As to Bispecific antibodies generally see Bispecific antibodies under structure and types

As to Be-specific antibody purificaiton see bi-specific antibodies under particular types of antibodies purified

see also Engineering of BiAbs to Facilitate Production/Purification 

Companies: Charles River (supporst bispecific antibody development from discovery of binders through to preclinical safety assessment). 

Difficulty in Production

The generation of bispecific IgG molecules is difficult due to the fact that the antigen-binding sites are built by the variable domains of the light and heavy chain. A bispecific antibody requries to different heavy chains, and two different light changs, and exhibits asymmetry due to the presence of at least two different Fc region. Promiscous pairing of H and L chians of two antibodies expressed in one cell can theorectically result in 16 different cominbations (10 different molecules) with only one being bispecific and the reamining pairings, resulting in non-functional or monospecific molecules. (Brinkmann “The making of bispecific antibodies” 9:2, 182-212, 2016). 

Production of BsIgG by co-expression of the two light and two heavy cahins in a single host cell can be highly challenging because of the low yield of desired BsIgG and the difficulty in removing closely related mispaired IgG contaminants. This reflects that H chains form homodimers as well as the desired heterodimers. Additionally, light chains can mispair with non-cognate heavy chains. Consequently, coexpression of two antibodies can give rise to up to nine unwanted IgG species. (Spies, Molecular Immunology 67 (2015) 95-106).

Currently the most widely used route to BiIgG is by separate expression of the component antibodies into two different host cells followed by purificaiton and assembly into BsIgG in vitro. (Spies, Molecular Immunology 67 (2015) 95-106).

A major challenge in developing BsApb drugs is the selection of the molecular formate from over 60 structurally diverse alternative. The BsAb format is best chosen to match the proposed mechanisms of action and the specific clinical application. Some applications of BsAb require both antigen-binding activities be included in a single molecule. Examples include co-engagement of tumor and cytotoxic T cells and dimerization of receptors to mimic a natural ligand. By contrast, other applications of BsAb do not require both binding activities to be present in the same moelcule. Examples include the blocking and neutroalization of independent targets or the decoration of a single target with multiple antibodies. Alterantive therapeutic strategies such as the co-administration of two or multiple antibodies may be the best strategy in this case. (Spies, Molecular Immunology 67 (2015) 95-106).

Fusing Two Hybridomas

Generation of bispecific antibodies by hybrid hybridoma technology is done by selecting two hybridomas of selected specificity and fusing them, resulting in the formation of a hybrid hybridoma or qudroma cell line. One clonal quadroma cell line is therorectically capable of producing up to 10 differnt antibody species, which can be divided into 4 categories: monovalent bispecific, bivalent monospecific, monovalent monspecific (partially mismatached) and completely mismathec, based on the different heavy and light chain associations. (krosen, Advanced Drug Delivery Reviews 31 (1998) 105-129)

Quadromas and triomas can be generated by fusing either two hybridomas or one hybridoma with a B lymphocytes, respectively. In this case, the simultaneous expression of two heavy and two light chains leads to the random assembly of 10 antibody combiantions and the desired bsAb resprent only a small fraction of the secreted antibodies. 

While bispecific antibodies were originally made by fusing two hybridomas, each capable of producgin a different immunoglobulin, the complexity of species (up to 10 different speceis) produced in cell culture made purificaiton difficult and expensive. (Baurin, US 20120251541). 

Chemical engineering of BsAbs

Chemical cross-linking: Antibody fragments generated form their respective parent antibodies by enzymatic digestion or generated through recombinant technologies are conjugated using bifunctional reagents. 

Antibodies derived form two different hybridomas can be coupled directly to each other using bifunctional cross-linking reagents such as N-succinimidyl 3-(2-pyridyldithiolpropionate (SPDP). However, cross linking Ig molecules as such may result in a poorly defined and difficult to reproduce end-product. To overcome this, a more sophisticated approach using 2-nitro benzoic acid (DTNB) of alpha-p-henyl-dimaleimide which are able to react with sulfhydryl groups present in the hing region of IgG has been used. (krosen, Advanced Drug Delivery Reviews 31 (1998) 105-129).

Reduction — Oxidation –Rejoining:

Hybrid antibodies of the IgG class having two binding sites for antigens have been achieved in low yields by means of reductive dissociation of each of two intact immunoglobulin molecules (H2L2) having different antigen binding specificites into half molecules (H1L1) followed by reoxidation of a mixture of these half molecules. (Hong, J. Biol. Chem 240, 3883 (1965).

Auditore-Hargreaves (US 4,470,925) discloses production of hybrid antibodies consisting essentially of two different heavy chain light chain half molecules wherein the first half molecule provide a binding site for a frist antigen and the second a binding site for the first or a second antigen by (a) selectively cleaving a first IgG molecule to a first antigen into its H and L chain half molecules by sulfitolysis of the inter heavy chain disulfide bond to produce S-sulfonated half molecule as with sodium sulfide in the presence of 5,5′-dithiobis (2-nitrobenzoic acid)  (b) selectively cleaving a second IgG to the first or a second antigen into its H and L chain half molecules by reduction of the inter heavy chain disulfide bond to produce reduced half molecuels and then (c) combining the S sulfonated half molecules. 

Labrlin (WO2011/131746) and Schuurman (WO 2008/119353) disclose an in vtiro method for producing bispecifc antibodies wehrein a bispecific antibody is formed by “Fab-arm” or “half-molecule” exchange (swapping of a H chain and attached L chain) between two monoscpeific IgG4 antibodies upon incubation under reducing conditions. The Fab-arm exchane is the result of a disulfide bond isomerization reaction where in the inter H chain disulfide bonds in the hinge regions of monoscpeic antibodies are reduced and the resulting free cysteins form a new inter H chain disulfulbe bond with cysteine residues of another antibody molecule with a different specificity. 

Gramer (WO2013/060867) discloses an in vitro method for the production of a heterodimeric protein which includes a) incubating a first homodimeric protein and a second homodimeric protein under reducing conditions with a reducing agent such as 2-mercaptoethylamine (2-MEA) sufficient to allow reduciton of the inter-chain disulfide bonds in the hinge region and b) subjecting the composition obtain from a to oxidizing conditions sufficient to alow oxidation of cysteines in the heterodimeric protein to inter-chain disulfide bonds. The production of hterodimeric proteins is based on the hterodimeric interaction between the CH3 region of the first and second homodimeric protein is stronger than each of the homodimeric interactions between said frist and second CH3 region. This effect is obtained by cH3 modifications of the first and/or second homodimeric protein. The 2-MEA induced Fab arm exchange between two antibodies. 

Genetic Engineering of BsAbs

Dual Variable Domain IgG connected by Peptide Linker:

Dual variable domain IgG have been generated by appending the VL and VH domains of an IgG with similar domains from a second antibody via short peptide linkers. DVD-Ig are bispecific and bivalent for each antigen specificity (2+2 antigen-binding valency). (Spies, Molecular Immunology 67 (2015) 95-106).

Bispecific antibody fragments:

Antibody fragments are preferred to intact antibodies for the treamtent of solid tumours because of their enhanced tissue penetraiton. Also, their rapid clearance in vivo is ideal for diagnostic imaging of cancerous, diseased or other specific tissues. Recombinant Fab and Fv fragments of antibodies can be secreted form bacterial. However, these fragments carry a single antigne-binding site. Recombinant fragments with two binding sites have been made in several ways; for example, by chemical cross-linking of the hinge cysteine reisdues, by including C temrinal peptides that promote dimerization, by using short linkers between VH and VL that do not allow the interaction of VH and VL domains from the same chain and therefore promote the formation of bivalent scFv dimers (diabodies) or by making bispecific scFv able to chelate two adjacent epitopdes on the same antigenic molecule (CRAbs). The dimerization or a higher odrder of polymerization of antibody fragmnets results in an increase of the total apparent affintiy (or avidity) of the molecule, by means of an overall increase in the valency. Li (Protein Engineering, 10(6), 731-736, 1997)

–ScFv fragments

ScFv fragments are a commonly used building block in generating BsAb. ScFv can be constructed in either VH-VL or VL-VH orientations. However, the V domain orientation can sometimes impact antigen binding. (Spies, Molecular Immunology 67 (2015) 95-106).

Various forms of genetically engineered BsAbs have been described. For example, scFv which are recombinant antibody fragments consisting of only the VL and VH domains covalently connected to each other by a flexible polypeptide linker have been described. (krosen, Advanced Drug Delivery Reviews 31 (1998) 105-129).

Duonor-Cerutti, “Design and validation of a novel generic platform for the production of tetravalent IgG1-like bispecific antibodeis”. J. of Immunology, 2016) discloses symthesis of IgG-1 like bispecific tetravelent Ab molecules comparrying a complete and functional Fc. The technology consists of the introduction of paired mutations at the CH1-CL interface of a first human IgG1 mA (mAb1) and fusing the VH-CH1 sequence of a second Ab (mAb2) at the N terminus of the H chain of the first Ab. The mAb1 CH1-CL mutations force the correct binding of the two different free light chains (CL-VL) to their respective VH-CH1 domains. a tetravelent IgG-like BsAb is described that maintains the natural Fab structures of both original mAbs as well as full human Fc. Specificities of the BsAb were against human CD5 (mAb1) and human HLA-DR (mAb2). The BsAb is composed of a fused H chain carrying VH CH1 domains of mAb1 fused in tandem with the VH-CH1-hinge domains of mAb2 through a peptide linker. The two different mAb1 and mAb2 L chains (both kappa) in contrast are free. 

—-Fusion of Two ScFv fragments via CH1 and CL:

Muller (The first constant domain (CH1 and CL) of an antibody used as heterodimerization domain for bispecific miniantibodies” FEBS Letters 422 91998) 29-264) discloses generation of bispecific miniantibodies which functionally assemble in E. coli by fusing the CH1 domain of an IgG1 to the C terminus of one ScFV specific for EGF recetpor and fusing the CL domain of a kappa light chain to the C terminus of a second sFv specific for CD2. The CL and CH1 domain themselves form a covalently linked heterodimer carrying the two different scFv specificities, named by the authors as a “CHCL miniantibody”. The bispecific miniantibodies are capable of binding to the EGF receptor and CD2 at the same time. EGF-R is overexpressed by a wide range of tumors whereas CD2 is expressed on cytotoxic T-cells (CTL) and natural Killer cells (NK). The advantage of the format is a longer reach to far apart antigens, compared to smaller bispecific variants, which is expected to be advantageous for briding tumor cells with effector cells. 

–—ScFv fragments –linker –CH3 domain:

Hollinger (US 6,492,123) discloses a diabody may be genetically fused to other proteins or antibody domains. For example, it can be fused to the CH3 domain of human IfGy4 with retention of the ability to bind antigen at both ends of the molecule, provided that a linker of sufficient size is inserted at the junction. 

Kontermann (FEBS Letters 454 (1999) 90-94) discloses a bispecific single-chain diabody (scDb) directed agaisnt carcinoembryonic antigen and E. coli beta-alactosidase as a model to generate bispecific IgG linke antibody molecules. Fusion of this single-chain diabody to the Fc (scDb-Fc) or CH3 (scDb-CH3) region of the human immunoglobuilin y1 chain results in the expression of dimeric fusion proteins exhibiting foru functional antigen binding sites with increaed functional affinity. 

Li (Protein Engineering, 10(6), 731-736, 1997) discloses scFv fragments connected through a short linker of four amino acids to the CH3 domain of the human immunoglobulin y1 H-chain. In one construct, a cysteine residue was included in the last amino acid of the flexible 15 amino acid long linker connecting the VL and VH domains, thus creating a disulphide bond stabilized molecule. 

Lu (J Immunol. Methods 279 (2003) 219-232) discloses production of a tetravelnt BsAb with two antigen binding sites to each of its target antigens by genetically fusing a bispecific/divalent diaboty to, vai the hinge region, the N-tmerinus of the CH3 domain of an IgG. The noevel BsAb, termed “di-diabody”, represents a tetravalent diabody dimer resulting form dimerizaiton between the inge region and the CH3 domains. 

Weiner (“Bispecific minibodies targeting HER2/neu and CD16 exhibit improved tumor lysis when placed in a divalent tumor antigen binding format” 279(52), 2004) dislose that the IgG1 CH3 constant domain serves as the oligomerization domain and is attached to an anti-CD16 and an Anti-HER2/nue single-chain Fv via 19 adn 29 amino aicd linkers, respectively. The bidspecific minibody can bind to HER2/neu and CD16, both individually and simultaneously. 

–Nanobodies:

Nanobodies are readily combined by short linkers providing a facile way to vary their antigen-bnding valency and introduce bispecificity or multispecificity. (Spies, Molecular Immunology 67 (2015) 95-106).

fusion of antibody fragment via polypeptide linkers: 

Another method for producing multispecific antagonists is to engineer recombinant fusion proteins linking two or more different single chain antibody or antibody fragment segments with the needed multiple specificies. (Coloma, Nature Biotech 15: 159-163, 1997). For example, a varient of bispecific fusion proteins can be produced using molecular engineering. In one form, the bispecific fusion protein is monovalnet, consisting of, for eample, a scFv with a single binding site for one antigen and a Fab fragment with a signle binding site for a secon antigen. In another form the bispecific fusion protein is divalent, consisting of, for example, an IgG with two binding sites for one antigen and two scFv with two binding sites for a second antigen. (Goldenberg, WO2006/063150). 

The majority of bispecific antibody techniques using antibody fragment such as scFV or Fab fragmetns as building blocks connected via polypeptide linkers. Formats based on linked antibody fragments include tandem scFV, diabodies and tandem-diabodies. These building blocks can further be linked to an immunoglobuilin Fc region given rise to IgG like molecules. 

For example, fragments compirising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a linker that is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragmetn are forced to pair with the ocmplementary VL and VH domains of another fragment, thereby forming two antigen binding sites. 

single domain antibodies: The immune systems of camelids use single V domains fused to an Fc demonstrating that a single domain can confer high affinity binding to an antigen. They can be reformatted into a classical IgG in which each arm has the potential to bind two targets eitehr via its VH of VL domain. 

Engineering of BiAbs to Facilitate Production/Purification (See outline)