WO2025006529A1 - Charge pair mutations to enable correct heavy-light chain pairing - Google Patents

Abstract

This application relates to the facilitation of selective binding between modified VH and VL domains by introducing amino acids of complementary charges in the two domains at novel locations within these domains. These alterations are particularly useful in designing and generating multispecific antibodies, such as bispecific antibodies, in which the charge pair mutations in VH and VL domains can facilitate desired pairing between particular heavy and light chain polypeptides. These charge pair mutations can be used on their own, or in combination with additional strategies to promote correct pairing of polypeptide chains.

Description

CHARGE PAIR MUTATIONS TO ENABLE CORRECT HEAVY-LIGHT CHAIN PAIRING

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0001] The present application contains a Sequence Listing, which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The computer readable format copy of the Sequence Listing, which was created on June 5, 2024. is named 10572-W001-SEC_ST26 and is 8.69 kilobytes in size.

TECHNICAL FIELD

[0002] The disclosure relates to the generation 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 particular desirable heavy and light chain polypeptides.

BACKGROUND

[0003] Multispecific antibodies such as bispecific antibodies are an exciting generation of biotherapeutics, enabling simultaneous or sequential targeting of two or more unique epitopes located on the same, or distinct, targets. This dual-recognition capability enables diverse applications such as recruiting immune cells to kill tumor cells, crosslinking distinct 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®) creates 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)) As of 2019, over 100 bispecific formats have been reported, with over 85 in development and three receiving US Food and Drug Administration approval. (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 major design strategies to generate bispecific molecules. The first approach is to encode two or more unique fragment variable (Fv) sequences on the same polypeptide chain(s), as with 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 bypass the challenges associated with assembling multiple polypeptide chains into a single molecule, but they also tend to show poor yields and suboptimal stability. Alternative strategies utilize biophysics and engineering to ‘steer’ individual chains to the correct orientation, while simultaneously disfavoring mispaired scenarios. Examples include technologies like Knob-into-Holes (KiH), Charge Pair Mutations (CPMs) and Strand-Exchange Engineered Domains (SEEDbody) and have been utilized in molecules requiring hetero-Fc pairing. (Davis JH et al., Protein Eng. Des. Sei. 23: 195-202 (2010); Dillon M et al. , MAbs 9:213-30 (2017); Gunasekaran K et al., J. Biol. Chem. 285: 19637-46 (2010); Ridgway JB et al. , Protein Eng. 9:617-21 (1996)). This second approach enables the production of molecules that more closely mimic the native structures of IgG molecules, allowing for increased stability and more diverse format design. (Wang Q et al., Antibodies (Basel) 8(3):43 (2019)) However, these engineering strategies are often imperfect, and chain-mispairing continues to pose a challenge. (Ha JH et al., Front. Immunol. 7:394 (2016))

[0005] Mispairing between heavy and light chains is a significant concern in the generation of multispecific antibodies. For example, if the incorrect light chain (LC) pairs with the undesired heavy chain (HC), the resulting mispaired molecule translates to an impurity that must be removed. Multiple 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 may not necessarily be applicable to the next. Accordingly, there is a need in the art for improved methods for facilitating the pairing between particular desirable heavy and light chain polypeptides. And there is a need in particular for pairings within the Fv interface.

SUMMARY

[0006] This application relates to the facilitation of selective binding between modified VH and VL domains by introducing amino acids of complementary charges in the two domains at novel locations within these domains. For example, alterations may be made at VH position 39 and VL position 85, at VH position 105 and VL position 105, or VH position 91 and VL position 38. These alterations are particularly useful in designing and generating multispecific antibodies, such as bispecific antibodies, in which the charge pair mutations in VH and VL domains can facilitate desired pairing between particular heavy and light chain polypeptides. These charge pair mutations can be used on their own, or in combination with additional strategies to promote correct pairing of polypeptide chains.

[0007] In some embodiments, an isolated protein comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH and VL are bound together, wherein the VH and VL comprise at least one of the following sets of charged amino acids: (a) the VH comprises a charged amino acid at position number 39, and the VL comprises a charged amino acid at position number 85 that is complementary in charge to the amino acid at position 39 of the VH; (b) the VH comprises a charged amino acid at position number 105, and the VL comprises a charged amino acid at position number 42 that is complementary in charge to the amino acid at position 105 of the VH; or (c) the VH comprises a charged amino acid at position number 91, and the VL compnses a charged amino acid at position number 38 that is complementary in charge to the amino acid at position 91 of the 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 particular embodiments, the isolated protein comprises a VH and VL that comprise a set of charged amino acids, such that the VH comprises a charged amino acid at position number 39, and the VL comprises a charged amino acid at position number 85 that is complementary in charge to the amino acid at position 39 of the VH. In some of these embodiments, the VH comprises a positively charged amino acid at position 39 and the VL comprises a negatively charged amino acid at position 85. In some embodiments in which the VH comprises a positively charged amino acid at position 39 and the VL comprises a negatively charged amino acid at position 85, the 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, the VH comprises a negatively charged amino acid at position 39 and the VL comprises a positively charged amino acid at position 85. In some embodiments in which the VH comprises a negatively charged amino acid at position 39 and the VL comprises a positively charged amino acid at position 85, the 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 particular embodiments, the isolated protein comprises a VH and VL that comprise a set of charged amino acids, such that the VH comprises a charged amino acid at position number 105, and the VL comprises a charged amino acid at position number 42 that is complementary' in charge to the amino acid at position 105 of the VH. In some of these embodiments, the VH comprises a positively charged amino acid at position 105 and the VL comprises a negatively charged amino acid at position 42. In some embodiments in which the VH comprises a positively charged amino acid at position 105 and the VL comprises a negatively charged amino acid at position 42, the 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, the VH comprises a negatively charged amino acid at position 105 and the VL comprises a positively charged amino acid at position 42. In some embodiments in which the VH comprises a negatively charged amino acid at position 105 and the VL comprises a positively charged amino acid at position 42, the 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 particular embodiments, the isolated protein comprises a VH and VL that comprise a set of charged amino acids, such that the VH comprises a charged amino acid at position number 91, and the VL comprises a charged amino acid at position number 38 that is complementary in charge to the amino acid at position 91 of the VH. In some of these embodiments, the VH comprises a positively charged amino acid at position 91 and the VL comprises a negatively charged amino acid at position 38. In some embodiments in which the VH comprises a positively charged amino acid at position 91 and the VL comprises a negatively charged amino acid at position 38, the 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, the VH comprises a negatively charged amino acid at position 91 and the VL comprises a positively charged amino acid at position 38. In some embodiments in which the VH comprises a negatively charged amino acid at position 91 and the VL compnses a positively charged amino acid at position 38, the 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 comprising the VH and an antibody light chain comprising 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 IgGl, 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 that comprises 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 number 39, and each VL comprises a charged amino acid at position number 85 that is complementary in charge to the amino acid at position 39 of a VH; (b) each VH comprises a charged amino acid at position number 105, and each VL comprises a charged amino acid at position number 42 that is complementary in charge to the amino acid at position 105 of a VH; or (c) each VH comprises a charged amino acid at position number 91. and each VL comprises a charged amino acid at position number 38 that is complementary in charge to the amino acid at position 91 of a VH.

[0012] In some embodiments, the isolated protein comprises an antibody that comprises a first human IgG CH3 domain (CH3) and a second human IgG CH3 domain (CH3¹), wherein the CH3 domain comprises replacement of the amino acids at positions 392, 409, and 439 with a negatively charged amino acid, wherein the CH3' domain comprises 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.

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

[0014] In some embodiments, a bispecific antibody comprises a first heavy chain variable domain and light chain variable domain pair (VH and VL) and a second heavy chain variable domain and light chain variable domain pair (VH' and VL'), wherein the VH and VL pair and the VH' and VL' pair comprise at least one of the following sets of charged amino acids: (a) the VH and VH' each comprise a charged amino acid at position number 39; the VL and VL' each comprise a charged amino acid at position number 85 that is complementary' in charge to the amino acid at position 39 of the VH and VH', respectively; and the VH and VH' comprise amino acids with complementary charges; (b) the VH and VH' each comprise a charged amino acid at position number 105; the VL and VL' each comprise a charged amino acid at position number 42 that is complementary in charge to the amino acid at position 105 of the VH and VH', respectively; and the VH and VH' comprise amino acids with complementary' charges; or (c) the VH and VH' each comprise a charged amino acid at position number 91; the VL and VL' each comprise a charged amino acid at position number 38 that is complementary in charge to the amino acid at position 91 of the VH and VH', respectively; and the 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 particular embodiments, a bispecific antibody comprises a first heavy chain variable domain and light chain variable domain pair (VH and VL) and a second heavy chain variable domain and light chain variable domain pair (VH¹ and VL'), wherein the VH and VH' each comprise a charged amino acid at position number 39; the VL and VL' each comprise a charged amino acid at position number 85 that is complementary in charge to the amino acid at position 39 of the VH and VH', respectively; and the VH and VH' comprise amino acids with complementary charges. In some embodiments, the VH and VL' comprise a positively charged amino acid, and wherein the 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, the VH and VL' comprise a negatively charged amino acid, and wherein the 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 particular embodiments, a bispecific antibody comprises a first heavy chain variable domain and light chain variable domain pair (VH and VL) and a second heavy chain variable domain and light chain variable domain pair (VH¹ and VL'), wherein the VH and VH' each comprise a charged amino acid at position number 105; the VL and VL' each comprise a charged amino acid at position number 42 that is complementary in charge to the amino acid at position 105 of the VH and VH', respectively: and the VH and VH' comprise amino acids with complementary charges. In some embodiments, the VH and VL' comprise a positively charged amino acid, and wherein the 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, the VH and VL' comprise a negatively charged amino acid, and wherein the 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 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.

[0017] In particular embodiments, a bispecific antibody comprises a first heavy chain variable domain and light chain variable domain pair (VH and VL) and a second heavy chain variable domain and light chain variable domain pair (VH' and VL'), wherein the VH and VH' each comprise a charged amino acid at position number 91; the VL and VL' each comprise a charged amino acid at position number 38 that is complementary in charge to the amino acid at position 91 of the VH and VH', respectively; and the VH and VH' comprise amino acids with complementary charges. In some embodiments the VH and VL' comprise a positively charged amino acid, and wherein the 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, the VH and VL' comprise a negatively charged amino acid, and wherein the 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 of producing bispecific antibody comprising expressing one or more polynucleotides encoding a bispecific antibody containing charge pair mutations described herein in a host cell, culturing the host cells in a culture media under conditions such that the component polypeptide chains are produced, and recovering the bispecific antibody from the cell or the culture media. Similarly, some embodiments are a method of producing isolated proteins comprising expressing one or more polynucleotides encoding a protein containing charge pair mutations described herein in a host cell, culturing the host cells in a culture media under conditions such that the component polypeptide chains are produced, and recovering the protein from the cell or the culture media. In some embodiments, the host cells comprise one or more plasmids that comprise 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 recovered bispecific antibody or protein is purified by protein A chromatography. In some embodiments, the recovered bispecific antibody or protein is further purified by ion exchange chromatography. In some embodiments, the recovered bispecific antibody or protein is purified by protein A chromatography and ion exchange chromatography.

BRIEF DESCRIPTION OF THE FIGURES

[0019] FIG. 1 A is a simplified diagram of an example bispecific antibody, which contains 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 attached through their respective Fc regions. The heavy chains of the antibody are connected through interactions in the CH2 and CH3 domains within the Fc region, and FIG 1A identifies the CH2-CH2 interface and the CH3-CH3 interface in which such interactions occur. Analogously, FIG 1 A shows how each heavy chain connects to its respective light chain through two different interfaces. The first of those contains interactions between the CHI domain of the heavy chain and the CL domain of the light chain (the CHI -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, FIG. 1 A shows the regions on each arm containing the CDR regions that are responsible for interacting with Antigen 1 and Antigen 2.

[0020] FIG. IB shows some of the undesired by-products that may form when producing a bispecific antibody. The depicted undesired by-products are those in which the wrong light chain is paired with one or both of the heavy chains of the bispecific antibody.

[0021] FIGS. 2A-2C relate to the process used to identify new charge pair mutations to introduce in the Fv interface of antibodies. FIG. 2A shows a rendering of the structural interface between a VH domain and a VL domain, with interactions shown as dotted lines. FIG. 2B shows a diagram of the intended result when making charge pair alterations at the interaction sites of FIG 2A. in which the introduced alterations lead to an unstable high energy state in undesired “mispaired” states, such that kinetics drive the antibody chains to assemble in the desired pairing orientation. FIG. 2C shows the energy level calculation (REU) for an exemplary charge pair mutation, which was predicted to have a higher energy state in each of its mispaired structures.

[0022] FIGS. 3A-3E show spectrographs of cation exchange chromatography of bispecific antibodies, after purification from HEK 293 cells using a protein A column. Each of FIGS 3 A-3E further provides a list of the charge pair mutations present in the bispecific antibody being produced. The locations of peaks containing particular antibody species are noted with diagrams of the species and arrows pointing to the pertinent peak. The desired bispecific antibody is also highlighted by a box surrounding its peak in each of FIGS 3A-3E.

[0023] FIGS. 4A-4C also show' spectrographs of cation exchange chromatography of bispecific antibodies, after purification from HEK 293 cells using a protein A column. Each of FIGS 4A-4C further provides a list of the charge pair mutations present in the bispecific antibody being produced. The location of the peak containing the desired antibody species is identified w ith a diagram of that species and arrow s pointing to the pertinent peak. The desired bispecific antibody is also highlighted by a box surrounding its peak in each of FIGS 4A-4C. [0024] FIG. 5 also shows a spectrographs of cation exchange chromatography of a bispecific antibody, after purification from HEK 293 cells using a protein A column. FIG. 5 further provides a list of the charge pair mutations present in the bispecific antibody being produced. The location of the peak containing the desired antibody species is identified with a diagram of that species and arrows pointing to the pertinent peak. The desired bispecific antibody is also highlighted by a box surrounding its peak.

[0025] FIG. 6 shows a plot of the recovery' of various monospecific and bispecific antibody species when purified after production in HEK 293 cells. FIG. 5 illustrates how the identified charge pair mutations facilitate production of bispecific antibodies at levels beyond the parental antibodies.

[0026] FIGS. 7A and 7B contain an exemplary' VH sequence and VL sequence respectively. These sequences are annotated with the Kabat numbering for each residue, and the figures also include boxed regions 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

[0027] The present application arises from the discovery' of new methods by which antibody VH and VL domains can be engineered for specific pairing such that they preferably assemble with each other rather than assembling as part of other VH-VL pairings. A structural analysis of a range of different antibody structures has revealed positions between VH and VL domains in which complementary charge-based alterations can be made to drive such preferential assembly consistently across different antibody sequences. The VL/VH interface residues selected for engineering are buried and spatially close within the VL/VH interface. The target residues are well conserved among different antibody families.

[0028] These engineered VH and VL domains are useful in a range of different contexts in which specific domain interactions are desired. In some aspects, these VH and VL alterations can be used to facilitate forming a heterodimeric pair of polypeptides, each containing either the engineered VH domain or the engineered VL domain. In some aspects, the VH and VL domains of two distinct antibody heavy chains and two distinct antibody light chains are engineered so that they form a four-chain heterodimeric antibody. Electrostatic steering achieved by engineering interface residues between the VL domain and the VH domain prevents mis-pairing of light chains to the non-cognate heavy chains when two different heavy chain and light chain pairs are assembling to form a desired four-chain heterodimeric antibody. As described herein, an exemplar}' strategy comprises introducing one or more negatively-charged residues (e.g., Asp or Glu) in a first VL (VL1) and one or more positively -charged residues (e.g., Lys, His or Arg) in the companion VH (VH1) at the VL1/VH1 interface while introducing one or more positively-charged residues (e.g., Lys, His or Arg) in a second VL (VL2) and one or more negatively-charged residues (e g., Asp or Glu) in the companion VH (VH2) at the VL2/VH2 interface. The electrostatic steering effect directs the VL1 to pair with VH1 and VL2 to pair with VH2, as the opposite charged residues (polarity) at the interface attract, while the same type of charged residues (polarity) at an interface causes repulsion, resulting in suppression of the unwanted VL/VH pairings. Therefore, in some aspects, these VH and VL alterations can be used to facilitate forming the desired structure of a bispecific antibody and minimizing the formation of contaminating alternative species.

[0029] In some aspects, these VH and VL alterations can be combined with other mechanisms to drive specific formation of a desired bispecific antibody species. For example, the VH and VL alterations are combined with known charge pair mutations in the heavy and light chains of antibodies. The combination of the VH and VL alterations with other mechanisms to drive specific pairing increases the production and purity of an expressed bispecific antibody relative to implementing either strategy alone.

[0030] Other ways of engineering the light and heavy chains to form specific heterodimers include replacing one pair of charged residues in the VL/VH interface with a pair of cysteine residues to form a disulfide bond to stabilize the Fab region, replacing one or more hydrophilic residues (e.g.. glycine) in the VL/VH interface with a hydrophobic residue (e.g., glutamine), or engineering a pair of bulky/small residues at the VL/VH interface to exert a knob-into-hole effect to accommodate the correct VL/VH pairing. The strategy⁷ described herein can be used to efficiently produce a full-length heterodimeric antibody from two preexisting antibodies without using artificial linkers. The resulting heterodimeric antibodies are stable and amenable to commercial manufacturing without excessive aggregation or loss of yield. Because heterodimeric antibodies can target two different antigens or two different epitopes on the same antigen simultaneously, they may have significant potential to uniquely treat many diseases.

[0031] Particular embodiments and further detail regarding the invention are provided below.

Definitions of general terms and expressions

[0032] In order that the present disclosure can be more readily understood, certain terms are first defined. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary' skill in the art to which this disclosure is related. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0033] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form.

[0034] As used in the present disclosure and claims, the singular forms “a,” “an,” and ■‘the” include plural forms unless the context clearly dictates otherwise. Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. The term “and/or” as used in a phrase such as “A and/or B” herein is intended to include both “A and B.” “A or B,” “A,” and “B.” Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass 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).

[0035] It is understood that wherever embodiments are described herein yvith the language “comprising.” otherwise analogous embodiments described in terms of “consisting of’ and/or “consisting essentially of’ are also provided. In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of’ or “consists essentially” likewise has the meaning ascribed in U.S. Patent law' and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. [0036] The terms “about” or “comprising essentially of’ refer to a value or composition that is within an acceptable error range for the value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e.. the limitations of the measurement system. For example, “about” or “comprising essentially of’ can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, “about” or “comprising essentially of’ can mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When values or compositions are provided in the application and claims, unless otherwise stated, the meaning of “about” or “comprising essentially of’ should be assumed to be wi thin an acceptable error range for that value or composition.

[0037] 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 combinations of the foregoing. As used herein, the term "antibody" encompasses polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, recombinant antibodies, multispecific antibodies, and bispecific antibodies. An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heav -chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. For example, a common configuration for an antibody has two full length antibody heavy chains and two full length antibody light chains.

[0038] 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 N-terminal to C- terminal direction of an antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1 (CHI), an antibody hinge 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 distinct type, e g., alpha (a), delta (5), epsilon (a), gamma (y), and mu (p), based on the amino acid sequence of the constant domain, which give rise to IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG (e.g., IgGl, IgG2, IgG3, and IgG4) and subclasses of IgA (e.g., IgAl and IgA2). Heavy chain amino acid sequences are know n in the art.

[0039] 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 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 reference to an antibody can refer to any distinct type, e.g., kappa (K) or lambda (X) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are known in the art.

[0040] The term "antibody fragment" refers to a portion of an intact antibody. An "antigen-binding fragment," "antigen-binding domain," or "antigen-binding region," refers to a portion of an intact antibody that binds to an antigen. An antigen-binding fragment can contain the antigenic determining regions of an intact antibody (e.g., the complementarity determining regions (CDR)). Examples of antigen-binding fragments of antibodies include, but are not limited to Fab, Fab', F(ab’)2, and Fv fragments, linear antibodies, and single chain antibodies. An antigen-binding fragment of an antibody can be derived from any animal species, such as rodents (e.g., mouse, rat, or hamster) or humans, or can be artificially produced.

[0041] The term “multispecific antibody” means that an antigen binding protein is capable of specifically binding to two or more different antigens. A subcategory of multispecific antibodies is "bispecific antibodies," which are capable of specifically binding to two different antigens. As used herein, an antibody “specifically binds” to a target antigen when it has a significantly higher binding affinity for, and consequently is capable of distinguishing, that antigen, compared to its affinity for other unrelated proteins, under similar binding assay conditions. Antigen binding proteins that specifically bind an antigen may have an equilibrium dissociation constant (KD) < 1 x IO^-6 M. The antigen binding protein specifically binds antigen with “high affinity” when the KD is < 1 x 10‘⁸ M. [0042] An "isolated antibody" refers to an antibody population that comprises a single species of antibody. For example, a particular isolated antibody consists of an antibody population having a single heavy chain amino acid sequence and a single light chain amino acid sequence, which binds to a single epitope. An isolated antibody can, however, have cross-reactivity to other antigens, such as related molecules from different species. Also, a population of antibodies may still be an "isolated antibody" when contaminated by small amounts of other antibody species. In particular, an isolated antibody may contain less than 5%. less than 4%, less than 3%, less than 2%, less than 1%, or no other antibody species.

[0043] A "monoclonal antibody" refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. Furthermore, "monoclonal" antibody refers to such antibodies made in any number of manners including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals.

[0044] The terms "variable region" or "variable domain" are used interchangeably and are common in the art. The variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids or 110 to 125 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen. The variability in sequence is concentrated in those regions called complementarity determining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR). FromN-terminus to C-terminus, naturally occurring light and heavy chain variable regions both typically conform with the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with antigen.

[0045] The terms "VL" and " VL domain" and "VH region" are used interchangeably to refer to the light chain variable region of an antibody. [0046] The terms "VH" and "VH domain" and "VH region" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0047] The term "Kabat numbering" and like terms are recognized in the art and refer to a system of 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, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system. CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35, which optionally can include one or two additional amino acids, following 35 (referred to in the Kabat numbering scheme as 35A and 35B) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs within an antibody light chain molecule are typically present 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 may 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, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).

[0048] Chothia refers instead to the location of the structural 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).

[0049] The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody labelling software.

[0050] 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 unifies numbering across antibody lambda and kappa light chains, heavy chains, and T-cell receptor chains.

[0051] Alternatively, the position of particular amino acids within the framework regions of the variable domains (described below) can be described using the Aho numbering system. Because antibody CDR amino acid sequence length varies from antibody to antibody, numbering residues based on the linear sequence (assuming the first residue as position 1) leads to framework residues having different position numbers between antibodies. Annemarie Honegger and Andreas Pluckthun developed a structure-based numbering scheme (Aho), which introduces gaps in the CDR regions to minimize deviation from the average structure of the aligned domains. (Honegger, A., and Pluckthun, A. (2001). J. Mol. Biol. 309, 657-670) This leads to structurally equivalent positions having the same residue number when two different antibodies are compared. This enables comparison of the effect of substitutions in the variable domain framework region between antibodies.

[0052] The terms "constant region" and "constant domain" are interchangeable and have their common meaning in the art. The constant region is an antibody portion, e.g., a carboxyl terminal portion of a light and/or heavy chain which is not directly involved in binding of an antibody to antigen, but which can exhibit various effector functions, such as interaction with the Fc receptor. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to an immunoglobulin variable domain.

[0053] As used herein, the terms "Fc region’" and “Fc domain" refer to a C-terminal region of an IgG heavy chain; in case of an IgGl antibody, the C-terminal region comprises -CH2-CH3 (see above).

[0054] The term "interface" refers to the association surface that results from interaction one or more amino acids in a first antibody domain with one or more amino acids of a second antibody domain. Exemplary interfaces include the CH1/CL, VH/VL, CH2-CH2, and CH3/CH3 interfaces. In some embodiments, the interface includes, for example, hydrogen bonds, electrostatic interactions, or salt bridges between the amino acids forming an interface.

[0055] The term "chimeric antibody" refers to an antibody wherein the amino acid sequence is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability' while the constant regions are homologous to the sequences in derived from another (usually human) to avoid eliciting an immune response in that species.

[0056] A "humanized antibody" refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human framework regions and constant regions. A humanized antibody may comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "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 residues from the CDRs are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, hamster) that have the desired specificity', affinity, and capability. Accordingly, humanized antibodies are also referred to as "CDR grafted" antibodies. Early examples of methods used to generate humanized antibodies are described in U.S. 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 additional examples and methods relating to humanization of antibodies have subsequently been published.

[0057] A "human antibody" refers to an antibody having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the disclosure can include 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 framework sequences. The terms "human antibodies" and "fully human antibodies" and are used synonymously. [0058] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labelling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that, because the polypeptides of this invention are based upon antibodies, in certain embodiments, the polypeptides can occur as single chains or associated chains.

[0059] By ‘‘complimentary amino acid substitutions,’' it is meant that a substitution to a positive-charge amino acid in the heavy chain is paired with a negative-charged amino acid substitution to an amino acid in the light chain that associates with the heavy chain residue. Likewise, a substitution to a negative-charge amino acid in the heavy chain is paired with a positive-charged amino acid substitution to an amino acid in the light chain that associates with the heavy chain residue.

[0060] As used herein, 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 parent cell transfected with the nucleic acid molecule, e.g., due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.

VH Domain and VL Domain Modifications

[0061] In some embodiments, the disclosed proteins comprise alterations within VH and VL domains that introduce charged amino acids that are electrostatically favorable to the altered VH and VL domains interacting with each other and disfavorable to the altered VH and VL domains interacting with unaltered VH and VL domains.

[0062] To drive specific association of an altered VH domain with an altered VL domain, the two domains may comprise complementary charge alterations. Thus, if a VH domain comprises an alteration to introduce a negatively charged amino acid, then it is paired with a complementary alteration in a VL domain to a positively charged amino acid. And conversely, if a VL domain comprises an alteration to introduce a negatively charged amino acid, then it is paired with a complementary alteration in a VH domain to a positively charged amino acid. Accordingly, the pair of complementarity charged residues may be referred to as a "charge pair alteration," a "charge pair mutation," or a "CPM." [0063] The altered amino acids in a charge pair alteration may be any natural or artificial amino acid that is charged at a physiological pH. In some embodiments, the negatively- charged amino acid is aspartic acid or glutamic acid. In some embodiments, the positively charged amino acid is lysine, histidine, or arginine.

[0064] One of the charge pair alterations that may be used to drive specific VH-VL association comprises an alteration at position 39 of the VH domain paired with an alteration at position 85 of the VL domain (as numbered according to the Kabat scheme). In some embodiments, the VH domain comprises a charged amino acid at position number 39, and the VL domain comprises a charged amino acid at position number 85 that is complementary in charge to the amino acid at position 39 of the VH domain. In some embodiments, the VH domain comprises a positively charged amino acid at position 39 (e.g., arginine, lysine, or histidine) and the VL domain comprises 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).

[0065] Another charge pair alteration that may be used to drive specific VH-VL association comprises an alteration at position 105 of the VH domain paired with an alteration at position 42 of the VL domain (as numbered according to the Kabat scheme). In some embodiments, the VH domain comprises a charged amino acid at position number 105, and the VL domain comprises a charged amino acid at position number 42 that is complementary in charge to the amino acid at position 105 of the VH domain. In some embodiments, the VH domain comprises a positively charged amino acid at position 105 (e g., arginine, lysine, or histidine) and the VL domain comprises 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).

[0066] Another charge pair alteration that may be used to drive specific VH-VL association comprises an alteration at position 91 of the VH domain paired with an alteration at position 38 of the VL domain (as numbered according to the Kabat scheme). In some embodiments, the VH domain comprises a charged amino acid at position number 91, and the VL domain comprises a charged amino acid at position number 38 that is complementary in charge to the amino acid at position 91 of the VH domain. In some embodiments, the VH domain comprises a positively charged amino acid at position 91 (e.g., arginine, lysine, or histidine) and the VL domain comprises 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).

[0067] Numerous VH domain and VL domain sequences are known in the art as part of antibody sequences. The VH and VL domain alterations disclosed herein may be applied to those VH and VL sequences know n in the art. In some embodiments, the VH and VL domain alterations disclosed herein may be applied to naturally occurnng human VH and VL domain sequences. In some embodiments, the VH and VL domain alterations disclosed herein may 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 that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%. at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a human germline VH domain. In some embodiments, the VL domain is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%. at least about 75%. at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%. at least about 97%, at least about 98%, or at least about 99% identical to a human germline VL domain.

Proteins Containing VH Domain and VL Domain Modifications

[0068] The altered VH and VL domains described herein may be used in a variety of circumstances in which the VH and VL domains are part of one or more polypeptides where specific VH-VL interaction is desirable. For example, the altered VH and VL domains may be integrated into heavy and light chain polypeptides as part of forming an antibody or an antibody fragment. In some embodiments, the altered VH and VL domains aid in creating specific VH-VL pairings in heterodimeric antibodies, such as bispecific antibodies. Applications are not limited to antibody or antibody-like molecules, however, as the VH and VL domains operate as independent domains or may be conjugated or fused to other proteins to facilitate specific interactions.

[0069] Bispecific antibodies are one context in which the altered VH and VL domains described herein may be used. In some forms, bispecific antibodies comprise two different heavy chains and two different light chains, and require specific arrangement of the antibody such that each heavy chain binds to a specific light chain. Accordingly, in some embodiments, the altered VH and VL domains described herein are components of a bispecific antibody comprising a first heavy chain variable domain and light chain variable domain pair (VH and VL) and a second heavy chain variable domain and light chain variable domain pair (VH' and VL'). In such a bispecific antibody, each VH-VL pair may comprise a distinct set of charge pair mutations. In some embodiments, the VH and VH' each comprise a charged amino acid at the same position; the VL and VL' each comprise a charged amino acid at the same position that is complementary in charge to the altered amino acid of the VH and VH'. respectively; and the VH and VH' comprise amino acids with complementary charges. In such embodiments, the VH and VL' comprise like charges (z.e., both positively charged or both negatively charged amino acids), and the VH' and VL comprise like charges (z.e., both positively charged or both negatively charged amino acids). In some embodiments, the VH-VL and VH'-VL' pairs are altered at the same amino acid positions. Tn some embodiments, the VH-VL and VH'- VL' pairs are altered at different positions.

[0070] In some embodiments, the antibody or antibody fragment comprises a VL domain that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%. at least about 90%, at least about 91%, at least about 92%, at least about 93%. at least about 94%. at least about 95%. at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a human germline lambda chain VL domain.

[0071] In some embodiments, the antibody or antibody fragment comprises a VL domain that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%. at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a human germline kappa chain VL domain.

[0072] In some embodiments, the altered VH and VL domains described herein are components of antibody fragments. In some embodiments, the antibody fragment is any antibody fragment comprising both a VH domain and a VL domain. In some embodiments, the antibody fragment is a Fab, Fab', F(ab')2, or Fv fragment.

[0073] In some embodiments, the antibody or antibody fragment optionally further comprises alterations in the VH-VL interface in addition to the modifications described in the "VH Domain and VL Domain Modifications" section above. VH-VL domain interface residues (i.e., amino acid residues that mediate 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 interface residues in the VL domain are substituted with a charged amino acid, preferably that has an opposite charge to those introduces into the cognate VH domain. In some embodiments, the amino acid at AHo position 46 (Kabat 38) of the VL domain is replaced with a positive-charged amino acid. In some embodiments, such as when the amino acid at AHo position 46 (Kabat 39) in the VH domain is substituted with a positive-charged amino acid), the amino acid at AHo position 46 (Kabat 38) of the VL domain is replaced with a negative-charged amino acid. In some embodiments, the amino acid at AHo positions 51 (Kabat 43) and/or AHo position 141 (Kabat 100) are substituted for a positive- or negative-charged amino acid. Such embodiments may further include having the amino acid at AHo position 46 substituted for a positive- or negative-charged amino acid. In some embodiments, the amino acid at AHo position 51 is substituted for a positive-charged amino acid, e.g., lysine. In alternative embodiments, the amino acid at AHo position 51 is substituted for a negative-charged amino acid, e.g., aspartic acid. In some embodiments, the amino acid at AHo position 141 is substituted for a positive-charged amino acid, e.g., lysine. In alternative embodiments, the amino acid at AHo position 141 is substituted for a negative-charged amino acid, e.g., aspartic acid. In some embodiments, the amino acid at AHo position 51 and AHo position 141 are substituted for a positive-charged amino acid, e.g., lysine, or a negative-charged amino acid, e.g., aspartic acid. Such embodiments may further comprise a substitution at AHo position 46 (Kabat 38) to a positive- or negative- charged amino acid.

[0074] In some embodiments, the antibodies or antibody fragment containing modified VH-VL domains comprises 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 Fl 16, Fl 18, S121, D122, E123, Q124, S 131, V133, L135, N137, N138, Q160, S162, T164, S174 and S176 are replaced with a charged amino acid. In some embodiments, an exemplary residue for substitution with a negative- or positive- 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 with a positive-charged amino acid. In alternative embodiments, the amino acid at position 176 of the CL domain is replaced with a negative-charged amino acid, e.g., aspartic acid. In some embodiments, the antibody or antibody fragment comprises a kappa chain that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%. at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%. at least about 93%, at least about 94%, at least about 95%. at least about 96%. at least about 97%, at least about 98%, or at least about 99% identical to a human germline kappa chain.

[0075] In some embodiments, the antibodies 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, Fl 18, S121, E123, E124, K129, T131, V133, L135, S137, E160, T162, S165, Q167, A174, S176 and Y178 are replaced with a charged amino acid. In some embodiments, an exemplary residue for substitution with a negative- or positive- 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 with a positive-charged amino acid. In alternative embodiments, the amino acid at position 176 of the CL domain is replaced with a negative-charged amino acid, e.g.. aspartic acid. In some embodiments, the antibody or antibody fragment comprises a lambda chain that is at least about 70%, at least about 71 %, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%. at least about 81%, at least about 82%, at least about 83%. at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a human germline lambda chain.

[0076] In some embodiments, the altered VH and VL domains described herein are components of an antibody or antibody fragment that comprises a CHI domain. In some embodiments, the antibody or antibody fragment comprises a heavy chain CHI region that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%. at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%. or at least about 99% identical to a human germline heavy chain CHI region.

[0077] Because the CHI domain complexes with the light chain constant region (CL), the CHI and CL domains can be engineered to increase the efficiency of a particular heavy chain pairing with its cognate light chain. Accordingly, in some embodiments, the altered VH and VL domains disclosed herein may be combined with alterations in the CHI and CL domains. For example, assembly may be facilitated by introducing a cysteine residue into the heavy and light chain at or near the CH1-CL interface to allow formation of disulfide bonds, altering amino acids to create a knobs-into-holes effect, and electrostatic engineering similar to that described herein for the variable regions. In some embodiments, assembly may be facilitated by introducing one or more charged amino acids in the CHI domain of the antibody or antibody fragment at an EU position selected from the group consisting ofF126, P127, L128, A141, L145, K147, D148, H168, F170, P171. V173, Q175. S176, S183, V185 and K213. In this regard, a particularly preferred residue for substitution with a negative- or positive-charged amino acid is S 183 (EU numbering system). In some embodiments, SI 83 is substituted with a positive-charged amino acid. In alternative embodiments, SI 83 is substituted with a negative-charged amino acid. In some embodiments, an alteration at position SI 83 of the CHI domain is paired with an alteration to a complementarily charged amino acid at position 176 of the CL domain.

[0078] In some embodiments, the altered VH and VL domains described herein are components of an antibody or antibody fragment that comprises a constant region. The antibodies described herein can comprise any constant region. The light chain constant region can be, for example, a kappa- or lambda-type light chain constant region, e.g.. a human kappa- or lambda-type light chain constant region. The heavy chain constant region can be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region, e.g., 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, derivative, variant, or mutein of a naturally occurring constant region. The antibodies described herein can comprise any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The antibodies described herein may comprise constant domains comprising a CH2 domain, a CH3 domain., or a CH2 and a CH3 domain.

[0079] In those antibodies or antibody fragments described herein that comprise CH3 domains as part of their heavy chains, the antibodies or antibody fragments optionally further comprise two CH3 domains in which 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 in the CH3 domain to disfavor homodimerization, and more preferably, favor 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 at one or more positions selected from the group consisting of 399, 356 and 357 (EU numbering system) of the CH3 domain are replaced with a negative-charged amino acid. In some embodiments, amino acids at one or more positions selected from the group consisting of 370, 392 and 409 (EU numbering system) are replaced with a positive-charged amino acid. In alternative embodiments, amino acids at one or more positions selected from the group consisting of 399, 356 and 357 (EU numbering system) of the CH3 domain are replaced with a positive-charged amino acid. In further embodiments, amino acids at one or more positions selected from the group 370, 392 and 409 (EU numbering system) are replaced with a negative-charged amino acid. In some embodiments, the heterodimeric antibody comprises a first heavy chain comprising positive-charged amino acid at positions 399 and 356 (e.g., D399K and E356K), and a second heavy chain comprising negative-charged amino acids at positions 392 and 409 (e.g., K392D and K409D). In some embodiments, the heterodimeric antibody comprises a first heavy chain comprising positive-charged amino acid at positions 356 and 399 (e.g., 356K and 409K), and a second heavy chain comprising negative-charged amino acids at positions 392, 409, and 439 (e.g., 392D, 409D, and 439D).

Fc Modifications

[0080] The heavy chains of the antibodies or antibody fragments described herein may further comprise one of more mutations that affect 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 to the immune system when the antibody binds its target. This is commonly referenced as ‘‘effector function.” Communication leads to antibodydependent cellular cytotoxicity (ADCC), antibody -dependent cellular phagocytosis (ADCP), and/or complement dependent cytotoxicity (CDC). ADCC and ADCP are mediated through the binding of the Fc to Fc receptors on the surface of cells of the immune system. CDC is mediated through the binding of the Fc with proteins of the complement system, e.g., Cl q.

[0081] The IgG subclasses vary in their ability' to mediate effector functions. For example, IgGl is superior to IgG2 and IgG4 at 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, having an Fc engineered to increase effector function (U.S. Pat. No. 7,317,091 and Strohl, Curr. Opin. Biotech.. 20:685-691, 2009; both incorporated herein by reference in its entirety). Exemplary IgGl Fc molecules having increased effector function include those having 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+Q31 1M+K334V, S298T+K334V, A330M+K334V, A330F+K334V, Q31 1M+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.

[0082] Further embodiments of the invention include antibodies and antibody fragments having an Fc engineered to decrease effector function. Exemplary Fc molecules having decreased effector function include those having one or more of the following substitutions [numbering based on the EU numbering scheme]: N297A (IgGl). L234A/L235A (IgGl), V234A/G237A (IgG2), L235A/G237A/E318A (IgG4), H268Q/V309L/A330S/A331S (IgG2), C220S/C226S/C229S/P238S (IgGl), C226S/C229S/E233P/L234V/L235A (IgGl), L234F/L235E/P331S (IgGl), S267E/L328F (IgGl).

[0083] Another method of increasing effector function of IgG Fc-containing proteins is by reducing the fucosylation of the Fc. Removal of the core fucose from the biantennary complex-type oligosaccharides attached to the Fc greatly increased ADCC effector function without altering antigen binding or CDC effector function. Several methods are known for reducing or abolishing fucosylation of Fc-containing molecules, e.g., antibodies. These include recombinant expression in certain mammalian cell lines including a FUT8 knockout cell line, variant CHO line Led 3, rat hybridoma cell line YB2/0, a cell line comprising a small interfering RNA specifically against the FUT8 gene, and a cell line coexpressing P-1.4-N-acetylglucosaminyltransferase III and Golgi [3- mannosidase II. Alternatively, the Fc-containing molecule may be expressed in anon- mammalian cell such as a plant cell, yeast, or prokaryotic cell, e.g., E. coli. Thus, in certain embodiments, a composition comprises an antibody having reduced fucosylation or lacking fucosylation altogether.

Polynucleotides Encoding Engineered Heavy or Light Chains

[0084] Encompassed within the invention are nucleic acids encoding the VH and VL domain-containing polypeptide chains described herein. Nucleic acid molecules of the invention include DNA and RNA in both single-stranded and double-stranded form, as well as the corresponding complementary sequences. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. The nucleic acid molecules of the invention include full-length genes or cDNA molecules as well as a combination of fragments thereof. The nucleic acids of the invention are preferentially derived from human sources, but the invention includes those derived from non-human species, as well.

[0085] In some embodiments, the nucleic acids of the invention are isolated nucleic acids. An '‘isolated nucleic acid” 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 isolated from naturally occurring sources. In the case of nucleic acids synthesized enzymatically from a template or chemically, such as PCR products, cDNA molecules, or oligonucleotides for example, it is understood that the nucleic acids resulting from such processes are isolated nucleic acids. An 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 one preferred embodiment, the nucleic acids are substantially free from contaminating endogenous material. The nucleic acid molecule has preferably been derived from DNA or RNA isolated at least once in substantially pure form and in a quantity or concentration enabling identification, manipulation, and recovery’ of its component nucleotide sequences by standard biochemical methods (such as those outlined in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)). Such sequences are preferably provided and/or constructed in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, that are typically present in eukaryotic genes. Sequences of non-translated DNA can be present 5' or 3' from an open reading frame, where the same do not interfere with manipulation or expression of the coding region.

[0086] Variants are ordinarily prepared by site specific mutagenesis of nucleotides in the DNA encoding the polypeptide, using cassette or PCR mutagenesis or other techniques known in the art, to produce DNA encoding the variant, and thereafter expressing the recombinant DNA in cell culture as outlined herein. However, antibodies or antibody fragments comprising variant CDRs having up to about 100-150 residues may be prepared by in vitro synthesis using established techniques. The variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, e.g., binding to antigen, although variants can also be selected which have modified characteristics as will be more fully outlined herein.

[0087] As will be appreciated by those in the art, due to the degeneracy of the genetic code, a large number of nucleic acids may be made, all of which encode the polypeptides of the invention. Thus, having identified a particular amino acid sequence, those skilled in the art could make any number of different nucleic acids, by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the encoded protein.

[0088] The invention also provides expression systems and constructs in the form of plasmids, expression vectors, transcription or expression cassettes which comprise at least one polynucleotide as above. In addition, the invention provides host cells comprising such expression systems or constructs.

[0089] Typically, expression vectors used in the host cells will contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as “flanking sequences,” in certain embodiments will typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional 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 inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. Each of these sequences is discussed below.

[0090] Optionally, the vector may contain a “tag”-encoding sequence, i.e., an oligonucleotide molecule located at the 5' or 3' end of the polypeptide coding sequence; the oligonucleotide sequence encodes polyHis (such as hexaHis), or another “tag” such as FLAG, HA (hemagglutinin influenza virus), or myc, for which commercially available antibodies exist. This tag is typically fused to the polypeptide upon expression of the polypeptide, and can sen e as a means for affinity purification or detection of the polypeptide from the host cell. Affinity purification can be accomplished, for example, by column chromatography using antibodies against the tag as an affinity matrix. Optionally, the tag can subsequently be removed from the purified polypeptide by various means such as using certain peptidases for cleavage. [0091] Flanking sequences may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from more than one source), synthetic or native. As such, the source of a flanking sequence may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequence is functional in, and can be activated by, the host cell machinery.

[0092] Flanking sequences useful in the vectors of this invention may be obtained by any of several methods known in the art. Typically, flanking sequences useful herein will have been previously identified by mapping and/or by restriction endonuclease digestion and can thus be isolated from the proper tissue source using the appropriate restriction endonucleases. In some cases, the full nucleotide sequence of a flanking sequence may be known. Here, the flanking sequence may be synthesized using the methods described herein for nucleic acid synthesis or cloning.

[0093] Whether all or only a portion of the flanking sequence is known, it may be obtained using polymerase chain reaction (PCR) and/or by screening a genomic library with a suitable probe such as an oligonucleotide and/or flanking sequence fragment from the same or another species. Where the flanking sequence is not known, a fragment of DNA containing a flanking sequence may be isolated from a larger piece of DNA that may contain, for example, a coding sequence or even another gene or genes. Isolation may be accomplished by restriction endonuclease digestion to produce the proper DNA fragment followed by isolation using agarose gel purification, Qiagen® column chromatography (Chatsworth, Calif.), or other methods known to the skilled artisan. The selection of suitable enzymes to accomplish this purpose will be readily apparent to one of ordinary skill in the art.

[0094] An origin of replication is typically a part of those prokary otic expression vectors purchased commercially, and the origin aids in the amplification of the vector in a host cell. If the vector of choice does not contain an origin of replication site, one may be chemically synthesized based on a known sequence, and ligated into the vector. For example, the origin of replication from the plasmid pBR322 (New' England Biolabs, Beverly, Mass.) is suitable for most gram-negative bacteria, and various viral origins (e.g.. SV40, polyoma, adenovirus, vesicular stomatitis virus (VSV). or papillomaviruses such as HPV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (for example, the SV40 origin is often used only because it also contains the virus early promoter).

[0095] A transcription termination sequence is typically located 3' to the end of a polypeptide coding region and serves to terminate transcription. Usually, a transcription termination sequence in prokaryotic cells is a G-C rich fragment followed by a poly-T sequence. While the sequence is easily cloned from a library or even purchased commercially as part of a vector, it can also be readily synthesized using methods for nucleic acid synthesis such as those described herein.

[0096] A selectable marker gene encodes a protein necessary for the survival and grow th of a host cell grown in a selective culture medium. Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells; (b) complement auxotrophic deficiencies of the cell; or (c) supply critical nutrients not available from complex or defined media. Specific selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. Advantageously, a neomycin resistance gene may also be used for selection in both prokaryotic and eukaryotic host cells.

[0097] Other selectable genes may be used to amplify the gene that will be expressed. Amplification is the process wherein genes that are required for production of a protein critical for growth or cell survival are reiterated in tandem within the chromosomes of successive generations of recombinant cells. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and promoterless thymidine kinase genes. Mammalian cell transformants are placed under selection pressure wherein only the transformants are uniquely adapted to survive by virtue of the selectable gene present in the vector. Selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of selection agent in the medium is successively increased, thereby leading to the amplification of both the selectable gene and the DNA that encodes another gene, such as an antibody light or heavy chain. As a result, increased quantities of a polypeptide are synthesized from the amplified DNA.

[0098] A ribosome-binding site is usually necessary for translation initiation of mRNA and is characterized by a Shine-Dalgamo sequence (prokaryotes) or a Kozak sequence (eukaryotes). The 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 may be operably linked to an internal ribosome binding site (IRES), allowing translation of two open reading frames from a single RNA transcript.

[0099] In some cases, such as where glycosylation is desired in a eukaryotic host cell expression system, one may manipulate the various pre- or prosequences to improve glycosylation or yield. For example, one may alter the peptidase cleavage site of a particular signal peptide, or add prosequences, which also may affect glycosylation. The final protein product may have, in the -1 position (relative to the first amino acid of the mature protein) one or more additional amino acids incident to expression, which may not have been totally removed. For example, the final protein product may have one or two amino acid residues found in the peptidase cleavage site, attached to the amino terminus. Alternatively, use of some enzyme cleavage sites may result in a slightly truncated form of the desired polypeptide if the enzyme cuts at such area within the mature polypeptide.

[0100] Expression and cloning vectors of the invention will typically contain a promoter that is recognized by the host organism and operably linked to the molecule encoding the polypeptide. Promoters are untranscribed sequences located upstream (i.e., 5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control transcription of the structural gene. Promoters are conventionally grouped into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of a nutrient or a change in temperature. Constitutive promoters, on the other hand, uniformly transcribe gene to which they are operably linked, that is, with little or no control over gene expression. A large number of promoters, recognized by a variety of potential host cells, are known in the art. A suitable promoter is operably linked to the DNA encoding e.g., heavy chain or light chain, by removing the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence into the vector.

[0101] Suitable promoters for use with yeast hosts are also known in the art. 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 obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis-B virus and most preferably Simian Virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, for example, heat-shock promoters and the actin promoter.

[0102] Additional promoters which may be of interest include, but are not limited to: SV40 early promoter (Benoist and Chambon, 1981, Nature 290:304-310); CMV promoter (Thomsen et al., 1984, Proc. Natl. Acad. U.S.A. 81:659-663); the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787- 797); herpes thymidine kinase promoter (Wagner et al., 1981. Proc. Natl. Acad. Sci. U.S.A. 78: 1444-1445); promoter and regulatory sequences from the metallothionine gene Prinster et al., 1982, Nature 296:39-42); and prokaryotic promoters such as the betalactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727- 3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25). Also of interest are the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: the elastase I gene control region that is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Omitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); the insulin gene control region that is active in pancreatic beta cells (Hanahan, 1985, Nature 315: 115-122); the immunoglobulin gene control region that 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. Cell. Biol. 7: 1436-1444); the mouse mammary tumor virus control region that is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495); the albumin gene control region that is active in liver (Pinkert et al., 1987, Genes and Devel. 1 :268-276); the alpha-feto-protein gene control region that is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5: 1639- 1648; Hammer et al.. 1987, Science 253:53-58); the alpha 1-antitrypsin gene control region that is active in liver (Kelsey et al., 1987, Genes and Devel. 1 : 161-171); the betaglobin gene control region that is 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 that is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); the myosin light chain-2 gene control region that is active in skeletal muscle (Sani, 1985, Nature 314:283-286); and the gonadotropic releasing hormone gene control region that is active in the hypothalamus (Mason et al., 1986, Science 234: 1372-1378).

[0103] An enhancer sequence may be inserted into the vector to increase transcription of DNA encoding light chain or heavy chain of the invention by higher eukaryotes.

Enhancers are cis-acting elements of DNA, usually about 10-300 bp in length, that act on the promoter to increase transcription. Enhancers are relatively orientation and position independent, having been found at positions both 5' and 3’ to the transcription unit. Several enhancer sequences available from mammalian genes are known (e.g., globin, elastase, albumin, alpha-feto-protein, and insulin). Typically, however, an enhancer from a virus is used. The SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and adenovirus enhancers known in the art are exemplary' enhancing elements for the activation of eukaryotic promoters. While an enhancer may be positioned in the vector either 5' or 3' to a coding sequence, it is typically located at a site 5' from the promoter. A sequence encoding an appropriate native or heterologous signal 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 depends on the type of host cells in which the antibody is to be produced, and a heterologous signal sequence can replace the native signal sequence. Examples of signal peptides that are functional in mammalian host cells include the following: the signal sequence for interleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195; the signal sequence for interleukin-2 receptor described in Cosman et al., 1984, Nature 312:768; the interleukin-4 receptor signal peptide described in EP Patent No. 0367 566; the type I interleukin-1 receptor signal peptide described in U.S. Pat. No. 4,968,607; the type II interleukin-1 receptor signal peptide described in EP Patent No. 0 460 846.

[0104] The vector may contain one or more elements that facilitate expression when the vector is 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 the chromatin and may insulate the integrated vector from “position"’ effect. Thus, MARs are particularly useful when the vector is used to create stable transfectants. A number of natural and synthetic MAR- containing nucleic acids are known in the art, e.g.. U.S. 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. [0105] Expression vectors of the invention may be constructed from a starting vector such as a commercially available vector. Such vectors may or may not contain all of the desired flanking sequences. Where one or more of the flanking sequences described herein are not already present in the vector, they may be individually obtained and ligated into the vector. Methods used for obtaining each of the flanking sequences are known to one skilled in the art.

[0106] After the vector has been constructed and a nucleic acid molecule encoding light chain, a heavy chain, or a light chain and a heavy chain sequence has been inserted into the proper site of the vector, the completed vector may be inserted into a suitable host cell for amplification and/or polypeptide expression. The transformation of an expression vector into a selected host cell may be accomplished by known methods including transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-dextran mediated transfection, or other known techniques. The method selected will in part be a function of the type of host cell to be used. These methods and other suitable methods are known to the skilled artisan, and are set forth, for example, in Sambrook et al., 2001, supra.

Methods of Producing Proteins

[0107] A host cell, when cultured under appropriate conditions, synthesizes heterodimeric antibody that can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted). The selection of an appropriate host cell will depend upon various factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation) and ease of folding into a biologically active molecule. A host cell may be eukaryotic or prokary otic.

[0108] Mammalian cell lines available as hosts for expression are known in the art 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 art can be used to make the recombinant polypeptides of the invention. In general, host cells are transformed with a recombinant expression vector that comprises DNA encoding a desired heterodimeric antibody. Among the host cells that may be employed are prokaryotes, yeast, or higher eukaryotic cells. Prokaryotes include gram negative or gram positive organisms, for example E. coli or bacilli. Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, Cl 27 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, or their derivatives such as Veggie CHO and related cell lines which grow in serum-free media (Rasmussen et al., 1998, Cytotechnology 28: 31), HeLa cells, BHK (ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) as described by McMahan et al., 1991, EMBO J. 10: 2821, human embryonic kidney cells such as 293. 293 EBN A 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. Optionally, mammalian cell lines such as HepG2/3B, KB, NIH 3T3 or S49, for example, can be used for expression of the polypeptide when 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 prokary otes such as bacteria. Suitable yeasts include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains. Candida, or any yeast strain capable of expressing heterologous polypeptides. Suitable bacterial strains include Escherichia coli. Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous polypeptides.

[0109] If the antibody or antibody fragment is made in yeast or bacteria, it may be desirable to modify the product produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain a functional product. Such covalent attachments can be accomplished using known chemical or enzy matic methods. A polypeptide can also be produced by operably linking the isolated nucleic acid of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif, U.S.A, (the MaxBac® kit), and such methods are known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), and Luckow and Summers, Bio/Technology 6:47 (1988). Cell-free translation systems could also be employed to produce polypeptides, such as antibodies or antibody fragments, using RNAs derived from nucleic acid constructs disclosed herein. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory- Manual, Elsevier, New York, 1985). A host cell that comprises an isolated nucleic acid of the invention, preferably operably linked to at least one expression control sequence, is a “recombinant host cell”.

[0110] In certain embodiments, cell lines may be selected through determining which cell lines have high expression levels and constitutively produce antigen binding proteins with the desired binding properties. In another embodiment, a cell line from the B cell lineage that does not make its own antibody but has a capacity to make and secrete a heterologous antibody can be selected.

FURTHER EMBODIMENTS

[0111] Particular embodiments of the invention include the following.

1. An isolated protein comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH and VL are bound together, wherein the VH and VL comprise at least one of the following sets of charged amino acids: a. the VH comprises a charged amino acid at position number 39, and the VL comprises a charged amino acid at position number 85 that is complementary in charge to the amino acid at position 39 of the VH; b. the VH comprises a charged amino acid at position number 105, and the VL comprises a charged amino acid at position number 42 that is complementary in charge to the amino acid at position 105 of the VH; or c. the VH comprises a charged amino acid at position number 91, and the VL comprises a charged amino acid at position number 38 that is complementary in charge to the amino acid at position 91 of the 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. The isolated protein of embodiment 1, wherein the VH and VL comprise the set of charged amino acids in (a). The isolated protein of embodiment 2, wherein the VH comprises a positively charged amino acid at position 39 and the VL comprises a negatively charged amino acid at position 85. The isolated protein of embodiment 3, wherein the 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. The isolated protein of embodiment 2, wherein the VH comprises a negatively charged amino acid at position 39 and the VL comprises a positively charged amino acid at position 85. The isolated protein of embodiment 5, wherein the 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. The isolated protein of any of embodiments 1-6, wherein the VH and VL comprise the set of charged amino acids in (b). The isolated protein of embodiment 7, wherein the VH comprises a positively charged amino acid at position 105 and the VL comprises a negatively charged amino acid at position 42. The isolated protein of embodiment 8, wherein the 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. The isolated protein of embodiment 7, wherein the VH comprises a negatively charged amino acid at position 105 and the VL comprises a positively charged amino acid at position 42. The isolated protein of embodiment 10, wherein the VH position 105 (VH105) and VL position 42 (VL42) comprise: (i) an aspartic acid at VH105 and a ly sine at VL42, (ii) a glutamic acid at VH105 and a lysine at VL42, (iii) an aspartic acid at VH 105 and an arginine at VL42, or (iv) a glutamic acid at VH105 and an arginine at VL42. The isolated protein of any of embodiments 1-11, wherein the VH and VL comprise the set of charged amino acids in (c). The isolated protein of embodiment 12, wherein the VH comprises a positively charged amino acid at position 91 and the VL comprises a negatively charged amino acid at position 38. The isolated protein of embodiment 13, wherein the 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. The isolated protein of embodiment 12, wherein the VH comprises a negatively charged amino acid at position 91 and the VL comprises a positively charged amino acid at position 38. The isolated protein of embodiment 15, wherein the 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. The isolated protein of any of embodiments 1-16, wherein the isolated protein comprises an antibody heavy chain comprising the VH and an antibody light chain comprising the VL. The isolated protein of embodiment 17, wherein the isolated protein is an antibody. The isolated protein of embodiment 18, wherein the antibody is an IgG antibody. The isolated protein of embodiment 19, wherein the antibody is an IgGL IgG2. lgG3, or

IgG4 antibody. The isolated protein of any of embodiments 17-20, wherein the isolated protein is a bispecific antibody. The 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). The isolated protein of any of embodiment 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 replacement of the amino acids at positions 392, 409, and 439 with a negatively charged amino acid, wherein the CH3' domain comprises 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. The isolated protein of any of embodiment 18-23. wherein the antibody comprises a first heavy chain constant domain and light chain constant domain pair (CHI and CL) and a second heavy chain constant domain and light chain constant domain pair (CHI' and CL'); wherein the CHI and CHT each comprise an alteration at position 183, as numbered according to the EU numbering scheme; wherein the CL and CL' comprise an alteration at position 176, as numbered according to the EU numbering scheme; wherein CHI position 183 and CL' position 176 each comprise a positively charged amino acid; and wherein CHI' position 183 and CL position 176 each comprise a negatively charged amino acid. The isolated protein of embodiment 24, wherein (i) CHI position 183 and CL' position 176 each comprise lysine, and CHI' position 183 and CL position 176 each comprise aspartic acid; (ii) CHI position 183 and CL' position 176 each comprise lysine, and CHT position 183 and CL position 176 each comprise glutamic acid; (iii) CHI position 183 and CL' position 176 each comprise arginine, and CHT position 183 and CL position 176 each comprise aspartic acid; or (iv) CHI position 183 and CL' position 176 each comprise arginine, and CHT position 183 and CL position 176 each comprise glutamic acid. A bispecific antibody comprising a first heavy chain variable domain and light chain variable domain pair (VH and VL) and a second heavy chain variable domain and light chain variable domain pair (VH¹ 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. the VH and VH' each comprise a charged amino acid at position number 39; the VL and VL' each comprise a charged amino acid at position number 85 that is complementary in charge to the amino acid at position 39 of the VH and VH', respectively; and the VH and VH' comprise amino acids with complementary charges; b. the VH and VH' each comprise a charged amino acid at position number 105; the VL and VL' each comprise a charged amino acid at position number 42 that is complementary in charge to the amino acid at position 105 of the VH and VH', respectively; and the VH and VH' comprise amino acids with complementary charges; or c. the VH and VH' each comprise a charged amino acid at position number 91; the VL and VL' each comprise a charged amino acid at position number 38 that is complementary in charge to the amino acid at position 91 of the VH and VH', respectively; and the 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. The 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). The bispecific antibody of embodiment 27, wherein the VH and VL' comprise a positively charged amino acid, and wherein the VH' and VL comprise a negatively charged amino acid. The 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. The bispecific antibody of embodiment 27, wherein the VH and VL' comprise a negatively charged amino acid, and wherein the VH' and VL comprise a positively charged amino acid. The 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. The bispecific antibody of any 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). The bispecific antibody of embodiment 32, wherein the VH and VL' comprise a positively charged amino acid, and wherein the VH' and VL comprise a negatively charged amino acid. The 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 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. The bispecific antibody of embodiment 32, wherein the VH and VL' comprise a negatively charged amino acid, and wherein the VH' and VL comprise a positively charged amino acid. The 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. The bispecific antibody of any 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). The bispecific antibody of embodiment 37, wherein the VH and VL' comprise a positively charged amino acid, and wherein the VH' and VL comprise a negatively charged amino acid. The 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. The bispecific antibody of embodiment 37, wherein the VH and VL' comprise a negatively charged amino acid, and wherein the VH' and VL comprise a positively charged amino acid. The 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. A method of producing bispecific antibody comprising expressing one or more polynucleotides encoding the bispecific antibody of any of embodiments 26-41 in host cells, culturing the host cells in a culture media under conditions such that the component polypeptide chains are produced, and recovering the bispecific antibody from the cell or the culture media. 43. The method of embodiment 42, wherein the host cells comprise one or more plasmids that comprise the one or more polynucleotides.

44. The 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. The method of embodiment 42, wherein the recovered bispecific antibody is purified byprotein A chromatography.

46. The method of any of embodiments 42-45, wherein the recovered bispecific antibody is further purified by ion exchange chromatography.

47. A method of producing an isolated protein comprising expressing one or more polynucleotides encoding protein of any of embodiments 1-25 in host cells, culturing the host cells in a culture media under conditions such that the component polypeptide chains are produced, and recovering the protein from the cell or the culture media.

48. The method of embodiment 47, wherein the host cells comprise one or more plasmids that comprise the one or more polynucleotides.

49. The method of embodiment 47 or embodiment 48, wherein the isolated protein is purified by ion exchange chromatography.

EXAMPLES

EXAMPLE 1 : ANALYSIS OF FV STRUCTURES FOR CANDIDATE CHARGE PAIR MUTATIONS

[0112] As the Fv interface can comprise up to 50% of the entire Heavy-Light interaction (FIGI), we hypothesized engineering solutions localized to the constant CH-CL interface may not be sufficient for attracting/repelling the complete polypeptide chain. Therefore, to develop improved methods for facilitating the pairing between particular desirable heavy and light chain polypeptides, a research effort was undertaken to identify locations within the heavy chain variable region (VH) and light chain variable region (VL) at which charge pair mutations could be placed. Broadly, the goal was to identify novel paired mutations which would contribute to an unstable high energy state when undesired VH and VL combinations are bound (i.e., when the VH and VL are mispaired). Conversely, there novel paired mutations should contribute to a stable low energy state when the desired VH and VL combination is bound.

[0113] FIG. 1A shows an exemplary bispecific antibody containing four distinct 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 illustrated in FIG. 1 A). In such a bispecific antibody, there are four distinct zones of interaction. First, the VH and VL regions interact via an Fv interface in each arm of the antibody. Second, the heavy chain constant domain 1 (CHI) and the light chain constant domain (CL) form a CH1-CL interface. Third, the heavy chain constant domain 2 (CH2) in each the heavy chain form a CH2-CH2 interface. And fourth, the heavy chain constant domain 3 (CH3) in each the heavy chain form a CH3-CH3 interface. The CH2-CH2 interface and the CH3-CH3 interface are responsible for holding the two arms of the antibody together, but have no impact on the pairing between each heavy chain and its corresponding light chain.

[0114] FIG. IB shows exemplary undesired by-products formed when the wrong light chain is paired with one or both of the heavy chains of the bispecific antibody. These byproducts generally reduce expression of a desired bispecific antibody by using protein chains that might otherwise be assembled into the desired product.

[0115] A series of structures of known VH-VL interactions were used as the 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 shown in FIG. 2A (showing the structure of the anti -HIV antibody Bl 2). That exemplary structure includes dotted lines between the domains showing distance-based interaction lines between atoms in the VH and VL domains, as calculated using PyMoL Each of the residues that participates in these interactions was a candidate for a potential charge pair mutation.

[0116] An in silico analysis was performed to identify novel sites for charge pair mutations. The goal of that analysis was to identify sites where paired mutations could be made, such that the introduced alterations lead to an unstable high energy state in undesired “mispaired” states and a stable lower energy state in the desired pairing pattern. FIG. 2B illustrates this dynamic. In FIG. 2B, the undesired states (“Mispaired Antibody 1” and “Mispaired Antibody 2”) contain unstable mispairings in which like-charged amino acid residues are forced into close proximity, as shown by the paired open circles or paired filled circles. These unstable mispairings result in a high energy state. Tn comparison, the desired states (Antibody 1 or Antibody 2) have a lower energy state because there are oppositely charged residues present at the same site. Accordingly, the difference in energy would drive the antibody chains to assemble in an equilibrium state with a large preponderance of the desired pairing state. In FIG. 2B, this is show n by the larger arrows pointing tow ards Antibody 1 and Antibody 2.

[0117] Nineteen different amino acids residues within the VH domain were selected based on their presence in the VH-VL interface, and amino acid residues within the VL domain were chosen for potential charge pairing mutations with each VH residue based on proximity. This resulted in between one and nine residues being selected for pairing with each VH residue, and a total of sixty -nine potential CPMs for in silico analysis.

[0118] As the first step in the in silico analysis, each of the identified potential charge pair mutations at selected positions in the Fv were designed and assessed with Rosetta via XML script (RosettaScripts) in the talaris2014 score function. Asp, Glu, Lys, and Arg mutations w ere made pairwise and minimized by cyclic coordinate descent of a 5-residue fragment including 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 process w as replicated using the structure of 18 different Fv sequences combined into 54 different Fv pairs as input structures. Thus, using this strategy⁷, thousands of pairing designs w ere screened in order to identify a subset of promising candidates for experimental validation. Desirable mutations were selected on the basis of having a consistently large difference in total Rosetta score between the correctly paired and mispaired chains across all input structures and having the total Rosetta score of the correctly paired combination be as low⁷ as possible as a prediction of Fv pair energy state. An exemplary⁷ output is shown in the chart in FIG. 2C. which shows that the energy state for one mispaired antibody was predicted to be much higher than that of either of the correctly paired antibodies, while the predicted energy state for the other mispaired antibody⁷ was somewhat higher than either of the correctly paired antibodies.

[0119] In the in silico analysis, a number of CPM designs w ere predicted to do well in a variety of different Fv frameworks. In addition to the previously reported VH:39-VL:38 design previously reported (Lewis. S.M., et al., Nat Biotechnol. 32(2): 191-198 (2014)), multiple additional pairings were identified that resulted in a stable interface. Specifically, introducing complementary charged mutations at sites VH:39-VL:85, VH: 105-VL:42, and VH:91-VL:38 (as numbered according to the Kabat scheme) were consistently predicted to do well regardless of the Fv framework analyzed. The positions of these newly identified CPMs are shown in the exemplary VH and VL sequences in FIGS. 7A and 7B, which are from the anti -HIV antibody Bl 2. In FIGS. 7 A and 7B, the VH and VL sequences are annotated with the Kabat numbering for each residue and the location of the CDR and framework regions. FIGS. 7A and 7B also include boxes highlighting the amino acids that would be altered if introducing 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

[0120] Unless otherwise indicated, the plasmid construction, cell culture, plasmid transfection, protein expression, and antibody purification referenced in subsequent examples were performed as described in this Example.

[0121] Plasmid Construction: genes to be expressed as polypeptides were synthesized by Twist Bioscience. Genes were cloned individually into vectors for mammalian transient expression using the Golden Gate assembly method. (See Engler C et al., PLoS One 3:e3647 (2008), which is incorporated herein by reference in its entirety) All chains containing Fc regions were constructed using a human IgGl scaffold (IgGl-SEFL2) carrying an aglycosylation mutation, as well as an engineered disulfide bond. (See Estes et al., iScience 24: 103447 (2021); and Jacobsen FW et al., J. Biol. Chem. 292: 1865-75 (2017); which are incorporated herein by reference in their entirety). All molecules requiring hetero-Fc pairing included charge pair mutations (“CPM”) v!03 in the CH3 regions to promote heterodimerization. (See Estes et al.. iScience 24: 103447 (2021)). Briefly, the v!03 pattern of mutations includes the 392D, 409D, and 439D mutations in one heavy chain, and the 356K and 399K mutations on the other heavy chain (as numbered according to the EU antibody numbering scheme). Other CPMs were also included in the heavy and light chain sequences as indicated below.

[0122] After synthesis and cloning, the plasmid sequences were confirmed by Sanger, and transfection-grade DNA was prepared using Maxi plasmid purification kits (Qiagen).

[0123] Cell Culture, Plasmid Transfection, and Protein Expression: proteins were transiently expressed in suspension human embryonic kidney 293-6E cells (NRC-BRI) using the PEImax transfection reagent. Prior to transfection, the cells were maintained in FreeStyle F-17 medium (Gibco, catalog # A1383502) with 0.1 % Kolliphor P188 (Sigma, catalog # K4894), 25 pg/ml G418 (Gibco, catalog # 10131027) and 6 mM L-glutamine (Gibco, catalog # 25030149). To achieve an optimal density of 2xl0⁶ viable cells per mL for transfection, cells were passaged 26 hours prior to transfection. The purified plasmids were mixed at a ratio of 1 : 1 : 1 : 1 for the tested four-chain Hetero-IgGs. For each mL of cell media, 0.5 pg DNA was incubated with 1.5 pL PEImax reagent (Polysciences, catalog # 24765-2) in 100 pL FreeStyle F-17 medium for 10 minutes, then added to the cell culture. 24 hours following transfection, cell cultures were fed with Tryptone N1 solution (Organotechnie. catalog # 19553) and glucose (Thermo Fisher, catalog # A2494001) to a final concentration of 2.5 g/L and 4.5 g/L, respectively. Seven days following transfection, the conditioned medium was harvested for purification.

[0124] Antibody purification with Protein A and cation exchange chromatography: Following harvest of 40 mL cultures, 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 mM Tris, 100 mM NaCl, pH 7.4. Protein was eluted with 100 mM sodium acetate, pH 3.6 followed immediately by buffer exchange into 10 mM sodium acetate, 150 mM NaCl, pH 5.2 using a 5 ml HiTrap Desalting column (GE Life Sciences, catalog # GE17-1408-01).

[0125] For analysis and separation of antibody species present in Protein A purifications, cation-exchange chromatography (CIEX) was performed. Protein A eluates were diluted with 20 ml of 20 mM MES, pH 6.2 and loaded onto a 1 ml cation ion-exchange column (HiTrap SP-HP, GE Life Sciences, catalog # GE29-0513-24) at 1 ml/min. After washing with 8 column volumes of dilution buffer at 1 ml/min, the samples were eluted with a linear 0-400 mM NaCl gradient over 40 column volumes at 0.4 ml/min.

[0126] To identify species within fractions gathered during CIEX or to analyze purity' of samples, samples were analyzed with non-reducing micro capillary electrophoresis (MCE) and analytical size exclusion chromatography (SEC). For non-reducing MCE, 6 pL of proteins were mixed with 21 pL 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 lodoacetamide), heated to 85°C for 10 min, and analyzed using a Caliper LabChip GXII Touch instrument (PerkinElmer). For analytical SEC. protein samples were analyzed on an Acquity HPLC instrument (Waters) using a Zenix-C column (300 A, 3 micron, 4.6x300 mm) (Sepax Technologies) with 100 mM sodium phosphate pH 6.8, 250 mM NaCl as the running buffer at 0.45 ml/min flow rate. Fraction pool concentrations were determined using Nanodrop 8000 (Thermo Fisher).

EXAMPLE 3: ANALYSIS OF CONTAMINATING SPECIES WHEN PRODUCING ANTIBODIES CONTAINING CHARGE PAIR MUTATIONS

[0127] Antibodies containing the complementary charged mutations at sites VH:91- VL:38, VH:39-VL:85, and VH: 105-VL:42 were tested in bispecific antibodies, with and without other CPMs present in the Fab region of the antibodies. Each set of complementary mutations was tested in the background of an IgGl antibody containing a set of known charge pair mutations present in the heavy chain Fc region. This background allowed for testing of the new complementary mutations on the heavy chain-light chain binding only, with reduced contaminants resulting from the mispairing of heavy chains. In addition, some tested antibodies contained a charge pair mutation in the CH1-CL interface. Tested antibodies are listed in TABLE 1 below, along with the CPMs present in each of the domains of those antibodies. Thus, all of the antibodies listed in Table 1 are IgGl antibodies, with the only differences between them being in the CDR regions (i.e., changing the specificity to bind antigen A or antigen C) and in the listed charge pair mutations.

TABLE 1

[0128] Constructs for expressing each of the bispecific antibodies in Table 1 were prepared according to the methods described in Example 2. Briefly, for expression of these antibodies, the four antibody chains for expressing a given antibody were transfected into HEK 293 cells in a 1 : 1: 1: 1 ratio (also as described in Example 2). Seven days following transfection, the conditioned medium was harvested for purification of secreted antibodies. Initial purification was accomplished using protein A affinity' capture. Subsequently, species of antibody were separated using cation exchange chromatography. FIGS. 3A-3E, 4A-4C. and 5 show chromatographs of the separation of purified antibody samples. As part of the CIEX chromatography process, individual fractions eluted from the CIEX column were sequentially collected and analyzed to determine the antibody species present in each major peak.

[0129] FIGS. 3A-3E’s chromatographs are from tests of five different bispecific antibody variants, which all target two antigens generically referred to as Antigen A and Antigen C (see TAB E 1 above). To distinguish the two arms of these bispecific antibodies, the domains that are in the peptide chains responsible for binding Antigen C are designated with the prime symbol ('), while the domains responsible for binding Antigen A lack such notation. Each of FIGS. 3 A-3E further provides a list of the charge pair mutations present in the bispecific antibody^ being produced. Outside of differences between the listed CPM alterations, the bispecific antibodies in FIGS. 3A-3E are identical in sequence. Within FIGS. 3A-3E, the locations of peaks containing particular antibody species are noted with diagrams of the species and arrows pointing to the pertinent peak. The desired bispecific antibody in each chromatograph is also highlighted by a box surrounding its peak. The bispecific antibody variants tested within FIGS. 3A-3E were designed to allow testing the efficacy of CPMs at the newly identified VH:91-VL:38 site at preventing antibody chain mispairing.

[0130] FIG. 3A shows a CIEX chromatograph of secreted antibody from cells expressing the components of ‘‘Bispecific Antibody' 1.” Bispecific Antibody 1 contains CPMs only in its Fc region, in residues that are part of the heavy chain CH3-CH3' interface in particular. 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 , FIG. 3 A contains peaks corresponding to at least three contaminating species. While the desired bispecific antibody peak is the largest in FIG. 3A, significant amounts of contaminating alternative antibody species are also present.

[0131] FIG. 3B shows a CIEX chromatograph of 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 in the CHI -CL and CH1'-CL' interfaces. The additional CPM is comprised of a 183E alteration in the CHI domain and 176K alteration in the CL domain, alongside a 183K alteration in the CHI' domain and a 176E alteration in the CL' domain. In both FIG. 3 A and FIG. 3B. the most prominent peak is the desired bispecific antibody. Both also contain peaks indicative of contaminating 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 in for Bispecific Antibody 1 (in FIG. 3A). Notably, however, expression and purification of an antibody containing both CPMs in the Fc interface and in the CHI -CL interface still resulted in significant contaminants.

[0132] To determine whether adding a newly identified CPM in the Fv interface (see Example 1 above) can reduce the presence of undesired contaminants when producing 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 in 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 alteration in the VH domain and an 85R alteration in the VL domain, alongside a 39R alteration in the VH' domain and an 85E alteration in the VL' domain. FIG. 3C shows a CIEX chromatograph of secreted antibody from cells expressing the components of Bispecific Antibody 3. In FIG. 3C, the most prominent peak is the desired bispecific antibody. Between approximately 108 and 115 mL (where contaminants were noted for Bispecific Antibody 2), little to no contaminating antibody was detected for Bispecific Antibody 3. Comparison of FIG. 3B and FIG. 3C shows that introduction of a CPM at the VH:39-VL:85 sites of each arm led to elimination of almost all contaminants observed in the Bispecific Antibody 2 purified antibody sample. This indicates that a CPM at the VH:39-VL:85 site can lead to more effective and complete pairing of antibody chains, and can improve pairing even in the presence of other CPMs in an antibody. [0133] “Bispecific Antibody 4” was also constructed by combining CPMs in the VH:39- VL:85 site with CPMs in the CH3-CH3' interface. The heavy chain CH3-CH3' interface in Bispecific Antibody 4 contains the 392D, 409D, and 439D alterations in the CH3 domain while the CH3' domain contains the 356K and 399K alterations (as numbered according to the EU antibody numbering scheme). Bispecific Antibody 4 is identical to Bispecific Antibody 3, except that Bispecific Antibody 4 lacks any CPM in its CHI -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 CHI -CL interfaces. FIG. 3D shows a C1EX chromatograph of secreted antibody from cells expressing the components of Bispecific Antibody 4. In FIG. 3D, the most prominent peak is the desired bispecific antibody. FIG. 3D shows that, between approximately 108 and 115 mL, Bispecific Antibody 4 contained fewer contaminating species than an antibody with only CPMs in the CH3-CH3' interface (as shown by comparison to Bispecific Antibody 2's chromatograph in FIG. 3A). Moreover, Bispecific Antibody 4 showed a different pattern of contaminants than Bispecific Antibody 2 — indicating that CPMs in the VH-VL interface, and at the VH:39-VL:85 site in particular, have a complementary effect to those in the CHI -CL interface.

[0134] To allow comparison of the CPMs with the VH:91-VL:38 format with those having the previously known VH:39-VL:38 format, “Bispecific Antibody 5” was constructed and tested. Bispecific Antibody 5 was constructed by combining CPMs in the VH:39-VL:38 site with CPMs in the CH3-CH3' interface. As with Bispecific Antibodies 1-4. Bispecific Antibody 5's 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). As with Bispecific Antibodies 2 and 3, Bispecific Antibody 5 contains CPMs in the CH1-CL and CH1'-CL' interfaces comprising a 183E alteration in the CHI domain and 176K alteration in the CL domain, alongside a 183K alteration in the CHT domain and a 176E alteration in the CL' domain. Therefore, comparison of Bispecific Antibody 3 and Bispecific Antibody 5 provides an indication of the relative effects of the VH:91-VL:38 CPM with the VH:39-VL:38 CPM. FIG. 3E shows a CIEX chromatograph of secreted antibody from cells expressing the components of “Bispecific Antibody 5.” Comparison of FIG. 3E with FIG. 3Cs results with Bispecific Antibody 3 shows that fewer contaminants are present in FIG. 3C between approximately 108 and 115 mL. This indicates that the VH:91-VL:38 CPM was better able to drive selective pairing that the VH:39-VL:38 CPM, when present in otherwise identical bispecific antibodies.

[0135] FIGS. 4A-4C’s chromatographs are from tests of three different bispecific antibody variants, which all target two antigens generically referred to as Antigen B and Antigen D (see TABLE 1 above). To distinguish the two arms of these bispecific antibodies, the domains that are in the peptide chains responsible for binding Antigen D are designated with the prime symbol ('), while the domains responsible for binding Antigen B lack such notation. Each of FIGS. 4A-4C further provides a list of the charge pair mutations present in the bispecific antibody being produced. Outside of differences between the listed CPM alterations, the bispecific antibodies in FIGS. 4A-4C are identical in sequence. Within FIGS. 4A-4C, the locations of the peak containing the desired antibody are noted with diagrams of the antibody and arrows pointing to the pertinent peak. The desired bispecific antibody in each chromatograph is also highlighted by a box surrounding its peak. The bispecific antibody variants tested within FIGS. 4A-4C were designed to allow testing the efficacy of CPMS at the newly identified VH: 105-VL:42 site at preventing antibody chain mispairing.

[0136] FIG. 4A shows a CIEX chromatograph of secreted antibody from cells expressing the components of “Bispecific Antibody 6.” Bispecific Antibody 6 contains CPMs only in its Fc region, in residues that are part of the heavy chain CH3-CH3' interface in particular. 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, FIG. 4A contains peaks corresponding to contaminating species 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, significant amounts of contaminating alternative antibody species are also present.

[0137] FIG. 4B shows a CIEX chromatograph of 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 in the CH1-CL and CH1'-CL' interfaces. The additional CPM is comprised of a 183E alteration in the CHI domain and 176K alteration in the CL domain, alongside a 183K alteration in the CHT domain and a 176E alteration in the CL' domain. In both FIG. 4A and FIG. 4B, the most prominent peak is the desired bispecific antibody. The addition of the CPMs in the CH1 - CL and CH1'-CL' interfaces led to the near elimination of the peak eluted before the desired species in FIG. 4B. where a prominent peak was observed in FIG. 4A. However. FIG. 4B shows a peak indicative of a contaminating alternative species on the shoulder of the desired peak at approximately 92-93 mL (as was also observed in FIG. 4A). Thus, expression and purification of the antibody containing both CPMs in the Fc interface and in the CHI -CL interface still resulted in a significant contaminant.

[0138] To determine whether adding a newly identified CPM in the Fv interface (see Example 1 above) can reduce the presence of undesired contaminants when producing 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 in 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 comprise a 105R alteration in the VH domain and a 42E alteration in the VL domain, alongside a 105E alteration in the VH' domain and a 42R alteration in the VL' domain. FIG. 4C shows a CIEX chromatograph of secreted antibody from cells expressing the components of Bispecific Antibody 8. In FIG. 4C, the most prominent peak is the desired bispecific antibody. As observed for Bispecific Antibody 7 in FIG. 4B, Bispecific Antibody 8’s CPMs led to the near elimination of the peak eluted before the desired species in FIG. 4C. Comparison of FIG. 4B and FIG. 4C shows that introduction of a CPM at the VH: 105- VL:42 sites of each arm led to reduction of the size of the contaminating peak on the shoulder of the desired peak at approximately 92-93 mL. This indicates that a CPM at the VH: 105-VL:42 site can lead to more effective and complete pairing of antibody chains, and can improve pairing even in the presence of other CPMs in an antibody.

[0139] FIG. 5’s chromatograph is from a test of a bispecific antibody called “Bispecific Antibody 9,” which targets two antigens generically referred to as Antigen E and Antigen F (see TABLE 1 above). To distinguish the two arms of the bispecific antibody, the domains that are in the peptide chains responsible for binding Antigen F are designated with the prime symbol ('), while the domains responsible for binding Antigen E lack such notation. FIG. 5 further provides a list of the charge pair mutations present in Bispecific Antibody 9. Within FIG. 5, the locations of the peak containing Bispecific Antibody 9 is noted with aa diagram of the antibody and an arrow pointing to the pertinent peak. The desired bispecific antibody in each chromatograph is also highlighted by a box surrounding its peak. The bispecific antibody variant tested within FIG. 5 was designed to test the efficacy of a CPM at the newly identified VH:91-VL:38 site at preventing antibody chain mispairing. Specifically, Bispecific Antibody 9 contains a CPM at the VH:91-VL:38 sites of each arm of the antibody. These CPMs comprise a 91R alteration in the VH domain and a 38E alteration in the VL domain, alongside a 91E alteration in the VH' domain and a 38R alteration in the VL' domain. As seen in FIG. 5, including CPMs at the VH:91-VL:38 sites led highly effective pairing of the antibody chains, with low amounts of contaminating mispaired antibody species.

EXAMPLE 4: PRODUCTION LEVELS OF ANTIBODIES CONTAINING CHARGE PAIR MUTATIONS

[0140] Experiments were also performed to test whether including charge pair mutations in the VH:VL interface could lead to increased production and purification of the desired antibody species. Tested antibodies are listed in TABLE 2 below, along with the CPMs present in each of the domains of those antibodies. All of the antibodies listed in Table 2 are IgGl antibodies, with the only differences between them being in the CDR regions (i.e., changing the specificity to bind antigen A or antigen C) and in the listed charge pair mutations.

TABLE 2

[0141] Plasmids encoding the antibodies in Table 2 were constructed and expressed in HEK 293-6E cells as described in Example 2 above. For the monospecific antibodies, plasmids were transfected at a 1 : 1 ratio of the heavy and light chain plasmids, while the four bispecific plasmids were transfected at a 1: 1 : 1: 1 ratio. Seven days following transfection, the conditioned medium was harvested for purification of secreted antibodies. Initial purification was accomplished using protein A affinity capture. Subsequently, species of antibody were separated using cation exchange chromatography. As part of the CIEX chromatography process, individual 1 ml fractions eluted from the CIEX column were sequentially collected and analyzed to determine the antibody species present in each major peak. Fractions containing the desired antibody were pooled and quantified by A280 measurement.

[0142] FIG. 6 shows the amount of pooled antibody recovered for each transfection. The

Anti-A Antibody and Anti-C Antibody tests serve as controls for the amount of antibody recovered when expressing and purifying unmodified TgGl antibodies. After purification, there was recovery⁷ of approximately 33 mg/L and 43 mg/L of Antibody A and Antibody C, respectively. Expression and purification of Bispecific Antibody 1 (containing Fc CPMs, but no Fab CPMs) led to recovery of 20 mg/L antibody. This decrease was likely the result of a significant proportion of the expressed antibody chains being used as part of unrecovered contaminating species. The effects of adding a single charge pair mutation in the Fab region (alongside Fc CMPs) were then tested by expressing either Bispecific Antibody 2 (CH1 : 183-CL: 176) or Bispecific Antibody 4 (VH:39-VL:85). Inclusion of either of these CPMs increased antibody recovery to levels equivalent to or better than either parent antibody. Bispecific Antibody 2 was recovered at approximately 43 mg/L and Bispecific Antibody 4 was recovered at approximately 45 mg/L.

[0143] The effects of combining multiple CPMs in the Fab region on antibody production was also tested (see FIG. 6). Bispecific Antibody 3 (CH1 : 183-CL: 176 plus VH:39-VL:85) and Bispecific Antibody 4 (CHL 183-CL: 176 plus VH:39-VL:38) both had increased recovery relative to any 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 translates to an increase in production for these two bispecifics of 27% relative to Anti-C Antibody and an increase of 65% relative to Anti-A Antibody.

[0144] The data in FIG. 6 demonstrates that, in addition to decreasing the number and amount of contaminating species present when producing antibodies, the newly discovered CPMs in the VH-VL interface facilitate increased production levels of antibodies.

Claims

1. An isolated protein comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH and VL are bound together, wherein the VH and VL comprise at least one of the following sets of charged amino acids: a. the VH comprises a charged amino acid at position number 39, and the VL comprises a charged amino acid at position number 85 that is complementary in charge to the amino acid at position 39 of the VH; b. the VH comprises a charged amino acid at position number 105, and the VL comprises a charged amino acid at position number 42 that is complementary in charge to the amino acid at position 105 of the VH; or c. the VH comprises a charged amino acid at position number 91, and the VL comprises a charged ammo acid at position number 38 that is complementary in charge to the amino acid at position 91 of the 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. The isolated protein of claim 1, wherein the VH and VL comprise the set of charged amino acids in (a).

3. The isolated protein of claim 2, wherein the VH comprises a positively charged amino acid at position 39 and the VL comprises a negatively charged amino acid at position 85.

4. The isolated protein of claim 3, wherein the 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. The isolated protein of claim 2, wherein the VH comprises a negatively charged amino acid at position 39 and the VL comprises a positively charged amino acid at position 85.

6. The isolated protein of claim 5, wherein the 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. The isolated protein of any of claims 1-6, wherein the VH and VL comprise the set of charged amino acids in (b).

8. The isolated protein of claim 7, wherein the VH comprises a positively charged amino acid at position 105 and the VL comprises a negatively charged amino acid at position 42.

9. The isolated protein of claim 8, wherein the 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, (hi) an arginine at VH105 and an aspartic acid at VL42, or (iv) an arginine at VH105 and a glutamic acid at VL42.

10. The isolated protein of claim 7, wherein the VH comprises a negatively charged amino acid at position 105 and the VL comprises a positively charged amino acid at position 42.

11. The isolated protein of claim 10, wherein the 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. The isolated protein of any of claims 1-11, wherein the VH and VL comprise the set of charged amino acids in (c).

13. The isolated protein of claim 12, wherein the VH comprises a positively charged amino acid at position 91 and the VL comprises a negatively charged amino acid at position 38.

14. The isolated protein of claim 13, wherein the 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. The isolated protein of claim 12, wherein the VH comprises a negatively charged amino acid at position 91 and the VL comprises a positively charged amino acid at position 38.

1 . The isolated protein of claim 15, wherein the 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. The isolated protein of any 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. The isolated protein of claim 17, wherein the isolated protein is an antibody.

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

20. The isolated protein of claim 19, wherein the antibody is an IgGl, IgG2, IgG3, or IgG4 antibody.

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

22. The isolated protein of 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. The isolated protein of any of claim 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 replacement of the amino acids at positions 392, 409, and 439 with a negatively charged amino acid, wherein the CH3' domain comprises 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. The isolated protein of any of claim 18-23, wherein the antibody comprises a first heavy chain constant domain and light chain constant domain pair (CHI and CL) and a second heavy chain constant domain and light chain constant domain pair (CHI' and CL'); wherein the CHI and CHI' each comprise an alteration at position 183, as numbered according to the EU numbering scheme; wherein the CL and CL' comprise an alteration at position 176, as numbered according to the EU numbering scheme; wherein CHI position 183 and CL' position 176 each comprise a positively charged amino acid; and wherein CHI' position 183 and CL position 176 each comprise a negatively charged amino acid.

25. The isolated protein of claim 24, wherein (i) CHI position 183 and CL' position 176 each comprise lysine, and CHI' position 183 and CL position 176 each comprise aspartic acid; (ii) CHI position 183 and CL' position 176 each comprise lysine, and CHI' position 183 and CL position 176 each comprise glutamic acid; (iii) CHI position 183 and CL' position 176 each comprise arginine, and CHI' position 183 and CL position 176 each comprise aspartic acid; or (iv) CHI position 183 and CL' position 176 each comprise arginine, and CHI' position 183 and CL position 176 each comprise glutamic acid.

26. A bispecific antibody comprising a first heavy chain variable domain and light chain variable domain pair (VH and VL) and a second heavy chain variable domain and light chain variable domain pair (VH¹ 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. the VH and VH' each comprise a charged amino acid at position number 39; the VL and VL' each comprise a charged amino acid at position number 85 that is complementary in charge to the amino acid at position 39 of the VH and VH', respectively; and the VH and VH' comprise amino acids with complementary charges; b. the VH and VH' each comprise a charged amino acid at position number 105; the VL and VL' each comprise a charged amino acid at position number 42 that is complementary’ in charge to the amino acid at position 105 of the VH and VH', respectively; and the VH and VH' comprise amino acids with complementary charges; or c. the VH and VH' each comprise a charged amino acid at position number 91; the VL and VL' each comprise a charged amino acid at position number 38 that is complementary’ in charge to the amino acid at position 91 of the VH and VH', respectively; and the 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. The bispecific antibody of claim 26. wherein the VH and VL pair and the VH' and VL' pair comprise the set of charged amino acids in (a).

28. The bispecific antibody of claim 27, wherein the VH and VL' comprise a positively charged amino acid, and wherein the VH' and VL comprise a negatively charged amino acid.

29. The bispecific antibody of 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. The bispecific antibody of claim 27, wherein the VH and VL' comprise a negatively charged amino acid, and wherein the VH' and VL comprise a positively charged amino acid.

31. The bispecific antibody of 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. The bispecific antibody of any 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. The bispecific antibody of claim 32, wherein the VH and VL' comprise a positively charged amino acid, and wherein the VH' and VL comprise a negatively charged amino acid.

34. The bispecific antibody of 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 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. The bispecific antibody of claim 32, wherein the VH and VL' comprise a negatively charged amino acid, and wherein the VH' and VL comprise a positively charged amino acid.

36. The bispecific antibody of 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. The bispecific antibody of any 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. The bispecific antibody of claim 37, wherein the VH and VL' comprise a positively charged amino acid, and wherein the VH' and VL comprise a negatively charged amino acid.

39. The bispecific antibody of 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. The bispecific antibody of claim 37, wherein the VH and VL' comprise a negatively charged amino acid, and wherein the VH' and VL comprise a positively charged amino acid.

41. The bispecific antibody of 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. A method of producing bispecific antibody comprising expressing one or more polynucleotides encoding the bispecific antibody of any of claims 26-41 in host cells, culturing the host cells in a culture media under conditions such that the component polypeptide chains are produced, and recovering the bispecific antibody from the cell or the culture media.

43. The method of claim 42, wherein the host cells comprise one or more plasmids that comprise the one or more polynucleotides.

44. The method of claim 42. wherein the host cells are a Chinese Hamster Ovary (CHO) cell line or a Human Embryonic Kidney (HEK) cell line.

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

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

47. A method of producing an isolated protein comprising expressing one or more polynucleotides encoding protein of any of claims 1-25 in host cells, culturing the host cells in a culture media under conditions such that the component polypeptide chains are produced, and recovering the protein from the cell or the culture media.

48. The method of claim 47, wherein the host cells comprise one or more plasmids that comprise the one or more polynucleotides.

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