approximately 73%, at least approximately 74%, at least approximately 75%, at least approximately 76%, at least approximately 77%, at least approximately 78%, at least approximately 79%, at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% identical to a Human germline kappa chain VL domain is.

[0064] In some embodiments, the modified VH and VL domains described herein are components of antibody fragments. In some embodiments, the antibody fragment is any antibody fragment that includes both a VH domain and a VL domain. In some embodiments, the antibody fragment is a Fab, Fab', F(ab')2, or Fv fragment.

[0065] In some embodiments, the antibody or antibody fragment may optionally include, in addition to the modifications described in the preceding section “VH domain and VL domain modifications”, changes in the VH-VL interface. VH-VL domain interface residues (i.e., amino acid residues mediating the assembly of the VH and VL domains) within the VL domain include AHo positions 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 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 a charged amino acid, preferably having the opposite charge to those introduced into the related VH domain. In some embodiments, the amino acid at AHo position 46 (Kabat 38) of the VL domain is replaced by a positively charged amino acid. In some embodiments, such as when the amino acid at AHo position 46 (Kabat 39) in the VH domain is substituted by a positively charged amino acid, the amino acid at AHo position 46 (Kabat 38) of the VL domain is replaced by a negatively charged amino acid. In some embodiments, the amino acids at AHo positions 51 (Kabat 43) and / or AHo position 141 (Kabat 100) are substituted by a positively or negatively charged amino acid.Such embodiments may further include the substitution of the amino acid at AHo position 46 by a positively or negatively charged amino acid. In some embodiments, the amino acid at AHo position 51 is substituted by a positively charged amino acid, e.g., lysine. In alternative embodiments, the amino acid at AHo position 51 is substituted by a negatively charged amino acid, e.g., aspartic acid. In some embodiments, the amino acid at AHo position 141 is substituted by a positively charged amino acid, e.g., lysine. In alternative embodiments, the amino acid at AHo position 141 is substituted by a negatively charged amino acid, e.g., aspartic acid. In some embodiments, the amino acids at AHo positions 51 and 141 are substituted by a positively charged amino acid, e.g., lysine, or a negatively charged amino acid, e.g., aspartic acid.Such embodiments may further include a substitution at AHo position 46 (Kabat 38) for a positively or negatively charged amino acid.

[0066] In some embodiments, the antibodies or antibody fragments containing modified VH-VL domains comprise 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 (EU and Kabat numbering in a kappa light chain) selected from the group consisting of F116, F118, S121, D122, E123, Q124, S131, V133, L135, N137, N138, Q160, S162, T164, S174, and S176 are replaced by 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 (EU and Kabat numbering system) of the CL domain. In some embodiments, the amino acid at position 176 of the CL domain is replaced by a positively charged amino acid.In alternative embodiments, the amino acid at position 176 of the CL domain is replaced by a negatively charged amino acid, e.g., aspartic acid. In some embodiments, the antibody or antibody fragment comprises a capacitive chain that is at least approximately 70%, at least approximately 71%, at least approximately 72%, at least approximately 73%, at least approximately 74%, at least approximately 75%, at least approximately 76%, at least approximately 77%, at least approximately 78%, at least approximately 79%, at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98% or at least approximately 99% identical to a human germline capacitive chain.

[0067] In some embodiments, the antibody or antibody fragment containing modified VH-VL domains comprises 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 (Kabat numbering in a lambda chain) selected from the group consisting of T116, F118, S121, E123, E124, K129, T131, V133, L135, S137, E160, T162, S165, Q167, A174, S176, and Y178 are replaced by 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 (EU and Kabat numbering system) of the CL domain. In some embodiments, the amino acid at position 176 of the CL domain is replaced by a positively charged amino acid.In alternative embodiments, the amino acid at position 176 of the CL domain is replaced by a negatively charged amino acid, e.g., aspartic acid. In some embodiments, the antibody or antibody fragment comprises a lambda chain that is at least approximately 70%, at least approximately 71%, at least approximately 72%, at least approximately 73%, at least approximately 74%, at least approximately 75%, at least approximately 76%, at least approximately 77%, at least approximately 78%, at least approximately 79%, at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98% or at least approximately 99% identical to a human germline lambda chain.

[0068] In some embodiments, the modified VH and VL domains described herein are components of an antibody or antibody fragment that includes a CH1 domain.In some embodiments, the antibody or antibody fragment comprises a heavy chain CH1 region that is at least approximately 70%, at least approximately 71%, at least approximately 72%, at least approximately 73%, at least approximately 74%, at least approximately 75%, at least approximately 76%, at least approximately 77%, at least approximately 78%, at least approximately 79%, at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% identical to a Human germline heavy chain CH1 region is.

[0069] Since the CH1 domain forms complexes with the light chain constant (CL) region, the CH1 and CL domains can be engineered to enhance the efficiency of a particular heavy chain pairing with its associated light chain. Accordingly, in some embodiments, the modified VH and VL domains disclosed herein can be combined with modifications in the CH1 and CL domains. For example, assembly can be facilitated by introducing a cysteine ​​residue into the heavy and light chains at or near the CH1-CL interface to enable the formation of disulfide bonds, by modifying amino acids to create a button-in-hole effect, and by performing electrostatic manipulation similar to that described herein for the variable regions.In some embodiments, assembly can be facilitated by introducing one or more charged amino acids into the CH1 domain of the antibody or antibody fragment at an 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 respect, a particularly preferred residue for substitution with a negatively or positively charged amino acid is S183 (EU numbering system). In some embodiments, S183 is substituted with a positively charged amino acid. In alternative embodiments, S183 is substituted with a negatively charged amino acid. In some embodiments, a modification at position S183 of the CH1 domain is paired with a modification to a complementary charged amino acid at position 176 of the CL domain.

[0070] In some embodiments, the modified VH and VL domains described herein are components of an antibody or antibody fragment that includes a constant region. The antibodies described herein may include any constant region. The light chain constant region may, for example, be a kappa- or lambda-type light chain constant region, such as a human kappa- or lambda-type light chain constant region. The heavy chain constant region may, for example, be an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region, such as a human alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region. In one embodiment, the light or heavy chain constant region is a fragment, a derivative, a variant, or a mutant of a naturally occurring constant region.The antibodies described herein may comprise any of the five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy chain constant domains, designated alpha, delta, epsilon, gamma, and mu, respectively. The antibodies described herein may include constant domains comprising a CH2 domain, a CH3 domain, or both a CH2 and a CH3 domain.

[0071] In the antibodies or antibody fragments described herein, which include CH3 domains as part of their heavy chains, the antibodies or antibody fragments optionally further comprise two CH3 domains, at least one of which contains one or more substitutions introducing a non-native charged amino acid into the domain. In some embodiments, each CH3 domain comprises one or more amino acid substitutions within the CH3 domain to prevent homodimerization and preferentially promote heterodimerization with the corresponding CH3 domain. International Publication No. WO 2009 / 089004 and US Patent No. 10,233,237 (incorporated herein by reference in their entirety) describe compositions and methods for engineering the CH3 domain interface to decrease homodimerization and increase heterodimerization between two CH3 domain-containing molecules.In some embodiments, amino acids selected from the group consisting of 399, 356, and 357 (EU numbering system) of the CH3 domain are replaced by a negatively charged amino acid. In some embodiments, amino acids selected from the group consisting of 370, 392, and 409 (EU numbering system) are replaced by a positively charged amino acid. In alternative embodiments, amino acids selected from the group consisting of 399, 356, and 357 (EU numbering system) of the CH3 domain are replaced by a positively charged amino acid. In further embodiments, amino acids selected from the group consisting of 370, 392, and 409 (EU numbering system) are replaced by a negatively charged amino acid.In some embodiments, the heterodimeric antibody comprises a first heavy chain containing positively charged amino acids at positions 399 and 356 (e.g., D399K and E356K), and a second heavy chain containing negatively charged amino acids at positions 392 and 409 (e.g., K392D and K409D). In some embodiments, the heterodimeric antibody comprises a first heavy chain containing positively charged amino acids at positions 356 and 399 (e.g., 356K and 409K), and a second heavy chain containing negatively charged amino acids at positions 392, 409, and 439 (e.g., 392D, 409D, and 439D). FC modifications

[0072] The heavy chains of the antibodies or antibody fragments described herein may further include one or more mutations that affect the binding of the antibody containing the heavy chains to one or more Fc receptors. One of the functions of the Fc portion of an antibody is to communicate with the immune system when the antibody binds to its target. This is usually referred to as the "effector function." Communication leads to antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). ADCC and ADCP are mediated by the binding of Fc to Fc receptors on the surface of immune system cells. CDC is mediated by the binding of Fc to proteins of the complement system, e.g., C1q.

[0073] The IgG subclasses vary in their ability to mediate effector functions. For example, IgG1 is superior to IgG2 and IgG4 in mediating ADCC and CDC. The effector function of an antibody can be increased or decreased by introducing one or more mutations into the Fc. Embodiments of the invention include heterodimeric antibodies with an Fc engineered to enhance effector function (US 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 with one or more of the following substitutions [numbering based on 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 / S298V, F243L / R292P / Y300L, F243L / R292P / Y300L / P396L, F243L / R292P / Y300L / V305I / P396L, G236A / S239D / I332E, K326A / E333A, K326W / E333S, K290E / S298G / T299A, K290N / S298G / T299A, K290E / S298G / T299A / K326E, K290N / 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.

[0074] Further embodiments of the invention include antibodies and antibody fragments with an Fc designed to lower the effector function. Examples of Fc molecules with reduced effector function include those with one or more of the following substitutions [numbering based on 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).

[0075] Another method for increasing the effector function of IgG Fc-containing proteins is to decrease the fucosylation of Fc. Removal of the core fucose from the biantennary complex oligosaccharides bound to Fc greatly increased the ADCC effector function without altering antigen binding or the CDC effector function. Several methods are known for decreasing or eliminating the fucosylation of Fc-containing molecules, such as antibodies. These include recombinant expression in certain mammalian cell lines, including a FUT8 knockout cell line, the CHO variant line Lec13, the rat hybridoma cell line YB2 / 0, a cell line containing a small interfering RNA specific for the FUT8 gene, and a cell line co-expressing β-1,4-N-acetylglucosaminyltransferase III and Golgi β-mannosidase II. Alternatively, the Fc-containing molecule can be expressed in a non-mammalian cell, such as...a plant cell, yeast or prokaryotic cell, e.g. E. coli. Thus, in certain embodiments, a composition comprises an antibody with reduced fucosylation or without any fucosylation. Polynucleotides that encode modified heavy or light chains

[0076] The invention comprises nucleic acids encoding the VH- and VL-domain-containing polypeptide chains described herein. Nucleic acid molecules according to the invention include DNA and RNA in both single-stranded and double-stranded forms, as well as the corresponding complementary sequences. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, PCR-amplified DNA, and combinations thereof. Nucleic acid molecules according to the invention include full-length genes or cDNA molecules, as well as combinations of fragments thereof. The nucleic acids according to the invention are preferably derived from human sources, but the invention also includes those derived from non-human species.

[0077] In some embodiments, the nucleic acids according to the invention are isolated nucleic acids. An “isolated nucleic acid,” in the case of nucleic acids isolated from naturally occurring sources, is a nucleic acid that has been separated from adjacent genetic sequences present in the genome of the organism from which the nucleic acid was isolated. In the case of nucleic acids synthesized enzymatically from a template or chemically, such as PCR products, cDNA molecules, or oligonucleotides, it is understood that the nucleic acids obtained by such methods are isolated nucleic acids. “Isolated nucleic acid molecule” refers to a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct. In a preferred embodiment, the nucleic acids are substantially free of contaminating endogenous material.The nucleic acid molecule is preferably derived from DNA or RNA that has been isolated at least once in substantially pure form and in an amount or concentration that allows the identification, manipulation, and reconstruction of its component nucleotide sequences by standard biochemical procedures (such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, 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, which are typically found in eukaryotic genes. Sequences of untranslated DNA may be located 5' or 3' from an open reading frame, provided that they do not interfere with the manipulation or expression of the coding region.

[0078] Variants are typically generated by site-specific mutagenesis of nucleotides in the DNA encoding the polypeptide using cassette or PCR mutagenesis, or other techniques known in the prior art, generating DNA encoding the variant, and subsequently expressing the recombinant DNA in the cell culture described herein. However, antibodies or antibody fragments comprising variant CDRs of up to approximately 100–150 residues can be prepared by in vitro synthesis using established procedures. The variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, e.g., binding to antigen, although variants with modified properties can also be selected, as described in more detail herein.

[0079] As is clear to those skilled in the art, due to the degeneration of the genetic code, a large number of nucleic acids can be produced, all of which code for the polypeptides according to the invention. Therefore, after identifying a specific amino acid sequence, a person skilled in the art could produce any number of different nucleic acids simply by modifying the sequence of one or more codons in a way that does not alter the amino acid sequence of the encoded protein.

[0080] The invention also provides expression systems and constructs in the form of plasmids, expression vectors, transcription or expression cassettes comprising at least one polynucleotide as described above. Furthermore, the invention provides host cells comprising such expression systems or constructs.

[0081] Expression vectors used in host cells typically contain sequences for plasmid maintenance and for the cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as "flanking sequences" in certain embodiments, typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, a replication origin, a transcription termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for the insertion of the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. Each of these sequences is described below.

[0082] Optionally, the vector may contain a “tag”-encoding sequence, i.e., an oligonucleotide molecule positioned at the 5' or 3' end of the polypeptide-encoding sequence. The oligonucleotide sequence may encode PolyHis (e.g., HexaHis) or another “tag,” such as FLAG, HA (hemaglutinin influenza virus), or Myc, for which commercially available antibodies exist. This tag is typically fused to the polypeptide upon expression and can serve as an affinity purifier or for recognizing the polypeptide from the host cell. Affinity purification can be performed, for example, by column chromatography using antibodies against the tag as the affinity matrix. Optionally, the tag can then be removed from the purified polypeptide by various means, such as the use of specific peptidases for cleavage.

[0083] Flanking sequences can be homologous (i.e., from the same species or strain as the host cell), heterologous (i.e., from a different species or strain than the host cell), hybrid (i.e., a combination of flanking sequences from more than one source), synthetic, or native. As such, the source of a flanking sequence can be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequence is functional within the host cell's machinery and can be activated by it.

[0084] The flanking sequences usable in the vectors of the present invention can be obtained by one of several methods known in the field. The flanking sequences usable herein will typically have been previously identified by mapping and / or restriction endonuclease digestion and can therefore be isolated from the appropriate tissue source using suitable restriction endonucleases. In some cases, the complete nucleotide sequence of a flanking sequence may be known. The flanking sequence can then be synthesized using the nucleic acid synthesis or cloning methods described herein.

[0085] Regardless of whether all or only part of the flanking sequence is known, it can be obtained from the same or a different species using polymerase chain reaction (PCR) and / or by screening a genome library with a suitable probe, such as an oligonucleotide and / or flanking sequence fragment. If the flanking sequence is unknown, a DNA fragment containing a flanking sequence can be isolated from a larger piece of DNA, which may contain, for example, a coding sequence or even another gene or genes. Isolation can be achieved by restriction endonuclease digestion to generate the correct DNA fragment, 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 this purpose will be obvious to those skilled in the art.

[0086] A replication origin is usually included in commercially available prokaryotic expression vectors, and the origin aids in vector amplification within a host cell. If the chosen vector does not contain a replication origin, one can be chemically synthesized based on a known sequence and ligated into the vector. For example, the replication origin of the plasmid pBR322 (New England Biolabs, Beverly, Mass.) is suitable for most Gram-negative bacteria, and various viral origins (e.g., SV40, polyomavirus, adenovirus, vesicular stomatitis virus (VSV), or papillomaviruses such as HPV or BPV) are suitable for cloning vectors into mammalian cells. In general, the replication origin component is not required for mammalian expression vectors (for example, the SV40 origin is often used simply because it also contains the virus's early promoter).

[0087] A transcription termination sequence is typically located 3' from the end of a polypeptide-coding region and serves to terminate transcription. Usually, in prokaryotic cells, a transcription termination sequence is a GC-rich fragment followed by a poly-T sequence. While the sequence can be readily cloned from a library or even purchased as part of a vector, it can also be easily synthesized using nucleic acid synthesis methods such as those described herein.

[0088] A selectable marker gene encodes a protein necessary for the survival and growth of a host cell cultivated in a selective culture medium. Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins in prokaryotic host cells, such as ampicillin, tetracycline, or kanamycin; (b) compensate for auxotrophic deficiencies in the cell; or (c) provide essential nutrients unavailable in complex or defined media. Specific selectable markers include the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. A neomycin resistance gene can also be advantageously used for selection in both prokaryotic and eukaryotic host cells.

[0089] Other selectable genes can be used to amplify the gene to be expressed. Amplification is the process by which genes required for the production of a protein essential for cell growth or survival are sequentially repeated in the chromosomes of successive generations of recombinant cells. Examples of suitable selectable markers for mammalian cells include the genes for dihydrofolate reductase (DHFR) and promoterless thymidine kinase. The mammalian cell transformants are subjected to selective pressure, with only those transformants appropriate for survival being adapted due to the presence of the selectable gene in the vector.Selection pressure is generated by culturing the transformed cells under conditions where the concentration of selection agent in the medium is successively increased. This leads to the amplification of both the selectable gene and the DNA encoding another gene, such as an antibody light or heavy chain. As a result, increased amounts of a polypeptide are synthesized from the amplified DNA.

[0090] To trigger mRNA translation, a ribosome binding site is usually required and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). This element is typically located 3' to the promoter and 5' to the coding sequence of the polypeptide to be expressed. In certain embodiments, one or more coding regions can be functionally linked to an internal ribosome binding site (IRES), enabling the translation of two open reading frames from a single RNA transcript.

[0091] In some cases, such as when glycosylation is desired in a eukaryotic host cell expression system, the various pre- or pro-sequences can be manipulated to improve glycosylation or yield. For example, the peptidase cleavage site of a particular signal peptide can be altered, or pro-sequences can be added, which can also affect glycosylation. The finished protein product may have one or more additional amino acids at position -1 (relative to the first amino acid of the mature protein) that are generated during expression and may not have been completely removed. For example, the finished protein product may have one or two amino acid residues bound to the amino terminus at the peptidase cleavage site.Alternatively, the use of certain enzyme cleavage sites can lead to a slightly shortened form of the desired polypeptide if the enzyme cuts at such a region within the mature polypeptide.

[0092] Expression and cloning vectors of the invention will typically include a promoter that is recognized by the host mechanism and functionally linked to the molecule encoding the polypeptide. Promoters are non-transcribed sequences located upstream (i.e., 5') of the start codon of a structural gene (generally within about 100 to 1000 bp) and that control the transcription of the structural gene. Promoters are usually classified into one of two classes: inducible promoters and constitutive promoters. Inducible promoters trigger increased transcription levels of the DNA they control in response to a change in culture conditions, such as the presence or absence of a nutrient or a change in temperature. In contrast, constitutive promoters uniformly transcribe a gene to which they are functionally linked, i.e., with little or no control over gene expression.Numerous promoters are known in this field, which are recognized by various potential host cells. A suitable promoter is functionally linked to the DNA that encodes, for example, a heavy chain or light chain, by removing the promoter from the original DNA via restriction enzyme digestion and inserting the desired promoter sequence into the vector.

[0093] Suitable promoters for use with yeast hosts are also known in this field. Yeast enhancers are advantageously used with yeast promoters. Suitable promoters for use with mammalian host cells are known and include, but are not limited to, those derived from the genomes of viruses such as polyomavirus, avian poxvirus, adenovirus (e.g., adenovirus 2), bovine papillomavirus, avian sarcoma 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 the actin promoter.

[0094] Other promoters that may be of interest include, but are not limited to: the SV40 early promoter (Benoist and Chambon, 1981, Nature 290:304-310); the CMV promoter (Thomsen et al., 1984, Proc. Natl. Acad. USA 81:659-663); the promoter contained in the 3' terminal repeat of the Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797); the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:1444-1445); and promoter and regulatory sequences from the metallothionin gene (Prinster et al., 1982, Nature 296:39-42). and prokaryotic promoters, such as the beta-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731); or the TAC promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25).Also of interest are the following animal transcriptional control regions, which exhibit tissue specificity and have been used in transgenic animals: the elastase I gene control region, which is 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 control region, which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122); the immunoglobulin gene control region, which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Zelle. Biol. 7:1436-1444); the mouse mammary tumor virus control region, which is active in testicular, breast, lymphoid, and mast cells (Leder et al., 1986, Cell 45:485-495); the albumin gene control region, which is active in the liver (Pinkert et al., 1987, Gene and Devel.1:268-276); the alpha-fetoprotein gene control region active in the liver (Krumlauf et al., 1985, Mol. Zelle. Biol. 5:1639-1648; Hammer et al., 1987, Science 253:53-58); the alpha 1-antitrypsin gene control region active in the liver (Kelsey et al., 1987, Gene and Devel. 1:161-171); the beta-globin gene control region active in myeloid cells (Magram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94); the myelin basic protein gene control region, which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); the myosin light chain 2 gene control region, which is active in skeletal muscle (Sani, 1985, Nature 314:283-286); and the gonadotropic hormone-releasing gene control region, which is active in the hypothalamus (Mason et al., 1986, Science, 234: 1372:1378).

[0095] An enhancer sequence can be introduced into the vector to increase the transcription of DNA encoding a light chain or heavy chain according to the invention by higher eukaryotes. Enhancers are cis-acting DNA elements, typically about 10–300 bp long, that act on the promoter to enhance transcription. Enhancers are relatively independent of orientation and position and have been found at positions both 5' and 3' relative to the transcription unit. Several enhancer sequences are known to be derived from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin). However, a viral enhancer is usually used. The SV40 enhancer, the early promoter enhancer of cytomegalovirus, the polyomavirus enhancer, and the adenovirus enhancers known in the field are exemplary enhancer elements for the activation of eukaryotic promoters.An enhancer can be positioned either 5' or 3' from a coding sequence in the vector, but is usually located 5' from the promoter. A sequence encoding a suitable native or heterologous signaling sequence (leader sequence or signal peptide) can be incorporated into an expression vector to promote extracellular secretion of the antibody. The choice of signal peptide or leader sequence depends on the type of host cells in which the antibody is to be produced, with a heterologous signaling sequence being able to replace the native signaling sequence. Examples of signal peptides that are functional in mammalian host cells include the following: the signaling sequence for interleukin-7 (IL-7), described in US Patent No. 4,965,195; the signaling sequence for the interleukin-2 receptor, described in Cosman et al., 1984, Nature 312:768; the interleukin-4 receptor signaling peptide, described in EP patent no.0367 566; the type I interleukin-1 receptor signaling peptide, described in US Patent No. 4,968,607; the type II interleukin-1 receptor signaling peptide, described in EP Patent No. 0 460 846.

[0096] The vector may contain one or more elements that facilitate expression when integrated into the host cell genome. Examples include an EASE element (Aldrich et al. 2003 Biotechnol Prog. 19:1433-38) and a matrix attachment region (MAR). MARs mediate structural organization of chromatin and can isolate the integrated vector from the positional effect. MARs are therefore particularly useful when the vector is used to generate stable transfectants. A number of natural and synthetic MAR-containing nucleic acids are known in the field, e.g., US Pat. 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; 7,129,062.

[0097] Expression vectors according to the invention can be constructed from a starting vector, such as a commercially available vector. Such vectors may, but need not, contain all desired flanking sequences. If one or more of the flanking sequences described herein are not already present in the vector, they can 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.

[0098] Once the vector has been constructed and a nucleic acid molecule encoding a light chain, a heavy chain, or both a light chain and a heavy chain has been inserted into the correct position within the vector, the completed vector can be introduced into a suitable host cell for amplification and / or polypeptide expression. The transformation of an expression vector into a selected host cell can be carried out by known methods, including transfection, infection, calcium phosphate coprecipitation, electroporation, microinjection, lipofection, DEAE-dextran-mediated transfection, or other known methods. The method chosen depends, in part, on the type of host cell 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, supra. Methods for the production of proteins

[0099] A host cell, when cultured under suitable conditions, synthesizes a heterodimeric antibody, which can then be extracted from the culture medium (if the host cell secretes it into the medium) or directly from the host cell that produces it (if it is not secreted). The selection of a suitable host cell depends on various factors, such as the desired level of expression, the modifications of the polypeptide desired or necessary for activity (such as glycosylation or phosphorylation), and the ease of folding into a biologically active molecule. A host cell can be eukaryotic or prokaryotic.

[0100] Mammalian cell lines available as hosts for expression are known in the field and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC) and any cell lines used in an expression system known in the field to produce the recombinant polypeptides of the invention. Generally, host cells are transformed with a recombinant expression vector comprising DNA encoding a desired heterodimeric antibody. Host cells that may be used include prokaryotes, yeast, or higher eukaryotic cells. Prokaryotes include Gram-negative or Gram-positive organisms, such as E. coli or Bacilli. Higher eukaryotic cells include insect cells and known 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 ovarian cells (CHO) or their derivatives such as veggie CHO and related cell lines grown in serum-free media (Rasmussen et al., 1998, Cytotechnology 28:31), HeLa cells, BHK cell lines (ATCC CRL 10), and the CV1 / EBNA cell line derived from the CV1 cell line of African green monkeys (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 strains derived from in vitro culture of primary tissue, primary explants, HL-60, U937, HaK or Jurkat cells. If appropriate, mammalian cell lines, such asHepG2 / 3B, KB, NIH 3T3, or S49 can be used to express the polypeptide if it is desirable to use the polypeptide in various signal transduction or reporter assays. Alternatively, it is possible to produce the polypeptide in lower eukaryotes, such as yeast, or in prokaryotes, such as bacteria. Suitable yeasts include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, and any yeast strain capable of expressing heterologous polypeptides. Suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, and any bacterial strain capable of expressing heterologous polypeptides.

[0101] If the antibody or antibody fragment is produced in yeast or bacteria, it may be desirable to modify the product produced therein, for example by phosphorylation or glycosylation of the relevant sites, to obtain a functional product. Such covalent linkages can be achieved using known chemical or enzymatic methods. A polypeptide can also be produced by functionally linking the isolated nucleic acid of the invention with suitable control sequences in one or more insect expression vectors and using an insect expression system. Materials and methods for baculovirus / insect cell expression systems are commercially available in kits, e.g., from Invitrogen, San Diego, CA, USA (the MaxBac® kit), and such methods are known in the prior 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 could also be used to produce polypeptides, such as antibodies or antibody fragments, using RNAs derived from nucleic acid constructs disclosed herein. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian host cells are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985). A host cell comprising an isolated nucleic acid according to the invention, preferably functionally linked to at least one expression control sequence, is a “recombinant host cell”.

[0102] In certain embodiments, cell lines can be selected by determining which cell lines exhibit high expression levels and constitutively produce antigen-binding proteins with the desired binding properties. In another embodiment, a cell line can be selected from the B-cell line that does not produce its own antibody but has the ability to produce and secrete a heterologous antibody. OTHER VERSIONS

[0103] The following are some special embodiments of the invention. 1. Isolated protein comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH and VL are linked to each other, and wherein the VH and VL comprise at least one of the following sets of charged amino acids: a. VH comprises a charged amino acid at position 39 and VL comprises a charged amino acid at position 85, which is charge complementary to the amino acid at position 39 of VH; b. the VH comprises a charged amino acid at position 105 and the VL comprises a charged amino acid at position 42, which is charge-complementary to the amino acid at position 105 of the VH; or c. VH comprises a charged amino acid at position 91 and VL comprises a charged amino acid at position 38, which is charge complementary to the amino acid at position 91 of VH; and wherein the position numbers of the charged amino acids in the VH and VL domains refer to their positions according to the Kabat numbering scheme. 2. Isolated protein of embodiment 1, wherein VH and VL comprise the set of charged amino acids in (a). 3. Isolated protein of embodiment 2, wherein VH comprises a positively charged amino acid at position 39 and VL comprises a negatively charged amino acid at position 85. 4. Isolated protein of embodiment 3, wherein VH position 39 (VH39) and VL position 85 (VL85) comprise: (i) a lysine at VH39 and an aspartic acid at VL85, (ii) a lysine at VH39 and a glutamic acid at VL85, (iii) an arginine at VH39 and an aspartic acid at VL85 or (iv) an arginine at VH39 and a glutamic acid at VL85. 5. Isolated protein of embodiment 2, wherein VH comprises a negatively charged amino acid at position 39 and VL comprises a positively charged amino acid at position 85. 6. Isolated protein of embodiment 5, wherein VH position 39 (VH39) and VL position 85 (VL85) comprise: (i) an aspartic acid at VH39 and a lysine at VL85, (ii) a glutamic acid at VH39 and a lysine at VL85, (iii) an aspartic acid at VH39 and an arginine at VL85 or (iv) a glutamic acid at VH39 and an arginine at VL85. 7. Isolated protein according to one of embodiments 1-6, wherein VH and VL comprise the set of charged amino acids in (b). 8. Isolated protein of embodiment 7, wherein VH comprises a positively charged amino acid at position 105 and VL comprises a negatively charged amino acid at position 42. 9. Isolated protein of embodiment 8, wherein VH position 105 (VH105) and VL position 42 (VL42) comprise: (i) a lysine at VH105 and an aspartic acid at VL42, (ii) a lysine at VH105 and a glutamic acid at VL42, (iii) an arginine at VH105 and an aspartic acid at VL42 or (iv) an arginine at VH105 and a glutamic acid at VL42. 10. Isolated protein of embodiment 7, wherein VH comprises a negatively charged amino acid at position 105 and VL comprises a positively charged amino acid at position 42. 11. Isolated protein of embodiment 10, wherein VH position 105 (VH105) and VL position 42 (VL42) comprise: (i) an aspartic acid at VH105 and a lysine at VL42, (ii) a glutamic acid at VH105 and a lysine at VL42, (iii) an aspartic acid at VH105 and an arginine at VL42 or (iv) a glutamic acid at VH105 and an arginine at VL42. 12. Isolated protein according to one of embodiments 1-11, wherein VH and VL comprise the set of charged amino acids in (c). 13. Isolated protein of embodiment 12, wherein VH comprises a positively charged amino acid at position 91 and VL comprises a negatively charged amino acid at position 38. 14. Isolated protein of embodiment 13, wherein VH position 91 (VH91) and VL position 38 (VL38) comprise: (i) a lysine at VH91 and an aspartic acid at VL38, (ii) a lysine at VH91 and a glutamic acid at VL38, (iii) an arginine at VH91 and an aspartic acid at VL38 or (iv) an arginine at VH91 and a glutamic acid at VL38. 15. Isolated protein of embodiment 12, wherein VH comprises a negatively charged amino acid at position 91 and VL comprises a positively charged amino acid at position 38. 16. Isolated protein of embodiment 15, wherein VH position 91 (VH91) and VL position 38 (VL38) comprise: (i) an aspartic acid at VH91 and a lysine at VL38, (ii) a glutamic acid at VH91 and a lysine at VL38, (iii) an aspartic acid at VH91 and an arginine at VL38 or (iv) a glutamic acid at VH91 and an arginine at VL38. 17. Isolated protein according to one of embodiments 1-16, wherein the isolated protein comprises an antibody heavy chain comprising the VH and an antibody light chain comprising the VL. 18. Isolated protein of embodiment 17, wherein the isolated protein is an antibody. 19. Isolated protein of embodiment 18, wherein the antibody is an IgG antibody. 20. Isolated protein of embodiment 19, wherein the antibody is an IgG1, IgG2, IgG3 or IgG4 antibody. 21. Isolated protein according to one of embodiments 17-20, wherein the isolated protein is a bispecific antibody. 22. Isolated protein of embodiment 21, wherein the bispecific antibody comprises two arms and each arm of the antibody comprises a VH-VL pair with the set of charged amino acids in (a), (b) or (c). 23. Isolated protein according to one of embodiments 18-22, wherein the antibody comprises a first human IgG-CH3 domain (CH3) and a second human IgG-CH3 domain (CH3'), wherein the CH3 domain comprises a replacement of the amino acids at positions 392, 409 and 439 by a negatively charged amino acid, wherein the CH3' domain comprises a replacement of the amino acids at positions 356 and 399, and wherein the position numbers of the charged amino acids in the CH3 and CH3' domains refer to their positions according to the EU numbering scheme. 24. Isolated protein according to one of embodiments 18-23, wherein the antibody comprises a first pair of heavy chain constant domain and light chain constant domain (CH1 and CL) and a second pair of heavy chain constant domain and light chain constant domain (CH1' and CL'); where CH1 and CH1' each comprise a change at position 183, as numbered according to the EU numbering scheme; where CL and CL' comprise a change at position 176, as numbered according to the EU numbering scheme; where CH1 position 183 and CL' position 176 each comprise a positively charged amino acid; and where CH1' position 183 and CL position 176 each comprise a negatively charged amino acid. 25. Isolated protein of embodiment 24, wherein (i) CH1 position 183 and CL position 176 each comprise lysine and CH1' position 183 and CL position 176 each comprise aspartic acid; (ii) CH1 position 183 and CL' position 176 each comprise lysine and CH1' position 183 and CL position 176 each comprise glutamic acid; (iii) CH1 position 183 and CL' position 176 each comprise arginine and CH1' position 183 and CL position 176 each comprise aspartic acid; or (iv) CH1 position 183 and CL' position 176 each comprise arginine and CH1' position 183 and CL position 176 each comprise glutamic acid. 26. Bispecific antibody comprising a first pair of heavy chain variable domain and light chain variable domain (VH and VL) and a second pair of heavy chain variable domain and light chain variable domain (VH' and VL'), wherein the VH and VL pair and the VH' and VL' pair comprise at least one of the following sets of charged amino acids: a. VH and VH' each comprise a charged amino acid at position 39; VL and VL' each comprise a charged amino acid at position 85, which is complementary to the amino acid at position 39 of the VH or VH'; and VH and VH' comprise amino acids with complementary charges; b. VH and VH' each comprise a charged amino acid at position 105; VL and VL' each comprise a charged amino acid at position 42, which is complementary to the amino acid at position 105 of the VH or VH'; and VH and VH' comprise amino acids with complementary charges; or c. VH and VH' each comprise a charged amino acid at position 91; VL and VL' each comprise a charged amino acid at position 38, which is complementary to the amino acid at position 91 of the VH or VH'; and VH and VH' comprise amino acids with complementary charges; and wherein the position numbers of the charged amino acids in the VH and VL domains refer to their positions according to the Kabat numbering scheme. 27. Bispecific antibody of embodiment 26, wherein the VH and VL pair and the VH' and VL' pair comprise the set of charged amino acids in (a). 28. Bispecific antibody of embodiment 27, wherein VH and VL' comprise a positively charged amino acid and wherein VH' and VL comprise a negatively charged amino acid. 29. Bispecific antibody of embodiment 28, wherein the VH and VL pair and the VH' and VL' pair comprise: (i) a lysine at VH39, a lysine at VL'85, an aspartic acid at VH'39 and an aspartic acid at VL85; (ii) a lysine at VH39, a lysine at VL'85, a glutamic acid at VH'39 and a glutamic acid at VL85; (iii) an arginine at VH39, an arginine at VL'85, an aspartic acid at VH'39 and an aspartic acid at VL85; or (iv) an arginine at VH39, an arginine at VL'85, a glutamic acid at VH'39 and a glutamic acid at VL85. 30. Bispecific antibody of embodiment 27, wherein VH and VL' comprise a negatively charged amino acid and wherein VH' and VL comprise a positively charged amino acid. 31. Bispecific antibody of embodiment 30, wherein the VH and VL pair and the VH' and VL' pair comprise: (i) an aspartic acid at VH39, an aspartic acid at VL'85, a lysine at VH'39 and a lysine at VL85; (ii) a glutamic acid at VH39, a glutamic acid at VL'85, a lysine at VH'39 and a lysine at VL85; (iii) an aspartic acid at VH39, an aspartic acid at VL'85, an arginine at VH'39 and an arginine at VL85; or (iv) a glutamic acid at VH39, a glutamic acid at VL'85, an arginine at VH'39 and an arginine at VL85. 32. Bispecific antibody according to one of embodiments 25-31, wherein the VH and VL pair and the VH' and VL' pair comprise the set of charged amino acids in (b). 33. Bispecific antibody of embodiment 32, wherein VH and VL comprise a positively charged amino acid and wherein VH' and VL comprise a negatively charged amino acid. 34. Bispecific antibody of embodiment 33, wherein the VH and VL pair and the VH' and VL' pair comprise: (i) a lysine at VH105, a lysine at VL42, an aspartic acid at VH'105 and an aspartic acid at VL42; (ii) a lysine at VH105, a lysine at VL'42, a glutamic acid at VH'105 and a glutamic acid at VL42; (iii) an arginine at VH105, an arginine at VL'42, an aspartic acid at VH'105 and an aspartic acid at VL42, or (iv) an arginine at VH105, an arginine at VL'42, a glutamic acid at VH'105 and a glutamic acid at VL42. 35. Bispecific antibody of embodiment 32, wherein VH and VL' comprise a negatively charged amino acid and wherein VH' and VL comprise a positively charged amino acid. 36. Bispecific antibody of embodiment 35, wherein the VH and VL pair and the VH' and VL' pair comprise: (i) an aspartic acid at VH105, an aspartic acid at VL'42, a lysine at VH'105 and a lysine at VL42; (ii) a glutamic acid at VH105, a glutamic acid at VL'42, a lysine at VH'105 and a lysine at VL42; (iii) an aspartic acid at VH105, an aspartic acid at VL'42, an arginine at VH'105 and an arginine at VL42; or (iv) a glutamic acid at VH105, a glutamic acid at VL'42, an arginine at VH'105 and an arginine at VL42. 37. Bispecific antibody according to one of embodiments 26-36, wherein the VH and VL pair and the VH' and VL' pair comprise the set of charged amino acids in (c). 38. Bispecific antibody of embodiment 37, wherein VH and VL' comprise a positively charged amino acid and wherein VH' and VL comprise a negatively charged amino acid. 39. Bispecific antibody of embodiment 38, wherein the VH and VL pair and the VH' and VL' pair comprise: (i) a lysine at VH91, a lysine at VL'38, an aspartic acid at VH'91 and an aspartic acid at VL38; (ii) a lysine at VH91, a lysine at VL'38, a glutamic acid at VH'91 and a glutamic acid at VL38; (iii) an arginine at VH91, an arginine at VL'38, an aspartic acid at VH'91 and an aspartic acid at VL38; or (iv) an arginine at VH91, an arginine at VL'38, a glutamic acid at VH'91 and a glutamic acid at VL38. 40. Bispecific antibody of embodiment 37, wherein VH and VL' comprise a negatively charged amino acid and wherein VH' and VL comprise a positively charged amino acid. 41. Bispecific antibody of embodiment 40, wherein the VH and VL pair and the VH' and VL' pair comprise: (i) an aspartic acid at VH91, an aspartic acid at VL'38, a lysine at VH'91 and a lysine at VL38; (ii) a glutamic acid at VH91, a glutamic acid at VL'38, a lysine at VH'91 and a lysine at VL38; (iii) an aspartic acid at VH91, an aspartic acid at VL'38, an arginine at VH'91 and an arginine at VL38; or (iv) a glutamic acid at VH91, a glutamic acid at VL'38, an arginine at VH'91 and an arginine at VL38. 42. Method for producing a bispecific antibody, comprising expressing one or more polynucleotides encoding the bispecific antibody according to one of embodiments 26-41 in host cells, cultivating the host cells in a culture medium under conditions in which the component polypeptide chains are produced, and obtaining the bispecific antibody from the cell or the culture medium. 43. Method of embodiment 42, wherein the host cells comprise one or more plasmids comprising the one or more polynucleotides. 44. Method of embodiment 42, wherein the host cells are a Chinese Hamster Ovary (CHO) cell line or a human embryonic kidney (HEK) cell line. 45. Method of embodiment 42, wherein the obtained bispecific antibody is purified by protein A chromatography. 46. ​​Method according to one of embodiments 42-45, wherein the obtained bispecific antibody is further purified by ion exchange chromatography. 47. Method for producing an isolated protein, comprising expressing one or more polynucleotides encoding a protein according to any embodiment 1-25 in host cells, cultivating the host cells in a culture medium under conditions in which the component polypeptide chains are produced, and obtaining the protein from the cell or the culture medium. 48. Method of embodiment 47, wherein the host cells comprise one or more plasmids comprising the one or more polynucleotides. 49. Method of embodiment 47 or embodiment 48, wherein the isolated protein is purified by ion exchange chromatography. EXAMPLE EXAMPLE 1: ANALYSIS OF FV STRUCTURES FOR CANDIDATE CHARGE PAIR MUTATIONS

[0104] Since the Fv interface accounts for up to 50% of the total heavy-light interaction ( Fig. 1) Since the polypeptide chain can encompass a wide range of components, we assume that manipulation solutions localized at the constant CH-CL interface may not be sufficient to attract / repel the entire polypeptide chain. To develop improved methods for facilitating pairing between certain desirable heavy- and light-chain polypeptides, research has been undertaken to identify sites within the heavy-chain variable region (VH) and the light-chain variable region (VL) where charge-pair mutations could be introduced. The overall goal was to identify novel paired mutations that would contribute to an unstable high-energy state when undesired VH and VL combinations bind (i.e., when VH and VL are mispaired). Conversely, novel paired mutations should contribute to a stable low-energy state when the desired VH and VL combination binds.

[0105] Fig. 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, with the CDR regions of each heavy-and-light chain pair combining to create a binding site for an antigen (antigen 1 or antigen 2, as shown in Figure 1). Fig. (Figure 1A shows this). In such a bispecific antibody, there are four different interaction zones. First, the VH and VL regions interact via an Fv interface in each arm of the antibody. Second, the heavy chain constant domain 1 (CH1) and the light chain constant domain (CL) form a CH1-CL interface. Third, the heavy chain constant domain 2 (CH2) forms a CH2-CH2 interface in each of the heavy chains. And fourth, the heavy chain constant domain 3 (CH3) forms a CH3-CH3 interface in each of the heavy chains. The CH2-CH2 and CH3-CH3 interfaces are responsible for holding the two arms of the antibody together but do not affect the pairing between each heavy chain and its corresponding light chain.

[0106] Fig. Figure 1B shows examples of unwanted byproducts formed when the incorrect light chain pairs with one or both of the heavy chains of the bispecific antibody. These byproducts generally reduce the expression of a desired bispecific antibody by using protein chains that could otherwise be assembled into the desired product.

[0107] A series of known VH-VL interaction structures were used as a starting point for in silico analysis. An exemplary structure showing the interaction between a VH domain (SEQ ID NO: 1) and a VL domain (SEQ ID NO: 5) is presented in Fig. Figure 2A shows the structure of the anti-HIV antibody B12. This exemplary structure includes dotted lines between the domains, representing distance-based interaction lines between atoms in the VH and VL domains, as calculated using PyMol. Each of the residues involved in these interactions was a candidate for a possible charge-pair mutation.

[0108] An in silico analysis was performed to identify new sites for charge-pair mutations. The aim of this analysis was to identify locations where paired mutations could be introduced such that the introduced changes would lead to an unstable high-energy state in undesired "mispaired" states and a stable lower-energy state in the desired pairing pattern. Fig. Figure 2B illustrates this dynamic. Fig. Figure 2B contains the undesired states (“mismatched antibody 1” and “mismatched antibody 2”), unstable mismatches in which similarly charged amino acid residues are forced into close proximity, as shown by the paired open circles or paired filled circles. These unstable mismatches lead to a high-energy state. In contrast, the desired states (antibody 1 or antibody 2) have a lower energy state because oppositely charged residues are present at the same position. Accordingly, the energy difference would drive the antibody chains to assemble into an equilibrium state with a strong predominance of the desired pairing state. Fig. In 2B, this is shown by the larger arrows pointing towards antibody 1 and antibody 2.

[0109] 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 based on their proximity for potential charge-pairing mutations with each VH residue. This resulted in the selection of between one and nine residues for pairing with each VH residue and a total of sixty-nine potential CPMs for in silico analysis.

[0110] As a first step in the in silico analysis, each of the identified potential charge-pair mutations at selected positions in the Fv was designed and evaluated using Rosetta via XML script (RosettaScripts) in the talaris2014 score function. Asp, Glu, Lys, and Arg mutations were performed in pairs 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 with mutations in both correctly paired (+ / -) and mispaired (+ / + and - / -) combinations. This procedure was repeated using the structure of 18 different Fv sequences, which were combined to form 54 different Fv pairs as input structures. Using this strategy, thousands of pairing designs were examined to identify a subset of promising candidates for experimental validation.Desirable mutations were selected based on the fact that they consistently exhibit a high difference in the total Rosetta score between the correctly paired and mispaired chains across all input structures, and that the total Rosetta score of the correctly paired combination is as low as possible as a prediction of the energy state of the Fv pair. An example output is shown in the diagram in . Fig. Figure 2C shows that the predicted energy state for one mismatched antibody is much higher than that of each of the correctly matched antibodies, while the predicted energy state for the other mismatched antibody is slightly higher than that of each of the correctly matched antibodies.

[0111] In silico analysis, several CPM designs were predicted to function well in various Fv scaffolds. In addition to the previously described VH:39-VL:38 design (Lewis, SM, et al., Nat Biotechnol, 32(2):191-198 (2014)), several additional pairings were identified that resulted in a stable interface. In particular, the introduction of complementary charged mutations at positions VH:39-VL:85, VH:105-VL:42, and VH:91-VL:38 (as numbered according to the Kabat scheme) was consistently predicted to function well, regardless of the Fv scaffold analyzed. The positions of these newly identified CPMs are shown in the exemplary VH and VL sequences in [reference missing]. Fig. 7A and Fig. 7B, which originate from the anti-HIV antibody B12, were shown. In Fig. 7A and Fig. 7B, the VH and VL sequences for each residue and the position of the CDR and scaffold regions are identified by the Kabat numbering. Fig. 7A and Fig. 7B also contains boxes that highlight the amino acids that would be changed upon introduction of a charge pair mutation at VH:39-VL:85 (CPM pair A), VH:105-VL:42 (CPM pair B) or VH:91-VL:38 (CPM pair C). EXAMPLE 2: PLASMID CONSTRUCTION, CELL CULTURE AND PROTEIN EXPRESSION

[0112] Unless otherwise stated, plasmid construction, cell culture, plasmid transfection, protein expression and antibody purification, which are referenced in subsequent examples, were performed as described in this example.

[0113] Plasmid construction: Genes intended for polypeptide expression were synthesized by Twist Bioscience. Genes were individually cloned into vectors for transient mammalian expression using the Golden Gate assembler method. (See Engler C et al., PLoS One 3:e3647 (2008), incorporated herein by reference in their entirety.) All chains containing Fc regions were constructed using a human IgG1 scaffold (IgG1-SEFL2) carrying an aglycosylation mutation and an engineered disulfide bond. (See Estes et al., iScience 24:103447 (2021); and Jacobsen FW et al., J. Biol. Chem. 292:1865-75 (2017), incorporated herein by reference in their entirety.) All molecules requiring hetero-Fc pairing contained charge-pair mutations (“CPM”) v103 in the CH3 regions to promote heterodimerization. (See Estes et al., iScience 24:103447 (2021)).In short, the v103 mutation patterns contain 392D, 409D, and 439D mutations in one heavy chain and 356K and 399K mutations in the other heavy chain (as numbered according to the EU antibody numbering scheme). Other CPMs were also incorporated into the heavy and light chain sequences, as indicated below.

[0114] Following synthesis and cloning, the plasmid sequences of Sanger were confirmed and transfection-quality DNA was produced using maxi-plasmid purification kits (Qiagen).

[0115] Cell culture, plasmid transfection, and protein expression: Proteins were transiently expressed in human embryonic kidney cells 293 6E (NRC-BRI) using the PEImax transfection reagent in suspension. Prior to transfection, the cells were maintained in FreeStyle F-17 medium (Gibco, catalog no. A1383502) containing 0.1% colliphore P188 (Sigma, catalog no. K4894), 25 µg / ml G418 (Gibco, catalog no. 10131027), and 6 µg L-glutamine (Gibco, catalog no. 25030149). To achieve an optimal density of 2 × 10 6To achieve a certain number of viable cells per ml for transfection, the cells were subcultured for 26 hours prior to transfection. The purified plasmids were mixed in a 1:1:1:1 ratio for the tested four-chain hetero-IgGs. For each ml of cell medium, 0.5 µg of DNA was incubated with 1.5 µl of PEImax reagent (Polysciences, catalog no. 24765-2) in 100 µl of FreeStyle F-17 medium for 10 minutes and then added to the cell culture. Twenty-four hours after transfection, cell cultures were treated with Tryptone N1 solution (Organotechnie, catalog no. 19553) and glucose (Thermo Fisher, catalog no. A2494001) to final concentrations of 2.5 g / l and 4.5 g / l, respectively. Seven days after transfection, the conditioned medium was harvested for purification.

[0116] Antibody purification with protein A and cation exchange chromatography: After harvesting 40 ml of culture, the conditioned medium was purified by protein A affinity capture (1 ml HiTrap MabSelect Sure, GE Life Sciences, catalog # GE11-0034-93), equilibrated with 25 mmol Tris, 100 mmol NaCl, pH 7.4. Protein was eluted with 100 mmol sodium acetate, pH 3.6, immediately followed by buffer exchange in 10 mmol sodium acetate, 150 mmol NaCl, pH 5.2, using a 5 ml HiTrap desalination column (GE Life Sciences, catalog # GE17-1408-01).

[0117] Cation exchange chromatography (CIEX) was performed to analyze and separate antibody species present in protein A purifications. Protein A eluates were diluted with 20 ml of 20 mm MES, pH 6.2, and loaded at 1 ml / min onto a 1 ml cation exchange column (HiTrap SP-HP, GE Life Sciences, catalog no. GE29-0513-24). After washing with 8 column volumes of dilution buffer at 1 ml / min, the samples were eluted with a linear gradient of 0–400 mm NaCl over 40 column volumes at 0.4 ml / min.

[0118] To identify species in fractions collected during CIEX or to analyze sample purity, samples were analyzed by non-reducing microcapillary electrophoresis (MCE) and analytical size exclusion chromatography (SEC). For non-reducing MCE, 6 µl of protein were 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 to 85 °C for 10 min, and analyzed using a LabChip GXII Touch caliper instrument (PerkinElmer). For analytical SEC analysis, protein samples were analyzed on an Acquity HPLC instrument (Waters) using a Zenix C column (300 Å, 3 micrometers, 4.6 × 300 mm) (Sepax Technologies) with 100 mmol sodium phosphate, pH 6.8, and 250 mmol NaCl as running buffer at a flow rate of 0.45 ml / min. Fraction pool concentrations were determined using NanoDrop 8000 (Thermo Fisher). EXAMPLE 3: ANALYSIS OF CONTAMINATING SPECIES IN THE PRODUCTION OF ANTIBODIES WITH CHARGE PAIR MUTATIONS

[0119] Antibodies containing complementary charged mutations at positions VH:91-VL:38, VH:39-VL:85, and VH:105-VL:42 were tested in bispecific antibodies, both with and without the presence of other complementary charge pair mutations (CPMs) in the antibody's Fab region. Each set of complementary mutations was tested against a background of an IgG1 antibody containing a set of known charge pair mutations in the heavy chain Fc region. This background allowed testing of the novel complementary mutations at the heavy chain-light chain interaction only, with reduced contamination from heavy chain mismatch. Additionally, some tested antibodies contained a charge pair mutation at the CH1-CL interface. Tested antibodies are listed in Table 1 below, along with the CPMs present in each of the antibody domains.Thus, all antibodies listed in Table 1 are IgG1 antibodies, with the only differences between them being in the CDR regions (i.e., changes in specificity for binding antigen A or antigen C) and in the listed charge pair mutations. TABLE 1 BispezifischerAntikörper Zielantigene CPMs 1 A (HC und LC)C (HC' und LC') Fv-CPMs: Keine CH1 / CL-CPMs: Keine Fc-CPMs: CH3: 392D; 409D; 439DCH3': 356K; 399K 2 A (HC und LC)C (HC' und LC') Fv-CPMs: Keine CH1 / CL-CPMs: CH1: 183E / CL: 176KCH1': 183K / CL': 176E Fc-CPMs: CH3: 392D; 409D; 439DCH3': 356K; 399K 3 A (HC und LC)C (HC' und LC') Fv-CPMs: VH: 39E / VL: 85RVH': 39R / VL': 85E CH1 / CL-CPMs: CH1: 183E / CL: 176KCH1': 183K / CL': 176E Fc-CPMs: CH3: 392D; 409D; 439DCH3': 356K; 399K 4 A (HC und LC)C (HC' und LC') Fv-CPMs: VH: 39E / VL: 85RVH': 39R / VL': 85E CH1 / CL-CPMs: Keine Fc-CPMs: CH3: 392D; 409D; 439DCH3': 356K; 399K 5 A (HC und LC)C (HC' und LC') Fv-CPMs: VH: 39E / VL: 38KVH': 39K / VL': 38E CH1 / CL-CPMs: CH1: 183E / CL: 176KCH1': 183K / CL': 176E Fc-CPMs: CH3: 392D; 409D; 439DCH3': 356K; 399K 6 B (HC und LC)D (HC' und LC') Fv-CPMs: Keine CH1 / CL-CPMs: Keine Fc-CPMs: CH3: 392D; 409D; 439DCH3': 356K; 399K 7 B (HC und LC)D (HC' und LC') Fv-CPMs: Keine CH1 / CL-CPMs: CH1: 183E / CL: 176KCH1': 183K / CL': 176E Fc-CPMs: CH3: 392D; 409D; 439DCH3': 356K; 399K 8 B (HC und LC)D (HC' und LC') Fv-CPMs: VH: 105R / VL: 42EVH': 105E / VL': 42R CH1 / CL-CPMs: CH1: 183E / CL: 176KCH1': 183K / CL': 176E Fc-CPMs: CH3: 392D; 409D; 439DCH3': 356K; 399K 9 E (HC und LC)F (HC' und LC') Fv-CPMs: VH: 91R / VL: 38EVH': 91E / VL': 38R CH1 / CL-CPMs: CH1: 183E / CL: 176KCH1': 183K / CL': 176E Fc-CPMs: CH3: 392D; 409D; 439DCH3': 356K; 399K

[0120] Constructs for the expression of each of the bispecific antibodies in Table 1 were prepared according to the procedures described in Example 2. In short, to express these antibodies, the four antibody chains for a given antibody were transfected into HEK-293 cells in a 1:1:1:1 ratio (also as described in Example 2). Seven days after transfection, the conditioned medium was harvested for purification of secreted antibodies. Initial purification was performed using protein A affinity capture. Subsequently, antibody species were separated by cation-exchange chromatography. Fig. Figures 3A-3E, 4A-4C, and 5 show chromatograms of the separation of purified antibody samples. As part of the CIEX chromatography procedure, individual fractions eluted from the CIEX column were collected sequentially and analyzed to determine the antibody species present in each major peak.

[0121] The chromatograms of Fig. 3A-3E are derived from tests of five different bispecific antibody variants, all targeting 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 domains present in the peptide chains responsible for binding antigen C are marked with the dash symbol ('), while the domains responsible for binding antigen A are not so marked. Each of Fig. 3A-3E further provides a list of charge-pair mutations present in the generated bispecific antibody. In addition to the differences between the listed CPM alterations, the bispecific antibodies in Fig. 3A-3E are identical in sequence. In Fig. 3A-3E are the locations of peaks containing specific antibody species, labeled with diagrams of the species and arrows pointing to the corresponding peak. In each chromatogram, the desired bispecific antibody is also highlighted by a box around its peak. The in Fig. The 3A-3E tested bispecific antibody variants were designed to allow testing of the efficacy of CPMs at the newly identified VH:91-VL:38 site to prevent antibody chain mismatch.

[0122] Fig. Figure 3A shows a CIEX chromatogram of a secreted antibody from cells expressing the components of bispecific antibody 1. Bispecific antibody 1 contains CPMs only in its Fc region, specifically in residues that are part of the heavy chain CH3-CH3' interface. Specifically, its CH3 domain contains the 392D, 409D, and 439D alterations, while its CH3' domain contains the 356K and 399K alterations (as numbered according to the EU antibody numbering scheme). In addition to a peak corresponding to bispecific antibody 1, it contains Fig. 3A peaks corresponding to at least three contaminating species. While the desired bispecific antibody peak is the largest in Fig. 3A also contains significant amounts of contaminating alternative antibody species.

[0123] Fig. Figure 3B shows a CIEX chromatogram of a secreted antibody 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 a CPM at the CH1-CL and CH1'-CL' interfaces. The additional CPM consists of a 183E alteration in the CH1 domain and a 176K alteration in the CL domain, along with a 183K alteration in the CH1' domain and a 176E alteration in the CL' domain. In both Fig. 3A and Fig. 3B is the most prominent peak of the desired bispecific antibody. Both also contain peaks indicating contamination by alternative species. The contamination peaks for bispecific antibody 2 (in Fig. 3B) are present between approximately 108 and 115 ml, but are lower than those for bispecific antibody 1 (in Fig. 3A). However, expression and purification of an antibody containing both CPMs at the Fc interface and at the CH1-CL interface still yielded significant contaminants.

[0124] To determine whether introducing a newly identified CPM at the Fv interface (see Example 1 above) could reduce the presence of unwanted impurities in the fabrication of bispecific antibodies, “bispecific antibody 3” was constructed. Bispecific antibody 3 is identical to bispecific antibody 2, except that bispecific antibody 3 also contains a CPM at the VH-VL and VH'-VL' interfaces. Specifically, bispecific antibody 3 contains a CPM at the VH:39-VL:85 sites of each arm of the antibody. These CPMs comprise a 39E modification in the VH domain and an 85R modification in the VL domain, along with a 39R modification in the VH' domain and an 85E modification in the VL' domain. Fig. Figure 3C shows a CIEX chromatogram of a secreted antibody from cells expressing the components of bispecific antibody 3. Fig. 3C is the most prominent peak of the desired bispecific antibody. Between approximately 108 and 115 ml (where impurities for bispecific antibody 2 were detected), little to no contaminating antibody for bispecific antibody 3 was detected. The comparison of Fig. 3B and Fig. Figure 3C shows that the introduction of a CPM at the VH:39-VL:85 sites of each arm led to the elimination of almost all impurities observed in the purified antibody sample of bispecific antibody 2. This indicates that a CPM at the VH:39-VL:85 site can lead to more effective and complete pairing of antibody chains and may even improve pairing in the presence of other CPMs in an antibody.

[0125] The “bispecific antibody 4” was also constructed by combining CPMs at the VH:39-VL:85 site with CPMs at the CH3-CH3' interface. The heavy chain CH3-CH3' interface in bispecific antibody 4 contains the 392D, 409D, and 439D modifications in the CH3 domain, while the CH3' domain contains the 356K and 399K modifications (as numbered according to the EU antibody numbering scheme). Bispecific antibody 4 is identical to bispecific antibody 3, except that bispecific antibody 4 lacks CPMs at its CH1-CL interfaces. Bispecific antibody 4 differs from bispecific antibody 2 in that bispecific antibody 4 has CPMs in its VH-VL interfaces, while bispecific antibody 2 has CPMs in its CH1-CL interfaces. Fig. 3D shows a CIEX chromatogram of secreted antibodies from cells expressing the components of bispecific antibody 4. Fig. 3D is the most prominent peak of the desired bispecific antibody. Fig. 3D shows that between approximately 108 and 115 ml of bispecific antibody 4 contained fewer contaminating species than an antibody with only CPMs at the CH3-CH3' interface (as compared to the chromatogram of bispecific antibody 2 in Fig. 3A shown). Furthermore, the bispecific antibody 4 showed a different pattern of impurities than the bispecific antibody 2 - indicating that CPMs at the VH-VL interface and especially at the VH:39-VL:85 site have a complementary effect to those at the CH1-CL interface.

[0126] To enable the comparison of CPMs with the format VH:91-VL:38 with those with the previously known format VH:39-VL:38, the “bispecific antibody 5” was constructed and tested. Bispecific antibody 5 was constructed by combining CPMs at the VH:39-VL:38 site with CPMs at the CH3-CH3' interface. As with bispecific antibodies 1-4, the CH3 domain of bispecific antibody 5 contains the 392D, 409D, and 439D alterations, while its CH3' domain contains the 356K and 399K alterations (as numbered according to the EU antibody numbering scheme). As with bispecific antibodies 2 and 3, the bispecific antibody contains 5 CPMs in the CH1-CL and CH1'-CL' interfaces, comprising a 183E modification in the CH1 domain and a 176K modification in the CL domain, together with a 183K modification in the CH1' domain and a 176E modification in the CL' domain.Therefore, the comparison of bispecific antibody 3 and bispecific antibody 5 provides an indication of the relative effects of VH:91-VL:38-CPM with VH:39-VL:38-CPM. Fig. Figure 3E shows a CIEX chromatogram of secreted antibodies from cells expressing the components of bispecific antibody 5. The comparison of Fig. 3E with results using the bispecific antibody 3 in IG. 3C' show that in Fig. 3C showed that between approximately 108 and 115 ml fewer contaminants were present. This indicates that the VH:91-VL:38-CPM was better able than the VH:39-VL:38-CPM to target selective pairing when present in otherwise identical bispecific antibodies.

[0127] The chromatograms of Fig. 4A-4C are derived from tests of three different bispecific antibody variants, each targeting 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 domains located in the peptide chains responsible for binding antigen D are marked with the dash symbol ('), while the domains responsible for binding antigen B are not marked in this way. Each of Fig. 4A-4C further provides a list of charge-pair mutations present in the generated bispecific antibody. In addition to the differences between the listed CPM alterations, the bispecific antibodies in Fig. 4A-4C are identical in sequence. In Fig. 4A-4C indicate the positions of the peak containing the desired antibody with diagrams of the antibody and arrows pointing to the corresponding peak. In each chromatogram, the desired bispecific antibody is also highlighted by a box around its peak. The in Fig. The 4A-4C tested bispecific antibody variants were designed to allow testing of the efficacy of CPMs at the newly identified VH:105-VL:42 site to prevent antibody chain mismatch.

[0128] Fig. Figure 4A shows a CIEX chromatogram of a secreted antibody from cells expressing components of bispecific antibody 6. Bispecific antibody 6 contains CPMs only in its Fc region, specifically in residues that are part of the heavy chain CH3-CH3' interface. Specifically, its CH3 domain contains the 392D, 409D, and 439D alterations, while its CH3' domain contains the 356K and 399K alterations (as numbered according to the EU antibody numbering scheme). In addition to a peak corresponding to bispecific antibody 6, it also contains Fig. 4A peaks, corresponding to contaminating species, occur at approximately 86-89 ml and on the shoulder of the desired peak at approximately 92-93 ml. While the desired bispecific antibody peak is the largest in Fig. 4A also contains significant amounts of contaminating alternative antibody species.

[0129] Fig. Figure 4B shows a CIEX chromatogram of a secreted antibody from cells expressing the components of "bispecific antibody 7". Bispecific antibody 7 is identical to bispecific antibody 6, except that bispecific antibody 7 also contains a CPM at the CH1-CL and CH1'-CL' interfaces. The additional CPM consists of a 183E alteration in the CH1 domain and a 176K alteration in the CL domain, along with a 183K alteration in the CH1' domain and a 176E alteration in the CL' domain. In both Fig. 4A and Fig. 4B is the most prominent peak of the desired bispecific antibody. The introduction of CPMs into the CH1-CL and CH1'-CL' interfaces almost completely eliminated the interference from the desired species in Fig. 4B eluted peaks, where in Fig. A prominent peak was observed at 4A. Fig. However, 4B shows a peak indicating a contaminating alternative species at the shoulder of the desired peak at approximately 92-93 ml (as also in Fig. 4A observed). Thus, expression and purification of the antibody containing both CPMs at the Fc interface and at the CH1-CL interface still resulted in significant contamination.

[0130] To determine whether introducing a newly identified CPM at the Fv interface (see Example 1 above) could reduce the presence of unwanted impurities in the manufacture of bispecific antibodies, “bispecific antibody 8” was constructed. Bispecific antibody 8 is identical to bispecific antibody 7, except that bispecific antibody 8 also contains a CPM at the VH-VL and VH'-VL' interfaces. Specifically, bispecific antibody 8 contains a CPM at the VH:105-VL:42 sites of each arm of the antibody. These CPMs include a 105R modification in the VH domain and a 42E modification in the VL domain, along with a 105E modification in the VH' domain and a 42R modification in the VL' domain. Fig. Figure 4C shows a CIEX chromatogram of secreted antibodies from cells expressing the components of bispecific antibody 8. Fig. 4C is the most prominent peak of the desired bispecific antibody. As for bispecific antibody 7 in Fig. 4B observed that the CPMs of bispecific antibody 8 almost led to the elimination of the target species in Fig. 4C eluting peaks. The comparison of Fig. 4B and Fig. Figure 4C shows that introducing a CPM at the VH:105-VL:42 sites of each arm resulted in a reduction of the size of the contaminating peak on the shoulder of the desired peak at approximately 92-93 ml. This suggests that a CPM at the VH:105-VL:42 site can lead to more effective and complete pairing of antibody chains and may even improve pairing in the presence of other CPMs in an antibody.

[0131] The chromatogram of Fig. 5 is derived from a test 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 domains present in the peptide chains responsible for binding antigen F are marked with the dash symbol ('), while the domains responsible for binding antigen E are not marked in this way. Fig. Section 5 further provides a list of the charge-pair mutations present in the bispecific antibody 9. Fig. Figure 5 shows the positions of the peak containing bispecific antibody 9, labeled with an aa diagram of the antibody and an arrow pointing to the respective peak. In each chromatogram, the desired bispecific antibody is also highlighted by a box around its peak. The tested bispecific antibody variant in Fig. 5 was developed to test the efficacy of a CPM at the newly identified VH:91-VL:38 site to prevent antibody chain mismatch. Specifically, the bispecific antibody 9 contains a CPM at the VH:91-VL:38 sites of each arm of the antibody. These CPMs comprise a 91R modification in the VH domain and a 38E modification in the VL domain, along with a 91E modification in the VH' domain and a 38R modification in the VL' domain. As described in Fig. As seen in Figure 5, the introduction of CPMs at the VH:91-VL:38 sites resulted in highly effective pairing of antibody chains with low levels of contaminating mismatched antibody species. EXAMPLE 4: PRODUCTION LEVELS OF ANTIBODIES CONTAINING CHARGE-PAIR MUTATIONS

[0132] Experiments were also conducted to investigate whether introducing charge-pair mutations into the VH:VL interface could lead to increased production and purification of the desired antibody species. The tested antibodies are listed in Table 2 below, along with the CPMs present in each of the domains of these antibodies. All antibodies listed in Table 2 are IgG1 antibodies, with the only differences between them being in the CDR regions (i.e., changes in specificity for binding antigen A or antigen C) and in the listed charge-pair mutations. TABLE 2 Antikörper Zielantigene CPMs Anti-A-Antikörper A Fv-CPMs: Keine CH1 / CL-CPMs: Keine Fc-CPMs: Keine Anti-C-Antikörper C Fv-CPMs: Keine CH1 / CL-CPMs: Keine Fc-CPMs: Keine BispezifischerAntikörper 1 A (HC und LC)C (HC' und LC') Fv-CPMs: Keine CH1 / CL-CPMs: Keine Fc-CPMs: CH3: 392D; 409D; 439DCH3': 356K; 399K BispezifischerAntikörper 2 A (HC und LC)C (HC' und LC') Fv-CPMs: Keine CH1 / CL-CPMs: CH1: 183E / CL: 176KCH1': 183K / CL': 176E Fc-CPMs: CH3: 392D; 409D; 439DCH3': 356K; 399K BispezifischerAntikörper 3 A (HC und LC)C (HC' und LC') Fv-CPMs: VH: 39E / VL: 85RVH': 39R / VL': 85E CH1 / CL-CPMs: CH1: 183E / CL: 176KCH1': 183K / CL': 176E Fc-CPMs: CH3: 392D; 409D; 439DCH3': 356K; 399K BispezifischerAntikörper 4 A (HC und LC) C (HC' und LC') Fv-CPMs: VH: 39E / VL: 85RVH': 39R / VL': 85E CH1 / CL-CPMs: Keine Fc-CPMs: CH3: 392D; 409D; 439DCH3': 356K; 399K BispezifischerAntikörper 5 A (HC und LC)C (HC' und LC') Fv-CPMs: VH: 39E / VL: 38KVH': 39K / VL": 38E CH1 / CL-CPMs: CH1: 183E / CL: 176KCH1': 183K / CL': 176E Fc-CPMs: CH3: 392D; 409D; 439DCH3': 356K; 399K

[0133] Plasmids encoding the antibodies in Table 2 were constructed as described in Example 2 above and expressed in HEK 293-6E cells. For the monospecific antibodies, plasmids were transfected in a 1:1 ratio of heavy-chain to light-chain plasmids, while the four bispecific plasmids were transfected in a 1:1:1:1 ratio. Seven days post-transfection, the conditioned medium was harvested for purification of secreted antibodies. Initial purification was performed using protein A affinity capture. Subsequently, antibody species were separated by cation-exchange chromatography (CIEX). As part of the CIEX chromatography procedure, individual 1-ml fractions eluted from the CIEX column were collected sequentially and analyzed to determine the antibody species present in each major peak. Fractions containing the desired antibody were pooled and quantified by A280 measurement.

[0134] Fig. Figure 6 shows the amount of composite antibody obtained for each transfection. The anti-A antibody and anti-C antibody assays serve as controls for the amount of antibody obtained by expression and purification of unmodified IgG1 antibodies. After purification, approximately 33 mg / L and 43 mg / L of antibody A and antibody C, respectively, were obtained. Expression and purification of bispecific antibody 1 (which contains Fc-CPMs but no Fab-CPMs) resulted in the recovery of 20 mg / L of antibody. This reduction was presumably the result of a significant proportion of the expressed antibody chains being used as part of unobtained contaminating species. Subsequently, the effects of introducing a single charge-pair mutation in the Fab region (in addition to Fc-CPMs) were tested by expressing either bispecific antibody 2 (CH1:183-CL:176) or bispecific antibody 4 (VH:39-VL:85).The introduction of each of these CPMs increased antibody recovery to levels equal to or better than each of the parent antibodies. Bispecific antibody 2 was recovered at approximately 43 mg / L and bispecific antibody 4 at approximately 45 mg / L.

[0135] The effects of combining multiple CPMs in the Fab region on antibody production were also examined (see Fig. 6) The bispecific antibody 3 (CH1:183-CL:176 plus VH:39-VL:85) and the bispecific antibody 4 (CH1:183-CL:176 plus VH:39-VL:38) both showed increased recovery compared to every other antibody tested; bispecific antibody 3 was recovered at approximately 55 mg / l and bispecific antibody 4 was recovered at approximately 54 mg / l. This results in an increase in production for these two bispecific antibodies of 27% compared to anti-C antibodies and an increase of 65% compared to anti-A antibodies.

[0136] The data in Fig. 6 show that the newly discovered CPMs in the VH-VL interface, in addition to reducing the number and quantity of contaminating species present during antibody production, enable increased antibody production levels. QUOTES INCLUDED IN THE DESCRIPTION

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[0113]

Claims

[1] Isolated protein comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH and VL are linked together, and wherein the VH and VL comprise at least one of the following sets of charged amino acids: a. VH comprises a charged amino acid at position 39 and VL comprises a charged amino acid at position 85, which is charge complementary to the amino acid at position 39 of VH; b. the VH comprises a charged amino acid at position 105 and the VL comprises a charged amino acid at position 42, which is charge-complementary to the amino acid at position 105 of the VH; or c. VH comprises a charged amino acid at position 91 and VL comprises a charged amino acid at position 38, which is charge complementary to the amino acid at position 91 of VH; and wherein the position numbers of the charged amino acids in the VH and VL domains refer to their positions according to the Kabat numbering scheme. [2] Isolated protein according to claim 1, wherein VH and VL comprise the set of charged amino acids in (a). [3] Isolated protein according to claim 2, wherein VH comprises a positively charged amino acid at position 39 and VL comprises a negatively charged amino acid at position 85. [4] Isolated protein according to claim 3, wherein VH position 39 (VH39) and VL position 85 (VL85) comprise: (i) a lysine at VH39 and an aspartic acid at VL85, (ii) a lysine at VH39 and a glutamic acid at VL85, (iii) an arginine at VH39 and an aspartic acid at VL85 or (iv) an arginine at VH39 and a glutamic acid at VL85. [5] Isolated protein according to claim 2, wherein VH comprises a negatively charged amino acid at position 39 and VL comprises a positively charged amino acid at position 85. [6] Isolated protein according to claim 5, wherein VH position 39 (VH39) and VL position 85 (VL85) comprise: (i) an aspartic acid at VH39 and a lysine at VL85, (ii) a glutamic acid at VH39 and a lysine at VL85, (iii) an aspartic acid at VH39 and an arginine at VL85 or (iv) a glutamic acid at VH39 and an arginine at VL85. [7] Isolated protein according to any one of claims 1-6, wherein VH and VL comprise the set of charged amino acids in (b). [8] Isolated protein according to claim 7, wherein VH comprises a positively charged amino acid at position 105 and VL comprises a negatively charged amino acid at position 42. [9] Isolated protein according to claim 8, wherein VH position 105 (VH105) and VL position 42 (VL42) comprise: (i) a lysine at VH105 and an aspartic acid at VL42, (ii) a lysine at VH105 and a glutamic acid at VL42, (iii) an arginine at VH105 and an aspartic acid at VL42 or (iv) an arginine at VH105 and a glutamic acid at VL42. [10] Isolated protein according to claim 7, wherein VH comprises a negatively charged amino acid at position 105 and VL comprises a positively charged amino acid at position 42. [11] Isolated protein according to claim 10, wherein VH position 105 (VH105) and VL position 42 (VL42) comprise: (i) an aspartic acid at VH105 and a lysine at VL42, (ii) a glutamic acid at VH105 and a lysine at VL42, (iii) an aspartic acid at VH105 and an arginine at VL42 or (iv) a glutamic acid at VH105 and an arginine at VL42. [12] Isolated protein according to any one of claims 1-11, wherein VH and VL comprise the set of charged amino acids in (c). [13] Isolated protein according to claim 12, wherein VH comprises a positively charged amino acid at position 91 and VL comprises a negatively charged amino acid at position 38. [14] Isolated protein according to claim 13, wherein VH position 91 (VH91) and VL position 38 (VL38) comprise: (i) a lysine at VH91 and an aspartic acid at VL38, (ii) a lysine at VH91 and a glutamic acid at VL38, (iii) an arginine at VH91 and an aspartic acid at VL38 or (iv) an arginine at VH91 and a glutamic acid at VL38. [15] Isolated protein according to claim 12, wherein VH comprises a negatively charged amino acid at position 91 and VL comprises a positively charged amino acid at position 38. [16] Isolated protein according to claim 15, wherein VH position 91 (VH91) and VL position 38 (VL38) comprise: (i) an aspartic acid at VH91 and a lysine at VL38, (ii) a glutamic acid at VH91 and a lysine at VL38, (iii) an aspartic acid at VH91 and an arginine at VL38 or (iv) a glutamic acid at VH91 and an arginine at VL38. [17] Isolated protein according to any one of claims 1-16, wherein the isolated protein comprises an antibody heavy chain comprising the VH and an antibody light chain comprising the VL. [18] Isolated protein according to claim 17, wherein the isolated protein is an antibody. [19] Isolated protein according to claim 18, wherein the antibody is an IgG antibody. [20] Isolated protein according to claim 19, wherein the antibody is an IgG1, IgG2, IgG3 or IgG4 antibody. [21] Isolated protein according to one of claims 17-20, wherein the isolated protein is a bispecific antibody. [22] Isolated protein according to claim 21, wherein the bispecific antibody comprises two arms and each arm of the antibody comprises a VH-VL pair with the set of charged amino acids in (a), (b) or (c). [23] Isolated protein according to one of claims 18-22, wherein the antibody comprises a first human IgG-CH3 domain (CH3) and a second human IgG-CH3 domain (CH3'), wherein the CH3 domain comprises the replacement of the amino acids at positions 392, 409 and 439 by a negatively charged amino acid, wherein the CH3' domain comprises the replacement of the amino acids at positions 356 and 399, and wherein the position numbers of the charged amino acids in the CH3 and CH3' domains refer to their positions according to the EU numbering scheme. [24] Isolated protein according to one of claims 18-23, wherein the antibody comprises a first pair of heavy chain constant domain and light chain constant domain (CH1 and CL) and a second pair of heavy chain constant domain and light chain constant domain (CH1' and CL'); where CH1 and CH1' each comprise a change at position 183, as numbered according to the EU numbering scheme; where CL and CL' comprise a change at position 176, as numbered according to the EU numbering scheme; where CH1 position 183 and CL' position 176 each comprise a positively charged amino acid; and where CH1' position 183 and CL position 176 each comprise a negatively charged amino acid. [25] Isolated protein according to claim 24, wherein (i) CH1 position 183 and CL' position 176 each comprise lysine and CH1' position 183 and CL' position 176 each comprise aspartic acid; (ii) CH1 position 183 and CL' position 176 each comprise lysine and CH1' position 183 and CL' position 176 each comprise glutamic acid; (iii) CH1 position 183 and CL' position 176 each comprise arginine and CH1' position 183 and CL' position 176 each comprise aspartic acid; or (iv) CH1 position 183 and CL' position 176 each comprise arginine and CH1' position 183 and CL' position 176 each comprise glutamic acid. [26] Bispecific antibody comprising a first pair of heavy chain variable domain and light chain variable domain (VH and VL) and a second pair of heavy chain variable domain and light chain variable domain (VH' and VL'), wherein the VH and VL pair and the VH' and VL' pair comprise at least one of the following sets of charged amino acids: a. VH and VH' each comprise a charged amino acid at position 39; VL and VL' each comprise a charged amino acid at position 85, which is complementary to the amino acid at position 39 of the VH or VH'; and VH and VH' comprise amino acids with complementary charges; b. VH and VH' each comprise a charged amino acid at position 105; VL and VL' each comprise a charged amino acid at position 42, which is complementary to the amino acid at position 105 of the VH or VH'; and VH and VH' comprise amino acids with complementary charges; or c. VH and VH' each comprise a charged amino acid at position 91; VL and VL' each comprise a charged amino acid at position 38, which is complementary to the amino acid at position 91 of the VH or VH'; and The VH and VH' comprise amino acids with complementary charges; and the position numbers of the charged amino acids in the VH and VL domains refer to their positions according to the Kabat numbering scheme. [27] Bispecific antibody according to claim 26, wherein the VH and VL pair and the VH' and VL' pair comprise the set of charged amino acids in (a). [28] Bispecific antibody according to claim 27, wherein VH and VL' comprise a positively charged amino acid and wherein VH' and VL comprise a negatively charged amino acid. [29] Bispecific antibody according to claim 28, wherein the VH and VL pair and the VH' and VL' pair comprise: (i) a lysine at VH39, a lysine at VL'85, an aspartic acid at VH'39 and an aspartic acid at VL85; (ii) a lysine at VH39, a lysine at VL'85, a glutamic acid at VH'39 and a glutamic acid at VL85; (iii) an arginine at VH39, an arginine at VL'85, an aspartic acid at VH'39 and an aspartic acid at VL85; or (iv) an arginine at VH39, an arginine at VL'85, a glutamic acid at VH'39 and a glutamic acid at VL85. [30] Bispecific antibody according to claim 27, wherein VH and VL' comprise a negatively charged amino acid and wherein VH' and VL comprise a positively charged amino acid. [31] Bispecific antibody according to claim 30, wherein the VH and VL pair and the VH' and VL' pair comprise: (i) an aspartic acid at VH39, an aspartic acid at VL'85, a lysine at VH'39 and a lysine at VL85; (ii) a glutamic acid at VH39, a glutamic acid at VL'85, a lysine at VH'39 and a lysine at VL85; (iii) an aspartic acid at VH39, an aspartic acid at VL'85, an arginine at VH'39 and an arginine at VL85; or (iv) a glutamic acid at VH39, a glutamic acid at VL'85, an arginine at VH'39 and an arginine at VL85. [32] Bispecific antibody according to one of claims 25-31, wherein the VH and VL pair and the VH' and VL' pair comprise the set of charged amino acids in (b). [33] Bispecific antibody according to claim 32, wherein VH and VL' comprise a positively charged amino acid and wherein VH' and VL comprise a negatively charged amino acid. [34] Bispecific antibody according to claim 33, wherein the VH and VL pair and the VH' and VL' pair comprise: (i) a lysine at VH105, a lysine at VL'42, an aspartic acid at VH105 and an aspartic acid at VL42; (ii) a lysine at VH105, a lysine at VL'42, a glutamic acid at VH'105 and a glutamic acid at VL42; (iii) an arginine at VH105, an arginine at VL'42, an aspartic acid at VH'105 and an aspartic acid at VL42, or (iv) an arginine at VH105, an arginine at VL'42, a glutamic acid at VH'105 and a glutamic acid at VL42. [35] Bispecific antibody according to claim 32, wherein VH and VL' comprise a negatively charged amino acid and wherein VH' and VL comprise a positively charged amino acid. [36] Bispecific antibody according to claim 35, wherein the VH and VL pair and the VH' and VL' pair comprise: (i) an aspartic acid at VH105, an aspartic acid at VL'42, a lysine at VH'105 and a lysine at VL42; (ii) a glutamic acid at VH105, a glutamic acid at VL'42, a lysine at VH'105 and a lysine at VL42; (iii) an aspartic acid at VH105, an aspartic acid at VL'42, an arginine at VH'105 and an arginine at VL42; or (iv) a glutamic acid at VH105, a glutamic acid at VL'42, an arginine at VH'105 and an arginine at VL42. [37] Bispecific antibody according to one of claims 26-36, wherein the VH and VL pair and the VH' and VL' pair comprise the set of charged amino acids in (c). [38] Bispecific antibody according to claim 37, wherein VH and VL' comprise a positively charged amino acid and wherein VH' and VL comprise a negatively charged amino acid. [39] Bispecific antibody according to claim 38, wherein the VH and VL pair and the VH' and VL' pair comprise: (i) a lysine at VH91, a lysine at VL'38, an aspartic acid at VH'91 and an aspartic acid at VL38; (ii) a lysine at VH91, a lysine at VL'38, a glutamic acid at VH'91 and a glutamic acid at VL38; (iii) an arginine at VH91, an arginine at VL'38, an aspartic acid at VH'91 and an aspartic acid at VL38; or (iv) an arginine at VH91, an arginine at VL'38, a glutamic acid at VH'91 and a glutamic acid at VL38. [40] Bispecific antibody according to claim 37, wherein VH and VL' comprise a negatively charged amino acid and wherein VH' and VL comprise a positively charged amino acid. [41] Bispecific antibody according to claim 40, wherein the VH and VL pair and the VH' and VL' pair comprise: (i) an aspartic acid at VH91, an aspartic acid at VL'38, a lysine at VH'91 and a lysine at VL38; (ii) a glutamic acid at VH91, a glutamic acid at VL'38, a lysine at VH'91 and a lysine at VL38; (iii) an aspartic acid at VH91, an aspartic acid at VL'38, an arginine at VH'91 and an arginine at VL38; or (iv) a glutamic acid at VH91, a glutamic acid at VL'38, an arginine at VH'91 and an arginine at VL38. [42] Method for producing a bispecific antibody, comprising expressing one or more polynucleotides encoding the bispecific antibody according to any one of claims 26-41 in host cells, cultivating the host cells in a culture medium under conditions in which the component polypeptide chains are produced, and obtaining the bispecific antibody from the cell or the culture medium. [43] Method according to claim 42, wherein the host cells comprise one or more plasmids comprising the one or more polynucleotides. [44] 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] Method according to claim 42, wherein the obtained bispecific antibody is purified by protein A chromatography. [46] Method according to one of claims 42-45, wherein the obtained bispecific antibody is further purified by ion exchange chromatography. [47] Method for producing an isolated protein, comprising expressing one or more polynucleotides encoding a protein according to any one of claims 1-25 in host cells, cultivating the host cells in a culture medium under conditions in which the component polypeptide chains are produced, and obtaining the protein from the cell or the culture medium. [48] ​​Method according to claim 47, wherein the host cells comprise one or more plasmids comprising the one or more polynucleotides. [49] Method according to claim 47 or claim 48, wherein the isolated protein is purified by ion exchange chromatography.

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