Bispecific antibodies

#The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled IMMB_001WO.TXT, created Aug. 30, 2017, which is 87 kb in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

#Bispecific antibodies can provide therapeutic benefits in the treatment of various conditions, including various cancers by targeting two different moieties. Accordingly, it is desirable to provide bispecific antibodies with therapeutically useful properties.

#Some embodiments provide a bispecific antibody including at least one synthetic linker, methods of preparing the bispecific antibody including at least one synthetic linker, and uses thereof, wherein a heavy chain antibody portion includes the amino acid sequence SPPC, CPPS, APPC or CPPA in the hinge region wherein the cysteine sulfur of the SPPC, CPPS, APPC or CPPA sequence is covalently bonded to at least one synthetic linker.

#FIG. 1 provides a bispecific antibody synthesized using six different bifunctional linker reagents.

#FIG. 2 provides an HIC-HPLC trace of the bispecific antibody of Table 12.

#FIG. 3 provides the HIC-HPLC trace of a mixture of the bispecific antibody of Table 12 with Adalimumab antibody and Anti-IL17 antibody.

#Some embodiments provide a bispecific antibody, comprising a light chain (LC1) and heavy chain (HC1) of an antibody (Ab1) targeting a first moiety; and a light chain (LC2) and heavy chain (HC2) of an antibody (Ab2) targeting a second moiety, wherein LC1 and HC1 may be connected to each other via a linker to provide a first subunit, wherein LC2 and HC2 may be connected to each other via a linker to provide a second subunit, and wherein the first subunit and the second subunit may be connected to each other via a linker.

#The term “antibody” as used herein refers to whole, monoclonal antibodies. Such whole antibodies consist of two pairs of a “light chain” (LC) and a “heavy chain” (HC) (such light chain (LC)/heavy chain pairs are abbreviated herein as LC/HC). The light chains and heavy chains of such antibodies are polypeptides consisting of several domains. In a whole antibody, each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises the heavy chain constant domains CH1, CH2 and CH3 (antibody classes IgA, IgD, and IgG) and optionally the heavy chain constant domain CH4 (antibody classes IgE and IgM). Each light chain comprises a light chain variable domain VL and a light chain constant domain CL. The structure of one naturally occurring whole antibody, the IgG antibody, is shown e.g. in FIG. 1. The variable domains VH and VL can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The two pairs of heavy chain and light chain (HC/LC) are capable of specifically binding to same antigen. Thus said whole antibody is a bivalent, monospecific antibody. Such “antibodies” include e.g. mouse antibodies, human antibodies, chimeric antibodies, humanized antibodies and genetically engineered antibodies (variant or mutant antibodies) as long as their characteristic properties are retained. Especially preferred are human or humanized antibodies, especially as recombinant human or humanized antibodies. In some embodiments, the antibody may be Adalimumab, Bevacizumab, Anti-IL17, Anti-PDGF, Anti-Ang2, Anti-HGF, Anti-CD3, Anti-CD20, anti-CLL1, Anti-mesothelin. Anti-CD47, Anti-5T4, Anti-Trop2 or Anti-GPC3.

#There are five types of mammalian antibody heavy chains denoted by the Greek letters: α, δ, ε, γ, and μ. The type of heavy chain present defines the class of antibody; these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively. Distinct heavy chains differ in size and composition; α and γ contain approximately 450 amino acids, while μ and ε have approximately 550 amino acids.