Bispecific heterodimeric diabodies and uses thereof

FIELD OF THE INVENTION

The present invention relates to bispecific heterodimeric diabodies and uses thereof in the treatment of cancer.

BACKGROUND OF THE INVENTION

Various strategies have been developed to generate bispecific molecules capable of T cell recruitment to mediate tumor cell killing. However, most efforts to produce such molecules have been limited because of inefficient production and poor stability properties. To address these problems, an Fv-derived strategy based on a covalently linked, bispecific heterodimeric diabody structure, also known as dual-affinity re-targeting (DART®) proteins, has been developed (US Patent Application Publications Nos. 2007/0004909, 2009/0060910, and 2010/0174053).

There remains a need for bispecific heterodimeric diabodies that overcome the challenges associated with the generation of bispecific antibodies and provide highly specific and potent agents that bind to tumor cells expressing a target antigen and recruit and activate T cells through CD3 activation, resulting in an innovative and effective treatment for cancer.

SUMMARY OF THE INVENTION

The present invention provides for bispecific heterodimeric diabodies, wherein the bispecific heterodimeric diabody is capable of specific binding to an epitope of P-cadherin and to an epitope of CD3, wherein the bispecific heterodimeric diabody comprises a first polypeptide chain and a second polypeptide chain, wherein: a. the first polypeptide comprises, in the N-terminal to C-terminal direction: i. a Domain 1, comprising a sub-Domain 1A and a sub-Domain 1B, and ii. a first heterodimer-promoting domain; and b. the second polypeptide chain comprises, in the N-terminal to C-terminal direction: i. a Domain 2, comprising a sub-Domain 2A and a sub-Domain 2B, and ii. a second heterodimer-promoting domain, and wherein sub-Domain 1A and sub-Domain 2A form a P-cadherin VL/VH binding domain comprising a variable heavy (VH) domain of an anti-P-cadherin antibody (P-CAD VH) and a variable light (VL) domain of an anti-P-cadherin antibody (P-CAD VL), and sub-Domain 1B and sub-Domain 2B form a CD3 VL/VH binding domain comprising a VL domain of an anti-CD3 antibody (CD3 VL) and a VH binding domain of an anti-CD3 antibody (CD3 VH); or wherein sub-Domain 1A and sub-Domain 2A form a CD3 VL/VH binding domain comprising a CD3 VL and a CD3 VH, and sub-Domain 1B and sub-Domain 2B form a P-cadherin VL/VH binding domain comprising a P-CAD VH and a P-CAD VL.

In another aspect of the invention, the sub-Domain 1A comprises a P-CAD VL or CD3 VL, and the sub-Domain 1B comprises a P-CAD VH, if the sub-Domain 1A comprises CD3 VL, or a CD3 VH, if the sub-Domain 1A comprises P-CAD VL; and wherein the sub-Domain 2B comprises a P-CAD VL or a CD3 VL depending on the VL domain selected for sub-Domain 1A, and the sub-Domain 2A comprises P-CAD VH, if the sub-Domain 2B comprises CD3 VL, or a CD3 VH, if the sub-Domain 2B comprises P-CAD VL.

In a particular aspect of the invention, the sub-Domain 1A comprises a P-CAD VL and the sub-Domain 1B comprises a CD3 VH, and the sub-Domain 2B comprises a CD3 VL and the sub-Domain 2A comprises a P-CAD VH; and wherein the P-CAD VL of the sub-Domain 1A and the P-CAD VH of the sub-Domain 2A form a VL/VH binding domain capable of specifically binding to an epitope of P-cadherin, and the CD3 VH of the sub-Domain 1B and the CD3 VL of the sub-Domain 2B form a VL/VH binding domain capable of specifically binding to an epitope of CD3.

In a particular aspect, sub-Domain 1A comprises a CD3 VL and the sub-Domain 1B comprises a P-CAD VH, and the sub-Domain 2B comprises a P-CAD VL and the sub-Domain 2A a CD3 VH; and wherein the CD3 VL of the sub-Domain 1A and the CD3 VH of the sub-Domain 2A form a VL/VH binding domain capable of specifically binding to an epitope of CD3, and the P-CAD VH of the sub-Domain 1B and the P-CAD VL of the sub-Domain 2B form a VL/VH binding domain capable of specifically binding to an epitope of P-cadherin.

In another aspect, the sub-Domain 1A comprises a VH binding domain of either an anti-P-cadherin antibody (P-CAD VH) or an anti-CD3 antibody (CD3 VH), and the sub-Domain 1B comprises a VL binding domain of either an anti-P-cadherin antibody (P-CAD VL), if the sub-Domain 1A comprises CD3 VH, or an anti-CD3 antibody (CD3 VL), if the sub-Domain 1A comprises a P-CAD VH; and wherein the sub-Domain 2B comprises a P-CAD VH or a CD3 VH depending on the VH domain selected for sub-Domain 1A, and the sub-Domain 2A comprises P-CAD VL, if the sub-Domain 2B comprises a CD3 VH, or a CD3 VL, if the sub-Domain 2B comprises a P-CAD VH.

In a particular aspect, the sub-Domain 1A comprises a P-CAD VH and the sub-Domain 1B comprises a CD3 VL, and the sub-Domain 2B comprises a CD3 VH and the sub-Domain 2A comprises a P-CAD VL; and wherein the P-CAD VH of the sub-Domain 1A and the P-CAD VL of the sub-Domain 2A form a VL/VH binding domain capable of specifically binding to an epitope of P-cadherin, and the CD3 VL of the sub-Domain 1B and the CD3 VH of the sub-Domain 2B form a VL/VH binding domain capable of specifically binding to an epitope of CD3.

In a particular aspect, the sub-Domain 1A comprises a CD3 VH and the sub-Domain 1B comprises a P-CAD VL, and the sub-Domain 2B comprises a P-CAD VH and the sub-Domain 2A comprises a CD3 VL; and wherein the CD3 VH of the sub-Domain 1A and the CD3 VL of the sub-Domain 2A form a VL/VH binding domain capable of specifically binding to an epitope of CD3, and the P-CAD VL of the sub-Domain 1B and the P-CAD VH of the sub-Domain 2B form a VL/VH binding domain capable of specifically binding to an epitope of P-cadherin.

The present invention further provides for bispecific heterodimeric diabodies wherein the first heterodimer-promoting domain and the second heterodimer-promoting domain comprise an IgG Fc region comprising a CH2 and a CH3 domain, wherein the amino acid sequence of the CH2 domain and/or the CH3 domain comprises at least one amino acid modification, as compared to a wild-type IgG Fc region, to form a knob or a hole. In one aspect, the first heterodimer-promoting domain and the second heterodimer-promoting domain are not both knobs or both holes; and/or wherein the first heterodimer-promoting domain and the second heterodimer-promoting domain form an IgG immunoglobulin Fc region. In another aspect, the IgG Fc region forming the knob comprises a sequence of SEQ ID NO: 63, and the IgG Fc region forming the hole comprises a sequence of SEQ ID NO: 64.

The present invention further provides for bispecific heterodimeric diabodies, wherein the first heterodimer-promoting domain and the second heterodimer-promoting domain comprise an E-coil region comprising glutamic acid and a negatively charged alpha-helical coil or a K-coil region comprising lysine and a positively charged helical coil; and wherein the first heterodimer-promoting domain and the second heterodimer-promoting domain are not both E-coil regions or both K-coil regions. In one aspect, the E-coil region comprises a sequence of SEQ ID NO: 61, and/or the K-coil region comprises a sequence of SEQ ID NO: 62.

The present invention further provides for bispecific heterodimeric diabodies, wherein the sub-Domain 1A and the sub-Domain 1B may be linked by a glycine-serine linker (Linker 1) and do not associate to form a VL/VH epitope binding domain, and the sub-Domain 2B and the sub-Domain 2A may be linked by a glycine-serine linker (Linker 1) and do not associate to form a VL/VH epitope binding domain. In one aspect of the invention, the glycine-serine linker (Linker 1) comprises a sequence of SEQ ID NO: 68 or SEQ ID NO: 69.

The present invention further provides for bispecific heterodimeric diabodies, wherein the first heterodimer-promoting domain comprises a cysteine linker (Linker 2) on sub-Domain 1B and/or the second heterodimer-promoting domain comprises a cysteine linker (Linker 2) on sub-Domain 2A. In one aspect, the Linker 2 of the first heterodimer-promoting domain and/or the second heterodimer-promoting domain further comprises at least one glycine residue. In another aspect, the Linker 2 of the first heterodimer-promoting domain and/or the second heterodimer-promoting domain comprises a sequence of GFNRGEC (SEQ ID NO: 70), GVEPKSC (SEQ ID NO: 71), GGCGGG (SEQ ID NO: 72), GCPPCP (SEQ ID NO: 73), GGTGGCPPCP (SEQ ID NO: 74), GEPKSSDKTHTCPPCP (SEQ ID NO: 75) or GGTGGGEPKSSDKTHTCPPCP (SEQ ID NO: 76). In another aspect, the Linker 2 of the first heterodimer-promoting domain comprises the sequence of GCPPCP (SEQ ID NO: 73), GGTGGCPPCP (SEQ ID NO: 74), GEPKSSDKTHTCPPCP (SEQ ID NO: 75) or GGTGGGEPKSSDKTHTCPPCP (SEQ ID NO: 76), and the Linker 2 of the second heterodimer-promoting domain comprises the sequence of GCPPCP (SEQ ID NO: 73), GGTGGCPPCP (SEQ ID NO: 74), GEPKSSDKTHTCPPCP (SEQ ID NO: 75) or GGTGGGEPKSSDKTHTCPPCP (SEQ ID NO: 76). In another aspect, the Linker 2 of the first heterodimer-promoting domain comprises the sequence of GGCGGG (SEQ ID NO: 72), and the Linker 2 of the second heterodimer-promoting domain comprises the sequence of GGCGGG (SEQ ID NO: 72). In another aspect, the Linker 2 of the first heterodimer-promoting domain comprises the sequence of GFNRGEC (SEQ ID NO: 70) and the Linker 2 of the second heterodimer-promoting domain comprises the sequence of GVEPKSC (SEQ ID NO: 71), or the Linker 2 of the first heterodimer-promoting domain comprises the sequence of GVEPKSC (SEQ ID NO: 71) and the Linker 2 of the second heterodimer-promoting domain comprises the sequence of GFNRGEC (SEQ ID NO: 70).

The present invention provides for bispecific heterodimeric diabodies, wherein the bispecific heterodimeric diabodies specifically bind to extracellular domain 3 (ECD3) of human P-cadherin. The present invention further provides for bispecific heterodimeric diabodies, wherein the bispecific heterodimeric diabody specifically bind to an epitope on P-cadherin but does not bind to an epitope on E-cadherin or VE-cadherin. The present invention also provides for bispecific heterodimeric diabodies, wherein the bispecific heterodimeric diabodies specifically bind to an epitope on human P-cadherin but do not bind to an epitope on mouse P-cadherin.

The present invention provides for bispecific heterodimeric diabodies, wherein the bispecific heterodimeric diabodies demonstrate an extended serum and tumor half-life. Pharmacokinetic analysis may conducted by various assays, such as ELISA. The present invention further provides for bispecific heterodimeric diabodies, wherein the bispecific heterodimeric diabodies demonstrate a lower EC50 in the presence of increased P-cadherin expression levels or increased receptor density levels. The EC50 may be determined by various in vitro and in vivo cytotoxicity assays

The present invention provides for bispecific heterodimeric diabodies, comprising a P-CAD VL CDR1, a P-CAD VL CDR2, and a P-CAD VL CDR3 of a P-CAD VL comprising a sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23; a CD3 VH CDR1, a CD3 VH CDR2, and a CD3 VH CDR3 of a CD3 VH comprising a sequence of SEQ ID NOS: 45 or 46; a CD3 VL CDR1, a CD3 VL CDR2, and a CD3 VL CDR3 of a CD3 VL comprising a sequence of SEQ ID NO: 47; and/or a P-CAD VH CDR1, a P-CAD VH CDR2, and a P-CAD VH CDR3 of a P-CAD VH comprising a sequence of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24.

The present invention provides for bispecific heterodimeric diabodies, comprising the P-CAD VL CDR1 comprises a sequence of SEQ ID NOS: 35 or 36, the P-CAD VL CDR2 comprises a sequence of SEQ ID NOS: 37 or 38, and the P-CAD VL CDR3 comprises a sequence of SEQ ID NOS: 39, 40, 41, 42, 43 or 44; the CD3 VH CDR1 comprises a sequence of SEQ ID NO: 48, 52 or 53, the CD3 VH CDR2 comprises a sequence of SEQ ID NOS: 49, 50 or 54 and the CD3 VH CDR3 comprises a sequence of SEQ ID NO: 51; the CD3 VL CDR1 comprises a sequence of SEQ ID NO: 55, the CD3 VL CDR2 comprises a sequence of SEQ ID NO: 56, and the CD3 VL CDR3 comprises a sequence of SEQ ID NO: 57; and/or the P-CAD VH CDR1 comprises a sequence of SEQ ID NOS: 25 or 33, the P-CAD VH CDR2 comprises a sequence of SEQ ID NOS: 26 or 34, and a P-CAD VH CDR3 comprising a sequence of SEQ ID NOS: 27, 28, 29, 30, 31 or 32.

In one aspect of the present invention, the P-CAD VL CDR1 comprises the sequence of SEQ ID NO: 35, the P-CAD VL CDR2 comprises the sequence of SEQ ID NO: 37, the P-CAD VL CDR3 comprises the sequence of SEQ ID NO: 41, the P-CAD VH CDR1 comprises the sequence of SEQ ID NO: 25, the P-CAD VH CDR2 comprises the sequence of SEQ ID NO: 26, and the P-CAD VH CDR3 comprises the sequence of SEQ ID NO: 28. In another aspect, the P-CAD VL CDR1 comprises the sequence of SEQ ID NO: 35; the P-CAD VL CDR2 comprises the sequence of SEQ ID NO: 37; and the P-CAD VL CDR3 comprises the sequence of SEQ ID NO: 42, the P-CAD VH CDR1 comprises the sequence of SEQ ID NO: 25, the P-CAD VH CDR2 comprises the sequence of SEQ ID NO: 26, and the P-CAD VH CDR3 comprises the sequence of SEQ ID NO: 29. In another aspect, the P-CAD VL CDR1 comprises the sequence of SEQ ID NO: 35; the P-CAD VL CDR2 comprises the sequence of SEQ ID NO: 37, the P-CAD VL CDR3 comprises the sequence of SEQ ID NO: 43, the P-CAD VH CDR1 comprises the sequence of SEQ ID NO: 25, the P-CAD VH CDR2 comprises the sequence of SEQ ID NO: 26, and the P-CAD VH CDR3 comprises the sequence of SEQ ID NO: 30. In another aspect, the P-CAD VL CDR1 comprises the sequence of SEQ ID NO: 35; the P-CAD VL CDR2 comprises the sequence of SEQ ID NO: 37, the P-CAD VL CDR3 comprises the sequence of SEQ ID NO: 39, the P-CAD VH CDR1 comprises the sequence of SEQ ID NO: 25, the P-CAD VH CDR2 comprises the sequence of SEQ ID NO: 26, and the P-CAD VH CDR3 comprises the sequence of SEQ ID NO: 31.

The present invention further provides for bispecific heterodimeric diabodies, comprising a P-CAD VL comprising a sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23; a CD3 VH comprising a sequence of SEQ ID NOS: 45 or 46; a CD3 VL comprising the sequence of SEQ ID NO: 47; and a P-CAD VH comprising a sequence of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24. In one aspect, the P-CAD VL comprises the sequence of SEQ ID NO: 5, and the P-CAD VH comprises the sequence of SEQ ID NO: 6. In another aspect, the P-CAD VL comprises the sequence of SEQ ID NO: 7, and the P-CAD VH comprises the sequence of SEQ ID NO: 8. In another aspect, the P-CAD VL comprises the sequence of SEQ ID NO: 9 and the P-CAD VH comprises the sequence of SEQ ID NO: 10. In another aspect, the P-CAD VL comprises the sequence of SEQ ID NO: 15 and the P-CAD VH comprises the sequence of SEQ ID NO: 16.

The present invention further provides for bispecific heterodimeric diabodies wherein the first and second polypeptide chains may be covalently bonded to one another by at least one disulfide bond.

The present invention provides for bispecific heterodimeric diabodies, wherein the bispecific heterodimeric diabodies may be conjugated to a detectable label, including but not limited to, fluorophore or a radionuclide.

The present invention provides for bispecific heterodimeric diabodies that bind to P-cadherin and compete for binding to bispecific heterodimeric diabodies disclosed herein.

The present invention further provides for a pharmaceutical composition comprising a therapeutically effective amount of a bispecific heterodimeric diabody disclosed herein and a pharmaceutically acceptable carrier.

The present invention further provides for a method of treating P-cadherin associated disorders in a patient in need thereof, comprising administering to the patient a bispecific heterodimeric diabody disclosed herein or a pharmaceutical composition comprising a bispecific heterodimeric diabody disclosed herein. The present invention further provides for a method of treating a P-cadherin associated disorders in a patient in need thereof, comprising administering to the patient the bispecific heterodimeric disclosed herein or a pharmaceutical composition comprising a bispecific heterodimeric diabody disclosed herein, wherein a cytolytic T cell response is activated or induced.

The present invention further provides bispecific heterodimeric diabodies disclosed herein for use in therapy. The present invention further provides the use of bispecific heterodimeric diabodies disclosed herein in the manufacture of a medicament for use in therapy. In another aspect, the therapy is treatment of P-cadherin associated disorder. The present invention further provides bispecific heterodimeric diabodies disclosed herein for use in therapy, wherein the therapy activates or induces a cytolytic T cell response.

In one aspect, the P-cadherin associated disorder is cancer. In another aspect, the cancer is a P-cadherin expressing or P cadherin positive cancer. The cancers include, but are not limited to, breast, colorectal, ovarian, gastric, thyroid, prostate, cervical, pancreatic, lung, bladder, liver, endometrial, head and neck, testicular, and glioblastoma cancer.

The present invention further provides isolated host cells that recombinantly produce bispecific heterodimeric diabodies described herein, isolated polynucleotides comprising nucleotide sequences encoding the bispecific heterodimeric diabodies disclosed herein, and vectors comprising the polynucleotides.

The present invention further provides methods of producing bispecific heterodimeric diabodies disclosed herein, comprising culturing the host cells disclosed herein under conditions that result in production of the bispecific heterodimeric diabodies, and purifying the bispecific heterodimeric diabodies from the culture supernatant.

The present invention further provides for bispecific heterodimeric diabodies disclosed herein, wherein a crystal structure assembles into a compact spherical structure stabilized by a disulfide linkage between a pair of cysteine residues consisting of a cysteine residue at position 239 (Cys²³⁹) of sub-Domain 1B and a cysteine residue at position 246 (Cys²⁴⁶) of sub-Domain 2A; and, wherein the crystal diffracts X-rays for determination of atomic coordinates to provide resolution of better than about 2.0 Angstroms.

The present invention further provides for bispecific heterodimeric diabodies capable of specific binding to an epitope of P-cadherin and to an epitope of CD3 comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises, in the N-terminal to C-terminal direction: a Domain 1, comprising a sub-Domain 1A which comprises a VL binding domain of an anti-CD3 antibody (CD3 VL) comprising a sequence of SEQ ID NO: 47, and a sub-Domain 1B which comprises a VH binding domain of either an anti-P-cadherin antibody (P-CAD VH) comprising a sequence of SEQ ID NO: 6, wherein the sub-Domain 1A and sub-Domain 1B may be covalently linked by a glycine-serine linker (Linker 1) and do not associate to form a VL/VH epitope binding domain; and the second polypeptide chain comprises, in the N-terminal to C-terminal direction: a Domain 2, comprising a sub-Domain 2B which comprises a P-CAD VL comprising a sequence of SEQ ID NO: 5, and a sub-Domain 2A which comprises a CD3 VH comprising a sequence of SEQ ID NO: 45, wherein the sub-Domain 2B and sub-Domain 2A may be covalently linked by a glycine-serine linker (Linker 1) and do not associate to form a VL/VH epitope binding domain; and wherein a crystal structure assembles into a compact spherical structure stabilized by a disulfide linkage between a pair of cysteine residues comprising a cysteine residue at position 239 (Cys²³⁹) of sub-Domain 1B and a cysteine residue at position 246 (Cys²⁴⁶) of sub-Domain 2A; and, wherein the crystal diffracts X-rays for determination of atomic coordinates to provide resolution of better than about 2.0 Angstroms.

In another aspect, the bispecific heterodimeric diabodies described herein contain two antigen binding sites separated from each other by about 40 Angstroms and the binding sites may be located on the orthogonally opposite sides of the bispecific heterodimeric diabody.

The present invention further provides methods of identifying additional locations for cysteine residues to form a disulfide linkage within bispecific heterodimeric diabodies based on the crystal structures disclosed herein, wherein the crystal structure is analyzed for a pair of amino acid residue locations for substitution with a pair of cysteine residues that form a disulfide linkage. In a particular aspect of the present invention, the pair of amino acid residue locations for substitution with a pair of cysteine residues that form a disulfide linkage may be selected from the group consisting of amino acid locations Gln¹²¹(VH1B)Gly¹⁶⁰(VL1A), Val¹²⁹(VH1B)Gly²⁴⁴(VL1A), Val¹²³(VH1B)Gly¹⁶⁰(VL1A), Gly¹²⁸(VH1B)Ser²⁴²(VL1A), and Ala¹²⁷(VH1B)Ser²⁴²(VL1A). The disulfide linkage may reduce solvent accessibility and reduce the length of linkers.

The present invention further provides methods of engineering bispecific heterodimeric diabody variants in an attempt to form a more stable interdomain association, wherein the engineering is through amino acid site-directed mutagenesis in the interdomain interface based on the crystal structure disclosed herein, wherein the amino acid site-directed mutagenesis fills up large interior voids/holes. In a particular aspect of the present invention, the site-directed mutagenesis may increase amino acid side chain volumes by replacing small amino acids for amino acids with bulky aromatic side-chains. In another aspect of the invention, the small amino acids may be selected from the group consisting of amino acids at positions Ala⁴⁴(VL1A), Val²¹³(VH2A), Leu²³⁸(VH2A), or Met²³¹(VL2B). In another aspect of the invention, the amino acids with bulky aromatic side-chains may be selected from the group consisting of phenylalanine, tyrosine, or tryptophan.

The present invention further provides for antibodies that binds to P-cadherin, comprising a CDR1, a CDR2, and a CDR3 of a light chain variable region (VL) comprising the sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23 and/or a CDR1, a CDR2, and a CDR3 of a heavy chain variable region (VH) comprising the sequence of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24.

In another aspect of the invention, the antibodies disclosed herein comprise a VL CDR1 comprising the sequence of SEQ ID NO: 35 or 36, a VLCDR2 comprising the sequence of SEQ ID NO: 37 or 38, a VL CDR3 comprising the sequence of SEQ ID NO: 39, 40, 41, 42, 43 or 44, a VH CDR1 comprising the sequence of SEQ ID NO: 25 or 33, a VH CDR2 comprising the sequence of SEQ ID NO: 26 or 34, and a VH CDR3 comprising the sequence of SEQ ID NO: 27, 28, 29, 30, 31 or 32.

In another aspect of the invention, the antibodies disclosed herein comprise a VL CDR1 comprising the sequence of SEQ ID NO: 35 or 36, a VL CDR2 comprising the sequence of SEQ ID NO: 37 or 38, a VL CDR3 comprising the sequence of SEQ ID NO: 39, 40, 41, 42, 43 or 44, a VH CDR1 comprising the sequence of SEQ ID NO: 25, a VH CDR2 comprising the sequence of SEQ ID NO: 26, and a VH CDR3 comprising the sequence of SEQ ID NO: 27, 28, 29, 30, 31 or 32.

In another aspect of the invention, the antibodies disclosed herein comprise a VL CDR1 comprising the sequence of SEQ ID NO: 35, a VL CDR2 comprising the sequence of SEQ ID NO: 37, a VL CDR3 comprising the sequence of SEQ ID NO: 42, a VH CDR1 comprising the sequence of SEQ ID NO: 25, a VH CDR2 comprising the sequence of SEQ ID NO: 26, and a VH CDR3 comprising the sequence of SEQ ID NO: 29.

The present invention further provides for antibodies that binds to P-cadherin, comprising a light chain variable region comprising the sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23, and/or a heavy chain variable region comprising the sequence of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24. In another aspect of the invention, the antibodies disclosed herein comprise a light chain variable region amino acid comprising the sequence of SEQ ID NO: 5, and/or a heavy chain variable region comprising the sequence of SEQ ID NO: 6. In another aspect of the invention, the antibodies disclosed herein comprise a light chain variable region comprising the sequence of SEQ ID NO: 7, and/or a heavy chain variable region comprising the sequence of SEQ ID NO: 8. In another aspect of the invention, the antibodies disclosed herein comprise a light chain variable region comprising the sequence of SEQ ID NO: 9 and/or a heavy chain variable region comprising the sequence of SEQ ID NO: 10. In another aspect of the invention, the antibodies disclosed herein comprise a light chain variable region comprising the sequence of SEQ ID NO: 15 and/or a heavy chain variable region comprising the sequence of SEQ ID NO: 16.

The present invention further provides for antibodies that bind to P-cadherin and compete for binding to P-cadherin with antibodies disclosed herein.

The present invention further provides for pharmaceutical compositions comprising a therapeutically effective amount of an antibody disclosed herein and a pharmaceutically acceptable carrier.

The present invention further provides a method of treating P-cadherin associated disorder in a patient in need thereof, comprising administering to the patient an antibody disclosed herein or a pharmaceutical composition comprising an antibody disclosed herein. The present invention further provides antibodies disclosed herein for use in therapy. The present invention further provides the use of antibodies disclosed herein in the manufacture of a medicament for use in therapy.

The present invention further provides nucleic acids that encode the antibodies disclosed herein. In another aspect of the invention, the nucleic acids may comprise a sequence that is selected from the group consisting of SEQ ID NOS: 98, 100, 102, 104, 106, 108, 110, 112, 114 and 116. In a further aspect of the invention, the nucleic acids may comprise a sequence that is selected from the group consisting of SEQ ID NOS: 97, 99, 101, 103, 105, 107, 109, 111, 113 and 115. The present invention further provides vectors comprising nucleic acids disclosed herein and host cells comprising vectors disclosed herein.

The present invention further provides for bispecific heterodimeric diabodies or antibodies disclosed herein, wherein the P-CAD VL comprises a P-CAD VL CDR1 sequence comprising X_L1.1-X_L1.2-X_L1.3-X_L1.4-XL_1.5-X_L1.6-X_L1.7-X_L1.8-X_L1.9-X_L1.10-X_L1.11-X_L1.2-X_L1.13, wherein X_L1.1to X_L1.13each independently is an amino acid residue according to column 3 of Table 57, Table 58, or Table 59; a P-CAD VL CDR2 sequence comprising X_L2.1-X_L2.2-X_L2.3-X_L2.4-X_L2.5-X_L2.6-X_L2.7, wherein X_L2.1to X_L2.7each independently is an amino acid residue according to column 3 of Table 60, Table 61, or Table 62; and a P-CAD VL CDR3 sequence comprising X_L3.1-X_L3.2-X_L3.3-X_L3.4-X_L3.5-X_L3.6-X_L3.7-X_L3.8-X_L3.9-X_L3.10-X_L3.11, wherein X_L3.1to X_L3.11each independently is an amino acid residue according to column 3 of Table 63, Table 64, or Table 65; and the P-CAD VH comprises: a P-CAD VH CDR1 sequence comprising X_H1.1-X_H1.2-X_H1.3-X_H1.4-X_H1.5-X_H1.6-X_H1.7-X_H1.8-X_H1.9-X_H1.10or X_H1.5, X_H1.6-X_H1.7-X_H1.8-X_H1.9-X_H1.10, wherein X_H1.1to X_H1.10each independently is an amino acid residue according to column 3 of Table 48, Table 49, or Table 50; a P-CAD VH CDR2 sequence comprising X_H2.1-X_H2.2-X_H2.3-X_H2.4-X_H2.5-X_H2.6-X_H2.7-X_H2.8-X_H2.9-X_H2.10-X_H2.11-X_H2.12-X_H2.13-X_H2.14-X_H2.15-X_H2.16-X_H2.17or X_H2.1-X_H2.2-X_H2.3-X_H2.4-X_H2.5-X_H2.6-X_H2.7-X_H2.8-X_H2.9-X_H2.10, wherein X_H2.1to X_H2.17each independently is an amino acid residue according to column 3 of Table 51, Table 52, or Table 53; and a P-CAD VH CDR3 sequence comprising X_H3.1-X_H3.2-X_H3.3-X_H3.4-X_H3.5-X_H3.6-X_H3.7-X_H3.8-X_H3.9, wherein X_H3.1to X_H3.9each independently is an amino acid residue according to column 3 of Table 54, Table 55, or Table 56.

In one aspect, X_L1.1to X_L1.13each independently is an amino acid residue according to column 3 of Table 57. In another aspect, X_L1.1to X_L1.13each independently is an amino acid residue according to column 3 of Table 58. In another aspect, X_L1.1to X_L1.13each independently is an amino acid residue according to column 3 of Table 59. In another aspect, X_L2.1to X_L2.7each independently is an amino acid residue according to column 3 of Table 60. In another aspect, X_L2.1to X_L2.7each independently is an amino acid residue according to column 3 of Table 61. In another aspect, X_L2.1to X_L2.7each independently is an amino acid residue according to column 3 of, or Table 62. In another aspect, X_L3.1to X_L3.11each independently is an amino acid residue according to column 3 of Table 63. In another aspect, X_L3.1to X_L3.11each independently is an amino acid residue according to column 3 of Table 64. In another aspect, X_L3.1to X_L3.11each independently is an amino acid residue according to column 3 of or Table 65.

In another aspect, X_H1.1to X_H1.10each independently is an amino acid residue according to column 3 of Table 48. In another aspect, X_H1.1to X_H1.10each independently is an amino acid residue according to column 3 of Table 49. In another aspect, X_H1.1to X_H1.10each independently is an amino acid residue according to column 3 of Table 50. In another aspect, X_H2.1to X_H2.17each independently is an amino acid residue according to column 3 of Table 51. In another aspect, X_H2.1to X_H2.17each independently is an amino acid residue according to column 3 of Table 52. In another aspect, X_H2.1to X_H2.17each independently is an amino acid residue according to column 3 of Table 53. In another aspect, X_H3.1to X_H3.9each independently is an amino acid residue according to column 3 of Table 54. In another aspect, X_H3.1to X_H3.9each independently is an amino acid residue according to column 3 of Table 55. In another aspect, X_H3.1to X_H3.9each independently is an amino acid residue according to column 3 of Table 56.

The present invention further provides for bispecific heterodimeric diabodies or antibodies disclosed herein, wherein the P-CAD VL comprises a P-CAD VL CDR1 sequence comprising X_L1.1-X_L1.2-X_L1.3-X_L1.4-X_L1.5-X_L1.6-X_L1.7-X_L1.8-X_L1.9-X_L1.10-X_L1.11-X_L1.2-X_L1.13, wherein X_L1.1to X_L1.13each independently is an amino acid residue according to column 3 or column 4 of Table 69; a P-CAD VL CDR2 sequence comprising X_L2.1-X_L2.2-X_L2.3-X_L2.4-X_L2.5-X_L2.6-X_L2.7, wherein X_L2.1to X_L2.7each independently is an amino acid residue according to column 3 or column 4 of Table 70; and a P-CAD VL CDR3 sequence comprising X_L3.1-X_L3.2-X_L3.3-X_L3.4-X_L3.5-X_L3.6-X_L3.7-X_L3.8-X_L3.9-X_L3.10-X_L3.11, wherein X_L3.1to X_L3.11each independently is an amino acid residue according to column 3 or column 4 of Table 71; and the P-CAD VH comprises a P-CAD VH CDR1 sequence comprising X_H1.1-X_H1.2-X_H1.3-X_H1.4-X_H1.5-X_H1.6-X_H1.7-X_H1.8-X_H1.9-X_H1.10or X^H1.5, X_H1.6-X_H1.7-X_H1.8-X_H1.9-X_H1.10, wherein X_H1.1to X_H1.10each independently is an amino acid residue according to column 3 or column 4 of Table 66; a P-CAD VH CDR2 sequence comprising X_H2.1-X_H2.2-X_H2.3-X_H2.4-X_H2.5-X_H2.6-X_H2.7-X_H2.8-X_H2.9-X_H2.10-X_H2.11-X_H2.12-X_H2.13-X_H2.14-X_H2.15-X_H2.16-X_H2.17or X_H2.1-X_H2.2-X_H2.3-X_H2.4-X_H2.5-X_H2.6-X_H2.7-X_H2.8-X_H2.9-X_H2.10, wherein X_H2.1to X_H2.17each independently is an amino acid residue according to column 3 or column 4 of Table 67, and a P-CAD VH CDR3 sequence comprising X_H3.1-X_H3.2-X_H3.3-X_H3.4-X_H3.5-X_H3.6-X_H3.7-X_H3.8-X_H3.9, wherein X_H3.1to X_H3.9each independently is an amino acid residue according to column 3 or column 4 of Table 68.

In one aspect, X_L1.1to X_L1.13each independently is an amino acid residue according to column 3 of Table 69. In another aspect, X_L1.1to X_L1.13each independently is an amino acid residue according to column 4 of Table 69. In another aspect, X_L2.1to X_L2.7each independently is an amino acid residue according to column 3 of Table 70. In another aspect, X_L2.1to X_L2.7each independently is an amino acid residue according to column 4 of Table 70. In another aspect, X_L3.1to X_L3.11each independently is an amino acid residue according to column 3 of Table 71. In another aspect, X_L3.1to X_L3.11each independently is an amino acid residue according to column 4 of Table 71. In another aspect, X_H1.1to X_H1.10each independently is an amino acid residue according to column 3 of Table 66. In another aspect, X_H1.1to X_H1.10each independently is an amino acid residue according to column 4 of Table 66. In another aspect, X_H2.1to X_H2.17each independently is an amino acid residue according to column 3 of Table 67. In another aspect, X_H2.1to X_H2.17each independently is an amino acid residue according to column 4 of Table 67. In another aspect, X_H3.1to X_H3.9each independently is an amino acid residue according to column 3 of Table 68. In another aspect, X_H3.1to X_H3.9each independently is an amino acid residue according to column 4 of Table 68.

Further, the present invention provides for X_H1.8is G, X_H2.5is Y, X_H3.1is I, X_H3.7is F, X_L3.3is W, X_H2.6is N, X_H2.16is Q, X_H3.5is N, X_H3.9is I, X_L1.8is G, X_L2.2is N, X_L2.3is N, X_L3.2is T, and/or X_L3.4is D. The present invention also provides for any combination of the aspects described herein.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B provide schematics of four alternative representations of LP-DART P-cadherin/CD3 bispecific heterodimeric diabodies having a first heterodimer-promoting domain and second heterodimer-promoting domain comprising an Fc region optimized to associate via a “knob-in-hole” association.

FIG. 2 provides a schematic representation of a VF-DART bispecific heterodimeric diabody that simultaneously targets and activates T cells (via CD3 antigen) and tumor cells (via P-cadherin antigen) having a first polypeptide chain (1) wherein a first heterodimer-promoting domain comprises a C-terminal peptide that forms a disulfide bond for stability with a C-terminal peptide of a second heterodimer-promoting domain of a second polypeptide chain (2) via a cysteine linker (Linker 2) having sequences, for example, GFNRGEC (SEQ ID NO: 70) or GVEPKSC (SEQ ID NO 71). The polypeptide chains of the bispecific heterodimeric diabodies may comprise a glycine-serine linker (Linker 1) having a sequence GGGSGGGG (SEQ ID NO: 68) as depicted herein.

FIG. 3 provides a schematic representation of an assembled VF-DART bispecific heterodimeric diabody having a first polypeptide chain (1) and second polypeptide chain (2) wherein a first heterodimer-promoting domain (such as SEQ ID NO: 70 or SEQ ID NO: 71, described in detail below) and a second heterodimer-promoting domain (such as SEQ ID NO: 71 or SEQ ID NO: 70, depending on the selection of the first heterodimer promoting domain) comprise a cysteine residue.

FIG. 4 provides a schematic representation of an assembled EK-DART bispecific heterodimeric diabody having a first polypeptide chain (1) wherein a first heterodimer-promoting domain comprises a glutamic acid-rich region that forms a negatively charged alpha-helical coil (E-coil), and a second polypeptide chain (2) wherein a second heterodimer-promoting domain comprises a lysine rich region that forms a positively charged helical coil (K-coil).

FIG. 5 provides a schematic representation of an assembled LP-DART bispecific heterodimeric diabody having a first heterodimer-promoting domain and a second heterodimer-promoting domain comprising an Fc region optimized to associate via a “knob-in-hole” association.

FIG. 6 provides the structures of an alternative bispecific heterodimeric diabody having an Fc domain termed MP3-DART. The bispecific heterodimeric diabody is attached to the N-terminal side of the Fc domain and is thus termed “N-terminal MP3-DART.” The N-terminal MP3-DART comprises a first, second and third polypeptide chain which are schematically represented. The final structure of the N-terminal MP3-DART is represented.

FIG. 7 provides the structures of an alternative bispecific heterodimeric diabody having an Fc domain termed MP3-DART. The bispecific heterodimeric diabody is attached to the C-terminal side of the Fc domain and is thus termed “C-terminal MP3 DART.” The C-terminal MP3-DART comprises a first, second and third polypeptide chain which are schematically represented. The final structure of the C-terminal MP3-DART is represented.

FIG. 8 provides a linear regression analysis comparing cytotoxic T lymphocyte (CTL) activity to relative cell surface P-cadherin expression.

FIG. 9 demonstrates the in vivo ability of 177 EK-DART to decrease tumor volume in murine HCT116 colorectal cancer model.

FIG. 10 demonstrates the in vivo ability of 153 EK-DART to decrease tumor volume in murine HCT116 colorectal cancer model.

FIG. 11 demonstrates the in vivo ability of 154 EK-DART to decrease tumor volume in murine HCT116 colorectal cancer model.

FIG. 12 demonstrates the in vivo ability of 35 EK-DART to decrease tumor volume in murine HCT116 colorectal cancer model.

FIG. 13 demonstrates the in vivo ability of 153 LP-DART to decrease tumor volume in murine HCT116 colorectal cancer model.

FIG. 14 demonstrates the in vivo ability of 153 LP-DART to decrease tumor volume in murine HCT116 colorectal cancer model.

FIG. 15 demonstrates the in vivo ability of 153 LP-DART to decrease tumor volume in murine Du145 prostate cancer model.

FIG. 16 demonstrates the in vivo ability of 153 LP-DART to decrease tumor volume in murine H1650 lung cancer model.

FIGS. 17A-17D demonstrate the in vivo ability of 153 LP-DART to decrease tumor volume in an established tumor+engrafted human T cell model, 17E demonstrates a one way ANOVA analysis of the tumor volume.

FIGS. 18A and 18B demonstrate the in vivo ability of 153 LP-DART to decrease tumor volume in an established tumor human T cell adoptive transfer model.

FIG. 19 demonstrates the in vivo ability of 153 LP-DART to decrease tumor volume in a P-cadherin positive patient derived colorectal tumor xenograft,

FIG. 20 shows a 153 LP-DART mediated dose dependent increase in tumor infiltrating lymphocytes (TILS).

FIGS. 21A-21D provide in vitro assays to evaluate the properties of fluorophore Labeled 153 LP-DART and T cells.

FIGS. 22A-22D provide biodistribution and targeting of 153 LP-DART in a HCT116 xenograft model using longitudinal FMT imaging.

FIGS. 23A-23C show the pharmacokinetic data (ELISA) from the FMT imaging study.

FIG. 24A shows the evaluation of T cell activity of fluorphore labeled and unlabeled T cells and 24B shows the cellular tracking kinetics of fluorophore labeled T cells in a HCT116 tumor model using FMT imaging (in vivo).

FIG. 25 provides a schematic representation of a DART construct designed for crystallography, having a C-terminal His tag designated “6×HIS” covalently linked to the P-CAD VH domain and a C-terminal FLAG designated “FLAG” covalently linked to the CD3 VH domain.

FIG. 26 provides a graphical depiction of the crystal structure of the crystallography 35 DART protein.

FIG. 27 provides a graphical depiction of the crystallography 35 DART protein showing the antigen binding sites for the anti-CD3 CDR regions and the anti-P-CAD CDR regions.

FIG. 28 provides a schematic of a crystallography 35 DART protein variant with a more stable interdomain association through site-directed mutagenesis in the interdomain interface containing large interior voids/holes that may be filled up with increasing side chain volumes with various amino acids.

FIG. 29 provides a graphical depiction of the crystal structure of a crystallography 35 DART protein with various disulfide bond locations (Cys-Cys sites) to reduce solvent accessibility of the disulfide bond, as well as, to reduce the length of linkers.

FIGS. 30A and 30B provide amino acid sequences for first and second polypeptide chains of VF-DART bispecific heterodimeric diabodies of the present invention.

FIGS. 31A and 31B provide amino acid sequences for first and second polypeptide chains of EK-DART bispecific heterodimeric diabodies of the present invention.

FIGS. 32A and 32B provide amino acid sequences for first and second polypeptide chains of LP-DART bispecific heterodimeric diabodies of the present invention.

FIGS. 33A and 33B provide amino acid sequences for first and second polypeptide chains of DART bispecific heterodimeric diabodies of the present invention.

FIG. 34 provides P-cadherin-extra cellular domain (ECD)-Fc fusion protein constructs for eptitope mapping of P-cadherin 153 LP-DART.

FIG. 35 provides the full length P-Cadherin epitope sequence (UniProt P22223, CADH3, Human Cadherin-3).

DETAILED DESCRIPTION

The present invention describes novel bispecific heterodimeric diabodies comprising an antigen binding domain that specifically binds P-cadherin, and an antigen binding domain that specifically binds CD3 for the recruitment of and activation of cytolytic T cells. The bispecific heterodimeric diabodies may further comprise a Fc domain. Moreover, it is demonstrated herein that the bispecific heterodimeric diabodies successfully direct and activate T cell cytotoxicity to tumor cells expressing P-cadherin. Further, the bispecific heterodimeric diabodies of the present invention have enhanced pharmacokinetic properties to extend in vivo half-life, and were designed to engage and activate polyclonal T cell populations via the CD3 complex in the presence of P-cadherin expressing tumors.

The present invention describes the isolation and characterization of fully human single-chain antibody binding domains against P-cadherin derived from human phage display libraries. The invention further provides covalently linking these P-cadherin binding domains to antigen binding domains of anti-CD3 antibodies in the previously described DART format (Moore et al., Blood, 117(17): 4542-4551, 2011) to create bispecific heterodimeric diabodies that are capable of simultaneous binding to P-cadherin and CD3. Further, in an aspect of the invention, the anti-P-cadherin/anti-CD3 DART proteins, also known as bispecific heterodimeric diabodies, successfully direct and activate T cell cytotoxicity of cells expressing P-cadherin.

Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Current Protocols in Immunology (J. E. Coligan et al., eds., 1999, including supplements through 2001); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987, including supplements through 2001); Molecular Cloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001).

P-cadherin (Cadherin-3, CDH3) belongs to the classical cadherin superfamily of transmembrane glycolproteins that regulate calcium-dependent cell-cell adhesion during development and tissue homeostasis (Gumbiner et al., J. Cell Biol., 148: 399-403, (2000)). The cadherin intracellular domains directly interact with cytoplasmic catenins that link to the actin cytoskeletal network, providing the molecular basis for stable cell interactions. The cadherin/catenin complex, as well as the signaling pathways controlled by this structure, represent a major regulatory mechanism that guide cell fate decisions, through its influence on cell growth, differentiation, motility, and survival (Cavallaro et al, 2011). Classical cadherins include the E-cadherin (CDH1), N-cadherin (CDH2) and P-cadherin (CDH3). P-cadherin expression in normal tissues is low and is restricted primarily to myoepithelial cells and the basal layers of stratified epithelium. Increased expression of P-cadherin has been reported in various tumors, including breast, gastric, endometrial, pancreatic and colorectal cancers and is a good prognosticator of poor survival of patients with advanced disease (Hardy et al, 2002; Imai et al, 2008; Parades et al, 2005; Stefansson et al, 2004; Taniuchi et al, 2005). Gene expression profiling and immunohistochemistry analyses further suggested lung (including but not limited to non-small cell lung and small cell lung cancers), ovarian, head and neck, thyroid and bladder cancers.

CD3 (cluster of differentiation 3) is a T cell co-receptor protein complex that comprising four distinct chains ε, δ, γ and ζ that form εδ, εγ and ζζ dimers.

These dimers associate with a molecule known as the T cell receptor (TCR) and generate an activation signal in T lymphocytes. The TCR and CD3 molecules together comprise the TCR complex. It is well known that anti-CD3 antibodies elicit the generation of cytotoxic T cells through the activation of endogenous lymphokine production and are capable of selectively killing tumor targets (Yun et al., Cancer Research, 49: 4770-4774 (1989)).

More specifically, T cells express TCR complexes that are able to induce antigen specific immune responses (Smith-Garvin et al, 2009). Antigens are peptides expressed by tumor cells and virally infected cells capable of stimulating immune responses. Intracellularly expressed antigens are bound to major histocompatibility class I (MHC class I) molecules and transported to the surface where they are exposed to T cells. If the binding affinity of the TCR to the MHC class I in complex with the antigen is sufficient the formation of an immune synapse will be initiated. Signaling through the immune synapse is mediated through the CD3 co-receptors that form εδ, εγ and ζζ dimers. These dimers associate with the TCR and generate an activation signal in T lymphocytes. This signaling cascade directs T cell mediated killing of the cell expressing the antigen. Cytotoxicity is mediated by release and transfer of granzyme B and perforin from the T cell to the target cell.

Without being bound by theory, it is believed that the bispecific heterodimer diabodies of the present invention may allow the T cell to circumvent the need for the interaction of the TCR and MHC class I in complex with antigen, and instead redirects T cells to target cells through direct co-engagement of CD3 (such as CD3 epsilon) expressed on the T cell and P-cadherin expressed on the tumor.

“Effector function” as used herein is meant as the capacity to support a biochemical event that results from the interaction of an anti-CD3 binding domain of a bispecific heterodimeric diabody with a cytotoxic T cell.

As used herein, the terms “antibody” and “antibodies” refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, camelized antibodies, single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab′) fragments, disulfide-linked bispecific Fvs (sdFv), intrabodies, and anti-idiotypic (anti-Id) antibodies (including, e.g. anti-Id and anti-anti-Id antibodies to antibodies of the invention), and epitope-binding fragments or binding domains of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Immunoglobulin molecules can be of any type (e.g. IgG, IgE, IgM, IgD, IgA and IgY), class (e.g. IgG₁, IgG₂, IgG₃, IgG₄, IgA₁and IgA₂) or subclass.

As used herein, the term “bispecific heterodimeric diabody” refers to a complex of two or more polypeptide chains or proteins, each comprising at least one antibody VL and one antibody VH domain or fragment thereof, wherein both antibody binding domains are comprised within a single polypeptide chain and wherein the VL and VH domains in each polypeptide chain are from different antibodies.

As used herein, the terms “immunospecifically binds,” “immunospecifically recognizes,” “specifically binds,” “specifically recognizes” and analogous terms refer to molecules e.g. binding domains, that specifically bind to an antigen (e.g. epitope or immune complex) and do not specifically bind to another molecule. A molecule that specifically binds to an antigen may bind to other peptides or polypeptides with lower affinity as determined by assays known in the art e.g. immunoassays, BIACORE®, or other assays. Preferably, molecules that specifically bind an antigen do not cross-react with other proteins.

As used herein, the terms “heavy chain”, “light chain”, “variable region” or “variable domain”, “framework region”, “constant domain”, and the like, have their ordinary meaning in the immunology art and refer to domains in naturally occurring immunoglobulins and the corresponding domains of recombinant binding proteins (e.g. humanized antibodies, single chain antibodies, chimeric antibodies, etc.). The basic structural unit of naturally occurring immunoglobulins is a tetramer having two light chains and two heavy chains, usually expressed as a glycoprotein of about 150,000 Da. The amino-terminal (N-terminal) portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal (C-terminal) portion of each chain defines a constant region. Each light chain is comprised a light chain variable domain (VL) and a light chain constant domain (CL). Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region, having CH1, hinge, CH2 and CH3 domains. The variable regions of an IgG molecule comprise regions of hypervariability, termed the complementarity determining regions (CDRs), which contain the residues in contact with antigen, and non-CDR segments, termed framework regains (FR), which generally maintain the structure and determine the positioning of the CDR loops (although certain framework residues may also contact antigen). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following structure: n-FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4-c. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY) and class (e.g., IgGI, IgG2, IgG 3, IgG4, IgAI and IgA2) or subclass.

The “hinge region” or “hinge domain” is generally defined as stretching from Glu216 to Pro230 of human IgG-1. Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine residues forming inter-heavy chain S—S binds in the same positions.

As used herein, the term “Fc region,” “Fc domain” or analogous terms are used to define a C-terminal region of an IgG heavy chain. The Fc region of an IgG comprises two constant domains, CH2 and CH3. The CH2 domain of a human IgG Fc region usually extends from amino acids 231 to amino acid 341 according to the numbering system of Kabat. The CH3 domain of a human IgG Fc region usually extends from amino acids 342 to 447 according to the numbering system of Kabat. The CH2 domain of a human IgG Fc region (also referred to as “Cγ 2” domain) is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG.

When referring to binding proteins, binding domains, or antibodies (as broadly defined herein), the assignment of amino acids to each domain is in accordance with the definitions of Kabat, Chothia, the accumulation of both Kabat and Chothia, AbM, contact, and/or conformational definitions or any method of CDR determination well known in the art. Antibody CDRs may be identified as the hypervariable regions originally defined by Kabat et al. See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C. The positions of the CDRs may also be identified as the structural loop structures originally described by Chothia and others. See, e.g., Chothia et al., 1989, Nature 342:877-883. Other approaches to CDR identification include the “AbM definition,” which is a compromise between Kabat and Chothia and is derived using Oxford Molecular's AbM antibody modeling software (Accelrys, San Diego, Calif.), or the “contact definition” of CDRs based on observed antigen contacts, set forth in MacCallum et al., 1996, J. Mol. Biol., 262:732-745. In another approach, referred to herein as the “conformational definition” of CDRs, the positions of the CDRs may be identified as the residues that make enthalpic contributions to antigen binding. See, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166. Still other CDR boundary definitions may not strictly follow one of the above approaches, but may nonetheless overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. As used herein, a CDR may refer to CDRs defined by any approach known in the art, including combinations of approaches. The methods used herein may utilize CDRs defined according to any of these approaches. For any given aspect containing more than one CDR, the CDRs may be defined in accordance with any of Kabat, Chothia, extended, AbM, contact, and/or conformational definitions.

A humanized antibody refers to forms of non-human (e.g. murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂or other antigen-binding subsequences or binding domains of antibodies) that contain minimal sequences derived from non-human immunoglobulins. Preferably, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.

The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Monoclonal antibodies from which binding domains of the bispecific heterodimeric diabodies of the invention can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art.

Antibodies can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular aspect, such phage can be utilized to display antigen binding domains, such as Fab and Fv or disulfide-bond stabilized Fv, expressed from a repertoire or combinatorial antibody library (e.g. human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g. using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage, including fd and M13. The antigen binding domains are expressed as a recombinantly fused protein to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the immunoglobulins, or fragments thereof, of the present invention include those disclosed in Brinkmann et al. (1995) “Phage Display Of Disulfide-Stabilized Fv Fragments,” J. Immunol. Methods, 182:41-50.

The functional characteristics of the multiple IgG isotypes, and domains thereof, are well known in the art. The amino acid sequences of IgG1, IgG2, IgG3 and IgG4 are known in the art. Selection and/or combinations of two or more domains from specific IgG isotypes for use in the methods of the invention may be based on any known parameter of the parent isotypes including affinity to FcγR. For example, use of regions or domains from IgG isotypes that exhibit limited or no binding to FcγRIIB, e.g. IgG2 or IgG4, may find particular use where a bispecific heterodimeric diabody is desired to be engineered to maximize binding to an activating receptor and minimize binding to an inhibitory receptor. Similarly, use of Fc regions or domains from IgG isotypes known to preferentially bind C1q or FcγRIIIA, e.g. IgG3 may be combined with Fc amino acid modifications known in the art to enhance antibody-dependent cell mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC), to engineer a bispecific heterodimeric diabody molecule such that effector function activity, e.g. complement activation or ADCC, is maximized. In a similar fashion, mutations may be made in the Fc regions or domains of IgG isotypes that minimize or eliminate the effector function of the Fc region.

The term “epitope” refers to that portion of a molecule capable of being recognized by and bound by an antibody at one or more of the antibody's antigen-binding regions. Epitopes often consist of a chemically active surface grouping of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics. The term “antigenic epitope” as used herein, is defined as a portion of a polypeptide to which an antibody can specifically bind as determined by any method well known in the art, for example, by conventional immunoassays. A “nonlinear epitope” or “conformational epitope” comprises noncontiguous polypeptides (or amino acids) within the antigenic protein to which an antibody specific to the epitope binds. Once a desired epitope on an antigen is determined, it is possible to generate antibodies to that epitope, e.g. using the techniques described herein. During the discovery process, the generation and characterization of antibodies may elucidate information about desirable epitopes. From this information, it is then possible to competitively screen antibodies for binding to the same epitope. An approach to achieve this is to conduct competition and cross-competition studies to find antibodies that compete or cross-compete with one another e.g. the antibodies compete for binding to the antigen.

One method is to identify the epitope to which antibodies bind, or “epitope mapping.” There are many methods known in the art for mapping and characterizing the location of epitopes on proteins, including solving the crystal structure of an antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. In an additional example, epitope mapping can be used to determine the sequence to which an antibody binds. Epitope mapping is commercially available from various sources, for example, Pepscan Systems (Edelhertweg 15, 8219 PH Lelystad, The Netherlands). In an additional example, mutagenesis of an antigen binding domain, domain swapping experiments and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and/or necessary for epitope binding. The binding affinity and the off-rate of an antigen-binding domain interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen, and the detection of the molecule bound to the labeled antigen. The affinity of the molecule of the present invention for an antigen and the binding off-rates can be determined from the saturation data by Scatchard analysis.

The affinities and binding properties of the molecules of the invention for an antigen may be initially determined using in vitro assays (biochemical or immunological based assays) known in the art for antigen-binding domain, including but not limited to enzyme-linked immunosorbent assay (ELISA) assay, surface plasmon resonance (SPR) assay, Bio-Layer Interferometry, or immunoprecipitation assays. The molecules of the invention may have similar binding properties in in vivo models (such as those described and disclosed herein) as those in in vitro based assays. However, the present invention does not exclude molecules of the invention that do not exhibit the desired phenotype in in vitro based assays but do exhibit the desired phenotype in vivo.

The term “binding affinity (K_D)” as used herein, is intended to refer to the dissociation rate of a particular antigen-antibody interaction. The K_Dis the ratio of the rate of dissociation, also called the “off-rate (k_off)”, to the association rate, or “on-rate (k_on)”. Thus, K_Dequals k_off/k_onand is expressed as a molar concentration (M). It follows that the smaller the K_D, the stronger the affinity of binding. Therefore, a K_Dof 1 μM indicates weak binding affinity compared to a K_Dof 1 nM. K_Dvalues for antibodies can be determined using methods well established in the art. One method for determining the K_Dof an antibody is by using surface plasmon resonance (SPR), typically using a biosensor system such as a BIACORE® system. BIAcore kinetic analysis comprises analyzing the binding and dissociation of an antigen from chips with immobilized molecules (e.g. molecules comprising epitope binding domains), on their surface. Another method for determining the K_Dof an antibody is by using Bio-Layer Interferometry, typically using OCTET® technology (Octet QK^esystem, ForteBio).

As used herein, the terms “nucleic acids” and “nucleotide sequences” include DNA molecules (e.g. cDNA or genomic DNA), RNA molecules (e.g. mRNA), combinations of DNA and RNA molecules or hybrid DNA/RNA molecules, and analogs of DNA or RNA molecules. Such analogs can be generated using, for example, nucleotide analogs, which include, but are not limited to, inosine or tritylated bases. Such analogs can also comprise DNA or RNA molecules comprising modified backbones that lend beneficial attributes to the molecules such as, for example, nuclease resistance or an increased ability to cross cellular membranes. The nucleic acids or nucleotide sequences can be single-stranded, double-stranded, may contain both single-stranded and double-stranded portions, and may contain triple-stranded portions, but preferably is double-stranded DNA.

The present invention also includes polynucleotides that encode the bispecific heterodimeric diabodies of the invention, including the polypeptides and binding regions of the antibodies. The polynucleotides encoding the molecules of the invention may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art.

The polynucleotides that encode the bispecific heterodimeric diabodies of the present invention may include the following: only the coding sequence for the variant, the coding sequence for the variant and additional coding sequences such as a functional polypeptide, or a signal or secretory sequence or a pro-protein sequence; the coding sequence for the antibody and non-coding sequence, such as introns or non-coding sequence 5′ and/or 3′ of the coding sequence for the antibody. The term “polynucleotide encoding a bispecific heterodimeric diabody” encompasses a polynucleotide which includes additional coding sequence for the variant but also a polynucleotide which includes additional coding and/or non-coding sequence. It is known in the art that a polynucleotide sequence that is optimized for a specific host cell/expression system can readily be obtained from the amino acid sequence of the desired protein (see GENEART® AG, Regensburg, Germany).

As used herein, the term “cancer” refers to a neoplasm or tumor resulting from abnormal uncontrolled growth of cells. In some aspects, cancer refers to a benign tumor, which has remained localized. In other aspects, cancer refers to a malignant tumor, which has invaded and destroyed neighboring body structures and spread to distant sites. In some aspects, the cancer is associated with a specific cancer antigen.

As used herein, a “therapeutically effective amount” refers to that amount of the therapeutic agent sufficient to treat or manage a disease or disorder. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease, e.g. delay or minimize the spread of cancer. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease. Further, a therapeutically effective amount with respect to a therapeutic agent of the invention means the amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease.

As used herein, the terms “prophylactic agent” and “prophylactic agents” refer to any agent(s) which can be used in the prevention of a disorder, or prevention of recurrence or spread of a disorder. A prophylactically effective amount may refer to the amount of prophylactic agent sufficient to prevent the recurrence or spread of hyperproliferative disease, particularly cancer, or the occurrence of such in a patient, including but not limited to those predisposed to hyperproliferative disease, for example those genetically predisposed to cancer or previously exposed to carcinogens. A prophylactically effective amount may also refer to the amount of the prophylactic agent that provides a prophylactic benefit in the prevention of disease. Further, a prophylactically effective amount with respect to a prophylactic agent of the invention means that amount of prophylactic agent alone, or in combination with other agents, that provides a prophylactic benefit in the prevention of disease.

As used herein, the terms “prevent”, “preventing” and “prevention” refer to the prevention of the recurrence or onset of one or more symptoms of a disorder in a subject as result of the administration of a prophylactic or therapeutic agent.

As used herein, the term “in combination” refers to the use of more than one prophylactic and/or therapeutic agents. The use of the term “in combination” does not restrict the order in which prophylactic and/or therapeutic agents are administered to a subject with a disorder. In other words, the combination therapy may be done by separately, sequentially, or simultaneously treating with the therapeutic agents.

Recombinant Expression of Molecules of the Invention

Once a nucleic acid sequence encoding molecules of the invention (i.e. binding domains) has been obtained, the vector for the production of the molecules may be produced by recombinant DNA technology using techniques well known in the art.

The polynucleotides encoding the bispecific heterodimeric diabody binding domains of the present invention may include an expression control polynucleotide sequence operably linked to the antibody coding sequences, including naturally-associated or heterologous promoter regions known in the art. The expression control sequences may be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, but control sequences for prokaryotic hosts may also be used. Once the vector has been incorporated into the appropriate host cell line, the host cell is propagated under conditions suitable for expressing the nucleotide sequences, and, as desired, for the collection and purification of the antibodies. Eukaryotic cell lines include the CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, transformed B-cells, or human embryonic kidney cell lines.

Bispecific Heterodimeric Diabodies

As stated previously, bispecific heterodimeric diabody refers to a complex of two or more polypeptide chains or proteins, each comprising at least one antibody VL domain and one antibody VH domain or fragment thereof, wherein both antibody binding domains are comprised within a single polypeptide chain and wherein the VL and VH domains in each polypeptide chain are from different antibodies. In specific aspects, bispecific heterodimeric diabody includes dimers or tetramers of polypeptide chains containing both a VL and VH domain. The individual polypeptide chains comprising the multimeric proteins may be covalently joined to at least one other peptide of the multimer by interchain disulfide bonds.

Bispecific heterodimeric diabodies of the present invention are comprised of anti-P-cadherin (P-CAD) binding domains covalently linked to anti-CD3 (CD3) binding domains utilizing a Dual-Affinity Re-Targeting (DART®) platform technology (US Patent Application Publications Nos. 2007/0004909, 2009/0060910 and 2010/0174053). The DART technology provides a covalent linkage which results in a molecule that may have superior stability, optimal heavy and light chain pairing and antigen recognition. Moreover, the minimal linker size and content may decrease the potential for immunogenicity. The DART proteins of the present invention simultaneously target T cells (CD3) and tumor cells (P-CAD) and successfully direct and activate T cell cytoxicity to tumor cells expressing P-cadherin.

Each polypeptide chain of the bispecific heterodimeric diabody comprises a VL domain and a VH domain, which may be covalently linked (Linker 1) such that the antibody binding domains are constrained from self-assembly. In addition, each polypeptide chain comprises a heterodimerization domain, which promotes heterodimerization of the multiple polypeptide chains and reduces the probability of homodimerization of the different polypeptide chains. The heterodimerization domain may be located at the N-terminus of the polypeptide chain or the C-terminus. The heterodimerization domain may comprise a cysteine linker (Linker 2) that is 1, 2, 3, 4, 5, 6, or more amino acid residues in length. Interaction of two of the polypeptide chains may produce two VL/VH pairings, forming two epitope binding domains, i.e., a bivalent molecule. Neither the VH or VL domain is constrained to any position within the polypeptide chain, i.e. restricted to the amino or carboxy terminus, nor are the domains restricted in their relative positions to one another, i.e. the VL domain may be N-terminal to the VH domain and vice versa. The only restriction is that a complimentary polypeptide chain be available in order to form a functional diabody. Where the VL and VH domains are derived from antibodies specific for different antigens, formation of a functional bispecific heterodimeric diabody requires the interaction of two different polypeptide chains, i.e. formation of a heterodimer. In contrast, where two differing polypeptide chains are free to interact, e.g. in a recombinant expression system, one comprising a VLA and a VHB (A, being a first epitope and B, being a second epitope) and the other comprising a VLB and a VHA, two differing binding sites may form: VLA-VHA and VLB-VHB. For all bispecific heterodimeric diabody polypeptide chain pairs, misalignment or mis-binding of the two chains is a possibility, e.g. interaction of VL-VL or VH-VH domains. However, purification of functional bispecific heterodimeric diabodies is easily managed based on the immunospecificity of the properly dimerized binding site using any affinity based method known in the art or exemplified herein, e.g. affinity chromatography.

The bispecific heterodimeric diabodies of the invention may simultaneously bind two separate and distinct epitopes. In certain aspects, at least one epitope binding site is specific for the CD3 determinant expressed on an immune effector cell e.g. expressed on T lymphocytes. In one aspect, the bispecific heterodimeric diabody molecule binds to the effector cell determinant and also activates the effector cell. In one aspect, the epitope-binding domain is capable of binding the P-cadherin tumor-associated antigen that is associated with breast, colorectal, ovarian, gastric, thyroid, prostate, cervical, pancreatic, lung (including but not limited to non-small cell lung cancer and small cell lunch cancer), bladder, liver, endometrial, head and neck, testicular, and glioblastoma cancer.

The bispecific heterodimeric diabodies of the present invention comprise antigen binding domains generally derived from immunoglobulins or antibodies. The antibodies from which the binding domains used in the methods of the invention are derived may be from any animal origin including birds and mammals (e.g. human, non-human primate, murine, donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken). Preferably, the antibodies are human or humanized monoclonal antibodies. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or libraries of synthetic human immunoglobulin coding sequences or from mice that express antibodies from human genes.

The bispecific heterodimeric diabodies of the present invention may be characterized in a variety of ways. In particular, molecules of the invention may be assayed for the ability to immunospecifically bind to an antigen. Molecules that have been identified to immunospecifically bind to an antigen can then be assayed for their specificity and affinity for the antigen.

The bispecific heterodimeric diabodies of the present invention may be produced using a variety of methods well known in the art, including de novo protein synthesis and recombinant expression of nucleic acids encoding the binding proteins. The desired nucleic acid sequences may be produced by recombinant methods (e.g. PCR mutagenesis of an earlier prepared variant of the desired polynucleotide) or by solid-phase DNA synthesis. Usually recombinant expression methods are used. In one aspect, the invention provides a polynucleotide that comprises a sequence encoding an anti-CD3 VH and/or VL; in another aspect, the invention provides a polynucleotide that comprises a sequence encoding an anti-P-cadherin VH and/or VL. Because of the degeneracy of the genetic code, a variety of nucleic acid sequences encode each immunoglobulin amino acid sequence, and the present invention includes all nucleic acids encoding the binding proteins described herein.

Linkers/Peptides

The polypeptide chains of the bispecific heterodimeric diabodies may be comprise various linkers and peptides. The linkers and peptides may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more amino acids. In certain aspects, the polypeptide chains of the bispecific heterodimeric diabodies may comprise a glycine-serine linker (Linker 1). In another aspect the Linker 1 may comprise, but is not limited to, the sequences of GGGSGGGG (SEQ ID NO: 68) and GGGGSGGGG (SEQ ID NO 69).

In certain aspects, the polypeptide chains of the bispecific heterodimeric diabodies may comprise a cysteine linker (Linker 2). In another aspect, the Linker 2 may further comprise at least one glycine residue. In one aspect, the Linker 2 may comprise, but is not limited to, the sequences selected of GFNRGEC (SEQ ID NO: 70), GVEPKSC (SEQ ID NO: 71), and GGCGGG (SEQ ID NO: 72). In certain aspects, the Linker 2 may comprise a truncated IgG1 hinge region having the sequence CPPCP (SEQ ID NO 60) and at least one glycine residue. In another aspect, the Linker 2 may comprise, but is not limited to, the sequences of GCPPCP (SEQ ID NO: 73), GGTGGCPPCP (SEQ ID NO: 74), GEPKSSDKTHTCPPCP (SEQ ID NO: 75) and GGTGGGEPKSSDKTHTCPPCP (SEQ ID NO: 76).

In certain aspects, the polypeptide chains of the bispecific heterodimeric diabodies may comprise a Linker 3. In one aspect, the Linker 3 may comprise, but is not limited to, the sequence of KAPSSSPME (SEQ ID NO 77).

In certain aspects, the polypeptide chains of the bispecific heterodimeric diabodies may comprise a Peptide 1. In certain aspects, the Peptide 1 may comprise a truncated IgG1 hinge region having the sequence CPPCP (SEQ ID NO 60). In one aspect, the Peptide 1 may comprise, but is not limited to, the sequence of DKTHTCPPCP (SEQ ID NO 96).

VF-DART

In an aspect of the present invention, a bispecific heterodimeric diabody, known as a VF-DART, as shown in FIGS. 2 and 3, comprises a first polypeptide chain (1) and a second polypeptide chain (2).

In one aspect, the first polypeptide chain (1) may comprise: a Domain 1, comprising (i) a sub-Domain 1A which comprises a VL binding region of either an anti-P-cadherin antibody (P-CAD VL) or an anti-CD3 antibody (CD3 VL), (ii) a sub-Domain 1B which comprises a VH binding region of either an anti-P-cadherin antibody (P-CAD VH), if such sub-Domain 1A comprises a CD3 VL, or an anti-CD3 antibody (CD3 VH), if the sub-Domain 1A comprises a P-CAD VL, and the sub-Domain 1A and sub-Domain 1B may be covalently linked by a glycine-serine linker (Linker 1) such that the sub-Domain 1B and sub-Domain 1A do not associate to form an epitope binding site; and may comprise a first heterodimer promoting domain comprising a cysteine linker (Linker 2) on the sub-Domain 1B.

In another aspect, the first polypeptide chain (1) may comprise: a Domain 1, comprising (i) a sub-Domain 1A which comprises a P-CAD VH or a CD3 VH, (ii) a sub-Domain 1B which comprises either a P-CAD VL, if the sub-Domain 1A comprises a CD3 VH, or a CD3 VL, if the sub-Domain 1A comprises a P-CAD VH, the sub-Domain 1A and sub-Domain 1B may be covalently linked by a glycine-serine linker (Linker 1) such that the sub-Domain 1B and sub-Domain 1A do not associate to form an epitope binding site; and may comprise a first heterodimer promoting domain comprising a cysteine linker (Linker 2) on the sub-Domain 1B.

In one aspect, the first polypeptide chain (1) may comprise: a Domain 1, comprising (i) a sub-Domain 1A which comprises a P-CAD VL and (ii) a sub-Domain 1B which comprises a CD3 VH. In another aspect, the first polypeptide chain (1) may comprise: a Domain 1, comprising (i) a sub-Domain 1A which comprises a CD3 VL and (ii) a sub-Domain 1B which comprises a P-CAD VH. In a further aspect, the first polypeptide chain (1) may comprise: a Domain 1, comprising (i) a sub-Domain 1A which comprises a P-CAD VH and (ii) a sub-Domain 1B which comprises a CD3 VL. In another aspect, the first polypeptide chain (1) may comprise: a Domain 1, comprising (i) a sub-Domain 1A which comprises a CD3 VH and (ii) a sub-Domain 1B which comprises a P-CAD VL.

In one aspect, the second polypeptide chain (2) may comprise: a Domain 2, comprising (i) a sub-Domain 2B, which comprises a VL binding region of either an anti-P-cadherin antibody (P-CAD VL) or an anti-CD3 antibody (CD3 VL) depending on the VL domain selected for sub-Domain 1A, (ii) a sub-Domain 2A which comprises a VH binding region of either an anti-P-cadherin antibody (P-CAD VH), if such sub-Domain 2B comprises a CD3 VL, or an anti-CD3 antibody (CD3 VH), if such sub-Domain 2B comprises a P-CAD VL, and the sub-Domain 2B and sub-Domain 2A may be covalently linked by a glycine-serine linker (Linker 1) such that the sub-Domain 2B and sub-Domain 2A do not associate to form an epitope binding site; and may comprise a second heterodimer promoting domain comprising a cysteine linker (Linker 2) on the sub-Domain 2A.

In one aspect, the second polypeptide chain (2) may comprise: a Domain 2, comprising (i) a sub-Domain 2B which comprises a P-CAD VL and (ii) a sub-Domain 2A which comprises a CD3 VH. In another aspect, the second polypeptide chain (2) may comprise: a Domain 2, comprising (i) a sub-Domain 2B which comprises a CD3 VL and (ii) a sub-Domain 2A which comprises a P-CAD VH. In a further aspect, the second polypeptide chain (2) may comprise: a Domain 2, comprising (i) a sub-Domain 2B which comprises a P-CAD VH and (ii) a sub-Domain 2A which comprises a CD3 VL. In another aspect, the second polypeptide chain (2) may comprise: a Domain 2, comprising (i) a sub-Domain 2B which comprises a CD3 VH and (ii) a sub-Domain 2A which comprises a P-CAD VL.

In another aspect of the VF-DART, the sub-Domain 1A and the sub-Domain 2A may associate to form a first binding site, having a VL/VH binding domain, that binds to an epitope on CD3 or P-cadherin, and the sub-Domain 1B and the sub-Domain 2B may associate to form a second binding site, having a VL/VH binding domain, that binds to an epitope on P-cadherin or CD3.

The first polypeptide chain (1) and second polypeptide chain (2) may be covalently bonded to one another, for example at the cysteine residues located within the first and second heterodimer promoting domain. In one aspect, the cysteine residue in Linker 2 of the first heterodimer promoting domain and the cysteine residue in the Linker 2 of the second heterodimer promoting domain may be linked by at least one disulfide bond.

In an aspect of the present invention, the CD3 VL may comprise the sequence of SEQ ID NO: 47 and the CD3 VH may be a sequence selected from the group consisting of SEQ ID NOS: 45 and 46. In another aspect, the P-CAD VL may be a sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 and the P-CAD VH may be a sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24. In one aspect, the glycine-serine linker (Linker 1) may comprise a sequence selected from the group consisting of GGGSGGGG (SEQ ID NO: 68) and GGGGSGGGG (SEQ ID NO 69). In an aspect of the present invention, the cysteine linker (Linker 2) of the first heterodimer promoting domain may comprise the sequence of GFNRGEC (SEQ ID NO: 70) and the cysteine linker (Linker 2) of the second heterodimer promoting domain may comprise the sequence of GVEPKSC (SEQ ID NO: 71). In another aspect of the present invention, the cysteine linker (Linker 2) of the first heterodimer promoting domain may comprise the sequence of GVEPKSC (SEQ ID NO: 71) and the cysteine linker (Linker 2) of the second heterodimer promoting domain may comprise the sequence of GFNRGEC (SEQ ID NO: 70).

The sequences for the first and second polypeptide chains of VF-DART proteins with the various P-cadherin and CD3 antibodies described herein are provided in FIGS. 30A and 30B.

EK-DART Containing E/K Coil Regions

Heterodimer formation of the two different polypeptide chains of a bispecific heterodimeric diabody may also be promoted by the use of domains that have affinity for the opposite member and repulsion towards itself. An example of such a domain is a coiled-coil domain. For example, a first polypeptide chain (1) may include a first heterodimer promoting domain having a glutamic acid-rich region that forms a negatively charged alpha-helical coil (E-coil), and a second polypeptide chain (2) may include a second heterodimer promoting domain having a lysine rich region that forms a positively charged helical coil (K-coil), allowing for an electrostatic interaction to promote heterodimer formation. Homodimer formation may be reduced as an E-coil would have repulsive force for another E-coil-containing polypeptide and vice-versa for the K-coil.

In an aspect of the present invention, a bispecific heterodimeric diabody, known as an EK-DART, as shown in FIG. 4, comprises a first polypeptide chain (1) and a second polypeptide chain (2).

In one aspect, the first polypeptide chain (1) may comprise: a Domain 1, comprising (i) a sub-Domain 1A which comprises a VL binding region of either an anti-P-cadherin antibody (P-CAD VL) or an anti-CD3 antibody (CD3 VL), (ii) a sub-Domain 1B which comprises a VH binding region of either an anti-P-cadherin antibody (P-CAD VH), if the sub-Domain 1A comprises a CD3 VL, or an anti-CD3 antibody (CD3 VH), if the sub-Domain 1A comprises a P-CAD VL, and the sub-Domain 1A and sub-Domain 1B may be covalently linked by a glycine-serine linker (Linker 1) such that the sub-Domain 1A and sub-Domain 1B do not associate to form an epitope binding site; and may comprise a first heterodimer promoting domain comprising (i) a cysteine linker (Linker 2) on the sub-Domain 1B, and (ii) an E-coil region covalently linked to the Linker 2.

In another aspect, the first polypeptide chain (1) may comprise: a Domain 1, comprising (i) a sub-Domain 1A which comprises a P-CAD VH or a CD3 VH, (ii) a sub-Domain 1B which comprises either a P-CAD VL, if the sub-Domain 1A comprises a CD3 VH, or a CD3 VL, if the sub-Domain 1A comprises a P-CAD VH, and the sub-Domain 1A and sub-Domain 1B may be covalently linked by a glycine-serine linker (Linker 1) such that the sub-Domain 1A and sub-Domain 1B do not associate to form an epitope binding site; and may comprise a first heterodimer promoting domain comprising (i) a cysteine linker (Linker 2) on the sub-Domain 1B, and (ii) an E-coil region covalently linked to the Linker 2.

In one aspect, the second polypeptide chain (2) may comprise: a Domain 2, comprising (i) a sub-Domain 2B which comprises a VL binding region of either an anti-P-cadherin antibody (P-CAD VL) or an anti-CD3 antibody (CD3 VL) depending on the VL domain selected for sub-Domain 1A, (ii) a sub-Domain 2A which comprises a VH binding region of either an anti-P-cadherin antibody (P-CAD VH), if the sub-Domain 2B comprises a CD3 VL, or an anti-CD3 antibody (CD3 VH), if the sub-Domain 2B comprises a P-CAD VL, and the sub-Domain 2B and sub-Domain 2A may be covalently linked by a glycine-serine linker (Linker 1) such that the sub-Domain 2B and sub-Domain 2A do not associate to form an epitope binding site; and may comprise a second heterodimer promoting domain comprising (i) a cysteine linker (Linker 2) on the sub-Domain 2A and (ii) a K-coil region covalently linked to the Linker 2.

In another aspect, the second polypeptide chain (2) may comprise: a Domain 2, comprising (i) a sub-Domain 2B which comprises a P-CAD VH or a CD3 VH, (ii) a sub-Domain 2A which comprises either a P-CAD VL, if the sub-Domain 2B comprises a CD3 VH, or a CD3 VL, if the sub-Domain 2B comprises a P-CAD VH, and the sub-Domain 2B and sub-Domain 2A may be covalently linked by a glycine-serine linker (Linker 1) such that the sub-Domain 2B and sub-Domain 2A do not associate to form an epitope binding site; and may comprise a second heterodimer promoting domain comprising (i) a cysteine linker (Linker 2) on the sub-Domain 2A and (ii) a K-coil region covalently linked to the Linker 2.

In another aspect of the EK-DART, the sub-Domain 1A and the sub-Domain 2A may associate to form a first binding site, having a VL/VH binding domain region, that binds to an epitope on CD3 or P-cadherin, and the sub-Domain 1B and the sub-Domain 2B may associate to form a second binding site, having a VL/VH binding domain region, that binds to an epitope on P-cadherin or CD3.

In an aspect of the present invention, the CD3 VL may comprise the sequence of SEQ ID NO: 47 and the CD3 VH may be a sequence selected from the group consisting of SEQ ID NOS: 45 and 46. In another aspect, the P-CAD VL may be a sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 and the P-CAD VH may be a sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24. In one aspect, the glycine-serine linker (Linker 1) may comprise a sequence selected from the group consisting of GGGSGGGG (SEQ ID NO: 68) and GGGGSGGGG (SEQ ID NO 69). In an aspect of the invention, the cysteine linker (Linker 2) of the first heterodimer promoting domain and the second heterodimer domain may comprise the sequence of GGCGGG (SEQ ID NO: 72). In one aspect, first heterodimer promoting domain further comprises a glutamic acid-rich region (E-coil) comprising the sequence of EVAALEKEVAALEKEVAALEKEVAALEK (SEQ ID NO: 61). In another aspect, the second heterodimer promoting domain further comprises a lysine rich region (K-coil) comprising the sequence of KVAALKEKVAALKEKVAALKEKVAALKE (SEQ ID NO: 62).

Table 1 and FIGS. 31A and 31B provide the sequences for the first and second polypeptide chains of EK-DART proteins with the various P-cadherin and CD3 antibodies described herein.

TABLE 1

Sequences of the first and second polypeptide

chains of EK-DART proteins.

EK-DART
Sequence

35
P-CAD 35VL X CD3-1VH-K-COIL SEQ ID NO: 80

QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPD

RFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAVVFGGGTKLTVLGGGSGGGGEV

QLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATY

YADSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTL

VTVSSGGCGGGKVAALKEKVAALKEKVAALKEKVAALKE

CD3-1-2VL X P-CAD 35VH-E-COIL SEQ ID NO: 81

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPW

TPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLGGGGSGGGG

EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTN

YAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCATIDTASAFDIWGQGTMVTVSSG

GCGGGEVAALEKEVAALEKEVAALEKEVAALEK

153
P-CAD 153VL X CD3-2VH-K-COIL SEQ ID NO: 82

QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPD

RFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSGVVFGGGTKLTVLGGGSGGGGEV

QLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATY

YADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTL

VTVSSGGCGGGKVAALKEKVAALKEKVAALKEKVAALKE

CD3-1-2VL X P-CAD 153VH-E-COIL SEQ ID NO: 83

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPW

TPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLGGGGSGGGG

EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTN

YAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCATIDTANAFGIWGQGTMVTVSSG

GCGGGEVAALEKEVAALEKEVAALEKEVAALEK

154
P-CAD 154VL X CD3-2VH-K-COIL SEQ ID NO: 84

QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPD

RFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSSYVFGTGTKVTVLGGGSGGGGEV

QLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATY

YADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTL

VTVSSGGCGGGKVAALKEKVAALKEKVAALKEKVAALKE

CD3-1-2VL X P-CAD 154VH-E-COIL SEQ ID NO: 85

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPW

TPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLGGGGSGGGG

EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTN

YAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCATIDTATAFDIWGQGTMVTVSSG

GCGGGEVAALEKEVAALEKEVAALEKEVAALEK

177
P-CAD 177VL X CD3-1VH-K-COIL SEQ ID NO: 86

QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPD

RFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAYVFGTGTKVTVLGGGSGGGGEV

QLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATY

YADSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTL

VTVSSGGCGGGKVAALKEKVAALKEKVAALKEKVAALKE

CD3-1-2VL X P-CAD 177VH-E-COIL SEQ ID NO: 87

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPW

TPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLGGGGSGGGG

EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTN

YAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCATIDTANAFDIWGQGTMVTVSSG

GCGGGEVAALEKEVAALEKEVAALEKEVAALEK

Bispecific Heterodimeric Diabodies Comprising Fc Regions or Portions Thereof

The invention encompasses bispecific heterodimeric diabodies comprising Fc regions or domains, or portions thereof. In certain aspects, the Fc region, or portion(s) thereof, comprises one or more constant domain(s) of the Fc region of IgG1, IgG2, IgG3 or IgG4 (e.g. a CH2 or CH3 domain). In other aspects, the invention encompasses molecules comprising an Fc region or portion thereof, wherein the Fc region or portion thereof comprises at least one amino acid modification (e.g. substitution) relative to a comparable wild-type Fc region or portion thereof. Variant Fc regions are well known in the art, and are primarily used to alter the phenotype of the antibody comprising the variant Fc region as assayed in any of the binding activity or effector function assays well known in the art, e.g. ELISA, SPR analysis, or ADCC. Such variant Fc regions, or portions thereof, may extend the plasma half-life and stability exhibited by a diabody molecule of the invention comprising an Fc region or portion thereof. In other aspects, the invention encompasses the use of any Fc variant known in the art.

In certain aspects, one or more modifications to the amino acids of the Fc region reduce the affinity and avidity of the Fc region and, thus, the diabody molecule of the invention, for one or more FcγR receptors. In a specific aspect, the invention encompasses diabodies comprising a variant Fc region, or portion thereof, wherein the variant Fc region comprises at least one amino acid modification relative to a wild type Fc region which variant Fc region only binds one FcγR, wherein the FcγR is FcγRIIIA. In another specific aspect, the invention encompasses diabodies comprising a variant Fc region, or portion thereof, wherein the variant Fc region comprises at least one amino acid modification relative to a wild type Fc region, which variant Fc region only binds one FcγR, wherein the FcγR is FcγRIIA. In another specific aspect, the invention encompasses diabodies comprising a variant Fc region or portion thereof, wherein the variant Fc region comprises at least one amino acid modification relative to a wild type Fc region, which variant Fc region only binds one FcγR, wherein the FcγR is FcγRIIB. In certain aspects, the invention encompasses molecules comprising a variant Fc region wherein the variant confers or mediates decreased ADCC activity (or other effector function) and/or an increased binding to FcγRIIB (CD32B), relative to a molecule comprising no Fc region or comprising a wild-type Fc region, as measured using methods known to one skilled in the art and described herein.

The invention also encompasses the use of an Fc region comprising domains or regions from two or more IgG isotypes. As known in the art, amino acid modification of the Fc region may profoundly affect Fc-mediated effector function and/or binding activity. However, these alterations in functional characteristics may be further refined and/or manipulated when implemented in the context of selected IgG isotypes. Similarly, the native characteristics of the isotype Fc may be manipulated by one or more amino acid modifications. The multiple IgG isotypes (i.e., IgG1, IgG2, IgG3 and IgG4) exhibit differing physical and functional properties including serum half-life, complement fixation, FcγR binding affinities and effector function activities (e.g. ADCC, CDC) due to differences in the amino acid sequences of their hinge and/or Fc regions. In certain aspects, the amino acid modification and IgG Fc region are independently selected based on their respective, separate binding and/or effector function activities in order to engineer a diabody with desired characteristics. In most aspects, the amino acid modifications and IgG hinge/Fc regions have been separately assayed for binding and/or effector function activity as described herein or known in the art in the context of an IgG1. In certain aspects, the amino acid modification and IgG hinge/Fc region display similar functionality, e.g. decreased ADCC activity (or other effector function) and/or an increased binding to FcγRIIB in the context of the diabody molecule or other Fc-containing molecule (e.g. and immunoglobulin). In other aspects, the invention encompasses variant Fc regions comprising combinations of amino acid modifications known in the art and selected IgG regions that exhibit novel properties, which properties were not detectable when the modifications and/or regions were independently assayed as described herein.

LP-DART

In an aspect of the present invention, a bispecific heterodimeric diabody having an Fc region, termed LP-DART, is shown in FIGS. 1 and 5. An LP-DART may be comprised of two scFv domains that bind a target antigen and CD3, which is fused to a Fc domain of IgG. In one aspect of the invention, the bispecific heterodimeric diabody may be comprised of two scFv domains that bind P-cadherin and CD3 epsilon, which is fused to a Fc domain of IgG1. In particular, FIGS. 1A and 1B show four alternative representations of anti-P-cadherin/anti-human CD3 LP-DART and FIG. 5 shows a general schematic of an assembled LP-DART. The LP-DART is similar to the structure described above for the VF-DART or EK-DART, except the first and second heterodimer promoting domains comprise an immunoglobulin Fc region having CH2 and/or CH3 domains of an immunoglobulin Fc region, wherein the CH2 and/or CH3 domains have been altered to comprise a knob (protuberance) or a hole (cavity). Modifications to the Fc portion of the LP-DART are described below.

In an aspect of the present invention, a LP-DART bispecific heterodimeric diabody, as shown in FIGS. 1A, 1B and 5, comprises a first polypeptide chain (1) and a second polypeptide chain (2).

In one aspect, the first polypeptide chain (1) may comprise: a Domain 1, comprising (i) a sub-Domain 1A which comprises a VL binding region of either an anti-P-cadherin antibody (P-CAD VL) or an anti-CD3 antibody (CD3 VL), (ii) a sub-Domain 1B which comprises a VH binding region of either an anti-P-cadherin antibody (P-CAD VH), if such sub-Domain 1A comprises a CD3 VL, or an anti-CD3 antibody (CD3 VH), if such sub-Domain 1A comprises a P-CAD VL, and the sub-Domain 1A and sub-Domain 1B may be covalently linked by a glycine-serine linker (Linker 1) such that the sub-Domain 1A and sub-Domain 1B do not associate to form an epitope binding site; and may comprise a first heterodimer promoting domain comprising (i) a cysteine linker (Linker 2) on the sub-Domain 1B and (ii) a Fc region having a CH2 and/or CH3 domain altered to comprise a knob Fc chain (protuberance) or a hole Fc chain (cavity) covalently linked to the Linker 2.

In another aspect, the first polypeptide chain (1) may comprise: a Domain 1, comprising (i) a sub-Domain 1A which comprises a P-CAD VH or a CD3 VH, (ii) a sub-Domain 1B which comprises either a P-CAD VL, if such sub-Domain 1A comprises a CD3 VH, or a CD3 VL, if such sub-Domain 1A comprises a P-CAD VH, and the sub-Domain 1A and sub-Domain 1B may be covalently linked by a glycine-serine linker (Linker 1) such that the sub-Domain 1A and sub-Domain 1B do not associate to form an epitope binding site; and may comprise a first heterodimer promoting domain comprising (i) a cysteine linker (Linker 2) on the sub-Domain 1B and (ii) a Fc region having a CH2 and/or CH3 domain altered to comprise a knob Fc chain (protuberance) or a hole Fc chain (cavity) covalently linked to the Linker 2.

In one aspect, the second polypeptide chain (2) may comprise: a Domain 2, comprising (i) a sub-Domain 2B which comprises a VL binding region of either an anti-P-cadherin antibody (P-CAD VL) or an anti-CD3 antibody (CD3 VL) depending on the VL domain selected for sub-Domain 1A, (ii) a sub-Domain 2A which comprises a VH binding region of either an anti-P-cadherin antibody (P-CAD VH), if such sub-Domain 2B comprises a CD3 VL, or an anti-CD3 antibody (CD3 VH), if such sub-Domain 2B comprises a P-CAD VL, and the sub-Domain 2B and sub-Domain 2A may be covalently linked by a glycine-serine linker (Linker 1) such that the sub-Domain 2B and sub-Domain 2A do not associate to form an epitope binding site; and may comprise a second heterodimer promoting domain comprising (i) cysteine linker (Linker 2) on the sub-Domain 2A and (ii) a Fc region having a CH2 and/or CH3 domain altered to comprise either a knob Fc chain (protuberance), if such first heterodimer region comprises a hole Fc chain (cavity), or a hole Fc chain (cavity), if such first heterodimer region comprises a knob Fc chain (protuberance), covalently linked to the Linker 2.

In another aspect, the second polypeptide chain (2) may comprise: a Domain 2, comprising (i) a sub-Domain 2B which comprises a P-CAD VH or a CD3 VH, (ii) a sub-Domain 2A which comprises either a P-CAD VL, if such sub-Domain 2B comprises a CD3 VH, or a CD3 VL, if such sub-Domain 2B comprises a P-CAD VH, and the sub-Domain 2B and sub-Domain 2A may be covalently linked by a glycine-serine linker (Linker 1) such that the sub-Domain 2B and sub-Domain 2A do not associate to form an epitope binding site; and may comprise a second heterodimer promoting domain comprising (i) a cysteine linker (Linker 2) on the sub-Domain 2A and (ii) a Fc region having a CH2 and/or CH3 domain altered to comprise either a knob Fc chain (protuberance), if such first heterodimer region comprises a hole Fc chain (cavity), or a hole Fc chain (cavity), if such first heterodimer region comprises a knob Fc chain (protuberance), covalently linked to the Linker 2.

In another aspect of the LP-DART, the sub-Domain 1A and the sub-Domain 2A may associate to form a first binding site, having a VL/VH binding domain region, that binds to an epitope on CD3 or P-cadherin, and the sub-Domain 1B and the sub-Domain 2B may associate to form a second binding site, having a VL/VH binding domain region, that binds to an epitope on P-cadherin or CD3.

In another aspect of the invention, the cysteine linker (Linker 2) may comprise a truncated IgG1 hinge region having the sequence CPPCP (SEQ ID NO 60) and at least one glycine residue preceding the hinge region. In one aspect, the Linker 2 may comprise a sequence selected from the group consisting of GCPPCP (SEQ ID NO: 73), GGTGGCPPCP (SEQ ID NO 74), GEPKSSDKTHTCPPCP (SEQ ID NO 75) and GGTGGGEPKSSDKTHTCPPCP (SEQ ID NO 76).

In one aspect, the first heterodimer promoting domain may comprise a Fc region having a CH2 and/or CH3 domain altered to comprise either a knob Fc chain (protuberance) comprising a sequence of SEQ ID NO: 63, or a hole Fc chain (cavity) comprising a sequence of SEQ ID NO: 64. In another aspect, the second heterodimer promoting domain may comprise a Fc region having a CH2 and/or CH3 domain altered to comprise either a knob Fc chain (protuberance) comprising a sequence of SEQ ID NO: 63, if such first heterodimer region comprises a hole Fc chain (cavity), or a hole Fc chain (cavity) comprising a sequence of SEQ ID NO: 64, if such first heterodimer region comprises a knob Fc chain (protuberance).

Table 2 and FIGS. 32A and 32B provide the amino acid sequences for the first and second polypeptide chains of LP-DART proteins with the various P-cadherin and CD3 antibodies described herein. SEQ ID NOs 117-124 provide the corresponding nucleotide sequences are referenced in parenthesis.

TABLE 2

Sequences of the first and second polypeptide chains of LP-DART proteins.

LP-DART
Sequence

35
P-CAD 35VL x CD3-1VH-Knob, SEQ ID NO: 88

QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPD

RFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAVVFGGGTKLTVLGGGSGGGGEV

QLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATY

YADSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTL

VTVSSGCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV

DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

GQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 117)

CD3-1-2VL x P-CAD 35VH-Hole, SEQ ID NO: 89

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPW

TPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLGGGGSGGGG

EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTN

YAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCATIDTASAFDIWGQGTMVTVSSG

CPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP

QVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL

VSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 118)

153
P-CAD 153VL x CD3-1VH-Knob (153 LP-DART B), SEQ ID NO: 90

QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPD

RFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSGVVFGGGTKLTVLGGGSGGGGEV

QLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATY

YADSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTL

VTVSSGCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV

DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

GQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 119)

CD3-1-2VL x P-CAD 153VH-Hole (153 LP-DART A), SEQ ID NO: 91

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPW

TPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLGGGGSGGGG

EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTN

YAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCATIDTANAFGIWGQGTMVTVSSG

CPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP

QVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL

VSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 120)

154
P-CAD 154VL x CD3-1VH-Knob, SEQ ID NO: 92

QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPD

RFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSSYVFGTGTKVTVLGGGSGGGGEV

QLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATY

YADSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTL

VTVSSGCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV

DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

GQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 121)

CD3-1-2VL x P-CAD 154VH-Hole, SEQ ID NO: 93

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPW

TPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLGGGGSGGGG

EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTN

YAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCATIDTATAFDIWGQGTMVTVSSG

CPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP

QVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL

VSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 122)

177
P-CAD 177VL x CD3-1VH-Knob, SEQ ID NO: 94

QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPD

RFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAYVFGTGTKVTVLGGGSGGGGEV

QLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATY

YADSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTL

VTVSSGCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV

DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

GQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 123)

CD3-1-2VL x P-CAD 177VH-Hole, SEQ ID NO: 95

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPW

TPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLGGGGSGGGG

EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTN

YAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCATIDTANAFDIWGQGTMVTVSSG

CPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP

QVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL

VSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 124)

Effector Null Mutations in Human IgG1 CH2-CH3 and Removal of C-Terminal Lys.

The Fc region of human IgG1 was modified to remove the C-terminal lysine residue often cleaved post-translationally, as well as, introducing mutations L234A, L235A and G237A using standard primer-directed PCR mutagenesis to oblate effector function due to binding to FcγRIII (effector null phenotype).

Knobs-in-Holes Mutations in Human IgG1 CH2-CH3

‘Knobs-in-holes’ is an effective design strategy known in the art for engineering antibody heavy chain homodimers for heterodimerization. In this approach a ‘knob’ variant was obtained by replacement of a small amino acid with a larger one in one chain of the Fc region of IgG1 e.g. Y349C and T366W. The ‘knob’ was designed to insert into a ‘hole’ in the CH3 domain of the complimentary chain of the Fc region created by replacement of a large residue with a smaller one e.g. S354C, T366S, L368A and Y407V.

The knob-in-hole designation is analogous to the protuberance and cavity designation and may be used interchangeably.

A “protuberance” or “knob” refers to at least one amino acid side chain which projects from the interface of the first polypeptide and is therefore positionable in a compensatory cavity in the adjacent interface (i.e. the interface of the second polypeptide) so as to stabilize the heterodimer, and thereby favor heterodimer formation over homodimer formation, for example. The protuberance may exist in the original interface or may be introduced synthetically (e.g. by altering nucleic acid encoding the interface). Normally, nucleic acid encoding the interface of the first polypeptide is altered to encode the protuberance. To achieve this, the nucleic acid encoding at least one “original” amino acid residue in the interface of the first polypeptide is replaced with nucleic acid encoding at least one “import” amino acid residue which has a larger side chain volume than the original amino acid residue. The upper limit for the number of original residues which are replaced is the total number of residues in the interface of the first polypeptide.

Certain import residues for the formation of a protuberance are generally naturally occurring amino acid residues and are preferably selected from arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W).

A “cavity” or “hole” refers to at least one amino acid side chain which is recessed from the interface of the second polypeptide and therefore accommodates a corresponding protuberance on the adjacent interface of the first polypeptide. The cavity may exist in the original interface or may be introduced synthetically (e.g. by altering nucleic acid encoding the interface). Normally, nucleic acid encoding the interface of the second polypeptide is altered to encode the cavity. To achieve this, the nucleic acid encoding at least one “original” amino acid residue in the interface of the second polypeptide is replaced with DNA encoding at least one “import” amino acid residue which has a smaller side chain volume than the original amino acid residue. The upper limit for the number of original residues which are replaced is the total number of residues in the interface of the second polypeptide.

Certain import residues for the formation of a cavity are usually naturally occurring amino acid residues and are preferably selected from alanine (A), serine (S), threonine (T) and valine (V).

An “original” amino acid residue is one which is replaced by an “import” residue which can have a smaller or larger side chain volume than the original residue. The import amino acid residue can be a naturally occurring or non-naturally occurring amino acid residue, but preferably is the former. “Naturally occurring” amino acid residues are those residues encoded by the genetic code. By “non-naturally occurring” amino acid residue is meant a residue which is not encoded by the genetic code, but which is able to covalently bind adjacent amino acid residue(s) in the polypeptide chain. Examples of non-naturally occurring amino acid residues are norleucine, ornithine, norvaline, homoserine and other amino acid residue analogues such as those described in Ellman et al., Meth. Enzym. 202:301-336 (1991), for example. The method of the instant invention involves replacing at least one original amino acid residue, but more than one original residue can be replaced. Normally, no more than the total residues in the interface of the first or second polypeptide may comprise original amino acid residues which are replaced.

The protuberance or knob is “positionable” in the cavity or hole which means that the spatial location of the protuberance and cavity on the interface of the first polypeptide and second polypeptide respectively and the sizes of the protuberance and cavity are such that the protuberance may be located in the cavity without significantly perturbing the normal association of the first and second polypeptides at the interface. Since protuberances such as Tyr, Phe and Trp do not typically extend perpendicularly from the axis of the interface, the alignment of a protuberance with a corresponding cavity relies on modeling the protuberance/cavity pair based upon a three-dimensional structure such as that obtained by X-ray crystallography or nuclear magnetic resonance (NMR). This can be achieved using widely accepted techniques in the art.

Therefore, to favor heterodimerization of the resultant Fc regions, complimentary mutations were introduced such that each Fc chain would carry one set of mutations, Y349C and T366W for the knob (or protuberance) Fc chain (SEQ ID NO: 63), or S354C, T366S, L368A and Y407V for the hole (or cavity) Fc chain (SEQ ID NO: 64), as provided in Table 3. When co-transfected into a suitable mammalian host the DNA encoding the amino acid sequences of SEQ ID NOS: 63 and 64 produce an Fc region that is predominantly bispecific heterodimeric possessing one knob (or protuberance) Fc chain associated with one hole (or cavity) Fc chain.

TABLE 3

Sequences of Fc regions.

SEQ ID NO:
Sequence

SEQ ID NO: 63
APEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

Knob Fc chain
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG

Mutations
QPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP

PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

SEQ ID NO: 64
APEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

Hole Fc chain
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG

Mutations
QPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTP

PVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Using overlapping primer extension PCR, a 5′ BspEI restriction enzyme recognition sequence TCCGGA (SEQ ID NO 67) was introduced for use in subcloning the knob (protuberance) and hole (cavity) Fc chains to each DART chain containing a 3′ BspEI site. This restriction site codes for an in-frame insertion of the amino acids serine and glycine (ser-gly) at the DART-Fc junction. Anti-P-cadherin/anti-CD3 LP-DART proteins were constructed as described in Example 6.

The anti-P-cadherin/anti-CD3 LP-DART proteins of the present invention are stable against aggregation in thermal stability studies and, potent bispecific diabody-Fc fusions targeting both human CD3 and P-Cadherin. The knob-in-hole Fc domain allows for improved expression in CHO cells and improved purification resulting in high purity of desired heterodimer. The mutations engineered within the Fc domain abrogate FcγR binding thus potentially avoiding ADCC mediated T cell depletion. Further, the incorporation of the Fc domain to a DART protein enhances stability of the molecule as shown by differential scanning calorimetry (DSC) and forced aggregation assays when compared to clinically approved monoclonal antibodies. Additionally, the Fc domain improves the pharmacokinetic profile.

MP3 DART

FIGS. 6 and 7 are schematic representations of an N-terminal MP3-DART and a C-terminal MP3-DART bispecific heterodimeric diabody, respectively. Such MP3-DART proteins are composed of three polypeptide chains: a first polypeptide chain (1), a second polypeptide chain (2) and a third polypeptide chain (3).

In an aspect of the present invention, an N-terminal MP3-DART, as shown in FIG. 6, may comprises, in an N-terminal to C-terminal orientation, the first polypeptide chain (1) comprising: a Domain 1, comprising (i) a sub-Domain 1A which comprises a VL binding region of either an anti-P-cadherin antibody (P-CAD VL) or an anti-CD3 antibody (CD3 VL), (ii) a sub-Domain 1B which comprises a VH binding region of either an anti-P-cadherin antibody (P-CAD VH), if such sub-Domain 1A comprises a CD3 VL, or an anti-CD3 antibody (CD3 VH), if such sub-Domain 1A comprises a P-CAD VL, and the sub-Domain 1A and sub-Domain 1B may be covalently linked by a glycine-serine linker (Linker 1) such that the sub-Domain 1A and sub-Domain 1B do not associate to form an epitope binding site; and may comprise a first heterodimer promoting domain comprising (i) a cysteine linker (Linker 2) on the sub-Domain 1B, (ii) a cysteine Peptide 1 on the Linker 2 and (iii) a Fc region having a CH2 and/or CH3 domain of an IgG domain covalently linked to the Peptide 1.

In another aspect of the present invention, a C-terminal MP3-DART, as shown in FIG. 7, may comprise, in an N-terminal to C-terminal orientation, the first polypeptide chain (1) comprising: a first heterodimer promoting domain, comprising (i) a cysteine Peptide 1, (ii) a Fc region having CH2 and/or CH3 domain of an IgG Fc domain, and (iii) a Linker 3 on the Fc region; and a Domain 1, comprising (i) a sub-Domain 1A which comprises a VL binding region of either an anti-P-cadherin antibody (P-CAD VL) or an anti-CD3 antibody (CD3 VL), (ii) a sub-Domain 1B which comprises a VH binding region of either an anti-P-cadherin antibody (P-CAD VH), if such sub-Domain 1A comprises a CD3 VL, or an anti-CD3 antibody (CD3 VH), if such sub-Domain 1A comprises a P-CAD VL, and the sub-Domain 1A and sub-Domain 1B may be covalently linked by a glycine-serine linker (Linker 1) such that the sub-Domain 1A and sub-Domain 1B do not associate to form an epitope binding site, and (iv) a cysteine linker (Linker 2) on the sub-Domain 1B.

In another aspect, the first polypeptide chain (1) of the N-terminal MP3-DART or C-terminal MP3-DART, may comprises: a Domain 1, comprising (i) a sub-Domain 1A which comprises a P-CAD VH or a CD3 VH, (ii) a sub-Domain 1B which comprises either a P-CAD VL, if such sub-Domain 1A comprises a CD3 VH, or a CD3 VL, if such sub-Domain 1A comprises a P-CAD VH.

In one aspect, the first polypeptide chain (1) of the N-terminal MP3-DART or C-terminal MP3-DART may comprise: a Domain 1, comprising (i) a sub-Domain 1A which comprises a P-CAD VL and (ii) a sub-Domain 1B having a CD3 VH. In another aspect, the first polypeptide chain (1) may comprise: a Domain 1, comprising (i) a sub-Domain 1A which comprises a CD3 VL and (ii) a sub-Domain 1B which comprises a P-CAD VH. In a further aspect, the first polypeptide chain (1) may comprise: a Domain 1, comprising (i) a sub-Domain 1A which comprises a P-CAD VH and (ii) a sub-Domain 1B which comprises a CD3 VL. In another aspect, the first polypeptide chain (1) may comprise: a Domain 1, comprising (i) a sub-Domain 1A having a CD3 VH and (ii) a sub-Domain 1B which comprises a P-CAD VL.

In one aspect, the second polypeptide chain (2) of an N-terminal MP3-DART or C-terminal MP3-DART may comprise: a Domain 2, comprising (i) a sub-Domain 2B which comprises a VL binding region of either an anti-P-cadherin antibody (P-CAD VL) or an anti-CD3 antibody (CD3 VL) depending upon the VL domain selected for the first polypeptide chain of the MP3-DART, (ii) a sub-Domain 2A which comprises a VH binding region of either an anti-P-cadherin antibody (P-CAD VH), if such sub-Domain 2B comprises a CD3 VL, or an anti-CD3 antibody (CD3 VH), if such sub-Domain 2B comprises a P-CAD VL, and the sub-Domain 2B and sub-Domain 2A may be covalently linked by a glycine-serine linker (Linker 1) such that the sub-Domain 2B and sub-Domain 2A do not associate to form an epitope binding site; and may comprise a second heterodimer promoting domain comprising a cysteine linker (Linker 2) on sub-Domain 2A.

In one aspect, the second polypeptide chain (2) may comprise: a Domain 2, comprises (i) a sub-Domain 2B which comprises a P-CAD VL and (ii) a sub-Domain 2A having a CD3 VH. In another aspect, the second polypeptide chain (2) may comprise: a Domain 2, comprising (i) a sub-Domain 2B which comprises a CD3 VL and (ii) a sub-Domain 2A which comprises a P-CAD VH. In a further aspect, the second polypeptide chain (2) may comprise: a Domain 2, comprising (i) a sub-Domain 2B which comprises a P-CAD VH and (ii) a sub-Domain 2A which comprises a CD3 VL. In another aspect, the second polypeptide chain (2) may comprise: a Domain 2, comprising (i) a sub-Domain 2B which comprises a CD3 VH and (ii) a sub-Domain 2A which comprises a P-CAD VL.

In one aspect, the third polypeptide chain (3) of an N-terminal MP3-DART or C-terminal MP3-DART, in an N-terminal to C-terminal orientation, may comprise: a third heterodimer promoting domain comprising (i) a cysteine Peptide 1 and (ii) a Fc region having a CH2 and/or CH3 domain of an IgG Fc domain.

In one aspect of the invention, the sub-Domain 1A and the sub-Domain 2A may associate to form a first binding site, having a VL/VH region, that binds to an epitope on CD3 or P-cadherin, and the sub-Domain 1B and the sub-Domain 2B may associate to form a second binding site, having a VL/VH region, that binds to an epitope on P-cadherin or CD3.

The first polypeptide chain (1) and second polypeptide chain (2) may be covalently bonded to one another, for example at the cysteine residues located within the first and second heterodimer promoting domains. In one aspect, the cysteine residue in Linker 2 on the first heterodimer promoting domain and the cysteine residue in the Linker 2 on the second heterodimer promoting region may be linked by at least one disulfide bond.

The first heterodimer promoting domain of the first polypeptide chain (1) and the third heterodimer promoting domain of the third polypeptide chain (3) may associate with one another to form an immunoglobulin Fc region. The first polypeptide chain (1) and third polypeptide chain (3) may be covalently bonded to one another, for example at cysteine residues located within the first and third heterodimer promoting domains. In another aspect, the cysteine residue in Peptide 1 on the first heterodimer promoting domain and the cysteine residue in the Peptide 1 on the third heterodimer promoting domain may be linked by at least one disulfide bond.

In an aspect of the present invention, the cysteine linker (Linker 2) may comprise a sequence selected from the group consisting of GFNRGEC (SEQ ID NO: 70) and GVEPKSC (SEQ ID NO: 71). In another aspect the cysteine linker (Linker 2) of the first heterodimer promoting domain may comprise the sequence of GFNRGEC (SEQ ID NO: 70) and the cysteine linker (Linker 2) of the second heterodimer promoting domain may comprise the sequence of GVEPKSC (SEQ ID NO: 71).

In certain aspects, the Linker 3 of the third heterodimer promoting region may comprise the sequence of KAPSSSPME (SEQ ID NO 77).

In certain aspects, the Peptide 1 on the first and third heterodimer promoting regions may comprise a truncated IgG1 hinge region having the sequence CPPCP (SEQ ID NO 60). In one aspect, the Peptide 1 may comprise, but is not limited to, the sequence of DKTHTCPPCP (SEQ ID NO 96).

The knob and hole strategy described herein may be utilized in the MP3-DART constructs to ensure proper pairing of the first and third polypeptide chains. Similar to the LP-DART described herein, the Fc region of human IgG1 may be modified to remove the C-terminal lysine residue often cleaved post-translationally, as well as, to introduce mutations L234A, L235A and G237A mutations to oblate effector function due to binding to FcγRIII (effector null phenotype). In one aspect a knob is created by modifying a native IgG Fc region to contain the modification T366W. In another aspect a hole is created by modifying a native IgG Fc region to contain the modification T366S, L368A and Y407V. Preferably the first polypeptide chain comprises the knob and the third polypeptide chain comprises the hole. To aid in purifying the MP3-DART™ from any homodimer of the third polypeptide chain, the protein A binding site of the CH2 and CH3 domains of the third polypeptide chain is preferably mutated by amino acid substitution at position 435 (H435R). Thus, any homodimer of the third polypeptide chain does not bind to protein A, whereas the MP3-DART™ retains its ability to bind protein A via the protein A binding site on the first polypeptide chain. Therefore, the knob (or protuberance) Fc chain may have the sequences of SEQ ID NO: 65 and the hole (or cavity) Fc chain may have sequence of SEQ ID NO: 66, as provided in Table 4.

TABLE 4

Sequences of Fc regions.

SEQ ID NO:
Sequence

SEQ ID 65
APEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

Knob Fc chain
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG

Mutations
QPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTP

PVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK

SEQ ID 66
APEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

Hole Fc chain
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG

mutations
QPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP

PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Table 5 describes the first (1), second (2) and third (3) polypeptide chains of exemplary N-terminal MP3-DART and C-terminal MP3-DART proteins that were constructed with the various P-cadherin and CD3-1 antibodies described herein. SEQ ID NOs.125-158 provide the amino acid and nucleotide sequences (referenced in parenthesis) for polypeptide chains 1, 2 and 3 for the N-terminal and C-terminal MP3-DART bispecific heterodimeric diabodies described in Table 5.

TABLE 5

First, second and third polypeptide chains of MP3- DART proteins.

First
Second
Third

polypeptide
polypeptide
polypeptide

chain (1)
chain (2)
chain (3)

sequences
sequences
sequences

C-terminal
P-CAD
CD3-1-2VLXP-
SEQ ID NO:

MP3-DART
35VLXCD3-1VH
CAD 35VH
125

P-CAD 35
SEQ ID NO:
SEQ ID NO:
(SEQ ID NO:

VH and VL
127
129
126)

(SEQ ID NO:
(SEQ ID NO:

128)
130)

C-terminal
P-CAD
CD3-1-2VLXP-
SEQ ID NO:

MP3-DART
153VLXCD3-1VH
CAD 153VH
125

P-CAD153
SEQ ID NO:
SEQ ID NO:
(SEQ ID NO:

VH and VL
131
133
126)

(SEQ ID NO:
(SEQ ID NO:

132)
134)

C-terminal
P-CAD
CD3-1-2VLXP-
SEQ ID NO:

MP3-DART
154VLXCD3-1VH
CAD 154VH
125

P-CAD154
SEQ ID NO:
SEQ ID NO:
(SEQ ID NO:

VH and VL
135
137
126)

(SEQ ID NO:
(SEQ ID NO:

136)
138)

C-terminal
P-CAD
CD3-1-2VLXP-
SEQ ID NO:

MP3-DART
177VLXCD3-1VH
CAD 177VH
125

P-CAD177
SEQ ID NO:
SEQ ID NO:
(SEQ ID NO:

VH and VL
139
141
126)

(SEQ ID NO:
(SEQ ID NO:

140)
142)

N-terminal
P-CAD
CD3-1-2VLXP-
SEQ ID NO:

MP3-DART
35VLXCD3-1VH
CAD 35VH
125

P-CAD 35
SEQ ID NO:
SEQ ID NO:
(SEQ ID NO:

VH and VL
143
145
126)

(SEQ ID NO:
(SEQ ID NO:

144)
146)

N-terminal
P-CAD
CD3-1-2VLXP-
SEQ ID NO:

MP3-DART
153VLXCD3-1VH
CAD 153VH
125

P-CAD 153
SEQ ID NO:
SEQ ID NO:
(SEQ ID NO:

VH and VL
147
149
126)

(SEQ ID NO:
(SEQ ID NO:

148)
150)

N-terminal
P-CAD
CD3-1-2VLXP-
SEQ ID NO:

MP3-DART
154VLXCD3-1VH
CAD 154VH
125

P-CAD 154
SEQ ID NO:
SEQ ID NO:
(SEQ ID NO:

VH and VL
151
153
126)

(SEQ ID NO:
(SEQ ID NO:

152)
154)

N-terminal
P-CAD
CD3-1-2VLXP-
SEQ ID NO:

MP3 DART
177VLXCD3-1VH
CAD 177VH
125

P-CAD 177
SEQ ID NO:
SEQ ID NO:
(SEQ ID NO:

VH and VL
155
157
126)

(SEQ ID NO:
(SEQ ID NO:

156)
158)

Engineered Cysteine Residues

In certain aspects, each polypeptide chain of the bispecific heterodimeric diabody may be engineered to comprise at least one cysteine residue that may interact with a counterpart cysteine residue on another polypeptide chain of the invention to form an inter-chain disulfide bond. The inter-chain disulfide bonds may serve to stabilize the diabody molecule, improving expression and recovery in recombinant systems, resulting in a stable and consistent formulation, as well as, improving the stability of the isolated and/or purified product in vivo. The cysteine residue or residues may be introduced as a single amino acid or as part of larger amino-acid sequence, e.g. hinge region, in any portion of the polypeptide chain. In a specific aspect, at least one cysteine residue is engineered to occur at the C-terminus of the polypeptide chain.

Cancer Treatment

The invention encompasses methods and compositions for treatment, prevention or management of cancer in a subject, comprising administering to the subject a therapeutically effective amount of a bispecific heterodimeric diabody engineered in accordance with the invention, which molecule further binds a cancer antigen. Molecules of the invention may be particularly useful for the prevention, inhibition, reduction of growth and/or regression of primary tumors and metastasis of cancer cells. Although not intending to be bound by a particular mechanism of action, molecules of the invention may mediate effector function which may result in tumor clearance, tumor reduction or a combination thereof.

Accordingly, the invention provides methods of preventing or treating cancer characterized by a P-cadherin cancer antigen by engineering the bispecific heterodimeric diabody to immunospecifically recognize the P-cadherin cancer antigen and a CD3 antigen on T cells. The bispecific heterodimeric diabodies that have been engineered according to the invention are useful for prevention or treatment of cancer, since they have a cytotoxic activity by anti-CD3 induced activated killer T cells.

Therefore an aspect of the present invention provides a method of treating P-cadherin positive cancer in a patient in need thereof, wherein the cancer is selected from the group consisting of breast, colorectal, ovarian, gastric, thyroid, prostate, cervical, pancreatic, lung (including but not limited to non-small cell lung cancer and small cell lunch cancer), bladder, liver, endometrial, head and neck, testicular, and glioblastoma cancer.

The invention also provides a bispecific heterodimeric diabody of the invention for use in the method of treating cancer defined herein. In addition, the invention provides the use of a bispecific heterodimeric diabody of the invention in the manufacture of a medicament for the treatment of cancer as defined herein.

In a specific aspect, a bispecific heterodimeric diabody of the invention inhibits or reduces the growth of cancer cells by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% relative to the growth of cancer cells in the absence of the bispecific heterodimeric diabody of the invention.

In a specific aspect, a bispecific heterodimeric diabody of the invention kills cells or inhibits or reduces the growth of cancer cells at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% better than in the absence of the bispecific heterodimeric diabody of the invention.

The invention further encompasses administering the molecules of the invention in combination with other therapies known to those skilled in the art for the treatment or prevention of cancer including but not limited to, current standard and experimental chemotherapies, biological therapies, immunotherapies, radiation therapies, or surgery. In some aspects, the molecules of the invention may be administered in combination with a therapeutically or prophylactically effective amount of one or more agents, therapeutic antibodies or other agents known to those skilled in the art for the treatment and/or prevention of cancer.

Accordingly, methods for treating cancer include administering to a patient in need thereof an effective amount of a bispecific heterodimeric diabody of the present invention in combination with a chemotherapeutic agent. Such combination treatment may be administered separately, sequentially, or simultaneously. Suitable chemotherapeutic agents include, but are not limited to, 5-fluorouracil, asparaginase, BCNU, bleomycin, calicheamicin, camptothecins, carboplatin, cisplatin, cyclophosphamide, cytarabine, cytoxan, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, etoposide, hydroxyurea, idarubicin, ifosfamide, irinotecan, L-asparaginase, mercaptopurine, methotrexate, mitomycin, mitoxantrone, nitrogen mustards, nitrosoureas, paclitaxel, plicamycin, procarbizine, taxol, thioguanine, topotecan, vinblastine, vincristine, and vinorelbine,

The dosage amounts and frequencies of administration provided herein are encompassed by the terms therapeutically effective and prophylactically effective. The dosage and frequency further may vary according to factors specific for each patient depending on the specific therapeutic or prophylactic agents administered, the severity and type of cancer, the route of administration, as well as age, body weight, response, and the past medical history of the patient. Suitable regimens may be selected by one skilled in the art by considering such factors and by following, for example, dosages reported in the literature and recommended in the Physician's Desk Reference (56^thed., 2002).

The bispecific heterodimeric diabodies of the present invention may be in the form of a pharmaceutical composition for administration that are formulated to be appropriate for the selected mode of administration, and pharmaceutically acceptable diluent or excipients, such as buffers, surfactants, preservatives, solubilizing agents, isotonicity agents, stabilizing agents, carriers, and the like. Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton Pa., 18^thed., 1995, provides a compendium of formulation techniques as are generally known to practitioners.

These pharmaceutical compositions may be administered by any means known in the art that achieve the generally intended purpose to treat cancer. The route of administration may be parenteral, defined herein as referring to modes of administration that include but not limited to intravenous, intramuscular, intraperitoneal, subcutaneous, and intraarticular injection and infusion. The dosage administered may be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

Compositions within the scope of the invention include all compositions wherein a bispecific heterodimeric diabody is present in an amount that is effective to achieve the desired medical effect for treating cancer. While individual needs may vary from one patient to another, the determination of the optimal ranges of effective amounts of all of the components is within the ability of the clinician of ordinary skill.

The present invention provides kits that may be used in the above methods. In one aspect, a kit comprises a bispecific heterodimeric diabody of the invention. In another aspect, a kit further comprises one or more other prophylactic or therapeutic agents useful for the treatment of cancer, in one or more containers. In certain aspects, the other prophylactic or therapeutic agent is a chemotherapeutic. In other aspects, the prophylactic or therapeutic agent is a biological or hormonal therapeutic.

Several aspects of the pharmaceutical compositions, prophylactic, or therapeutic agents of the invention are preferably tested in vitro, in a cell culture system, and in an animal model organism, such as a rodent animal model system, for the desired therapeutic activity prior to use in humans.

Toxicity and efficacy of the prophylactic and/or therapeutic protocols of the instant invention maybe determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀(the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it may be expressed as the ratio LD₅₀/ED₅₀. Prophylactic and/or therapeutic agents that exhibit large therapeutic indices are preferred.

Further, any assays known to those skilled in the art may be used to evaluate the prophylactic and/or therapeutic utility of the therapies or combinatorial therapies disclosed herein for treatment or prevention of cancer.

Biological Deposits

Representative materials of the present invention were deposited in the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, USA (ATCC) on Dec. 20, 2013. Vector 153 LP-DART A having ATCC Accession No. PTA-120809 comprises a recombinant human DNA insert encoding P-cadherin LP-DART (Fc-fusion diabody) Chain A, and vector 153 LP-DART B having ATCC Accession No. PTA-120810 comprises a recombinant human DNA insert encoding P-cadherin LP-DART (Fc-fusion diabody) Chain B. The deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and Regulations thereunder (Budapest Treaty). This assures maintenance of a viable culture of the deposit for 30 years from the date of deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Pfizer Inc and ATCC, which assures permanent and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 U.S.C. Section 122 and the Commissioner's rules pursuant thereto (including 37 C.F.R. Section 1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

The following examples of specific aspects for carrying out the present invention are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

Example 1
Generation of P-Cadherin Antibodies and Bispecific Heterodimeric Diabodies

Bispecific heterodimeric diabodies were constructed to assess the recombinant production, purification and binding characteristics of each. The affinity purified bispecific heterodimeric diabodies were produced by the recombinant expression systems described herein. ELISA and SPR analysis further revealed that the covalent bispecific heterodimeric diabodies exhibited affinity for both target antigens, P-cadherin and CD-3, and could bind both antigens simultaneously.

A. Phage Display

Single chain fragment variable (scFv) moieties that bind to the extracellular domain (ECD) of P-cadherin were identified with a phage display library composed of scFv derived from non-immunized human donors utilizing techniques known in the art. The binding of the scFvs expressed on the surface of phage was measured on P-cadherin protein constructs and P-cadherin-expressing cells by standard ELISA techniques.

B. Sequencing Analysis

ScFv fragments were sequenced with primers 5′ GGAGATTTTCAACGTGAA 3′ (SEQ ID NO: 58) and 5′ CTCTTCTGAGATGAGTTTTTG 3′ (SEQ ID NO: 59) as either unpurified bacterial glycerol stock or as purified plasmid using conventional methods.

C. Conversion to DART

ScFv fragments that demonstrated strong recombinant P-cadherin or cell surface binding (>3× background or negative cell binding) were selected for subcloning into DART proteins. DART design and cloning methods were previously described (Johnson et al, J Mol Biol, 399:436-449, 2010; Moore et al, Blood, 117(17):4542-51, 2011). Methods for adapting novel anti-P-cadherin scFv from the phage display library are as follows: fragments were amplified by standard polymerase chain reaction (PCR) with primers incorporating BamHI/BspEI for VH and BssHII/BamHI for VL. Fragments were cut with corresponding restriction enzymes according to manufacturer's specifications (New England Biolabs).

To generate a DART expression vector, anti-P-cadherin VH (P-CAD VH) or VL (P-CAD VL) scFv were gel purified (QIAGEN® Gel Purification Kit) and ligated separately into mammalian expression vectors containing either the anti-CD3 VL (CD3 VL) or VH (CD3 VH) scFv.

For the generation of a VF-DART, as shown in FIGS. 2 and 3, the DART expression vector further comprised a first and second heterodimer promoting domain each having a cysteine linker (Linker 2), such as GFNRGEC (SEQ ID NO: 70) or GVEPKSC (SEQ ID NO: 77).

To generate an EK-DART, as shown in FIG. 4, the DART expression vector further comprised a first and second heterodimer promoting domain each having a cysteine linker (Linker 2), such as GGCGGG (SEQ ID NO: 72), and either an E-coil domain (SEQ ID NO: 61) or a K-coil domain (SEQ ID NO: 62).

To generate a LP-DART, as shown in FIGS. 1 and 5, the DART expression vectors further comprised a first and second heterodimer promoting domain each having a cysteine linker (Linker 2), such as GCPPCP (SEQ ID NO: 73), GGTGGCPPCP (SEQ ID NO 74), GEPKSSDKTHTCPPCP (SEQ ID NO 75), or GGTGGGEPKSSDKTHTCPPCP (SEQ ID NO 76), and either a “knob” Fc chain (SEQ ID NO: 63) or a “hole” Fc chain (SEQ ID NO: 64).

In addition to novel anti-P-cadherin scFv from the phage display library, the VH and VL domains from the anti-P-cadherin clinical mAb PF-03732010 (Zhang et al, Clin Cancer Res. 16(21), 5177-5188, 2010) were also sub-cloned into the various DART expression vectors described above to act as a positive control.

The DART proteins were then transiently expressed in mammalian cells by co-transfecting expression vectors into HEK 293 cells. DART proteins retaining P-cadherin affinity were affinity purified using anti-coiled-coil mAb 15F1 coupled to CNBr-activated sepharose 4B (GE Healthcare, Piscataway, N.J.). The biochemical properties of the purified DART proteins were assessed by SDS-PAGE and SEC.

Table 6 provides the VL and VH amino acid sequences for the anti-P-cadherin scFv clones generated by the techniques described herein. CDRs for clones 20 and 30 are underlined according to Kabat. SEQ ID NOs 97-116 provide the corresponding nucleotide sequences referenced in parenthesis.

TABLE 6

P-CAD VL and VH sequences.

SEQ ID NO.

P-CAD Clone #
SEQUENCE

SEQ ID NO: 1
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKR

P-CAD 33 VL
PSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAYVFGTGTKVTVL

SEQ ID NO: 2
QMQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISA

P-CAD 33 VH
YNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCALIGSGVAFDIW

GQGTMVTVSS

SEQ ID NO: 3
QSVVTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKR

P-CAD 34 VL
PSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

SEQ ID NO: 4
EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAY

P-CAD 34 VH
NGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCATIDTASAFDIWG

QGTMVTVSS

SEQ ID NO: 5
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKR

P-CAD 35 VL
PSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAVVFGGGTKLTVL

(SEQ ID NO: 97)

SEQ ID NO: 6
EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAY

P-CAD 35 VH
NGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCATIDTASAFDIWG

(SEQ ID NO: 98)
QGTMVTVSS

SEQ ID NO: 7
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKR

P-CAD 153 VL
PSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSGVVFGGGTKLTVL

(SEQ ID NO: 99)

SEQ ID NO: 8
EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAY

P-CAD 153 VH
NGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCATIDTANAFGIWG

(SEQ ID NO: 100)
QGTMVTVSS

SEQ ID NO: 9
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKR

P-CAD 154 VL
PSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSSYVFGTGTKVTVL

(SEQ ID NO: 101)

SEQ ID NO: 10 P-
EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAY

CAD 154 VH
NGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCATIDTATAFDIWG

(SEQ ID NO: 102)
QGTMVTVSS

SEQ ID NO: 11
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKR

P-CAD 163 VL
PSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAYVFGTGTKVTVL

(SEQ ID NO: 103)

SEQ ID NO: 12
EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGFSWMRQAPGQGLEWMGWISA

P-CAD 163 VH
YNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCATIDTASAFDIW

(SEQ ID NO: 104)
GQGTMVTVSS

SEQ ID NO: 13
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKR

P-CAD 165 VL
PSGIPDRFSGSKSGTSATLGITGLQAGDEADYYCGTWDSSLSAYVFGTGTKVTVL

(SEQ ID NO: 105)

SEQ ID NO: 14
EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAY

P-CAD 165 VH
NGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCATIDTANAFDIWG

(SEQ ID NO: 106)
QGTMVTVSS

SEQ ID NO: 15
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKR

P-CAD 177 VL
PSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAYVFGTGTKVTVL

(SEQ ID NO: 107)

SEQ ID NO: 16
EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAY

P-CAD 177 VH
NGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCATIDTANAFDIWG

(SEQ ID NO: 108)
QGTMVTVSS

SEQ ID NO: 17
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKR

P-CAD 178 VL
PSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAVVFGGGTKLTVL

(SEQ ID NO: 109)

SEQ ID NO: 18
EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAY

P-CAD 178 VH
NGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCATIDTANAFDIWG

(SEQ ID NO: 110)
QGTMVTVSS

SEQ ID NO: 19
QSVLTQPPSVSAAPGQKVTISCSGSRSNIGNNYVSWYQQLPGTAPKLLIYDSNKR

P-CAD 179 VL
PSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSSWVFGGGTKLTVL

(SEQ ID NO: 111)

SEQ ID NO: 20
EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAY

P-CAD 179 VH
NGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCATIDTANAFDIWG

(SEQ ID NO: 112)
QGTMVTVSS

SEQ ID NO: 21
QSVVTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKR

P-CAD 180 VL
PSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAYVFGTGTKVTVL

(SEQ ID NO: 113)

SEQ ID NO: 22
EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAY

P-CAD 180 VH
NGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCATIDTANAFDIWG

(SEQ ID NO: 114)
QGTMVTVSS

SEQ ID NO: 23
QSVVTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKR

P-CAD 281 VL
PSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

(SEQ ID NO: 115)

SEQ ID NO: 24
EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAY

P-CAD 281 VH
NGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCATINAPNNFDIWG

(SEQ ID NO: 116)
QGTMVTVSS

SEQ ID NO: 236
AIQMTQSPSSLSASVGDRVTITCRASQSIGKYLNWYQQIPGKAPKLLIYTASTLQTG

P-CAD 20 VL
VPSRFSGSGSGTDFTLTIVSLQPEDFATYYCQQSFNTPRTFGQGTKVEIK

SEQ ID NO: 237
EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGRINP

P-CAD 20 VH

NSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAKGSGSGAFDI

WGQGTMVTVSS

SEQ ID NO: 238
DIVMTQSPLSLPVTLGQPASISCRSSQSLVHSDGNTYLHWFQQRPGQSPRRLIYK

P-CAD 30 VL

VSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPLFGQGTKV

EIK

SEQ ID NO: 239
EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISY

P-CAD 30 VH

DGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSSDRTFDYW

GQGTLVTVSS

Tables 7a and 7b provide the CDR sequences for the anti-P-cadherin VH scFv clones generated, using the Kabat and AbM numbering designations, respectively.

TABLE 7a

P-CAD VH CDR sequences using Kabat numbering.

P-CAD

scFv

Clone

#
P-CAD VH CDR1
P-CAD VH CDR2
P-CAD VH CDR3

33
SYGIS
WISAYNGNTNYAQKLQG
IGSGVAFDI

SEQ ID NO: 25
SEQ ID NO: 26
SEQ ID NO: 27

34
SYGIS
WISAYNGNTNYAQKLQG
IDTASAFDI

SEQ ID NO: 25
SEQ ID NO: 26
SEQ ID NO: 28

35
SYGIS
WISAYNGNTNYAQKLQG
IDTASAFDI

SEQ ID NO: 25
SEQ ID NO: 26
SEQ ID NO: 28

153
SYGIS
WISAYNGNTNYAQKLQG
IDTANAFGI

SEQ ID NO: 25
SEQ ID NO: 26
SEQ ID NO: 29

154
SYGIS
WISAYNGNTNYAQKLQG
IDTATAFDI

SEQ ID NO: 25
SEQ ID NO: 26
SEQ ID NO: 30

163
SYGIS
WISAYNGNTNYAQKLQG
IDTASAFDI

SEQ ID NO: 25
SEQ ID NO: 26
SEQ ID NO: 28

165
SYGIS
WISAYNGNTNYAQKLQG
IDTANAFDI

SEQ ID NO: 25
SEQ ID NO: 26
SEQ ID NO: 31

177
SYGIS
WISAYNGNTNYAQKLQG
IDTANAFDI

SEQ ID NO: 25
SEQ ID NO: 26
SEQ ID NO: 31

178
SYGIS
WISAYNGNTNYAQKLQG
IDTANAFDI

SEQ ID NO: 25
SEQ ID NO: 26
SEQ ID NO: 31

179
SYGIS
WISAYNGNTNYAQKLQG
IDTANAFDI

SEQ ID NO: 25
SEQ ID NO: 26
SEQ ID NO: 31

180
SYGIS
WISAYNGNTNYAQKLQG
IDTANAFDI

SEQ ID NO: 25
SEQ ID NO: 26
SEQ ID NO: 31

281
SYGIS
WISAYNGNTNYAQKLQG
INAPNNFDI

SEQ ID NO: 25
SEQ ID NO: 26
SEQ ID NO: 32

TABLE 7b

P-CAD VH CDR sequences using AbM numbering.

P-CAD

scFv

Clone #
P-CAD VH CDR1
P-CAD VH CDR2
P-CAD VH CDR3

33
GYTFTSYGIS
WISAYNGNTN
IGSGVAFDI

SEQ ID NO: 33
SEQ ID NO: 34
SEQ ID NO: 27

34
GYTFTSYGIS
WISAYNGNTN
IDTASAFDI

SEQ ID NO: 33
SEQ ID NO: 34
SEQ ID NO: 28

35
GYTFTSYGIS
WISAYNGNTN
IDTASAFDI

SEQ ID NO: 33
SEQ ID NO: 34
SEQ ID NO: 28

153
GYTFTSYGIS
WISAYNGNTN
IDTANAFGI

SEQ ID NO: 33
SEQ ID NO: 34
SEQ ID NO: 29

154
GYTFTSYGIS
WISAYNGNTN
IDTATAFDI

SEQ ID NO: 33
SEQ ID NO: 34
SEQ ID NO: 30

163
GYTFTSYGIS
WISAYNGNTN
IDTASAFDI

SEQ ID NO: 33
SEQ ID NO: 34
SEQ ID NO: 28

165
GYTFTSYGIS
WISAYNGNTN
IDTANAFDI

SEQ ID NO: 33
SEQ ID NO: 34
SEQ ID NO: 31

177
GYTFTSYGIS
WISAYNGNTN
IDTANAFDI

SEQ ID NO: 33
SEQ ID NO: 34
SEQ ID NO: 31

178
GYTFTSYGIS
WISAYNGNTN
IDTANAFDI

SEQ ID NO: 33
SEQ ID NO: 34
SEQ ID NO: 31

179
GYTFTSYGIS
WISAYNGNTN
IDTANAFDI

SEQ ID NO: 33
SEQ ID NO: 34
SEQ ID NO: 31

180
GYTFTSYGIS
WISAYNGNTN
IDTANAFDI

SEQ ID NO: 33
SEQ ID NO: 34
SEQ ID NO: 31

281
GYTFTSYGIS
WISAYNGNTN
INAPNNFDI

SEQ ID NO: 33
SEQ ID NO: 34
SEQ ID NO: 32

Tables 7c provides the CDR sequences for the anti-P-cadherin VL scFv clones generated.

TABLE 7c

P-CAD VL CDR sequences using Kabat/AbM numbering.

P-CAD

scFv

Clone #
P-CAD VL CDR1
P-CAD VL CDR2
P-CAD VL CDR3

33
SGSSSNIGNNYVS
DNNKRPS
GTWDSSLSAYV

SEQ ID NO: 35
SEQ ID NO: 37
SEQ ID NO: 39

34
SGSSSNIGNNYVS
DNNKRPS
GTWDSSLSAWV

SEQ ID NO: 35
SEQ ID NO: 37
SEQ ID NO: 40

35
SGSSSNIGNNYVS
DNNKRPS
GTWDSSLSAVV

SEQ ID NO: 35
SEQ ID NO: 37
SEQ ID NO: 41

153
SGSSSNIGNNYVS
DNNKRPS
GTWDSSLSGVV

SEQ ID NO: 35
SEQ ID NO: 37
SEQ ID NO: 42

154
SGSSSNIGNNYVS
DNNKRPS
GTWDSSLSSYV

SEQ ID NO: 35
SEQ ID NO: 37
SEQ ID NO: 43

163
SGSSSNIGNNYVS
DNNKRPS
GTWDSSLSAYV

SEQ ID NO: 35
SEQ ID NO: 37
SEQ ID NO: 39

165
SGSSSNIGNNYVS
DNNKRPS
GTWDSSLSAYV

SEQ ID NO: 35
SEQ ID NO: 37
SEQ ID NO: 39

177
SGSSSNIGNNYVS
DNNKRPS
GTWDSSLSAYV

SEQ ID NO: 35
SEQ ID NO: 37
SEQ ID NO: 39

178
SGSSSNIGNNYVS
DNNKRPS
GTWDSSLSAVV

SEQ ID NO: 35
SEQ ID NO: 37
SEQ ID NO: 41

179
SGSRSNIGNNYVS
DSNKRPS
GTWDSSLSSWV

SEQ ID NO: 36
SEQ ID NO: 38
SEQ ID NO: 44

180
SGSSSNIGNNYVS
DNNKRPS
GTWDSSLSAYV

SEQ ID NO: 35
SEQ ID NO: 37
SEQ ID NO: 39

281
SGSSSNIGNNYVS
DNNKRPS
GTWDSSLSAWV

SEQ ID NO: 35
SEQ ID NO: 37
SEQ ID NO: 40

Table 8 shows the amino acid mutations found in affinity-matured P-cadherin clones. The data suggests potential effects on VL/VH folding and ligand interaction based on the solved crystal structure. Mutations within the CDR binding pocket showed potential for improved ligand interaction. Mutations found outside the CDR binding pocket suggested a role for improved VL/VH folding.

TABLE 8

Amino acid mutations of affinity-matured P-cadherin clones.

P-CAD
VL mutations P-CAD 35
VH mutations P-CAD 35

Clone #
Sequential
VL chain
VL Chothia
Sequential
VH chain
VH Chothia

153
A98G
A98G
A95BG
S103N,
S221N,
S99N,

D106G
D224G
D101G

154
A98S (V99Y,
A98S V99Y
A95BS V96Y
S103T
S221T
S99T

G103T,
G103T L107V
G100T L104V

L107V)

165
T81A (V99Y,
T81A V99Y
T80A V96Y
S103N
S221N
S99N

G103T,
G103T L107V
G100T L104V

L107V)

177
(V99Y, G103T,
V99Y G103T
V96Y G100T
S103N
S221N
S99N

L107V)
L107V
L104V

178
—
—
—
S103N
S221N
S99N

179
S26R, N52S,
S26R N52S
S27R N51S
S103N
S221N
S99N

A98S, V99W
A98S V99W
A95BS V96W

180
L4V, (V99Y,
L4V V99Y
L4V V96Y
S103N
S221N
S99N

G103T,
G103T L107V
G100T L104V

L107V)

Table 9 provides the VL and VH amino acid sequences for the anti-CD3 scFv clones generated by the techniques described herein.

TABLE 9

CD3 VL and VH amino acids sequences.

SEQ ID NO.
SEQUENCE

SEQ ID NO: 45
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWV

Anti hu CD3 humanized
GRIRSKYNNYATYYADSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCV

CD3-1 VH
RHGNFGNSYVSWFAYWGQGTLVTVSS

SEQ ID NO: 46
EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWV

Anti hu CD3 humanized
ARIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCV

CD3-2 VH
RHGNFGNSYVSWFAYWGQGTLVTVSS

SEQ ID NO: 47
QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLI

Anti hu CD3 humanized
GGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVF

CD3-1 VL and CD3-2 VL
GGGTKLTVL

(CD3-1-2)

Tables 10a and 10b provide the CDR sequences for the anti-CD3 VH scFv clones generated, using the Kabat and AbM numbering designations, respectively.

TABLE 10a

CD3 VH CDR sequences using Kabat numbering.

CD3
CD3 VH

Clone #
CDR1
CD3 VH CDR2
CD3 VH CDR3

CD3-1
TYAMN
RIRSKYNNYATYYADSVKG
HGNFGNSYVSWFAY

SEQ ID
SEQ ID NO: 49
SEQ ID NO: 51

NO: 48

CD3-2
TYAMN
RIRSKYNNYATYYADSVKD
HGNFGNSYVSWFAY

SEQ ID
SEQ ID NO: 50
SEQ ID NO: 51

NO: 48

TABLE 10b

CD3 VH CDR sequences using AbM numbering.

CD3

Clone #
CD3 VH CDR1
CD3 VH CDR2
CD3 VH CDR3

CD3-1
GFTFSTYAMN
RIRSKYNNYATY
HGNFGNSYVSWFAY

SEQ ID NO: 52
SEQ ID NO: 54
SEQ ID NO: 51

CD3-2
GFTFNTYAMN
RIRSKYNNYATY
HGNFGNSYVSWFAY

SEQ ID NO: 53
SEQ ID NO: 54
SEQ ID NO: 51

Tables 10c provides the CDR sequences for the anti-CD3 VL scFv clones generated.

TABLE 10c

CD3-1 and CD3-2 VL CDR sequences using

Kabat/AbM numbering.

CD3

Clone #
CD3 VL CDR1
CD3 VL CDR2
CD3 VL CDR3

CD3-1 and
RSSTGAVTTSNYAN
GTNKRAP
ALWYSNLWV

CD3-2
SEQ ID NO: 55
SEQ ID NO: 56
SEQ ID NO: 57

Example 2
Construction of an Anti-P-Cadherin/Anti-Human CD3 LP-DART

Anti-P-cadherin/anti-human CD3 DART proteins were amplified by PCR such that each DART protein contained a restriction enzyme cloning site at each end for cloning into the mammalian expression vector, see Table 11 and FIGS. 33A and 33B.

TABLE 11

Anti-P-cadherin/anti-human CD3 DART proteins.

DART
SEQUENCES

35
P-CAD 35VL(SEQ ID NO: 5)-Linker 1

(SEQ ID NO: 68)-CD3-1 VH(SEQ ID NO: 45 or 46), and

CD3-1-2VL(SEQ ID NO: 47)-Linker 1

(SEQ ID NO: 69)-P-CAD 35VH(SEQ ID NO: 6)

153
P-CAD 153 VL (SEQ ID NO: 7)-Linker 1

(SEQ ID NO: 68)-CD3 VH (SEQ ID NO: 45 or 46), and

CD3 VL (SEQ ID NO: 47)-Linker 1

(SEQ ID NO: 69)-P-CAD 153 VH (SEQ ID NO: 8)

154
P-CAD 154 VL (SEQ ID NO: 9)-Linker 1

(SEQ ID NO: 68)-CD3 VH (SEQ ID NO: 45 or 46), and

CD3 VL (SEQ ID NO: 47)-Linker 1

(SEQ ID NO: 69)-P-CAD 153 VH (SEQ ID NO: 10)

Thereafter, nucleic acids encoding the DART protein sequences were fused to each nucleic acid encoding the modified Fc constructs e.g. the “knob” Fc chain (SEQ ID NO: 63) and the “hole” Fc chain (SEQ ID NO: 64), each connected to the C-terminus of the DART with a cysteine linker, such as GCPPCP (SEQ ID NO: 73), GGTGGCPPCP (SEQ ID NO 74), GEPKSSDKTHTCPPCP (SEQ ID NO 75) and GGTGGGEPKSSDKTHTCPPCP (SEQ ID NO 76).

After PCR amplification, both the DART and Fc chain nucleic acids were digested with BspEI and either HindIII (5′ DART cloning site) or EcoRI (3′ Fc cloning site). Digested DNAs were isolated by excision from agarose gels and subsequent purification prior to ligation into the HindIII-EcoRI digested expression vector for 30 minutes at room temperature. Ligations were then transformed into competent E. coli DH5α and grown overnight at 37° C. on agarose plates containing 100 μg/mL carbenicillin. Colonies were counted, picked and grown overnight in YT-broth+100 μg/mL Carbenicillin and DNA isolated using standard methods. All DNA constructs were then sequenced on both strands prior to mammalian cell expression. Complimentary construct pairs were then co-transfected into 1 L log phase cultures containing 1 million cells/ml HEK 293 cells. 24 hours post-transfection, tryptone was added to a final concentration of 0.75% and cells were allowed to grow an additional 5 days before harvesting. Spent cultures were then collected, centrifuged to remove cell debris then passed through a 20 μm filter. Protein purification was then performed utilizing techniques known in the art. Schematic examples of anti-P-cadherin/anti-human CD3 LP-DARTs are shown in FIGS. 1A and 1B.

Example 3
Binding Properties of P-Cadherin scFv-Fc and DART Proteins

The binding properties of P-cadherin scFv-Fc constructs (P-cadherin VH and VL fused to a human IgG Fc region) and DART proteins to P-cadherin protein constructs and to P-cadherin expressing cells were analyzed using standard ELISA techniques. The data in Table 12 demonstrates that the phage derived P-cadherin scFv-Fc constructs had strong binding to recombinant human P-cadherin and no detectable binding to murine P-cadherin.

TABLE 12

Binding properties of P-cadherin scFv-Fc

to P-cadherin protein constructs.

huP-cad-ECD

P-CAD Clone
huP-cad-ECD
biotinylated
muP-cad-ECD

#
EC50 (nM)
EC50(nM)
EC50 (nM)

33 scFv-Fc
3.464
0.1012
NB

34 scFv-Fc
11.16
0.1943
NB

35 scFv-Fc
2.5
0.01762
NB

PF03732010
1.936
0.0188
2.333

Neg Control
NB
NB
NB

NB = no detectable binding

The binding properties of P-cadherin scFv-Fc to various P-cadherin expressing cell types in cell-based ELISA is shown in Table 13. The phage derived P-cadherin scFv-Fc constructs demonstrated strong binding to H1650 cells and to the SW480-huPcad and CHO-huPcad cell types.

TABLE 13

Binding properties of P-cadherin scFv-Fc to P-cadherin expressing cells.

CHO-
CHO

P-CAD
SW480-huPcad
H1650 EC50
huPcad
Parental

Clone #
EC50 (nM)
(nM)
EC50(nM)
EC50 (nM)

33 scFv-Fc
0.4243
0.5669
0.4931
NB

34 scFv-Fc
0.4422
0.494
0.3938
NB

35 scFv-Fc
0.1734
0.1631
0.2761
NB

PF-03732010
0.1665
0.2092
0.3293
NB

Neg Control
NB
NB
NB
NB

NB = no detectable binding

As shown in Table 14, anti-P-cadherin EK-DART proteins of the present invention demonstrated strong binding in ELISA format to cell surface expressed P-cadherin on H1650 cells and to recombinant human P-cadherin-Fc but no binding to other cadherin family members, E-cadherin and VE-cadherin. The EK-DART is an E/K coil domain construct. The PF EK-DART is a positive control utilizing the VH and VL from anti-P-cadherin clinical mAb PF-03732010 as described in Example 1. All EK-DART proteins in Table 14 comprise CD3-2.

TABLE 14

Binding properties of P-cadherin EK-DART proteins.

Hu P-

VE-

CHO

cadherin-
E-cadherin-
cadherin-
H1650
Parental

Fc EC50
Fc EC50
Fc EC50
EC50
EC50

DART
(nM)
(nM)
(nM)
(nM)
(nM)

33 EK-DART
1.8
NB
NB
60.1
NB

34 EK-DART
2.85
NB
NB
67.95
NB

35 EK-DART
1.92
NB
NB
24.87
NB

PF EK-DART
2.1
NB
NB
19.69
NB

20 EK-DART
12.3
NB
NB
~114.7
NB

30 EK-DART
3.68
NB
NB
141.9
NB

Neg Control
NB
NB
NB
NB
NB

EK-DART

NB = no detectable binding

Example 4
Surface Plasmon Resonance (SPR) Analysis of DART Proteins

Binding affinity of EK-DART proteins to P-cadherin and CD3 was analyzed by SPR using a BIACORE® 3000 biosensor (GE Healthcare). The antigens huCD3_εδ and either human P-cadherin-Fc or human/cynomolgus monkey/murine P-cadherin-ECD were immobilized on the CM-5 sensor chip by amine coupling kit as recommended by the manufacturer. Table 15 shows the binding affinity of phage derived EK-DART proteins against cynomolgus monkey (“cyno”) P-cadherin ECD and Table 16 shows the binding affinity of phage derived EK-DART proteins against human soluble CD3 protein. All EK-DART proteins in Tables 15 and 16 comprise CD3-2.

TABLE 15

Binding affinity of EK-DART proteins against cyno P-cadherin ECD.

DART
ka (1/Ms)
kd (1/s)
K_D(nM)

33 EK-DART
1.15E+06
5.78E−02
46.4

34 EK-DART
7.05E+05
8.33E−02
129.3

35 EK-DART
3.18E+05
1.38E−02
43.4

PF EK-DART
2.11E+05
2.51E−03
11.90

20 EK-DART
3.46E+06
7.04E−03
2.03

30 EK-DART
4.72E+06
6.48E−03
1.37

TABLE 16

Binding affinity of EK-DART proteins against human soluble CD3.

DART
ka (1/Ms)
kd (1/s)
K_D(nM)

33 EK-DART
2.28E+05
4.03E−03
17.7

34 EK-DART
1.95E+05
4.45E−03
22.8

35 EK-DART
1.89E+05
4.47E−03
23.6

PF EK-DART
4.11E+05
3.95E−03
9.61

20 EK-DART
9.79E+05
6.09E−03
5.19

30 EK-DART
3.53E+05
4.57E−03
13.6

In Table 17, the Biacore data demonstrates the binding affinity of optimized EK-DART proteins, with CD3-2, to cyno P-cadherin ECD compared with the parental 35 EK-DART with CD3-2. All EK-DART proteins in Table 17 comprise CD3-2.

TABLE 17

Binding affinity of EK-DART proteins against cyno P-cadherin ECD.

DART
ka (1/Ms)
kd(1/s)
KD (pM)

35 EK-DART
6.14E+05
2.15E−02
35050 ± 2950

153 EK-DART
7.32E+05
1.72E−05
231.5 ± 17.5

154 EK-DART
1.04E+06
3.90E−03
3930.0 ± 910

165 EK-DART
8.34E+05
3.09E−04
370.0 ± 87

177 EK-DART
8.02E+05
3.83E−04
483.0 ± 82

178 EK-DART
1.67E+05
2.16E−04
1310.0 ± 140

179 EK-DART
1.11E+06
4.58E−03
4165.0 ± 255

180 EK-DART
7.71E+05
7.44E−04
965.5 ± 19.5

In Table 18, the Biacore data demonstrates the binding affinity of the parental 35 EK-DART and optimized EK-DART proteins to soluble CD3_δε. All EK-DART proteins in Table 18 comprise CD3-2.

TABLE 18

Binding affinity of EK-DART proteins against human soluble CD3.

DART
ka (1/Ms)
kd(1/s)
KD (nM)

35 EK-DART
1.89E+05
4.47E−03
23.6

153 EK-DART
4.03E+05
6.31E−03
15.65 ± 1.75

154 EK-DART
4.56E+05
7.68E−03
17.10 ± 0.90

165 EK-DART
2.78E+05
4.17E−03
14.90 ± 2.80

177 EK-DART
2.53E+05
4.43E−03
19.95 ± 6.55

178 EK-DART
2.30E+05
5.26E−03
23.10 ± 1.10

179 EK-DART
3.06E+05
6.41E−03
25.75 ± 9.25

180 EK-DART
7.06E+05
1.11E−02
15.9 ± 1.00

The binding affinity versus cyno P-cadherin at pH7.4 of various DART formats is shown in Table 19. DART proteins in the VF, EK, LP, MP3-C-terminal and MP3-N-terminal formats with P-cadherin clone 153 VH and VL binding domains were compared. 153 VF-DART demonstrated the highest binding affinity and fastest on rate. The KDs were similar to the 153 EK-DART and 153 LP-DART proteins. 153 MP3-DART proteins had slightly lower binding affinities to cyno P-cadherin as indicated by slower on rates. The % activity (Rmax) was low and similar for all 153 DART proteins. All DART proteins in Table 19 comprise CD3-1, except 153 EK-DART which comprises CD3-2.

TABLE 19

Binding affinity of various DART formats against cyno P-cadherin.

ka
kd
t½
Rmax
*%
% Chi2/
KD

DART
Ligand
(1/Ms)
(1/s)
(min)
(RU)
Rmax
Rmax
(nM)

153 EK-DART
Cyno P-
5.24E+05
2.11E−04
51.56
23.2
17.3
0.24
0.403

CD3-2
cadherin

153 EK-DART
Cyno P-
5.17E+05
2.22E−04
48.98
14.0
14.9
0.33
0.430

cadherin

16.1
AVG
0.417 ± 0.014

153VF-DART
Cyno P-
7.22E+05
2.40E−04
45.35
22.66
15.0
0.32
0.332

CD3-1
cadherin

153VF-DART
Cyno P-
7.52E+05
2.29E−04
47.59
21.68
14.4
0.43
0.304

cadherin

14.7
AVG
0.318 ± 0.014

153 LP-DART
Cyno P-
2.93E+05
2.00E−04
54.36
36.6
12.1
0.16
0.683

cadherin

153 LP-DART
Cyno P-
5.81E+05
2.09E−04
52.20
75.1
11.7
0.37
0.359

cadherin

11.9
AVG
0.521 ± 0.162

153-MP3 C-
Cyno P-
6.80E+04
1.06E−04
102.38
129.1
19.5
0.15
1.56

terminal DART
cadherin

153-MP3 C-
Cyno P-
8.46E+04
1.79E−04
60.90
73.9
16.3
0.19
2.11

terminal DART
cadherin

17.9
AVG
1.84 ± 0.28

153-MP3 N-
Cyno P-
1.22E+05
2.96E−04
36.82
72.4
16.1
1.34
2.43

terminal DART
cadherin

153-MP3 N-
Cyno P-
8.19E+04
2.28E−04
47.78
149.6
22.7
0.71
2.78

terminal DART
cadherin

19.4
AVG
2.610.18

The binding affinity versus shCD3 (soluble human CD3) at pH7.4 of various DART formats is shown in Table 20. DART proteins in the VF, EK, LP, MP3 C-terminal and MP3 N-terminal formats with P-cadherin clone #153 VH and VL binding domains were compared. 153 VF-DART demonstrated the fastest on rate and highest binding affinity to shCD3. The 153 EK, LP and MP3 DART proteins had similar affinities to shCD3. The % activity (Rmax) for shCD3 was better than that observed for cyno P-cadherin. All DART proteins in Table 20 comprise CD3-1, except 153 EK-DART which comprises CD3-2.

TABLE 20

Binding affinity of various DART formats against human soluble CD3.

ka
kd
t½
Rmax
*%
% Chi2/
KD

DART
Ligand
(1/Ms)
(1/s)
(min)
(RU)
Rmax
Rmax
(nM)

153 EK-DART
shCD3
2.77E+05
6.18E−03
1.76
80.94
55.5
0.09
22.3

CD3-2

153 EK-DART
shCD3
2.83E+05
6.80E−03
1.60
62.68
31.1
0.04
24.0

CD3-2

43.3
AVG
23.15 ± 0.85

153 VF-DART
shCD3
1.13E+06
5.25E−03
2.07
90.92
69.1
0.85
4.65

CD3-1

153 VF-DART
shCD3
1.16E+06
5.55E−03
1.96
89.81
68.2
0.94
4.77

68.7
AVG
4.71 ± 0.06

153 LP-DART
shCD3
4.18E+05
5.20E−03
2.09
170.3
64.7
1.07
12.4

153 LP-DART
shCD3
5.37E+05
5.72E−03
1.90
129.8
35.7
0.64
10.6

50.2
AVG
11.5 ± 0.9

153 MP3-C-
shCD3
4.20E+05
7.14E−03
1.52
133
35.3
0.29
17.0

terminal DART

153 MP3-C-
shCD3
5.65E+05
7.19E−03
1.51
113.9
30.2
0.40
12.7

terminal DART

32.8
AVG
14.9 ± 2.2

153 MP3-N-
shCD3
4.74E+05
6.62E−03
1.65
120.7
32.3
0.65
14.0

terminal DART

153 MP3-N-
shCD3
5.32E+05
5.82E−03
1.87
128.1
34.3
0.42
11.0

terminal DART

33.3
AVG
12.5 ± 1.5

Example 5
Characterization of P-Cadherin DART Proteins

Differential scanning calorimetry (DSC) analysis was used to characterize the thermal stability of phage derived EK-DART proteins was conducted. Table 21 demonstrates that the P-cadherin EK-DART proteins had favorable thermal profiles with Tm1 transitions above 65° C. Forced aggregation analysis demonstrates the resistance to thermal stress of P-cadherin EK-DART proteins at 40° C. All EK-DART proteins in Table 21 comprise CD3-2.

TABLE 21

DSC analysis of P-cadherin EK-DART proteins.

Forced
pH

Aggregation
Reversibility

DSC
DSC
(% aggregate
(% reduction in

DART
T_onset
T_m1
at 40 C. ON)
monomer)

35 EK-DART
NA
67.15 ± 0.09
NA
NA

153 EK-DART
59.8
67.82 ± 0.02
41
6

154 EK-DART
63.06
68.40 ± 0.01
40
3

165 EK-DART
63.28
68.53 ± 0.01
39
<1

177 EK-DART
63.31
68.54 ± 0.01
39
1

178 EK-DART
61.96
66.92 ± 0.02
32
<1

179 EK-DART
61.56
67.15 ± 0.03
31
7

180 EK-DART
63.36
68.52 ± 0.01
32
1.5

Additional biophysical properties of affinity-matured P-cadherin EK-DART proteins are shown in Table 22a. The theoretical pI of these molecules is around 8.5. All EK-DART proteins in Table 22a comprise CD3-2.

TABLE 22a

Biophysical properties of P-cadherin EK-DART proteins.

Calculated DART Biochemical Properties

Residue
Molar Mass

Charge at
ε₂₈₀
ε₂₈₀

DART
Count
(g/mol)
pl
pH 7.00
(M⁻¹cm⁻¹)
(L-g⁻¹cm⁻¹)

35 EK-DART
547
57223.56
8.5
6.67
116365
2.0335

153 EK-DART
547
57179.54
8.62
7.67
116365
2.0351

154 EK-DART
547
57347.65
8.5
6.67
117855
2.0551

165 EK-DART
547
57314.63
8.5
6.67
117855
2.0563

177 EK-DART
547
57344.66
8.5
6.67
117855
2.0552

178 EK-DART
547
57250.59
8.5
6.67
116365
2.0326

179 EK-DART
547
57396.76
8.62
7.67
121865
2.1232

180 EK-DART
547
57330.63
8.5
6.67
117855
2.0557

Table 22b shows functional characterization of affinity-matured P-cadherin VF-DART proteins. Data from two types of cytotoxic T lymphocyte (CTL) assays showed retained cytotoxicity of a bispecific heterodimeric diabody without E-K coils. Higher affinity to ligands corresponded to higher potency in CTL, as was seen in the EK-DART. All VF-DART proteins in Table 22b comprise CD3-1.

TABLE 22b

Functional characterization of P-cadherin VF-DART proteins.

Biacore
Biacore

HCT116

affinity P-CAD
affinity
DU145 CTL
CTL (Luc)

Cyno monomeric
soluble
(LDH)
EC50

DART
ECD (nM)
CD3 (nM)
EC50 (nM)
(nM)

153 VF-DART
0.31
4.71
0.0004
0.0005

154 VF-DART
2.85
3.69
0.0024
0.008

35 VF-DART
NT
NT
0.012
.041

Table 22c contains biophysical characterization data of affinity-matured P-cadherin VF-DART proteins. Analysis of VF-DART proteins indicated that no significant increase in aggregation was detected until 65° C. Data suggests that the VF-DART format improved forced aggregation profile at low temperatures while not impacting Tm1. LC/MS data indicated proper heterodimer formation following expression and purification. All VF-DART proteins in Table 22c comprise CD3-1.

TABLE 22c

Biophysical characterization data of P-cadherin VF-DART proteins.

Capillary

pH Sensitivity -
Mass Spec
Electrophoresis

DSC

% Increase in
Observations
pI and

DART
Tm1
Forced Aggregation
Aggregation
(final pool)
Observations

153 VF-DART
69.82
Increase in HMW >55 C. O/N;
0
>99% correct
8.18

similar profile to clinical mAb

154 VF-DART
70.08
Increase in HMW >55 C. O/N;
0
>99% correct
NT

similar profile to clinical mAb

35 VF-DART
68.68
Increase in HMW >55 C. O/N;
0
>99% correct
NT

similar profile to clinical mAb

Example 6
Characterization of P-Cadherin LP-DART Proteins

Table 23 summarizes various functional and biophysical data for eight different P-cadherin LP-DART constructs (#1-#8) generated with the parental clone P-CAD 35 and CD3-2.

For example, the first polypeptide chain (1) of LP-DART #1 has the designation: CD3 VL×P-CAD 35 VH-knob, where the CD3 VL refers to the anti-CD3-2 antibody VL binding region of SEQ ID NO: 47, P-CAD 35 VH refers to the anti-P-cadherin antibody clone 35 VH binding region of SEQ ID NO: 6, and “knob” refers to the Fc knob chain of SEQ ID NO: 63. The second polypeptide chain (2) of LP-DART #1 has the designation: P-CAD 35VL×CD3VH-hole, where the P-CAD 35VL refers to the anti-P-cadherin antibody clone 35 VL binding region of SEQ ID NO: 5, CD3 VH refers to the anti-CD3-2 antibody VH binding region of SEQ ID NO: 46, and “hole” refers to the Fc hole chain of SEQ ID NO: 64. Various cysteine linkers (Linker 2) with a truncated IgG1 hinge having the sequence CPPCP (SEQ ID NO 60) and at least one glycine residue were analyzed. The LP-DART constructs #1-#8 were compared to parental 35 EK-DART.

The data demonstrated that a shorter linker (Linker 2) between the DART portion and the Fc domain correlated to higher affinity to each ligand and higher potency in the CTL assay. This suggested that a shorter linker (Linker 2) prevented the Fc domain from interfering with the ligand binding sites on the DART portion. The data also suggested that the orientation: CD3 VL×P-CAD 35 VH-hole and P-CAD 35 VL×CD3 VH-knob, as shown in LP-DART #5 through LP-DART #8, was optimal for LP-DART generation.

TABLE 23

Functional and biophysical data for P-cadherin LP-DART proteins.

LP-
LP-
LP-
LP-
LP-
LP-
LP-
LP-
35

DART #1
DART #2
DART #3
DART #4
DART #5
DART #6
DART #7
DART #8
EK-DART

First
CD3VLx
CD3VLx
CD3VLx
CD3VLx
CD3VLx
CD3VLx
CD3VLx
CD3VLx

Chain
P-CAD
P-CAD
P-CAD
P-CAD
P-CAD
P-CAD
P-CAD
P-CAD

35VH-
35VH-
35VH-
35VH-
35VH-
35VH-
35VH-
35VH-

knob
knob
knob
knob
hole
hole
hole
hole

Second
P-CAD
P-CAD
P-CAD
P-CAD
P-CAD
P-CAD
P-CAD
P-CAD

Chain
35VLx
35VLx
35VLx
35VLx
35VLx
35VLx
35VLx
35VLx

CD3VH-
CD3VH-
CD3VH-
CD3VH-
CD3VH-
CD3VH-
CD3VH-
CD3VH-

hole
hole
hole
hole
knob
knob
knob
knob

Linker
GCPPCP
GGTGG
GEPKSS
GGTGG
GCPPCP
GGTGG
GEPKSS
GGTGG

(Linker 2)
SEQ ID
CPPCP
DKTHTC
GEPKSS
SEQ ID
CPPCP
DKTHTC
GEPKSS

NO: 73
SEQ ID
PPCP
DKTHTC
NO: 73
SEQ ID
PPCP
DKTHTC

NO: 74
SEQ ID
PPCP

NO: 74
SEQ ID
PPCP

NO: 75
SEQ ID

NO: 75
SEQ ID

NO: 76

NO: 76

Capture
24
23
35
28
20
24
27
34
5

Yield

(mg/L)

% Agg
40%
37%
33%
39%
33%
40%
40%
45%
7%

Post

capture

Cyno-
108.0
73.5
85.7
128.0
62.1
86.6
96.6
104.0
42.8

Pcad KD

(nM)

shCD3 KD
32.9
47.9
47.2
55.1
30.2
42.1
49.1
55.6
17.1

(nM)

T cell Cytox-
0.79
0.92
1.60
2.45
0.74
1.29
2.85
2.27
0.33

icity (nM)

Table 24 shows the in vitro functional characterization of several P-cadherin LP-DART proteins incorporating the high affinity P-cadherin VH and VL binding domains as well as the high affinity anti-CD3 VH and VL binding domains. The data demonstrate that an increased affinity correlates to increased CTL potency; similar to the correlation observed for the P-cadherin VF-DART and EK-DART formats. All LP-DART proteins in Table 24 comprise CD3-1. As with the 33, 34 and 35 parental clones, 153 LP-DART did not bind to murine P-cadherin.

TABLE 24

Functional characterization of P-cadherin LP-DART proteins.

Biacore affinity Cyno
Biacore affinity
Biacore
DU145 CTL

P-cadherin
murine P-
affinity soluble
(LDH) EC50
HCT116 CTL

DART
monomeric ECD (nM)
cadherin ECD
CD3 (nM)
(nM)
(Luc) EC50 (nM)

153 LP-
0.521
NB
11.5
0.0006
0.0004

DART

154 LP-
5.5
NT
7.18
0.0017
0.0072

DART

35 LP-DART
NT
NT
NT
0.0073
0.107

NT = not tested;

NB = no binding

Table 25 shows the biophysical characterization of several P-cadherin LP-DART proteins. DSC of the P-cadherin LP-DART proteins showed a similar Tm1 value as was obtained with the P-cadherin EK-DART and VF-DART proteins. Forced aggregation analysis suggested an improved stability at lower temperatures over the P-cadherin EK-DART format and an overall profile similar to clinical mAbs. LC/MS data indicated proper heterodimer formation following expression and purification. All LP-DART proteins in Table 25 comprise CD3-1.

TABLE 25

Biophysical characterization of P-cadherin LP-DART proteins.

Capillary

pH Sensitivity -
Mass Spec
Electrophoresis

DSC

% Increase in
Observations
pl and

Construct
Tm1
Forced Aggregation
Aggregation
(final pool)
Observations

153 LP-DART
68.91
Increase in HMW
0
>99% correct
8.26, Shouldering

>55 C. O/N; similar

profile to clinical mAb

154 LP-DART
68.63
Increase in HMW
0
>99% correct
NT

>55 C. O/N; similar

profile to clinical mAb

35 LP-DART
68.39
Increase in HMW
0
>99% correct
NT

>55 C. O/N; similar

profile to clinical mAb

NT = not tested

Table 26 provides the Biacore SPR data of P-cadherin LP-DART proteins, 153 LP-DART and 154 LP-DART, binding to cyno P-cadherin ECD and soluble CD3_δε. The data demonstrate that there was a minor decrease in affinity to each ligand compared to the affinity of the corresponding EK-DART. All LP-DART proteins in Table 26 comprise CD3-1.

TABLE 26

Binding properties of P-cadherin LP-DART proteins cyno P-cadherin and soluble CD3.

ka
kd
t½
Rmax
*%
% Chi2/
KD

DART
Ligand
(1/Ms)
(1/s)
(min)
(RU)
Rmax
Rmax
(nM)

153 LP-DART
Cyno P-
2.93E+05
2.00E−04
54.36
36.6
12.1
0.16
0.683

Cadherin
5.81E+05
2.09E−04
52.20
75.1
11.7
0.37
0.359

11.9
AVG
0.521 ± 0.162

154 LP-DART
Cyno P-
6.07E+05
3.32E−03
3.27
46.29
32.3
0.49
5.47

Cadherin
6.20E+05
3.43E−03
3.18
46.38
32.4
0.56
5.53

32.35
AVG
5.50 ± 0.03

153 LP-DART
shCD3
4.18E+05
5.20E−03
2.09
170.3
64.7
1.07
12.4

5.37E+05
5.72E−03
1.90
129.8
35.7
0.64
10.6

50.2
AVG
11.5 ± 0.9

154 LP-DART
shCD3
6.13E+05
4.42E−03
2.47
476.7
42.5
5.73
7.20

6.37E+05
4.56E−03
2.39
475.0
42.3
5.41
7.16

42.4
AVG
7.18 ± 0.02

Example 7
Quantification of P-Cadherin Expression

A. Quantification of P-Cadherin Expression on Cancer Cell Lines Engineered to Express Luciferase for In Vitro Studies

Flow cytometry experiments were conducted to evaluate anti-P-cadherin mAb (PF-03732010) binding to determine the relative level of endogenous cell surface P-cadherin expression across a panel of 10 cancer cell lines. All tumor cells lines for in vitro testing were engineered to express luciferase. Tumor cell lines demonstrating a range of P-cadherin receptor expression were collected as described for saturation FACS binding. 2.5×10⁵cells per sample were transferred to 96-well round bottom polypropylene plates. Cells were stained with 5 μg/ml and 10 μg/ml phycoerythrin (PE) labeled anti-human P-cadherin mAb (PF-03732010) conjugated at a ratio of 1:1 mAb to PE (eBioscience) or with PE conjugated to a control mouse IgG1 mAb (Biolegend) for 30 minutes at room temperature. Cells were washed and resuspended with FACS buffer plus 10 ng/ml propidium iodide prior to acquisition using LSRII with FACS Diva software. QuantiBRITE PE beads (BD Pharmingen) were reconstituted using 0.5 mL FACS buffer and acquired using the LSRII with the same voltage settings as the tumor cell samples. The PE geometric mean fluorescent intensity of both beads and tumor cells was used to calculate the number of PE labeled antibodies bound per cell (ABC) following the manufacturer prescribed protocol for the QuantiBRITE PE Bead Kit. A linear regression curve fit between receptor density and cytotoxicity EC50 was determined by plotting the Log₁₀value of the average receptor density determined by Quantibrite assay at 5 μg/mL and 10 μg/mL PE labeled anti-P-cadherin mAb for each tumor cell line versus the Log₁₀value of the average in vitro cytotoxicity EC50 for that same target tumor cell line using GraphPad Prism 5.0 software.

A broad range of cell surface P-cadherin expression was observed across the panel and the anti-P-cadherin mAb bound to cell lines that were derived from a variety of cancer types, including breast cancer, lung cancer and colorectal cancer, as shown in Table 27. In contrast, the control mAb did not show appreciable binding. MFI=Mean fluorescence intensity; ABC=Antibodies bound per cell.

TABLE 27

Endogenous cell surface P-cadherin expression level on luciferase

expressing cancer cell lines

MFI,
MFI,
ABC,

Control
P-cadherin
P-cadherin

Cell Line
Tumor Type
mAb
mAb
mAb

H1650_Luc
Lung Cancer
254
7,958
37,582

HCT116_Luc
Colorectal cancer
229
5,661
26,421

BT20_Luc
Breast Cancer
225
5,235
24,349

SUM149_Luc
Breast Cancer
213
3,717
16,982

H322_Luc
Lung Cancer
242
3,233
16,576

H1975_Luc
Lung Cancer
373
3,155
15,429

LoVo_Luc
Colorectal cancer
175
1,389
5,805

SW480_Luc
Colorectal cancer
151
1,256
5,304

HT29_Luc
Colorectal cancer
282
787
2,421

Ls174T_Luc
Colorectal cancer
94
290
929

Colo205_Luc
Colorectal cancer
173
357
874

SW620_Luc
Colorectal cancer
125
276
715

B. Quantification of P-Cadherin Expression on Cancer Cell Lines for in vivo Studies

Flow cytometry experiments were conducted to evaluate anti-P-cadherin mAb (Pfizer PF-03732010) binding to determine the relative level of endogenous cell surface P-cadherin expression across a panel of cancer cell lines for in vivo studies. Tumor cell lines demonstrating a range of P-cadherin receptor expression were collected as described for saturation FACS binding. 2.5×10⁵cells per sample were transferred to 96 well round bottom polypropylene plates. Cells were stained with 5 μg/ml and 10 μg/ml phycoerythrin (PE) labeled anti-human P-cadherin mAb conjugated at a ratio of 1:1 mAb to PE (eBioscience) or with 7.5 μg/mL PE conjugated to a control mouse IgG1 mAb (Biolegend) for 30 minutes at room temperature. Cells were washed and resuspended with FACS buffer plus 10 ng/mL propidium iodide prior to acquisition using LSRII with FACS Diva software. QuantiBRITE PE beads (BD Pharmingen) were reconstituted using 0.5 ml FACS buffer and acquired using the LSRII with the same voltage settings as the tumor cell samples. The average PE geometric mean fluorescent intensity of both beads and tumor cells was used to calculate the number of PE labeled antibodies bound per cell (ABC) following the manufacturer prescribed protocol for the QuantiBRITE PE Bead Kit.

A broad range of cell surface P-cadherin expression was observed on cell lines derived from colorectal and breast cancer and ranked accordingly; HCT116>SUM149>SW480>Ls174T>SW620, as shown in Table 28. In contrast, the isotype control mAb did not show appreciable binding to any of the cell lines.

TABLE 28

Endogenous cell surface P-cadherin expression level on cancer cell lines.

MFI,
MFI,
ABC,

Control
P-cadherin
P-cadherin

Cell Line
Tumor Type
mAb
mAb
mAb

HCT116
Colorectal cancer
239
8,033
43,801

SW480
Colorectal cancer
171
2,468
12,665

Ls174T
Colorectal cancer
222
986
4,160

SW620
Colorectal cancer
88
297
1,117

SUM149
Breast Cancer
68
2,872
15,426

Example 8
In Vitro Cytotoxicity Assays

A. EK-DART Cytotoxic T-Lymphocyte (CTL) Assays

EK-DART proteins showing strong recombinant P-cadherin/CD3 protein binding as well as a strong cell binding signal (>3× background or EC50≈PF03732010-DART) were screened in T cell directed cell killing assays as previously described (Moore et al., Blood, 117(17): 4542-4551, 2011). Table 29 demonstrates effective T cell directed cytotoxicity for P-cadherin EK-DART proteins of the present invention. In the presence of human PBMC at an E:T ratio of 30:1, CHO cells engineered to over express human P-cadherin and a cancer cell line that expresses endogenously high levels of P-cadherin (H1650) were both effectively lysed by the EK-DART proteins following 24 hour incubation as measured using an LDH release assay. Extending incubation time to 40 hours further increased the potency of cell killing as anticipated by the mechanism of action of redirected T cell killing with the P-cadherin EK-DART molecules. In contrast, no killing was observed on the parental CHO cell line as anticipated based on lack of P-cadherin expression. All EK-DART proteins in Table 29 comprise CD3-2.

TABLE 29

T cell directed cytotoxicity for P-cadherin EK-DART proteins.

CHO-CDh3 (24 h)
H1650 (24 h)
H1650 (40 h)

DART
EC50 (nM)
EC50 (nM)
EC50 (nM)

33 EK-DART
0.309
4.104
1.124

34 EK-DART
0.7365
5.604
1.6

35 EK-DART
0.0398
0.5014
0.0532

PF EK-DART
0.0284
0.4038
0.505

20 EK-DART
168.64
36.44
NT

30 EK-DART
NR
NR
NT

T cell directed cytotoxicity of optimized P-cadherin EK-DART proteins against tumor cell expressing human P-cadherin and a constitutively-expressed luciferase reporter construct is shown in Table 30. All EK-DART proteins in Table 30 comprise CD3-2.

TABLE 30

T cell directed cytotoxicity for P-cadherin EK-DART proteins.

HCT116 Luc
H1650 LDH

48 hr 10:1 D43905
24 hr 30:1 D41183

DART
EC50 nM
EC50 nM

153 EK-DART
0.0005
0.0008

154 EK-DART
0.0166
0.0107

165 EK-DART
0.0015
0.0011

177 EK-DART
0.0007
0.0011

178 EK-DART
0.0018
0.0008

179 EK-DART
0.0432
0.0178

180 EK-DART
0.0018
0.0016

35 EK-DART
0.2051
0.1016

B. LP-DART Cytotoxic T-Lymphocyte (CTL) Assays

Mediated cell killing of 153 LP-DART was assessed by cytotoxic T-lymphocyte (CTL) assay. CHO parental and CHO P-cadherin cells at a 10:1 effector (T cell) to target (CHO cell) ratio were incubated with CD3+ T cells in the presence of increasing concentrations of 153 LP-DART. Cell viability was measured after 48 hours and a half maximal effective concentration (EC50) was calculated to be 0.41 pM. The results showed that 153 LP-DART demonstrated potent killing on CHO P-cadherin cells, however no activity was detectable above the negative control on the CHO parental cells. Therefore, CTL activity was dependent on P-cadherin expression.

Mediated tumor killing of 153 LP-DART was assessed by cytotoxic T-lymphocyte (CTL) assay. T cell directed cytotoxicity of optimized 153 LP-DART proteins against tumor cell expressing human P-cadherin and a constitutively-expressed luciferase reporter construct was analyzed. CD3+ T cells were incubated with a panel of cancer cell lines at a 3:1 effector (T cell) to target (cancer cell) ratio in the presence of increasing concentrations of 153 LP-DART. Cell viability was measured after 72 hours and a half maximal effective concentration (EC50) for each cancer cell line was calculated, as shown in Table 31. The data demonstrates that 153 LP-DART mediates potent anti-tumor activity in the cancer models. EC50=half maximal effective concentration; S.D.=standard deviation; pM=picomolar; NA=no activity.

TABLE 31

Cytotoxic T-lymphocyte activity of 153 LP-DART

in luciferase expressing cancer cell lines.

Cell Line
Tumor Type
EC50 ± S.D. (pM)

H1650_Luc
Lung Cancer
4.24 ± 0.66

HCT116_Luc
Colorectal cancer
5.6 ± 3.61

BT20v_Luc
Breast Cancer
4.72 ± 2.43

SUM149_Luc
Breast Cancer
0.35 ± 0.10

H1975_Luc
Lung Cancer
0.96 ± 0.31

H322_Luc
Lung Cancer
5.78 ± 3.09

LoVo_Luc
Colorectal cancer
1.65 ± 0.91

SW480_Luc
Colorectal cancer
53.37 ± 30.73

HT29_Luc
Colorectal cancer
41.19 ± 22.17

Ls174T_Luc
Colorectal cancer
1439.07 ± 2462.25

Colo205_Luc
Colorectal cancer
191.28 ± 203.58

SW620_Luc
Colorectal cancer
NA

C. Comparison of CTL Activity to Cell Surface P-Cadherin Expression Levels

Tumor cell lines from section A demonstrating a range of P-cadherin receptor expression were collected and 2.5×10⁵cells per sample were transferred to 96-well round bottom polypropylene plates. Cells were stained with 5 μg/ml and 10 μg/ml phycoerythrin (PE) labeled anti-human P-cadherin mAb (Pfizer PF-03732010) conjugated at a ratio of 1:1 mAb to PE (eBioscience) or with PE conjugated to a control mouse IgG1 mAb (Biolegend) for 30 minutes at room temperature. Cells were washed and resuspended with FACS buffer plus 10 ng/ml propidium iodide prior to acquisition using LSRII with FACS Diva software. QuantiBRITE PE beads (BD Pharmingen) were reconstituted using 0.5 mL FACS buffer and acquired using the LSRII with the same voltage settings as the tumor cell samples. The PE geometric mean fluorescent intensity of both beads and tumor cells was used to calculate the number of PE labeled antibodies bound per cell (ABC) following the manufacturer prescribed protocol for the QuantiBRITE PE Bead Kit.

A linear regression curve fit between receptor density and cytotoxicity EC50 was determined by plotting the Log₁₀value of the average receptor density determined by Quantibrite assay at 5 μg/mL and 10 μg/mL PE labeled anti-P-cadherin mAb for each tumor cell line versus the Log₁₀value of the average in vitro cytotoxicity EC50 for that same target tumor cell line using GraphPad Prism 5.0 software. As shown in FIG. 8, there was a significant relationship between lower EC50s and increased binding of anti-P-cadherin mAb to the cell surface, P value=0.0127. This indicates that CTL activity was correlated with P-cadherin expression levels.

Example 9
Human PBMC Reconstitution Xenograft Studies

Various DART proteins demonstrating potent T cell directed cytotoxicity (<10 ng/mL EC50) were tested in vivo in xenograft reconstitution experiments as described below.

A. Isolation of PBMCs and T Cells from Human Whole Blood

PBMCs from healthy human donors were isolated from whole blood by using Ficoll gradient centrifugation. In brief, whole blood was diluted 1:1 with sterile PBS. Thirty-five mL of the diluted blood was layered onto 15 mL FICOLL-PAQUE™ Plus in 50-mL tubes and the tubes were centrifuged at 1400 rpm for 20 min with the brake off. The buffy coat layer between the two phases was collected into a 50 mL tube and washed with 45 mL PBS by centrifuging the tubes at 600×g (1620 rpm) for 5 min. The supernatant was discarded and the cell pellet was washed once with PBS and viable cell count was determined by Trypan Blue dye exclusion. The PBMCs were resuspended to a final concentration of 2.5×10⁶cells/mL in complete medium (RPMI 1640, 10% FBS, 2 mM Glutamine, 10 mM HEPES, 100μ/100μ/mL penicillin/Streptomycin (P/S).

B. T Cell Isolation and Activation

Human T cells were isolated from heparinized whole blood according to the manufacturer's protocol provided in the RosettaSep T cell isolation kit. The purified T cells were subsequently activated by exposing the cells to anti-CD3 (OKT-3; 1 μg/mL) and anti-CD28 (66 μg/mL) antibodies for a period of 48 hours. Following stimulation, the cells were grown in RPMI 1640 medium with 10% FBS and 1% penicillin/streptomycin in the presence of IL-2 (7.6 ng/mL) for up to 3 weeks.

C. Tumor Model

Human T cells and tumor cells (HCT116, Du145 or H1650) were combined at a ratio of 1:5 (1×10⁶and 5×10⁶, respectively) and suspended in 200 μL of sterile saline and injected subcutaneously (SC) on Study Day 0 (SD0). Various P-cadherin EK-DART (CD3-2), LP-DART (CD3-1) or a control DART (CD3-2) proteins, along with a control, were administered intravenously (IV) via tail vein injections in 100 μL at SD0, 1, 2, and 3. Each treatment group contained eight animals.

P-CAD 177 EK-DART in a HCT116 Tumor Model: P-CAD 177 EK-DART was administered at a dose of 0.5, 0.2, 0.1 or 0.05 mg/kg to four treatment groups and a vehicle control (HCT116 cells alone implanted or +T cells) was administered to one treatment group (0 mg/kg).

P-CAD 153 EK-DART in a HCT116 Tumor Model: P-CAD 153 EK-DART was administered at a dose of 0.5, 0.2, 0.1 or 0.05 mg/kg to four treatment groups and a vehicle control (HCT116 cells alone implanted or +T cells) was administered to one treatment group (0 mg/kg).

P-CAD 154 EK-DART in a HCT116 Tumor Model: P-CAD 154 EK-DART was administered at a dose of 0.5, 0.2, 0.1 or 0.05 mg/kg to four treatment groups and a vehicle control (HCT116 cells alone implanted or +T cells) was administered to one treatment group (0 mg/kg).

P-CAD 35 EK-DART in a HCT116 Tumor Model: P-CAD 35 EK-DART was administered at a dose of 0.5, 0.2, 0.1 or 0.05 mg/kg to four treatment groups and a vehicle control (HCT116 cells alone implanted or +T cells) was administered to one treatment group (0 mg/kg).

P-CAD 153 LP-DART in a HCT116 Tumor Model: Treatment groups were administered P-CAD 153 LP-DART at a dose of 0.5, 0.2, 0.1 or 0.05 mg/kg, or a vehicle control (HCT116 cells alone implanted or +T cells).

P-CAD 153 LP-DART in a HCT116 Tumor Model: Treatment groups were administered P-CAD 153 LP-DART at a dose of 100, 10, 1, 0.1, or 0.01 μg/kg, or a vehicle control (HCT116 cells implanted alone), a vehicle control (HCT116 cells implanted +T cells) or a 4420-hXR32 LP-DART (100 μg/kg) control.

P-CAD 153 LP-DART in a Du145 Tumor Model: Treatment groups were administered P-CAD 153 LP-DART at a dose of 100, 10, 1, 0.1, or 0.01 μg/kg or a vehicle control (Du145 cells implanted alone), a vehicle control (HCT116 cells implanted +T cells) or a 4420-hXR32 LP-DART (100 μg/kg) control.

P-CAD 153 LP-DART in an H1650 Tumor Model: Treatment groups were administered P-CAD 153 LP-DART at a dose of 100, 10, 1, 0.1, or 0.01 μg/kg, or a vehicle control (H1650 cells implanted alone), a vehicle control (H1650 cells implanted +T cells) or a 4420-hXR32 LP-DART (100 μg/kg) control.

C. Data Collection and Statistical Analysis

Animal weights: Individual animal weights were recorded twice weekly until study completion beginning at the time of tumor cell injection.

Moribundity/Mortality: Animals were observed twice weekly for general moribundity and daily for mortality. Animal deaths were assessed as drug-related or technical based on factors including gross observation and weight loss; animal deaths were recorded daily.

Tumor volume: Individual tumor volumes were recorded twice weekly beginning within one week of tumor implantation and continuing until study completion.

$Tumor Volume ({mm}^{3}) = \frac{Length (mm) \times {width}^{2}}{2}$

Animals experiencing technical or drug-related deaths were censored from the data calculations.

Tumor growth inhibition: Tumor growth inhibition (TGI) values were calculated for each group containing treated animals using the formula:

$1 - \frac{Mean Final Tumor Volume (Treated) - Mean Initial Tumor Volume (Treated)}{Mean Final Tumor Volume (Control) - Mean Initial Tumor Volume (Control)} \times 100$

Animals experiencing a partial or complete response, or animals experiencing technical or drug-related deaths were censored from the TGI calculations. The National Cancer Institute criteria for compound activity is TGI>58% (Corbett et al. (2004) Anticancer Drug Development Guide; Totowa, N.J.: Humana 99-123).

Partial/Complete Tumor Response: Individual mice possessing tumors measuring less than 1 mm³on Day 1 were classified as having partial response (PR) and a percent tumor regression (% TR) value was determined using the formula:

$1 - \frac{Final Tumor Volume ({mm}^{3})}{Initial Tumor Volume ({mm}^{3})} \times 100 %$

Individual mice lacking palpable tumors were classified as undergoing a complete response (CR).

Tumor Volume Statistics: Statistical analyses were carried out between treated and control groups comparing tumor volumes. For these analyses, a two-way analysis of variance followed by a Bonferroni post-test were employed. All analyses were performed using GraphPad PRISM® software (version 5.02). Weight and tumor data from individual animals experiencing technical or drug-related deaths were censored from analysis. However, tumor data from animals reporting partial or complete responses were included in these calculations.

D. HCT116 Tumor Model Results

The cell line HCT116 was pre-mixed with activated T cells and implanted subcutaneous (SC) in NOD/SCID gamma knockout mice (N=8/group) on study day 0 (SD0) as detailed above. Treatment with the various P-cadherin EK-DART, LP-DART or a control DART proteins was initiated on the same day the tumor cell/T cell mixture was implanted [(SD0)] and proceeded subsequently with daily injections for an additional 3 days for a total of 4 daily injections. The animals were treated with P-cadherin EK-DART, LP-DART or a control DART proteins at 4 dose levels (0.5, 0.2, 0.1, and 0.05 mg/kg). Results are shown in FIGS. 9-14.

The HCT116 tumors in the vehicle-treated group (HCT116 cells alone or plus T cells) demonstrated a relatively aggressive growth profile in vivo. At study day 20 (SD20), the average volume of the tumors in the vehicle-treated group was approximately 125 mm³and by study day 35 (SD35) the tumors had reached an average volume of approximately 450 mm³. By the end of the experiment on study day 45 (SD45), the tumors had reached an average volume of approximately 750 mm³. The growth of the HCT116 tumors was significantly inhibited at all dose levels for most of the P-cadherin DART proteins tested. By the end of the experiment on SD45, the average volume of the tumors ranged from around 100 to 0 mm³for all treatments except the 177 EK-DART which ranged from around 500 to 100 mm³.

E. Du145 Results

The cell line Du145 was pre-mixed with activated T cells and implanted SC in NOD/SCID gamma knockout mice (N=8/group) on SD0 as detailed above. Treatment with 153 LP-DART was initiated on the same day the tumor cell/T cell mixture was implanted [(SD0)] and proceeded subsequently with daily injections for an additional 3 days for a total of 4 daily injections. The animals were treated with 153 LP-DART at 5 dose levels (100, 10, 1, 0.1, and 0.01 μg/kg). Results are shown in FIG. 15.

The Du145 tumors in the vehicle-treated group (Du145 cells alone or plus T cells) demonstrated a relatively aggressive growth profile in vivo. The growth of the Du145 tumors was significantly inhibited at 100, 10 and 1 μg/kg dose level of 153 LP-DART and by the end of the experiment (SD74) the average volume of the tumors was 0 mm³. At the 0.1 μg/kg dose level of 153 LP-DART the growth of the Du145 tumors was significantly inhibited and by the end of the experiment (SD74) the average volume of the tumors was approximately 500 mm³.

F. H1650 Results

The cell line H1650 was pre-mixed with activated T cells and implanted SC in NOD/SCID gamma knockout mice (N=8/group) on SD0 as detailed above. Treatment with 153 LP-DART was initiated on the same day the tumor cell/T cell mixture was implanted [(SD0)] and proceeded subsequently with daily injections for an additional 3 days for a total of 4 daily injections. The animals were treated with 153 LP-DART at 5 dose levels (100, 10, 1, 0.1, and 0.01 μg/kg). Results are shown in FIG. 16.

The H1650 tumors in the vehicle-treated group (H1650 cells alone or plus T cells) demonstrated a relatively aggressive growth profile in vivo. The growth of the H1650 tumors was significantly inhibited at 100, 10 and 1 μg/kg dose level of 153 LP-DART and by the end of the experiment (SD49) the average volume of the tumors was 50 mm³. At the 0.1 μg/kg dose level of 153 LP-DART the growth of the H1650 tumors was significantly inhibited and by the end of the experiment (SD49) the average volume of the tumors was approximately 400 mm³.

The various EK-DART, LP-DART and control DART proteins effectively inhibited the growth of HCT116, Du145 and H1650 tumors implanted SC in NOD/SCID mice in the context of the Winn model when dosing was initiated on the day of implantation and continued for 3 or more consecutive days. Based on the criteria established by the National Cancer Institute, the various DART proteins at the 0.1 mg/kg dose level and higher (TGI>58) is considered active in the three models.

Example 10
Established Tumor Human PBMC Engrafted Model

Four separate xenograft tumor models of human colorectal cancer classified with P-cadherin high (HCT116), medium (SW480), low (Ls174T) and negative (SW620) expression based upon quantitative flow cytometry analysis were established. HCT116 demonstrates the highest relative level of cell surface P-cadherin expression (43,801 ABC, Table 28), SW480 demonstrates a relatively moderate level of cell surface P-cadherin expression (12,665 ABC, Table 28) and Ls174T demonstrates a relatively low level of cell surface P-cadherin expression (4,160 ABC, Table 28).

NOD scid gamma (NSG) animals were subcutaneously implanted into the right flank with 5×10⁶HCT116, 5×10⁶SW480, 2×10⁶Ls174T or 5×10⁶SW620 tumor cells on day 0. Seven days prior to randomization (day 5) based upon anticipated tumor growth, animals were inoculated with 5×10⁶freshly isolated human peripheral blood mononuclear cells (PBMC) via intraperitoneal injection of 0.2 mL cell suspension in PBS. One week following PBMC implant (day 12) the tumor volume was measured for all study animals and blood samples collected for flow cytometry. The red blood cells from each sample were lysed with BD Pharmlyse solution (BD Pharmingen) and resulting cell pellets stained for human CD3, human CD4, and human CD8 and analyzed by flow cytometry using a BD Pharmingen LSRII with FACS Diva software. The human CD3+ cells as a percent of total lymphocytes determined by forward scatter versus side scatter gating was established for each animal. Tumor measurements were collected using a digital Vernier caliper (Mitutoyo America, Aurora, Ill.), and volumes were calculated by use of the modified ellipsoid formula ½(width²×length).

Two parameter randomization was used to establish treatment groups of n=10 animals per group providing equal weighting between tumor volume and human CD3+ cell engraftment values. Animals were dosed intravenously with 0.05 mg/kg or 0.5 mg/kg 153 LP-DART (CD3-1), 0.5 mg/kg negative control bispecific (heterodimeric diabody Fc fusion protein against FITC and CD3 epsilon) or DPBS as vehicle to the lateral tail vein of each animal. Biweekly tumor measurements were collected to assess tumor growth inhibition versus controls, along with continuous monitoring for signs of graft versus host disease (e.g. decreased body weight, hair loss, hunched posture).

Tumor growth plots demonstrating the tumor volume (mm³) plotted versus time are shown in FIGS. 17A-17D and corresponding data tables are shown below in Tables 32-35. The data demonstrates that weekly 153 LP-DART administrations resulted in tumor regression at the highest dose (0.5 mg/kg) for the HCT116 (high), SW480 (medium) tumor models and moderate efficacy at the highest dose (0.5 mg/kg) for the Ls174T (low) tumor model. Administration of the negative control bispecific showed no increase in tumor inhibition compared to vehicle treated mice. In the HCT116 tumor model, FIG. 17A shows a 61.9% and 105.1% tumor growth inhibition (TGI) at 0.05 and 0.5 mg/kg doses of 153 LP-DART, respectively. FIGS. 17A-17D further demonstrate that tumor growth inhibition of the established xenograft tumors was dependent on P-cadherin level expression.

TABLE 32

Tumor growth inhibition in HCT116 tumor models.

0.5 mg/kg
0.05 mg/kg
0.5 mg/kg

Vehicle
Control LP-DART
P-cadherin LP-DART
P-cadherin LP-DART

Days from
Mean tumor

Mean tumor

Mean tumor

Mean tumor

Study Start
volume (mm³)
SEM
volume (mm³)
SEM
volume (mm³)
SEM
volume (mm³)
SEM

0
107
13
106
13
105
12
106
12

3
436
58
522
35
426
74
608
73

6
759
92
766
71
410
52
511
95

9
871
124
929
115
444
56
241
49

13
1005
127
1017
93
446
42
60
10

TABLE 33

Tumor growth inhibition in SW480 tumor models.

0.5 mg/kg
0.05 mg/kg
0.5 mg/kg

Vehicle
Control LP-DART
P-cadherin LP-DART
P-cadherin LP-DART

Days from
Mean tumor

Mean tumor

Mean tumor

Mean tumor

Study Start
volume (mm³)
SEM
volume (mm³)
SEM
volume (mm³)
SEM
volume (mm³)
SEM

0
67
6
68
7
68
6
64
7

4
132
14
130
18
123
13
101
13

7
167
12
180
37
129
8
81
13

11
232
31
216
40
157
20
26
3

14
305
39
278
42
240
41
10
3

TABLE 34

Tumor growth inhibition in Ls174T tumor models.

0.5 mg/kg
0.05 mg/kg
0.5 mg/kg

Vehicle
Control LP-DART
P-cadherin LP-DART
P-cadherin LP-DART

Days from
Mean tumor

Mean tumor

Mean tumor

Mean tumor

Study Start
volume (mm³)
SEM
volume (mm³)
SEM
volume (mm³)
SEM
volume (mm³)
SEM

0
121
12
121
14
118
13
126
18

3
301
43
328
40
366
51
393
51

7
686
103
801
59
619
104
420
107

10
1142
175
1099
116
796
110
451
106

14
1565
253
1456
206
1072
152
751
158

TABLE 35

Tumor growth inhibition with engrafted human T cells of SW620

0.5 mg/kg
0.05 mg/kg
0.5 mg/kg

Vehicle
Control LP-DART
P-cadherin LP-DART
P-cadherin LP-DART

Days from
Mean tumor

Mean tumor

Mean tumor

Mean tumor

Study Start
volume (mm³)
SEM
volume (mm³)
SEM
volume (mm³)
SEM
volume (mm³)
SEM

0
308
29
305
29
319
28
304
28

3
467
74
605
76
440
37
441
64

7
622
102
628
91
480
47
528
106

10
1014
219
930
125
790
117
767
106

FIG. 17E shows one way ANOVA analysis of the tumor volume (mm³) collected at day 25 post implant of the HCT116 tumor model with Dunnet's multiple comparison test to the vehicle control group. The data demonstrates significant tumor growth inhibition (*p<0.05) in both the 0.05 mg/kg and 0.5 mg/kg dosed groups with 153 LP-DART.

The results of pharmacokinetic analysis of P-cadherin 153 LP-DART treated HCT116 tumor bearing NSG animals is shown in Table 36. Animals were administered a single IV dose of 0.05 or 0.5 mg/kg 153 LP-DART and euthanized in n=3 groups at the indicated time-points following dosing to collect serum and tumor. Assay of the LP-DART was performed by sandwich ELISA against a standard curve. The 153 LP-DART demonstrated extended serum and tumor half-life.

TABLE 36

Pharmacokinetic analysis of 153 LP- DART treated animals.

IV Dose

Co
Cmax
Tmax
AUC(0-Tlast)
AUC(0, inf)
CL (0-t)
Vdss
T½
T½

(mg/kg)
Tissue
(μg/mL)
(μg/mL)
(hours)
(μg*hours/mL)
(μg*hours/mL)
(mL/kg/hours)
(L/kg)
(hours)
(days)

0.05
Serum
0.675
0.648
0.25
43.7
61
1.14
150
144
6

0.5
Serum
11.5
11
0.25
647
827
0.773
89.9
115
4.79

0.05
Tumor
nd
0.0561
48
7.48
10.8
nd
nd
130
5.42

0.5
Tumor
nd
1.04
48
167
199
nd
nd
88.4
3.68

Example 11
Established Tumor Human T Cell Adoptive Transfer Model

Tumor growth inhibition with adoptive transfer of human T cells of HCT116 (colon) and SUM149 (breast) was analyzed. HCT116 demonstrates the highest relative level of cell surface P-cadherin expression (43,801 ABC, Table 28) and SUM149 demonstrates a relatively moderate level of cell surface P-cadherin expression (15,426 ABC, Table 28). T cells were resuspended to 0.5×10⁶cells/ml with OpTmizer CTS T Cell Expansion Serum-Free Media supplemented with 2× GlutaMax-1, 1% PenStrep, and 20 ng/ml recombinant human IL-2 (Life Technologies, Carlsbad, Calif.). Dynabeads Human T-Expander CD3/CD28 magnetic beads (Life Technologies) were then added to T cells at 1×10e6 beads/ml (2 beads/cell), and the cells were cultured for one week according to the manufacturer's protocol. At the time of harvest, beads were removed with a magnet, and cells were resuspended in DPBS at 1×10⁷cells/ml for in vivo inoculation.

For xenograft studies, NSG mice were inoculated with either 5×10⁶HCT116 cells in the flank or 5×10⁶SUM149 cells in the mammary fat pad in a total injection volume of 0.2 mL. HCT116 cells were suspended in DPBS, while SUM149 cells were suspended in growth media and mixed 1:1 with Matrigel Basement Membrane Matrix (BD Biosciences, San Jose, Calif.).

Tumor measurements were collected using a digital Vernier caliper (Mitutoyo America, Aurora, Ill.), and volumes were calculated by use of the modified ellipsoid formula ½(width²×length). Mice were randomized and received the initial dose once tumors had reached growth phase, and 2×10⁶cultured T cells/mouse were inoculated the following day. Mice were dosed in 0.2 mL bolus injection weekly up to 5 times, and all compound and T cell administrations were intravenous via the lateral tail vein of each animal. Biweekly tumor measurements were collected along with continuous monitoring for signs of graft versus host disease (e.g. decreased body weight, hair loss, hunched posture).

Tumor growth inhibition with adoptive transfer of human T cells of HCT116 (colon) is shown in FIG. 18A (Table 37) and SUM149 (breast) is shown in FIG. 18B (Table 38). Weekly administration of 153 LP-DART (CD3-1) resulted in a dose response with tumor regressions at the highest dose (0.5 mg/kg) and tumor stasis with eventual relapse at the lower dose (0.05 mg/kg). Administration of the negative control LP-DART showed no increase in tumor inhibition compared to vehicle treated mice. Compared to the above engrafted T cell model of Example 10, the adoptive T cell transfer model allows for prolonged 153 LP-DART dosing before the mice succumb to GVHD (2 weeks and 6 weeks, respectively).

TABLE 37

Tumor growth inhibition with adoptive transfer of human T cells of HCT116.

0.05 mg/kg
0.5 mg/kg

Vehicle
P-cadherin 153 LP-DART
P-cadherin 153 LP-DART

Days from
Mean tumor

Mean tumor

Mean tumor

Randomization
Volume (mm³)
SEM
Volume (mm³)
SEM
Volume (mm³)
SEM

0
144
11
147
10
147
12

4
310
22
333
19
333
34

8
573
37
429
31
430
34

11
803
38
365
35
354
35

15
1074
48
357
29
114
22

18
1340
53
473
30
65
22

22
1577
48
720
49
18
13

25
1655
53
911
56
0
0

29
—
—
1175
46
0
0

32
—
—
1339
44
0
0

35
—
—
1523
50
0
0

38
—
—
—
—
0
0

42
—
—
—
—
0
0

45
—
—
—
—
0
0

TABLE 38

Tumor growth inhibition with adoptive transfer of human T cells of SUM149.

0.5 mg/kg
0.05 mg/kg
0.5 mg/kg

Vehicle
Control LP-DART
P-cadherin 153 LP-DART
P-cadherin 153 LP-DART

Days from
Mean tumor

Mean tumor

Mean tumor

Mean tumor

Randomization
volume (mm³)
SEM
Volume (mm³)
SEM
Volume (mm³)
SEM
Volume (mm³)
SEM

0
208
5
207
5
209
6
207
13

4
348
13
342
41
393
18
400
22

8
528
18
541
30
424
17
306
27

11
733
36
782
42
389
24
183
15

14
889
39
856
36
330
18
101
7

17
1132
42
1137
49
435
26
50
10

23
1464
61
1524
57
620
47
0
0

25
1671
81
1696
75
694
60
0
0

29
—
—
—
—
874
89
0
0

32
—
—
—
—
—
—
0
0

36
—
—
—
—
—
—
0
0

39
—
—
—
—
—
—
0
0

42
—
—
—
—
—
—
0
0

Example 12
P-Cadherin Positive Patient Derived Xenograft (PDX)

In vivo tumor growth inhibition of a P-cadherin positive PDX was analyzed. P-cadherin positive patient derived colorectal tumor xenograft, PDX-CRX-11260, was identified by positive staining of FFPE tumor samples with anti-P-cadherin antibody. PDX-CRX-11260 tumor tissue was implanted to NSG animals and grown to roughly 100 mm³. The animals were randomized into dose groups of n=7 and dosed with vehicle, 0.05 mg/kg 153 LP-DART (CD3-1), or 0.5 mg/kg 153 LP-DART (CD3-1) weekly. One day following the initial dose all animals received 2×10⁶in vitro expanded human T cells.

Tumor growth inhibition of PDX-CRX-11260 is shown in FIG. 19A (Table 39). The 0.05 mg/kg 153 LP-DART dose group demonstrated 3 of 7 animals having complete responses and the 0.5 mg/kg 153 LP-DART dose group demonstrated 7 of 7 animals having complete responses. The data demonstrates that 153 LP-DART exhibits potent tumor growth inhibition of a P-cadherin positive colon tumor PDX.

TABLE 39

Tumor growth inhibition P-cadherin positive PDX-CRX-11260.

0.05 mg/kg
0.5 mg/kg

Vehicle
P-cadherin 153 LP-DART
P-cadherin 153 LP-DART

Days from
Mean tumor

Mean tumor

Mean tumor

Randomization
Volume (mm³)
SEM
Volume (mm³)
SEM
Volume (mm³)
SEM

0
107
6
106
5
106
6

5
206
22
226
23
215
22

8
311
20
290
37
186
16

13
579
52
224
52
104
28

16
591
77
227
93
33
17

20
725
67
165
87
25
17

23
884
79
172
89
0
0

27
1101
99
292
161
0
0

30
1318
136
294
179
0
0

34
1463
161
370
240
0
0

37
1473
181
132
101
0
0

40
—
—
181
141
0
0

Example 13
Dose Dependent Accumulation of Tumor Infiltrating Lymphocytes (TILs)

HCT116 tumor bearing mice engrafted with human peripheral blood mononuclear cells (PBMC) and dosed with varying levels of 153 LP-DART (CD3-1) (n=3) were euthanized six days following dosing to assess tumor infiltrating human CD3+ lymphocytes. Tumor samples were collected into gentleMACS C tubes containing human tumor cell dissociation buffer (Miltenyi Biotech) and processed to single cell suspension using the manufacturer suggested protocol for soft human tumors using the gentleMACS tissue dissociator (Miltenyi Biotech). The cell suspensions were then washed with DPBS containing 2 mM EDTA and viable cell counts determined by trypan blue exclusion and hemocytometer. 1×10⁶live cells from each sample were collected into PBS containing 2% FBS and 0.02% sodium azide and stained with CD3 FITC (BD Pharmingen) for 30 min on ice. Following washing, propidium iodide was added to each sample immediately prior to analysis using LSRII with FACS Diva software (BD Pharmingen).

The data was analyzed by FlowJo software (Treestar) and the percent CD3+ cells of total live cell events was plotted in a grouped plot with significance determined via 1 way ANOVA with Dunnett's Multiple Comparison test versus untreated control samples using GraphPad Prism 5.0 software. The results shown in FIG. 20 and Table 40 demonstrate a dose dependent increase in tumor infiltrating lymphocytes (TILS). Further, immunohistochemistry (IHC) staining of human CD3 of the tumor tissue (not shown) demonstrated that 153 LP-DART treatment of animals having P-cadherin positive tumors and human T cells resulted in intra-tumoral accumulation of CD3+ lymphocytes.

TABLE 40

Dose dependent accumulation of tumor

infiltrating lymphocytes (TILs).

Dose of 153 LP-DART
Mouse
% CD3+ of all viable cells

0.01 mg/kg
1
7.60

2
6.48

3
12.40

0.05 mg/kg
1
43.00

2
21.70

3
25.70

0.5 mg/kg
1
61.30

2
63.40

3
50.30

Example 14
Biodistribution and Tumor Targeting of P-Cadherin LP-DART Using FMT Imaging

NSG or athymic nude mice with established subcutaneous HCT116 xenografts were used. Studies that included engraftment of T cells received T cells isolated from healthy human volunteers. The bio-distribution studies were initiated when the tumors reached 300-500 mm³. P-cadherin 153 LP-DART (CD3-1) or a negative control-DART (non-targeted domain×CD3 binding domain) was conjugated with a near-infrared fluorophore VivoTag680XL (VT680), with ratios of fluorphore to bispecific labeling between 1 to 2.75, resulting in: P-Cadherin 153 LP-DART-VivoTag680XL and Control-LP-DART-VivoTag680XL.

The labeling efficiency was determined by spectrophotometer. T cells used in trafficking studies were labeled with CellVue815 (CV815). Cell surface P-cadherin expression and P-cadherin 153 LP-DART binding was determined by flow cytometry. T cell activity was measured with cytotoxic T-lymphocyte (CTL) assays. FMT imaging was performed longitudinally post injection of labeled bispecifics. Data was analyzed using TrueQuant software. Plasma and tissues were collected at various time points for PK analysis by ELISA.

A. In vitro Characterization of Labeled Biologic Molecule

To determine whether the VT680 labeling affected the molecule properties, quality control studies were performed: in vitro binding to cells by FACS analysis and cytotoxicity assays for molecule activity. The properties were compared for VT680 labeled and unlabeled control LP-DART and P-cadherin 153 LP-DART. For T cells labeled with CV815, T cell expansion and also cytotoxicity on effector cells were evaluated. Table 41 shows the molecules used for the biodistribution study. DOL=degree of labeling.

TABLE 41

VivoTag680XL labeled molecules.

Molecules
DOL
~Ab/Dose

P-Cadherin 153 LP-DART-VivoTag680X
2.62
0.925 mg/kg

Control-LP-DART-VivoTag680XL
2.75
0.9 mg/kg

Direct soluble P-cadherin ELISA to evaluate the binding capability: FIG. 21A demonstrates that VT680 labeling to P-cadherin 153 LP-DART had minimal effect on P-cadherin binding. The minimal decrease was DOL dependent. Control-LP-DART (labeled or unlabeled) did not bind to P-cadherin.

Direct soluble CD3 ELISA to evaluate the binding capability: FIG. 21B demonstrates that VT680 labeling to P-cadherin 153 LP-DART significantly reduced binding to CD3 epsilon/delta protein. The decrease in binding was DOL dependent. Control LP-DART had lower binding than P-cadherin 153 LP-DART. VT680 labeling significantly reduced the binding to CD3 epsilon/delta.

HCT116 cells expressing luciferase were utilized as “target” cells for in vitro cytotoxicity with purified human T lymphocyte “effector” cells in a 24 hour assay at a 10:1 effector to target ratio against a titration of P-cadherin 153 LP-DART. The cytotoxicity plot shown in FIG. 21C illustrates that the cytotoxicity was reduced when P-Cadherin 153 LP-DART was labeled with VT680 at 2.0 DOL.

Comparison of cell viability and expansion of T cells after labeling with cell labeling dyes: Human T cells isolated from donors were labeled with fluorescent cell tracking dyes—VivoTrack680 and CellVue815 (CV815). FIG. 21D demonstrates that neither VivoTrack680 or CV815 affected the cell viability or expansion of T cells.

B. In vivo FMT Imaging and PK Analysis Data

Biodistribution and targeting of P-cadherin 153 LP-DART in HCT116 xenograft model was analyzed using longitudinal FMT imaging. One million HCT116 cells in 50% matrigel were injected into the SQ flanks of the female nu/nu mice (8 weeks old). The tumors were allowed to grow up to ˜300-500 mm³in size. The animals were injected with probes (VT680 labeled biologic molecules) equivalent to 1 nmoles of VT680 (Table 41). In vivo FMT Imaging (whole body) was performed longitudinally at 5 min, 24 hr, 48 hr, 96 hr and 240 hr post injection. Blood (plasma) samples were collected at each time point. At intermittent time point and end of the study, the animals were euthanized and perfused with PBS/Saline to remove the blood from vascular compartment. The tissues (tumor, liver, spleen, kidney, lungs and brain) were imaged ex vivo. The FMT data was analyzed using TrueQuant software. After ex vivo imaging, the tissues were flash frozen for future PK analysis. PK analysis of plasma and tissue samples was performed by ELISA. T cell engraftment was not performed in the biodistribution study.

FMT imaging showed that P-cadherin 153 LP-DART specifically targeted HCT116 tumors. Non-invasive in vivo FMT imaging revealed high levels of P-cadherin 153 LP-DART accumulation in the tumors (images not shown) compared to control LP-DART. The in vivo kinetics revealed that the peak accumulation in tumors was about 96 hours post injection, as shown in FIG. 22A. Whole body distribution and clearance data demonstrating the composite of accumulation and clearance from various tissues, tumor and vascular compartments showed no overall significant difference between P-cadherin 153 LP-DART and control-LP-DART, as shown in FIG. 22B.

As shown in FIGS. 22C and 22D, ex vivo analysis of tumor and selected tissues at 48, 96 and 240 hours demonstrated that the P-cadherin 153 LP-DART penetrated into the tissue. Tumors showed 20-30 fold higher accumulation of P-cadherin 153 LP-DART compared to control-LP-DART at all time points. At 240 hours post-injection, there was still measurable P-cadherin 153 LP-DART detected in tumors. Ex vivo comparison of accumulation in various organs (liver (FIG. 22D), kidney, spleen, lungs and brain (data not shown) showed no difference between P-cadherin 153 LP-DART or negative control DART

A comparison of FMT data with pharmacokinetic methods was conducted to confirm the profiles of FMT imaging. Samples from the FMT study were evaluated by routine pharmacokinetic method, ELISA. The PK data from tumor and liver showed similar trends as FMT imaging data described above. As shown in FIG. 23A, the plasma profile showed ˜2-3 fold longer plasma exposure of P-Cad-LP-DART compared to Control-LP-DART. As shown in FIG. 23B, the evaluation of Ex vivo tumor sample showed ˜7 fold increase in accumulation of P-Cad-LP-DART. As shown in FIG. 23C, the evaluation of ex vivo liver samples showed increased accumulation at 48 hours, however there was no significant difference at 96 and 240 hours.

C. Cellular Tracking of Engrafted T Cells

Cellular tracking of engrafted T cells in a HCT116 model using FMT imaging was conducted. One million HCT116 cells in 50% matrigel were injected into the SQ flanks of the female NSG mice (8 weeks old). When the tumors were ˜300-500 mm³in size, P-cadherin 153 LP-DART (labeled or unlabeled with VT680) was injected via SQ route. After 24 hours of injecting the drugs, five million T cells labeled with CV815 were injected via IV route. FMT imaging was performed longitudinally after cell engraftment. Sequential imaging was performed using 680 nm and 800 nm laser for P-cadherin 153 LP-DART-VT680 and T Cell-CV815 groups.

The cell trafficking studies with CellVue labeled T cells showed the co-localization of T cells and P-cadherin 153 LP-DART in tumors. As shown in FIG. 24A, the evaluation of T cell activity of CV815 labeled and unlabeled T cells demonstrates that the labeling of T cells with fluorophore did not have an effect on the cytotoxic ability of T cells (in vitro). As shown in FIG. 24B, the in vivo trafficking kinetics of fluorophore labeled T cells in tumor by FMT imaging demonstrates significantly increased T cell trafficking and accumulation on day 7 post cell engraftment in the P-cadherin 153 LP-DART injected group.

FMT images (not shown) demonstrated targeting of VivoTag680 labeled P-cadherin 153 LP-DART and trafficking of CV815 labeled T cells to tumor on day 5 and day 7 post cell engraftment. This group received CV815 labeled T cells+P-Cadherin 153 LP-DART labeled with VivoTag680. P-Cadherin 153 LP-DART-VT680 with a DOL of 1.0 was used. DOL of 1.0 has shown to minimally affect the binding to P-Cadherin and CD3 proteins.

Example 15
Crystallization and Structure Determination

For crystallization trials a purified DART protein shown in FIG. 25 was utilized. In particular, the DART protein (having a clone 35 P-CAD VL/VH binding domain and a clone CD3 binding domain) was designed for crystallography without heterodimer promoting domains and having a C-terminal His tag HHHHHH (SEQ ID NO: 78) designated “6×HIS” covalently linked to the P-CAD 35 VH domain and a C-terminal FLAG sequence GGCGGDYKDDDDK (SEQ ID NO: 79) designated “FLAG” covalently linked to a CD3 VH domain (herein after designated “crystallography 35 DART”).

The purified crystallography 35 DART was concentrated to 9.6 mg/ml in a protein solution containing TBS. The crystals were obtained by hanging-drop vapor-diffusion method from a condition containing 15% PEG 8K and 0.5 M lithium sulfate. The hexagonal plate-like crystals had symmetry consistent with trigonal space group P321 with cell parameters a=b=142.81 Å; c=62.69 Å and with one crystallography 35 DART molecule in the crystallographic asymmetric unit. The crystals were cryo-protected using reservoir solution containing 25% ethylene glycol and were flash frozen in liquid nitrogen. A data set to a 2.0 Å resolution was collected from a single frozen crystal at IMCA beamline 17-ID at the Argonne National Laboratory (APS). The data were processed and scaled using autoPROC and SCALA. The final data set was 96.8% complete with average redundancy of 9.9 and with R_symof 14.2%.

The structure was determined by molecular replacement with PHASER starting with the single chain Fv fragment models prepared from the Brookhaven PDB entry code, 1 moe. The solution was obtained by searching for each of the four subunits of the crystallography 35 DART molecule separately. Several iterative rounds of manual adjustment and model rebuilding using COOT and crystallographic refinement using autoBUSTER yielded the final crystallography 35 DART model with a crystallographic R_workof 17.6% and R_freeof 20.5%, where R_work=∥F_obs|−|F_calc∥/|F_obs| and R_freeis equivalent to R_work, but calculated for a randomly chosen 5% of reflections omitted from the refinement process.

The final crystallography 35 DART model comprised two chains, heavy (H) and light (L), with residues 1-110, 117-239 of chain H and residues 2-110, 116-246 of chain L. Non-protein atoms present in the model included 574 water molecules and 5 sulfate ions. Missing amino acids in the linker regions (11 residues) were not modeled into the structure because of the lack of electron density, very likely due to disorder.

FIG. 26 shows a graphical depiction of the crystal structure of the crystallography 35 DART protein. In the crystal, crystallography 35 DART assembled into a very compact spherical structure that differed considerably from the previously published diabody structures (FIG. 27) (see Carmichael et al., J. Mol. Biol. 326: 341-351 (2003); Perisic, et al., Structure, 2(12): 1217-1226, (1994)). It has an unexpectedly extensive interface between subunits and clear evidence for stabilization of the structure by a disulfide linkage between the two polypeptide chains, Cys²³⁹(VH1B)-Cys²⁴⁶(VL2A). The four subunits, VL1A VH1B, VL2B and VH2A, all contributed to the binding interface, with the most contribution coming from the framework amino acid residues of both chains. The latter observation suggested that other DART proteins, constructed with other CDR sequences, with binding to diverse tumor-associated antigens, may share a similar architecture and a similar domain interface with the crystallography 35 DART.

Despite the relatively tight packing, the crystallography 35 DART interface was not as highly enriched and as shaped complementary as naturally occurring interfaces in Fab molecules, characterized by a high degree of interchain association between the heavy and light chain domains. In crystallography 35 DART, a few depressions in one chain or domain that were not complemented by protrusions in the other resulted in small interior packing defects and large voids, filled with bound water in the crystal structure (FIG. 28).

The two antigen binding sites on the crystallography 35 DART were separated from each other by about 40 Å and were located on the orthogonally opposite sides of the molecule. In this context, “orthogonally opposite sides” means the two binding sites on the crystallography 35 DART were opposing each other at an angle of ˜90°.

The anti-CD3 CDR regions were positioned remotely from the subunit interface whereas the anti-P-CAD CDR regions were within a more adjacent area (see FIG. 27).

In addition, novel single chain DART (scDART) variants were generated to address the problem of two chain diabody mispairing (various VH and VL chain directionalities may be used based on the crystal structure). Additional cysteine residues were engineered to induce the covalent linkage within scDART proteins to further improve the stability of the single chain diabody. The method was a variation of Dani et al. (Prot. Eng. 16(3): 187-193 (2003)) utilizing further analysis to rank the quality of each site. Various disulfide bond locations were also engineered using this method to reduce solvent accessibility of the disulfide bond as well as to reduce the length of linkers. For example, modeling positions for the introduction of stereochemically optimal disulfides suggested the following amino acid residue-pairs for cysteine mutagenesis (see FIG. 29): Gln¹²¹(VH1B)Gly¹⁶⁰(VL1A), Val¹²⁹(VH1B)Gly²⁴⁴(VL1A), Val¹²³(VH1B)Gly¹⁶⁰(VL1A), Gly¹²⁶(VH1B)Ser²⁴²(VL1A), and Ala¹²⁷(VH1B)Ser²⁴²(VL1A).

Further, using the Discovery Studio (Accelrys Software Inc) stability prediction protocols and the Rosetta v2.3 stability protocol, DART variants (including scDART variants) were engineered through site-directed mutagenesis in the interdomain interface to fill up large interior voids/holes (FIG. 26) in an attempt to further improve the stability of interdomain association. For example, increasing side chain volumes at positions Ala⁴⁴(VL1A), Val²¹³(VH2A), Leu²³⁸(VH2A), or Met²³¹(VL2B) by replacing the relatively small amino acids for amino acids with bulky aromatic side-chains like phenylalanine, tyrosine, or tryptophan. Further engineered were single surface cysteine mutants probing various positions based on the crystal structure for a site specific bio-conjugation approach.

Example 16
Eptitope Mapping of P-Cadherin 153 LP-DART

To identify the binding epitope of the P-cadherin 153 LP-DART, soluble P-cadherin-extra cellular domain (ECD)-Fc fusion protein constructs were generated. Each P-cadherin-Fc construct comprised a signal peptide, pro-peptide and a P-cadherin ECD subdomain region (either ECD1, ECD1-2, ECD1-3, ECD1-4, or ECD1-5) genetically fused to the hinge and CH2 and CH3 domains of human IgG1 via cleavable linker, as shown in FIG. 34. The ECD1, ECD2, ECD3, ECD4, and ECD5 P-cadherin subdomain fragments used to generate the ECD-Fc constructs correspond with the sequences identified in the full length p-cadherin epitope (UniProt P22223, CADH3, Human Cadherin-3), shown in FIG. 35 and further characterized in Table 42.

TABLE 42

P-cadherin domains and subdomains generated.

SEQ ID

Domain
Residues
NO:

Full length P-cadherin (UniProt P22223)
1-829
159

P-cadherin Signal Peptide
1-24
160

P-cadherin Propetide
25-107
161

P-cadherin Extra Cellular Domain 1
108-215
162

P-cadherin Extra Cellular Domain 2
216-328
163

P-cadherin Extra Cellular Domain 3
329-440
164

P-cadherin Extra Cellular Domain 4
441-546
165

P-cadherin Extra Cellular Domain 5
547-650
166

P-cadherin Transmembrane and Cytoplasmic
651-829
167

P-cadherin Extra Cellular Domain 1-2
108-328
168

P-cadherin Extra Cellular Domain 1-3
108-440
169

P-cadherin Extra Cellular Domain 1-4
108-546
170

P-cadherin Extra Cellular Domain 1-5
108-650
171

All constructs were confirmed by DNA sequencing and transiently transfected into FreeStyle™ 293 HEK cells (Life Technologies, Grand Island, N.Y.) according to the manufacturer's method and expressed over 5-7 days. For enhanced processing of the propeptide, an expression vector containing the PACE cleavage enzyme was co-transfected along with the P-cadherin-containing vector. Soluble protein of interest was purified using standard Protein A chromatographic techniques (Protein A FF, GE Healthcare, Piscataway, N.J.) followed by gel filtration size exclusion chromatography (Superdex200, GE Healthcare, Piscataway, N.J.). Purified protein was characterized for purity and activity by binding ELISA using commercially-available anti-human P-cadherin monoclonal and polyclonal antibodies. Purified P-cadherin ECD-Fc constructs were used in a protein ELISA to determine which ECD subdomain(s) of P-cadherin the 153 LP-DART bound to.

Table 43 shows the binding results of the 153 LP-DART, and various other anti-P-cadherin DART molecules to the P-cadherin ECD-Fc constructs generated, along with a polyclonal Ab (R&D Systems, Minneapolis, Minn.) used to confirm proper expression and folding the ECD-Fc constructs. The results show that 153 LP-DART did not bind to ECD1-Fc or ECD1-2-Fc, but did bind to all constructs that contain ECD3 (ECD1-3-Fc, ECD1-4-Fc and ECD1-5-Fc). This data demonstrates that 153 LP-DART binds to human P-cadherin within ECD3.

TABLE 43

Binding of P-cadherin DARTs to P-cadherin ECD-Fc constructs

P-cad ECD -Fc
35 EK-
153 EK-
153 LP-
PF EK-
20 EK-
30 EK-
Polyclonal

Construct
DART
DART
DART
DART
DART
DART
Ab (AF-761)

ECD 1-Fc
No
No
No
No
No
Yes
Yes

ECD 1-2-Fc
No
No
No
Yes
Yes
Yes
Yes

ECD 1-3-Fc
Yes
Yes
Yes
Yes
Yes
Yes
Yes

ECD 1-4-Fc
Yes
Yes
Yes
Yes
Yes
Yes
Yes

ECD 1-5-Fc
Yes
Yes
Yes
Yes
Yes
Yes
Yes

As shown in Table 24 (above), 153 LP-DART does not bind to murine P-cadherin, therefore 153 LP-DART recognizes an epitope on ECD3 of human P-cadherin (SEQ ID NO: 164) where it differs in sequence from murine P-cadherin ECD3 (SEQ ID NO: 172). Table 44 shows an alignment of human and murine P-cadherin ECD-3 illustrates the differences between these orthologs. (;) represents similar, (.) represents different and (—) represents very different residues.

TABLE 44

Alignment of human and mouse P-cadherin extracellular domain 3 (ECD3).

ECD3
Sequence

huPcadECD3
DPQKYEAHVPENAVGHEVQRLTVTDLDAPNSPAWRATYLIMGGDDGDHFTITTHPESNQGILTTRK

muPcadECD3
EPQKYEAWVPENEVGHEVQRLTVTDLDVPNSPAWRATYHIVGGDDGDHFTITTHPETNQGVLTTKK

:******-****-**************.**********-*:***************:***:***:*

huPcadECD3
GLDFEAKNQHTLYVEVTNEAPFVLKLPTSTATIVVHVEDVNEAPVF (SEQ ID NO: 164)

muPcadECD3
GLDFEAQDQHTLYVEVTNEAPFAVKLPTATATVVVHVKDVNEAPVF (SEQ ID NO: 172)

******::**************.:****:***:****:********

To determine whether binding epitope and binding affinity had an effect on potency in T-cell retargeting cytotoxicity assays, the results of the cytotoxic T-lymphocyte (CTL) assays for various anti-P-cadherin EK-DARTs was analyzed. The cytotoxicity values (see Table 29) were compared to the binding affinity to soluble and cell surface-expressed P-cadherin by Biacore and ELISA (see Tables 14-16, 24). The PF-DART binds to P-cadherin in ECD2 with single digit nM binding affinity and has sub-nM CTL cytotoxic activity. The 35 EK-DART (parental clone of 153 EK-DART) binds to P-cadherin in ECD3 with a binding affinity of approximately 35 nM, almost 35 times less than that of the PF-DART but still kills with similar potency (sub-nM). This data suggests that binding in ECD3, which is closer to the cell membrane, results in higher CTL potency. The 153 LP-DART also binds to P-cadherin in ECD3 with much stronger binding affinity than 35 EK-DART (0.5 nM), and shows the highest CTL activity (sub-pM).

Example 17
Paratope Mapping of Anti-P-Cadherin CDRs

To further understand which amino acid (AA) residues in the complementarity determining regions (CDRs) are involved in scFv 153 binding to human P-cadherin, all non-alanine CDR AAs were individually mutated to alanine by parallel site-directed mutagenesis.

Site-Directed Mutagenesis

Sixty three oligos (33-45 bases in length) were designed (Table 45). Oligo length was adjusted for each oligo to have comparable predicted melting temperature (Tm) values. Each oligo was designed to be the reverse complement of the sense strand of clone 153 scFv and to be flanked by 15-21 bases on either side of the “AGC” (reverse complement of alanine codon, GCT).

TABLE 45

Designed oligos.

Sequence

SEQ ID

Name
Sequence
Tm
NO.

pcdAlaH1-1
GCT GGT AAA GGT GTA AGC AGA AGC CTT GCA GGA
66
173

pcdAlaH1-2
ACC GTA GCT GGT AAA GGT AGC ACC AGA AGC CTT GCA GGA
69
174

pcdAlaH1-3
GAT ACC GTA GCT GGT AAA AGC GTA ACC AGA AGC CTT GCA
66
175

pcdAlaH1-4
GAT ACC GTA GCT GGT AGC GGT GTA ACC AGA AGC
65
176

pcdAlaH1-5
CCA GCT GAT ACC GTA GCT AGC AAA GGT GTA ACC AGA AGC
66
177

pcdAlaH1-6
CAC CCA GCT GAT ACC GTA AGC GGT AAA GGT GTA ACC AGA
67
178

pcdAlaH1-7
TCG CAC CCA GCT GAT ACC AGC GCT GGT AAA GGT GTA ACC
70
179

pcdAlaH1-8
TCG CAC CCA GCT GAT AGC GTA GCT GGT AAA GGT
67
180

pcdAlaH1-9
CTG TCG CAC CCA GCT AGC ACC GTA GCT GGT AAA
68
181

pcdAlaH1-10
GGC CTG TCG CAC CCA AGC GAT ACC GTA GCT GGT
71
182

pcdAlaH2-1
ACC ATT GTA AGC GCT GAT AGC TCC CAT CCA CTC AAG CCC
69
183

pcdAlaH2-2
GTT ACC ATT GTA AGC GCT AGC CCA TCC CAT CCA CTC AAG
67
184

pcdAlaH2-3
TGT GTT ACC ATT GTA AGC AGC GAT CCA TCC CAT CCA CTC
66
185

pcdAlaH2-5
TGC ATA GTT TGT GTT ACC ATT AGC AGC GCT GAT CCA TCC CAT CCA
68
186

pcdAlaH2-6
TGC ATA GTT TGT GTT ACC AGC GTA AGC GCT GAT CCA TCC
67
187

pcdAlaH2-7
CTG TGC ATA GTT TGT GTT AGC ATT GTA AGC GCT GAT CCA
65
188

pcdAlaH2-8
GAG CTT CTG TGC ATA GTT TGT AGC ACC ATT GTA AGC GCT GAT CCA
67
189

pcdAlaH2-9
GAG CTT CTG TGC ATA GTT AGC GTT ACC ATT GTA AGC GCT
65
190

pcdAlaH2-10
CTG GAG CTT CTG TGC ATA AGC TGT GTT ACC ATT GTA AGC
65
191

pcdAlaH2-11
GCC CTG GAG CTT CTG TGC AGC GTT TGT GTT ACC ATT GTA
68
192

pcdAlaH2-13
GAC TCT GCC CTG GAG CTT AGC TGC ATA GTT TGT GTT ACC
66
193

pcdAlaH2-14
GGT GAC TCT GCC CTG GAG AGC CTG TGC ATA GTT TGT GTT
69
194

pcdAlaH2-15
CAT GGT GAC TCT GCC CTG AGC CTT CTG TGC ATA GTT TGT
68
195

pcdAlaH2-16
GGT CAT GGT GAC TCT GCC AGC GAG CTT CTG TGC ATA GTT
69
196

pcdAlaH2-17
GGT CAT GGT GAC TCT AGC CTG GAG CTT CTG TGC
67
197

pcdAlaH3-1
ACC AAA AGC ATT AGC TGT ATC AGC AGT CGC ACA GTA ATA CAC GGC
67
198

pcdAlaH3-2
AAA AGC ATT AGC TGT AGC GAT AGT CGC ACA GTA
62
199

pcdAlaH3-3
GAT ACC AAA AGC ATT AGC AGC ATC GAT AGT CGC ACA GTA
64
200

pcdAlaH3-5
GCC CCA GAT ACC AAA AGC AGC AGC TGT ATC GAT AGT CGC
68
201

pcdAlaH3-7
TTG GCC CCA GAT ACC AGC AGC ATT AGC TGT ATC
65
202

pcdAlaH3-8
CCC TTG GCC CCA GAT AGC AAA AGC ATT AGC TGT
66
203

pcdAlaH3-9
CAT TGT CCC TTG GCC CCA AGC ACC AAA AGC ATT AGC TGT
69
204

pcdAlaL1-1
GTT GGA GCT GCT TCC AGC GCA GGA GAT GGT GAC
69
205

pcdAlaL1-2
AAT GTT GGA GCT GCT AGC AGA GCA GGA GAT GGT
66
206

pcdAlaL1-3
ATT CCC AAT GTT GGA GCT AGC TCC AGA GCA GGA GAT GGT
68
207

pcdAlaL1-4
ATT ATT CCC AAT GTT GGA AGC GCT TCC AGA GCA GGA GAT
66
208

pcdAlaL1-5
ATA ATT ATT CCC AAT GTT AGC GCT GCT TCC AGA GCA GGA
65
209

pcdAlaL1-6
GGA TAC ATA ATT ATT CCC AAT AGC GGA GCT GCT TCC AGA GCA GGA
67
210

pcdAlaL1-7
GGA TAC ATA ATT ATT CCC AGC GTT GGA GCT GCT TCC AGA
65
211

pcdAlaL1-8
CCA GGA TAC ATA ATT ATT AGC AAT GTT GGA GCT GCT TCC
62
212

pcdAlaL1-9
GTA CCA GGA TAC ATA ATT AGC CCC AAT GTT GGA GCT GCT
65
213

pcdAlaL1-10
CTG GTA CCA GGA TAC ATA AGC ATT CCC AAT GTT GGA GCT
65
214

pcdAlaL1-11
CTG CTG GTA CCA GGA TAC AGC ATT ATT CCC AAT GTT GGA
65
215

pcdAlaL1-12
GAG CTG CTG GTA CCA GGA AGC ATA ATT ATT CCC AAT GTT
64
216

pcdAlaL1-13
TGG GAG CTG CTG GTA CCA AGC TAC ATA ATT ATT CCC AAT
65
217

pcdAlaL2-1
TGA GGG TCG CTT ATT ATT AGC ATA AAT GAG GAG TTT GGG
63
218

pcdAlaL2-2
CCC TGA GGG TCG CTT ATT AGC GTC ATA AAT GAG GAG TTT
65
219

pcdAlaL2-3
AAT CCC TGA GGG TCG CTT AGC ATT GTC ATA AAT GAG GAG
65
220

pcdAlaL2-4
GTC AGG AAT CCC TGA GGG TCG AGC ATT ATT GTC ATA AAT GAG GAG
66
221

pcdAlaL2-5
TCG GTC AGG AAT CCC TGA GGG AGC CTT ATT ATT GTC ATA AAT GAG
66
222

pcdAlaL2-6
TCG GTC AGG AAT CCC TGA AGC TCG CTT ATT ATT GTC ATA
64
223

pcdAlaL2-7
TCG GTC AGG AAT CCC AGC GGG TCG CTT ATT ATT
66
224

pcdAlaL3-1
GCT GCT ATC CCA TGT AGC GCA GTA ATA ATC GGC
64
225

pcdAlaL3-2
CAG GCT GCT ATC CCA AGC TCC GCA GTA ATA ATC
64
226

pcdAlaL3-3
ACC ACT CAG GCT GCT ATC AGC TGT TCC GCA GTA ATA ATC
66
227

pcdAlaL3-4
ACC ACT CAG GCT GCT AGC CCA TGT TCC GCA GTA
69
228

pcdAlaL3-5
CAC ACC ACT CAG GCT AGC ATC CCA TGT TCC GCA
68
229

pcdAlaL3-6
GAA TAC CAC ACC ACT CAG AGC GCT ATC CCA TGT TCC GCA
68
230

pcdAlaL3-7
GCC GAA TAC CAC ACC ACT AGC GCT GCT ATC CCA TGT TCC
69
231

pcdAlaL3-8
GCC GAA TAC CAC ACC AGC CAG GCT GCT ATC CCA
70
232

pcdAlaL3-9
TCC GCC GAA TAC CAC AGC ACT CAG GCT GCT ATC
68
233

pcdAlaL3-10
CCC TCC GCC GAA TAC AGC ACC ACT CAG GCT GCT
71
234

pcdAlaL3-11
GGT CCC TCC GCC GAA AGC CAC ACC ACT CAG GCT
72
235

Single-stranded DNA was produced using a published protocol (Tonikian R et al, Nat. Protoc. 2007; 2(6):1368-86). Briefly, phage expression vector was transformed into CJ236 electro-competent cells according to the manufacturer's protocol (Lucigen, Middleton, Wis.) and 0.05% of cells were plated on LB/Amp plates. After overnight growth, six 1-mL cultures, grown in 2× TY/2% glucose supplemented with 100 μg/mL ampicillin, 10 ug/mL chloramphenicol, and 1e+10 M13KO7 helper phage particles, were inoculated with independent colonies and grown at 37° C. for 2 hours before kanamycin was added to 25 μg/mL. After 4 more hours of shaking at 37° C., cultures showing turbidity were pooled and expanded into a 120 mL culture (2× TY/2% glucose supplemented with 100 μg/mL ampicillin, 25 μg/mL kanamycin, 0.25 μg/mL uridine) in a 2-L baffled flask. Following overnight growth, culture supernatant was rescued via centrifugation (6000 rpm, 10 minutes) and 20 mL PEG/NaCl was added to 100 mL supernatant and phage was purified by PEG-precipitation. Purified phage (resuspended in 1 mL PBS) was lysed and single-stranded uracil DNA was purified (Qiaprep spin M13 kit, Cat. #: 27704) according to manufacturer's protocol. DNA was quantified by Nanodrop (Thermo).

Oligos were ordered in normalized form in tris-EDTA buffer (50 μM in 200 μL). Next, 200 pmoles of each oligo (4 μL) were phosphorylated in 96-well format at 37° C. for 90 minutes and heat inactivated 65° C. for 20 minutes. The reaction mixture contained the following: 4 μL oligo, 2 μL 10×PNK buffer, 2 μL 10 mM ATP, 1 μL 100 mM DTT, 11 μL water, 2 μL T4 PNK (T4 Polynucleotide Kinase). Site-directed mutagenesis reactions were set up in 96-well format as follows: 2.2 μL single-stranded uracil template DNA (200 ng), 2 μL Pfu Turbo Cx buffer, 2 μL 100 mM DTT, 0.4 μL NAD+, 0.16 μL dNTPs, 10 μL water, 0.2 μL (0.5 units of Pfu turbo Cx), 1 μL Taq DNA Ligase, 2 μL of 20-fold diluted phosphorylation reaction (0.9 pmoles). The reaction was incubated as follows: 95° C. for 3 min, 55° C. for 90 sec, 68° C. for 15 min, 45° C. for 15 min. Next, 1 μL (1 pmole) 5′ phosphorylated OmpA oligo was added and the reaction was incubated further as follows: 95° C. for 30 sec, 55° C. for 45 sec, 68° C. for 10 min, 45° C. for 15 min. Following these incubations, 2 μL or 2 units UDG: Uracil DNA glycosylase and 5 units ExonucleaseIII enzyme mix were added to digest the template, first at 37° C. for 60 min then at 65° C. for 20 min. Finally, 2 μL from each reaction (63 total reactions) were pooled and 0.3 μL was electroporated into ER2738 electro-competent cells for sequencing.

Preparation of Phage Expressing scFv for Use in ELISAs

ScFvs may be expressed on the surface of a phage particle. To prepare phage expressing scFv on their surface, 96-deep well plates containing 1 mL 2× TY media with 2% glucose/100 μg/mL ampicillin were inoculated with 0.5-1 μL from thawed glycerol stocks (one clone per well) using the QPix™2 Colony picker (Molecular Devices, Sunnyvale Calif.) and grown at 37° C. (900 rpm) for ˜4 hours. Next, 5 μL of a 1:29 dilution of helper phage (8.3×10¹³pfu) was added and the plates and incubated for a further 30 minutes at 37° C. with no shaking then 1 hour at 300 rpm. Plates were centrifuged and the media was replaced with a kanamycin/non-glucose containing media (2× TY with 50 μg/mL kanamycin and 100 μg/mL ampicillin). Plates were grown overnight at 25° C. (900 rpm), and phage were harvested in the supernatant following centrifugation.

ELISA to Measure Binding of scFv Proteins to Human P-Cadherin-Fc.

P-cadherin-ECD1-3-Fc protein or negative control protein was coated overnight at 4° C. on 96-well Nunc Maxisorp® plates (Thermo Fisher Scientific, Madison, Conn.) at a concentration of 2 μg/mL in PBS+Ca²⁺ and Mg²⁺. Plates were washed three times using PBS+Ca²⁺ and Mg²⁺ and blocked for 1 hour at room temperature in 3% milk/PBS+Ca²⁺ and Mg²⁺. Phage samples prepared as described above were added to the blocked plates for 1 hour at room temperature. Plates were washed three times with PBS+Ca²⁺ and Mg²⁺ prior to the addition of secondary antibody (anti M13-HRP 1:2000, GE Healthcare, Piscataway, N.J.). Plates were incubated for a further 1 hour at room temperature and washed six times with PBS+Ca²⁺ and Mg²⁺. Signal was developed using TMB (SurModics, Eden Prairie, Minn.), the reaction stopped with H₂SO₄and the absorbance read at 450 nm on an EnVision® plate reader (Perkin Elmer, Waltham, Mass.).

Assessment of Binding to Human P-Cadherin

Binding of phage preparations of anti-P-cadherin scFv 153 alanine-mutants to human P-cadherin-Fc protein were tested by ELISA. Data was plotted as total binding signal (OD450) and as a percent of binding compared to the parental clone 153. A Z-score was then calculated based on the standard deviation of the percent binding of clone 153. Data for this assay is presented in Table 46 and Table 47.

Results indicate good expression of scFv on phage and demonstrate expected binding activity of clone 153. Certain CDR residues are deemed important because mutation to Ala negatively impacts human P-cadherin binding and has a Z-score of lower than −1. Certain CDR residues are deemed moderately important because mutation to Ala results in a Z-score between −0.2 and −1.0. These results suggest that H_1.8, H_2.5, H_3.1, H_3.7and L_3.3are important AAs for human P-cadherin binding, while H_2.6, H_2.16, F_3.5, F_3.9, L_1.8, L_2.2, L_2.3, L_3.2and L_3.2are moderately important for binding.

TABLE 46

Binding of anti-P-cadherin clone 153 alanine-mutants.

Human
% of binding of Clone

Parental
P-cadherin-Fc
#153 to human
Z

CDR-VH
sequence
binding (OD450)
P-cadherin-Fc
Score

H1.1-Ala
G
1.79
135
0.7

H1.2-Ala
Y
2.36
179
1.7

H1.3-Ala
T
2.30
174
1.6

H1.4-Ala
F
2.33
177
1.6

H1.5-Ala
T
2.05
155
1.2

H1.6-Ala
S
1.97
149
1.0

H1.7-Ala
Y
1.71
129
0.6

H1.8-Ala
G
0.36
27
−1.5

H1.9-Ala
I
2.21
167
1.4

H1.10-Ala
S
2.13
161
1.3

H2.1-Ala
W
1.37
103
0.1

H2.2-Ala
I
2.20
166
1.4

H2.3-Ala
S
1.83
138
0.8

H2.4-Ala
A

H2.5-Ala
Y
0.45
34
−1.4

H2.6-Ala
N
1.06
80
−0.4

H2.7-Ala
G
1.50
114
0.3

H2.8-Ala
N
2.14
162
1.3

H2.9-Ala
T
2.03
154
1.1

H2.10-Ala
N
2.27
172
1.5

H2.11-Ala
Y
2.15
163
1.3

H2.12-Ala
A

H2.13-Ala
Q
2.01
152
1.1

H2.14-Ala
K
1.54
117
0.4

H2.15-Ala
L
1.34
101
0.0

H2.16-Ala
Q
0.90
68
−0.7

H2.17-Ala
G
2.14
162
1.3

H3.1-Ala
I
0.17
13
−1.8

H3.2-Ala
D
2.22
168
1.4

H3.3-Ala
T
2.26
171
1.5

H3.4-Ala
A

H3.5-Ala
N
0.77
58
−0.9

H3.6-Ala
A

H3.7-Ala
F
0.31
23
−1.6

H3.8-Ala
G
1.36
103
0.1

H3.9-Ala
I
0.80
61
−0.8

P-cad 153
—
1.32
100
0.0

TABLE 47

Binding of anti-P-cadherin clone 153 alanine-mutants.

Human
% of binding of Clone

Parental
P-cadherin-Fc
#153 to human
Z

CDR-VL
sequence
binding (OD450)
P-cadherin-Fc
Score

L1.1-Ala
S
2.19
166
1.4

L1.2-Ala
G
1.28
97
−0.1

L1.3-Ala
S
2.19
165
1.4

L1.4-Ala
S
2.31
175
1.6

L1.5-Ala
S
1.70
129
0.6

L1.6-Ala
N
1.16
88
−0.3

L1.7-Ala
I
1.42
108
0.2

L1.8-Ala
G
1.00
75
−0.5

L1.9-Ala
N
1.71
129
0.6

L1.10-Ala
N
1.81
137
0.8

L1.11-Ala
Y
1.84
139
0.8

L1.12-Ala
V
2.29
173
1.6

L1.13-Ala
S
2.26
171
1.5

L2.1-Ala
D
1.45
110
0.2

L2.2-Ala
N
1.35
102
0.0

L2.3-Ala
N
0.98
74
−0.5

L2.4-Ala
K
2.13
161
1.3

L2.5-Ala
R
2.25
170
1.5

L2.6-Ala
P
2.28
172
1.5

L2.7-Ala
S
2.16
163
1.3

L3.1-Ala
G
2.02
153
1.1

L3.2-Ala
T
0.81
61
−0.8

L3.3-Ala
W
0.18
14
−1.8

L3.4-Ala
D
0.92
70
−0.6

L3.5-Ala
S
2.16
163
1.3

L3.6-Ala
S
2.29
173
1.5

L3.7-Ala
L
2.29
173
1.6

L3.8-Ala
S
2.28
173
1.5

L3.9-Ala
G
2.19
166
1.4

L3.10-Ala
V
1.86
141
0.9

L3.11-Ala
V
1.38
104
0.1

P-cad 153
—
1.32
100
0.0

The Alanine scan data and structural information (Example 15) provided herein suggest that a significant number of CDR residues in anti-P-cadherin scFv 153 could be substituted without significantly impact antigen binding. Thus further analyses were conducted to determine which CDR residues may be substituted without significant impact on P-cadherin binding. Tables 48-65 show the predicted Delta Delta G (DDG) stability for each position using the Discovery Studio 4.0 Stability prediction algorithm. Stability score (DDG) indicate the predicted effect of the mutation on protein stability.

TABLES 48

Alternative VH CDR1 residues (DDG <=1.0 kcal/mol)

CDR
Residue from

position
P-cad 153
Alternative residues

(column 1)
(column 2)
(column 3)

H1.1
G
R, Q, I, L, C, F, T, G, V, W, M, Y, N, E,

P, K, H, D, S, or A

H1.2
Y
H, R, C, Q, Y, I, F, K, M, V, L, N, or T

H1.3
T
N, C, R, V, L, T, I, S, K, Q, M, W, A, F,

Y, H, or D

H1.4
F
F or Y

H1.5
T
W, R, I, C, K, Y, M, V, F, T, S, L, Q, E,

A, N, H, D, or G

H1.6
S
R, I, M, N, Y, C, H, Q, L, K, F, V, T, E,

A, S, D, W, or G

H1.7
Y
K, R, L, H, F, Q, N, C, Y, M, I, T, V,

or A

H1.8
G
P, R, F, W, H, C, Y, I, M, K, G, S, Q, A,

L, N, T, or D

H1.9
I
Y, F, K, I, W, or L

H1.10
S
R, C, L, N, S, V, T, or A

TABLES 49

Alternative VH CDR1 residues (DDG <= 0.5 kcal/mol)

CDR
Residue from

position
P-cad 153
Alternative residues

(column 1)
(column 2)
(column 3)

H1.1
G
R, Q, I, L, C, F, T, G, V, W, M, Y,

N, or E

H1.2
Y
H, R, C, Q, Y, I, F, K, M, V, or L

H1.3
T
N, C, R, V, L, T, I, S, K, Q, or M

H1.4
F
F or Y

H1.5
T
W, R, I, C, K, Y, M, V, F, T, S, L,

Q, E, A, N, or H

H1.6
S
R, I, M, N, Y, C, H, Q, L, K, F, V,

T, E, A, S, D, or W

H1.7
Y
K, R, L, H, F, Q, N, C, Y, M, I, or T

H1.8
G
P, R, F, W, H, C, Y, I, M, K, G, S,

Q, A, L or N

H1.9
I
Y, F, K, I, W, or L

H1.10
S
R, C, L, N, S, V, or T

TABLES 50

Alternative VH CDR1 residues (DDG <= 0 kcal/mol)

CDR
Residue from

position
P-cad 153
Alternative residues

(column 1)
(column 2)
(column 3)

H1.1
G
R, Q, I, L, C, F, T, or G

H1.2
Y
H, R, C, a or Y

H1.3
T
N, C, R, V, L, or T

H1.4
F
F

H1.5
T
W, R, , I, C, K, Y, M, V, F, or T

H1.6
S
R, I, M, N, Y, C, H, Q, L, K, F,

V, T, E, A, or S

H1.7
Y
K, R, L, H, F, Q, N, C, or Y

H1.8
G
P, R, F, W, H, C, Y, I, M, K, or G

H1.9
I
Y, F, K, or I

H1.10
S
R, C, L, N, or S

TABLES 51

Alternative VH CDR2 residues (DDG <= 1.0 kcal/mol)

CDR
Residue from

position
P-cad 153
Alternative residues

(column 1)
(column 2)
(column 3)

H2.1
W
W

H2.2
I
W, F, Y, I, V, H, L, or Q

H2.3
S
W, F, L, R, C, H, T, N, Y, Q, K, A, I,

V, S, M, E, or D

H2.4
A
I, Y, V, Q, F, H, K, C, N, T, E, L, M,

D, P, S, A, or W

H2.5
Y
C, P, T, S, L, V, M, F, W, Q, K, G, I,

A, N, H, E, D, Y, R

H2.6
N
I, Q, W, V, C, L, K, E , T, M, F, Y, R,

S, A, N, H, D, or G

H2.7
G
G, C, L, N, W, F, S, Y, T, D, I, or M

H2.8
N
Q, C, I, Y, W, L, T, V, K, M, H, F, S,

R, A, E, N, D, or G

H2.9
T
F, L, V, W, H, C, Y, K, I, Q, R, T, M,

N, A, S, or D

H2.10
N
R, W, K, F, C, T, L, Y, N, Q, V, H, M,

A, S, or D

H2.11
Y
L, F, W, N, Y, H, K, R, I, M, V, C, Q,

or E

H2.12
A
W, F, I, Y, L, R, V, M, H, N, C, Q K,

T, D, E, A, or S

H2.13
Q
P, R, Q, C, T, I, W, F, K, L, Y, S, V,

M, D, or H

H2.14
K
W, C, I, F, T, R, Y, L, Q, S, V, D, N,

M, K, A, H, E, or G

H2.15
L
W, I, L, V, F, K, R, H, Y, or M

H2.16
Q
W, F, C, T, L, R, N, Q, A, S, M, K, E,

H, Y, or G

H2.17
G
P, G, T, M, C, E, or V

TABLES 52

Alternative VH CDR2 residues (DDG <= 0.5 kcal/mol)

CDR
Residue from

position
P-cad 153
Alternative residues

(column 1)
(column 2)
(column 3)

H2.1
W
W

H2.2
I
W, F, Y, I, or V

H2.3
S
W, F, L, R, C, H, T, N, Y, Q, K, A,

I, V, S, M, or E

H2.4
A
I, Y, V, Q, F, H, K, C, N, T, E, L,

M, D, P, S, A, or W

H2.5
Y
C, P, T, S, L, V, M, F, W, Q, K, G,

I, A, N, H, E, D, or Y

H2.6
N
I, Q, W, V, C, L, K, E, T, M, F, Y,

R, S, A, N, H, D, or G

H2.7
G
G, C, L, or N

H2.8
N
Q, C, I, Y, W, L, T, V, K, M, H, F,

S, R, A, E, N, or D

H2.9
T
F, L, V, W, H, C, Y, K, I, Q, R, T,

M, N, A, or S

H2.10
N
R, W, K, F, C, T, L, Y, N, Q, V, H,

M, A, or S

H2.11
Y
L, F, W, N, Y, H, K, R, I, M, or V

H2.12
A
W, F, I, Y, L, R, V, M, H, N, C, Q,

K, T, D, E, A, or S

H2.13
Q
P, R, Q, C, T, I, W, F, K, L, Y, or S

H2.14
K
W, C, I, F, T, R, Y, L, Q, S, V, D,

N, M, K, A, H, or E

H2.15
L
W, I, L, V, F, K, R, or H

H2.16
Q
W, F, C, T, L, R, N, Q, A, S, M, or K

H2.17
G
P or G

TABLES 53

Alternative VH CDR2 residues (DDG <= 0 kcal/mol)

CDR
Residue from

position
P-cad 153
Alternative residues

(column 1)
(column 2)
(column 3)

H2.1
W
W

H2.2
I
W, F, Y, or I

H2.3
S
W, F, L, R, C, H, T, N, Y, Q, K, A, I,

V, or S

H2.4
A
I, Y, V, Q, F, H, K, C, N, T, E, L, M,

D, P, S, or A

H2.5
Y
C, P, T, S, L, V, M, F, W, Q, K, G, I,

A, N, H, E, D, or Y

H2.6
N
I, Q, W, V, C, L, K, E, T, M, F, Y, R,

S, A, N, or H

H2.7
G
G

H2.8
N
Q, C, I, Y, W, L, T, V, K, M, H, F, S,

R, A, E, or N

H2.9
T
F, L, V, W, H, C, Y, K, I, Q, R, or T

H2.10
N
R, W, K, F, C, T, L, Y, or N

H2.11
Y
L, F, W, N, or Y

H2.12
A
W, F, I, Y, L, R, V, M, H, N, C, Q, K,

T, D, E, or A

H2.13
Q
P, R, or Q

H2.14
K
W, C, I, F, T, R, Y, L, Q, S, V, D, N,

M, or K

H2.15
L
W, I, or L

H2.16
Q
W, F, C, T, L, R, N, or Q

H2.17
G
P or G

TABLES 54

Alternative VH CDR3 residues (DDG <= 1.0 kcal/mol)

CDR
Residue from

position
P-cad 153
Alternative residues

(column 1)
(column 2)
(column 3)

H3.1
I
F, Y, I, R, W, or L

H3.2
D
F, I, C, M, W, Q, L, H, V, N, R, T, D,

S, E, A, Y, or K

H3.3
T
H, W, C, I, S, L, T, F, Q, Y, V, A, M,

K, N, G, D, or E

H3.4
A
Y, I, M, W, R, H, V, F, C, K, N, Q, L,

D, T, S, A, or E

H3.5
N
W, L, V, I, P, C, Y, M, H, T, N, F, D,

A, or S

H3.6
A
F, W, C, H, N, R, Y, L, A, Q, E, M, T,

I, S, K, or D

H3.7
F
Y, F, or W

H3.8
G
W, F, Y, I, L, Q, R, H, M, N, V, C, T,

K, E, D, S, A, P, or G

H3.9
I
W, Y, F, R, I, L, C, V, H, N, K, T, Q,

M, or D

TABLES 55

Alternative VH CDR3 residues (DDG <= 0.5 kcal/mol)

CDR
Residue from

position
P-cad 153
Alternative residues

(column 1)
(column 2)
(column 3)

H3.1
I
F, Y, I, or R

H3.2
D
F, I, C, M, W, Q, L, H, V, N, R, T, D, S,

E, A, Y, or K

H3.3
T
H, W, C, I, S, L, T, F, Q, Y, V, A, or M

H3.4
A
Y, I, M, W, R, H, V, F, C, K, N, Q, L, D,

T, S, A, or E

H3.5
N
W, L, V, I, P, C, Y, M, H, T, N, F, or D

H3.6
A
F, W, C, H, N, R, Y, L, A, Q, E, M, T, I,

or S

H3.7
F
Y, F, or W

H3.8
G
W, F, Y, I, L, Q, R, H, M, N, V, C, T, K,

E, D, S, A, P, or G

H3.9
I
W, Y, F, R, I, L, C, V, H, N, or K

TABLES 56

Alternative VH CDR3 residues (DDG <= 0 kcal/mol)

CDR
Residue from

position
P-cad 153
Alternative residues

(column 1)
(column 2)
(column 3)

H3.1
I
F, Y, I, or R

H3.2
D
F, I, C, M, W, Q, L, H, V, N, R, T,

or D

H3.3
T
H, W, C, I, S, L, or T

H3.4
A
Y, I, M, W, R, H, V, F, C, K, N, Q,

L, D, T, S, or A

H3.5
N
W, L, V, I, P, C, Y, M, H, T, or N

H3.6
A
F, W, C, H, N, R, Y, L, or A

H3.7
F
Y or F

H3.8
G
W, F, Y, I, L, Q, R, H, M, N, V, C,

T, K, E, D, S, A, P, or G

H3.9
I
W, Y, F, R, or I

TABLE 57

Alternative VL CDR1 residues (DDG <= 1.0 kcal/mol)

CDR
Residue from

position
P-cad 153
Alternative residues

(column 1)
(column 2)
(column 3)

L1.1
S
W, C, I, F, Y, T, H, N, M, Q, K, L,

S, A, V, D, R, E, or G

L1.2
G
G

L1.3
S
R, Q, Y, L, K, F, M, N, W, C, I, T,

V, S, E, A, H, D, or G

L1.4
S
C, L, F, S, I, T, W, N, Y, V, D, A,

K, Q, M, H, or R

L1.5
S
F, C, I, T, V, N, H, L, Y, S, R, Q,

W, A, M, E, K, or D

L1.6
N
L, Q, F, M, N, C, K, H, or T

L1.7
I
I, Q, H, F, L, or V

L1.8
G
R, Q, L, T, G, C, E, D, M, Y, or S

L1.9
N
C, W, I, L, F, R, V, T, K, N, S, M,

Q, Y, E, A, H, or D

L1.10
N
Q, F, N, L, C, Y, H, R, W, or M

L1.11
Y
M, H, K, V, Q, L, Y, C, I, N, F, T,

S, E, or W

L1.12
V
I, N, V, L, C, or T

L1.13
S
W, R, F, K, I, H, V, C, T, L, N, M,

Q, Y, S, A, D, or E

TABLE 58

Alternative VL CDR1 residues (DDG <= 0.5 kcal/mol)

CDR
Residue from

position
P-cad 153
Alternative residues

(column 1)
(column 2)
(column 3)

L1.1
S
W, C, I, F, Y, T, H, N, M, Q, K, L,

S, A, V, D, R, or E

L1.2
G
G

L1.3
S
R, Q, Y, L, K, F, M, N, W, C, I, T,

V, S, E, A, H, or D

L1.4
S
C, L, F, S, I, T, W, N, Y, or V

L1.5
S
F, C, I, T, V, N, H, L, Y, S, R, Q,

W, A, M, or E

L1.6
N
L, Q, F, M, N, or C

L1.7
I
I, Q, or H

L1.8
G
R, Q, L, T, G, or C

L1.9
N
C, W, I, L, F, R, V, T, K, N, S, M,

Q, Y, E, A, H, or D

L1.10
N
Q, F, N, L, or C

L1.11
Y
M, H, K, V, Q, L, Y, C, or I

L1.12
V
I, N, or V

L1.13
S
W, R, F, K, I, H, V, C, T, L, N, M,

Q, Y, S, or A

TABLE 59

Alternative VL CDR1 residues (DDG <= 0 kcal/mol)

CDR
Residue from

position
P-cad 153
Alternative residues

(column 1)
(column 2)
(column 3)

L1.1
S
W, C, I, F, Y, T, H, N, M, Q, K, L,

or S

L1.2
G
G

L1.3
S
R, Q, Y, L, K, F, M, N, W, C, I, T,

V, or S

L1.4
S
C, L, F, or S

L1.5
S
F, C, I, T, V, N, H, L, Y, or S

L1.6
N
L, Q, F, M, or N

L1.7
I
I

L1.8
G
R, Q, L, T, or G

L1.9
N
C, W, I, L, F, R, V, T, K, or N

L1.10
N
Q, F, or N

L1.11
Y
M, H, K, V, Q, L, or Y

L1.12
V
I, N, or V

L1.13
S
W, R, F, K, I, H, V, C, T, L, N, M,

Q, Y, or S

TABLE 60

Alternative VL CDR2 residues (DDG <= 1.0 kcal/mol)

CDR
Residue from

position
P-cad 153
Alternative residues

(column 1)
(column 2)
(column 3)

L2.1
D
I, L, V, F, Y, W, C, R, Q, N, M, H, E,

T, P, K, D, S, A, or G

L2.2
N
W, L, F, I, C, N, R, V, Q, Y, E, D, H,

or M

L2.3
N
C, Q, L, F, Y, W, E, R, M, H, A, N, T,

K, D, S, I, V, or G

L2.4
K
R, K, I, L, C, Q, V, W, Y, M, F, or H

L2.5
R
R, Q, L, W, K, or H

L2.6
P
R, K, I, W, F, L, Q, C, M, Y, H, V, N,

T, A, E, S, P, or D

L2.7
S
C, L, I, T, S, K, M, R, V, F, P, A, W,

Q, D, N, G, or Y

TABLE 61

Alternative VL CDR2 residues (DDG <= 0.5 kcal/mol)

CDR
Residue from

position
P-cad 153
Alternative residues

(column 1)
(column 2)
(column 3)

L2.1
D
I, L, V, F, Y, W, C, R, Q, N, M, H, E,

T, P, K, D, S, or A

L2.2
N
W, L, F, I, C, N, R, V, or Q

L2.3
N
C, Q, L, F, Y, W, E, R, M, H, A, N, T,

K, D, S, or I

L2.4
K
R, K, I, L, C, Q, or V

L2.5
R
R or Q

L2.6
P
R, K, I, W, F, L, Q, C, M, Y, H, V, N,

T, A, E, S, or P

L2.7
S
C, L, I, T, S, K, M, R, V, F, P, or A

TABLE 62

Alternative VL CDR2 residues (DDG <= 0 kcal/mol)

CDR
Residue from

position
P-cad 153
Alternative residues

(column 1)
(column 2)
(column 3)

L2.1
D
I, L, V, F, Y, W, C, R, Q, N, M, H, E,

T, P, K, or D

L2.2
N
W, L, F, I, C, or N

L2.3
N
C, Q, L, F, Y, W, E, R, M, H, A, or N

L2.4
K
R or K

L2.5
R
R

L2.6
P
R, K, I, W, F, L, Q, C, M, Y, H, V, N,

T, A, E, S, or P

L2.7
S
C, L, I, T, or S

TABLE 63

Alternative VL CDR3 residues (DDG <= 1.0 kcal/mol)

CDR
Residue from

position
P-cad 153
Alternative residues

(column 1)
(column 2)
(column 3)

L3.1
G
W, R, Y, F, L, N, Q, C, M, I, H, T, A, D,

E, S, K, V, or G

L3.2
T
N, C, V, T, S, or A

L3.3
W
W

L3.4
D
F, N, D, E, H, Q, Y, R, or W

L3.5
S
T, I, R, L, C, F, Y, W, Q, M, H, E, N, V,

S, D, K, or A

L3.6
S
I, H, C, M, N, L, V, Q, E, S, T, A, K, D,

F, Y, G, R, or W

L3.7
L
L, C, R, I, H, Q, E, T, V, M, K, F, or Y

L3.8
S
W, L, C, F, T, N, V, Y, I, M, H, Q S, K,

E, D, A, R, or G

L3.9
G
L, Y, Q, F, H, I, C, R, M, N, D, W, E, K,

T, V, A, S, or G

L3.10
V
I, F, K, L, H, Y, M, V, Q, W, T, N, or C

L3.11
V
Q, W, R, F, T, C, Y, V, E, L, N, I, K,

or M

TABLE 64

Alternative VL CDR3 residues (DDG <= 0.5 kcal/mol)

CDR
Residue from

position
P-cad 153
Alternative residues

(column 1)
(column 2)
(column 3)

L3.1
G
W, R, Y, F, L, N, Q, C, M, I, H, T, A, D,

E, S, K, V, or G

L3.2
T
N, C, V, or T

L3.3
W
W

L3.4
D
F, N, D, or E

L3.5
S
T, I, R, L, C, F, Y, W, Q, M, H, E, N, V,

S, D, K, or A

L3.6
S
I, H, C, M, N, L, V, Q, E, S, T, A, or K

L3.7
L
L, C, R, I, H, Q, E, T, or V

L3.8
S
W, L, C, F, T, N, V, Y, I, M, H, Q, S, K,

E, D, A, or R

L3.9
G
L, Y, Q, F, H, I, C, R, M, N, D, W, E, K,

T, V, A, S, or G

L3.10
V
I, F, K, L, H, Y, M, V, Q, W, or T

L3.11
V
Q, W, R, F, T, C, Y, V, E, L, or N

TABLE 65

Alternative VL CDR3 residues (DDG <= 0 kcal/mol)

CDR
Residue from

position
P-cad 153
Alternative residues

(column 1)
(column 2)
(column 3)

L3.1
G
W, R, Y, F, L, N, Q, C, M, I, H, T, A, D,

E, S, K, V, or G

L3.2
T
N, C, V, or T

L3.3
W
W

L3.4
D
F, N, D, or E

L3.5
S
T, I, R, L, C, F, Y, W, Q, M, H, E, N, V,

or S

L3.6
S
I, H, C, M, N, L, V, Q, E, or S

L3.7
L
L

L3.8
S
W, L, C, F, T, N, V, Y, I, M, H, Q, or S

L3.9
G
L, Y, Q, F, H, I, C, R, M, N, D, W, E, K,

T, V, A, S, or G

L3.10
V
I, F, K, L, H, Y, M, or V

L3.11
V
Q, W, R, F, T, C, Y, or V

Tables 66-71 show the results of Abysis analysis of human sequences at each CDR position. Each CDR from anti-P-cadherin 153 was compared with the corresponding CDR from human antibody sequences from a database. This database combined all human sequence from several databases including: the PDB, Kabat database, IMGT database, VBASE, and Pfizer's internal database. Human antibody sequences in this database were Kabat numbered with Abysis, and the frequencies of CDR residues were calculated only using CDRs with the same length as those found in the anti-P-cadherin clone 153. Tables 66-71 show alternative amino acids at each position wherein the listed residues are present in at least 5% or 10% of the human sequences. Original P-cadherin 153 residues are also included in column 3 and column 4 even if they do not fall in above 5% or 10% category.

TABLES 66

Alternative VH CDR1 residues (Abysis)

Alternative residues
Alternative residues

CDR
Residue from
(>5% of human
(>10% of human

position
P-cad 153
sequences)
sequences)

(column 1)
(column 2)
(column 3)
(column 4)

H1.1
G
G
G

H1.2
Y
Y, F, or G
Y, F, or G

H1.3
T
T or S
T or S

H1.4
F
F
F

H1.5
T
S or T
S or T

H1.6
S
S, G, N, or D
S, G, or N

H1.7
Y
Y
Y

H1.8
G
A, Y, W, or G
A, Y, W, or G

H1.9
I
M, I, or W
M or I

H1.10
S
S, H, N, or G
S, H, N, or G

TABLES 67

Alternative VH CDR2 residues (Abysis)

Alternative residues
Alternative residues

CDR
Residue from
(>5% of human
(>10% of human

position
P-cad 153
sequences)
sequences)

(column 1)
(column 2)
(column 3)
(column 4)

H2.1
W
W, I, G, A, V, R,
W, I, G, or A

or S

H2.2
I
I
I

H2.3
S
S, N, Y, or I
S, N, Y, or I

H2.4
A
P, G, Y, A, or S
P, G, or A

H2.5
Y
S, N, D, G, I, or Y
S, N, D, G, I, or Y

H2.6
N
G, S, D, N, or F
G, S, D, or N

H2.7
G
G or S
G or S

H2.8
N
N, S, T, D, G, or Y
N, S, T, D, or G

H2.9
T
T, K, A, or I
T, K, or A

H2.10
N
Y, N, R, S, or G
Y, N, or R

H2.11
Y
Y
Y

H2.12
A
A or S
A or S

H2.13
Q
Q, D, or P
Q, D, or P

H2.14
K
S or K
S or K

H2.15
L
F, V, or L
F, V, or L

H2.16
Q
Q or K
Q or K

H2.17
G
G
G

TABLES 68

Alternative VH CDR3 residues (Abysis)

Alternative residues
Alternative residues

CDR
Residue from
(>5% of human
(>10% of human

position
P-cad 153
sequences)
sequences)

(column 1)
(column 2)
(column 3)
(column 4)

H3.1
I
G, D, P, V, F, S,
G, D, or I

or I

H3.2
D
G, R, I, L, V, S, Q,
G, R, or D

or D

H3.3
T
G, R, V, S, L, K, Y,
G or T

or T

H3.4
A
G, I, Y, R, F, L, S,
G, I, Y, R, or A

or A

H3.5
N
G, S, Y, D, V, E, R,
G, S, Y, or N

or N

H3.6
A
G, A, R, Y, or P
G, A, or R

H3.7
F
F, M, P, or L
F, M, or P

H3.8
G
D, E, or G
D, E, or G

H3.9
I
Y, V, or I
Y, V, or I

TABLES 69

Alternative VL CDR1 residues (Abysis)

Alternative residues
Alternative residues

CDR
Residue from
(>5% of human
(>10% of human

position
P-cad 153
sequences)
sequences)

(column 1)
(column 2)
(column 3)
(column 4)

L1.1
S
S or T
S

L1.2
G
G or R
G

L1.3
S
S
S

L1.4
S
S, R, or T
S

L1.5
S
S or G
S

L1.6
N
N or S
N

L1.7
I
I or V
I

L1.8
G
G or A
G

L1.9
N
S, N, or G
S or N

L1.10
N
N or Y
N

L1.11
Y
Y, T, or S
Y or T

L1.12
V
V
V

L1.13
S
S, N, Y, or Q
S, N, or Y

TABLES 70

Alternative VL CDR2 residues (Abysis)

Alternative residues
Alternative residues

CDR
Residue from
(>5% of human
(>10% of human

position
P-cad 153
sequences)
sequences)

(column 1)
(column 2)
(column 3)
(column 4)

L2.1
D
D, G, E, A, S, or R
D, G, E, A, or S

L2.2
N
A, N, V, D, or T
A, N, or V

L2.3
N
S, N, or T
S or N

L2.4
K
N, S, Q, K, or T
N, S, Q, or K

L2.5
R
R or L
R or L

L2.6
P
P, A, Q, or E
P, A, or Q

L2.7
S
S or T
S or T

TABLES 71

Alternative VL CDR3 residues (Abysis)

Alternative residues
Alternative residues

CDR
Residue from
(>5% of human
(>10% of human

position
P-cad 153
sequences)
sequences)

(column 1)
(column 2)
(column 3)
(column 4)

L3.1
G
A, Q, G, or S
A, Q, G, or S

L3.2
T
S, A, T, or V
S, A, or T

L3.3
W
W, Y, or R
W or Y

L3.4
D
D, A, or T
D

L3.5
S
S, D, or G
S or D

L3.6
S
S
S

L3.7
L
L, S, or G
L or S

L3.8
S
S, N, T, R, or D
S, N, or T

L3.9
G
G, A, H, or L
G or A

L3.10
V
V, W, Y, G, or P
V, W, or Y

L3.11
V
V, L, or I
V

The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention can be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims include all such embodiments and equivalent variations.

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	Number	Date	Country
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	62019762	Jul 2014	US

Bispecific heterodimeric diabodies and uses thereof

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