Bispecific antibodies against plasma kallikrein and factor XII

Abstract

Provided are bispecific antibodies that bind to plasma kallikrein (pKal) and factor XII, as well as methods of making and using such bispecific antibodies to treat diseases or disorders associated with the contact system (e.g., hereditary angioedema or thrombosis). The present invention provides a first polypeptide comprising a light chain of a first antibody, the light chain comprising a light chain variable region (V L ) and the light chain constant region (C L a first polypeptide comprising a heavy chain of the first antibody, wherein the heavy chain comprises a heavy chain variable region (V H ) and the heavy chain constant region (C H and a second polypeptide comprising the first polypeptide or the second polypeptide. The bispecific antibody further comprises a second antibody that is a single-chain antibody, wherein the first polypeptide or the second polypeptide is fused to the C-terminus of either the first polypeptide or the second polypeptide, and one of the first antibody and the second antibody binds to plasma kallikrein (pKal) and the other antibody binds to factor XII.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing dates of U.S. Provisional Application No. 62 / 099,236, filed January 2, 2015, U.S. Provisional Application No. 62 / 200,363, filed August 3, 2015, and U.S. Provisional Application No. 62 / 261,609, filed December 1, 2015. The entire contents of these referenced applications are incorporated herein by reference. [Background technology]

[0002] Factor XII (FXII) is the major activator that converts prekallikrein to plasma kallikrein (pKal). Activated plasma kallikrein cleaves high molecular weight kininogen (HMWK) to liberate bradykinin (BK). pKal can also activate latent factor XII to active factor XII (factor XIIa). In disease states associated with abnormal activation of the contact system, such as hereditary angioedema, uncontrolled BK levels can induce seizures in patients. Summary of the Invention

[0003] One aspect of the present disclosure is a first polypeptide comprising a light chain of a first antibody, the light chain comprising a light chain variable region (V L ) and the light chain constant region (C L ) (e.g., a κ light chain or a λ light chain), and a second polypeptide comprising a heavy chain of the first antibody, wherein the heavy chain comprises a heavy chain variable region (V H ) and the heavy chain constant region (C Hand a second polypeptide comprising the first polypeptide, the second polypeptide comprising ..., the second polypeptide, the second polypeptide, the second polypeptide, the second polypeptide, the second polypeptide, the second polypeptide, the second polypeptide, the second polypeptide, the second polypeptide, the second

[0004] In some embodiments, the first antibody is an IgG. In one example, the IgG comprises a mutant heavy chain in which the C-terminal lysine residue is deleted or mutated compared to its wild-type counterpart. For example, the mutant heavy chain of the first antibody may comprise a C-terminal glycine residue instead of the lysine residue as in the wild-type IgG heavy chain. In one example, the bispecific antibody may be tetravalent.

[0005] In some embodiments, the second polypeptide in the bispecific antibody comprises a peptide linker between the heavy chain of the first antibody and the second antibody. In one example, the peptide linker can be SGGGS (SEQ ID NO: 22).

[0006] In the second antibody which is an scFv antibody, V H V L Alternatively, V H V L In some instances, the second antibody is fused to the C-terminus of H Area and V L A peptide linker between the domains, e.g. (G 4 S) 4 (SEQ ID NO: 23). In some embodiments, the scFc antibody comprises a linker H Chain and V L It contains disulfide bonds formed between the V chains. H The chain may include a cysteine ​​residue at position 44 (C44), LThe chain may contain a cysteine ​​residue at position 100, where V H C 44 and V L C 100 In some examples, the second antibody does not comprise a KR motif at its C-terminus.

[0007] In any of the bispecific antibodies described herein, the V of the first antibody H has the same complementarity determining regions (CDRs) as in SEQ ID NO: 1. In some examples, the V of the first antibody H comprises the amino acid sequence of SEQ ID NO: 1. In one example, the heavy chain of the first antibody comprises the amino acid sequence of residues 20 to 470 of SEQ ID NO: 9. In one example, the heavy chain of the first antibody comprises the amino acid sequence of SEQ ID NO: 9, 149 or 150. Alternatively, or additionally, the V of the first antibody L has the same CDRs as in SEQ ID NO: 2. In some examples, V of the first antibody L comprises the amino acid sequence of SEQ ID NO:2.

[0008] In addition, the V H may have the same CDRs as in any of SEQ ID NOs: 3, 4, and 123-126. H Alternatively, or in addition, the V of the second antibody comprises any of the amino acid sequences of SEQ ID NOs: 3, 4, and 123 to 126. L has the same CDRs as in any of SEQ ID NOs: 5-8 and 127-130. L comprises residues 1-111 of any one of the amino acid sequences of SEQ ID NOs: 5-8 and 127. L comprises any one of the amino acid sequences of SEQ ID NOs: 5 to 8 and 127 to 130.

[0009] In some examples, the bispecific antibodies described herein comprise a first polypeptide comprising the amino acid sequence of SEQ ID NO: 10, and a second polypeptide comprises any of the amino acid sequences of SEQ ID NOs: 11-20, 47-122, 141-148, and 151-158.

[0010] In another aspect, the disclosure provides a bispecific antibody comprising a first antibody that binds to plasma kallikrein (pKal) and a second antibody that binds to factor XII, e.g., the first antibody binds to activated pKal and / or the second antibody binds to activated factor XII (FXIIa). In some embodiments, the first antibody comprises a V sequence that includes complementarity determining regions (CDRs) identical to those in SEQ ID NO:1. H V chains and / or V containing CDRs identical to those in SEQ ID NO:2 L For example, the V of the first antibody H comprises the amino acid sequence of SEQ ID NO: 1, and / or V of the first antibody L comprises the amino acid sequence of SEQ ID NO:2.

[0011] Alternatively, or in addition, the second antibody comprises a V sequence comprising the same CDRs as in SEQ ID NO: 3 or 4. H V chains and / or Vs containing CDRs identical to those in SEQ ID NO: 5, 6, 7 or 8 L For example, the V of the second antibody H The V chain comprises the amino acid sequence of SEQ ID NO: 3 or 4, and / or L comprises the amino acid sequence of SEQ ID NO:5, 6, 7 or 8.

[0012] Alternatively, or in addition, the second antibody comprises a V sequence that includes a CDR identical to any of SEQ ID NOs: 123-126. H V chains and / or V sequences containing CDRs identical to those in SEQ ID NO: 127 L For example, the V of the second antibody H The V chain comprises any one of the amino acid sequences of SEQ ID NOs: 123 to 126, and / or the V Lcomprises residues 1 to 111 of any one of the amino acid sequences of SEQ ID NOs: 5 to 8 and 127.

[0013] In yet another aspect, the present disclosure provides an isolated nucleic acid or set of nucleic acids, comprising a first nucleotide sequence encoding a first polypeptide or a first antibody as described herein and a second nucleotide sequence encoding a second polypeptide or a second antibody as described herein.In some embodiments, the first nucleotide sequence and the second nucleotide sequence are located on two separate nucleic acid molecules (e.g., two vectors (such as expression vectors)).Alternatively, the first nucleotide sequence and the second nucleotide sequence are located on one nucleic acid molecule (e.g., a vector such as an expression vector).

[0014] The nucleic acid or set of nucleic acids described herein may be a set of vectors, including a first vector comprising a first nucleotide sequence and a second vector comprising a second nucleotide sequence. In some examples, the first vector and the second vector are expression vectors, in which the first nucleotide sequence and the second nucleotide sequence are operably linked to a promoter. In other examples, the nucleic acid described herein is a vector that comprises both the first nucleotide sequence and the second nucleotide sequence. Any of the vectors described herein may be an expression vector. For example, the expression vector may comprise the first nucleotide sequence and the second nucleotide sequence operably linked to a promoter. Also within the scope of this disclosure is a host cell that comprises the vector or set of vectors described herein.

[0015] Further, the present disclosure provides a composition comprising any of the bispecific antibodies or nucleic acids / sets of nucleic acids as described herein and a pharma- ceutically acceptable carrier. Such compositions can be used to treat diseases associated with the contact activation system (e.g., hereditary angioedema (HAE) or thrombosis). The treatment methods described herein include administering to a subject in need thereof an effective amount of a pharmaceutical composition as described herein. The present disclosure also provides a pharmaceutical composition for use in treating diseases as described herein, comprising any of the bispecific antibodies or nucleic acids / sets of nucleic acids encoding said bispecific antibodies as described herein and a pharma- ceutical acceptable carrier, and the use of such pharmaceutical compositions in the manufacture of a medicament for use in treating such diseases (such as HAE or thrombosis). In some embodiments, the thrombosis is associated with atrial fibrillation, deep vein thrombosis (DVT), pulmonary embolism, stroke, or an arterial or venous thrombotic event.

[0016] In yet another aspect, the disclosure features a method for preparing a bispecific antibody, the method including: (a) culturing a host cell or set of host cells as described herein under conditions that allow for expression of a first polypeptide and a second polypeptide, and (b) isolating the bispecific antibody comprising the first polypeptide and the second polypeptide. In some examples, the host cell comprises an expression vector comprising a first nucleotide sequence encoding the first polypeptide and a second nucleotide sequence encoding the second polypeptide.

[0017] The details of one or more embodiments of the disclosure are set forth in the description below. Other features or advantages of the disclosure will become apparent from the following drawings and detailed description of certain embodiments, as well as the appended claims.

[0018] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 is a graph showing the activity of various bispecific antibody clones, including clones X120-A01 (scFv=559C-M184-B04 H4L), X121-E01 (scFv=559C-M184-G03 H4L), X122-A01 (scFv=559C-M71-F06 H4L) and X122-C01 (scFv=559C-M71-F06 L4H), for inhibition of pKal. [Figure 2-1] FIG. 2 includes graphs showing the FXIIa inhibitory activity of clones X120-A01 (A and B), X122-A01 (C), X121-E01 (D), X122-C01 (E) and the control clone M71-F06 IgG (F). [Figure 2-2] FIG. 2 includes graphs showing the FXIIa inhibitory activity of clones X120-A01 (A and B), X122-A01 (C), X121-E01 (D), X122-C01 (E) and the control clone M71-F06 IgG (F). [Figure 2-3] FIG. 2 includes graphs showing the FXIIa inhibitory activity of clones X120-A01 (A and B), X122-A01 (C), X121-E01 (D), X122-C01 (E) and the control clone M71-F06 IgG (F). [Figure 3-1]3 includes graphs showing analytical size exclusion chromatography (SEC) traces of five exemplary bispecific molecules. The front peak indicates that these clones have high molecular weight aggregates, with % HMW ranging from 16.5 to 33.8. A: 620I-X136-C11. B: 620I-X136-C05. C: 620I-X136-G05. D: 620I-X136-D12. E: 620I-X136-A01. [Figure 3-2] 3 includes graphs showing analytical size exclusion chromatography (SEC) traces of five exemplary bispecific molecules. The front peak indicates that these clones have high molecular weight aggregates, with % HMW ranging from 16.5 to 33.8. A: 620I-X136-C11. B: 620I-X136-C05. C: 620I-X136-G05. D: 620I-X136-D12. E: 620I-X136-A01. [Figure 4-1] Figure 4 includes graphs showing the reduction of high molecular weight aggregates for 620I-X0173-A11 (620I-X0136-D12 with H44 / L100 engineered disulfide bonds) over a range of concentrations. A: 620I-X0173-A11 at 1 mg / mL. B: 620I-X0173-A11 at 10 mg / mL. C: 620I-X0173-A11 at 20 mg / ml. D: 620I-X0173-A11 at 45 mg / ml. [Figure 4-2] Figure 4 includes graphs showing the reduction of high molecular weight aggregates for 620I-X0173-A11 (620I-X0136-D12 with H44 / L100 engineered disulfide bonds) over a range of concentrations. A: 620I-X0173-A11 at 1 mg / mL. B: 620I-X0173-A11 at 10 mg / mL. C: 620I-X0173-A11 at 20 mg / ml. D: 620I-X0173-A11 at 45 mg / ml. [Figure 5-1]5 includes graphs showing the inhibitory activity of anti-pKal, anti-FXIIa, a combination of anti-pKal and anti-FXIIa, and bispecific antibody D12 as determined by reconstituted plasma assay. A: DX-2930 in the presence of single-chain HMWK. B: anti-FXIIa in the presence of single-chain HMWK. C: DX-2930 + anti-FXIIa in the presence of single-chain HMWK. D: bispecific clone 620I-X0136-D12 in the presence of single-chain HMWK. [Figure 5-2] 5 includes graphs showing the inhibitory activity of anti-pKal, anti-FXIIa, a combination of anti-pKal and anti-FXIIa, and bispecific antibody D12 as determined by reconstituted plasma assay. A: DX-2930 in the presence of single-chain HMWK. B: anti-FXIIa in the presence of single-chain HMWK. C: DX-2930 + anti-FXIIa in the presence of single-chain HMWK. D: bispecific clone 620I-X0136-D12 in the presence of single-chain HMWK. [Figure 6-1] 6 includes graphs showing the inhibitory activity of anti-pKal, anti-FXIIa, a combination of anti-pKal and anti-FXIIa, and bispecific antibody D12 as determined by reconstituted plasma assay. A: DX-2930 in the absence of HMWK. B: anti-FXIIa in the absence of HMWK. C: DX-2930 + anti-FXIIa in the absence of HMWK. D: bispecific clone 620I-X0136-D12 in the absence of HMWK. [Figure 6-2] 6 includes graphs showing the inhibitory activity of anti-pKal, anti-FXIIa, a combination of anti-pKal and anti-FXIIa, and bispecific antibody D12 as determined by reconstituted plasma assay. A: DX-2930 in the absence of HMWK. B: anti-FXIIa in the absence of HMWK. C: DX-2930 + anti-FXIIa in the absence of HMWK. D: bispecific clone 620I-X0136-D12 in the absence of HMWK. [Figure 7]Figure 7 includes graphs showing the inhibitory activity of anti-pKal, anti-FXIIa, a combination of anti-pKal and anti-FXIIa, and bispecific antibody D12 as determined by plasma assay. A: DX-2930 in the absence of HMWK. B: anti-FXIIa in the absence of HMWK. C: DX-2930 + anti-FXIIa in the absence of HMWK. D: bispecific clone D12 in the absence of HMWK. [Figure 8] FIG. 8 is a graph showing the effect of a bispecific antibody (D12) at three concentrations in an activated partial thromboplastin time (APTT) assay in comparison to an anti-FXIIa antibody (D06) and an anti-pKal antibody (H03). [Figure 9] FIG. 9 contains graphs showing the binding of 620I-X0173-A11 (620I-X0136-D12 with disulfide) to pKal (upper sensorgram) and FXIIa (lower sensorgram) at Biacore. A: Binding to pKal (upper curve) is higher than to the blank surface (middle) and prekallikrein (lower). The original FXIIa isolate showed non-specific binding to the Biacore chip. This explains the binding signals observed in the case of preKal and the blank. B: Binding to FXIIa (upper curve) is clearly higher than to FXII (lower) and the blank (middle). [Figure 10-1] Figure 10 includes graphs showing calculation of IC50 and apparent Ki for three disulfide-constrained bispecific antibodies in a Plasma Inhibition Assay. A and B: Clone 620I-X0173-A11. C and D: Clone 620I-X0173-C07. E and F: Clone 620I-X0173-G11. [Figure 10-2]Figure 10 includes graphs showing calculation of IC50 and apparent Ki for three disulfide-constrained bispecific antibodies in a Plasma Inhibition Assay. A and B: Clone 620I-X0173-A11. C and D: Clone 620I-X0173-C07. E and F: Clone 620I-X0173-G11. [Figure 10-3] Figure 10 includes graphs showing calculation of IC50 and apparent Ki for three disulfide-constrained bispecific antibodies in a Plasma Inhibition Assay. A and B: Clone 620I-X0173-A11. C and D: Clone 620I-X0173-C07. E and F: Clone 620I-X0173-G11. [Figure 11] FIG. 11 is a graph showing the inhibitory properties of bispecific antibody 620I-X0177-A01 (also known as 620I-X0173-A11) as determined in a plasma inhibition assay. [Figure 12-1] Figure 12 contains graphs showing that a decrease in affinity is observed between parental IgG and bispecific antibodies. A: Binding properties of parental clone 559C-X0211-A01 (left panel) and bispecific antibody 620I-X0177-A01 (right panel). B: Binding properties of parental clone DX-2930 (left panel) and bispecific antibody A01 (right panel). [Figure 12-2] Figure 12 contains graphs showing that a decrease in affinity is observed between parental IgG and bispecific antibodies. A: Binding properties of parental clone 559C-X0211-A01 (left panel) and bispecific antibody 620I-X0177-A01 (right panel). B: Binding properties of parental clone DX-2930 (left panel) and bispecific antibody A01 (right panel). [Figure 13]Figure 13 is a chart showing the dose-dependent delay of APTT by various antibodies as indicated. Clones D06, 1A01 and F12 are anti-FXIIa antibodies. Clone H03 is an anti-pKal antibody. Clones D12 and 7A01 are bispecific antibodies without and with disulfide bonds, respectively. [Figure 14] FIG. 14 is a chart showing the dose-dependent delay of fibrin deposition by clones 1A01 (559C-X211-A01) and 7A01 (620I-X0177-A01). [Figure 15] FIG. 15 is an SDS-PAGE protein gel showing samples of bispecific antibody 620I-X0177-A01 under reducing (lanes 2-4) and non-reducing (lanes 6-8) conditions. [Figure 16] Figure 16 contains graphs showing analytical size exclusion chromatography (SEC) traces of bispecific antibody 620I-X0177-A01 demonstrating pH-dependent cleavage. The peak between 15.7 and 16.1 min represents correctly formed bispecific antibody. The peak at 17 min represents DX-2930 IgG1. The peak at 22 min represents cleaved single chain antibody. A: pH 6.0. B: pH 7.0. C: 8.0. [Figure 17] Figure 17 includes SDS-PAGE protein gels of the indicated bispecific antibodies modified to mutate or delete the C-terminal lysine of the IgG at t=0. A: Non-reducing conditions. B: Reducing conditions. [Figure 18] 18 shows an SDS-PAGE protein gel containing the indicated bispecific antibodies modified to mutate or delete the C-terminal lysine of the IgG at t=48 h under reducing conditions. The positive control is bispecific antibody 620I-X0177-A01. [Figure 19]Figure 19 contains graphs showing analytical size exclusion chromatography (SEC) traces of control bispecific antibody 620I-X0177-A01. The peak between 15.7 and 16.1 min represents correctly formed bispecific antibody. The peak at 17 min represents DX-2930 IgG1. The peak at 22 min represents cleaved single chain antibody. A: t=0. B: t=48 hrs. [Figure 20-1] 20 includes graphs showing analytical size exclusion chromatography (SEC) traces of the reengineered bispecific antibody after 48 hours at room temperature at pH=7.5. A: 620I-X180-E07. B: 620I-X180-G03. C: 620I-X180-A05. D: 620I-X180-E06. E: 620I-X180-C11. F: 620I-X179-C01. G: 620I-X179-G05. H: 620I-X179-A09. [Figure 20-2] 20 includes graphs showing analytical size exclusion chromatography (SEC) traces of the reengineered bispecific antibody after 48 hours at room temperature at pH=7.5. A: 620I-X180-E07. B: 620I-X180-G03. C: 620I-X180-A05. D: 620I-X180-E06. E: 620I-X180-C11. F: 620I-X179-C01. G: 620I-X179-G05. H: 620I-X179-A09. [Figure 21] 21 shows an SDS-PAGE protein gel showing the indicated bispecific antibodies modified to mutate or delete the C-terminal lysine of the heavy chain IgG under non-reducing conditions. The positive control is 620I-X0177-A01. Samples were incubated with endoproteinase Lys C for 1 hour at 37° C. [Figure 22]Figure 22 includes graphs showing the inhibition of pKal by exemplary bispecific antibodies. Plate 1 shows the inhibitory properties of bispecific antibodies 620I-X0179-A09 (open circle), 620I-X0179-C01 (filled triangle) and 620I-X0179-E05 (open triangle). Plate 2 shows the inhibitory properties of bispecific antibodies 620I-X0179-G05 (open circle), 620I-X0180-E07 (filled triangle) and 620I-X0180-G03 (open triangle). Plate 3 shows the inhibitory properties of bispecific antibodies 620I-X0180-A05 (open circle) and 620I-X0180-C11 (filled triangle). Antibody DX-2930 was used as a control in each of the plates. [Diagram 23] Figure 23 includes graphs showing the inhibition of FXIIa by exemplary bispecific antibodies. Plate 1 shows the inhibitory properties of bispecific antibodies 620I-X0179-A09 (open circle), 620I-X0179-C01 (filled triangle) and 620I-X0179-E05 (open triangle). Plate 2 shows the inhibitory properties of bispecific antibodies 620I-X0179-G05 (open circle), 620I-X0180-E07 (filled triangle) and 620I-X0180-G03 (open triangle). Plate 3 shows the inhibitory properties of bispecific antibodies 620I-X0180-A05 (open circle) and 620I-X0180-C11 (filled triangle). Antibody DX-4012 (559C-M0192-H11) was used as a control in each of the plates. [Figure 24]Figure 24 includes graphs showing inhibition of activated plasma by exemplary bispecific antibodies. The top left panel shows the inhibitory profile of control plates 1, 2 and 3, DX-2930, and DX-4012. The top right panel shows the inhibitory profile of bispecific antibodies 620I-X0179-A09 (filled circles), 620I-X0179-C01 (open circles). The bottom left panel shows the inhibitory profile of bispecific antibodies 620I-X0179-E05 (filled circles), 620I-X0179-G05 (open circles) and 620I-X0180-E07 (filled triangles). The bottom right panel shows the inhibitory properties of bispecific antibodies 620I-X0180-G03 (filled circles), 620I-X0180-A05 (open circles) and 620I-X0180-C11 (filled triangles). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The contact activation system initiates the intrinsic pathway of coagulation through the release of the proinflammatory peptide bradykinin (BK). The release of BK is promoted by a series of enzymatic activation steps in the contact activation system. Factor XIIa (FXIIa) converts prekallikrein to plasma kallikrein (pKal). Activated pKal then cleaves high molecular weight kininogen (HMWK) to release bradykinin (BK). Importantly, pKal can also activate latent factor XII to give additional activated factor XIIa. A positive feedback loop is thought to be formed, where pKal activates FXII to FXIIa, which in turn activates prekallikrein to pKal.

[0021] In diseases associated with the contact activation system (such as hereditary angioedema (HAE) or thrombosis), unregulated BK levels can induce inflammatory responses (such as HAE attacks in patients). Thus, agents to control the levels of BK (e.g., inhibitors of pKal and FXII) may have important therapeutic value.

[0022] Described herein are bispecific antibodies that bind to both pKal and FXII (e.g., activated pKal and / or FXIIa) and the use of the bispecific antibodies in inhibiting both pKal and FXII to treat diseases associated with the contact activation system (such as hereditary angioedema (HAE) and thrombosis). As shown in the Examples below, several exemplary bispecific antibodies as described herein have been shown to inhibit both pKal and FXIIa activity. Without wishing to be bound by theory, it is expected that the bispecific antibodies described herein will exhibit superior therapeutic efficacy in treating diseases associated with the contact activation system compared to agents that can inhibit either pKal or FXII. This is because the bispecific antibodies can inhibit the activity of both pKal and FXII, thereby synergistically reducing BK levels, for example, by blocking the positive feedback loop between pKal and FXII.

[0023] A bispecific antibody that binds pKal and FXII As used herein, an antibody (used interchangeably in the plural) is an immunoglobulin molecule or a functional fragment thereof that is capable of binding to a target antigen (such as a carbohydrate, polynucleotide, lipid, or polypeptide) via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. A multispecific antibody (e.g., a bispecific antibody) is an immunoglobulin molecule or a functional fragment / variant thereof that is capable of binding to multiple target antigens (e.g., two antigens, or two epitopes of one antigen). The bispecific antibodies described herein can bind to both plasma kallikrein (pKal) and factor XII. In some embodiments, the bispecific antibody can bind to and inhibit both active pKal and FXIIa.

[0024] As used herein, an antigen refers to any molecule (e.g., a protein, a nucleic acid, a polysaccharide, or a lipid) that has the ability to raise an antibody. An epitope is the part of an antigen (e.g., a part of pKal or FXII) to which an antibody binds. Epitopes usually consist of chemically active (such as polar, non-polar, or hydrophobic) surface groups of moieties such as amino acids or polysaccharide side chains, and may have unique three-dimensional structural properties as well as unique charge properties. Epitopes may be essentially linear or discontinuous epitopes (e.g., conformational epitopes formed by the spatial relationship between non-adjacent amino acids of an antigen rather than a linear string of amino acids). Conformational epitopes include epitopes that arise from folding of an antigen, where amino acids from separate parts of the antigen's linear sequence are brought into close proximity in three-dimensional space.

[0025] The bispecific antibodies described herein comprise two antibody moieties: a first antibody moiety that binds to pKal (e.g., activated pKal) and a second antibody moiety that binds to FXII (e.g., FXIIa). The first antibody moiety and the second antibody moiety may be derived from two parent antibodies capable of binding to the desired antigen (i.e., pKal (e.g., activated pKal) and FXII (e.g., FXIIa)). One or both of the parent antibodies for constructing a bispecific antibody as described herein may be a naturally occurring antibody (e.g., an antibody derived from a suitable donor such as a human, mouse, rat, rabbit, horse or sheep), a genetically engineered antibody (e.g., a humanized antibody, a chimeric antibody), or an antibody derived from a library of natural or synthetic antibodies. In some embodiments, one parent antibody is an IgG antibody (e.g., an IgG antibody that binds to pKal (such as DX2930) or an IgG antibody that binds to FXIIa), and the other parent antibody is an scFv antibody (e.g., an scFv antibody that binds to FXIIa (such as the anti-FXIIa clones described herein) or an scFv antibody that binds to pKal).

[0026] The heavy chain of a naturally occurring IgG molecule typically contains a lysine residue at the C-terminus. In some embodiments, this C-terminal lysine residue may be deleted or mutated (e.g., to a glycine residue) in the bispecific antibodies disclosed herein. Alternatively, or additionally, the KR motif normally present at the junction between the light chain variable region and the light chain constant region may be deleted from the light chain of the first antibody, the second antibody, or both, in the bispecific antibodies described herein. In some examples, the KR motif is deleted from the scFv portion of any of the bispecific antibodies described herein (e.g., at the C-terminus of the scFv). These mutations may reduce proteolytic cleavage of the bispecific antibody and / or improve expression, production and / or manufacturing of the bispecific antibody.

[0027] In some examples, at least one parent antibody may be an affinity matured antibody, which refers to an antibody that has one or more modifications in one or more CDRs or framework regions (FRs) compared to the unmodified parent antibody, which result in improved affinity of the antibody for the target antigen. Preferred affinity matured antibodies may have nanomolar or picomolar affinities for the target antigen. Affinity maturation of antibodies can be carried out by a variety of methods known in the art, including by variable domain shuffling (see, e.g., Marks et al. 1992, Bio / Technology 10:779-783), random mutagenesis of CDR and / or FR residues (see, e.g., Barbas et al., 1994, Proc Nat. Acad. Sci, USA 91:3809-3813; Schier et al., 1995, Gene 169:147-155; Yelton et al., 1995, J. Immunol. 155:1994-2004; Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al, 1992, J. Mol. Biol. 226:889-896). The parent antibody may be of any class (such as IgD, IgE, IgG, IgA or IgM), or a subclass thereof, or a single chain antibody (such as scFv).

[0028] Each antibody moiety in a bispecific antibody as described herein may be, but is not limited to, an intact (i.e., full-length) antibody, an antigen-binding fragment thereof (Fab, Fab', F(ab') 2 The antibody may be in any form, including a single chain antibody (such as a Fv), a single chain antibody (such as a scFv antibody) and a tetravalent antibody. In some embodiments, the bispecific antibody is tetravalent, which contains two pKal binding sites and two FXII binding sites.

[0029] In some embodiments, the anti-pKal moiety, the anti-FXII moiety, or both in the bispecific antibody described herein specifically bind to the corresponding target antigen or epitope thereof. An antibody that "specifically binds" to an antigen or epitope is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" when it reacts or associates with a particular target antigen more frequently, more rapidly, with a longer duration, and / or with a higher affinity than it reacts or associates with another target. An antibody "specifically binds" to a target antigen or epitope when it binds with higher affinity, avidity, more rapidly, and / or with a longer duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to an antigen (e.g., human pKal or FXII) or an antigenic epitope therein is an antibody that binds to the target antigen with higher affinity, avidity, more rapidity, and / or longer duration than it binds to other antigens or other epitopes within the same antigen. By reading this definition, it is also understood that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. Thus, "specific binding" or "preferential binding" does not necessarily require (but may encompass) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding. In some instances, an antibody that "specifically binds" to a target antigen or an epitope thereof may not bind to other antigens or other epitopes within the same antigen. In some embodiments, the bispecific antibodies described herein specifically bind to both active pKal and FXIIa.

[0030] In some embodiments, a bispecific antibody as described herein has suitable binding affinity for its target antigen (e.g., pKal or FXIIa) or one or both of its antigenic epitopes. As used herein, "binding affinity" refers to the apparent association constant or K ARefers to. A is the dissociation constant (K D The bispecific antibodies described herein have a binding affinity of at least 10 to one or both of their target antigens or antigen epitopes. -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 Binding affinity of M (K D ), or a lower binding affinity (K D ), the increase in binding affinity can be attributed to the K D The fact that an antibody binds with higher affinity to a first antigen and a second antigen than to a third antigen corresponds to a decrease in the K A (or K D K for binding to the first antigen and the second antigen compared to A is higher (or K D In such cases, the antibody has specificity for a first antigen and a second antigen (e.g., a first protein or mimetic thereof in a first conformation and a second protein or mimetic thereof in a first conformation) over a third antigen (e.g., the same first or second protein or mimetic thereof in a second conformation; or a third protein). The difference in binding affinity (e.g., with respect to specificity or other comparison) is at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000, or 10 5 It may be double.

[0031] Binding affinity (or binding specificity) can be determined by a variety of methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v / v) Surfactant P20). Using these techniques, the concentration of bound binding protein can be measured as a function of the concentration of the target protein. The concentration of bound binding protein ([Bound]) can be calculated according to the following formula: [Bound]=[N][Free] / (Kd+[Free]) is related to the concentration of free target protein ([Free]) and the concentration of binding protein binding sites on the target by, where (N) is the number of binding sites per target molecule.

[0032] However, the K A It is not necessarily the case that the K is a function of affinity, since it may be sufficient to obtain a quantitative measure of affinity that is proportional to K and can therefore be used for comparison (such as determining whether the higher affinity is, for example, two-fold higher) or to obtain an estimate of affinity, for example, from activity in a functional assay (e.g., an in vitro or in vivo assay). A It is not necessary to determine exactly.

[0033] (i) Anti-pKal moiety Any antibody capable of binding to pKal, such as active pKal, can be used to construct the bispecific antibody described herein. In some examples, the anti-pKal antibody portion in the bispecific antibody can bind to human pKal and inhibit its activity by at least 50% (e.g., 60%, 70%, 80%, 90%, 95% or more than 95%). The inhibition constant (Ki) provides a measure of the potency of the inhibitor. The inhibition constant (Ki) is the concentration of inhibitor required to reduce the enzyme activity by half and is independent of the concentration of enzyme or substrate. The inhibitory activity of the anti-pKal antibody portion in the bispecific antibody described herein can be determined by conventional methods. In some examples, a bispecific antibody as described herein has an anti-pKal K activity of less than 1 nM (e.g., 0.5 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, 0.01 nM, or less than 0.01 nM). i,app The antibody has a K value i,app The value can be estimated according to methods known in the art.

[0034] In some embodiments, the anti-pKal portion of the bispecific antibody binds to the following residues of human pKal: V410, L412, T413, A414, Q415, R416, L418, C419, H434, C435, F436, D437, G438, L439, W445, Y475, K476, V477, S478, E479, G480, D483, F524, E527, K528, Y552, D554, Y555, A564, D572, A573, C574, K575, G576, S578, T596, S597, W598, G599, E600, G601, C602, A603, R604, Q607, P608, G609, V610, and Y611, The amino acid sequence of the C-terminal fragment of human pKal, including the involved amino acid residues (bold and underlined), is shown below (SEQ ID NO:21).

[0035] [ka]

[0036] In some examples, the anti-pKal antibody portion is an epitope of pKal that is the following segment of SEQ ID NO:21 above: V410-C419, H434-L439, Y475-G480, F524-K528, Y552-Y555, D572-S578, T596-R604, or Q607-Y611, It can bind to an epitope that includes one of the following:

[0037] In one example, the anti-pKal portion of the bispecific antibody described herein is derived from the antibody DX-2930 described in US Application Publication No. 20120201756, which is incorporated herein by reference. The heavy and light chain variable regions of DX-2930 and the full-length heavy and light chains of this antibody are presented below (CDR regions: bold and underlined; signal sequence: italics).

[0038] [ka]

[0039] In some examples, the anti-pKal portion of the bispecific antibody comprises a heavy chain variable region comprising an amino acid sequence that is at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 1, and / or a light chain variable region comprising an amino acid sequence that is at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 2. The "percent identity" for two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed using the XBLAST program (score=50, wordlength=3) to obtain amino acid sequences homologous to a protein molecule of interest. When gaps exist between the two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0040] In another example, the anti-pKal moiety in a bispecific antibody as described herein comprises a heavy chain variable region that contains three CDRs identical to those in SEQ ID NO: 1, and / or three CDRs identical to those in SEQ ID NO: 2. Two heavy chain variable regions (or two light chain variable regions) with identical CDRs means that the CDRs in the two heavy chain variable regions (or light chain variable regions) are identical, as determined by the same numbering scheme. Exemplary numbering schemes for determining the CDRs of an antibody include the "Kabat" numbering scheme (Kabat et al. (1991), 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.), the "Chothia" numbering scheme (Al-Lazikani et al., (1997) JMB 273, 927-948), the "Contact" numbering scheme (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)), the "IMGT" numbering scheme (Lefranc MP et al., Dev Comp Immunol, 2003 January;27(1):55-77), and the "AHo" numbering scheme (Honegger A and Pluckthun A, J Mol Biol, 2001 June 8;309(3):657-70). As will be appreciated by one of skill in the art, the CDR regions of the exemplary anti-pKal and anti-FXII antibodies identified herein are determined by the "Chothia" numbering scheme, which is used as an example.

[0041] Alternatively, the anti-pKal moiety may comprise one or more (e.g., up to 2, 3, 4, 5, 6, 7, or 8) mutations in one or more of the CDRs compared to SEQ ID NO:1 and / or SEQ ID NO:2. Such mutations may be conservative amino acid substitutions. As used herein, "conservative amino acid substitutions" refer to amino acid substitutions that do not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Conservative amino acid substitutions include substitutions made between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0042] In any of the examples described herein, the anti-pKal portion of the bispecific antibody may contain one or more (e.g., one, two, three, four, five, or more than six) mutations or deletions compared to a reference antibody. Such mutations may be introduced, for example, to reduce proteolytic cleavage of the bispecific antibody and / or to improve expression, production and / or manufacturing of the bispecific antibody. In some embodiments, the anti-pKal portion of the bispecific antibody is an IgG, the heavy chain of which has a removed or mutated C-terminal lysine residue compared to its wild-type counterpart. In some embodiments, the C-terminal lysine of the IgG heavy chain is mutated to a neutral amino acid residue (e.g., a glycine or alanine residue).

[0043] Exemplary sequences of such mutant heavy chains of the anti-pKal portion of a bispecific antibody are provided below (using the heavy chain of DX-2930 as an example).

[0044] [ka]

[0045] The italicized portions of the sequences presented above refer to the signal peptides. The anti-pKal portions of the bispecific antibodies disclosed herein may contain the same signal peptide, or the signal peptide may be removed or replaced with a different signal peptide. Signal peptides for use in producing secreted proteins are well known in the art.

[0046] The anti-pKal moiety in the bispecific antibody may be, but is not limited to, an intact (i.e., full-length) antibody, an antigen-binding fragment thereof (Fab, Fab', F(ab') 2 In some examples, the heavy chain variable region of the anti-pKal moiety as described herein may be a heavy chain constant region (C H ) and this heavy chain constant region (C H ) can be a full-length heavy chain constant region or a portion thereof (e.g., H 1. C H 2. C H The heavy chain constant region may be any C, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C31, C42, C53, C64, C7, C8, C9, C12, C13, C14, C15, C16, H In some embodiments, the C H Alternatively, or in addition, the light chain variable region of the anti-pKal moiety can be a light chain constant region (C L ) and this light chain constant region (C L ) is any C known in the art L In some examples, C L In another example, C is a kappa light chain. L is a lambda light chain. Antibody heavy and light chain constant regions are well known in the art (e.g., those provided in the IMGT database (www.imgt.org) or www.vbase2.org / vbstat.php., both of which are incorporated herein by reference). In some examples, the anti-pKal moiety is an IgG that may include the same heavy chain as DX-2930 (SEQ ID NO: 9) and / or the same light chain as DX-2930 (SEQ ID NO: 10).

[0047] Alternatively, the anti-pKal moiety in a bispecific antibody as described herein may be linked via, for example, a peptide linker ((GGGGS) 4 (SEQ ID NO: 23)) in which the heavy chain variable region and the light chain variable region are fused together. In one example, the heavy chain variable region and the light chain variable region are fused together in the H→L orientation. In another example, the heavy chain variable region and the light chain variable region are fused together in the L→H orientation. In some embodiments, the light chain portion of the ScFv does not contain a Lys-Arg (KR) motif at its C-terminus.

[0048] In one example, the anti-pKal portion in the bispecific antibody described herein is DX-2930 (an IgG antibody) described herein comprising a heavy chain of SEQ ID NO: 9 and a light chain of SEQ ID NO: 10, or an antigen-binding fragment thereof.

[0049] (ii) anti-FXII moiety Any antibody capable of binding to FXII, such as activated FXII (FXIIa), can be used to construct the bispecific antibodies described herein. In some examples, the anti-FXII antibody portion in the bispecific antibody can bind to human FXIIa and inhibit its activity by at least 50% (e.g., 60%, 70%, 80%, 90%, 95% or more than 95%). The inhibitory activity of the anti-FXII antibody portion in the bispecific antibody described herein can be determined by routine methods. In some examples, the bispecific antibody as described herein has an anti-pFXIIa K of less than 1 nM (e.g., 0.5 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, 0.01 nM, or less than 0.01 nM). i,app The antibody has a K value i,app The value can be estimated according to methods known in the art.

[0050] In some examples, the anti-FXII portion of the bispecific antibodies described herein is derived from anti-FXII clones 559C-M0071-F06, 559C-M0179-D04, 559C-M0181-C02, 559C-M0180-G03 and 559C-M0184-B04. The heavy and light chain variable regions of these clones are presented below (CDRs: bold and underlined).

[0051] [ka] TIFF0007635285000005.tif241165TIFF0007635285000006.tif35160

[0052] The light chain variable regions of clones 620I-X0173-A11 (sequence number: 127), 620I-X0173-C07 (sequence number: 128), 620I-X0173-E07 (sequence number: 129) and 620I-X0173-G11 (sequence number: 130) are identical.

[0053] In some examples, the anti-FXIIa portion of the bispecific antibody comprises a heavy chain variable region comprising an amino acid sequence at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to SEQ ID NO:3 or SEQ ID NO:4, and / or a light chain variable region comprising an amino acid sequence at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to any of SEQ ID NOs:5-8. For example, the heavy chain variable region may comprise an amino acid sequence at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to SEQ ID NO:3, and the light chain variable region may comprise an amino acid sequence at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to any of SEQ ID NOs:5-8. Alternatively, the heavy chain variable region may comprise an amino acid sequence that is at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to SEQ ID NO:4, and the light chain variable region may comprise an amino acid sequence that is at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to SEQ ID NO:5.

[0054] In some examples, the anti-FXIIa portion of the bispecific antibody comprises a heavy chain variable region comprising an amino acid sequence at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to any of SEQ ID NOs: 123-126, and / or a light chain variable region comprising an amino acid sequence at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to any of SEQ ID NOs: 127-130. For example, the heavy chain variable region may comprise an amino acid sequence at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 123, and the light chain variable region may comprise an amino acid sequence at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 127. Alternatively, the heavy chain variable region may comprise an amino acid sequence at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 124, and the light chain variable region may comprise an amino acid sequence at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 128. Alternatively, the heavy chain variable region may comprise an amino acid sequence at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 125, and the light chain variable region may comprise an amino acid sequence at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 129. Alternatively, the heavy chain variable region may comprise an amino acid sequence that is at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to SEQ ID NO:126, and the light chain variable region may comprise an amino acid sequence that is at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to SEQ ID NO:130.

[0055] In other examples, the anti-FXIIa portion of a bispecific antibody as described herein comprises a heavy chain variable region and / or a light chain variable region that comprises three CDRs identical to those of clones 559C-M0071-F06, 559C-M0179-D04, 559C-M0181-C02, 559C-M0180-G03, 559C-M0184-B04, 620I-X0173-A11, 620I-X0173-C07, 620I-X0173-E07, or 620I-X0173-G11, and / or three CDRs identical to those of these clones. See Table 1 below.

[0056] [Table 1]

[0057] Alternatively, the anti-FXIIa portion may comprise one or more (e.g., up to two, three, four, five, six, seven, or eight) mutations in one or more of the heavy and / or light chain CDRs listed in Table 1 above, compared to any of clones 559C-M0071-F06, 559C-M0179-D04, 559C-M0181-C02, 559C-M0180-G03, 559C-M0184-B04, 620I-X0173-A11, 620I-X0173-C07, 620I-X0173-E07, or 620I-X0173-G11. Such mutations may be conservative amino acid substitutions as described herein.

[0058] In some embodiments, the anti-FXIIa moiety in the bispecific antibody described herein is an IgG molecule, which may be a naturally occurring IgG or a variant, e.g., containing one or more (e.g., 1, 2, 3, 4, 5, or 6 or more) mutations or deletions, e.g., to reduce proteolytic cleavage of the bispecific antibody, to reduce charge heterogeneity of the bispecific antibody, and / or to improve expression, production, and / or manufacturing of the bispecific antibody. In some embodiments, the heavy chain of the IgG has a removed or mutated C-terminal lysine residue compared to its wild-type counterpart. In some embodiments, the C-terminal lysine of the IgG heavy chain is mutated to a neutral amino acid residue (e.g., a glycine or alanine residue).

[0059] The anti-FXIIa portion of the bispecific antibody may be, but is not limited to, an intact (i.e., full-length) antibody, an antigen-binding fragment thereof (Fab, Fab', F(ab') 2 In some examples, the heavy chain variable region of the anti-pKal moiety as described herein may be a heavy chain constant region (C H ) and this heavy chain constant region (C H ) can be a full-length heavy chain constant region or a portion thereof (e.g., H 1. C H 2. C H The heavy chain constant region may be any C, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C31, C42, C53, C64, C7, C8, C9, C12, C13, C14, C15, C16, H In some embodiments, the C H Alternatively, or in addition, the light chain variable region of the anti-pKal moiety can be a light chain constant region (C L ) and this light chain constant region (C L ) is any C known in the art L In some examples, C L In another example, C is a kappa light chain. L is a λ light chain. Antibody heavy and light chain constant regions are well known in the art (e.g., as described herein).

[0060] Alternatively, the anti-FXIIa moiety in a bispecific antibody as described herein may be linked via, for example, a flexible peptide linker ((GGGGS) 4 (SEQ ID NO: 23)) in which the heavy chain variable region and the light chain variable region are fused together. The heavy chain variable region and the light chain variable region may be fused in the H→L orientation or in the L→H orientation. In some embodiments, the light chain portion of the ScFv does not contain a KR motif at its C-terminus.

[0061] In some embodiments, the anti-FXIIa portion is an scFv comprising a heavy chain variable region of SEQ ID NO: 3 or SEQ ID NO: 4, and / or a light chain variable region of any of SEQ ID NOs: 5-8. In one example, the anti-FXIIa portion is an scFv antibody comprising a heavy chain variable region of SEQ ID NO: 3 and a light chain variable region of any of SEQ ID NOs: 5-8 in either H→L or L→H orientation. In another example, the anti-FXIIa portion is an scFv antibody comprising a heavy chain variable region of SEQ ID NO: 4 and a light chain variable region of SEQ ID NO: 5 in either H→L or L→H orientation. In some embodiments, the anti-FXIIa portion is an scFv comprising a heavy chain variable region of any of SEQ ID NOs: 123-126, and / or a light chain variable region of any of SEQ ID NOs: 127-130. In one example, the anti-FXIIa portion is an scFv antibody comprising a heavy chain variable region of SEQ ID NO: 123 and a light chain variable region of SEQ ID NO: 127 in either H→L or L→H orientation. In one example, the anti-FXIIa portion is an scFv antibody comprising the heavy chain variable region of SEQ ID NO: 123 and the light chain variable region of SEQ ID NO: 127 in either H→L or L→H orientation. In another example, the anti-FXIIa portion is an scFv antibody comprising the heavy chain variable region of SEQ ID NO: 124 and the light chain variable region of SEQ ID NO: 128 in either H→L or L→H orientation. In another example, the anti-FXIIa portion is an scFv antibody comprising the heavy chain variable region of SEQ ID NO: 125 and the light chain variable region of SEQ ID NO: 129 in either H→L or L→H orientation. In another example, the anti-FXIIa portion is an scFv antibody comprising the heavy chain variable region of SEQ ID NO: 126 and the light chain variable region of SEQ ID NO: 130 in either H→L or L→H orientation.

[0062] In some embodiments, the heavy and light chain variable regions of any of the scFv antibodies described herein are bonded together via, e.g., disulfide bonds (V H Residues 44 and V L The nucleotides are further linked via a nucleotide sequence (such as between residue 100 of

[0063] (iii) Construction of anti-pKal / anti-FXII bispecific antibodies Anti-pKal / anti-FXIIa bispecific antibodies as described herein may be of any configuration for bispecific antibodies as known in the art (e.g., those described in Klein et al., mAbs 4(6):653-663, 2012; Kontermann et al., mAbs 4(2):182-197, 2012; and Coloma et al., Nature Biotechnology 15:159-163, 1997). In some examples, the bispecific antibody may be a full-length hybrid antibody (also known as a quadroma antibody or trifunctional antibody) that contains one arm (heavy chain / light chain complex) that binds pKal and another arm (heavy chain / light chain complex) that binds FXII. In some examples, the bispecific antibody may be a bispecific Fab' antibody that contains one Fab fragment that binds pKal and another Fab fragment that binds FXII. 2 or a tri-Fab molecule that contains two copies of a Fab fragment that binds one target antigen (e.g., pKal or FXIIa) and one copy of a Fab fragment that binds the other target antigen (e.g., FXIIa or pKal). Alternatively, the bispecific antibody is a tandem scFv molecule that contains at least one copy of an scFv that binds pKal and one copy of another scFv that binds FXIIa. The bispecific antibodies described herein may also be diabodies or single chain diabodies as known in the art. Other examples include, but are not limited to, IgG 2 , F(ab') 2 , CovX-body, scFv 4 -Ig, IgG-scFv, scFv-IgG, DVD-Ig, IgG-sVD, sVD-IgG, 2-in-1-IgG, mAb 2, Tandemab common LC, kih IgG, kih IgG common LC, CrossMab, kih IgG-scFab, mAb-Fv, charge pairs, SEED-body, Diabody(Db), dsDd, scDb, tandemAbs, tandem scFv, triple body, Fab-scFv and F(ab') 2 -scFv 2 See, for example, Fig. 2 in Kontermann et al., mAbs 4(2):182-197, 2012.

[0064] In some embodiments, the scaffold of the bispecific antibodies described herein is designed to include an IgG antibody portion and an scFv portion fused to the C-terminus of either the heavy or light chain of the IgG portion (e.g., the C-terminus of the heavy chain of the IgG; see, e.g., Coloma, MJ & Morrison, SL Design and production of novel tetravalent bispecific antibodies. Nature Biotechnology. 15(2):159-163. 1997). The IgG heavy or light chain may be fused to the scFv via a short peptide linker (such as a peptide rich in Gly and Ser residues). In one example, the peptide linker comprises the amino acid sequence SGGGS (SEQ ID NO: 22).

[0065] Such bispecific antibodies are characterized by the light chain variable region (V L ) and the light chain constant region (C L a first polypeptide comprising a light chain of a first antibody comprising, from N-terminus to C-terminus, a heavy chain variable region (V H ) and heavy chain constant region (C H ) and a second polypeptide comprising a fusion protein comprising a heavy chain of a first antibody, the heavy chain comprising a heavy chain variable region (V H ) and heavy chain constant region (C Ha heavy chain comprising, from N-terminus to C-terminus, a light chain variable region (V L ) and the light chain constant region (C L ) and a second polypeptide comprising a fusion protein comprising a light chain of a first antibody comprising an active site for pKal (e.g., activated pKal) and a second antibody that is a single chain antibody. In some examples, the first antibody can bind to pKal (e.g., activated pKal) and the second antibody can bind to FXII (e.g., FXIIa). In other examples, the first antibody can bind to FXIIa and the second antibody can bind to pKal.

[0066] C of the light chain of the first antibody L is any C known in the art L In some embodiments, C L is a κ light chain. L is the λ light chain. H is any C known in the art H In some embodiments, C H is a gamma heavy chain. Such heavy and light chain constant regions are well known in the art, for example as described herein.

[0067] In one example, the bispecific comprises an IgG antibody derived from DX-2930 in either H→L or L→H orientation, and an scFv antibody derived from clones 559C-M0071-F06, 559C-M0179-D04, 559C-M0181-C02, 559C-M0180-G03, 559C-M0184-B04, 620I-X0173-A11, 620I-X0173-C07, 620I-X0173-E07 or 620I-X0173-G11. See disclosure above. Antibodies derived from a parent antibody may comprise heavy and light chains substantially similar (having at least 80%, 85%, 90%, 95% or 98% sequence identity) to those of the parent antibody. In some examples, such antibodies comprise heavy and light chain CDRs identical to those of the parent antibody, while in other examples, such antibodies comprise heavy and / or light chain CDRs that are substantially identical to those of the parent antibody (e.g., containing up to 5, 4, 3, 2 or 1 amino acid residue changes (such as conservative amino acid residue substitutions) compared to the CDRs of the parent antibody).

[0068] In some embodiments, the scFv antibody in the bispecific antibody is L V fused to the N-terminus of H In other embodiments, the scFv antibody comprises L V fused to the C-terminus of H In any of the scFv antibodies described herein, V H Area and V L The domains may be fused via a linker, such as a peptide linker.

[0069] A peptide linker as described herein may comprise, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 10 or more amino acid residues. In some embodiments, the peptide linker may comprise 2-50 amino acids, 5-25 amino acids, or 5-20 amino acids. In some embodiments, the peptide linker is SGGGS. In some embodiments, the peptide linker is (G 4S) x, where x can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 11. In some embodiments, x is 4.

[0070] A peptide linker as described herein (e.g., a SGGGS (SEQ ID NO: 22) linker or a (GGGGS) 4Any of the (SEQ ID NO:23) linkers) may include naturally occurring and / or non-naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamic acid (Glu), glutamine (Gin), glycine (Gly), histidine (His), isoleucine (He), leucine (Leu), lysine (Lys), methionine (Met), ornithine (Orn), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val). Non-naturally occurring amino acids may include protected amino acids, such as naturally occurring amino acids protected with groups such as acetyl, formyl, tosyl, nitro, etc. Non-limiting examples of non-naturally occurring amino acids include azidohomoalanine, homopropargylglycine, homoallylglycine, p-bromophenylalanine, p-iodophenylalanine, azidophenylalanine, acetylphenylalanine or ethynylphenylalanine, amino acids containing an internal alkene such as trans-crotylalkene, serine allyl ether, allylglycine, propargylglycine, vinylglycine, pyrrolysine, N-sigma-o-azidobenzyloxycarbonyl-L-lysine (AzZLys), N-sigma-propargyloxy Carbonyl-L-lysine, N-sigma-2-azidoethoxycarbonyl-L-lysine, N-sigma-tert-butyloxycarbonyl-L-lysine (BocLys), N-sigma-allyloxycarbonyl-L-lysine (AlocLys), N-sigma-acetyl-L-lysine (AcLys), N-sigma-benzyloxycarbonyl-L-lysine (ZLys), N-sigma-cyclopentyloxycarbonyl-L-lysine (CycLys), N-sigma-D-prolyl-L-lysine, N-sigma-nicotinoyl-L-lysine (NicLys), N-sigma-N-Me-anthraniloyl-L-lysine (NmaLys), N-sigma-biotinyl-L-lysine,N-sigma-9-fluorenylmethoxycarbonyl-L-lysine, N-sigma-methyl-L-lysine, N-sigma-dimethyl-L-lysine, N-sigma-trimethyl-L-lysine, N-sigma-isopropyl-L-lysine, N-sigma-dansyl-L-lysine, N-sigma-o,p-dinitrophenyl-L-lysine, N-sigma-p-toluenesulfonyl-L-lysine, N-sigma-DL-2-amino-2-carboxyethyl-L-lysine, N-sigma-phenyl pyruvamido-L-lysine, N-sigma-pyruvamido-L-lysine, azidohomoalanine, homopropargylglycine, homoallylglycine, p-bromophenylalanine, p-iodophenylalanine, azidophenylalanine, acetylphenylalanine or ethynylphenylalanine, amino acids containing internal alkenes such as trans-crotylalkene, serine allyl ether, allyl glycine, propargylglycine, and vinyl glycine.

[0071] In some embodiments, the scFv portion of the bispecific antibodies described herein may be a VFv fragment that is heterodimerized due to the formation of one or more disulfide bonds, which may reduce the formation of high molecular weight aggregates. H Chain and V L In some instances, the cysteine ​​residues may be modified to introduce cysteine ​​residues into both V H Alternatively, or in addition, a cysteine ​​residue may be introduced at residue 44 of the V L It may be introduced at residue 100 of the chain.

[0072] Exemplary anti-pKal / anti-FXIIa bispecific antibodies include clones X0120-A01, X0120-C01, X0120-E01, X0120-G01, X0121-A03, X0121-C01, X0121-E01, X0121-G01, X0122-A01, X0122-C01, 620I-X0173-A11, 620I-X0173-C07, 620I-X0173-E07 and 620I-X0173-G11, as described in the Examples below. Other exemplary anti-pKal / anti-FXIIa bispecific antibodies include clones 620I-X138-A08, 620I-X136-B02, 620I-X139-A12, 620I-X137-B08, 620I-X142-A04, 620I-X142-B11, 620I-X138-B01, 620I-X136-C01, 620I-X138-A12, 620I-X136-A12, 620I-X138-A0 2, 620I-X136-A05, 620I-X138-C07, 620I-X136-E07, 620I-X142-B02, 620I-X136-F11, 620I-X142-A05, 620I-X136-C0 9, 620I-X138-B10, 620I-X136-C08, 620I-X139-A11, 620I-X136-D05, 620I-X138-D04, 620I-X136-G08, 620I-X142-B07 , 620I-X142-A11, 620I-X138-G12, 620I-X142-A10, 620I-X138-D03, 620I-X137-C08, 620I-X142-E02, 620I-X136-E05 , 620I-X138-B06, 620I-X136-A09, 620I-X138-A06, 620I-X137-A10, 620I-X139-B10, 620I-X136-A04, 620I-X138-D06 , 620I-X136-C11, 620I-X138-B07, 620I-X136-A02, 620I-X139-G02, 620I-X136-B07, 620I-X138-E03, 620I-X136-G05 , 620I-X139-D12, 620I-X136-A01, 620I-X138-C12, 620I-X136-G10, 620I-X138-D05, 620I-X136-F07, 620I-X138-A01,620I-X142-E09, 620I-X138-D11, 620I-X136-C05, 620I-X142-A02, 620I-X136 -C04, 620I-X138-F02, 620I-X136-G04, 620I-X139-G12, 620I-X136-B11, 620I -X142-D04, 620I-X136-D06, 620I-X139-A01, 620I-X136-D12, 620I-X138-F05, 620I-X136-A11, 620I-X139-E05, 620I-X136-C12 and 620I-X138-E05.

[0073] Preparation of bispecific antibodies For the preparation of the bispecific antibodies described herein, any suitable method known in the art (e.g., standard recombinant techniques) can be used. An example is provided below.

[0074] Suitable parent antibody heavy and light chain genes can be obtained by conventional techniques (e.g., PCR amplification from a suitable source). In one example, DNA encoding a monoclonal antibody specific to a target antigen can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the monoclonal antibody). Cells such as hybridoma cells can serve as a source of such DNA. In another example, the sequence of DNA encoding a monoclonal antibody specific to a target antigen can be obtained, for example, from a database or other publicly available source, and the DNA can be synthesized. Parent antibody genes can also be obtained by screening a suitable antibody library with the antigen of interest.

[0075] The heavy and light chain genes of the antibodies thus obtained may be analyzed to identify the complementarity determining region (CDR) regions according to conventional techniques. Any of the polypeptides in a bispecific antibody as described herein may be prepared by conventional recombinant techniques and inserted into a suitable expression vector for production in a suitable host cell.

[0076] The nucleotide sequences encoding one or more of the polypeptides of the bispecific antibody as described herein may be cloned into one expression vector, with each nucleotide sequence operably linked to a suitable promoter. Alternatively, the nucleotide sequences may be operably linked to a single promoter, such that both sequences are expressed from the same promoter. In some examples, the expression of the two polypeptides is controlled by a common promoter. In other examples, the expression of each of the two polypeptides is under the control of a separate promoter. In another alternative, the nucleotide sequences encoding the two polypeptides are cloned into two vectors that may be introduced into the same cell or into separate cells. When the two polypeptides are expressed in separate cells, each of the two polypeptides may be isolated from the host cell that expresses them, and the two isolated heavy chains may be mixed and incubated under suitable conditions that allow the formation of the bispecific antibody.

[0077] In general, the nucleic acid sequence encoding one or all chains of the bispecific antibody can be cloned into a suitable expression vector, operably linked to a suitable promoter, using methods known in the art. For example, the nucleotide sequence and the vector can be contacted with a restriction enzyme under suitable conditions to generate complementary ends on each molecule that can pair with each other and be joined by a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the ends of the genes. These synthetic linkers contain nucleic acid sequences that correspond to specific restriction sites in the vector. The choice of expression vector / promoter will depend on the type of host cell to be used to produce the antibody.

[0078] A variety of promoters can be used for expression of the bispecific antibodies described herein, including but not limited to the cytomegalovirus (CMV) intermediate early promoter, viral LTRs (such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR), simian virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and herpes simplex tk virus promoter.

[0079] Regulatable promoters can also be used. Such regulatable promoters include those that use the lac repressor from E. coli as a transcriptional modulator that regulates transcription from mammalian cell promoters carrying the lac operator [Brown, M. et al., Cell, 49:603-612 (1987)] and those that use the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimers, VP16 or p65 using astradiol, RU486, diphenol murislerone or rapamycin. Inducible systems are available from Invitrogen, Clontech and Ariad.

[0080] Regulatable promoters that include a repressor together with an operon can be used. In one embodiment, the lac repressor from E. coli can function as a transcriptional modulator that regulates transcription from a mammalian cell promoter carrying a lac operator [M. Brown et al., Cell, 49:603-612 (1987)]. Gossen and Bujard (1992) [M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)] combined the tetracycline repressor (tetR) with a transcriptional activator (VP16) to create the tetR-mammalian cell transcriptional activator fusion protein tTa (tetR-VP16), in combination with a minimal promoter carrying tetO from the human cytomegalovirus (hCMV) major immediate early promoter to create the tetR-tet operator system that controls gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. When the tetracycline operator is appropriately positioned downstream of the TATA element of the CMVIE promoter, the tetracycline repressor (tetR) alone, but not tetR-mammalian cell transcription factor fusion derivatives, can function as a potent trans-modulator to regulate gene expression in mammalian cells (Yao et al., Human Gene Therapy). One particular advantage of this tetracycline-inducible switch is that it does not require the use of fusion proteins of the tetracycline repressor and mammalian cell transactivators or repressors (which may be toxic to cells in some cases (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995))) to achieve its regulatable effect.

[0081] In addition, the vector may contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene, for selection of stable or transient transfectants in mammalian cells; an enhancer / promoter sequence derived from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals derived from SV40 for mRNA stability; an SV40 polyoma origin of replication and ColE1 for proper episomal replication; an internal ribosome binding site (IRES); a versatile multiple cloning site; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for generating vectors containing transgenes are well known and available in the art.

[0082] Examples of polyadenylation signals useful in practicing the methods described herein include, but are not limited to, the human collagen I polyadenylation signal, the human collagen II polyadenylation signal, and the SV40 polyadenylation signal.

[0083] Another aspect of the present disclosure relates to a method for preparing a bispecific antibody comprising culturing a host cell or set of host cells described herein under conditions allowing expression of a first polypeptide and a second polypeptide, and isolating the bispecific antibody comprising the first and second polypeptides. In some embodiments, the host cell comprises an expression vector comprising a first nucleotide sequence encoding the first polypeptide as described herein and a second nucleotide sequence encoding the second polypeptide as described herein.

[0084] Host cells suitable for use in the preparation of the bispecific antibodies described herein may be any host cell known in the art that can be used for protein production, including but not limited to bacterial cells, yeast cells, insect cells, plant cells or mammalian cells.

[0085] The bispecific antibodies described herein may be produced in bacterial cells (e.g., E. coli cells). Alternatively, the bispecific antibodies may be produced in eukaryotic cells. In one embodiment, the antibodies are expressed in yeast cells such as Pichia (see, e.g., Powers et al., 2001, J. Immunol. Methods. 251:123-35), Hanseula, or Saccharomyces. In another embodiment, the bispecific antibodies may be produced in mammalian cells. Mammalian host cells for expressing the antibody include, but are not limited to, 293 cells (see, e.g., ATCC CRL-1573 (American Type Culture Collection®), and Expi293F™ cells (Life Technologies™)), Chinese hamster ovary (CHO) cells (used with the DHFR selection marker, e.g., as described in Kaufman and Sharp, 1982, Mol. Biol. 159:601-621, dhfr-FcR1 cells as described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, used with the DHFR selection marker). - Included are human CHO cells, lymphoid cell lines (e.g., NS0 myeloma cells and SP2 cells), COS cells, and cells derived from transgenic animals (e.g., transgenic mammals). For example, the cell is a mammary epithelial cell.

[0086] In an exemplary system for recombinant expression of a bispecific antibody as described herein, a recombinant expression vector encoding both of the polypeptides in the bispecific antibody is transfected with dhfrRNA by calcium phosphate mediated transfection. -The recombinant expression vector is introduced into CHO cells. In the recombinant expression vector, the nucleic acid encoding the two polypeptides is operably linked to enhancer / promoter regulatory elements (e.g., from SV40, CMV, adenovirus, etc.; such as CMV enhancer / AdMLP promoter regulatory elements or SV40 enhancer / AdMLP promoter regulatory elements) to drive high levels of gene transcription. The recombinant expression vector also carries a DHFR gene, which allows for the selection of CHO cells transfected with the vector using methotrexate selection / amplification. The selected transformed host cells are cultured to express the two polypeptides. The tetrameric molecules formed thereby can be recovered from the culture medium. Another exemplary system for recombinant expression is described in Example 2.

[0087] Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography using a Protein A or Protein G-bound matrix.

[0088] The utility of bispecific antibodies The bispecific antibodies or encoding nucleic acid or sets of nucleic acids described herein can be used for diagnostic and therapeutic purposes. They can also be used as research tools in basic and therapeutic research.

[0089] (i) Pharmaceutical Composition The bispecific antibody (or encoding nucleic acid or set of nucleic acids) as described herein can be mixed with a pharma- ceutically acceptable carrier (excipient) (including a buffer) to make a pharmaceutical composition for use in treating a target disease. By "acceptable" it is meant that the carrier must be compatible with (and preferably capable of stabilizing) the active ingredient of the composition and not deleterious to the subject being treated. Pharmaceutically acceptable excipients (carriers) (including buffers) are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover.

[0090] The pharmaceutical compositions used in the methods of the invention may contain pharma- ceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants (including ascorbic acid and methionine); preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); polypeptides of low molecular weight (less than about 10 residues); serum alginates, such as glycerol, glycerol, tert-butyl alcohol ... The surfactants may include proteins such as albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates (including glucose, mannose or dextran); chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium:metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0091] In some examples, the pharmaceutical compositions described herein include liposomes containing bispecific antibodies, which can be prepared by methods known in the art, such as those described in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with improved circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be made by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter of defined pore size to obtain liposomes with desired diameter.

[0092] The bispecific antibodies or encoding nucleic acid(s) may also be encapsulated in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules, and poly(methylmethacylate) microcapsules, respectively), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, prepared, for example, by coacervation techniques or interfacial polymerization. Such techniques are known in the art. See, for example, Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[0093] In another example, the pharmaceutical compositions described herein can be formulated into sustained release form.Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles (e.g., films or microcapsules).Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0094] Pharmaceutical compositions used for in vivo administration must be sterile. This is easily accomplished, for example, by filtration through sterile filtration membranes. Therapeutic antibody compositions are generally placed into a container having a sterile access port (for example, an intravenous solution bag or a vial having a stopper that can be pierced by a hypodermic injection needle).

[0095] The pharmaceutical compositions described herein may be in unit dosage form, such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation. To prepare solid compositions, such as tablets, the primary active ingredient can be mixed with pharmaceutical carriers (e.g., conventional tableting ingredients, such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gums) and other pharmaceutical diluents (e.g., water) to produce a solid preformulation composition containing a homogenous mixture of the compound of the present invention or a non-toxic pharmaceutically acceptable salt thereof. When these preformulation compositions are referred to as homogenous, it is meant that the active ingredient is uniformly dispersed throughout the composition, such that the composition can be readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above, containing from 0.1 to about 500 mg of the active ingredient of the present invention. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form that provides the advantage of prolonged action.For example, the tablet or pill can comprise an inner dosage component and an outer dosage component, the latter being in the form of an envelope over the former.These two components can be separated by an enteric layer, which acts to prevent disintegration in the stomach and allows the inner component to pass intact into the duodenum or to be delayed in release.Various materials can be used for such enteric layers or coatings, including a number of polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol and cellulose acetate.

[0096] Suitable surfactants include, inter alia, non-ionic surfactants such as polyoxyethylene sorbitans (e.g., Tween™ 20, 40, 60, 80 or 85) and other sorbitans (e.g., Span™ 20, 40, 60, 80 or 85). Compositions having a surfactant will conveniently contain 0.05 to 5% surfactant, and may be 0.1 to 2.5%. It will be appreciated that other ingredients may be added as required, such as mannitol or other pharma- ceutical acceptable vehicle.

[0097] Suitable emulsions may be prepared using commercially available fat emulsions such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ and Lipiphysan™. The active ingredient may be dissolved in a premixed emulsion composition or the active ingredient may be dissolved in oil (e.g. soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and mixed with phospholipids (e.g. egg phospholipids, soybean phospholipids or soybean lecithin) and water to form an emulsion. It will be understood that other ingredients (e.g. glycerol or glucose) may be added to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil (e.g. 5-20%). Fat emulsions may contain 0.1-1.0.im (especially 0.1-0.5.im) fat droplets and may have a pH in the range of 5.5-8.0.

[0098] The emulsion composition may be prepared by mixing the bispecific antibody with Intralipid™ or its components (soybean oil, egg phospholipids, glycerol and water).

[0099] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharma- ceutically acceptable aqueous or organic solvents or mixtures thereof, as well as powders. Liquid or solid compositions may contain suitable pharma- ceutically acceptable excipients as described above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.

[0100] Compositions, preferably in sterile pharma- ceutically acceptable solvents, may be nebulized by the use of gas. Nebulized solutions may be directly inhaled from the nebulizing device, or the nebulizing device may be attached to a face mask, tent, or intermittent positive pressure respirator. Solution, suspension, or powder compositions may be administered (preferably orally or nasally) from a device that delivers the formulation in an appropriate manner.

[0101] (ii) Disease treatment The bispecific antibodies (or encoding nucleic acids or sets of nucleic acids) described herein are useful for treating diseases or disorders associated with one or both of the antigens to which the bispecific antibodies bind. For example, if the bispecific antibody is capable of binding to pKal and FXIIa and blocking the activity of pKal and FXIIa, the bispecific antibody can be used to treat diseases associated with dysregulation of the contact activation system (e.g., HAE and thrombosis).

[0102] HAE (including types I, II, and III HAE) is a disorder characterized by recurrent episodes of severe swelling, for example in the limbs, face, intestines, and airways. HAE attacks (HAE attach) can be triggered by minor trauma or stress. Intestinal swelling resulting from an HAE attack can cause severe abdominal pain, nausea, and vomiting. Airway swelling can restrict breathing and cause life-threatening airway obstruction.

[0103] Thrombosis (e.g., venous or arterial thrombosis) refers to the formation of a blood clot in a blood vessel that can impede blood flow through the circulatory system. Thrombosis can include thrombosis associated with atrial fibrillation, DVT, pulmonary embolism, stroke, or other arterial or venous thrombotic events.

[0104] To practice the methods disclosed herein, an effective amount of the pharmaceutical composition described above may be administered to a subject (e.g., a human) in need of treatment via a suitable route, such as intravenous administration (e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, inhalation or topical routes). Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations can be nebulized as is, and lyophilized powders can be nebulized after reconstitution. Alternatively, bispecific antibodies as described herein can be aerosolized using fluorocarbon formulations and metered dose inhalers, or inhaled as lyophilized and milled powders.

[0105] The subject treated by the methods described herein may be a mammal (more preferably a human). Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. The human subject in need of treatment may be a human patient who has, is at risk for, or is suspected of having a target disease / disorder, such as HAE or thrombosis. In some embodiments, the thrombosis is associated with atrial fibrillation, deep vein thrombosis (DVT), pulmonary embolism, stroke, or arterial or venous thrombotic events. Subjects with a target disease or disorder can be identified by routine medical examination (e.g., laboratory tests, organ function tests, CT scans, or ultrasound examinations). Subjects suspected of having any of such target diseases / disorders may exhibit one or more symptoms of the disease / disorder. Subjects at risk of a disease / disorder may be subjects who have one or more of the risk factors for the disease / disorder.

[0106] As used herein, "effective amount" refers to the amount of each active agent required to provide a therapeutic effect to a subject, either alone or in combination with one or more other active agents. As will be recognized by those skilled in the art, the effective amount will vary according to the specific condition being treated, the severity of the condition, the parameters of the individual patient (including age, health, size, sex and weight), the duration of treatment, the nature of concomitant therapy (if any), the specific route of administration, and similar factors within the knowledge and expertise of medical practitioners. These factors are well known to those skilled in the art and can be addressed with no more than routine experimentation. It is generally preferred to use the maximum dose of each component or combination thereof, i.e., the maximum safe dose according to sound medical judgment. However, those skilled in the art will understand that a patient may require a lower dose or tolerated dose for medical reasons, psychological reasons, or virtually any other reason.

[0107] Experimental considerations, such as half-life, will generally contribute to the determination of dosage. For example, to extend the half-life of the antibody and to prevent the antibody from being attacked by the host's immune system, an antibody compatible with the human immune system, such as a humanized antibody or an antibody that is fully human, may be used. The frequency of administration may be determined and adjusted during treatment, and is generally, but not necessarily, based on the treatment and / or suppression and / or remission and / or delay of the target disease / disorder. Alternatively, a sustained continuous release formulation of the bispecific antibody may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0108] In one example, the dosage of a bispecific antibody as described herein may be determined empirically in an individual who is administered one or more doses of the antibody. The individual is given increasing doses of the antagonist. Indicators of disease / disorder may be followed to assess the effectiveness of the antagonist.

[0109] Generally, for administration of any of the antibodies described herein, the initial candidate dosage may be about 2 mg / kg. For purposes of this disclosure, a typical daily dosage may range from about 0.1 μg / kg to 3 μg / kg to 30 μg / kg to 300 μg / kg to 3 mg / kg, up to 30 mg / kg to 100 mg / kg or more, depending on the factors mentioned above. When administered repeatedly over several days or more depending on the condition, treatment is sustained until a desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved to alleviate the targeted disease or disorder or symptoms thereof. An exemplary dosing regimen includes administering an initial dose of about 2 mg / kg, followed by a weekly maintenance dose of about 1 mg / kg of antibody or every other week maintenance dose of about 1 mg / kg. However, other dosing regimens may be useful depending on the pattern of pharmacokinetic decay the practitioner wishes to achieve. For example, administration 1 to 4 times per week is contemplated. In some embodiments, a dose in the range of about 3 μg / mg to about 2 mg / kg (such as about 3 μg / mg, about 10 μg / mg, about 30 μg / mg, about 100 μg / mg, about 300 μg / mg, about 1 mg / kg, and about 2 mg / kg) may be used. In some embodiments, the frequency of administration is once every 1, 2, 4, 5, 6, 7, 8, 9, or 10 weeks, or once every 1, 2, or 3 months or more than 3 months. The progress of this therapy is easily monitored by conventional techniques and assays. The dosing regimen (including the antibody used) may vary over time.

[0110] In some embodiments, for a normal weight adult patient, a dose ranging from about 0.3 to 5.00 mg / kg may be administered. The particular dosing regimen (i.e., dose, timing and frequency) will depend on the particular individual and that individual's medical history as well as the characteristics of the individual drug (such as the drug's half-life and other considerations well known in the art).

[0111] For purposes of this disclosure, the appropriate dosage of a bispecific antibody as described herein will depend on the specific antibody (or composition thereof) used, the type and severity of the disease / disorder, whether the antibody is administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the antagonist, and the discretion of the attending physician. Typically, the clinician will administer the bispecific antibody until a dosage is reached that achieves the desired result. Administration of one or more bispecific antibodies may be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to those of skill in the art. Administration of the bispecific antibody may be essentially continuous over a preselected period of time, or may be a series of spaced doses (e.g., before, during, or after the onset of the target disease or disorder).

[0112] As used herein, the term "treatment" refers to the application or administration of a composition comprising one or more active agents to a subject having a target disease or disorder, a symptom of the disease / disorder, or a predisposition to the disease / disorder, for the purpose of curing, healing, alleviating, mitigating, altering, ameliorating, ameliorating, reversing, or affecting the disorder, the symptom of the disease, or the predisposition to the disease or disorder.

[0113] Alleviating the target disease / disorder includes delaying the onset or progression of the disease, or reducing the severity of the disease. Alleviating the disease does not necessarily require a curative outcome. As used in this context, "delaying" the onset of the target disease or disorder means delaying, preventing, slowing, slowing, stabilizing, and / or postponing the progression of the disease. This delay may be of various lengths of time, depending on the disease being treated and / or the medical history of the individual. A method of "delaying" or alleviating the onset of a disease or delaying the onset of a disease is a method that reduces the likelihood of developing one or more symptoms of the disease within a given time frame and / or reduces the severity of symptoms within a given time frame, compared to not using the method. Such comparisons are typically based on clinical studies using a sufficient number of subjects to produce statistically significant results.

[0114] "Onset" or "progression" of a disease refers to the initial manifestation and / or subsequent progression of the disease. Onset of a disease can be detected and evaluated using standard clinical techniques well known in the art. However, onset also refers to progression that may be undetectable. For the purposes of this disclosure, onset or progression refers to the biological course of a symptom. "Onset" includes occurrence, recurrence, and onset. As used herein, "onset" or "onset" of a target disease or disorder includes initial onset and / or recurrence.

[0115] In some embodiments, the bispecific antibodies described herein are administered to a subject in need of treatment in an amount sufficient to inhibit the activity of one or both of the target antigens in vivo by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90%). In other embodiments, the antibodies are administered in an amount effective to reduce the levels of one or both of the target antigens by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90%).

[0116] The pharmaceutical composition can be administered to a subject using conventional methods known to those skilled in the art of medicine, depending on the type of disease or the site of the disease to be treated.The composition can also be administered via other conventional routes, such as orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, intravaginally or via implanted reservoirs.As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.In addition, the composition can be administered to a subject via injectable depot administration routes, such as those using 1, 3 or 6 month depot injections or biodegradable materials and methods.

[0117] Injectable compositions may contain various carriers, such as salad oil, dimethylactamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies can be administered by drip infusion, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. Intramuscular preparations (e.g., a sterile formulation of a suitable soluble salt form of the antibody) can be administered dissolved in a pharmaceutical excipient, such as water for injection, 0.9% saline, or 5% glucose solution.

[0118] In one embodiment, bispecific antibodies are administered via site-specific delivery techniques or targeted local delivery techniques.Examples of site-specific delivery techniques or targeted local delivery techniques include various implantable depot sources or local delivery catheters (such as injection catheters, indwelling catheters or needle catheters) of bispecific antibodies, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site-specific carriers, direct injection, or direct application.See, for example, PCT International Publication No. WO00 / 53211 and U.S. Patent No. 5,981,568.

[0119] Targeted delivery of therapeutic compositions comprising antisense polynucleotides, expression vectors or subgenomic polynucleotides can also be used.Receptor-mediated DNA delivery technology is described, for example, in Findeis et al., Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (JA Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA (1990) 87:3655; Wu et al., J. Biol. Chem. (1991) 266:338.

[0120] Therapeutic compositions containing polynucleotides (e.g., those encoding bispecific antibodies described herein) are administered in the range of about 100 ng to about 200 mg of DNA for local administration in gene therapy protocols. In some embodiments, concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA or higher may be used during gene therapy protocols.

[0121] The therapeutic polynucleotides and polypeptides described herein can be delivered using gene delivery vehicles. Gene delivery vehicles can be of viral or non-viral origin (see generally Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148). The expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters and / or enhancers. The expression of coding sequences can be constitutive or regulated.

[0122] Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT International Publication Nos. WO 90 / 07936; WO 94 / 03622; WO 93 / 25698; WO 93 / 25234; WO 93 / 11230; WO 93 / 10218; WO 91 / 02805; U.S. Pat. Nos. 5,219,740 and 4,777,127; British Patent No. 2,200,651; and European Patent No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-2111), and the like). VR-1246), and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532), and adeno-associated virus (AAV) vectors (see, e.g., PCT International Publication Nos. WO94 / 12649, WO93 / 03769; WO93 / 19191; WO94 / 28938; WO95 / 11984 and WO95 / 00655). Administration of DNA linked to killed adenovirus, such as that described in Curiel, Hum. Gene Ther. (1992) 3:147, can also be used.

[0123] Non-viral delivery vehicles and methods can also be used, including but not limited to polycation-condensed DNA (see, for example, Curiel, Hum. Gene Ther. (1992) 3:147), with or without being linked to killed adenovirus alone; ligand-linked DNA (see, for example, Wu, J. Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vehicle cells (see, for example, U.S. Patent No. 5,814,482; PCT International Publication Nos. WO95 / 07994; WO96 / 17072; WO95 / 30763; and WO97 / 42338), and nuclear charge neutralization or fusion with cell membranes. Naked DNA can also be used. Exemplary naked DNA introduction methods are described in PCT International Publication No. WO90 / 11092 and U.S. Patent No. 5,580,859. Liposomes that can serve as gene delivery vehicles are described in U.S. Patent No. 5,422,120; PCT International Publication No. WO95 / 13796; WO94 / 23697; WO91 / 14445; and European Patent No. 0524968. Further techniques are described in Philip, Mol.Cell.Biol.(1994)14:2411 and Woffendin, Proc.Natl.Acad.Sci.(1994)91:1581.

[0124] The particular dosing regimen (i.e., dose, timing and frequency) used in the methods described herein will depend on the particular subject and the subject's medical history. In some embodiments, a subject in need of treatment may be administered two or more bispecific antibodies, or a combination of a bispecific antibody and another suitable therapeutic agent. Bispecific antibodies may also be used with other agents that serve to enhance and / or complement the effectiveness of the agents.

[0125] The efficacy of a treatment against a target disease / disorder can be assessed by methods well known in the art.

[0126] Kits for use in treating a target disease The present disclosure also provides kits for use in alleviating a target disease or disorder. Such kits may include one or more containers containing one or more of the bispecific antibodies described herein and / or one or more isolated nucleic acids or sets of nucleic acids.

[0127] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The included instructions may include instructions for administration of the bispecific antibody to treat, delay the onset of, or alleviate a target disease, such as HAE or thrombosis. The kit may further include instructions for selecting an individual suitable for treatment based on identifying whether the individual has the target disease. In yet other embodiments, the instructions include instructions for administering the antibody to an individual at risk for the target disease.

[0128] Instructions for use of bispecific antibodies as described herein typically include information regarding dosages, dosing schedules and routes of administration for the intended treatment. Containers may be unit dose, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions provided in the kits of the invention are typically instructions written on a label or insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0129] The label or package insert indicates that the composition is used for treating, delaying the onset of, and / or ameliorating the targeted disease or disorder. Instructions may be provided for practicing any of the methods described herein.

[0130] The kit of the present invention is in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with specific devices, such as inhalers, nasal administration devices (e.g., nebulizers) or injection devices (mini-pumps, etc.). The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is a bispecific antibody, such as those described herein.

[0131] The kit may optionally provide additional components such as buffers and instructional information. Typically, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiments, the invention provides an article of manufacture comprising the contents of the kit described above.

[0132] General Technology The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the ordinary skill in the art. Such techniques are described in Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JECellis, ed., 1998) Academic Press; Culture(RIFreshney,ed.,1987);Introduction to Cell and Tissue Culture(JPMather and PERoberts,1998)Plenum Press;Cell and Tissue Culture:Laboratory Procedures(A.Doyle,JBGriffiths,and DGNewell,eds.,1993-8)J.Wiley and Sons;Methods in Enzymology(Academic Press,Inc.);Handbook of Experimental Immunology(DMWeir and CCBlackwell, eds.);Gene Transfer Vectors for Mammalian Cells (JMMiller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FMAusubel, et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology (JEColigan et al., eds.,1991);Short Protocols in Molecular Biology(Wiley and Sons,1999);Immunobiology(CA Janeway and P. Travers,1997);Antibodies(P. Finch,1997);Antibodies:a practical approach(D. Catty.,ed.,IRL Press,1988-1989);Monoclonal antibodies:a practical approach(P. Shepherd and C. Dean,eds.,Oxford University Press,2000);Using antibodies:a laboratory manual(E. Harlow and D. Lane(Cold Spring Harbor Laboratory Press,1999);The Antibodies(M. Zanetti and JD Capra,eds.,Harwood Academic Publishers,1995) and other publications.

[0133] Without further elaboration, it is believed that one skilled in the art can utilize the present invention to its fullest extent based on the above description. Accordingly, the following specific embodiments are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way. All publications cited herein are incorporated by reference for the purposes or subject matter discussed herein. EXAMPLES

[0134] Example 1: Construction and characterization of an exemplary bispecific antibody that binds pKal and Factor XIIa Using DX-2930 as the parent antibody and one of the anti-FXIIa clones 559C-M0071-F06, 559C-M0184-B04, 559C-M0179-D04, 559C-M0181-C02 and 559C-M0180-G03, several exemplary anti-pKal / anti-FXIIa bispecific antibodies were constructed, including clones X0120-A01, X0120-C01, X0120-E01, X0120-G01, X0121-A03, X0121-C01, X0121-E01, X0121-G01, X0122-A01 and X0122-C01. See Table 2 below.

[0135] [Table 2]

[0136] Among the anti-FXIIa clones, 559C-M0071-F06 is the parent clone, 559C-M0184-B04 is derived from affinity maturation of HCDR1+2, and 559C-M0179-D04, 559C-M0181-C02 and 559C-M0180-G03 are clones derived from affinity maturation of the light chain.

[0137] All of the exemplary bispecific antibody clones listed in Table 2 above are tetravalent molecules containing four polypeptide chains, including two polypeptide chains of the light chain of DX-2930 (SEQ ID NO: 10 presented above) and two fusion polypeptide chains of the heavy chain of DX-2930 (excluding the lysine residues in the hinge domain of the constant chain) fused to the scFv chain of one of the FXIIa clones. The scFv chains of each of the five anti-FXIIa clones were synthesized in both the heavy-light (H→L) and light-heavy (L→H) orientations. In all examples of the scFv chains, the internal (GGGGS) 4 A linker (SEQ ID NO: 23) was used. The scFvs were constructed such that clones in the light-heavy orientation contained the first two amino acids (RT) that start the constant region before the start of the linker sequence. Clones in the heavy-light orientation contained only the first amino acid (R) from the light chain constant region before the stop codon.

[0138] The amino acid sequences of each fusion polypeptide of an exemplary bispecific antibody are provided below.

[0139] [ka] TIFF0007635285000010.tif242159TIFF0007635285000011.tif99162

[0140] To construct the expression cassettes for the above exemplary bispecific antibodies, the coding sequences for the heavy and light chains of DX-2930 were modified with a C-terminal SGGGS linker leading to the scFv coding sequence and cloned into the pRh1-CHO vector. The linker region contained a BamHI restriction site for efficient cloning of the scFv. Five anti-factor XIIa clones were selected for insertion into the construct via the BamHI / XbaI restriction sites.

[0141] The italicized portions of the sequences presented above refer to the signal peptides. The anti-pKal portions of the bispecific antibodies disclosed herein may contain the same signal peptide or the signal peptide may be removed or replaced with a different signal peptide. Signal peptides for use in the production of secreted proteins are well known in the art.

[0142] The nucleotide sequence encoding the bispecific antibody (in a cis-tronic operon format) is shown below.

[0143] [ka] TIFF0007635285000013.tif242166TIFF0007635285000014.tif245160TIFF00076352850 00015.tif239166TIFF0007635285000016.tif245161TIFF0007635285000017.tif245163 TIFF0007635285000018.tif235161TIFF0007635285000019.tif244166TIFF00076352850 00020.tif243161TIFF0007635285000021.tif245162TIFF0007635285000022.tif237166

[0144] A pRh1 expression plasmid was generated encoding the bispecific antibody described above. After 0.2 μm sterile filtration, the plasmid was transfected into a 60 mL d1 culture of Expi293F™ cells grown in Expi293™ Expression Medium using ExpiFectamine™ as the transfection reagent as described in the LifeTech protocol (Life Technologies™, Carlsbad, CA). Expifectamine™ Transfection Enhancers 1 and 2 were added to the day 2 culture as described in the LifeTech protocol. Cultures were incubated at 37°C and 8% CO. 2 The cells were incubated at 140 rpm for up to 7 days. Cultures were harvested by centrifugation, 0.2 μm sterile filtered, and stored at 4° C. Clones were batch purified using a Protein A column.

[0145] Varying concentrations of the bispecific antibody were incubated with each of the FXIIa and pKal samples, and the ability of these proteases to cleave the peptide substrate was monitored over time by measuring the change in fluorescence of a chemical moiety covalently attached to the peptide substrate. The slope of this kinetic data, which equates to the rate of proteolysis by the enzyme, is then plotted versus the concentration of inhibitor. The resulting plot was then fitted by nonlinear regression to the equation for a tight-binding inhibitor (Equation 1) to obtain the apparent inhibition constant (K i app ) is obtained.

number

[0146] Figures 1 and 2 show plots of pKal and FXIIa inhibitory activity for each of the bispecific antibodies tested. All clones tested were able to inhibit both pKal and FXIIa. The K i app pKal and K i app FXIIa are listed in Table 3 below.

[0147] [Table 3]

[0148] Example 2: Construction and characterization of an exemplary bispecific antibody that binds pKal and Factor XIIa Another exemplary set of anti-pKal / anti-FXIIa bispecific antibodies was constructed as follows. The IgG portion of the molecule was the same as that used in Example 1 (i.e., DX-2930). For the anti-FXIIa component, 36 isolates were selected and converted to scFvs in both the light / heavy and heavy / light orientations. The scFvs were fused to DX-2930 IgG using the SGGGS (SEQ ID NO: 22) linker. In constructing the scFvs, the (G 4 S) 4The variable heavy and light domains of anti-FXIIa were fused to each other using a linker. The sequence of the bispecific antibody is presented below.

[0149] The constructed bispecific molecules showed anti-pKal activity generally consistent with values ​​previously determined for DX-2930 (Table 4). Some values ​​showed lower potency for pKal, possibly due to errors in concentration calculations or possibly due to aggregation. Anti-FXIIa activity of the scFv components was typically lower than previously determined values, possibly due to inherent instability associated with scFvs (Table 4). Activity of the bispecific molecules in the plasma assay showed a marked improvement compared to DX-2930 and anti-FXIIa IgG. While DX-2930 showed a range of 70-100 nM in this assay, the anti-FXIIa parent antibody showed inhibition in the range of approximately 100 nM. The panel of bispecific molecules tested shows inhibition in the range of 1-10 nM (Table 5).

[0150] [Table 4] TIFF0007635285000026.tif245164TIFF0007635285000027.tif210166

[0151] [Table 5] TIFF0007635285000029.tif48159

[0152] Five exemplary candidates (620I-X0136-D12, 620I-X0136-C05, 620I-X0136-C11, 620I-X0136-G05, and 620I-X0136-A01) were selected for further analysis. Of these five potential candidates, 620I-X0136-A01 was excluded due to low expression values ​​and the presence of multiple species in size-exclusion chromatography (SEC) traces. For the remaining four potential candidates, each isolate contained varying degrees of high molecular weight aggregates (16-35%) (Figure 3). The aggregates were determined to be concentration-dependent and were hypothesized to be dimeric structures interacting via the scFv domain.

[0153] Exemplary bispecific antibody 620I-X0136-D12 (D12) was evaluated for its ability to inhibit plasma pKal activity by plasma inhibition assay. Briefly, reconstituted plasma containing a fixed amount of pre-pKal and FXII in the presence or absence of HMWK was diluted 1:40 with assay buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% PEG-8000 and 0.1% Triton X-100). The concentrations of pre-pKal, FXII and HMWK are equivalent to their normal concentrations in plasma. Inhibitors were added to reconstituted plasma at various concentrations in a 96-well microplate at room temperature. Contact activation was then initiated by the addition of a dilute ellagic acid solution at 25% (final concentration 2.5%), the microplate was gently shaken to mix and allowed to proceed for 2 minutes at room temperature, whereupon 100 nM CTI was added. 10 μl of this mixture was then transferred to a duplicate microplate containing 80 μl of assay buffer pre-equilibrated at 30° C. The dilution plate was then incubated for an additional 5 min at 30° C. and proteolysis of PFR-AMC was assessed as above, except that the back-calculated inhibitor concentration was used on the x-axis for curve fitting to a modified Morrison equation for tight binding inhibitors (plasma was diluted 1:400 for the final assay reading). The results of this study are shown in FIG. 5 (in the presence of single-chain HMWK) and FIG. 6 (in the absence of HMWK). The bispecific antibody performed better than the sum of the parental IgGs, especially in the presence of HMWK. Using the equation for tight binding inhibitors, the apparent Ki value of D12 was determined to be 8.8 nM in the presence of HMWK and 2.6 nM in the absence of HMWK.

[0154] Bispecific antibody candidate 620I-X0136-D12 (D12) was also evaluated for its ability to delay the activated partial thromboplastin time (APTT) in an APTT assay compared to anti-FXIIa (D06) and anti-pKal (H03) antibodies (Figure 7). Briefly, pure plasma was spiked with three concentrations (25, 50, 100) of inhibitor molecules (or control dilution buffer = 25 mM HEPES, pH 7.5, 125 mM NaCl) in a 1:1 mixture and pre-equilibrated for 5 min at 37°C. This mixture was dispensed in 2 x 50 μl into two separate KC4 Delta assay cuvettes (with metal balls). After 60 s, 50 μl of aPTT reagent (activator, Pacific Hemostasis APTT-XL) was added to the rotating cuvette and 180 s after the addition of aPTT (at t = 0 s), 50 μl of CaCl 2 The KC4 Delta instrument recorded clotting times in seconds.

[0155] The bispecific antibody candidate 620I-X0136-D12 (D12) was also evaluated for its ability to inhibit fibrin formation (Figure 8).

[0156] Antibody sequences: All bispecific molecules described in this example contained a first polypeptide comprising the heavy chain of DX-2930, a SGGGS linker, and an anti-FXIIa scFv in either heavy / light or light / heavy orientation. The light chain of DX-2930 was also expressed using the same vector. Only the heavy chain + scFv sequences are listed for each isolate.

[0157] [ka]

[0158] 36 Exemplary Anti-FXIIa IgG-Derived Bispecifics [ka] TIFF0007635285000032.tif244164TIFF0007635285000033.tif245163TIFF000 7635285000034.tif244164TIFF0007635285000035.tif245160TIFF00076352850 00036.tif245160TIFF0007635285000037.tif211159TIFF0007635285000038.t if238160TIFF0007635285000039.tif239160TIFF0007635285000040.tif228160 TIFF0007635285000041.tif235160TIFF0007635285000042.tif236160TIFF000 7635285000043.tif245159TIFF0007635285000044.tif240161TIFF00076352850 00045.tif240161TIFF0007635285000046.tif236161TIFF0007635285000047.t if242161TIFF0007635285000048.tif240161TIFF0007635285000049.tif117164

[0159] Example 3: Construction and characterization of exemplary bispecific antibodies containing disulfide bonds To combat these aggregates, residue 44 (C 44 ) and residue 100 of VL (C 100Disulfide bonds between the scFv and the scFv domains were engineered into the scFv region of four clones, 620I-X0177-A01 (620I-X0173-A11), 620I-X0177-C01 (620I-X0173-C07), 620I-X0177-E01 (620I-X0173-E07) and 620I-X0177-G01 (620I-X0173-G11). SEC analysis of bispecifics containing the disulfide-bearing scFvs showed a dramatic reduction in the high molecular weight peak, which fell to 1-2% (Figure 4). This reduction in aggregation was true across all concentrations tested. Biacore of these bispecific clones showed strong specific binding to pKal and FXIIa (Figure 9). Plasma inhibition of these isolates ranged from 0.5 to 8 nM (Figure 10).

[0160] A plasma inhibition assay as described herein was performed to determine the inhibitory activity of bispecific antibody 620I-X0177-A01. Plasma was diluted 1:40 and inhibitors were added to the diluted plasma. A dilute ellagic acid solution of 2.5% (final concentration) was added to the plasma. After about 2 minutes, plasma activation was quenched by addition of CTI. pKal activity in plasma was measured by addition of a profluorescent substrate as described herein. The results thus obtained are shown in FIG. 11.

[0161] The inhibitory activity of clone 620I-X0177-A01 was compared to that of either the parent antibodies alone or in combination. A decrease in affinity was observed between the parent IgG and the bispecific antibody (Figure 12).

[0162] Furthermore, the ability of various bispecific antibodies against APTT was evaluated according to the methods described herein. All tested antibodies showed a dose-dependent delay of APTT (FIG. 13).

[0163] The ability of antibody clones 1A01 (anti-FXIIa) and 7A01 (bispecific for both pKal and FXII) to inhibit fibrin deposition was also examined. The results are shown in Figure 14. A dose-dependent inhibition of fibrin deposition was observed.

[0164] Overall, enzyme inhibition assays determined that the apparent Ki values ​​of each of the anti-pKal and anti-FXIIa components of the exemplary bispecific antibody 620I-X0177-A01 are similar to those of the parent molecules, with apparent Ki values ​​of 389 pM and 73 pM, respectively. Surprisingly, further experiments in contact-activated diluted plasma reveal that this bispecific antibody is more than 5-fold more effective at blocking pKal production than a 1:1 combination of the parent antibodies and more than 20-fold more effective than either of the parent antibodies alone. These data suggest that bispecific antibodies are uniquely potent in their ability to block the positive feedback loop of contact system activation.

[0165] The sequences of bispecific antibodies with disulfide-constrained scFvs are provided below.

[0166] [ka] TIFF0007635285000051.tif97165

[0167] Example 4: Construction and characterization of exemplary bispecific antibodies with C-terminal mutations and / or deletions Exemplary bispecific antibodies (620I-X0177-A01, 620I-X0177-C01, 620I-X0177-E01, 620I-X0177-G01) were evaluated for production and manufacturability. Samples of bispecific antibodies were incubated at room temperature for 48 hours at various pHs prior to analysis. Samples were then separated on SDS-PAGE protein gels or analyzed by size exclusion chromatography, as shown for example in FIG. 15 for bispecific antibody 620I-X0177-A01 (FIG. 16A-FIG. 16C). Under reducing conditions, a pH-dependent increase in the 30 kDa and 50 kDa bands and a decrease in the 80 kDa band were observed (FIG. 15, lanes 2-6). The appearance of these unexpected bands indicated that the bispecific antibodies were undergoing unexpected proteolytic cleavage. The appearance of the same 30 kDa species under non-reducing conditions indicated that the cleaved species was monomeric (Figure 15, lanes 6-8). By SEC analysis, peaks representing the correctly formed bispecific antibody were observed at 15.7-16.1 min, DX-2930 at 17 min, and the cleaved single chain antibody at 22 min (Figure 16).

[0168] Exemplary bispecific antibodies were designed such that the IgG1 heavy chain C-terminal lysine residue was either removed or mutated to a glycine residue.

[0169] The amino acid sequences of the first polypeptides of bispecific antibodies comprising a deletion of the C-terminal lysine residue of the heavy chain of the first antibody or a mutation of the C-terminal lysine to a glycine residue are provided below. These first polypeptides may be paired with the light chain of DX-2930.

[0170] [ka] TIFF0007635285000053.tif239161TIFF0007635285000054.tif237165

[0171] Each of the exemplary bispecific antibodies comprising a deletion of the C-terminal lysine residue of the heavy chain of the first antibody or a mutation of the C-terminal lysine to a glycine residue was evaluated by separating the bispecific antibody on an SDS-PAGE gel at t=0 ( FIG. 17 ). Samples of each of the exemplary bispecific antibodies were also concentrated to approximately 10 mg / mL in 50 mM HEPES (pH 7.5) using an Amicon 10 kDa molecular weight cut-off spin filter and incubated at room temperature for 48 hours. The samples were then evaluated by SDS-PAGE gel ( FIG. 18 ). In each case, the deletion or mutation of the C-terminal lysine reduced or eliminated cleavage of the scFv from the bispecific antibody.

[0172] Bispecific samples were also evaluated by analytical size exclusion chromatography, demonstrating that deletion or mutation of the C-terminal lysine reduces bispecific antibody cleavage (Figures 19-20). Bispecific antibody cleavage was also assessed by incubating the antibodies with EndoLysC for 1 hour at 37°C followed by separation on an SDS-PAGE gel (Figure 21). The protein band at 50 kDa corresponds to the Fab portion of DX-2930 and the band at 100 kDa corresponds to the Fc-scFv homodimer, further demonstrating that deletion or mutation of the heavy chain C-terminal lysine reduces bispecific antibody cleavage.

[0173] Exemplary bispecific antibodies containing C-terminal lysine deletions or mutations were also characterized for their ability to inhibit pKal, FXIIa, and activated plasma compared to DX2930 and DX-4012 controls (Table 1, Figures 22-24). Each of the exemplary bispecific antibodies was found to be functionally equivalent to the parent bispecific antibody (a bispecific antibody that does not contain an IgG1 heavy chain C-terminal lysine deletion or mutation). The mutations may reduce the charge heterogeneity of the bispecific antibody.

[0174] [Table 6] TIFF0007635285000056.tif140166

[0175] Alternatively, or additionally, the C-terminal Lys-Arg(KR) motif of the anti-FXIIa scFv described above can be removed. The amino acid sequences of exemplary bispecific antibody polypeptides comprising a deletion or mutation of the C-terminal lysine residue of the heavy chain of the first antibody to a glycine residue and a deletion of the C-terminal lysine-arginine residue of the scFv are provided below.

[0176] [ka] TIFF0007635285000058.tif245164TIFF0007635285000059.tif220166

[0177] The above polypeptides can be paired with the light chain of DX2930 to form bispecific antibodies that are also within the scope of the present disclosure.

[0178] Other embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced with an alternative feature serving the same, equivalent or similar purpose. Thus, unless otherwise stated, each disclosed feature is only an example of a generic series of equivalent or similar features.

[0179] From the above description, those skilled in the art can easily ascertain the essential features of the present invention, and can make various changes and modifications to the present invention to suit various applications and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments also fall within the scope of the appended claims.

Claims

1. 1. A bispecific antibody comprising a first polypeptide comprising a light chain of a first antibody that binds to plasma kallikrein (pKal) and a second polypeptide comprising a heavy chain of said first antibody that binds to pKal and a second antibody that binds to factor XIIa (FXIIa), The first polypeptide comprises the sequence of SEQ ID NO: 46, and the second polypeptide comprises the sequence of any one of SEQ ID NOs: 51-122; Bispecific antibodies.

2. 1. An isolated nucleic acid or set of nucleic acids comprising a first nucleotide sequence encoding a first polypeptide described in claim 1 and a second nucleotide sequence encoding a second polypeptide described in claim 1.

3. 3. The nucleic acid or set of nucleic acids of claim 2, wherein the first nucleotide sequence and the second nucleotide sequence are located on two separate nucleic acid molecules.

4. 3. The nucleic acid or set of nucleic acids according to claim 2, wherein the first nucleotide sequence and the second nucleotide sequence are located on a single nucleic acid molecule.

5. 4. The nucleic acid or set of nucleic acids according to claim 3, which is a set of vectors comprising a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

6. 6. The nucleic acid or set of nucleic acids of claim 5, wherein the first vector and the second vector are expression vectors, and the first nucleotide sequence and the second nucleotide sequence are operably linked to a common promoter or separate promoters, respectively.

7. The nucleic acid or set of nucleic acids according to claim 4, which is a vector comprising both the first nucleotide sequence and the second nucleotide sequence.

8. 8. The nucleic acid or set of nucleic acids of claim 7, wherein the vector is an expression vector and the first nucleotide sequence and the second nucleotide sequence are each operably linked to a common promoter or separate promoters.

9. A host cell or a set of host cells comprising a nucleic acid or a set of nucleic acids according to any one of claims 2 to 8.

10. 1. A method for preparing a bispecific antibody, comprising: Culturing the host cell or set of host cells of claim 9 under conditions allowing expression of said first polypeptide and said second polypeptide; isolating a bispecific antibody comprising said first polypeptide and said second polypeptide; The method includes:

11. (i) a bispecific antibody according to claim 1, or a nucleic acid or a set of nucleic acids according to any one of claims 2 to 8, and (ii) a pharma- ceutically acceptable carrier; 23. A pharmaceutical composition comprising:

12. 1. A pharmaceutical composition for use in the treatment of a disease associated with the contact activation system, comprising: (i) a bispecific antibody according to claim 1, or a nucleic acid or a set of nucleic acids according to any one of claims 2 to 8, and (ii) a pharma- ceutically acceptable carrier; 23. A pharmaceutical composition comprising:

13. The pharmaceutical composition according to claim 12, wherein the disease associated with the contact activation system is hereditary angioedema.

14. The pharmaceutical composition according to claim 12, wherein the disease associated with the contact activation system is thrombosis.

Images