Multispecific thyroxine transporter immunoglobulin fusion

Abstract

The present invention relates to multispecific thyroxine transporter (TTR) complexes useful as multispecific binding proteins. The multispecific TTR complexes described herein are particularly useful in binding to one, two or more epitopes that may be present on one or more proteins. Methods of treating diseases using the TTR complexes of the invention are described herein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 871,247, filed on July 8, 2019; which is incorporated herein by reference in its entirety.

[0003] Reference sequence list

[0004] This application contains a sequence listing in computer readable form. The sequence listing is provided as a text file entitled A--2414-WO-PCT_SeqList_ST25.txt, which was created on July 1, 2020 and is 101,660 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. Technical Field

[0005] The present invention relates to multispecific thyroxine transporter (TTR) complexes useful as multispecific binding proteins. The multispecific TTR complexes described herein are particularly useful in binding to one, two or more epitopes that may be present on one or more proteins. Methods of treating diseases using the TTR complexes of the invention are described herein. Background Art

[0006] Monospecific antibodies, i.e. antibodies that bind a single antigen, are a well-established class of compounds that have been approved in various therapeutic areas. In fact, over the past decade, many monospecific antibody-based drugs have been approved in multiple countries.

[0007] Multispecific proteins, such as multispecific antibodies, have become the subject of increasing research. Multispecific proteins are able to bind to two or more different antigens on the same or different proteins. This allows the possibility to affect two different biological pathways simultaneously.

[0008] Transthyretin (TTR) is a non-covalent tetrameric human serum and cerebrospinal fluid protein that plays a role in carrying a portion of circulating thyroxine and in the serum half-life of retinol binding protein. TTR normally exists as a tetrameric (approximately 56 kDa) serum protein, with each monomer unit having a molecular weight of approximately 14 kDa.

[0009] Previous efforts to polymerize proteins include the use of streptavidin (Kipriyanov et al., Protein Engineering, 9(2):203-211 (1996)), helix-turn-helix constructs (Kriangkum et al., Biomolecular Engineering, 18:31-40 (2001)), leucine zippers (Kruif et al., The Journal of Biological Chemistry, 271(13):7630-7634, 1996 (1996)), barnase / barnase inhibitor complexes (Deyev et al., Nature Biotechnology, 21(12):1486-1492 (2003)), and the Dock N Lock technique (protein kinase and A kinase anchor protein anchor domain interaction) (Goldenberg et al., Journal of Nuclear Medicine, 49(1):158-163 (2008).

[0010] However, there remains a need for an efficient method to generate multispecific proteins (eg, whole antibodies and antibody fragments) that can bind to multiple epitopes on the same or different proteins. Summary of the invention

[0011] The present invention relates to a TTR protein complex, wherein

[0012] The TTR protein complex contains TTR subunits A, B, C, and D;

[0013] TTR subunits A and B dimerize to form TTR dimer AB;

[0014] TTR subunits C and D dimerize to form TTR dimer CD;

[0015] TTR dimers AB and CD further dimerize to form TTR tetramer ABCD; and

[0016] Each of A, B, C, and D comprises the amino acid sequence of SEQ ID NO: 1, except that at least one amino acid in the interface between TTR dimer AB and TTR dimer CD is mutated to favor the formation of an ABCD tetramer over any other tetramer (e.g., an ABAB tetramer or a CDCD tetramer).

[0017] Each of the A, B, C, and D subunits of the TTR protein complex can comprise the amino acid sequence of SEQ ID NO: 1 having the following mutations: C10A, K15A, or C10A and K15A.

[0018] 104, 106, 108, 110, 112, 113, 114, 115, 117, 119, 121, 123, 124, 125, 126, and 127 of SEQ ID NO: 1.

[0019] In another embodiment, the invention relates to a TTR protein complex wherein both A and B, both C and D, or all four of A, B, C and D comprise a mutation at one or more amino acid positions selected from the following list, the list comprising: 15, 17, 20, 21, 22, 23, 24, 51, 52, 84, 106, 108, 112, 114, 115, 119, 121 and 123 of SEQ ID NO:1.

[0020] In another embodiment, the invention relates to a TTR protein complex, wherein both A and B, both C and D, or all four of A, B, C and D comprise a mutation at one or more amino acid positions selected from the following list, the list comprising: 15, 17, 20, 21, 22, 23, 24, 51, 52, 84, 106, 108, 112, 114, 115, 119, 121 and 123 of SEQ ID NO: 1, wherein the amino acid is mutated to aspartic acid, glutamic acid, arginine, lysine or histidine.

[0021] In another embodiment, the invention relates to a TTR protein complex, wherein A and B comprise mutations at one or more amino acid positions selected from the following list, the list comprising: 15, 17, 20, 21, 22, 23, 24, 51, 52, 84, 106, 108, 112, 114, 115, 119, 121 and 123 of SEQ ID NO: 1, wherein the amino acid is mutated to aspartic acid or glutamic acid.

[0022] In yet another embodiment, the invention relates to a TTR protein complex, wherein C and D comprise mutations at one or more amino acid positions selected from the following list, the list comprising: 15, 17, 20, 21, 22, 23, 24, 51, 52, 84, 106, 108, 112, 114, 115, 119, 121 and 123 of SEQ ID NO: 1, wherein the amino acid is mutated to arginine, lysine or histidine.

[0023] In a specific embodiment, A and B comprise a mutation at one or more amino acid positions selected from the following list, the list comprising: 15, 17, 20, 21, 22, 23, 24, 51, 52, 84, 106, 108, 112, 114, 115, 119, 121 and 123 of SEQ ID NO: 1, wherein the amino acid is mutated to aspartic acid or glutamic acid; and C and D comprise a mutation at one or more amino acid positions selected from the following list, the list comprising: 15, 17, 20, 21, 22, 23, 24, 51, 52, 84, 106, 108, 112, 114, 115, 119, 121 and 123 of SEQ ID NO: 1, wherein the amino acid is mutated to arginine, lysine or histidine.

[0024] In some embodiments, A and B comprise at least one mutation in SEQ ID NO: 1, wherein the mutation is selected from the list consisting of K15D, L17D, V20D, R21D, G22D, S23D, P24D, S52D, I84D, T106D, A108D, S112D, Y114D, S115D, T119D, V121D, S123D, K15E, L17E, V20E, R21E, G22E, S23E, P24E, D51E, S52E, I84E, T106E, A108E, S112E, Y114E, S115E, T119E, V121E, and S123E. The present invention also relates to a TTR protein complex, wherein A and B comprise at least one mutation in SEQ ID NO: 1, wherein the mutation is selected from the list consisting of: L17D, L17E, V20D, V20E, G22D, G22E, S112D, S112E, T119D, T119E, V121D and V121E.

[0025] In some embodiments, C and D comprise at least one mutation in SEQ ID NO: 1, wherein the mutation is selected from the list consisting of K15R, L17R, V20R, G22R, S23R, P24R, D51R, S52R, I84R, T106R, A108R, S112R, Y114R, S115R, T119R, V121R, S123R, L17K, V20K, R21K, G22K, S23R, P24K, D51K , S52K, I84K, T106K, A108K, S112K, Y114K, S115K, T119K, V121K, S123K, K15H, L17H, V20H, R21H, G22H, S23H, P24H, D51H, S52H, I84H, T106K, A108H, S112H, Y114H, S115H, T119H, V121H, and S123H. The present invention also relates to a TTR protein complex, wherein C and D comprise at least one mutation in SEQ ID NO: 1, wherein the mutation is selected from the list consisting of L17R, L17K, L17H, V20R, V20K, V20H, G22R, G22K, G22H, S112R, S112K, S112H, T119R, T119K, T119H, V121R, V121K and V121H.

[0026] In other embodiments, both A and B, both C and D, or all four of A, B, C, and D independently comprise one of the above mutations. In yet other embodiments, both A and B, both C and D, or all four of A, B, C, and D independently comprise two of the above mutations.

[0027] In a specific embodiment, the present invention relates to a TTR protein complex, wherein each of A, B, C and D comprises the amino acid sequence of SEQ ID NO: 1 with the following mutations:

[0028] A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / L17R (or vice versa);

[0029] A and B contain C10A / K15A / L17E, and C and D contain C10A / K15A / L17R (or vice versa);

[0030] A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / L17R (or vice versa);

[0031] A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / L17R (or vice versa);

[0032] A and B contain C10A / K15A / G22D, and C and D contain C10A / K15A / L17R (or vice versa);

[0033] A and B contain C10A / K15A / G22E, and C and D contain C10A / K15A / L17R (or vice versa);

[0034] A and B contain C10A / K15A / S112D, and C and D contain C10A / K15A / L17R (or vice versa);

[0035] A and B contain C10A / K15A / S112E, and C and D contain C10A / K15A / L17R (or vice versa);

[0036] A and B contain C10A / K15A / T119D, and C and D contain C10A / K15A / L17R (or vice versa);

[0037] A and B contain C10A / K15A / T119E, and C and D contain C10A / K15A / L17R (or vice versa);

[0038] A and B contain C10A / K15A / V121D, and C and D contain C10A / K15A / L17R (or vice versa);

[0039] A and B contain C10A / K15A / V121E, and C and D contain C10A / K15A / L17R (or vice versa);

[0040] A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / L17K (or vice versa);

[0041] A and B contain C10A / K15A / L17E, and C and D contain C10A / K15A / L17K (or vice versa);

[0042] A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / L17K (or vice versa);

[0043] A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / L17K (or vice versa);

[0044] A and B contain C10A / K15A / G22D, and C and D contain C10A / K15A / L17K (or vice versa);

[0045] A and B contain C10A / K15A / G22E, and C and D contain C10A / K15A / L17K (or vice versa);

[0046] A and B contain C10A / K15A / S112D, and C and D contain C10A / K15A / L17K (or vice versa);

[0047] A and B contain C10A / K15A / S112E, and C and D contain C10A / K15A / L17K (or vice versa);

[0048] A and B contain C10A / K15A / T119D, and C and D contain C10A / K15A / L17K (or vice versa);

[0049] A and B contain C10A / K15A / T119E, and C and D contain C10A / K15A / L17K (or vice versa);

[0050] A and B contain C10A / K15A / V121D, and C and D contain C10A / K15A / L17K (or vice versa);

[0051] A and B contain C10A / K15A / V121E, and C and D contain C10A / K15A / L17K (or vice versa);

[0052] A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / V20R (or vice versa);

[0053] A and B contain C10A / K15A / L17E, and C and D contain C10A / K15A / V20R (or vice versa);

[0054] A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / V20R (or vice versa);

[0055] A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / V20R (or vice versa);

[0056] A and B contain C10A / K15A / G22D, and C and D contain C10A / K15A / V20R (or vice versa);

[0057] A and B contain C10A / K15A / G22E, and C and D contain C10A / K15A / V20R (or vice versa);

[0058] A and B contain C10A / K15A / S112D, and C and D contain C10A / K15A / V20R (or vice versa);

[0059] A and B contain C10A / K15A / S112E, and C and D contain C10A / K15A / V20R (or vice versa);

[0060] A and B contain C10A / K15A / T119D, and C and D contain C10A / K15A / V20R (or vice versa);

[0061] A and B contain C10A / K15A / T119E, and C and D contain C10A / K15A / V20R (or vice versa);

[0062] A and B contain C10A / K15A / V121D, and C and D contain C10A / K15A / V20R (or vice versa);

[0063] A and B contain C10A / K15A / V121E, and C and D contain C10A / K15A / V20R (or vice versa);

[0064] A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / V20K (or vice versa);

[0065] A and B contain C10A / K15A / L17E, and C and D contain C10A / K15A / V20K (or vice versa);

[0066] A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / V20K (or vice versa);

[0067] A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / V20K (or vice versa);

[0068] A and B contain C10A / K15A / G22D, and C and D contain C10A / K15A / V20K (or vice versa);

[0069] A and B contain C10A / K15A / G22E, and C and D contain C10A / K15A / V20K (or vice versa);

[0070] A and B contain C10A / K15A / S112D, and C and D contain C10A / K15A / V20K (or vice versa);

[0071] A and B contain C10A / K15A / S112E, and C and D contain C10A / K15A / V20K (or vice versa);

[0072] A and B contain C10A / K15A / T119D, and C and D contain C10A / K15A / V20K (or vice versa);

[0073] A and B contain C10A / K15A / T119E, and C and D contain C10A / K15A / V20K (or vice versa);

[0074] A and B contain C10A / K15A / V121D, and C and D contain C10A / K15A / V20K (or vice versa);

[0075] A and B contain C10A / K15A / V121E, and C and D contain C10A / K15A / V20K (or vice versa);

[0076] A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / G22R (or vice versa);

[0077] A and B contain C10A / K15A / L17E, and C and D contain C10A / K15A / G22R (or vice versa);

[0078] A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / G22R (or vice versa);

[0079] A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / G22R (or vice versa);

[0080] A and B contain C10A / K15A / G22D, and C and D contain C10A / K15A / G22R (or vice versa);

[0081] A and B contain C10A / K15A / G22E, and C and D contain C10A / K15A / G22R (or vice versa);

[0082] A and B contain C10A / K15A / S112D, and C and D contain C10A / K15A / G22R (or vice versa);

[0083] A and B contain C10A / K15A / S112E, and C and D contain C10A / K15A / G22R (or vice versa);

[0084] A and B contain C10A / K15A / T119D, and C and D contain C10A / K15A / G22R (or vice versa);

[0085] A and B contain C10A / K15A / T119E, and C and D contain C10A / K15A / G22R (or vice versa);

[0086] A and B contain C10A / K15A / V121D, and C and D contain C10A / K15A / G22R (or vice versa);

[0087] A and B contain C10A / K15A / V121E, and C and D contain C10A / K15A / G22R (or vice versa);

[0088] A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / G22K (or vice versa);

[0089] A and B contain C10A / K15A / L17E, and C and D contain C10A / K15A / G22K (or vice versa);

[0090] A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / G22K (or vice versa);

[0091] A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / G22K (or vice versa);

[0092] A and B contain C10A / K15A / G22D, and C and D contain C10A / K15A / G22K (or vice versa);

[0093] A and B contain C10A / K15A / G22E, and C and D contain C10A / K15A / G22K (or vice versa);

[0094] A and B contain C10A / K15A / S112D, and C and D contain C10A / K15A / G22K (or vice versa);

[0095] A and B contain C10A / K15A / S112E, and C and D contain C10A / K15A / G22K (or vice versa);

[0096] A and B contain C10A / K15A / T119D, and C and D contain C10A / K15A / G22K (or vice versa);

[0097] A and B contain C10A / K15A / T119E, and C and D contain C10A / K15A / G22K (or vice versa);

[0098] A and B contain C10A / K15A / V121D, and C and D contain C10A / K15A / G22K (or vice versa);

[0099] A and B contain C10A / K15A / V121E, and C and D contain C10A / K15A / G22K (or vice versa);

[0100] A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / S112R (or vice versa);

[0101] A and B contain C10A / K15A / L17E, and C and D contain C10A / K15A / S112R (or vice versa);

[0102] A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / S112R (or vice versa);

[0103] A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / S112R (or vice versa);

[0104] A and B contain C10A / K15A / G22D, and C and D contain C10A / K15A / S112R (or vice versa);

[0105] A and B contain C10A / K15A / G22E, and C and D contain C10A / K15A / S112R (or vice versa);

[0106] A and B contain C10A / K15A / S112D, and C and D contain C10A / K15A / S112R (or vice versa);

[0107] A and B contain C10A / K15A / S112E, and C and D contain C10A / K15A / S112R (or vice versa);

[0108] A and B contain C10A / K15A / T119D, and C and D contain C10A / K15A / S112R (or vice versa);

[0109] A and B contain C10A / K15A / T119E, and C and D contain C10A / K15A / S112R (or vice versa);

[0110] A and B contain C10A / K15A / V121D, and C and D contain C10A / K15A / S112R (or vice versa);

[0111] A and B contain C10A / K15A / V121E, and C and D contain C10A / K15A / S112R (or vice versa);

[0112] A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / S112K (or vice versa);

[0113] A and B contain C10A / K15A / L17E, and C and D contain C10A / K15A / S112K (or vice versa);

[0114] A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / S112K (or vice versa);

[0115] A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / S112K (or vice versa);

[0116] A and B contain C10A / K15A / G22D, and C and D contain C10A / K15A / S112K (or vice versa);

[0117] A and B contain C10A / K15A / G22E, and C and D contain C10A / K15A / S112K (or vice versa);

[0118] A and B contain C10A / K15A / S112D, and C and D contain C10A / K15A / S112K (or vice versa);

[0119] A and B contain C10A / K15A / S112E, and C and D contain C10A / K15A / S112K (or vice versa);

[0120] A and B contain C10A / K15A / T119D, and C and D contain C10A / K15A / S112K (or vice versa);

[0121] A and B contain C10A / K15A / T119E, and C and D contain C10A / K15A / S112K (or vice versa);

[0122] A and B contain C10A / K15A / V121D, and C and D contain C10A / K15A / S112K (or vice versa);

[0123] A and B contain C10A / K15A / V121E, and C and D contain C10A / K15A / S112K (or vice versa);

[0124] A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / T119R (or vice versa);

[0125] A and B contain C10A / K15A / L17E, and C and D contain C10A / K15A / T119R (or vice versa);

[0126] A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / T119R (or vice versa);

[0127] A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / T119R (or vice versa);

[0128] A and B contain C10A / K15A / G22D, and C and D contain C10A / K15A / T119R (or vice versa);

[0129] A and B contain C10A / K15A / G22E, and C and D contain C10A / K15A / T119R (or vice versa);

[0130] A and B contain C10A / K15A / S112D, and C and D contain C10A / K15A / T119R (or vice versa);

[0131] A and B contain C10A / K15A / S112E, and C and D contain C10A / K15A / T119R (or vice versa);

[0132] A and B contain C10A / K15A / T119D, and C and D contain C10A / K15A / T119R (or vice versa);

[0133] A and B contain C10A / K15A / T119E, and C and D contain C10A / K15A / T119R (or vice versa);

[0134] A and B contain C10A / K15A / V121D, and C and D contain C10A / K15A / T119R (or vice versa);

[0135] A and B contain C10A / K15A / V121E, and C and D contain C10A / K15A / T119R (or vice versa);

[0136] A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / T119K (or vice versa);

[0137] A and B contain C10A / K15A / L17E, and C and D contain C10A / K15A / T119K (or vice versa);

[0138] A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / T119K (or vice versa);

[0139] A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / T119K (or vice versa);

[0140] A and B contain C10A / K15A / G22D, and C and D contain C10A / K15A / T119K (or vice versa);

[0141] A and B contain C10A / K15A / G22E, and C and D contain C10A / K15A / T119K (or vice versa);

[0142] A and B contain C10A / K15A / S112D, and C and D contain C10A / K15A / T119K (or vice versa);

[0143] A and B contain C10A / K15A / S112E, and C and D contain C10A / K15A / T119K (or vice versa);

[0144] A and B contain C10A / K15A / T119D, and C and D contain C10A / K15A / T119K (or vice versa);

[0145] A and B contain C10A / K15A / T119E, and C and D contain C10A / K15A / T119K (or vice versa);

[0146] A and B contain C10A / K15A / V121D, and C and D contain C10A / K15A / T119K (or vice versa);

[0147] A and B contain C10A / K15A / V121E, and C and D contain C10A / K15A / T119K (or vice versa);

[0148] A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / V121R (or vice versa);

[0149] A and B contain C10A / K15A / L17E, and C and D contain C10A / K15A / V121R (or vice versa);

[0150] A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / V121R (or vice versa);

[0151] A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / V121R (or vice versa);

[0152] A and B contain C10A / K15A / G22D, and C and D contain C10A / K15A / V121R (or vice versa);

[0153] A and B contain C10A / K15A / G22E, and C and D contain C10A / K15A / V121R (or vice versa);

[0154] A and B contain C10A / K15A / S112D, and C and D contain C10A / K15A / V121R (or vice versa);

[0155] A and B contain C10A / K15A / S112E, and C and D contain C10A / K15A / V121R (or vice versa);

[0156] A and B contain C10A / K15A / T119D, and C and D contain C10A / K15A / V121R (or vice versa);

[0157] A and B contain C10A / K15A / T119E, and C and D contain C10A / K15A / V121R (or vice versa);

[0158] A and B contain C10A / K15A / V121D, and C and D contain C10A / K15A / V121R (or vice versa);

[0159] A and B contain C10A / K15A / V121E, and C and D contain C10A / K15A / V121R (or vice versa);

[0160] A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / V121K (or vice versa);

[0161] A and B contain C10A / K15A / L17E, and C and D contain C10A / K15A / V121K (or vice versa);

[0162] A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / V121K (or vice versa);

[0163] A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / V121K (or vice versa);

[0164] A and B contain C10A / K15A / G22D, and C and D contain C10A / K15A / V121K (or vice versa);

[0165] A and B contain C10A / K15A / G22E, and C and D contain C10A / K15A / V121K (or vice versa);

[0166] A and B contain C10A / K15A / S112D, and C and D contain C10A / K15A / V121K (or vice versa);

[0167] A and B contain C10A / K15A / S112E, and C and D contain C10A / K15A / V121K (or vice versa);

[0168] A and B contain C10A / K15A / T119D, and C and D contain C10A / K15A / V121K (or vice versa);

[0169] A and B contain C10A / K15A / T119E, and C and D contain C10A / K15A / V121K (or vice versa);

[0170] A and B comprise C10A / K15A / V121D, and C and D comprise C10A / K15A / V121K (or vice versa); or

[0171] A and B contain C10A / K15A / V121E, and C and D contain C10A / K15A / V121K (or vice versa).

[0172] In other specific embodiments, the present invention relates to a TTR protein complex, wherein each of A, B, C and D comprises the amino acid sequence of SEQ ID NO: 1 with the following mutations:

[0173] A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / V121R (or vice versa);

[0174] A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / V121K (or vice versa);

[0175] A and B contain C10A / K15A / L17E, and C and D contain C10A / K15A / V121R (or vice versa);

[0176] A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / V20R (or vice versa);

[0177] A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / V20K (or vice versa);

[0178] A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / V20R (or vice versa);

[0179] A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / V20K (or vice versa);

[0180] A and B contain C10A / K15A / T119D, and C and D contain C10A / K15A / L17R (or vice versa);

[0181] A and B comprise C10A / K15A / T119D, and C and D comprise C10A / K15A / L17K (or vice versa); or

[0182] A and B comprise C10A / K15A / V121E, and C and D comprise C10A / K15A / L17K (or vice versa).

[0183] In some embodiments, A and B comprise two mutations in SEQ ID NO: 1, wherein the mutations are selected from the list consisting of L17D / V20D, L17D / V20E, L17E / V20D, L17E / V20E, L17D / T119D, L17D / V121E, L17E / T119D, L17E / V121E, V20D / T119D, V20D / V121E, V20E / T119D, and V20E / V121E.

[0184] In some embodiments, C and D comprise two mutations in SEQ ID NO:1, wherein the mutations are selected from the list consisting of L17K / V20K, L17K / V20R, L17R / V20K, L17R / V20R, L17K / V121K, L17K / V121R, L17R / V121K, L17R / V121R, V20K / V121K, V20K / V121R, V20R / V121K and V20R / V121R.

[0185] The present invention also includes an embodiment, wherein each of A, B, C and D in the TTR protein complex comprises an amino acid sequence of SEQ ID NO: 1 having the following mutations:

[0186] A and B contain C10A / K15A / L17D / V20D and C and D contain C10A / K15A / L17K / V20K (or vice versa);

[0187] A and B contain C10A / K15A / L17D / V20E and C and D contain C10A / K15A / L17K / V20R (or vice versa);

[0188] A and B contain C10A / K15A / L17E / V20D and C and D contain C10A / K15A / L17R / V20K (or vice versa);

[0189] A and B contain C10A / K15A / L17E / V20E and C and D contain C10A / K15A / L17R / V20R (or vice versa);

[0190] A and B contain C10A / K15A / L17D / T119D and C and D contain C10A / K15A / L17K / V121K (or vice versa);

[0191] A and B contain C10A / K15A / L17D / V121E and C and D contain C10A / K15A / L17K / V121R (or vice versa);

[0192] A and B contain C10A / K15A / L17E / T119D and C and D contain C10A / K15A / L17R / V121K (or vice versa);

[0193] A and B contain C10A / K15A / L17E / V121E and C and D contain C10A / K15A / L17R / V121R (or vice versa);

[0194] A and B contain C10A / K15A / V20D / T119D and C and D contain C10A / K15A / V20K / V121K (or vice versa);

[0195] A and B contain C10A / K15A / V20D / V121E and C and D contain C10A / K15A / V20K / V121R (or vice versa);

[0196] A and B contain C10A / K15A / V20E / T119D and C and D contain C10A / K15A / V20R / V121K (or vice versa); or

[0197] A and B contain C10A / K15A / V20E / V121E and C and D contain C10A / K15A / V20R / V121R (or vice versa)

[0198] In some embodiments, the TTR protein complex is attached to 1, 2, 3, 4, 5, 6, 7 or 8 biologically active proteins, peptides or small molecules. In some embodiments, the TTR protein complex is attached to 1, 2, 3, 4, 5, 6, 7 or 8 antigen binding proteins or peptides. In other embodiments, the TTR protein complex is attached to 1, 2, 3 or 4 antigen binding proteins or peptides. The antigen binding protein or peptide can be attached to the TTR protein complex at the C-terminus of the TTR subunit or the N-terminus of the TTR subunit. In addition, the TTR protein complex can be directly attached to 1, 2, 3, 4, 5, 6, 7 or 8 antigen binding proteins or peptides; or it can be attached to 1, 2, 3, 4, 5, 6, 7 or 8 antigen binding proteins or peptides through a linker. In specific embodiments, the TTR protein complex is directly attached to 1, 2, 3 or 4 antigen binding proteins or peptides; or it is attached to 1, 2, 3 or 4 antigen binding proteins or peptides through a linker.

[0199] The linker can be an amino acid based linker comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids. In other embodiments, the linker is an amino acid based linker comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids. In other embodiments, the linker is an amino acid based linker comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In other embodiments, the linker is an amino acid based linker comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In specific embodiments, the linker is G, GG, GGG, GGGG, GGGGG, GGGGGG, GGGGGGG, GGGGGGGG, GGGGGGGGG, or GGGGGGGGGGG. In other specific embodiments, the linker is selected from the list comprising: GG, GGGG, GGGSGG, GGGSGGGG, and GGAGGGAGGG.

[0200] Other suitable linkers include G(G x B y ) r G z A linker wherein G = glycine; B = any amino acid; x = 1-15; y = 1-5; z = 1-15; and r = 1-20. In another embodiment, the linker is G (G x B y ) r G z A linker wherein B=Q, S, A, E, P, T, K, R, D, or N; x=4; y=1; z=4; and r=1.

[0201] In some embodiments of the invention, the TTR protein complex is attached to two antigen binding proteins, wherein each antigen binding protein binds a different antigen. In other embodiments of the invention, the TTR protein complex is attached to four antigen binding proteins, wherein these antigen binding proteins bind at least two different antigens (e.g., one antigen binding protein binds a first antigen and three antigen binding proteins bind a second antigen; or two antigen binding proteins bind a first antigen and two antigen binding proteins bind a second antigen).

[0202] The antigen binding protein can be an antibody. In other embodiments, the antigen binding protein is a Fab or scFv. In specific embodiments, the antigen binding protein is a Fab. In other embodiments, the antigen binding protein is a mixture of an antibody and a Fab.

[0203] The invention also includes pharmaceutical compositions comprising any of the TTR protein complexes discussed herein.

[0204] In addition, the invention includes methods of treating cancer using any of the TTR protein complexes discussed herein. The TTR protein complexes of the invention can be used to treat cancer. The invention also includes any of the TTR protein complexes discussed herein for use in treating cancer.

[0205] In another embodiment, the invention includes isolated one or more nucleic acids encoding any of the TTR protein complexes discussed herein. In addition, the invention includes one or more expression vectors comprising nucleic acids encoding any of the TTR protein complexes discussed herein. The invention further includes recombinant host cells comprising such one or more nucleic acids or one or more vectors. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell, an E5 cell, a baby hamster kidney (BHK) cell, a monkey kidney (COS) cell, a human hepatocellular carcinoma cell, or a human embryonic kidney 293 (HEK293) cell.

[0206] In some embodiments, the invention relates to a method of preparing a TTR protein complex described herein, wherein the method comprises: a) culturing a recombinant host cell; b) isolating the TTR protein complex from the culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0207] Figure 1 . Figure 1 is a schematic diagram of a TTR protein complex (construct or fusion protein) of the present invention. Figure 1 a Depicts the four TTR subunits that make up the TTR complex of the present invention. Figure 1 b is an exemplary TTR antibody heterodimer fusion protein, in which the C-termini of the two antibody heavy chains are linked to the N-termini of each TTR subunit. In this example, the two antibodies bind to different epitopes on the same or different proteins. Figure 1c is an exemplary TTR antibody / Fab heterotrimeric fusion protein. In this example, the antibody binds to a first epitope, while the Fab binds to a second epitope on the same or different protein. Figure 1 d is an exemplary TTR Fab heterotetrameric fusion protein, in which the C-terminus of each Fab is linked to the N-terminus of each TTR subunit. In this example, two Fabs bind to a first epitope, and two Fabs bind to a second epitope on the same or different protein. Figure 1 Each of b-1d shows an optional linker between the heavy chain and TTR.

[0208] Figure 2 . Figure 2 ae represents exemplary heteromeric TTR Ab, TTR Fab, and TTR Ab / Fab protein complexes. Any antigen binding protein (e.g., Fab, antibody, scFv, scFab) or protein such as an enzyme can be used in the TTR protein complex of the present invention. Figure 2 The “+” and “-” symbols in e represent Fc charge pairs, which allow each intact antibody to always be linked to one TTR subunit. Figure 2 ae also lists an abbreviated description of each protein complex (e.g., [[Fab "A"]-[Negative TTR]]2: [[Positive TTR]-[Fab "B"]]2) where "A" refers to the first target and "B" refers to the second target. "-" indicates a single point of attachment between two parts (e.g., [Positive TTR] and [Fab "B"]), while "=" indicates two points of attachment between two parts (e.g., [655-341Ab] and [Negative TTR]). It is worth noting that the abbreviation does not imply the N→C or C→N direction, but is an abbreviated description of the molecule from "left to right". A second form of the abbreviation is also described. For example, Figure 2 The construct a can be labeled "4X-Fab-TTR", indicating that 4 Fabs are attached to the TTR complex. Similarly, the construct Figure 2 b and Figure 2 e can be labeled as “2X-Ab-TTR” and “4X-Ab-TTR” respectively.

[0209] Figure 3 . Figure 3 The interface between TTR monomers forming two sets of TTR dimers (left) and the interface between TTR dimers forming a TTR tetramer (right) are depicted. As can be seen, the interface between TTR monomers is different from the interface between TTR dimers.

[0210] Figure 4 . Figure 4Depicted are 18 TTR charge variants (C10A / K15A / XX) of TTR (SEQ ID NO: 1) that were used to assess whether charge mutations would result in substantial repulsion of the TTR dimer / dimer interface.

[0211] Figure 5 . Figure 5 Depicted is how various TTR dimer / dimer interfaces (with mutations) respond to the presence of SDS (chaotropic agent) with and without heating.

[0212] Figure 6 . Figure 6 The effect of non-denaturing conditions on TTR variants is depicted. TTR variants, yield, whether TTR exists as a tetramer or dimer (based on SEC and SDS-PAGE analysis), and TTR melting temperature are recorded.

[0213] Figure 7 . Figure 7 Depicted are assessments of TTR heterotetramer formation by SEC (upper) and SDS-PAGE (lower). SEC (upper) analysis indicated that many variant pairs had a propensity to form heteromultimers, as indicated by non-zero values ​​(% of molecules with retention times consistent with TTR tetramers). Furthermore, as indicated by SDS-PAGE results, many TTR heterotetramers were resistant to degradation by chaotropic SDS.

[0214] Figure 8 . Figure 8 Further SDS-PAGE evaluation of the positive / negative pairings is depicted, showing a high propensity to form stable TTR tetramers. TTR heterotetramers are resistant to SDS-induced denaturation, an indicator of good stability. For each pairing, [1] the negative (i.e., basic) variant; [2] the positive (i.e., acidic) variant; [3] a combination of the negative and positive variants (which should form tetramers); and [4] a combination of the negative and positive variants exposed to caspases were evaluated.

[0215] Fig. 9 . Fig. 9 Depicted is the evaluation of TTR heterotetramers comprising L17R / T119D, L17K / T119D, L17K / V121E, V20R / V20D, V20R / V20E, V20K / V20D, V20K / V20E, V121R / L17D, V121R / L17E, and V121K / L17D pairings when exposed to pH 5.0 conditions to determine whether they can maintain a tetrameric state (by SEC) under conditions similar to those found in pharmaceutical formulations.

[0216] Fig.10 . Fig.10Depicted are evaluations of the melting temperatures of TTR heterotetramers. In each case (mutants noted in the figure), the TTR heterotetramers were stable at at least 92°C, indicating that the heterotetramers are thermostable.

[0217] Fig.11 . Fig.11 Depicted is the construction of a bispecific TTR heterotetrameric Ab construct. An exemplary bispecific TTR heterotetrameric Ab construct is shown, wherein each heavy chain of the 655-341 Ab (line fill) is attached to the N-terminus of a negative TTR monomer (together forming a negative TTR dimer) and each heavy chain of the DNP-3B1 Ab (solid fill) is attached to the N-terminus of a positive TTR monomer (together forming a positive TTR dimer). Four negative TTR variants are fused to the 655-341 Ab, and four positive TTR variants are fused to the DNP-3B1 Ab. All Ab-TTR fusions were generated without a linker between the Ab and the TTR monomer.

[0218] Fig.12 . Fig.12 Depicted that when the [655-341Ab] = [negative TTR]2 and [positive TTR]2 = [DNP-3B1 Ab] tetramer portions were expressed in separate mammalian cells (293-6E HEK cells), TTR heterotetrameric Ab constructs could not be efficiently generated.

[0219] Fig.13 . Fig.13 Depicted is the construction of a bispecific TTR heterodimeric Ab construct used to evaluate the effect of adding a linker between the Ab heavy chain and the TTR monomer on the expression of the two tetrameric moieties in two different mammalian cells.

[0220] Fig.14 . Fig.14 Depicted are the expression results of the two Ab-TTR heterodimer parts of a bispecific TTR heterotetrameric Ab construct with a linker between the Ab heavy chain and the TTR monomer in two different mammalian cells (sorted by linker).

[0221] Fig.15 . Fig.15 Depicted are additional results of expression of the two Ab-TTR heterodimer portions of a bispecific TTR heterotetrameric Ab construct with a linker between the Ab heavy chain and the TTR monomer in two different mammalian cells (sorted by linker).

[0222] Fig.16 . Fig.16 Depicted Fig.14 and 15 The average result.

[0223] Fig.17 . Fig.17 Depicted is the construction of a bispecific TTR heterotetrameric Fab construct used to evaluate Fab TTR fusions in a mammalian cell line (CHO K1).

[0224] Fig.18 . Fig.18 Depicted Fig.17 Evaluation results of bispecific TTR heterotetrameric Fab constructs in .

[0225] Fig.19 . Fig.19 Depicted are the retention (SEC) of the heteromultimeric molecules 15524 ([655-341Fab]-[GG]-[TTR(C10A / K15A / L17D)] and [TTR(C10A / K15A / V121R)]-[GG]-[DNP-3B1 Fab]) compared to homomultimeric Ab- and Fab-TTR fusions and unfused Ab (all as standards).

[0226] Fig. 20 . Fig. 20 Confirmation of elution species by SEC coupled to MS (from Fig.19 ) is consistent with expectations for molecule 15524 (as a heteromultimer).

[0227] Fig.21 . Fig.21 The construction of Ab-containing TTR fusions was depicted to determine whether co-expression of Ab-containing TTR fusions would result in the production of the desired TTR heterotetramers [655-341Ab]=[[LX]-[minus TTR]]2:[[plus TTR]-[LX]]2=[DNP-3B1 Ab].

[0228] Fig. 22 . Fig. 22 Describes Fig.21 For many combinations of , a large number of the desired TTR heterotetramers were formed.

[0229] Fig.23 . Fig.23 Depicted is the retention (SEC) of the heteromultimeric molecule 15539 ([655-341Ab] = [[GGAGGGAGGG]-[TTR(C10A / K15A / L17D]]2:[[TTR(C10A / K15A / V121K)]-[GGAGGGAGGG]]2 = [DNP-3B1 Ab]) compared to homomultimeric Ab- and Fab-TTR fusions and unfused Ab (all as standards).

[0230] Fig.24 . Fig.24 Confirmation of elution species by SEC coupled to MS (from Fig.23 ) is consistent with expectations for molecule 15539 (as a heteromultimer).

[0231] Fig.25 . Fig.25 Depicted Fig. 22 The average result.

[0232] Fig.26 . Fig.26 Depicted is an evaluation of whether co-expression of negative and positive TTR variants (4 of each mutation) fused to Ab and Fab in the same cell line would result in the generation of Ab-Fab-TTR constructs (i.e., [Ab "A"] = [negative TTR]2: [[positive TTR] = [Fab "B"]]2 constructs).

[0233] Fig. 27 . Fig. 27 Depicted Fig.26 Results of expression, purification and analysis of Ab-Fab-TTR constructs.

[0234] Fig.28 . Fig.28 Depicted is the retention (by SEC) of the heteromultimeric molecule 15545 ([655-341Ab]=[[GGGG]-[TTR(C10A / K15A / V20D)]]2:[[TTR(C10A / K15A / V20R)]-[GG]-[DNP-3B1-Fab]]2) compared to homomultimeric Ab- and Fab-TTR fusions and unfused Ab (all used as standards).

[0235] Fig.29 . Fig.29 Confirmation of elution species by SEC coupled to MS (from Fig.28 ) is consistent with expectations for molecule 15545 (as a heteromultimer).

[0236] Fig.30 . Fig.30 Depicted Fig. 27 The average result.

[0237] Fig.31 . Fig.31 Depicted is a TTR double-charged variant (C10A / K15A / XX / YY) of TTR (SEQ ID NO: 1) that was prepared to assess whether the double-charged mutations could achieve a selective increase in heteromultimers over homomultimers.

[0238] Fig.32 . Fig.32Expression yields, purification yields, and SEC properties of individually expressed single and double interface mutants are depicted.

[0239] Fig.33 . Fig.33 SEC profiles of exemplary single- and double-interface mutants are depicted.

[0240] Fig.34 . Fig.34 Depicted is a TTR double-charged variant (C10A / K15A / XX / YY) of TTR (SEQ ID NO: 1) that was prepared to assess whether the double-charged mutations could achieve a selective increase in heteromultimers over homomultimers.

[0241] Fig.35 . Fig.35 Depicted are the SEC properties of purified pools of single and double interface mutants sorted by negative mutation.

[0242] Fig.36 . Fig.36 Depicted are the SEC properties of individual purified and mixed single and double interface mutants sorted by positive mutation.

[0243] Fig.37 . Fig.37 Depicted are SEC profiles of exemplary single and double interface mutants individually and after post-purification mixing.

[0244] Fig.38 . Fig.38 Purification yields and SEC profiles of single and double interface mutants produced by co-culture of cell lines are depicted.

[0245] Fig.39 . Fig.39 Depicted Fig.38 Continuation of the data presented in .

[0246] Fig.40 . Fig.40 Depicted are SEC profiles of exemplary molecules produced by co-culturing the dual interface mutants. DETAILED DESCRIPTION

[0247] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0248] Unless otherwise defined herein, scientific and technical terms used in conjunction with this application have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, singular terms will include pluralities and plural terms will include the singular.

[0249] Generally, the nomenclature used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and the techniques thereof are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present application can be carried out according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) [Molecular Cloning: Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York State (2001)]; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992) [Molecular Biology Modern Methods, Greene Publishing Associates (1992)]; and Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990) [Antibodies: Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1990)], which are incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer's instructions, as commonly accomplished in the art, or as described herein. The nomenclature and the laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0250] It should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein and thus may vary. The terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present disclosure, which will be limited only by the claims.

[0251] Except in the operating examples, or where otherwise indicated, all numbers expressing amounts of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term “about.” The term “about” when used in conjunction with a percentage may mean ±1%.

[0252] All embodiments that are narrower in any way than the scope of variation defined by a particular paragraph herein should be considered to be included in the present disclosure. For example, certain aspects are described as a class, and it should be understood that each member belonging to a class can be an embodiment. Similarly, aspects described as a class or members selected to belong to a class should be understood to include combinations of two or more members in the class. It should also be understood that although multiple embodiments in the specification are presented using "comprising" language, in many cases, "consisting of" or "consisting essentially of" language can also be used to describe related embodiments.

[0253] In this application, the use of "or" means "and / or" unless otherwise specified. In addition, the use of the term "including", as well as other forms such as "includes" and "included", is not limiting. Likewise, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components comprising more than one subunit, unless otherwise expressly specified.

[0254] definition

[0255] "Amino acid" includes its standard meaning in the art. Twenty natural amino acids and their abbreviations follow conventional usage. See, Immunology-A Synthesis [immunology-A synthesis], 2nd edition, (ES Golub and DR Green, ed.), Sinauer Associates: Sunderland, Massachusetts. (1991), which is incorporated herein by reference for any purpose. Twenty conventional amino acids, non-natural amino acids (e.g., [α]-, [α]-disubstituted amino acids), N-alkyl amino acids, and other unconventional amino acid stereoisomers (e.g., D-amino acids) can also be suitable components of polypeptides and are included in the term "amino acid". Examples of unconventional amino acids include: 4-hydroxyproline, [γ]-carboxyglutamate, [ε]-N,N,N-trimethyllysine, [ε]-N-acetylysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, [σ]-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl terminal direction in accordance with standard usage and convention.

[0256] As used herein, "antagonists" generally refer to molecules that can bind to an antigen and inhibit, reduce or eliminate biological signaling associated with the antigen, such as the antigen binding proteins provided herein.

[0257] As used herein, the term "antibody" refers to a protein having a conventional immunoglobulin form, comprising a heavy chain and a light chain and comprising a variable region and a constant region. For example, an antibody can be an IgG, which is a "Y-shaped" structure of two pairs of identical polypeptide chains, each pair having a "light" chain (typically having a molecular weight of about 25 kDa) and a "heavy" chain (usually having a molecular weight of about 50-70 kDa). Antibodies have a variable region and a constant region. In the IgG form, the variable region is typically about 100-110 or more amino acids, comprises three complementary determining regions (CDRs), is primarily responsible for antigen recognition, and is very different from other antibodies that bind to different antigens. The constant region allows antibodies to recruit cells and molecules of the immune system. The variable region is composed of the N-terminal region of each light chain and heavy chain, while the constant region is composed of the C-terminal portion of each heavy chain and light chain. (Janeway et al., "Structure of the Antibody Molecule and the Immunoglobulin Genes", Immunobiology: The Immune System in Health and Disease, 4th ed. Elsevier Science Ltd. / Garland Publishing, (1999)).

[0258] The antibody may comprise any constant region known in the art. Human light chains are classified as κ light chains and λ light chains. Heavy chains are classified as μ, δ, γ, α or ε, and the isotypes of the antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3 and IgG4. IgM has subclasses, including but not limited to IgM1 and IgM2. The embodiments disclosed herein include all such antibody classes or isotypes. The light chain constant region may be, for example, a κ-type or λ-type light chain constant region, such as a human κ-type or λ-type light chain constant region. The heavy chain constant region may be, for example, an α-type, δ-type, ε-type, γ-type or μ-type heavy chain constant region, such as a human α-type, δ-type, ε-type, γ-type or μ-type heavy chain constant region. Therefore, in an exemplary embodiment, the antibody is an antibody of isotype IgA, IgD, IgE, IgG or IgM, including any one of IgG1, IgG2, IgG3 or IgG4.

[0259] The term "antigen" refers to a molecule or portion of a molecule that can be bound by a binding agent such as an antigen binding protein (including, for example, an antibody) and can additionally be used in an animal to produce antibodies that can bind to the antigen. An antigen may have one or more epitopes that can interact with different antigen binding proteins (e.g., antibodies).

[0260] As used herein, "antigen binding protein" means any protein that specifically binds to a particular target antigen. The term includes polypeptides that include at least one antigen binding region. The term also encompasses intact antibodies that include at least two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and mutations thereof. Antigen binding proteins also include Fab, Fab', F(ab')2, Fv fragments, domain antibodies (e.g. ) and scFv, as described in more detail below.

[0261] "Antigen binding region" or "antigen binding domain" means the portion of a protein (e.g., an antibody or fragment, derivative, or variant thereof) that specifically binds to, interacts with, or recognizes a given epitope or site on a molecule (e.g., an antigen). For example, the portion of an antigen binding protein that contains amino acid residues that interact with an antigen and impart its specificity and affinity to the antigen is called an "antigen binding region." An antigen binding region may include one or more "complementarity determining regions" ("CDRs"). Certain antigen binding regions also include one or more "framework" regions. The "framework" region may directly contribute to the specific binding of the antigen binding protein, but generally helps to maintain the proper conformation of the CDRs to promote binding between the antigen binding region and the antigen.

[0262] The terms "cancer," "tumor," "cancerous," and "malignant" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, epithelial cancers, including adenocarcinomas, lymphomas, blastomas, melanomas, sarcomas, and leukemias. More specific examples of such cancers include melanoma, lung cancer, head and neck cancer, renal cell carcinoma, colon cancer, colorectal cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's lymphoma and non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer (e.g., hepatocellular carcinoma and hepatoma), bladder cancer, breast cancer, endometrial cancer, myeloma (e.g., multiple myeloma), salivary gland cancer, kidney cancer (e.g., renal cell carcinoma and Wilms' tumor), basal cell carcinoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, and esophageal cancer.

[0263] The term "CDR" and its plural "multiple CDRs" (also called "hypervariable regions") refer to the complementary determining regions of a protein, such as an antibody, or a fragment, derivative, or variant thereof. The light chain variable region and the heavy chain variable region each contain three CDRs. For example, the light chain variable region contains the following CDRs: CDR-L1, CDR-L2, and CDR-L3; and the heavy chain variable region contains the following CDRs: CDR-H1, CDR-H2, and CDR-H3. CDRs contain most of the residues that are responsible for the specific interaction of the antibody with the antigen, and therefore contribute to the functional activity of the antibody molecule. CDRs are the main determinants of antigen specificity.

[0264] Accurately defined CDR boundaries and lengths are subject to different classifications and numbering systems. Therefore, CDRs can be referenced by Kabat, Chothia, contact or any other boundary definition (including numbering systems described herein). The Kabat numbering scheme (system) is a widely used standard for numbering the amino acid residues of antibody variable domains in a consistent manner, and is a preferred embodiment of the present invention, as also mentioned elsewhere herein. Other structural considerations can also be used to determine the canonical structure of antibodies. For example, those differences that are not fully reflected by Kabat numbering can be described by the numbering system of Chothia et al., and / or revealed by other techniques (such as crystallography and two-dimensional or three-dimensional computational modeling). Despite different boundaries, each of these systems has a certain degree of overlap in terms of constituting the "CDR" in the variable sequence. Therefore, the CDR definitions according to these systems can be different in length and boundary regions relative to adjacent framework regions. See, e.g., Kabat (a method based on cross-species sequence variability), Chothia (a method based on crystallographic studies of antigen-antibody complexes), and / or MacCallum (Kabat et al., loc. cit.; Chothia et al., J. Mol. Biol., 1987, 196:901-917; and MacCallum et al., J. Mol. Biol., 1996, 262:732). Yet another standard for characterizing antigen binding sites is the AbM definition used by the AbM antibody modeling software of Oxford University Molecular. See, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains In: Antibody Engineering Lab Manual (Editors: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). For a review of antibody structure, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Harlow et al., eds., 1988.

[0265] Typically, CDR forms a loop structure that can be classified as a canonical structure. The term "canonical structure" refers to the main chain conformation used by the antigen binding (CDR) loop. From comparative structural studies, it has been found that five of the six antigen binding loops have only a limited set of available conformational repertoires. Each canonical structure can be characterized by the torsion angle of the polypeptide backbone. Therefore, the corresponding loops between antibodies may have very similar three-dimensional structures, but most parts of the loops have high amino acid sequence variability (Chothia and Lesk, J.MoI.Biol. [Molecular Biology Journal], 1987, 196: 901; Chothia et al., Nature [Nature], 1989, 342: 877; Martin and Thornton, J.MoI.Biol [Molecular Biology Journal], 1996, 263: 800). In addition, there is a relationship between the loop structure used and the amino acid sequence around it. The conformation of a specific canonical class is determined by the length of the loop and the amino acid residues located at key positions within the loop and within the conservative framework (i.e., outside the loop). Therefore, assignment to a specific canonical class can be made based on the presence of these key amino acid residues.

[0266] When used in the context of antigen binding proteins (e.g., antibodies or fragments thereof) that compete for the same epitope, the term "competition" means competition between the antigen binding proteins and is determined by an assay in which the antigen binding protein (e.g., antibody or fragment thereof) being tested prevents or inhibits specific binding of a reference antigen binding protein to a common antigen. Many types of competitive binding assays can be used, for example: solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619); solid phase direct label assays, solid phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, 1996 ... Press); solid phase direct labeling RIA using I-125 labeling (see, e.g., Morel et al., 1988, Molec. Immunol. [Molecular Immunology] 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung et al., 1990, Virology [Virology] 176:546-552); and direct labeling RIA (Moldenhauer et al., 1990, Scand. J. Immunol. [Scandinavian Journal of Immunology] 32:77-82). Typically, such assays involve the use of purified antigen or cells expressing antigen bound to a solid surface, an unlabeled test antigen binding protein, and a labeled reference antigen binding protein. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen binding protein. Typically, the test antigen binding protein is present in excess. Antigen binding proteins identified by competitive assays include: antigen binding proteins that bind to the same epitope as the reference antigen binding proteins and antigen binding proteins that bind to the epitope that is close enough to the reference antigen binding proteins (to cause steric hindrance). Other details about the method for determining competitive binding are provided herein. For example, in one embodiment, competition is determined according to BiaCore determination. Generally, when competitive antigen binding proteins are present in excess, they will suppress the specific binding of the reference antigen binding proteins to the common antigen by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some cases, the binding is suppressed by at least 80%, 85%, 90%, 95%, or 97% or more.

[0267] The term "control sequence" refers to a polynucleotide sequence that can affect the expression and processing of the coding sequence connected thereto. The nature of such control sequences may depend on the host organism. In a specific embodiment, the control sequence of a prokaryotic organism may include a promoter, a ribosome binding site, and a transcription termination sequence. For example, the control sequence of a eukaryotic organism may include a promoter, a transcription enhancer sequence, and a transcription termination sequence that include one or more transcription factor recognition sites. A "control sequence" may include a leader sequence and / or a fusion partner sequence.

[0268] A "derivative" of a polypeptide is one that has been modified (eg, chemically modified) in some manner other than insertion, deletion, or substitution variants, such as by conjugation to another chemical moiety.

[0269] "Domain antibodies" are immunologically functional immunoglobulin fragments that contain only the variable region of a heavy chain or the variable region of a light chain. Examples of domain antibodies include In some cases, two or more V H The two V regions of the bivalent domain antibody are H The regions may target the same or different antigens.

[0270] An "effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate symptoms and / or potential causes, prevent the occurrence of symptoms and / or their potential causes, and / or improve or cure the damage caused by or associated with cancer. In some embodiments, an effective amount is a therapeutically effective amount or a preventive effective amount. A "therapeutically effective amount" is an amount sufficient to treat a disease state (e.g., cancer) or symptom, a state or symptom particularly associated with the disease state, or otherwise prevent, hinder, delay or reverse the disease state or any other undesirable symptom associated with the disease in any way. A "preventive effective amount" is an amount of a pharmaceutical composition that will have a predetermined preventive effect when administered to a subject, such as preventing or delaying the onset (or recurrence) of cancer, or reducing the likelihood of the onset (or recurrence) of cancer or cancer symptoms. A single dose may not result in a complete therapeutic or preventive effect, and may only occur after a series of doses are administered. Therefore, a therapeutic or preventive effective amount may be administered once or multiple times.

[0271] The term "epitope" refers to an antigenic part that can be recognized and specifically bound by an antigen binding protein (e.g., an antibody). In the case of a polypeptide, an epitope can be formed from continuous amino acids or non-continuous amino acids juxtaposed by the tertiary folding of a protein. Epitopes formed by continuous amino acids are usually retained when the protein is denatured, while epitopes formed by tertiary folding are usually lost after protein denaturation. An epitope usually contains at least 3, more usually at least 5 or 8-10 amino acids in a unique spatial conformation. A "linear epitope" or "sequential epitope" is an epitope that an antigen binding protein (e.g., an antibody) recognizes by its linear sequence or primary structure of amino acids. A "conformational epitope" or "non-sequential epitope" is an epitope that an antigen binding protein (e.g., an antibody) recognizes by its tertiary structure. The residues that make up these epitopes may not be continuous in the primary amino acid sequence, but are close to each other in the tertiary structure of the molecule. When a protein is denatured, broken, or reduced, linear and conformational epitopes usually behave differently.

[0272] The term "expression vector" or "expression construct" refers to a vector suitable for transforming a host cell and containing a nucleic acid sequence that directs and / or controls (combines with a host cell) the expression of one or more heterologous coding regions operably linked thereto. An expression construct may include, but is not limited to, sequences that affect or control transcription, translation, and, when introns are present, RNA splicing of a coding region operably linked thereto.

[0273] The "Fab fragment" or "Fab" consists of a light chain and a heavy chain C H 1 and the variable region. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0274] A "Fab' fragment" or "Fab" contains one light chain and one heavy chain. H Domain and C H 1 domain and between C H 1 and C H The portion of the region between the two Fab' domains allows for the formation of an interchain disulfide bond between the two heavy chains of the two Fab' fragments, thereby forming a F(ab')2 molecule.

[0275] A "F(ab')2 fragment" or "F(ab')2" contains two light chains and two heavy chains, wherein the heavy chains contain C H 1 and C H A portion of the constant region between the two heavy chains allows the formation of an interchain disulfide bond between the two heavy chains. The F(ab')2 fragment is thus composed of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0276] The "Fc" region contains two C H 2 and C HThe two heavy chain fragments are linked by two or more disulfide bonds and C H The 3 domains are held together by hydrophobic interactions.

[0277] The "Fv region" comprises the variable regions from the heavy and light chains, but lacks the constant regions.

[0278] The term "heavy chain" when used with respect to an antigen binding protein, antibody or fragment thereof includes a full-length heavy chain. A full-length heavy chain includes the variable region domain (V H ) and three constant region domains (C H 1. C H 2. and C H 3). V H The domain is located at the amino terminus of the polypeptide, and the C H The domain is located at the carboxyl terminus, where C H 3 is closest to the carboxyl terminus of the polypeptide. The heavy chain may be of any isotype, such as IgG (including IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE. The heavy chain fragment has sufficient variable region sequence to confer binding specificity.

[0279] "Hematological cancers" are cancers that begin in blood-forming tissues (such as the bone marrow) or in cells of the immune system. Examples of blood cancers are leukemias, lymphomas, and multiple myeloma.

[0280] The term "heterodimer fusion protein" or "heterodimer protein complex" refers to a fusion protein comprising two different proteins (e.g., an antigen binding protein; a peptide, such as an agonist peptide; and an agonist protein domain). In a specific example, the heterodimer can be a TTR heterodimer fusion protein comprising two different antigen binding proteins (e.g., two different antibodies) connected by a TTR protein as described herein. In another example, the heterodimer can be a TTR heterodimer fusion protein comprising an antibody and a Fab connected by a TTR protein as described herein. Figure 1 Exemplary heterodimeric fusion proteins are depicted in b and 2b.

[0281] The term "heterotrimeric fusion protein" or "heterotrimeric protein complex" refers to a fusion protein comprising three different proteins (e.g., an antigen binding protein; a peptide, such as an agonist peptide; and an agonist protein domain). In a specific example, the heterotrimer can be a TTR heterotrimeric fusion protein comprising an antibody and two Fabs connected by a TTR protein as described herein (see, e.g., Figure 2 c).

[0282] The term "heterotetrameric fusion protein" or "heterotetrameric protein complex" refers to a fusion protein comprising four different proteins (e.g., an antigen binding protein; a peptide, such as an agonist peptide; and an agonist protein domain). In a specific example, the heterotetrameric fusion protein is a TTR heterotetrameric fusion protein, wherein, for example, an antibody, a Fab, or a mixture thereof is linked by a TTR protein as described herein. Examples include wherein the antigen binding protein is an antibody (see, e.g., Figure 2 e) or Fab (see, e.g., Figure 1 d and 2a). In a specific example, the heterotetrameric fusion protein is a TTR heterotetrameric fusion protein, wherein, for example, an antibody, a Fab or a mixture thereof is linked by a TTR protein as described herein.

[0283] The term "host cell" means a cell that has been transformed with a nucleic acid sequence and thereby expresses a gene of interest. The term includes progeny of a parent cell, whether or not the progeny is identical to the original parent cell in morphology or genetic makeup, as long as the gene of interest is present.

[0284] The term "identity" means a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules determined by alignment and comparison of sequences. "Percent identity" means the percentage of identical residues between amino acids or nucleotides in the molecules being compared, and is calculated based on the size of the smallest molecule being compared. For these calculations, gaps in the alignment, if any, must be resolved using a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM and Griffin, HG, ed.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer [Primer of Sequence Analysis], (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[0285] When calculating the percent consistency, the sequences compared are compared in a manner that achieves the maximum match between each sequence. The computer program used to determine the percent identity is the GCG software package, which includes GAP (Devereux et al., 1984 Nucl. Acid Res. [Nucleic Acids Research] 12: 387; Genetics Computer Group, University of Wisconsin, Madison, Wisconsin). The computer algorithm GAP is used to compare two polypeptides or polynucleotides whose percent sequence identity is to be determined. Align the sequences so that their respective amino acids or nucleotides reach the best match (providing the "matching range" determined by the algorithm). The gap opening penalty (which is calculated as 3x diagonal averages, wherein "diagonal averages" are the averages of the diagonals of the comparison matrix used; "diagonals" are the scores or values ​​assigned to each complete amino acid match by a specific comparison matrix) and the gap extension penalty (which is typically 1 / 10 × gap opening penalty) and the comparison matrix (such as PAM 250 or BLOSUM 62) are used in conjunction with the algorithm. In certain embodiments, the algorithm also uses a standard comparison matrix (for the PAM 250 comparison matrix, see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352; for the BLOSUM 62 comparison matrix, see Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919).

[0286] Recommended parameters for determining percent identity of polypeptide or nucleotide sequences using the GAP program are as follows:

[0287] Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453;

[0288] Comparison matrix: BLOSUM 62 from Henikoff et al., 1992, supra;

[0289] Gap Penalty: 12 (but no penalty for end gaps)

[0290] Gap length penalty: 4

[0291] Similarity threshold: 0

[0292] Certain alignment schemes used to align two amino acid sequences can match only shorter regions of the two sequences, and this smaller alignment region can have a very high sequence identity, even if there is no obvious relationship between the two full-length sequences. Therefore, if necessary, the selected alignment method (GAP program) can be adjusted to align over at least 50 consecutive amino acids of the target polypeptide.

[0293] The phrase "immunomodulator" refers to a molecule that induces, enhances, or suppresses an immune response. An immunoactivator is a molecule that induces or amplifies an immune response. An immunosuppressant is a molecule that reduces or suppresses an immune response. Thus, activation immunotherapy is a therapy that involves administering one or more molecules to induce or enhance a subject's immune system. Suppression immunotherapy is a therapy that treats a subject with one or more molecules to reduce or suppress the subject's immune system.

[0294] As used herein, the term "fragment" of an antibody or immunoglobulin chain (heavy chain or light chain) is an antigen-binding protein that includes a portion of an antibody that lacks at least some of the amino acids present in the full-length chain but is capable of specific binding to an antigen (regardless of how the portion is obtained or synthesized). Such fragments have biological activity because they specifically bind to a target antigen and can compete with other antigen-binding proteins (including complete antibodies) for binding to a given epitope. In one aspect, such fragments will retain at least one CDR present in a full-length light chain or heavy chain, and in some embodiments will include a single heavy chain and / or light chain or a portion thereof. These biologically active fragments can be produced by recombinant DNA technology, or can be produced by enzymatic or chemical cleavage of an antigen-binding protein (including a complete antibody). Immunoglobulin fragments with immune function include, but are not limited to, Fab, Fab', F(ab')2, Fv, domain antibodies, and scFv, and can be derived from any mammalian source, including, but not limited to, humans, mice, rats, camelids, or rabbits. It is further contemplated that a functional portion of the antigen binding proteins disclosed herein, such as one or more CDRs, can be covalently bound to a second protein or small molecule to generate a therapeutic agent directed to a specific target in vivo or with a prolonged serum half-life.

[0295] "Isolated nucleic acid molecule" means a DNA or RNA of genomic, mRNA, cDNA or synthetic origin or some combination thereof, which is not associated with all or part of a polynucleotide (where the isolated polynucleotide is found in nature) or is linked to a polynucleotide to which it is not linked in nature. For the purposes of this disclosure, it is understood that a "nucleic acid molecule" that "comprising" a specific nucleotide sequence does not encompass a complete chromosome. An isolated nucleic acid molecule that "comprising" a specified nucleic acid sequence may include, in addition to these specified sequences, coding sequences for up to ten or even up to twenty other proteins or portions thereof, or may include operably linked regulatory sequences that control the expression of the coding region of the described nucleic acid sequence, and / or may include vector sequences.

[0296] As used herein, the terms "isolated polypeptide," "purified polypeptide," "isolated protein," or "purified protein" are intended to refer to a composition separated from other components, wherein the polypeptide is purified to any degree relative to its naturally accessible state. Thus, a purified polypeptide also refers to a polypeptide that is not in the environment in which it is naturally present. Generally, "purified" will refer to a polypeptide composition that has been fractionated to remove various other components and that substantially retains the biological activity expressed by it. When the term "substantially purified" is used, the name will refer to a peptide or polypeptide composition in which the polypeptide or peptide forms the major component of the composition, e.g., constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more of the protein in the composition.

[0297] The term "light chain" when used with respect to an antigen binding protein, antibody or fragment thereof includes a full-length light chain. A full-length light chain includes a variable region domain (V L ) and constant region domain (C L ). The variable region domain of the light chain is located at the amino terminus of the polypeptide. Light chains include kappa chains and lambda chains. Light chain fragments have sufficient variable region sequence to confer binding specificity.

[0298] The term "naturally occurring" as used throughout this specification in conjunction with biological materials such as polypeptides, nucleic acids, host cells, and the like, refers to materials found in nature.

[0299] The term "oligonucleotide" means a polynucleotide comprising 200 or fewer nucleotides. In certain embodiments, the length of the oligonucleotide is 10 to 60 bases. In other embodiments, the length of the oligonucleotide is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 to 40 nucleotides. The oligonucleotide can be single-stranded or double-stranded, for example, to construct a mutant gene. The oligonucleotide can be a sense or antisense oligonucleotide. The oligonucleotide can include a label for detecting an assay, including a radioactive label, a fluorescent label, a hapten or an antigen label. The oligonucleotide can be used as, for example, a PCR primer, a cloning primer or a hybridization probe.

[0300] As used herein, "operably linked" means that the components to which the term is applied are in a relationship that allows them to perform their inherent functions under appropriate conditions. For example, a control sequence "operably linked" to a protein coding sequence in a vector is linked to the coding sequence so that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequence.

[0301] The term "polynucleotide" or "nucleic acid" includes both single-stranded and double-stranded nucleotide polymers. The nucleotides comprising the polynucleotide can be ribonucleotides and deoxyribonucleotides or modified forms of either type of nucleotide. Modifications include base modifications, such as bromouridine and inosine derivatives; ribose modifications, such as 2',3'-dideoxyribose; and internucleotide bond modifications, such as phosphorothioates, phosphorodithioates, selenophosphates, diselenphosphates, phenylamidophosphorothioates, phenylamidophosphorothioates, and phosphoramidates.

[0302] Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence discussed herein is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as 5'. The 5' to 3' direction of addition of a nascent RNA transcript is referred to as the transcription direction; the sequence region that is the 5' end of the 5' end of the RNA transcript on the same DNA strand as the RNA transcript is referred to as the "upstream sequence"; the sequence region that is the 3' end of the 3' end of the RNA transcript on the same DNA strand as the RNA transcript is referred to as the "downstream sequence".

[0303] The terms "polypeptide" or "protein" are used interchangeably herein to refer to polymers of amino acid residues. These terms are also applicable to amino acid polymers in which one or more amino acid residues are analogs or mimetics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers. These terms may also encompass amino acid polymers that have been modified, for example, by adding carbohydrate residues (to form glycoproteins) or by phosphorylation. Polypeptides and proteins may be produced by naturally occurring and non-recombinant cells or by genetically engineered or recombinant cells, and may include molecules having the amino acid sequence of a native protein or molecules having one or more amino acids of a native sequence that are deleted, added and / or substituted. The term "polypeptide fragment" refers to a polypeptide having an amino terminal deletion, a carboxyl terminal deletion and / or an internal deletion compared to the full-length protein. Such fragments may also contain modified amino acids compared to the full-length protein. In certain embodiments, the length of the fragment is about 5 to 500 amino acids. For example, a fragment can be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length.

[0304] A "recombinant protein," including a recombinant TTR protein, is a protein produced using recombinant technology, ie, by expressing a recombinant nucleic acid as described herein. Methods and techniques for producing recombinant proteins are well known in the art.

[0305] "Single-chain Fv" (scFv) is an Fv molecule in which the heavy and light chain variable regions are connected by a flexible linker to form a single polypeptide chain, which forms the antigen binding region. scFv is discussed in detail in International Patent Application Publication No. WO 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203.

[0306] "Solid tumor" refers to an abnormal growth or mass of tissue that usually does not contain cysts or areas of fluid. Solid tumors can be benign (noncancerous) or malignant (cancerous). Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, epithelial cancers, and lymphomas. Leukemias (cancers of the blood) do not usually form solid tumors.

[0307] An antigen binding protein "specifically binds" an antigen when it exhibits little to no binding to molecules other than the antigen. However, an antigen binding protein that specifically binds to an antigen may cross-react with antigens from different species. Typically, the dissociation constant (K) is measured as measured by surface plasmon resonance technology (e.g., BIACore, GE Healthcare, Uppsala, Sweden). D )≤10 -7When the antigen binding protein is at K, the antigen binding protein specifically binds to the antigen. D ≤5x10 -8 When the antigen binding protein binds to the antigen with K, the antigen binding protein specifically binds to the antigen with "high affinity", and when the antigen binding protein binds to the antigen with K, the antigen binding protein specifically binds to the antigen with "high affinity". D Yes ≤5x10 -9 When M binds to an antigen, the antigen binding protein specifically binds to the antigen with "very high affinity" (as measured using methods such as BIACore).

[0308] As used herein, a "subject" or "patient" can be any mammal. In a typical embodiment, the subject or patient is a human.

[0309] As used herein, "substantially pure" means that the molecule of the described species is the predominant species present, i.e., on a molar basis, it is more abundant than any other individual species in the same mixture. In certain embodiments, a substantially pure molecule is a composition in which the target species comprises at least 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition will constitute at least 80%, 85%, 90%, 95%, or 99% of all macromolecular species present in the composition. In other embodiments, the target species is purified to substantial homogeneity, wherein contaminating species cannot be detected in the composition by conventional detection methods and thus the composition consists of a single detectable macromolecular species.

[0310] The term "treatment" refers to any sign of successful treatment or improvement of an injury, lesion or condition, including any objective or subjective parameter, such as alleviation; relief; weakening symptoms or making an injury, lesion or condition more tolerable to the patient; slowing the rate of degeneration or weakness; making the degeneration endpoint less debilitating; improving the patient's physical or mental health. The basis for the treatment or relief of symptoms can be objective or subjective parameters; including the results of physical examinations, neuropsychiatric examinations and / or psychiatric assessments. For example, certain methods proposed herein successfully treat cancer and tumors by, for example, reducing the progression or spread of cancer, inhibiting tumor growth, causing remission of tumors and / or alleviating symptoms associated with cancer or tumors. Similarly, other methods provided herein treat infectious diseases by reducing the progression or spread of infection, reducing the extent of infection and / or alleviating symptoms associated with infection.

[0311] As used herein, the term "TTR" refers to "transthyretin". Human TTR is described in Mita et al., Biochem. Biophys. Res. Commun., 124(2):558-564 (1984) (which is incorporated herein by reference). The amino acid sequence of human TTR is also described in the UniProt knowledgebase (www.uniprot.org / uniprot / P02766#sequences) and is cited herein as SEQ ID NO: 1. The nucleic acid sequence of human TTR is available at NCBI ( www.ncbi.nlm.nih.gov / gene / 7276 ). See also GenBank Accession No. K02091.1. The nucleic acid sequence of human TTR is referenced herein as SEQ ID NO:44. The amino acid and nucleic acid sequences of murine TTR are set forth in SEQ ID NOs:2 and 3, respectively. In some embodiments, the human TTR nucleic acid is a nucleic acid encoding the human TTR protein of SEQ ID NO:1. In other embodiments, the murine TTR nucleic acid is a nucleic acid encoding the murine TTR protein of SEQ ID NO:2.

[0312] The term "TTR variant" refers to a protein having an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to TTR having SEQ ID NO: 1. The invention also includes nucleic acids encoding such TTR variants. Specific variants include, for example, TTR proteins that are truncated at the C-terminus or N-terminus.

[0313] A "tumor" is a mass of tissue that forms as cancer cells grow and multiply, and can invade and destroy normal adjacent tissue. Cancer cells can break away from a malignant tumor and enter the blood or lymphatic system, allowing the cancer cells to spread from the primary tumor to form new tumors in other organs.

[0314] A "variant" of a polypeptide comprises an amino acid sequence in which one or more amino acid residues are inserted into, deleted from and / or substituted into the amino acid sequence relative to another polypeptide sequence. Variants include fusion proteins.

[0315] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop in which another DNA segment can be connected. Another type of vector is a viral vector, in which another DNA segment can be connected to the viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors and free mammalian vectors with bacterial replication origins). Other vectors (e.g., non-free mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. In addition, some vectors can guide the expression of genes operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA technology are usually in the form of plasmids. In this specification, "plasmid" and "vector" can be used interchangeably because plasmids are the most commonly used vector forms. However, the present invention is intended to include expression vectors of other forms with equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0316] TTR variants

[0317] As mentioned above, human TTR is a non-covalent tetrameric protein. The TTR tetrameric protein is composed of dimers of dimers ( Figure 3 Interestingly, the interface between TTR monomers that form the TTR dimer ( Figure 3 , left) and the interface between TTR dimers that form a TTR tetramer ( Figure 3 , right). The differences between the two interfaces allow the design of TTR variants that modulate the interactions between TTR dimers without disrupting the interface between TTR monomers.

[0318] In one aspect of the invention, each of the four TTR monomers that make up the tetrameric protein can be described as TTR subunit A, B, C, or D - wherein TTR subunits A and B form a first AB dimer and TTR subunits C and D form a second CD dimer ( Figure 3 ). TTR dimer AB and TTR dimer CD combine to form TTR tetramer ABCD. The TTR monomer of the present invention comprises at least one amino acid mutation (relative to SEQ ID NO: 1) in the interface between TTR dimer AB and TTR dimer CD so as to form ABCD tetramers in preference to any other tetramers (e.g., ABAB tetramers or CDCD tetramers).

[0319] Therefore, the present invention relates to a TTR protein complex wherein

[0320] The TTR protein complex contains TTR subunits A, B, C, and D;

[0321] TTR subunits A and B dimerize to form TTR dimer AB;

[0322] TTR subunits C and D dimerize to form TTR dimer CD;

[0323] TTR dimers AB and CD further dimerize to form TTR tetramer ABCD; and

[0324] Each of A, B, C, and D comprises the amino acid sequence of SEQ ID NO: 1, except that at least one amino acid in the interface between TTR dimer AB and TTR dimer CD is mutated to favor the formation of an ABCD tetramer over any other tetramer (e.g., an ABAB tetramer or a CDCD tetramer).

[0325] Each of A, B, C, and D of the TTR protein complex can comprise the amino acid sequence of SEQ ID NO: 1 having the following mutations: C10A, K15A, or C10A and K15A.

[0326] Thus, in one embodiment, the invention relates to a TTR protein complex wherein both A and B, both C and D, or all four of A, B, C, and D comprise a mutation at one or more amino acid positions selected from the list consisting of 6, 7, 8, 9, 10, 13, 15, 17, 19, 20, 21, 22, 23, 24, 26, 50, 51, 52, 53, 54, 56, 57, 60, 61, 62, 63, 78, 82, 83, 84, 85, 100, 101, 102, 103, 104, 106, 108, 110, 112, 113, 114, 115, 117, 119, 121, 123, 124, 125, 126, and 127 of SEQ ID NO: 1. In some embodiments, the mutation is complementary to C10A and K15A.

[0327] In another embodiment, the invention relates to a TTR protein complex wherein both A and B, both C and D, or all four of A, B, C, and D comprise a mutation at one or more amino acid positions selected from the following list, the list comprising: 15, 17, 20, 21, 22, 23, 24, 51, 52, 84, 106, 108, 112, 114, 115, 119, 121, and 123 of SEQ ID NO: 1. In some embodiments, the mutation is complementary to C10A and K15A.

[0328] In another embodiment, the invention relates to a TTR protein complex, wherein both A and B, both C and D, or all four of A, B, C and D comprise a mutation at one or more amino acid positions selected from the following list, the list comprising: 15, 17, 20, 21, 22, 23, 24, 51, 52, 84, 106, 108, 112, 114, 115, 119, 121 and 123 of SEQ ID NO: 1, wherein the amino acid is mutated to aspartic acid, glutamic acid, arginine, lysine or histidine. In some embodiments, the mutation is complementary to C10A and K15A.

[0329] In another embodiment, the invention relates to a TTR protein complex, wherein A and B comprise mutations at one or more amino acid positions selected from the following list, the list comprising: 15, 17, 20, 21, 22, 23, 24, 51, 52, 84, 106, 108, 112, 114, 115, 119, 121 and 123 of SEQ ID NO: 1, wherein the amino acid is mutated to aspartic acid or glutamic acid. In some embodiments, the mutation is complementary to C10A and K15A.

[0330] In yet another embodiment, the invention relates to a TTR protein complex, wherein C and D comprise mutations at one or more amino acid positions selected from the following list, the list comprising: 15, 17, 20, 21, 22, 23, 24, 51, 52, 84, 106, 108, 112, 114, 115, 119, 121 and 123 of SEQ ID NO: 1, wherein the amino acid is mutated to arginine, lysine or histidine. In some embodiments, the mutation is a complement of C10A and K15A.

[0331] In certain embodiments, A and B comprise mutations at one or more amino acid positions selected from the following list, the list comprising: 15, 17, 20, 21, 22, 23, 24, 51, 52, 84, 106, 108, 112, 114, 115, 119, 121, and 123 of SEQ ID NO: 1, wherein the amino acid is mutated to aspartic acid or glutamic acid; and C and D comprise mutations at one or more amino acid positions selected from the following list, the list comprising: 15, 17, 20, 21, 22, 23, 24, 51, 52, 84, 106, 108, 112, 114, 115, 119, 121, and 123 of SEQ ID NO: 1, wherein the amino acid is mutated to arginine, lysine, or histidine. In some embodiments, the mutations are in addition to C10A and K15A.

[0332] In some embodiments, A and B comprise at least one mutation in SEQ ID NO: 1, wherein the mutation is selected from the list consisting of K15D, L17D, V20D, R21D, G22D, S23D, P24D, S52D, I84D, T106D, A108D, S112D, Y114D, S115D, T119D, V121D, S123D, K15E, L17E, V20E, R21E, G22E, S23E, P24E, D51E, S52E, I84E, T106E, A108E, S112E, Y114E, S115E, T119E, V121E, and S123E. The present invention also relates to a TTR protein complex, wherein A and B comprise at least one mutation in SEQ ID NO: 1, wherein the mutation is selected from the list consisting of L17D, L17E, V20D, V20E, G22D, G22E, S112D, S112E, T119D, T119E, V121D and V121E. In some embodiments, the mutation is in addition to C10A and K15A.

[0333] In some embodiments, C and D comprise at least one mutation in SEQ ID NO: 1, wherein the mutation is selected from the list consisting of K15R, L17R, V20R, G22R, S23R, P24R, D51R, S52R, I84R, T106R, A108R, S112R, Y114R, S115R, T119R, V121R, S123R, L17K, V20K, R21K, G22K, S23R, P24K, D51K , S52K, I84K, T106K, A108K, S112K, Y114K, S115K, T119K, V121K, S123K, K15H, L17H, V20H, R21H, G22H, S23H, P24H, D51H, S52H, I84H, T106K, A108H, S112H, Y114H, S115H, T119H, V121H, and S123H. The invention also relates to a TTR protein complex, wherein C and D comprise at least one mutation in SEQ ID NO: 1, wherein the mutation is selected from the list consisting of L17R, L17K, L17H, V20R, V20K, V20H, G22R, G22K, G22H, S112R, S112K, S112H, T119R, T119K, T119H, V121R, V121K and V121H. In some embodiments, the mutation is complementary to C10A and K15A.

[0334] The TTR protein complexes of the invention may comprise TTR subunits wherein both A and B, both C and D, or all four of A, B, C, and D independently comprise one or two mutations discussed herein. In some embodiments, the TTR protein complexes of the invention may comprise TTR subunits wherein both A and B, both C and D, or all four of A, B, C, and D independently comprise one mutation discussed herein. In some embodiments, the mutations are complementary to C10A and K15A.

[0335] In specific embodiments, the TTR protein complexes of the invention comprise TTR subunits, wherein each of A, B, C, and D comprises the amino acid sequence of SEQ ID NO: 1 with the following mutations in Table 1 (or vice versa):

[0336] Table 1

[0337]

[0338]

[0339]

[0340] Any TTR variant and variant pairing in Table 1 is suitable for use in the present invention. Table 2 indicates the amount of TTR tetramer formation observed for certain variants and pairs (see Examples 2 and Figure 7 ).

[0341] Table 2: TTR tetramer formation

[0342]

[0343]

[0344] * Grade 1 = Strong tetramer according to SEC and SDS resistant

[0345] Grade 2 = strong tetramer >60% by SEC, slight or no resistance to SDS

[0346] Level 3 = significant tetramers by SEC >10% and <=60%

[0347] The TTR protein complexes of the invention may comprise TTR subunits wherein both A and B, both C and D, or all four of A, B, C, and D independently comprise two mutations discussed herein. In some embodiments, A and B comprise two mutations in SEQ ID NO: 1, wherein the mutations are selected from the list comprising: L17D / V20D, L17D / V20E, L17E / V20D, L17E / V20E, L17D / T119D, L17D / V121E, L17E / T119D, L17E / V121E, V20D / T119D, V20D / V121E, V20E / T119D, and V20E / V121E. In some embodiments, the mutations are complementary to C10A and K15A.

[0348] In some embodiments, C and D comprise two mutations in SEQ ID NO: 1, wherein the mutations are selected from the list consisting of L17K / V20K, L17K / V20R, L17R / V20K, L17R / V20R, L17K / V121K, L17K / V121R, L17R / V121K, L17R / V121R, V20K / V121K, V20K / V121R, V20R / V121K, and V20R / V121R. In some embodiments, the mutations are complementary to C10A and K15A.

[0349] In specific embodiments, the TTR protein complexes of the invention comprise TTR subunits, wherein each of A, B, C, and D comprises the amino acid sequence of SEQ ID NO: 1 with the following mutations in Table 3 (or vice versa):

[0350] Table 3

[0351]

[0352] In some embodiments, a TTR protein complex of the invention comprises TTR subunits wherein A and B or C and D comprise the amino acid sequence of SEQ ID NO: 1 with the following mutations: C10A / K15A / V20E / T119D, C10A / K15A / L17D / T119D, C10A / K15A / L17E / T119D, C10A / K15A / L17R / V20K, C10A / K15A / L17K / V20K, C10A / K15A / L17R / V121R, or C10A / K15A / L17R / V121K.

[0353] As described above, TTR variants can also be used in the present invention. Any of the TTR variants discussed herein can be used in combination with each other. TTR variants include proteins having an amino acid sequence that is at least 80%, 81%, 82%, 83%, 86%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a TTR protein having a mutation relative to SEQ ID NO: 1.

[0354] The cysteines present in human TTR (SEQ ID NO: 1) can be used as sites for conjugation to biologically active proteins, peptides, or small molecules. In some embodiments, the cysteines present in human TTR (SEQ ID NO: 1) can be used as sites for conjugation to antigen binding proteins (e.g., antibodies and Fabs). In addition, TTR variants capable of site-specific conjugation, such as TTR variants with engineered cysteines, can be used in the present invention. See, e.g., USP 8,633,153, which is incorporated herein by reference. For example, a TTR variant can comprise one or more of the following cysteine ​​mutations: A37C, D38C, A81C, or G83C.

[0355] Other variants useful in the present invention include, for example, TTR proteins with truncations at the C-terminus or N-terminus. Such TTR proteins include those in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids are removed from the C-terminus or N-terminus of the TTR protein. In some embodiments, the fusion protein of the present invention comprises a TTR protein in which 1, 2, 3, 4, 5, 6, 7, or 8 amino acids are removed from the C-terminus or N-terminus of the TTR protein. In other embodiments, the fusion protein of the present invention comprises a TTR protein in which 1, 2, 3, 4, 5, 6, 7, or 8 amino acids are removed from the N-terminus of the TTR protein.

[0356] Other TTR variants useful in the present invention include those that reduce or block TTR binding to thyroxine. Each TTR tetramer contains two thyroxine binding sites located in the central channel of the TTR tetramer. For example, such variants can avoid interfering with the patient's thyroxine biology and can avoid thyroxine metabolic pathways from contributing to TTR fusion. Other TTR variants useful in the present invention include those that reduce or eliminate TTR proteolytic activity.

[0357] In addition, TTR-His tag fusions can be used in the present invention. For example, TTR-His tag fusions can be used to purify TTR Fab constructs (where the Fab lacks Fc), or to purify TTR Ab constructs (where it is advantageous to avoid the low pH purification environment of a protein A affinity column). In some embodiments, the His tag is removed after purification. The His tag can also be present in the final therapeutic molecule (i.e., the tag can remain after purification). In some embodiments, the His tag is His, (His)2, (His)3, (His)4, (His)5, (His)6, (His)7, (His)8, (His)9, or (His) 10 Tag. In a particular embodiment, the His tag is a (His)6 or (His)7 tag. In a specific embodiment, the His tag is a (His)6 tag. In some embodiments, the His tag comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 glycine amino acids as a linker. In a specific embodiment, the His tag comprises two glycines (e.g., GGHHHHHH).

[0358] In some embodiments, two glycine amino acid linkers can be inserted between the TTR variant and the heavy or light chain.

[0359] In addition, the TTR variants of the invention may include variants that incorporate glycosylation sites, which may help modulate the PK or solubility of the TTR fusion. Additionally, the TTR variants or TTR fusion proteins of the invention may be modified to include moieties that confer beneficial PK properties, such as triazine-containing moieties (contained in a construct having a terminal group capable of reacting with a protein; see, e.g., PCT Publication No. WO / 2017 / 083604, which is incorporated herein by reference in its entirety).

[0360] In some embodiments, the TTR protein complex is attached to 1, 2, 3, 4, 5, 6, 7, or 8 antigen binding proteins or peptides. In other embodiments, the TTR protein complex is attached to 1, 2, 3, or 4 antigen binding proteins or peptides. The antigen binding protein or peptide can be attached to the TTR protein complex at the C-terminus of the TTR subunit or the N-terminus of the TTR subunit. In addition, the TTR protein complex can be directly attached to 1, 2, 3, 4, 5, 6, 7, or 8 antigen binding proteins or peptides; or it can be attached to 1, 2, 3, 4, 5, 6, 7, or 8 antigen binding proteins or peptides through a linker. In specific embodiments, the TTR protein complex is directly attached to 1, 2, 3, or 4 antigen binding proteins or peptides; or it is attached to 1, 2, 3, or 4 antigen binding proteins through a linker or peptide.

[0361] Heterodimer fusion protein (complex)

[0362] As described herein, the present invention relates in part to the use of TTR in the multimerization of antigen binding proteins such as antibodies. Because TTR is a human extracellular protein found in human serum, its content in the human body is relatively high, making it less likely to elicit an immune response when present in the multimeric constructs of the present invention (compared to, for example, non-human, intracellular and rare proteins). Therefore, its use in the multimerization technology of the present invention is advantageous.

[0363] For example, TTR can be used to dimerize antibodies that bind to different epitopes, where these epitopes are present, for example, on the same or different proteins. In such heterodimeric fusion proteins, TTR (SEQ ID NO: 1) or a variant thereof is present in tetrameric form, where the TTR subunits are linked to the C-termini of the antibody heavy chains to form TTR antibody heterodimers. For example, the C-termini of each antibody heavy chain (each antibody contains two such C-termini) can be linked to the N-termini of each TTR subunit (see Figure 1 and 2 ). Thus, each antibody is linked to two TTR subunits in the TTR tetramer, generating TTR antibody heterodimers.

[0364] Thus, the present invention relates to heterodimeric fusion proteins comprising two antigen binding proteins, wherein each antigen binding protein binds to a different epitope, wherein the epitope is present, for example, on the same or different proteins. In some embodiments, the heterodimeric fusion protein comprises an antigen binding protein linked to a protein complex. In some embodiments, the protein complex is a TTR protein complex, wherein the TTR protein complex is a TTR tetramer. In some embodiments, the antigen binding protein is an antibody.

[0365] In certain embodiments, the invention relates to heterodimeric fusion proteins comprising two antibodies linked to a TTR tetramer, wherein each antigen binding protein binds to a different epitope, wherein the epitopes are present, for example, on the same or different proteins. The antibody can be linked to the TTR tetramer without a linker (i.e., the antibody is directly linked to TTR).

[0366] In other embodiments, the antibody is connected to the TTR tetramer via a linker. For example, an amino acid linker can be used to connect the C-terminus of the antibody heavy chain to the N-terminus of the TTR subunit. In some embodiments, the length of the linker is 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 1-35 or 1-40 amino acids. In some embodiments, the length of the linker is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acids. In other embodiments, the length of the linker is 0, 1, 5, 10, 15, 20, 25, 30, 35 or 40 amino acids. In other embodiments, the length of the linker is up to 5, 10, 15, 20, 25, 30, 35 or 40 amino acids. In some embodiments, the length of the linker is up to 5, 10, 15 or 20 amino acids. In specific embodiments, the length of the linker is 0, 5, 10, 15 or 20 amino acids.

[0367] In some embodiments, the linker is GGGGS, GGGGSGGGGS (i.e. (GGGGS) 2), GGGGSGGGGSGGGGS (i.e. (GGGGS) 3), GGGGSGGGGSGGGGSGGGGS (i.e. (GGGGS) 4), GGGGSGGGGSGGGGSGGGGSGGGGS (i.e. (GGGGS) 5), or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (i.e. (GGGGS) 6). In other embodiments, it is GGGGS, GGGGSGGGGS (i.e. (GGGGS) 2), GGGGSGGGGSGGGGS (i.e. (GGGGS) 3), or GGGGSGGGGSGGGGSGGGGSGGGGS (i.e. (GGGGS) 4).

[0368] Other suitable amino acid linkers include, for example, disulfide bonds, (Gly) n (n=1-10), (EAAAK) n (n=1-5), A(EAAAK)4ALEA(EAAAK)4A, PAPAP, AEAAAKEAAAKA, (Ala-Pro) n (n=1-20), VSQTSKLTRAETVFPDV, PLGLWA, RVLAEA, EDVVCCSMSY, GGIEGRGS, TRHRQPRGWE, AGNRVRRSVG, RRRRRRRRR, GFLG and LE. Suitable non-amino acid linkers include polyethylene glycol (PEG).

[0369] In some embodiments, the antibody is linked to a truncated TTR subunit with or without a linker. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids can be removed from the N-terminus of one or more TTR subunits, and the antibody can be attached to the N-terminus of the truncated TTR subunit.

[0370] The present invention also relates to nucleic acid molecules encoding the heterodimeric fusion proteins described herein.Details on exemplary methods of producing heterodimeric fusion proteins can be found in the Examples.

[0371] Heterotrimeric and heterotetrameric fusion proteins (complexes)

[0372] The present invention also relates in part to the use of TTR in the trimerization or tetramerization of antigen binding proteins, such as antibodies.

[0373] In heterotetrameric fusion proteins, TTR (SEQ ID NO: 1) or its variants are again present in tetrameric form. However, in the case of TTR antibody heterotetramers, a single antibody heavy chain (i.e., only one of the two heavy chains present in a single antibody) is attached to each TTR subunit, thereby allowing four antibodies to be attached to a TTR tetramer (see Figure 2 e). One of the two heavy chains at the C-terminus of the antibody can be linked to the N-terminus of each TTR subunit (see Figure 2 e) Thus, each antibody is linked to one TTR subunit in the TTR tetramer, resulting in a TTR antibody heterotetramer.

[0374] In this heterotetrameric fusion protein, the formation of Fc heterodimers (as described above) is unfavorable by mutations in Fc. Such modifications include Fc mutations such as knob-hole, DuoBodies, Azymetric, charge pairs, HA-TF, SEEDbody, and modifications with differential protein A affinity. See, e.g., Spiess et al., Molecular Immunology, 67(2, Part A), 2015, pp. 95-106. Knob-hole mutations include T366W in the first heavy chain, and T366S, L368A, and / or Y407V in the second heavy chain. See, e.g., Ridgway et al., Protein Eng., 9(1996), pp. 617-621; and Atwell et al., J. Mol. Biol., 270(1997), pp. 26-35. DuoBody mutations include F405L in the first heavy chain and K409R in the second heavy chain. See, e.g., Labrijn et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States], 110 (2013), pp. 5145-5150. Azymetric mutations include T350V, L351Y, F405A and / or Y407V in the first heavy chain and T350V, T366L, K392L and / or T394W in the second heavy chain. See, e.g., Von Kreudenstein et al., mAbs, 5 (2013), pp. 646-654. HA-TF mutations include S364H and / or F405A in the first heavy chain, and Y349T and / or T394F in the second heavy chain. See, e.g., Moore et al., mAbs, 3 (2011), pp. 546-557. SEEDbody mutations include IgG / A chimeric mutations in the first heavy chain and IgG / A chimeric mutations in the second heavy chain. See, e.g., Davis et al., Protein Eng. Des. Sel. [Protein Engineering Design and Selection], 23 (2010), pp. 195-202. Differential protein A affinity mutations include H435R in one heavy chain and no mutation in the other heavy chain. See, e.g., U.S. Patent No. 8,586,713. Each of these documents is incorporated by reference in its entirety.

[0375] In certain embodiments, it is possible to drive heterotetramerization of antibodies by using Fc charge pairs that disfavor dimerization of antibody heavy chains and thus favor heavy chain dimerization between one antibody heavy chain attached to a TTR subunit and one antibody heavy chain not attached to TTR (see Figure 1 c) For example, a set of charged mutations can be incorporated into the C HIn the 3 domains, one heavy chain carries a negative charge and the corresponding heavy chain carries a positive charge, or a mixture of negative and positive charges on one heavy chain (paired with the corresponding positive and negative charges on the corresponding heavy chain). Exemplary negative charges include K392D and K409D, and exemplary positive charges include E356K and D399K. H 3. Similar charges on the interface repel each other while heterogeneous charges attract, so homodimerization is unfavorable, while heterodimerization is favorable. TTR is fused to heavy chains of only one charge type (positive or negative, but not both); thus, one TTR subunit / complete antibody is produced, consisting of four chains (two light chains, one unfused heavy chain, and one TTR-fused heavy chain). Additional charge pair mutations are discussed, for example, in USP 9,546,203. Charge pair mutations (including D221E, P228E, and / or L368E in the first heavy chain and D221R, P228R, and / or K409R in the second heavy chain) are also described, for example, in Strop et al., J. Mol. Biol [Journal of Molecular Biology]., 420 (2012), pp. 204-219. Each of these documents is incorporated by reference in its entirety.

[0376] In a heterotrimeric fusion protein, TTR (SEQ ID NO: 1) or a variant thereof is again present in tetrameric form. Examples of heterotrimeric fusion proteins include those comprising one antibody and two Fabs. In the case of a TTR antibody heterotrimer, the C-terminus of each antibody heavy chain can be linked to the N-terminus of each of the two TTR subunits, while the C-terminus of each of the two Fabs is linked to the N-terminus of each of the two TTR subunits to form a TTR Ab / Fab heterotrimer (see Figure 2 c and 2d). Thus, the antibody is linked to two TTR subunits in the TTR tetramer, and each Fab is linked to a TTR subunit, resulting in a TTR Ab / Fab heterotrimer comprising a TTR tetramer, one antibody, and two Fabs.

[0377] The present invention also relates in part to the use of TTR in tetramerization of Fab. In such heterotetrameric fusion proteins, TTR (SEQ ID NO: 1), or a variant thereof, is again present in tetrameric form, wherein each TTR subunit is linked to the C-terminus of each Fab to form a TTR Fab heterotetramer (see Figure 2 a) Thus, each Fab is linked to a single TTR subunit in the TTR tetramer, thereby generating a TTR Fab heterotetramer.

[0378] Thus, the present invention relates to heterotrimers and heterotetramer fusion proteins comprising three or four antigen binding proteins (e.g., Ab / Fab trimers, Fab tetramers, or Ab tetramers). In some embodiments, heterotrimers and heterotetramer fusion proteins comprise an antigen binding protein connected to a protein complex. In some embodiments, the protein complex is a TTR protein complex, wherein the TTR protein complex is a TTR tetramer. In some embodiments, the antigen binding protein is an antibody. In other embodiments, the antigen binding protein is a Fab. In some embodiments, the heterotetramer fusion protein comprises a mixture of an antibody and a Fab.

[0379] In specific embodiments, the present invention relates to heterotetrameric fusion proteins comprising four antibodies linked to a TTR tetramer. In other embodiments, the present invention relates to heterotetrameric fusion proteins comprising four Fabs linked to a TTR tetramer via a linker. In other embodiments, the present invention relates to heterotrimeric fusion proteins comprising one Ab and two Fabs linked to a TTR tetramer via a linker. In some embodiments, the antibody or Fab is linked to a TTR tetramer without a linker (i.e., the antibody or Fab is directly linked to TTR).

[0380] The linker can be an amino acid based linker comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids. In other embodiments, the linker is an amino acid based linker comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids. In other embodiments, the linker is an amino acid based linker comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In other embodiments, the linker is an amino acid based linker comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In specific embodiments, the linker is G, GG, GGG, GGGG, GGGGG, GGGGGG, GGGGGGG, GGGGGGGG, GGGGGGGGG, or GGGGGGGGGGG. In other specific embodiments, the linker is selected from the list comprising: GG, GGGG, GGGSGG, GGGSGGGG, and GGAGGGAGGG.

[0381] In some embodiments, the linker is GGGGS, GGGGSGGGGS (i.e. (GGGGS) 2), GGGGSGGGGSGGGGS (i.e. (GGGGS) 3), GGGGSGGGGSGGGGSGGGGS (i.e. (GGGGS) 4), GGGGSGGGGSGGGGSGGGGSGGGGS (i.e. (GGGGS) 5), or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (i.e. (GGGGS) 6). In other embodiments, it is GGGGS, GGGGSGGGGS (i.e. (GGGGS) 2), GGGGSGGGGSGGGGS (i.e. (GGGGS) 3), or GGGGSGGGGSGGGGSGGGGSGGGGS (i.e. (GGGGS) 4).

[0382] Other suitable linkers include G(G x B y ) r G zA linker wherein G = glycine; B = any amino acid; x = 1-15; y = 1-5; z = 1-15; and r = 1-20. In another embodiment, the linker is G (G x B y ) r G z A linker wherein B=Q, S, A, E, P, T, K, R, D, or N; x=4; y=1; z=4; and r=1.

[0383] Additional suitable amino acid linkers include, for example, disulfide bonds, (Gly) n (n=1-10), (EAAAK) n (n=1-5), A(EAAAK)4ALEA(EAAAK)4A, PAPAP, AEAAAKEAAAKA, (Ala-Pro) n (n=1-20), VSQTSKLTRAETVFPDV, PLGLWA, RVLAEA, EDVVCCSMSY, GGIEGRGS, TRHRQPRGWE, AGNRVRRSVG, RRRRRRRRR, GFLG and LE. Suitable non-amino acid linkers include polyethylene glycol (PEG) and triazine-containing moieties (contained in constructs having terminal groups capable of reacting with proteins; see, e.g., PCT Publication No. WO / 2017 / 083604, which is incorporated herein by reference in its entirety).

[0384] In some embodiments, the antibody or Fab is linked to a truncated TTR subunit with or without a linker. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids can be removed from the N-terminus of one or more TTR subunits, and the antibody or Fab can be attached to the truncated TTR subunit N-terminus.

[0385] The present invention also relates to nucleic acid molecules encoding the heterotrimeric and heterotetrameric fusion proteins described herein.Details on exemplary methods for producing heterotrimeric and heterotetrameric fusion proteins can be found in the Examples.

[0386] Antigen Binding Protein

[0387] Any antigen binding protein (eg, Fab, antibody, scFv, scFab) can be used in the TTR fusion protein of the present invention. In addition, proteins (eg, enzymes) can be used in combination with antigen binding proteins in the TTR fusion protein of the present invention.

[0388] Because the fusion proteins of the invention allow for binding to different epitopes (e.g., on the same or different proteins), fusion proteins are useful in situations where it is beneficial to bring different targets into close proximity. Examples of successful implementations of such technology include imiclizumab, which works by binding activated factor IX and factor X together, thereby allowing the clotting process to continue without the need to replace factor VIII to treat hemophilia.

[0389] The fusion proteins of the present invention may also be used in the field of oncology. For example, depending on the mechanism of action associated with the oncology target, cross-linking of target cells (e.g., cancer cells) with effector cells (e.g., T cells) may be desirable. Such approaches have proven successful in the context of (bispecific T cell engager) antibody constructs. Other examples include trispecifics that can bind two different tumor markers (e.g., via Abs and / or Fabs of the TTR fusion proteins of the invention) as well as CD3 (e.g., via anti-CD3 scFvs, Abs or Fabs).

[0390] The fusion proteins of the present invention can also address the complexities associated with regulatory evaluation / approval of combination therapies. Clinical trials of combination therapies may require more complex clinical trial strategies to assess safety and efficacy, especially when the individual components have not been evaluated before. The fusion proteins of the present invention address this complexity by combining multiple components into a single construct.

[0391] Method for preparing TTR heterodimer, heterotrimer and heterotetramer fusion proteins

[0392] Methods for making TTR heteromultimer (eg, heterodimer, heterotrimer, and heterotetramer) fusions of the invention are discussed in the Examples.

[0393] Typically, TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) fusions of the present invention can be produced using recombinant methods. Therefore, the present invention includes polynucleotides encoding TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) fusions. On the other hand, the present invention includes expression vectors comprising polynucleotides encoding TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) fusions. In certain embodiments, the expression vector comprises a control sequence (e.g., a promoter, an enhancer) operably linked to a polynucleotide encoding a TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) fusion to support expression in a suitable host cell. In certain embodiments, the expression vector further comprises a polynucleotide sequence that allows chromosome-independent replication in a host cell. Exemplary vectors include, but are not limited to, plasmids, cosmids, and YACS. In a specific embodiment, the vector is pTT5.

[0394] Typically, mammalian host cells are used when generating TTR heterodimer, heterotrimer or heterotetramer fusion constructs. Mammalian host cells are also suitable for generating Fab TTR fusion constructs, although non-mammalian cells, such as prokaryotic (bacterial) and non-mammalian (e.g., yeast) host cells, can also be used.

[0395] In another aspect, the present invention includes host cells comprising expression vectors of the present invention. Methods of transfecting host cells with expression vectors and culturing transfected host cells under conditions suitable for expressing TTR heteromultimers (e.g., heterodimers, heterotrimers, and heterotetramers) fusions are known in the art. The transfection procedure used may depend on the host to be transformed. Certain methods for introducing heterologous polynucleotides into mammalian cells are known in the art and include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of one or more polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Certain mammalian cell lines that can be used as hosts for expression are well known in the art and include, but are not limited to, many immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, Chinese hamster ovary (CHO, e.g., CHO-K1) cells, E5 cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human embryonic kidney cells 293 (HEK 293), and a variety of other cell lines. In certain embodiments, cell lines can be selected by determining which cell lines have high expression levels and produce TTR heterodimers, heterotrimers, and heterotetramer fusions.

[0396] Therefore, the present invention also relates to methods for preparing the TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) fusion proteins described herein. For example, the TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) fusion proteins can be prepared by:

[0397] a) culturing a recombinant host cell comprising a polynucleotide encoding a TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) fusion; and

[0398] b) isolating the TTR heteromultimer (eg, heterodimer, heterotrimer and heterotetramer) fusion protein from the culture.

[0399] Pharmaceutical composition

[0400] In some embodiments, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of one or more TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) fusion proteins of the present invention and a pharmaceutically effective diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen, and lyophilized compositions.

[0401] Preferably, acceptable formulation materials are nontoxic to recipients at the dosages and concentrations employed. In specific embodiments, pharmaceutical compositions comprising a therapeutically effective amount of a TTR heteromultimer (eg, heterodimer, heterotrimer, and heterotetramer) fusion protein are provided.

[0402] In certain embodiments, pharmaceutical compositions may contain formulation materials to adjust, maintain or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, proline, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); bulking agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptide; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerol, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan, polysorbates (such as polysorbate 20, polysorbate), tritons, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. See, REMINGTON'S PHARMACEUTICAL SCIENCES, 18th ed. (AR Genrmo, ed.), 1990, Mack Publishing Company.

[0403] In certain embodiments, the optimal pharmaceutical composition will be determined by those skilled in the art according to, for example, the expected route of administration, delivery form, and desired dosage. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, the same. In certain embodiments, such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the antigen-binding proteins of the present invention. In certain embodiments, the main vehicle or carrier in the pharmaceutical composition may be aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, physiological saline solution, or artificial cerebrospinal fluid, which may be supplemented with other substances common in the composition for parenteral administration. Neutral buffered saline or saline mixed with serum albumin is another exemplary vehicle. In a specific embodiment, the pharmaceutical composition comprises a Tris buffer of pH about 7.0-8.5 or an acetate buffer of pH about 4.0-5.5, and may further include sorbitol or a suitable substitute thereof. In certain embodiments of the invention, TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) compositions can be prepared for storage by mixing selected constituent components having a desired purity with optional formulating agents (REMINGTON'S PHARMACEUTICAL SCIENCES, supra) in the form of a lyophilized cake or aqueous solution. In addition, in certain embodiments, TTR heteromultimers (e.g., heterodimer, heterotrimer, and heterotetramer) can be formulated as a lyophilizate using an appropriate excipient (e.g., sucrose).

[0404] The pharmaceutical composition of the present invention can be selected for parenteral delivery. Alternatively, the composition can be selected for inhalation or delivery by the digestive tract, such as oral administration. The preparation of such pharmaceutically acceptable compositions is within the technical scope of those skilled in the art. The formulation components are preferably present in an acceptable concentration at the administration site. In certain embodiments, the composition is maintained at a physiological pH or a slightly lower pH using a buffer, typically a pH range of from about 5 to about 8.

[0405] When parenteral administration is contemplated, the therapeutic compositions for use in the present invention can be provided in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired TTR heteromultimers (e.g., heterodimers, heterotrimers, and heterotetramers) in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, wherein the TTR heteromultimers (e.g., heterodimers, heterotrimers, and heterotetramers) are formulated into a sterile, isotonic solution that is appropriately preserved. In certain embodiments, the preparation may involve formulating the desired molecule with an agent that can provide a controlled or sustained release of a product that can be delivered via reservoir injection, such as an injectable microsphere, a bioerodible particle, a polymer compound (e.g., polylactic acid or polyglycolic acid), a bead, or a liposome. In certain embodiments, hyaluronic acid may also be used, which has the effect of promoting circulation duration. In certain embodiments, an implantable drug delivery device may be used to introduce the desired antigen binding protein.

[0406] The pharmaceutical compositions of the invention can be formulated for inhalation. In these embodiments, the TTR heteromultimers (e.g., heterodimers, heterotrimers, and heterotetramers) are advantageously formulated as dry, inhalable powders. In specific embodiments, TTR heteromultimers (e.g., heterodimers, heterotrimers, and heterotetramers) inhalation solutions can also be formulated with a propellant for aerosol delivery. In certain embodiments, the solution can be atomized. Thus, International Patent Application No. PCT / US94 / 001875 further describes pulmonary administration and formulation methods, which is incorporated by reference and describes pulmonary delivery of chemically modified proteins.

[0407] It is also contemplated that the formulations may be administered orally. TTR heteromultimers (e.g., heterodimers, heterotrimers, and heterotetramers) administered in this manner may be formulated in the presence or absence of carriers commonly used to compound solid dosage forms (e.g., tablets and capsules). In certain embodiments, the capsules may be designed to release the active portion of the formulation in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents may be included to promote absorption of TTR heteromultimers (e.g., heterodimers, heterotrimers, and heterotetramers). Diluents, flavoring agents, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and binders may also be employed.

[0408] Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations involving TTR heteromultimers (e.g., heterodimers, heterotrimers, and heterotetramers) in the form of sustained or controlled delivery formulations. Techniques for formulating various other sustained or controlled delivery modes (e.g., liposomal carriers, bioerodible microparticles or porous beads, and reservoir injections) are also known to those skilled in the art. See, e.g., International Patent Application No. PCT / US93 / 00829, which is incorporated herein by reference and describes controlled release of porous polymer microparticles for delivery of pharmaceutical compositions. Sustained release formulations may include a semipermeable polymer matrix in the form of a shaped article (e.g., a film or microcapsule). Sustained release matrices may include polyesters, hydrogels, polylactides (as disclosed in U.S. Pat. No. 3,773,919 and European Patent Application Publication No. EP 058481, each of which is incorporated herein by reference), copolymers of L-glutamic acid and γ-L-glutamic acid ethyl ester (Sidman et al., 1983, Biopolymers [Biopolymers] 2: 547-556), poly (2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. [Journal of Biomedical Materials Research] 15: 167-277 and Langer, 1982, Chem. Tech. [Chemical Technology] 12: 98-105), ethylene vinyl acetate (Langer et al., 1981, supra) or poly-D (-) -3-hydroxybutyric acid (European Patent Application Publication No. EP 133,988). Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art. See, for example, Eppstein et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-3692; European Patent Application Publication Nos. EP 036,676; EP 088,046 and EP 143,949, which are incorporated by reference.

[0409] Pharmaceutical compositions for in vivo administration are typically provided as sterile preparations. Sterilization can be accomplished by filtration through a sterile filter membrane. When the composition is freeze-dried, sterilization using the method can be performed before or after freeze-drying and reconstitution. Compositions for parenteral administration can be stored in a freeze-dried form or in a solution. Parenteral compositions are usually placed in a container with a sterile access port (e.g., an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle).

[0410] Aspects of the invention include self-buffering TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) formulations that can be used as pharmaceutical compositions as described in International Patent Application WO 06138181A2 (PCT / US2006 / 022599), which is incorporated herein by reference in its entirety.

[0411] As described above, certain embodiments provide TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) compositions, particularly pharmaceutical TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) compositions, which, in addition to the heteromultimer, also contain one or more excipients, such as those illustratively described in this section and elsewhere herein. In this regard, excipients can be used in the present invention for a variety of purposes, such as adjusting the physical, chemical, or biological properties of the formulation, such as adjusting the viscosity and / or the methods of the present invention to improve effectiveness and / or stabilize such formulations and methods to resist degradation and spoilage due to stresses that occur during, for example, manufacturing, transportation, storage, preparation before use, administration, and thereafter.

[0412] There are numerous discussions of protein stabilization and formulation materials and methods useful in this regard, e.g., Arakawa et al., "Solvent interactions in pharmaceutical formulations," Pharm Res. 8(3):285-91 (1991); Kendrick et al., "Physical stabilization of proteins in aqueous solution," in RATIONAL DESIGN OF STABLE PROTEIN FORMULATIONS: THEORY AND PRACTICE, Carpenter and Manning, eds. Pharmaceutical Biotechnology. 13:61-84 (2002), and Randolph et al., "Surfactant-protein interactions," Pharm Biotechnol. 13:159-75 (2002), each of which is incorporated herein by reference in its entirety, particularly with respect to the portions relating to excipients for self-buffering protein formulations according to the present invention and methods for their preparation, especially protein pharmaceutical products and methods for veterinary and / or human medical use.

[0413] According to certain embodiments of the invention, salts may be used, for example, to adjust the ionic strength and / or isotonicity of the formulation and / or to improve the solubility and / or physical stability of proteins or other ingredients of the compositions according to the invention.

[0414] As is well known, ions can stabilize the native state of proteins by binding to charged residues on the protein surface and by shielding the charged groups and polar groups in the protein and reducing the intensity of their electrostatic interactions, attraction and repulsion interactions. Ions can also stabilize the denatured state of proteins by binding to the denatured peptide bonds (--CONH) of proteins in particular. In addition, the ionic interactions with the charged groups and polar groups in the protein can also reduce the intermolecular electrostatic interactions, and thereby prevent or reduce protein aggregation and insolubility.

[0415] Ionic species have significantly different effects on proteins. A variety of ion classification ratings for the ions that can be used to prepare pharmaceutical compositions according to the present invention and their effects on proteins have been developed. An example is the Hofmeister series, which rates ions and polar non-ionic solutes by their effects on the conformational stability of proteins in solution. Stable solutes are called "lyophilic." Unstable solutes are called "chaotropic." High concentrations of lyophiles (e.g., >1 molar ammonium sulfate) are often used to precipitate proteins from solution ("salting out"). Chaotropic agents are often used to denature and / or dissolve proteins ("salting out"). The relative effectiveness of ions for "salting out" and "salting out" defines their position in the Hofmeister series.

[0416] According to various embodiments of the invention, free amino acids can be used as bulking agents, stabilizers, and antioxidants in TTR heteromultimer (e.g., heterodimers, heterotrimers, and heterotetramers) formulations, as well as other standard uses. Lysine, proline, serine, and alanine can be used to stabilize the protein in the formulation. Glycine can be used for lyophilization to ensure the correct cake structure and properties. Arginine can be used to inhibit protein aggregation in both liquid formulations and lyophilized formulations. Methionine can be used as an antioxidant.

[0417] Polyols include sugars such as mannitol, sucrose and sorbitol and polyols such as, for example, glycerol and propylene glycol, and for the purposes of this article, polyethylene glycol (PEG) and related substances. Polyols are lyophilic. They are useful stabilizers in both liquid formulations and lyophilized formulations to protect proteins from physical and chemical degradation processes. Polyols can also be used to adjust the tonicity of the formulation.

[0418] A useful polyol in the selected embodiment of the present invention is mannitol, which is generally used to ensure the structural stability of the cake in the freeze-dried formulation. It ensures the structural stability of the cake. It is generally used together with a lyophilization protective agent (e.g., sucrose). Sorbitol and sucrose are preferred agents for adjusting tension and as stabilizers to prevent freeze-thaw stress during transportation or to prevent the preparation of agglomerates during manufacturing. Reducing sugars (containing free aldehyde or ketone groups), such as glucose and lactose, can glycosylate surface lysine and arginine residues. Therefore, they are generally not preferred polyols for use according to the present invention. In addition, in this regard, sugars that form such reactive substances are also not preferred polyols of the present invention, such as sucrose, which is hydrolyzed to fructose and glucose under acidic conditions and thus produces glycosylation. PEG can be used to stabilize proteins and act as a cryoprotectant, and can be used in this regard for the present invention.

[0419] Embodiments of TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) formulations further comprise a surfactant. Protein molecules can readily adsorb on surfaces and denature and subsequently aggregate at air-liquid, solid-liquid, and liquid-liquid interfaces. These effects are generally inversely proportional to protein concentration. These deleterious interactions are generally inversely proportional to protein concentration and are typically exacerbated by physical oscillations, such as those generated during product transportation and handling.

[0420] Surfactants are routinely used to prevent, minimize or reduce surface adsorption. In this regard, surfactants useful in the present invention include polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan polyethoxylates, and poloxamer 188.

[0421] Surfactants are also commonly used to control protein conformational stability. The use of surfactants in this regard is protein specific, in that any given surfactant will typically stabilize some proteins and destabilize others.

[0422] Polysorbates are susceptible to oxidative degradation and are usually supplied with sufficient amounts of peroxide to cause oxidation of protein residue side chains, especially methionine. Therefore, polysorbates should be used with caution and, when used, should be used at their lowest effective concentration. In this regard, polysorbates exemplify the general rule that excipients should be used at their lowest effective concentration.

[0423] Embodiments of TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) formulations further comprise one or more antioxidants. Harmful oxidation of proteins in pharmaceutical formulations can be prevented to some extent by maintaining appropriate levels of ambient oxygen and temperature and avoiding exposure to light. Antioxidant excipients can also be used to prevent oxidative degradation of proteins. In this regard, useful antioxidants are reducing agents, oxygen / free radical scavengers, and chelating agents. Antioxidants used in therapeutic protein formulations according to the present invention are preferably water-soluble and maintain their activity throughout the shelf life of the product. In this regard, EDTA is a preferred antioxidant according to the present invention.

[0424] Antioxidants can damage proteins. For example, reducing agents, such as glutathione in particular, can damage intramolecular disulfide bonds. Therefore, the antioxidants selected for use in the present invention are particularly useful for eliminating or sufficiently reducing the possibility that they themselves will damage proteins in the formulation.

[0425] The formulations according to the invention may contain metal ions which are protein cofactors and are necessary for the formation of protein coordination complexes, such as zinc which is necessary for the formation of certain insulin suspensions. Metal ions may also inhibit some processes that degrade proteins. However, metal ions also catalyze physical and chemical processes that degrade proteins.

[0426] Magnesium ions (10-120 mM) can be used to inhibit the isomerization of aspartate to isoaspartate. +2 ions (up to 100 mM) can increase the stability of human DNAse. +2 , Mn +2 and Zn +2 can destabilize rhDNase. Similarly, Ca +2 and Sr +2 It can stabilize factor VIII, which can be affected by Mg +2 , Mn +2 and Zn +2 , Cu +2 and Fe +2 destabilization, and its aggregation can be achieved by Al +3 Ions increase.

[0427] Embodiments of TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) formulations further include one or more preservatives. When developing a multidose parenteral formulation involving extraction from the same container more than once, a preservative is necessary. Its primary function is to inhibit microbial growth and ensure product sterility throughout the shelf life or service life of the drug product. Commonly used preservatives include benzyl alcohol, phenol, and metacresol. Although preservatives have a long history in small molecule parenteral use, the development of protein formulations containing preservatives may be challenging. Preservatives almost always have an unstable effect (aggregation) on proteins, and this has become a major factor limiting their use in multidose protein formulations. To date, most protein drugs are formulated only for single use. However, when multidose formulations are possible, they have the added advantage of making patient convenience and increased marketability. A good example is human growth hormone (hGH), where the development of preservative formulations has contributed to the commercialization of more convenient, multiple-use injection pen displays. At least four such pen devices containing preserved formulations of hGH are currently available on the market. Norditropin (liquid, Novo Nordisk), Nutropin AQ (liquid, Genentech), and Genotropin (lyophilized - dual chamber cartridge, Pharmacia & Upjohn) contain phenol, while Somatrope (Eli Lilly) is formulated with m-cresol.

[0428] Several aspects need to be considered during the formulation and development of a preservative dosage form. Effective preservative concentrations in the drug product must be optimized. This requires testing a given preservative in the dosage form at a concentration range that imparts antimicrobial effectiveness without compromising protein stability.

[0429] As might be expected, the development of liquid formulations containing preservatives is more challenging than lyophilized formulations. Freeze-dried products can be lyophilized without preservatives and reconstituted with a diluent containing a preservative at the time of use. This shortens the time that the preservative is in contact with the protein, thereby significantly minimizing the associated stability risks. In the case of liquid formulations, preservative effectiveness and stability should be maintained throughout the product shelf life (e.g., about 18 to 24 months). An important point to note is that preservative effectiveness should be demonstrated in the final formulation containing the active drug and all excipient components.

[0430] TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) formulations will typically be designed for a specific route and method of administration, a specific dosage and frequency of administration, a specific treatment for a specific disease, having a range of bioavailability and persistence, etc. Thus, formulations can be designed in accordance with the present invention for delivery by any suitable route, including, but not limited to, oral, intraaural, ophthalmic, rectal, and vaginal, as well as by parenteral routes, including intravenous and intraarterial injections, intramuscular injections, and subcutaneous injections.

[0431] Once the pharmaceutical composition is formulated, it can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, crystal or as a dehydrated or lyophilized powder. Such preparations can be stored in a ready-to-use form or in a form (e.g., lyophilized form) reconstructed before administration. The present invention also provides a kit for producing a single-dose administration unit. The kit of the present invention can each contain a first container with dry protein and a second container with an aqueous preparation. In certain embodiments of the present invention, a kit containing a single-chamber and multi-chamber prefilled syringe (e.g., a liquid syringe and a lyophilized syringe) is provided.

[0432] The therapeutically effective amount of the pharmaceutical composition containing TTR heteromultimers (e.g., heterodimers, heterotrimers, and heterotetramers) to be employed will depend, for example, on the therapeutic situation and goals. Those skilled in the art will appreciate that the appropriate dosage level for treatment will depend in part on the delivered molecule, the indication for which the TTR heteromultimers (e.g., heterodimers, heterotrimers, and heterotetramers) are used, the route of administration, and the size (weight, body surface area, or organ size) and / or condition (age and general health) of the patient. In certain embodiments, the clinician may titrate the dose and modify the route of administration to obtain the best therapeutic effect. Depending on the above factors, a typical dosage range may be from about 0.1 μg / kg to up to about 30 mg / kg or more. In specific embodiments, the dosage range may be from 1.0 μg / kg to about 20 mg / kg, optionally from 10 μg / kg to up to about 10 mg / kg or from 100 μg / kg to up to about 5 mg / kg.

[0433] A therapeutically effective amount of a TTR heteromultimer (eg, heterodimer, heterotrimer, and heterotetramer) preferably results in a reduction in the severity of disease symptoms, an increase in the frequency or duration of disease symptom-free periods, or prevention of impairment or disability due to disease affliction.

[0434] The pharmaceutical composition can be administered using a medical device. Examples of medical devices for administering pharmaceutical compositions are described in U.S. Pat. Nos. 4,475,196; 4,439,196; 4,447,224; 4,447,233; 4,486,194; 4,487,603; 4,596,556; 4,790,824; 4,941,880; 5,064,413; 5,312,335; 5,312,335; 5,383,851; and 5,399,163, all of which are incorporated herein by reference.

[0435] Therapeutic uses of TTR heterodimer, heterotrimer and heterotetramer fusion proteins

[0436] As shown in the Examples, it has been found that the multi-specific TTR fusion proteins of the invention are capable of binding to two or more epitopes on one or more proteins. Such multi-specific TTR fusions are particularly useful because they can engage multiple biological pathways, thereby more effectively treating disease states (e.g., cancer) than traditional therapeutic modalities.

[0437] The multispecific TTR fusion proteins of the present invention are superior to many known bispecific / multispecific approaches. For example, the present invention provides bivalent bispecific presentation of antigen binding domains, which reduces or eliminates avidity loss compared to, for example, hetero-IgG constructs. Other benefits compared to hetero-IgG constructs include the ability to generate the multispecific TTR fusion proteins of the present invention without the need for Fc charge pair mutations (CPMs) (which are required to drive heavy chain heterodimerization in hetero-IgG constructs), as well as reducing or eliminating unwanted byproducts, such as half antibodies and light chain mispairing (present in hetero-IgG and IgG-Fab constructs). In fact, the TTR fusion proteins of the present invention reduce much (and in some cases, all) Ab or Fab engineering required in other constructs.

[0438] Compared to IgG-Fab and IgG-scFv constructs, the antigen binding domains of the TTR fusion proteins of the present invention are optimally oriented such that the N-terminal antigen binding region is exposed and sterically induced affinity loss is reduced or eliminated.

[0439] Another benefit of the TTR fusion proteins of the present invention derives from the use of the native IgG format, which helps to reduce the loss of affinity and increased aggregation tendency observed when converting mAbs to scFv constructs. Gil and Schrum, Advances in Bioscience and Biotechnology, 4:73-84 (2013).

[0440] Furthermore, because the TTR fusion proteins of the invention allow for the efficient incorporation of multiple antigen binding domains, rapid scanning of bispecific (or multispecific) combinations is possible.

[0441] Compared to single antibodies and / or Fabs, TTR heteromultimers (e.g., heterodimers, heterotrimers, and heterotetramers) fusion proteins also exhibit improved antigen clustering. When an antibody (e.g., an IgG antibody) binds to an antigen on a target cell (e.g., a tumor cell), the clustered Fc domain formed will engage with the FcγR on immune effector cells (e.g., NK cells and macrophages). This clustering helps signal transduction through FcγR, thereby initiating cell-mediated effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Therefore, TTR heteromultimers (e.g., heterodimers, heterotrimers, and heterotetramers) fusion proteins are particularly useful in targeting ligands, where high antibody or Fab affinity / avidity leads to enhanced biological effects. The TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) constructs of the present invention enhance cell-mediated effector functions resulting in enhanced ability to kill cells, which can be used, for example, for the treatment of cancer.

[0442] Therefore, the present invention also relates to methods of treating cancer using the heterodimeric fusion proteins and heterotetrameric fusion proteins described herein.

[0443] In other embodiments, the present invention relates to the use of the heterodimeric fusion proteins and heterotetrameric fusion proteins described herein in the treatment of cancer.

[0444] In other embodiments, the present invention relates to heterodimeric fusion proteins and heterotetrameric fusion proteins described herein for use in treating cancer.

[0445] Examples

[0446] The following examples are provided to illustrate specific embodiments or features of the present invention and are not intended to limit the scope thereof.

[0447] Example 1: General Technology

[0448] Example 1 describes the general techniques used to make and characterize the TTR minus and plus constructs discussed in the remaining examples.

[0449] The following techniques were used to generate TTR minus and plus constructs containing a TTR variant ("C10A / K15A / XX") with one TTR dimer / dimer interface mutation per TTR subunit:

[0450] Cloning of TTR minus and plus variants in E. coli

[0451] Amgen's macromolecular registration construct C37979 (pAMG21: huTTR (opt-C10A, K15A)) was used as a template for all TTR negative and positive variants (including C10A / K15A / XX). TTR negative and positive variants were generated using standard molecular biology techniques, including polymerase chain reaction (PCR), site-directed PCR mutagenesis, restriction endonuclease digestion, and enzymatic ligation into bacterial expression plasmids. TTR negative and positive variants containing MKH6GG at the TTR N terminus were also generated.

[0452] The techniques are generally performed according to the methods referenced in Molecular Cloning: A Laboratory Manuel, 3rd ed., Sambrook et al., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

[0453] Expression of TTR minus and plus variants in E. coli

[0454] BL21 cells containing pAMG21 vectors encoding TTR minus and plus variants were cultured overnight in a 250 ml baffled shake flask in a 50 ml volume of Terrific Broth (Teknova T7060) containing 20 μg / ml kanamycin at 30-37° C. The next day, 35 ml of the overnight culture was added to 1 L of Terrific Broth containing 20 μg / ml kanamycin and 50 μl of Sigma Y-30 defoamer and incubated at 33° C. until the OD at 600 nm reached 0.4. 1 ml of Sigma K-3255 N-(β-ketohexanoyl)-DL-homoserine lactone autoinducer (stock solution dissolved in ethanol) was added to the culture and allowed to express for four hours at 30 to 33° C.

[0455] Purification of TTR minus and plus variants from Escherichia coli

[0456] Using Omni TH (Omni International, Kennesaw, Georgia, the United States) handheld homogenizer, the frozen E. coli cell paste was homogenized in a 50mM sodium phosphate, 300mM NaCl, pH 8.0 solution with a weight to volume ratio of 1:10. The resulting suspension was then processed twice at 13,800PSI by an M-110S microfluidizer (Microfluidics Corporation, Irvine, California, the United States). The lysate was then centrifuged at 22,000RCF for 1 hour at 4°C. The soluble fraction was filtered through a 0.45μm cellulose acetate filter (Corning Life Sciences, Tewksbury, Massachusetts, the United States) and retained as the starting material for FPLC purification; the insoluble fraction was treated as waste.

[0457] Connect to (GE Healthcare Bio-Sciences, Marlborough, MA, USA) A 5 mL Ni-NTA SuperFlow FPLC column (Qiagen, Hilden, Germany) was equilibrated with 5 column volumes (CV) of 50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 8.0 before loading. The filtered soluble lysate was injected onto the column, washed with 15 CV of 50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 8.0, and eluted stepwise with 10 CV of 50 mM sodium phosphate, 300 mM NaCl, 250 mM imidazole, pH 8.0.

[0458] The purified pool was concentrated using VivaSpin 10 kDA MWCO (Sartorius AG, Gottingen, Germany) centrifugal filters by centrifugation at 3,000 RCF until the desired volume was reached.

[0459] The concentrated sample was dialyzed against 10 mM tris-HCl, pH 8.0, 150 mM NaCl using Slide-a-lyzer 10 kDa MWCO (Thermo Fisher Scientific, Waltham, MA, USA) dialysis cassettes until the starting buffer was calculated to be less than 1%.

[0460] PC analysis of TTR minus and plus variants (E. coli)

[0461] Protein quantification was performed by measuring UV absorbance at 280 nm using a Nanodrop 2000c (Thermo Fisher Scientific).

[0462] Non-reducing SDS-PAGE analysis was performed with and without heating the samples. In both cases, samples were treated with SDS-PAGE sample buffer and run on 4-20% Tris-Gly SDS-PAGE (Thermo Fisher Scientific) according to the manufacturer's protocol. In the heating experiments, samples and sample buffer were heated at 85°C for 5 minutes before loading on the gel; after adding sample buffer, non-heated samples were loaded directly on the gel. Gels were stained using SimplyBlue SafeStain (Thermo Fisher Scientific) according to the manufacturer's microwave protocol.

[0463] HPLC SEC analysis was performed on a SEC-3000, 7.8 x 300 mm column (Phenomenex, Torrance, CA, USA) connected to an Agilent 1290 Infinity HPLC system (Agilent Technologies, Santa Clara, CA, USA) running an isocratic 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 mobile phase at a flow rate of 1 mL / min and observing UV absorbance at 280 nm.

[0464] TTR negative and positive variants dimerize

[0465] Purified TTR samples were normalized to the lowest common molar concentration of the experimental cohort by dilution with 10 mM tris-HCl, pH 8.0, 150 mM NaCl. Samples were combined in equal volumes and incubated overnight at 4°C.

[0466] The part of the mixed sample is processed by caspase cleavage as follows. By diluting with 10mM tris-HCl, pH 8.0, 150mM NaCl, the purified protein sample concentration is adjusted to 2.5mg / mL. Prepare 5x digestion buffer consisting of 250mM NaCl, 15mM 2-mercaptoethanol, pH 8.0, and heat to 25 ° C in a water bath, and prepare 1x digestion buffer by diluting 5x buffer with water. The storage aliquot of caspase-3 (Amgen Inc., Thousand Oaks, California, the United States) is diluted to 0.1mg / mL using 1x digestion buffer. 4 parts of protein, 4 parts of diluted caspase-3, 8 parts of 5x digestion buffer and 20 parts of water are mixed, and incubated in 25 ° C water baths for 2 hours. Remove digestion solution and add 20 parts of SDS-PAGE sample buffer (same as the reagent previously specified for SDS-PAGE analysis). The cleavage reactions were run on SDS-PAGE using the same protocol specified previously.

[0467] The resulting mixture of molecules, caspase and non-caspase processes, were analyzed by non-reducing, non-heated SDS-PAGE and HPLC SEC.

[0468] Cloning of (1) [Ab "A"] = [Negative TTR] 2: [Positive TTR] 2 = [Ab "B"] (2X Ab-TTR); (2) [[Ab "A"]-[Negative TTR]] 2: [[Positive TTR]-[Ab "B"]] 2 (4X Ab-TTR); and (3) [[Fab "A"]-[Negative TTR]] 2: [[Positive TTR]-[Fab "B"]] 2 (4X Fab-TTR) molecules (without linkers)

[0469] TTR was fused to several engineered variants of hybridoma-derived anti-CB1, anti-GITR, and anti-TR2 antibody heavy chains (HC) using standard molecular biology techniques, including polymerase chain reaction (PCR), site-directed PCR mutagenesis, restriction endonuclease digestion, and enzymatic ligation into mammalian expression plasmids. His-tagged Fab-TTR molecules were also generated. These cloned TTR fusion variant heavy chain and Fab DNAs were combined with their respective cloned anti-CB1, anti-GITR, and anti-TR2 antibody light chain (LC) DNAs and used to transfect mammalian cells to express 2X Ab-TTR, 4X Ab-TTR, and 4X Fab-TTR. The techniques were generally performed according to the methods referenced in Molecular Cloning: A Laboratory Manuel, 3rd ed., Sambrook et al., 2001, Cold Spring Harbor Laboratory Press, cold Spring Harbor, NY.

[0470] TTR antibody and Fab fusion sequences were Seamless cloning (GSC) or Golden Gate Assembly (GGA) generation. Combined DNA fragments are generated by splicing overlap extension PCR (SOE-PCR) or ordered synthetically from external suppliers. The SOE-PCR products used in GSC cloning are generated using flanking primers paired with mutagenic palindromic primers, which produce two PCR products that share a 15 bp overlapping region around the codon for the desired amino acid change site. The SOE-PCR products used in GGA are designed to include directional unique 4 base pair overhangs generated by BsmBI digestion.

[0471] Briefly, GGA relies on type II restriction enzymes and T4 DNA ligase to cut and seamlessly join multiple DNA fragments together. (Engler et al., PLOS One, Vol. 3(11): e3647, 2008). In this example, the multiple DNA fragments consisted of: (i) a synthetic nucleic acid sequence encoding a Kozak consensus sequence, a signal peptide sequence, a complete antibody gene, a linker, and a TTR sequence (GeneByte, Gen9, Cambridge, Massachusetts), and (ii) an expression vector backbone. The GGA reaction consisted of: 50 ng GeneByte, 20 ng expression vector, 1 μl 10x fast digestion reaction buffer + 0.5 mM ATP (Thermo Fisher, Waltham, Massachusetts), 0.5 μl fast digestion Esp3I (Thermo Fisher, Waltham, Massachusetts), 1 μl T4 DNA ligase (5 U / μl, Thermo Fisher, Waltham, Massachusetts), and water (to 10 μl). These reactions were performed for 15 cycles, each cycle consisting of a 2-min digestion step at 37° C. and a 3-min ligation step at 16° C. After 15 cycles, a final 5-min digestion step at 37° C. and an enzyme inactivation step at 80° C. for 5 min were performed.

[0472] Expression of [Ab "A"] = [negative TTR] 2: [positive TTR] 2 = [Ab "B"] molecule (without linker)

[0473] HEK 293-6E cells were cultured in shake flasks in an incubator (5% CO2, 80%-90% humidity and 120 rpm agitation) on a 25 mm shaking diameter shaker at 36°C in FreeStyle F17 medium (Thermo Fisher Scientific) supplemented with 0.1% (w / v) poloxamer 188 (Sigma-Aldrich), 6 mM L-glutamine (Thermo Fisher Scientific), 25 μg / ml G418 (Thermo Fisher Scientific). 293-6E cells were cultured at 0.4×10 2 days before transfection. 6 On the day of transfection, the cells were in the exponential growth phase (approximately 1.5 x 10 6Cells / ml, >95% viability). Transient transfection was performed at 20% gene dose by adding a mixture of 0.5mg / L DNA (0.1mg / L target gene construct DNA + 0.4mg / L carrier DNA) and 2mg / L PEI Max (Polyethyleneimine Max, Polysciences, Cat#24765-2) to the cell culture. Proprietary feed {yeastate (0.5% w / v) and glucose (3g / L)} was added 4 hours after transfection. Six days after transfection, the product was harvested by centrifuging the cells at 4000rpm (3485x g) for 40 minutes. The supernatant was filtered with a 0.45μM PES (polyethersulfone) filter.

[0474] Purification of [Ab "A"] = [minus TTR] 2: [plus TTR] 2 = [Ab "B"] molecules (without linker)

[0475] Connect to The rProtein A Fast Flow column (General Health Biosciences, Marlborough, Massachusetts, USA) of (General Health Biosciences, Inc.) FPLC was balanced with Dulbecco's PBS (DPBS) before loading. The filtered cell culture medium was injected onto the column, washed with 5 column volumes (CV) of DPBS, and eluted progressively with 8 CV of 50 mM HOAc, pH 3.2. The eluent was titrated to pH 5.0 using 1 M tris and then filtered through a 0.45 μm cellulose acetate vacuum filter (Corning Inc., Corning, New York, USA). The titrated and filtered rProtein A pool was divided into two separate pools for additional purification.

[0476] Half of the rProtein A pool was diluted 1:5 by volume with 20 mM MES, pH 5.0 and injected into the (General Health Biosciences) FPLC on a SP Sepharose high performance column (General Health Biosciences) pre-equilibrated with 20 mM NaOAc, pH 5.0. The column was washed with 5 CV of 20 mM NaOAc, pH 5.0 and eluted with a 20 CV gradient from 20 mM NaOAc, pH 5.0 to 20 mM NaOAc, 500 mM NaCl, pH 5.0.

[0477] SP Sepharose fractions were analyzed on a Caliper LabChip GXII microcapillary electrophoresis system using the ProteinExpress Assay LabChip (Perkin Elmer, Waltham, MA, USA) according to the manufacturer's protocol. Fractions were selected for enrichment of bands at the approximate molecular weight of monomeric Ab-TTR compared to unqualified MW species and then pooled.

[0478] The SP Sepharose pool was dialyzed against 10 mM MES, 150 mM NaCl, pH 6.5 using Slide-a-lyzer 10 kDa MWCO (Thermo Fisher Scientific, Waltham, MA, USA) dialysis cassettes until the starting buffer was calculated to be less than 1%.

[0479] The other half of the rProtein A pool was injected into the (General Health Biosciences) FPLC on a Sephadex G-25 (General Health Biosciences) column pre-equilibrated with 20 mM MES, pH 6.5. The column was eluted isocratically in 10 mM MES, 150 mM NaCl, pH 6.5.

[0480] PC analysis of [Ab "A"] = [negative TTR]2: [positive TTR]2 = [Ab "B"] molecule (without linker)

[0481] Protein quantification was performed by measuring UV absorbance at 280 nm using a Nanodrop 2000c (Thermo Fisher Scientific).

[0482] Non-reduced SDS-PAGE analysis was performed by treating the samples with SDS-PAGE sample buffer (Thermo Fisher Scientific) containing 100 mM iodoacetamide (Sigma-Aldrich, St. Louis, Missouri, USA), then directly loading onto 10% Tris-Gly gel and running according to the manufacturer's protocol. Reducing SDS-PAGE analysis was performed by treating the samples with SDS-PAGE sample buffer and sample reducing agent (Thermo Fisher Scientific). The samples were incubated at 85°C for 5 minutes, then loaded onto 10% Tris-Gly gel and running according to the manufacturer's protocol. According to the manufacturer's microwave protocol, SimplyBlue SafeStain (Thermo Fisher Scientific) was used to stain the gel.

[0483] HPLC SEC analysis was performed on a Zenix-C SEC-300, 7.8 x 300 mm column (Sepax Technologies Inc., Newark, TX, USA) connected to an Agilent 1290 Infinity HPLC system (Agilent Technologies, Santa Clara, CA, USA) running an isocratic 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 mobile phase at a flow rate of 1 mL / min and observing UV absorbance at 280 nm.

[0484] DAS analysis of [Ab "A"] = [negative TTR]2: [positive TTR]2 = [Ab "B"] molecules (without linker)

[0485] Denaturing LC-MS: All LC-MS data were acquired on an Agilent 6230TOF LC / MS system equipped with a 1290 Infinity Lc system. Chromatographic separation was achieved using a Zorbax SB300-C8 3.5 μm 2.1x50 mm column operated at 75 °C. The solvents used were as follows: Mobile phase A was water containing 0.1% v / v TFA. Mobile phase B was 90% n-propanol containing 0.1% v / v TFA. The initial gradient conditions were 20% mobile phase B from 0.0 to 1.0 min; 20-70% mobile phase B from 1.0 to 9.0 min; 70-100% mobile phase B from 9.0 to 10.0 min, where it was held at 100% for another 1 min. The flow rate was 0.2 mL / min. Approximately 5 μg of IgG1-biotin conjugate was loaded onto the LC-MS system for each analysis. Data were collected in the m / z range 1000-7000. Source fragmentor, skimmer and octopole 1RF values ​​were: 460 V, 95 V and 800 V (peak-to-peak), respectively. ESI capillary voltage was 5.9 kV. Gas temperature was 340°C. Drying gas was 13 L / min. Nebulizer was 25 psig. Calibration was performed using Agilent TuneMix, and Oa-ToF was performed using the autocalibration procedure implemented by MassHunter Data Acquisition B.06.01 version 6.01.6157.

[0486] Expression of the [655-341Ab]=[[LX]-[Negative TTR]]2:[[Positive TTR]-[LX]]2=[655-341Ab] molecule was expressed as described for the [Ab"A"]=[Negative TTR]2:[Positive TTR]2=[Ab"B"] molecule.

[0487] Purification of [655-341Ab]=[[LX]-[negative TTR]]2:[[positive TTR]-[LX]]2=[655-341Ab] molecules

[0488] The filtered cell culture medium was injected into The rProtein A Fast Flow HiTrap column (General Health Biosciences) and the desalting HiTrap column (General Health Biosciences) were equilibrated in tandem with DPBS and 10 mM MES 150 mM NaCl, pH 6.5, respectively. The rProtein A column was washed with DPBS and eluted stepwise with 100 mM HOAc, pH 3.6. The rProtein A eluate was buffer exchanged with 10 mM MES, 150 mM NaCl, pH 6.5 on the desalting HiTrap column.

[0489] DAS analysis of [655-341Ab]=[[LX]-[negative TTR]]2:[[positive TTR]-[LX]]2=[655-341Ab] molecules

[0490] Denaturing LC-MS was performed as described for the [Ab"A"] = [negative TTR]2: [positive TTR]2 = [Ab"B"] molecule.

[0491] Expression (co-expression) of (1) [[Fab "A"]-[Negative TTR]]2: [[Positive TTR]-[Fab "B"]]2, (2) [Ab "A"] = [Negative TTR]2: [Positive TTR]2 = [Ab "B"] and (3) [Ab "A"] = [Negative TTR]2: [[Positive TTR]-[Fab "B"]]2 molecules

[0492] CHO-K1 growth medium consists of 50% CS9 medium (non-selected, proprietary to Amgen) + 50% ExCell302 (SAFC Biosciences #14324C) + 2mM L-glutamine (Gibco #25030-081). Selection medium consists of growth medium + 10ug / ml puromycin (Gibco #A11138-03) + 500μg / ml hygromycin (Invitrogen #10687-010). Production medium consists of CHO-K1 6DCD (ATO Media Lab, proprietary to Amgen).

[0493] The transfection reagent consists of Lipofectamine LTX (Gibco #15338-100 (p / n 94756)) and Opti-MEMI Reduced Serum Medium (Gibco #31985-070). Growth conditions are suspension growth in a humidified incubator at 36°C + 5% CO2, which uses a ventilated shaker at 120RPM. The transfection procedure is as follows. One day before transfection, the host culture is split into 7-10 e 5 VCD / ml. DNA / Lipofectamine LTX complex is prepared as follows. 4μg nonlinear DNA is diluted in 0.5ml Opti-MEM medium in 24DWB (2.0μg GOI (target gene) and 2.0μg PB200 (super active transposase)). For four chain transfections, 0.5ug of each chain and 2.0μg PB200 (highly active transposase) are used, for a total of 4.0μg / transfection. For three chain transfections, 0.66μg of each chain and 2.0μg PB200 (highly active transposase) are used, for a total of 4.0μg / transfection. 10μl Lipofectamine LTX is diluted in 0.5ml Opti-MEM medium in a 15ml polypropylene tube and allowed to stand for 5 minutes. The diluted DNA is then combined with Lipofectamine LTX and thoroughly mixed by pipetting. The mixture is incubated at room temperature for 15-20 minutes, mixing from time to time. Then 2e 6 Transfer each live cell / transfection to a 15-50 ml polypropylene tube, spin at 1200 rpm for 5 minutes, and aspirate the medium. Then wash the cells with 1x PBS by completely resuspending and spin at 1200 rpm for 5 minutes. Then aspirate the 1x PBS and resuspend the cells in 1 ml Opti-MEM (per transfection). Then add 1 ml of cells to each well, and then add the DNA / LTX complex dropwise to each well. Incubate the cells at 235 rpm, 36°C + 5% CO. 2 The cells were incubated with shaking for 5-6 hours. Then 2.0 ml non-selective growth medium (CHO-K1 medium) was added to the cells. The selection of 72 hours after transfection was completed by placing the cells in 4 ml selection medium and resuspending completely. The amplification scale of the 6th day was amplified by directly adding 1.6 ml from the DWB culture to the 12 ml in the 50 ml ventilated centrifuge tube. The production of the 10th day was completed by inoculating 40 ml batch production by resuspending about 13 ml N-1 culture in the production medium. The harvest of the 17th day was carried out by the centrifugation of the cells and then the sterile filtration of the conditioned medium.

[0494] Purification (co-expression) of [[Fab "A"]-[Negative TTR]]2:[[Positive TTR]-[Fab "B"]]2 molecules

[0495] The Fab-TTR fusion protein included a C-terminal 6x his-tag and was captured from CM by IMAC affinity chromatography (1 ml HisTrapExcel, GE Healthcare; 17-3712-05) at a flow rate of 2 ml / min. The IMAC column was then washed with 5 CV of 20 mM sodium phosphate, 250 mM sodium chloride, pH 7.4 at a flow rate of 4 ml / min, and the protein was eluted stepwise with 20 mM sodium phosphate, 250 mM sodium chloride, 0.5 M imidazole, pH 7.4 at 2 ml / min. Prior to the next loading, the IMAC column was desorbed with 6 M guanidine hydrochloride, 50 mM Tris, pH 8, and equilibrated with 20 mM sodium phosphate, 250 mM sodium chloride, pH 7.4.

[0496] All purified proteins were finally buffer exchanged into 10 mM MES, 150 mM NaCl, pH 6 by passing through a 5 ml HiTrap desalting column (General Healthcare; 17-1408-01) at a flow rate of 2 ml / min. All preparative chromatography runs were performed using Purifiers (GE Healthcare Inc.) were used. A combination of analytical methods were then used to characterize the quantity and quality of the protein produced, including A280 protein quantification, size exclusion chromatography (SEC), microcapillary electrophoresis (MCE), and SDS-PAGE.

[0497] Purification (co-expression) of [Ab "A"] = [Negative TTR] 2: [Positive TTR] 2 = [Ab "B"] and [Ab "A"] = [Negative TTR] 2: [[Positive TTR] - [Fab "B"]] 2 molecules

[0498] Antibody-TTR fusion protein was captured from CM by protein A affinity chromatography (1 ml MabSelect SuRe HiTrap, GE Healthcare, Bio-Sciences, Marlborough, MA, USA; 11-0034-93) at a flow rate of 2 ml / min. The protein A column was then washed with 5 CV of 25 mM Tris, 100 mM sodium chloride, pH 7.4 at a flow rate of 4 ml / min, and the protein was eluted by stepwise protein elution with 100 mM acetic acid, pH 3.6 at 2 ml / min. Prior to the next loading, the column was desorbed with 6 M guanidine hydrochloride, 50 mM Tris, pH 8, and equilibrated with 25 mM Tris, 100 mM sodium chloride, pH 7.4.

[0499] All purified proteins were finally buffer exchanged into 10 mM MES, 150 mM NaCl, pH 6 by passing through a 5 ml HiTrap desalting column (General Healthcare; 17-1408-01) at a flow rate of 2 ml / min. All preparative chromatography runs were performed using Purifiers (a GE Healthcare company).

[0500] PC analysis of (1) [[Fab "A"]-[Negative TTR]]2: [[Positive TTR]-[Fab "B"]]2, (2) [Ab "A"] = [Negative TTR]2: [Positive TTR]2 = [Ab "B"] and (3) [Ab "A"] = [Negative TTR]2: [[Positive TTR]-[Fab "B"]]2 molecules (co-expression)

[0501] A280 Quantification—Protein quantification was performed by measuring UV absorbance at 280 nm using a Multiskan Go (Thermo Fisher Scientific, Waltham, MA, USA).

[0502] The SEC-TTR fusion protein sample was applied to an ACQUITY UPLC BEH 1.7 μm, 4.6 x 300 mm SEC column (Waters, Milford, MA, USA; 186005226) and observed UV absorbance at 280 nm. Analytical SEC was performed using a 1290 Infinity HPLC (Agilent Technologies, Santa Clara, CA, USA). Due to the large MW (249-347 kDa) of these TTR fusion molecules and product-related impurities, a MW benchmark molecule was used to measure the approximate SEC retention time of the expected MW of a specific fusion molecule. These benchmark molecules are those produced by Amgen, Inc., USA, and include 2 different antibodies (145 kDa each; protein lots BR4214-1 and PL41591), antibody-TTR heterotetramer (635 kDa; protein lot PL38002), antibody-TTR heterodimer (265 kDa; protein lot PL46796), and Fab-TTR heterotetramer (248 kDa; protein lot PL38000).

[0503] TTR fusion protein samples were characterized by MCE using a LabChip GXII (Caliper LifeSciences, Mountain View, CA, USA). Reduced and non-reduced samples were prepared according to the manufacturer's guidelines. Microfluidic chip technology automates the staining, destaining, electrophoretic separation, and analysis of protein samples.

[0504] SDS-PAGE - TTR fusion protein samples were run on various Tris-glycine, one-dimensional gels, including 8%, 10%, and 4-20% (Invitrogen, Carlsbad, CA, USA; Wedge Well: XP00080, XP00100, XP04200, respectively). Unreduced samples were prepared (no heating or heating at 85°C for 10 minutes). Gels were stained with SimpyBlue SafeStain (Invitrogen; LC6060) and compared to MW reference standards to identify the expected product bands.

[0505] DAS analysis of (1) [[Fab "A"]-[Negative TTR]]2: [[Positive TTR]-[Fab "B"]]2, (2) [Ab "A"] = [Negative TTR]2: [Positive TTR]2 = [Ab "B"] and (3) [Ab "A"] = [Negative TTR]2: [[Positive TTR]-[Fab "B"]]2 molecules (co-expression)

[0506] Denaturing LC-MS was performed as described for the [Ab"A"] = [negative TTR]2: [positive TTR]2 = [Ab"B"] molecule.

[0507] SEC-native-MS: All QToF experiments were performed on a Synapt G1 HDMS instrument operating in positive ESI mode. The instrument was converted to an RF limited drift tube instrument similar to that described in Bush et al., Anal Chem 2010, 82:9557-9565. All critical instrument voltages and pressures were as follows: capillary voltage 3.1 kV; sample cone 200 V, extraction cone 1 V; source block temperature 25°C; trap collision energy 50 V; transmission collision energy 20 V; trap inlet 2.0 V; trap bias 5 V; trap outlet 0.0 V; IMS inlet -20 V; IMS outlet 21 V; transmission inlet 1.0 V; transmission outlet 1.0 V; transmission speed 248 m / sec; transmission amplitude 3.0 V; source RF amplitude (peak-to-peak) 450 V; triple wave RF amplitude (peak-to-peak) trap 380 V, IMS 250 V, transmission 380 V; source back pressure 6.0 mbar; trap / transmission pressure cC4F8, 2.00e-2 mbar (Pirani gauge indicated; flow rate 4.0 mL / min). Instrument control and data acquisition were performed by MassLynx 4.1SCN 872.

[0508] SEC uses an Agilent 1200 pump system and a 2.1x50mm, The Waters BEH was operated at a flow rate of 75 μL / min at ambient temperature. The mobile phase was 200 mM ammonium acetate. SEC separations were performed by an isocratic 6-min method. 25-50 μg of material were injected for analysis. 200 mM ammonium acetate was used because it is a volatile buffer and therefore compatible with the mass spectrometer. Instrument control was performed via ChemStation.

[0509] The following techniques were used to generate TTR minus and plus constructs containing a TTR variant with two TTR dimer / dimer interface mutations per TTR subunit ("C10A / K15A / XX / YY"): Cloning of Fabs of TTR variants containing two TTR dimer / dimer interface mutations:

[0510] Cloning of Fab TTR dimers with TTR variants containing two TTR dimer / dimer interface mutations was accomplished using methods similar to those described in the sections describing the cloning of (1) [Ab"A"]=[Negative TTR]2:[Positive TTR]2=[Ab"B"] (2X Ab-TTR); (2) [[Ab"A"]-[Negative TTR]]2:[[Positive TTR]-[Ab"B"]]2 (4X Ab-TTR); and (3) [[Fab"A"]-[Negative TTR]]2:[[Positive TTR]-[Fab"B"]]2 (4X Fab-TTR) molecules (without linkers).

[0511] Expression of TTR dimers with Fabs of TTR variants containing two TTR dimer / dimer interface mutations

[0512] Transfection was performed on a 50 ml scale. HEK 293-6E cells were cultured in FreeStyle F17 medium (Thermo Fisher Scientific) (supplemented with 0.1% (w / v) poloxamer 188 (Sigma-Aldrich), 6 mM L-glutamine (Thermo Fisher Scientific), 25 μg / ml G418 (Thermo Fisher Scientific)) in a shake flask in an incubator (5% CO2, 80%-90% humidity and 120 rpm agitation) on a shaker with a 25 mm shaking diameter at 36°C. 293-6E cells were cultured at 0.4 x 10 6 On the day of transfection, the cells were in the exponential growth phase (approximately 1.5 x 10 6 Cells / ml, >95% viability). Transient transfection was performed by adding a mixture of 0.5 mg / L DNA and 2 mg / L PEI Max (Polyethyleneimine Max, Polysciences, Cat# 24765-2) to the cell culture. Proprietary feed {yeastate (0.5% w / v) and glucose (3 g / L)} was added 4 hours after transfection. Six days after transfection, the product was harvested by centrifuging the cells at 4000 rpm (3485 x g) for 40 minutes. The supernatant was filtered with a 0.45 μM PES (polyethersulfone) filter.

[0513] Purification of TTR dimers from Fabs with TTR variants containing two TTR dimer / dimer interface mutations

[0514] The filtered cell culture medium was injected into (General Health Biosciences) on a HisTrap excel column (General Health Biosciences) and a desalting HiTrap column (General Health Biosciences) tandem purification system (equilibrated with 20 mM sodium phosphate, 500 mM NaCl, pH 7.4 and 10 mM MES 150 mM NaCl, pH 6.0, respectively). The HisTrap excel column was washed with 20 mM sodium phosphate, 500 mM NaCl, pH 7.4, and then eluted stepwise with 20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4. The HisTrap xcel eluate was buffer exchanged with 10 mM MES, 150 mM NaCl, pH 6.0 on the desalting HiTrap column.

[0515] PC of TTR dimer with Fab of TTR variants containing two TTR dimer / dimer interface mutations

[0516] Protein quantification was performed by measuring UV absorbance at 280 nm using a MultiSkan FC microplate photometer (Thermo Fisher Scientific).

[0517] Non-reducing and reducing microcapillary electrophoresis analyses were performed on a Caliper LabChip GXII system using the Protein Express Assay LabChip (PerkinElmer) according to the manufacturer's protocol.

[0518] HPLC SEC analysis was performed as follows: on an ACQUITY UPLC BEH450 SEC 2.5 μm 7.8 x 300 mm column (Waters Corp., Milford, MA, USA) connected to an Agilent 1290 Infinity HPLC system (Agilent Technologies), running an isocratic 100 mM NaH2PO4, 50 mM NaCl, 7.5% EtOH, pH 6.9 mobile phase at a flow rate of 0.4 mL / min and observing UV absorbance at 280 nm.

[0519] Heterodimerization of [[Fab "A"]-[Double Negative TTR]] and [[Double Positive TTR]-[Fab "B"]]: Mixing and analysis of separately generated constructs

[0520] Purified TTR-Fab samples were normalized to 0.2 mg / mL by dilution with 10 mM MES, 150 mM NaCl, pH 6.0. Samples were combined in equal volumes and incubated overnight at 4°C. The resulting molecular mixture was analyzed by HPLC-SEC.

[0521] Co-expression of [[Fab "A"]-[Double Negative TTR]] and [[Double Positive TTR]-[Fab "B"]]

[0522] Transfections were performed separately and after completion (1-4 hours), [[Fab "A"]-[Double Negative TTR]] and [[Double Positive TTR]-[Fab "B"]] constructs were combined and produced together at a 4 ml scale. HEK 293-6E cells were cultured in shake flasks in an incubator (5% CO2, 80%-90% humidity and 120 rpm agitation) on a 25 mm shaking diameter shaker at 36°C in FreeStyle F17 medium (Thermo Fisher Scientific) supplemented with 0.1% (w / v) poloxamer 188 (Sigma-Aldrich), 6 mM L-glutamine (Thermo Fisher Scientific), 25 μg / ml G418 (Thermo Fisher Scientific). 293-6E cells were seeded at 0.4x106 cells / ml 2 days before transfection. On the day of transfection, cells were in exponential growth phase (about 1.5x106 cells / ml,>95% survival rate). Transient transfection was carried out by adding a mixture of 0.5mg / L DNA and 2mg / L PEI Max (polyethyleneimine Max, Polysciences, Cat#24765-2) to cell culture. Proprietary feed {yeast acid salt (0.5% w / v) and glucose (3g / L)} were added 4 hours after transfection. 6 days after transfection, the product was harvested by centrifuging the cells at 4000rpm (3485x g) for 40 minutes. The supernatant was filtered with a 0.45μM PES (polyethersulfone) filter.

[0523] Purification of co-expressed [[Fab "A"]-[Double Negative TTR]] and [[Double Positive TTR]-[Fab "B"]]

[0524] The filtered cell culture medium was injected into (General Health Biosciences) on a HisTrap excel column (General Health Biosciences) and a desalting HiTrap column (General Health Biosciences) tandem purification system (equilibrated with 20 mM sodium phosphate, 500 mM NaCl, pH 7.4 and 10 mM MES 150 mM NaCl, pH 6.0, respectively). The HisTrap excel column was washed with 20 mM sodium phosphate, 500 mM NaCl, pH 7.4, and then eluted stepwise with 20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4. The HisTrap xcel eluate was buffer exchanged with 10 mM MES, 150 mM NaCl, pH 6.0 on the desalting HiTrap column.

[0525] PC analysis of co-expressed [[Fab "A"]-[Double Negative TTR]] and [[Double Positive TTR]-[Fab "B"]]

[0526] Protein quantification was performed by measuring UV absorbance at 280 nm using MultiSkan FC microplate photometer (Thermo Fisher Scientific). Non-reduced and reduced microcapillary electrophoresis analysis was performed on Caliper LabChip GXII system using Protein Express Assay LabChip (Perkin Elmer) according to the manufacturer's protocol. HPLCSEC analysis was performed as follows: on an ACQUITYUPLC BEH450 SEC 2.5 μm 7.8 x 150 mm column (Waters Corp., Milford, Massachusetts, USA) connected to an Agilent 1290 Infinity HPLC system (Agilent Technologies), an isocratic 100 mM NaH2PO4, 50 mM NaCl, 7.5% EtOH, pH 6.9 mobile phase was run at a flow rate of 0.4 mL / min and UV absorbance was observed at 280 nm.

[0527] Example 2: Evaluation of TTR heterotetramers containing a TTR variant with one TTR dimer / dimer interface mutation per TTR subunit ("C10A / K15A / XX") - produced in E. coli

[0528] Eighteen TTR charge variants (C10A / K15A / XX) of TTR (SEQ ID NO: 1) were prepared to determine which charge mutations would result in substantial repulsion of the TTR dimer / dimer interface (see Figure 4 ). Each TTR variant contained the C10A and K15A mutations and a third mutation denoted "XX". In these experiments, XX was K15R, L17R, V20R, R21E, G22R, S23R, P24R, D51R, S52R, I84R, T106R, A108R, S112R, Y114R, S115R, T119R, V121R, or S123R.

[0529] Nine of the TTR variants (P24R, I84R, L17R, V121R, V20R, G22R, S112R, T119R, Y114R, and S115R) showed a significant weakening of the TTR dimer / dimer interface, as indicated by the fact that in the presence of SDS (a chaotropic agent), TTR tetramers dissociate into the corresponding TTR dimers without heating (see Figure 5 , "non-heated" gels). Six of the variants (V20R, G22R, S112R, T119R, Y114R, and S115R) also interfered with dimer / dimer interactions under non-denaturing (native) conditions as assessed by SEC (see Figure 6In addition, four variants (P24R, I84R, L17R, and V121R) were observed to form predominantly TTR tetramers under native SEC conditions and displayed lower melting temperatures, again suggesting a weakened dimer / dimer interface in these variants.

[0530] The six most favorable dimer / dimer interface mutation sites were selected ( Figure 6 , red) to generate additional TTR variants to evaluate the formation of TTR heterotetramers containing two different TTR monomer sequences. In these experiments, the desired TTR heterotetramers contained [1] a TTR dimer, which itself was composed of two TTR monomers, each of which was a "negative" TTR variant; and [2] a TTR dimer, which itself was composed of two TTR monomers, each of which was a "positive" TTR variant. Six favorable dimer / dimer interface mutation sites were selected based on their ability to form heterotetramers under SEC and SDS-PAGE conditions. Mutation sites that predominantly resulted in dimer formation under SDS-PAGE conditions were selected as initial cutoffs (i.e., L17R, V121R, V20R, G22R, S112R, T119R, Y114R, and S115R). Y114R and S115R were no longer considered due to significantly lower protein yields.

[0531] The minus TTR variants comprised C10A / K15A / XX mutations, wherein each XX was L17D, L17E, V20D, V20E, G22D, G22E, S112D, S112E, T119D, T119E, V121D, or V121E. Similarly, the plus TTR variants comprised C10A / K15A / XX mutations, wherein each XX was L17R, L17K, V20R, V20K, G22R, G22K, S112R, S112K, T119R, T119K, V121R, or V121K. Thus, a total of 24 charge interface variants (12 minus and 12 plus) were generated ( Figure 7 ).

[0532] Positive variants were mixed with negative variants (in pairs) and TTR heterotetramer formation was assessed by SDS-PAGE and SEC. Many of the variant pairs showed some tendency to form the desired heterotetramers, such as Figure 7 The non-zero SEC values ​​in (which represent % heterotetramer formation) are shown. In fact, some pairs showed a very high propensity to form heterotetramers, with 40-100% tetramer formation.

[0533] As shown by SDS-PAGE results, many of these TTR heterotetramers are resistant to degradation by chaotropic SDS. Figure 7Positive / negative pairs that showed a high propensity to form stable heterotetramers (cross-referenced to SEC and SDS-PAGE data) included: L17R / T119D, L17K / T119D, L17K / V121E, V20R / V20D, V20R / V20E, V20K / V20D, V20K / V20E, V121R / L17D, V121R / L17E, and V121K / L17D.

[0534] Positive / negative pairs that showed a high propensity to form stable TTR tetramers were further evaluated by SDS-PAGE. In this experiment, for each pair, [1] the negative (i.e., basic) variant; [2] the positive (i.e., acidic) variant; [3] the combination of the negative and positive variants (which should form tetramers); and [4] the combination of the negative and positive variants exposed to caspases ( Figure 8 ).like Figure 8 As shown, the separate negative and positive variants migrated to the bottom of the gel, while the combination of the negative and positive variants migrated only part way down in the gel. This further suggests that the negative and positive variants form high molecular weight species (HMW) upon combination - likely the desired heterotetramer. Treatment of the combination of the negative and basic variants with caspase (which only cleaves the polyhistidine tag from the positive variant due to the inclusion of the DEVD sequence) produced a substantially uniform band that ran slightly lower than the uncleaved tetramer, indicating the presence of a positive component and showing that the tetramer is mostly uniform and likely a heterotetramer.

[0535] Heterotetramers containing L17R / T119D, L17K / T119D, L17K / V121E, V20R / V20D, V20R / V20E, V20K / V20D, V20K / V20E, V121R / L17D, V121R / L17E, and V121K / L17D pairings were then exposed to conditions at pH 5.0 to determine if they could remain tetrameric (by SEC) under conditions similar to those found in pharmaceutical formulations. Fig. 9 ). In fact, Fig. 9 The single peak in indicates that the heterotetramer is able to maintain its tetrameric state.

[0536] The melting temperatures of the three heterotetramers were evaluated. In each case, the heterotetramer was stable to at least 92°C, indicating that the heterotetramer is very thermostable ( Fig.10 ).

[0537] Example 3: Evaluation of TTR heterotetrameric Fab, Ab, and mixed Fab / Ab constructs containing a TTR variant with one TTR dimer / dimer interface mutation per TTR subunit ("C10A / K15A / XX") - produced in mammalian cells

[0538] The ability to form TTR heterotetrameric Fabs, Abs, and mixed Fab / Ab constructs was evaluated. In these constructs, a TTR tetramer containing two positive TTR variants and two negative TTR variants (as described above) can be used to generate TTR heterotetramers attached to four Fabs, 2 Abs, or 1 Ab and 2 Fabs. See, respectively Figure 2 a, 2b and 2c. Charge pair mutations in Fab constructs can be used to drive correct HC / LC Fab pairing. One benefit of such TTR heterotetramer constructs is that they allow assembly of multiple antigen targeting moieties (e.g., Ab and / or Fab) by fusing TTR monomer units to the C-terminus of Ab and / or Fab. This orients the Ab and / or Fab so that their antigen binding domains are less likely to suffer from the steric interference seen in other bivalent bispecific platforms (e.g., IgG-Fab and IgG scFv constructs), which is typically due to the fusion of the Fab or scFv N-terminus to the C-terminus of IgG.

[0539] TTR heterotetrameric Ab constructs

[0540] Bispecific TTR heterotetrameric Ab constructs were generated. In these constructs, one Ab (655-341Ab) is specific for the extracellular domain of human TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) receptor 2 (TR-2, death receptor 5), while the other Ab (DNP-3B1) is specific for DNP. Exemplary bispecific TTR heterotetrameric Ab constructs are shown in Fig.11 , in which each heavy chain of the 655-341 Ab (line fill) is attached to the N-terminus of a negative TTR monomer (together forming a negative TTR dimer) and each heavy chain of the DNP-3B1 Ab (solid fill) is attached to the N-terminus of a positive TTR monomer (together forming a positive TTR dimer).

[0541] Four negative TTR variants were fused to 655-341 Ab, and four positive TTR variants were fused to DNP-3B1 Ab ( Fig.11 ). All Ab-TTR fusions were produced without a linker between the Ab and TTR monomers. The variants were produced in mammalian cells (293-6E HEK cells) for secretion into the culture medium. Two groups of transformed cells were generated: one producing 655-341 Ab / minus TTR fusions ([655-341Ab]=[minus TTR]2) and one producing DNP-3B1 Ab / positive TTR fusions ([positive TTR]2=[DNP-3B1 Ab]).

[0542] Interestingly, in contrast to the TTR tetramer produced in E. coli (Example 2), a large portion of the secreted constructs were either self-associating 655-341Ab / minus TTR fusions (i.e., [655-341Ab] = [minus TTR] 2: [minus TTR] 2 = [655-341Ab]), self-associating DNP-3B1 Ab / positive TTR fusions (i.e., [DNP-3B1 Ab] = [positive TTR] 2: [positive TTR] 2 = [DNP-3B1Ab]), or other HMW species. Free 655-341Ab / minus TTR fusions ([655-341Ab] = [minus TTR] 2) and DNP-3B1Ab / positive TTR fusions ([positive TTR] 2 = [DNP-3B1Ab]) accounted for 11-32% of the secreted constructs ( Fig.12 ). Rapid buffer exchange reduced the number of HMW species, although this mainly resulted in an increase in self-associated species.

[0543] The effect of adding a linker between the Ab heavy chain and the TTR monomer was investigated using the same eight charge variants. Five linkers ranging in size from 2 to 10 amino acids were evaluated ( Fig.13 The major secreted linker-containing Ab / TTR ​​fusion species were again self-associating (e.g., [655-341Ab]=[[LX]:[minus TTR]]2:[[minus TTR]:[LX]]2=[655-341Ab]) and HMW species. Free 655-341Ab / minus TTR fusions and DNP-3B1 Ab / plus TTR fusions ([655-341Ab]=[[LX]:[minus TTR]]2 and [[plus TTR]:[LX]]2=[DNP-3B1 Ab]) accounted for up to 31% of the secreted constructs ( Fig.14 and 15 ). Notably, shorter linkers generally lead to higher titers and yields (i.e., more protein produced) and lower levels of HMW species ( Fig.16 ). In addition, the minus TTR fusions produced moderately higher yields and lower levels of HMW species. None of the Ab-TTR fusions containing a linker formed free 655-341 Ab / minus TTR fusions or DNP-3B1 Ab / positive TTR fusions as major products. Other observations included that the shorter linker produced higher levels of self-associating Ab / TTR ​​fusions that were resistant to SDS; V20K, V20R, and V121K produced higher levels of self-associating Ab / TTR ​​fusions that were susceptible to SDS; T119D, V20D, V121E, and L17D produced fusions with higher titers / yields and reduced amounts of free Ab / TTR ​​fusions; and the minus variants appeared to produce lower numbers of HMW species.

[0544] Based on these observations, it was hypothesized that mammalian cells might not be able to efficiently produce "untetramerized" TTR. That is, mammalian cells might have difficulty producing [655-341Ab]=[minus TTR]2 or [plus TTR]2=[DNP-3B1 Ab] (with or without a linker) because such constructs contain only two TTR monomers (compared to native TTR, which has four TTR monomers). To investigate this, five combinations of 655-341Fab / minus TTR fusions and DNP-3B1Fab / plus TTR fusions were co-produced in a mammalian cell line (CHO K1). In these constructs, the Fab HC was attached to TTR ( Fig.17 ).

[0545] Co-production of [655-341Fab]-[GG]-[negative TTR] and [positive TTR]-[GG]-[DNP-3B1Fab] in CHO K1 resulted in significant formation of the desired [[655-341Fab]-[GG]-[negative TTR]]2:[[positive TTR]-[GG]-[DNP-3B1Fab]]2 (78-88%) and reduced amounts of HMW species (less than 5% in each case) ( Fig.18 ). Fab formation was found to be very efficient for all mutant pairings, although L17D / V121R showed a slight benefit. In addition, it appears that the V20R / V20D pairing may form a weaker heterotetramer that can be disrupted by SDS.

[0546] Using Ab- and Fab-TTR fusions as well as unfused Ab as SEC standards, it can be seen that molecules 15524 ([655-341Fab]-[GG]-[TTR(C10A / K15A / L17D)] and [TTR(C10A / K15A / V121R)]-[GG]-[DNP-3B1 Fab]) have retention times similar to those of the 4X-Fab homomultimers and significantly shorter than what would be expected for the 2X-Fab-TTR fusions (which should elute after the control Ab). Fig.19 ). In addition, when 15524 was evaluated by SEC coupled to MS, the molecular weight of the eluted species was consistent with the expected ( Fig. 20 ).

[0547] The same approach was taken to determine whether co-expression of TTR fusions containing Abs would result in the production of the desired TTR heterotetramer [655-341Ab] = [[LX]-[minus TTR]]2: [[plus TTR]-[LX]]2 = [DNP-3B1 Ab]. A total of five TTR charge variants and three linker lengths (X = 0, 4, and 10 amino acids) were tested, for a total of 15 combinations ( Fig.21For many of the composition combinations, a significant amount of the desired TTR heterotetramer was formed, up to 70% ( Fig. 22 ). In addition, the 4 amino acid linker produced higher titers and yields compared to the 0 amino acid linker. Using Ab- and Fab-TTR fusions and unfused Ab as SEC standards, it can be seen that the main peak retention time observed for molecule 15539 ([655-341Ab]=[[GGAGGGAGGG]-[TTR(C10A / K15A / L17D]]2:[[TTR(C10A / K15A / V121K)]-[GGAGGGAGGG]]2=[DNP-3B1 Ab]) was almost identical to the main peak retention time of the 2X-Ab-TTR homomultimer ( Fig.23 Furthermore, when the construct was evaluated by SEC coupled to MS, the molecular mass of the eluted species was consistent with that expected for the TTR heterotetramer ( Fig.24 ). It was observed that, on average, the L4 linker seemed to result in good yields and good preferential production of the desired product. In addition, the L17K / T119D, V20K / V20D, and V20R / V20D mutation combinations seemed to result in high expression and yields. Finally, the L17K / V121E, V121K / L17D, and V20K / V20D mutation combinations seemed to best drive the desired TTR heterotetrameric assembly. See Fig.25 .

[0548] We next sought to determine whether co-expression of negative and positive TTR variants (4 of each mutation) fused to Ab and Fab in the same cell line would result in the generation of Ab-Fab-TTR constructs (i.e., [Ab "A"] = [negative TTR] 2 : [[positive TTR] = [Fab "B"]] 2 constructs). A total of five TTR variants were tested in combination with three linker lengths (X = 0, 4, and 10 amino acids) and two Ab / Fabs - a total of 30 combinations ( Fig.26 For many of these combinations, the number of Ab fusions that formed the desired Ab-Fab-TTR constructs based on SEC was as high as 45.6% ( Fig. 27). In addition, the 4 and 10 amino acid linkers appeared to produce more of the desired Ab-Fab-TTR constructs (based on potency and purification yield) compared to the 0 amino acid linker. Using Ab- and Fab-TTR fusions as well as unfused Ab as SEC standards, it was observed that molecule 15545 ([655-341Ab] = [[GGGG]-[TTR(C10A / K15A / V20D)]]2: [[TTR(C10A / K15A / V20R)]-[GG]-[DNP-3B1-Fab]]2) produced a peak with retention between the 2X-Ab-TTR homomultimer and the 4X-Fab-TTR homomultimer as expected ( Fig.28 Furthermore, when the molecule was evaluated by SEC coupled to MS, the molecular weight of the eluted species was consistent with the expected construct ( Fig.29 ). It was observed that, on average, the L10 linker was preferred in the Ab-Fab-TTR construct. In addition, the L17K / T119D TTR mutation appeared to be preferred in the Ab-Fab-TTR construct. See Fig.30 .

[0549] Example 4: Evaluation of TTR heterotetrameric Fab, Ab, and mixed Fab / Ab constructs containing a TTR variant with two TTR dimer / dimer interface mutations per TTR subunit ("C10A / K15A / XX / YY") - in mammalian cells

[0550] Ab- and Fab-TTR fusions with two charged interface mutations were produced separately in mammalian cells as previously described for single charged interface mutations. Potency, post-affinity chromatography yield, and SEC performance were evaluated as previously described for single charged interface mutations. The purified single and double charged interface variants were then mixed in matrix mode and the mixture was evaluated by SEC as previously described for single charged variants.

[0551] Ab- and Fab-TTR fusions with singly and doubly charged interface mutations were generated by incubating mammalian cells with oppositely charged TTR variants as described previously for singly charged interface mutations. Post-affinity chromatography yield and SEC performance were assessed as previously described for singly charged interface mutations.

[0552] The results of these experiments can be found in Figure 31-40 Found in. Fig.35 and 36 As can be seen, certain combinations of variants produced a significant increase in 4X-Fab formation compared to that produced by a single mixture of the molecules produced individually before mixing (determined by taking the average of the 4X-Fab levels before mixing and subtracting that value from the observed 4X-Fab). Fig.37 This is illustrated by showing that the 4X-Fab SEC peak of the mixture is much larger than the 4X-Fab peak before mixing. In addition, certain co-culture conditions lead to the formation of higher amounts of 4X-Fab ( Fig.38 and 39 ). Fig.40 The co-incubated 4X-Fab peak was shown to be significantly larger than that of the molecules incubated alone, suggesting that 4X-Fab formation may be enhanced in the presence of the charge-opposite variants.

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[0563] Underline =Signal peptide (the signal peptide can be removed from any amino acid or nucleic acid sequence disclosed herein. Thus, for each amino acid and nucleic acid sequence described herein, sequences with and without a signal peptide are specifically contemplated).

[0564] Bold = CDR

[0565] Bold+Underline= Connectors

[0566] Italics = TTR portion of the fusion protein Sequence Listing <110> Amgen <120> Multispecific thyroxine transporter immunoglobulin fusion <130> A-2414-WO-PCT <150> 62 / 871,247 <151> 2019-07-08 <160> 44 <170> PatentIn 3.5 Edition <210> 1 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Human TTR AA <400> 1 Gly Pro Thr Gly Thr Gly Glu Ser Lys Cys Pro Leu Met Val Lys Val 1 5 10 15 Leu Asp Ala Val Arg Gly Ser Pro Ala Ile Asn Val Ala Val His Val 20 25 30 Phe Arg Lys Ala Ala Asp Asp Thr Trp Glu Pro Phe Ala Ser Gly Lys 35 40 45 Thr Ser Glu Ser Gly Glu Leu His Gly Leu Thr Thr Glu Glu Glu Phe 50 55 60 Val Glu Gly Ile Tyr Lys Val Glu Ile Asp Thr Lys Ser Tyr Trp Lys 65 70 75 80 Ala Leu Gly Ile Ser Pro Phe His Glu His Ala Glu Val Val Phe Thr 85 90 95 Ala Asn Asp Ser Gly Pro Arg Arg Tyr Thr Ile Ala Ala Leu Leu Ser 100 105 110 Pro Tyr Ser Tyr Ser Thr Thr Ala Val Val Thr Asn Pro Lys Glu 115 120 125 <210> 2 <211> 127 <212> PRT <213> Artificial sequence <220> <223> Mouse TTR AA <400> 2 Gly Pro Ala Gly Ala Gly Glu Ser Lys Cys Pro Leu Met Val Lys Val 1 5 10 15 Leu Asp Ala Val Arg Gly Ser Pro Ala Val Asp Val Ala Val Lys Val 20 25 30 Phe Lys Lys Thr Ser Glu Gly Ser Trp Glu Pro Phe Ala Ser Gly Lys 35 40 45 Thr Ala Glu Ser Gly Glu Leu His Gly Leu Thr Thr Asp Glu Lys Phe 50 55 60 Val Glu Gly Val Tyr Arg Val Glu Leu Asp Thr Lys Ser Tyr Trp Lys 65 70 75 80 Thr Leu Gly Ile Ser Pro Phe His Glu Phe Ala Asp Val Val Phe Thr 85 90 95 Ala Asn Asp Ser Gly His Arg His Tyr Thr Ile Ala Ala Leu Leu Ser 100 105 110 Pro Tyr Ser Tyr Ser Thr Thr Ala Val Val Ser Asn Pro Gln Asn 115 120 125 <210> 3 <211> 615 <212> DNA <213> Artificial sequence <220> <223> Rat TTR NA <400> 3 acagaagtcc actcattctt ggcaggatgg cttctcatcg tctgctcctc ctctgccttg 60 ctggactggt atttgtgtct gaggctggcc ctacgggcac cggtgaatcc aagtgtcctc 120 tgatggtcaa agttctagat gctgtccgag gcagtcctgc catcaatgtg gccgtgcatg 180 tgttcagaaa ggctgctgat gacacctggg agccatttgc ctctgggaaa accagtgagt 240 ctggagagct gcatgggctc acaactgagg aggaatttgt agaagggata tacaaagtgg 300 aaatagacac caaatcttac tggaaggcac ttggcatctc cccattccat gagcatgcag 360 aggtggtatt cacagccaac gactccggcc cccgccgcta caccattgcc gccctgctga 420 gcccctactc ctattccacc acggctgtcg tcaccaatcc caaggaatga gggacttctc 480 ctccagtgga cctgaaggac gagggatggg atttcatgta accaagagta ttccattttt 540 actaaagcac tgttttcacc tcatatgcta tgttagaagt ccaggcagag acaataaaac 600 attcctgtga aaggc 615 <210> 4 <211> 236 <212> PRT <213> Artificial Sequence <220> <223> DNP 3B1 Ab LC AA <400> 4 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala Ser Glu Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45 Gln Gly Ile Arg Asn Asp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Arg Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val 65 70 75 80 Pro Leu Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln 100 105 110 Tyr Asn Ser Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 5 <211> 708 <212> DNA <213> Artificial Sequence <220> <223> DNP Ab LC NA <400> 5 atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct gagaggtgcg 60 cgctgtgaca tccagatgac ccagtctcca tcctccctgt ctgcatctga aggagacaga 120 gtcaccatca cttgccgggc aagtcagggc attagaaatg atttaggctg gtatcagcag 180 aaaccaggga aagcccctaa gcgcctgatc tatgctgcat ccagtttgca aagtggggtc 240 ccattaaggt tcagcggcag tggatctggg acagaattca ctctcacaat cagcagcctg 300 cagcctgaag attttgcaac ttattactgt ctacagtata atagttaccc gtggacgttc 360 ggccaaggga ccaaggtgga aatcaaacgt acggtggctg caccatctgt cttcatcttc 420 ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac 480 ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac 540 tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc 600 ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat 660 cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgt 708 <210> 6 <211> 236 <212> PRT <213> Artificial Sequence <220> <223> DNP 3B1 Ab LC AA w / CPM S230E (EU S176E) <400> 6 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala Ser Glu Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45 Gln Gly Ile Arg Asn Asp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Arg Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val 65 70 75 80 Pro Leu Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln 100 105 110 Tyr Asn Ser Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Glu Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 7 <211> 708 <212> DNA <213> Artificial Sequence <220> <223> DNP 3B1 Ab LC NA w / CPM S230E (EU S176E) <400> 7 atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct gagaggtgcg 60 cgctgtgaca tccagatgac ccagtctcca tcctccctgt ctgcatctga aggagacaga 120 gtcaccatca cttgccgggc aagtcagggc attagaaatg atttaggctg gtatcagcag 180 aaaccaggga aagcccctaa gcgcctgatc tatgctgcat ccagtttgca aagtggggtc 240 ccattaaggt tcagcggcag tggatctggg acagaattca ctctcacaat cagcagcctg 300 cagcctgaag attttgcaac ttattactgt ctacagtata atagttaccc gtggacgttc 360 ggccaaggga ccaaggtgga aatcaaacgt acggtggctg caccatctgt cttcatcttc 420 ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac 480 ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac 540 tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cgaaagcacc 600 ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat 660 cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgt 708 <210> 8 <211> 474 <212> PRT <213> Artificial Sequence <220> <223> DNP 3B1 Ab HC AA <400> 8 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Gln Glu Ser Gly Pro Gly 20 25 30 Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly 35 40 45 Gly Ser Ile Ser Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly 50 55 60 Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Asn Thr Asn 65 70 75 80 Ser Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser 85 90 95 Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Arg Thr Tyr Tyr Asp Ser Ser Gly Tyr Tyr 115 120 125 Tyr Arg Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser 130 135 140 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 145 150 155 160 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 165 170 175 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 180 185 190 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 195 200 205 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 210 215 220 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 225 230 235 240 Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 245 250 255 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 260 265 270 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 275 280 285 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 290 295 300 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 305 310 315 320 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 325 330 335 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 340 345 350 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 355 360 365 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 370 375 380 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 385 390 395 400 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 405 410 415 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 420 425 430 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 435 440 445 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 450 455 460 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 465 470 <210> 9 <211> 1422 <212> DNA <213> Artificial Sequence <220> <223> DNP 3B1 Ab HC NA <400> 9 atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct gagaggtgcg 60 cgctgtcagg tgcagctgca ggagtcgggc ccaggactgg ttaagccttc ggagaccctg 120 tccctcacct gcactgtctc tggtggctcc atcagtagtt actactggag ctggatccgg 180 cagcccccag ggaagggact ggagtggatt gggtatatct attacagtgg gaacaccaac 240 tccaacccct ccctcaagag tcgagtcacc atatcagtag acacgtccaa gaaccagttc 300 tccctgaagc tgagctctgt gaccgctgcg gacacggccg tgtattactg tgcgagaacc 360 tactatgata gtagtggtta ctactaccgt gcttttgata tctggggcca agggacaatg 420 gtcaccgtct ctagtgcctc caccaagggc ccatcggtct tccccctggc accctcctcc 480 aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 540 ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct 600 gtcctacagt cctcaggact ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc 660 ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtggac 720 aagaaagttg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacct 780 gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 840 atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 900 gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 960 gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1080. gagaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc ccatcccggg aggagac caagaccag gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtggggag agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctatagcaa gctcaccgtg 1320 1380. ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc ctgtctccgg gt <210> 10 <211> 474 <212> PRT <213> The snowstorm <220> <223> DNP Ab HC AA w / CPM S230K (EU S183K) <400> 10 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Gln Glu Ser Gly Pro Gly 20 25 30 Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly 35 40 45 Gly Ser Ile Ser Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly 50 55 60 Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Asn Thr Asn 65 70 75 80 Ser Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser 85 90 95 Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Arg Thr Tyr Tyr Asp Ser Ser Gly Tyr Tyr 115 120 125 Tyr Arg Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser 130 135 140 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 145 150 155 160 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 165 170 175 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 180 185 190 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 195 200 205 Ser Leu Lys Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 210 215 220 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 225 230 235 240 Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 245 250 255 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 260 265 270 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 275 280 285 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 290 295 300 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 305 310 315 320 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 325 330 335 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 340 345 350 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 355 360 365 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 370 375 380 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 385 390 395 400 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 405 410 415 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 420 425 430 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 435 440 445 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 450 455 460 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 465 470 <210> 11 <211> 1422 <212> DNA <213> Artificial Sequence <220> <223> DNP Ab HC NA w / CPM S230K (EU S183K) <400> 11 atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct gagaggtgcg 60 cgctgtcagg tgcagctgca ggagtcgggc ccaggactgg ttaagccttc ggagaccctg 120 tccctcacct gcactgtctc tggtggctcc atcagtagtt actactggag ctggatccgg 180 cagcccccag ggaagggact ggagtggatt gggtatatct attacagtgg gaacaccaac 240 tccaacccct ccctcaagag tcgagtcacc atatcagtag acacgtccaa gaaccagttc 300 tccctgaagc tgagctctgt gaccgctgcg gacacggccg tgtattactg tgcgagaacc 360 tactatgata gtagtggtta ctactaccgt gcttttgata tctggggcca agggacaatg 420 gtcaccgtct ctagtgcctc caccaagggc ccatcggtct tccccctggc accctcctcc 480 aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa540 ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc aagagcgtgg tgaccgtgcc ctccagcagc 660 ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtggac aagaaagttg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacct 780 gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 840 atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 900 gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 960 gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1080. gagaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc ccatcccggg aggagac caagaccag gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1260 accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctatagcaa gctcaccgtg 1320 gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1380 cacaaccact acacgcagaa gagcctctcc ctgtctccgg gt 1422 <210> 12 <211> 248 <212> PRT <213> Artificial Sequence <220> <223> DNP Fab HC AA <400> 12 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Gln Glu Ser Gly Pro Gly 20 25 30 Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly 35 40 45 Gly Ser Ile Ser Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly 50 55 60 Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Asn Thr Asn 65 70 75 80 Ser Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser 85 90 95 Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Arg Thr Tyr Tyr Asp Ser Ser Gly Tyr Tyr 115 120 125 Tyr Arg Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser 130 135 140 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 145 150 155 160 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 165 170 175 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 180 185 190 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 195 200 205 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 210 215 220 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 225 230 235 240 Lys Lys Val Glu Pro Lys Ser Cys 245 <210> 13 <211> 744 <212> DNA <213> Artificial Sequence <220> <223> DNP Fab HC AA <400> 13 atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct gagaggtgcg 60 cgctgtcagg tgcagctgca ggagtcgggc ccaggactgg ttaagccttc ggagaccctg 120 tccctcacct gcactgtctc tggtggctcc atcagtagtt actactggag ctggatccgg 180 cagcccccag ggaagggact ggagtggatt gggtatatct attacagtgg gaacaccaac 240 tccaacccct ccctcaagag tcgagtcacc atatcagtag acacgtccaa gaaccagttc 300 tccctgaagc tgagctctgt gaccgctgcg gacacggccg tgtattactg tgcgagaacc 360 tactatgata gtagtggtta ctactaccgt gcttttgata tctggggcca agggacaatg 420 gtcaccgtct ctagtgcctc caccaagggc ccatcggtct tccccctggc accctcctcc 480 aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 540 ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct 600 gtcctacagt cctcaggact ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc 660 ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtggac 720 aagaaagttg agcccaaatc ttgt 744 <210> 14 <211> 248 <212> PRT <213> Artificial Sequence <220> <223> DNP Fab HC AA w / CPM S230K (EU S183K) <400> 14 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Gln Glu Ser Gly Pro Gly 20 25 30 Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly 35 40 45 Gly Ser Ile Ser Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly 50 55 60 Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Asn Thr Asn 65 70 75 80 Ser Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser 85 90 95 Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Arg Thr Tyr Tyr Asp Ser Ser Gly Tyr Tyr 115 120 125 Tyr Arg Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser 130 135 140 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 145 150 155 160 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 165 170 175 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 180 185 190 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 195 200 205 Ser Leu Lys Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 210 215 220 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 225 230 235 240 Lys Lys Val Glu Pro Lys Ser Cys 245 <210> 15 <211> 744 <212> DNA <213> Artificial sequence <220> <223> DNP Fab HC NA w / CPM S230K (EU S183K) <400> 15 atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct gagaggtgcg 60 cgctgtcagg tgcagctgca ggagtcgggc ccaggactgg ttaagccttc ggagaccctg 120 tccctcacct gcactgtctc tggtggctcc atcagtagtt actactggag ctggatccgg 180 cagcccccag ggaagggact ggagtggatt gggtatatct attacagtgg gaacaccaac 240 tccaacccct ccctcaagag tcgagtcacc atatcagtag acacgtccaa gaaccagttc 300 tccctgaagc tgagctctgt gaccgctgcg gacacggccg tgtattactg tgcgagaacc 360 tactatgata gtagtggtta ctactaccgt gcttttgata tctggggcca agggacaatg 420 gtcaccgtct ctagtgcctc caccaagggc ccatcggtct tccccctggc accctcctcc 480 aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 540 ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct 600 gtcctacagt cctcaggact ctactccctc aagagcgtgg tgaccgtgcc ctccagcagc 660 ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtggac 720 aagaaagttg agcccaaatc ttgt 744 <210> 16 <211> 235 <212> PRT <213> Artificial Sequence <220> <223> 655-341 Ab LC AA <400> 16 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser 20 25 30 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly 35 40 45 Ile Ser Arg Ser Glu Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 55 60 Pro Ser Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro 65 70 75 80 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90 95 Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe 100 105 110 Gly Ser Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 115 120 125 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 130 135 140 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 145 150 155 160 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 165 170 175 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 180 185 190 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 195 200 205 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 210 215 220 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 17 <211> 705 <212> DNA <213> Artificial Sequence <220> <223> 655-341 Ab LC NA <400> 17 atggaaaccc cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 120 ctctcctgca gggccagtca gggtattagt agaagcgaat tagcctggta ccagcagaaa 180 cctggccagg ctcccagcct cctcatctat ggtgcatcca gcagggccac tggcatccca 240 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 300 cctgaagatt ttgcagtgta ttactgtcaa caatttggta gttcaccgtg gacgttcggc 360 caagggacca aggtggaaat caaacgaact gtggctgcac catctgtctt catcttcccg 420 ccatctgatg agcagttgaa atctggaact gctagcgttg tgtgcctgct gaataacttc 480 tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 540 caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 600 acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 660 ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgt 705 <210> 18 <211> 235 <212> PRT <213> Artificial Sequence <220> <223> 655-341 Ab LC AA w / CPM S230K (EU S176K) <400> 18 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser 20 25 30 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly 35 40 45 Ile Ser Arg Ser Glu Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 55 60 Pro Ser Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro 65 70 75 80 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90 95 Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe 100 105 110 Gly Ser Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 115 120 125 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 130 135 140 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 145 150 155 160 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 165 170 175 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 180 185 190 Thr Tyr Ser Leu Lys Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 195 200 205 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 210 215 220 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 19 <211> 705 <212> DNA <213> Artificial Sequence <220> <223> 655-341 Ab LC NA w / CPM S230K (EU S176K) <400> 19 atggaaaccc cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 120 ctctcctgca gggccagtca gggtattagt agaagcgaat tagcctggta ccagcagaaa 180 cctggccagg ctcccagcct cctcatctat ggtgcatcca gcagggccac tggcatccca 240 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 300 cctgaagatt ttgcagtgta ttactgtcaa caatttggta gttcaccgtg gacgttcggc 360 caagggacca aggtggaaat caaacgaact gtggctgcac catctgtctt catcttcccg 420 ccatctgatg agcagttgaa atctggaact gctagcgttg tgtgcctgct gaataacttc 480 tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 540 caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcaa gagcaccctg 600 acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 660 ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgt 705 <210> 20 <211> 470 <212> PRT <213> Artificial Sequence <220> <223> 655-341 Ab HC AA <400> 20 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile 35 40 45 Ser Ser Gly Asp Tyr Phe Trp Ser Trp Ile Arg Gln Leu Pro Gly Lys 50 55 60 Gly Leu Glu Trp Ile Gly His Ile His Asn Ser Gly Thr Thr Tyr Tyr 65 70 75 80 Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys 85 90 95 Lys Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ala Arg Asp Arg Gly Gly Asp Tyr Ala Tyr Gly Met 115 120 125 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 145 150 155 160 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 225 230 235 240 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 370 375 380 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly 465 470 <210> 21 <211> 1410 <212> DNA <213> Artificial Sequence <220> <223> 655-341 Ab HC NA <400> 21 atgaagcacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt cctgtcccag 60 gtgcagctgc aggagtcggg cccaggactg gtgaagcctt cacagaccct gtccctcacc 120 tgcactgtct ctggtggctc catcagcagt ggtgattact tctggagctg gatccgccag 180 ctcccaggga agggcctgga gtggattggg cacatccata acagtgggac cacctactac 240 aatccgtccc tcaagagtcg agttaccata tcagtagaca cgtctaagaa gcagttctcc 300 ctgaggctga gttctgtgac tgccgcggac acggccgtat attactgtgc gagagatcga 360 gggggtgact acgcttatgg tatggacgtc tggggccaag ggaccacggt caccgtctcc 420 tcagcctcca ccaagggccc atccgtcttc cccctggcac cctcctccaa gagcacctct 480 ggggggcacag cggccctggg ctgcctggtc areactact tccccgaacc ggtgacggtg 540 tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc 600 tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt gggcacccag 660 acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gagagttgag 720 cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 780 ggaccgtcag tcttctctctt ccccccaaaaa cccaaggaca ccctcatgat ctcccggacc 840 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 900 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 960 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1020 aaggagtca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaccatc 1080 tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggag 1140 gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1200 atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1260 gtgctggact ccgacggctc cttcttcctc tatagcaagc tcaccgtgga caagagcagg 1320 tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1380 acgcagaaga gcctctccct gtctccgggt 1410 <210> 22 <211> 470 <212> PRT <213> Artificial Sequence <220> <223> 655-341 Ab HC AA w / CPM S230E (EU S183E) <400> 22 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile 35 40 45 Ser Ser Gly Asp Tyr Phe Trp Ser Trp Ile Arg Gln Leu Pro Gly Lys 50 55 60 Gly Leu Glu Trp Ile Gly His Ile His Asn Ser Gly Thr Thr Tyr Tyr 65 70 75 80 Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys 85 90 95 Lys Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ala Arg Asp Arg Gly Gly Asp Tyr Ala Tyr Gly Met 115 120 125 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 145 150 155 160 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Glu Ser 195 200 205 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 225 230 235 240 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 370 375 380 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly 465 470 <210> 23 <211> 1410 <212> DNA <213> Artificial Sequence <220> <223> 655-341 Ab HC NA w / CPM S230E (EU S183E) <400> 23 atgaagcacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt cctgtcccag 60 gtgcagctgc aggagtcggg cccaggactg gtgaagcctt cacagaccct gtccctcacc 120 tgcactgtct ctggtggctc catcagcagt ggtgattact tctggagctg gatccgccag 180 ctcccaggga agggcctgga gtggattggg cacatccata acagtgggac cacctactac 240 aatccgtccc tcaagagtcg agttaccata tcagtagaca cgtctaagaa gcagttctcc 300 ctgaggctga gttctgtgac tgccgcggac acggccgtat attactgtgc gagagatcga 360 gggggtgact acgcttatgg tatggacgtc tggggccaag ggaccacggt caccgtctcc 420 tcagcctcca ccaagggccc atccgtcttc cccctggcac cctcctccaa gagcacctct 480 gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg 540 tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc 600 tcaggactct actccctcga gagcgtggtg accgtgccct ccagcagctt gggcacccag 660 acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gagagttgag 720 cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 780 ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 840 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 900 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 960 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1020 aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1080 tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggag 1140 gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1200 atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1260 gtgctggact ccgacggctc cttcttcctc tatagcaagc tcaccgtgga caagagcagg 1320 tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1380 acgcagaaga gcctctccct gtctccgggt 1410 <210> 24 <211> 244 <212> PRT <213> Artificial Sequence <220> <223> 655-341 Fab HC AA <400> 24 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile 35 40 45 Ser Ser Gly Asp Tyr Phe Trp Ser Trp Ile Arg Gln Leu Pro Gly Lys 50 55 60 Gly Leu Glu Trp Ile Gly His Ile His Asn Ser Gly Thr Thr Tyr Tyr 65 70 75 80 Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys 85 90 95 Lys Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ala Arg Asp Arg Gly Gly Asp Tyr Ala Tyr Gly Met 115 120 125 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 145 150 155 160 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 225 230 235 240 Pro Lys Ser Cys <210> 25 <211> 732 <212> DNA <213> Artificial Sequence <220> <223> 655-341 Fab HC NA <400> 25 atgaagcacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt cctgtcccag 60 gtgcagctgc aggagtcggg cccaggactg gtgaagcctt cacagaccct gtccctcacc 120 tgcactgtct ctggtggctc catcagcagt ggtgattact tctggagctg gatccgccag 180 ctcccaggga agggcctgga gtggattggg cacatccata acagtgggac cacctactac 240 aatccgtccc tcaagagtcg agttaccata tcagtagaca cgtctaagaa gcagttctcc 300 ctgaggctga gttctgtgac tgccgcggac acggccgtat attactgtgc gagagatcga 360 gggggtgact acgcttatgg tatggacgtc tggggccaag ggaccacggt caccgtctcc 420 tcagcctcca ccaagggccc atccgtcttc cccctggcac cctcctccaa gagcacctct 480 gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg 540 tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc 600 tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt gggcacccag 660 acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gagagttgag 720 cccaaatctt gt 732 <210> 26 <211> 244 <212> PRT <213> Artificial Sequence <220> <223> 655-341 Fab HC AA w / CPM S230E (EU S183E) <400> 26 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile 35 40 45 Ser Ser Gly Asp Tyr Phe Trp Ser Trp Ile Arg Gln Leu Pro Gly Lys 50 55 60 Gly Leu Glu Trp Ile Gly His Ile His Asn Ser Gly Thr Thr Tyr Tyr 65 70 75 80 Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys 85 90 95 Lys Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ala Arg Asp Arg Gly Gly Asp Tyr Ala Tyr Gly Met 115 120 125 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 145 150 155 160 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Glu Ser 195 200 205 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 225 230 235 240 Pro Lys Ser Cys <210> 27 <211> 732 <212> DNA <213> Artificial sequence <220> <223> 655-341 Fab HC NA w / CPM S230E (EU S183E) <400> 27 atgaagcacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt cctgtcccag 60 gtgcagctgc aggagtcggg cccaggactg gtgaagcctt cacagaccct gtccctcacc 120 tgcactgtct ctggtggctc catcagcagt ggtgattact tctggagctg gatccgccag 180 ctcccaggga agggcctgga gtggattggg cacatccata acagtgggac cacctactac 240 aatccgtccc tcaagagtcg agttaccata tcagtagaca cgtctaagaa gcagttctcc 300 ctgaggctga gttctgtgac tgccgcggac acggccgtat attactgtgc gagagatcga 360 gggggtgact acgcttatgg tatggacgtc tggggccaag ggaccacggt caccgtctcc 420 480. tcagcctcca ccaagggccc atccgtcttc cccctggcac cctcctccaa gagcacctct gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg 540 tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc 600 tcaggactct actccctcga gagcgtggtg accgtgccct ccagcagctt gggcacccag 660 acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gagagttgag cccaaatctt gt <210> 28 <211> 708 <212> DNA <213> The snowstorm <220> <223> DNP Ab LC NA (TTR license plate) <400> 28 atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct gagaggtgcg cgctgtgaca tccagatgac ccagtctcca tcctccctgt ctgcatctga aggagacaga gtcaccatca cttgccgggc aagtcagggc attagaaatg atttaggctg gtatcagcag 240. aaaccagggga aagcccctaa gcgcctgatc tatgctgcat ccagtttgca aagtggggtc ccattaaggt tcagcggcag tggatctggg acagaattca ctctcacaat cagcagcctg 300 cagcctgaag attttgcaac ttattactgt ctacagtata atagttaccc gtggacgttc 360 ggccaaggga ccaaggtgga aatcaaacgt acggtggctg caccatctgt cttcatcttc 420 ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac 480 ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac 540 tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc 600 ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat 660 cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgt 708 <210> 29 <211> 236 <212> PRT <213> Artificial Sequence <220> <223> DNP Ab LC AA (TTR construct) <400> 29 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala Ser Glu Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45 Gln Gly Ile Arg Asn Asp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Arg Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val 65 70 75 80 Pro Leu Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln 100 105 110 Tyr Asn Ser Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 30 <211> 1809 <212> DNA <213> Artificial sequence <220> <223> DNP Ab HC NA (TTR construct) <400> 30 atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct gagaggtgcg 60 cgctgtcagg tgcagctgca ggagtcgggc ccaggactgg ttaagccttc ggagaccctg 120 tccctcacct gcactgtctc tggtggctcc atcagtagtt actactggag ctggatccgg 180 cagcccccag ggaagggact ggagtggatt gggtatatct attacagtgg gaacaccaac 240 tccaacccct ccctcaagag tcgagtcacc atatcagtag acacgtccaa gaaccagttc 300 tccctgaagc tgagctctgt gaccgctgcg gacacggccg tgtattactg tgcgagaacc 360 tactatgata gtagtggtta ctactaccgt gcttttgata tctggggcca agggacaatg 420 gtcaccgtct ctagtgcctc caccaagggc ccatcggtct tccccctggc accctcctcc 480 aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 540 ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct 600 gtcctacagt cctcaggact ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc 660 ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtggac 720 aagaaagttg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacct 780 gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 840 atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 900 gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 960 gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1020 tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1080 gagaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc ccatcccggg aggagac caagaccag gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtggggag agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctatagcaa gctcaccgtg 1320 1380. ttctcatgct ccgtgatgca tgaggctctg 1440. cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtggaggcgg tccaactggt actggtgaat ctaaagctcc tcttatggtt gcagtcaaag atgctgttcg tggttccccg gcaattaatg ttgctgtaca tgttttccgt aaagctgctg acgacacctg ggaaccgttc gctagcggta aaacctccga atccggtga ctgcacggtc tgaccaccga agaagaattc gttgaaggta tctacaaagt tgaaatcgac accaaatcct actggaaagc tttgggtatc tccccgttcc acgaacacgc tgaagttgtt ttcaccgcta acgactccgg tccgcgtcgt 1740 1800. Tacacgatcg ctgctctgct gtccccgtac tcctactcca ccaccgctgt tgttaccaac ccgaaagaa 1809 <210> 31 <211> 603 <212> PRT <213> artificial sequence <220> <223> DNP Ab LC AA (TTR construct <400> 31 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Gln Glu Ser Gly Pro Gly 20 25 30 Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly 35 40 45 Gly Ser Ile Ser Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly 50 55 60 Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Asn Thr Asn 65 70 75 80 Ser Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser 85 90 95 Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Arg Thr Tyr Tyr Asp Ser Ser Gly Tyr Tyr 115 120 125 Tyr Arg Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser 130 135 140 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 145 150 155 160 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 165 170 175 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 180 185 190 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 195 200 205 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 210 215 220 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 225 230 235 240 Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 245 250 255 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 260 265 270 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 275 280 285 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 290 295 300 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 305 310 315 320 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 325 330 335 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 340 345 350 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 355 360 365 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 370 375 380 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 385 390 395 400 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 405 410 415 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 420 425 430 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 435 440 445 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 450 455 460 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Pro Thr Gly 465 470 475 480 Thr Gly Glu Ser Lys Ala Pro Leu Met Val Ala Val Lys Asp Ala Val 485 490 495 Arg Gly Ser Pro Ala Ile Asn Val Ala Val His Val Phe Arg Lys Ala 500 505 510 Ala Asp Asp Thr Trp Glu Pro Phe Ala Ser Gly Lys Thr Ser Glu Ser 515 520 525 Gly Glu Leu His Gly Leu Thr Thr Glu Glu Glu Phe Val Glu Gly Ile 530 535 540 Tyr Lys Val Glu Ile Asp Thr Lys Ser Tyr Trp Lys Ala Leu Gly Ile 545 550 555 560 Ser Pro Phe His Glu His Ala Glu Val Val Phe Thr Ala Asn Asp Ser 565 570 575 Gly Pro Arg Arg Tyr Thr Ile Ala Ala Leu Leu Ser Pro Tyr Ser Tyr 580 585 590 Ser Thr Thr Ala Val Val Thr Asn Pro Lys Glu 595 600 <210> 32 <211> 705 <212> DNA <213> Artificial sequence <220> <223> 655-341 Ab LC NA (TTR construct) <400> 32 atggaaaccc cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 120 ctctcctgca gggccagtca gggtattagt agaagcgaat tagcctggta ccagcagaaa 180 cctggccagg ctcccagcct cctcatctat ggtgcatcca gcagggccac tggcatccca 240 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 300 cctgaagatt ttgcagtgta ttactgtcaa caatttggta gttcaccgtg gacgttcggc 360 caagggacca aggtggaaat caaacgaact gtggctgcac catctgtctt catcttcccg 420 ccatctgatg agcagttgaa atctggaact gctagcgttg tgtgcctgct gaataacttc 480 tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 540 caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 600 acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 660 ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgt 705 <210> 33 <211> 235 <212> PRT <213> artificial sequence <220> <223> 655-341 Ab LC AA (TTR) <400> 33 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser 20 25 30 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly 35 40 45 Ile Ser Arg Ser Glu Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 55 60 Pro Ser Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro 65 70 75 80 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90 95 Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe 100 105 110 Gly Ser Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 115 120 125 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 130 135 140 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 145 150 155 160 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 165 170 175 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 180 185 190 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 195 200 205 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 210 215 220 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 34 <211> 1797 <212> DNA <213> Artificial Sequence <220> <223> 655 - 341 Ab HC NA (TTR construct) <400> 34 atgaagcacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt cctgtcccag 60 gtgcagctgc aggagtcggg cccaggactg gtgaagcctt cacagaccct gtccctcacc 120 tgcactgtct ctggtggctc catcagcagt ggtgattact tctggagctg gatccgccag 180 ctcccaggga agggcctgga gtggattggg cacatccata acagtgggac cacctactac 240 aatccgtccc tcaagagtcg agttaccata tcagtagaca cgtctaagaa gcagttctcc 300 ctgaggctga gttctgtgac tgccgcggac acggccgtat attactgtgc gagagatcga 360 gggggtgact acgcttatgg tatggacgtc tggggccaag ggaccacggt caccgtctcc 420 tcagcctcca ccaagggccc atccgtcttc cccctggcac cctcctccaa gagcacctct 480 gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg 540 tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc 600 tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt gggcacccag 660 acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gagagttgag 720 cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 780 ggaccgtcag tcttctctctt ccccccaaaaa cccaaggaca ccctcatgat ctcccggacc 840 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 900 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 960 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1020 aaggagtca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaccatc 1080 tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggag 1140 gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1200 atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1260 gtgctggact ccgacggctc cttcttcctc tagatcaagc tcaccgtgga caagagcagg 1320 tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1380 your acgcaaga gcctctccct gtctccgggt ggaggcggtc caactggtac tggtgaatct 1440 aaagctcctc ttatggttgc agtcgatgat gctgttcgtg gttccccggc aattaatgtt 1500 gctgtacatg ttttccgtaa agctgctgac gacacctggg aaccgttcgc tagcggtaaa 1560 acctccgaat ccggtgaact gcacggtctg accaccgaag aagaattcgt tgaaggtatc 1620 tacaaagttg aaatcgacac caaatcctac tggaaagctt tgggtatctc cccgttccac 1680 gaacacgctg aagttgtttt caccgctaac gactccggtc cgcgtcgtta cacgatcgct 1740 gctctgctgt ccccgtactc ctactccacc accgctgttg ttaccaaccc gaaagaa 1797 <210> 35 <211> 599 <212> PRT <213> Artificial Sequence <220> <223> 655-341 Ab LC AA (TTR construct) <400> 35 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile 35 40 45 Ser Ser Gly Asp Tyr Phe Trp Ser Trp Ile Arg Gln Leu Pro Gly Lys 50 55 60 Gly Leu Glu Trp Ile Gly His Ile His Asn Ser Gly Thr Thr Tyr Tyr 65 70 75 80 Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys 85 90 95 Lys Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ala Arg Asp Arg Gly Gly Asp Tyr Ala Tyr Gly Met 115 120 125 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 145 150 155 160 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 225 230 235 240 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 370 375 380 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Gly Gly Gly Pro Thr Gly Thr Gly Glu Ser 465 470 475 480 Lys Ala Pro Leu Met Val Ala Val Asp Asp Ala Val Arg Gly Ser Pro 485 490 495 Ala Ile Asn Val Ala Val His Val Phe Arg Lys Ala Ala Asp Asp Thr 500 505 510 Trp Glu Pro Phe Ala Ser Gly Lys Thr Ser Glu Ser Gly Glu Leu His 515 520 525 Gly Leu Thr Thr Glu Glu Glu Phe Val Glu Gly Ile Tyr Lys Val Glu 530 535 540 Ile Asp Thr Lys Ser Tyr Trp Lys Ala Leu Gly Ile Ser Pro Phe His 545 550 555 560 Glu His Ala Glu Val Val Phe Thr Ala Asn Asp Ser Gly Pro Arg Arg 565 570 575 Tyr Thr Ile Ala Ala Leu Leu Ser Pro Tyr Ser Tyr Ser Thr Thr Ala 580 585 590 Val Val Thr Asn Pro Lys Glu 595 <210> 36 <211> 705 <212> DNA <213> Artificial sequence <220> <223> 655-341 Fab LC NA (TTR construct) <400> 36 atggaaaccc cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 120 ctctcctgca gggccagtca gggtattagt agaagcgaat tagcctggta ccagcagaaa 180 cctggccagg ctcccagcct cctcatctat ggtgcatcca gcagggccac tggcatccca 240 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 300 cctgaagatt ttgcagtgta ttactgtcaa caatttggta gttcaccgtg gacgttcggc 360 caagggacca aggtggaaat caaacgaact gtggctgcac catctgtctt catcttcccg 420 ccatctgatg agcagttgaa atctggaact gctagcgttg tgtgcctgct gaataacttc 480 tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 540 caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 600 acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 660 ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgt 705 <210> 37 <211> 235 <212> PRT <213> Artificial Sequence <220> <223> 655 - 341 Fab LC AA (TTR construct) <400> 37 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser 20 25 30 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly 35 40 45 Ile Ser Arg Ser Glu Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 55 60 Pro Ser Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro 65 70 75 80 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90 95 Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe 100 105 110 Gly Ser Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 115 120 125 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 130 135 140 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 145 150 155 160 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 165 170 175 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 180 185 190 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 195 200 205 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 210 215 220 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 38 <211> 1143 <212> DNA <213> Artificial Sequence <220> <223> 655-341 Fab HC NA (TTR construct) <400> 38 atgaagcacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt cctgtcccag 60 gtgcagctgc aggagtcggg cccaggactg gtgaagcctt cacagaccct gtccctcacc 120 tgcactgtct ctggtggctc catcagcagt ggtgattact tctggagctg gatccgccag 180 ctcccaggga agggcctgga gtggattggg cacatccata acagtgggac cacctactac 240 aatccgtccc tcaagagtcg agttaccata tcagtagaca cgtctaagaa gcagttctcc 300 ctgaggctga gttctgtgac tgccgcggac acggccgtat attactgtgc gagagatcga 360 gggggtgact acgcttatgg tatggacgtc tggggccaag ggaccacggt caccgtctcc 420 tcagcctcca ccaagggccc atccgtcttc cccctggcac cctcctccaa gagcacctct 480 gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg 540 tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc 600 tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt gggcacccag 660 acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gagagttgag 720 cccaaatctt gtggaggagg tccaactggt actggtgaat ctaaagctcc tcttatggtt 780 gcagtcgatg atgctgttcg tggttccccg gcaattaatg ttgctgtaca tgttttccgt 840 aaagctgctg acgacacctg ggaaccgttc gctagcggta aaacctccga atccggtgaa 900 ctgcacggtc tgaccaccga agaagaattc gttgaaggta tctacaaagt tgaaatcgac 960 accaaatcct actggaaagc tttgggtatc tccccgttcc acgaacacgc tgaagttgtt 1020 ttcaccgcta acgactccgg tccgcgtcgt tacacgatcg ctgctctgct gtccccgtac 1080 tcctactcca ccaccgctgt tgttaccaac ccgaaagaag gcggtcacca tcaccatcac 1140 cac 1143 <210> 39 <211> 381 <212> PRT <213> Artificial Sequence <220> <223> 655-341 Fab HC AA (TTR construct) <400> 39 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile 35 40 45 Ser Ser Gly Asp Tyr Phe Trp Ser Trp Ile Arg Gln Leu Pro Gly Lys 50 55 60 Gly Leu Glu Trp Ile Gly His Ile His Asn Ser Gly Thr Thr Tyr Tyr 65 70 75 80 Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys 85 90 95 Lys Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ala Arg Asp Arg Gly Gly Asp Tyr Ala Tyr Gly Met 115 120 125 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 145 150 155 160 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 225 230 235 240 Pro Lys Ser Cys Gly Gly Gly Pro Thr Gly Thr Gly Glu Ser Lys Ala 245 250 255 Pro Leu Met Val Ala Val Asp Asp Ala Val Arg Gly Ser Pro Ala Ile 260 265 270 Asn Val Ala Val His Val Phe Arg Lys Ala Ala Asp Asp Thr Trp Glu 275 280 285 Pro Phe Ala Ser Gly Lys Thr Ser Glu Ser Gly Glu Leu His Gly Leu 290 295 300 Thr Thr Glu Glu Glu Phe Val Glu Gly Ile Tyr Lys Val Glu Ile Asp 305 310 315 320 Thr Lys Ser Tyr Trp Lys Ala Leu Gly Ile Ser Pro Phe His Glu His 325 330 335 Ala Glu Val Val Phe Thr Ala Asn Asp Ser Gly Pro Arg Arg Tyr Thr 340 345 350 Ile Ala Ala Leu Leu Ser Pro Tyr Ser Tyr Ser Thr Thr Ala Val Val 355 360 365 Thr Asn Pro Lys Glu Gly Gly His His His His His His 370 375 380 <210> 40 <211> 708 <212> DNA <213> Artificial sequence <220> <223> DNP Fab LC NA (TTR construct) <400> 40 atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct gagaggtgcg 60 cgctgtgaca tccagatgac ccagtctcca tcctccctgt ctgcatctga aggagacaga 120 gtcaccatca cttgccgggc aagtcagggc attagaaatg atttaggctg gtatcagcag 180 aaaccaggga aagcccctaa gcgcctgatc tatgctgcat ccagtttgca aagtggggtc 240 ccattaaggt tcagcggcag tggatctggg acagaattca ctctcacaat cagcagcctg 300 cagcctgaag attttgcaac ttattactgt ctacagtata atagttaccc gtggacgttc 360 ggccaaggga ccaaggtgga aatcaaacgt acggtggctg caccatctgt cttcatcttc 420 ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac 480 ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac 540 tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc 600 ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat 660 cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgt 708 <210> 41 <211> 236 <212> PRT <213> Artificial Sequence <220> <223> DNP Fab LC AA (TTR construct <400> 41 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala Ser Glu Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45 Gln Gly Ile Arg Asn Asp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Arg Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val 65 70 75 80 Pro Leu Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln 100 105 110 Tyr Asn Ser Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 42 <211> 1155 <212> PRT <213> Artificial Sequence <220> <223> DNP Fab HC NA (TTR construct) <400> 42 Ala Thr Gly Gly Ala Cys Ala Thr Gly Ala Gly Gly Gly Thr Gly Cys 1 5 10 15 Cys Cys Gly Cys Thr Cys Ala Gly Cys Thr Cys Cys Thr Gly Gly Gly 20 25 30 Gly Cys Thr Cys Cys Thr Gly Cys Thr Gly Cys Thr Gly Thr Gly Gly 35 40 45 Cys Thr Gly Ala Gly Ala Gly Gly Thr Gly Cys Gly Cys Gly Cys Thr 50 55 60 Gly Thr Cys Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Gly Cys Ala 65 70 75 80 Gly Gly Ala Gly Thr Cys Gly Gly Gly Cys Cys Cys Ala Gly Gly Ala 85 90 95 Cys Thr Gly Gly Thr Thr Ala Ala Gly Cys Cys Thr Thr Cys Gly Gly 100 105 110 Ala Gly Ala Cys Cys Cys Thr Gly Thr Cys Cys Cys Thr Cys Ala Cys 115 120 125 Cys Thr Gly Cys Ala Cys Thr Gly Thr Cys Thr Cys Thr Gly Gly Thr 130 135 140 Gly Gly Cys Thr Cys Cys Ala Thr Cys Ala Gly Thr Ala Gly Thr Thr 145 150 155 160 Ala Cys Thr Ala Cys Thr Gly Gly Ala Gly Cys Thr Gly Gly Ala Thr 165 170 175 Cys Cys Gly Gly Cys Ala Gly Cys Cys Cys Cys Cys Ala Gly Gly Gly 180 185 190 Ala Ala Gly Gly Gly Ala Cys Thr Gly Gly Ala Gly Thr Gly Gly Ala 195 200 205 Thr Thr Gly Gly Gly Thr Ala Thr Ala Thr Cys Thr Ala Thr Thr Ala 210 215 220 Cys Ala Gly Thr Gly Gly Gly Ala Ala Cys Ala Cys Cys Ala Ala Cys 225 230 235 240 Thr Cys Cys Ala Ala Cys Cys Cys Cys Thr Cys Cys Cys Thr Cys Ala 245 250 255 Ala Gly Ala Gly Thr Cys Gly Ala Gly Thr Cys Ala Cys Cys Ala Thr 260 265 270 Ala Thr Cys Ala Gly Thr Ala Gly Ala Cys Ala Cys Gly Thr Cys Cys 275 280 285 Ala Ala Gly Ala Ala Cys Cys Ala Gly Thr Thr Cys Thr Cys Cys Cys 290 295 300 Thr Gly Ala Ala Gly Cys Thr Gly Ala Gly Cys Thr Cys Thr Gly Thr 305 310 315 320 Gly Ala Cys Cys Gly Cys Thr Gly Cys Gly Gly Ala Cys Ala Cys Gly 325 330 335 Gly Cys Cys Gly Thr Gly Thr Ala Thr Thr Ala Cys Thr Gly Thr Gly 340 345 350 Cys Gly Ala Gly Ala Ala Cys Cys Thr Ala Cys Thr Ala Thr Gly Ala 355 360 365 Thr Ala Gly Thr Ala Gly Thr Gly Gly Thr Thr Ala Cys Thr Ala Cys 370 375 380 Thr Ala Cys Cys Gly Thr Gly Cys Thr Thr Thr Thr Gly Ala Thr Ala 385 390 395 400 Thr Cys Thr Gly Gly Gly Gly Cys Cys Ala Ala Gly Gly Gly Ala Cys 405 410 415 Ala Ala Thr Gly Gly Thr Cys Ala Cys Cys Gly Thr Cys Thr Cys Thr 420 425 430 Ala Gly Thr Gly Cys Cys Thr Cys Cys Ala Cys Cys Ala Ala Gly Gly 435 440 445 Gly Cys Cys Cys Ala Thr Cys Gly Gly Thr Cys Thr Thr Cys Cys Cys 450 455 460 Cys Cys Thr Gly Gly Cys Ala Cys Cys Cys Thr Cys Cys Thr Cys Cys 465 470 475 480 Ala Ala Gly Ala Gly Cys Ala Cys Cys Thr Cys Thr Gly Gly Gly Gly 485 490 495 Gly Cys Ala Cys Ala Gly Cys Gly Gly Cys Cys Cys Thr Gly Gly Gly 500 505 510 Cys Thr Gly Cys Cys Thr Gly Gly Thr Cys Ala Ala Gly Gly Ala Cys 515 520 525 Thr Ala Cys Thr Thr Cys Cys Cys Cys Gly Ala Ala Cys Cys Gly Gly 530 535 540 Thr Gly Ala Cys Gly Gly Thr Gly Thr Cys Gly Thr Gly Gly Ala Ala 545 550 555 560 Cys Thr Cys Ala Gly Gly Cys Gly Cys Cys Cys Thr Gly Ala Cys Cys 565 570 575 Ala Gly Cys Gly Gly Cys Gly Thr Gly Cys Ala Cys Ala Cys Cys Thr 580 585 590 Thr Cys Cys Cys Gly Gly Cys Thr Gly Thr Cys Cys Thr Ala Cys Ala 595 600 605 Gly Thr Cys Cys Thr Cys Ala Gly Gly Ala Cys Thr Cys Thr Ala Cys 610 615 620 Thr Cys Cys Cys Thr Cys Ala Gly Cys Ala Gly Cys Gly Thr Gly Gly 625 630 635 640 Thr Gly Ala Cys Cys Gly Thr Gly Cys Cys Cys Thr Cys Cys Ala Gly 645 650 655 Cys Ala Gly Cys Thr Thr Gly Gly Gly Cys Ala Cys Cys Cys Ala Gly 660 665 670 Ala Cys Cys Thr Ala Cys Ala Thr Cys Thr Gly Cys Ala Ala Cys Gly 675 680 685 Thr Gly Ala Ala Thr Cys Ala Cys Ala Ala Gly Cys Cys Cys Ala Gly 690 695 700 Cys Ala Ala Cys Ala Cys Cys Ala Ala Gly Gly Thr Gly Gly Ala Cys 705 710 715 720 Ala Ala Gly Ala Ala Ala Gly Thr Thr Gly Ala Gly Cys Cys Cys Ala 725 730 735 Ala Ala Thr Cys Thr Thr Gly Thr Gly Gly Ala Gly Gly Ala Gly Gly 740 745 750 Thr Cys Cys Ala Ala Cys Thr Gly Gly Thr Ala Cys Thr Gly Gly Thr 755 760 765 Gly Ala Ala Thr Cys Thr Ala Ala Ala Gly Cys Thr Cys Cys Thr Cys 770 775 780 Thr Thr Ala Thr Gly Gly Thr Thr Gly Cys Ala Gly Thr Cys Ala Ala 785 790 795 800 Ala Gly Ala Thr Gly Cys Thr Gly Thr Thr Cys Gly Thr Gly Gly Thr 805 810 815 Thr Cys Cys Cys Cys Gly Gly Cys Ala Ala Thr Thr Ala Ala Thr Gly 820 825 830 Thr Thr Gly Cys Thr Gly Thr Ala Cys Ala Thr Gly Thr Thr Thr Thr 835 840 845 Cys Cys Gly Thr Ala Ala Ala Gly Cys Thr Gly Cys Thr Gly Ala Cys 850 855 860 Gly Ala Cys Ala Cys Cys Thr Gly Gly Gly Ala Ala Cys Cys Gly Thr 865 870 875 880 Thr Cys Gly Cys Thr Ala Gly Cys Gly Gly Thr Ala Ala Ala Ala Cys 885 890 895 Cys Thr Cys Cys Gly Ala Ala Thr Cys Cys Gly Gly Thr Gly Ala Ala 900 905 910 Cys Thr Gly Cys Ala Cys Gly Gly Thr Cys Thr Gly Ala Cys Cys Ala 915 920 925 Cys Cys Gly Ala Ala Gly Ala Ala Gly Ala Ala Thr Thr Cys Gly Thr 930 935 940 Thr Gly Ala Ala Gly Gly Thr Ala Thr Cys Thr Ala Cys Ala Ala Ala 945 950 955 960 Gly Thr Thr Gly Ala Ala Ala Thr Cys Gly Ala Cys Ala Cys Cys Ala 965 970 975 Ala Ala Thr Cys Cys Thr Ala Cys Thr Gly Gly Ala Ala Ala Gly Cys 980 985 990 Thr Thr Thr Gly Gly Gly Thr Ala Thr Cys Thr Cys Cys Cys Cys Gly 995 1000 1005 Thr Thr Cys Cys Ala Cys Gly Ala Ala Cys Ala Cys Gly Cys Thr 1010 1015 1020 Gly Ala Ala Gly Thr Thr Gly Thr Thr Thr Thr Cys Ala Cys Cys 1025 1030 1035 Gly Cys Thr Ala Ala Cys Gly Ala Cys Thr Cys Cys Gly Gly Thr 1040 1045 1050 Cys Cys Gly Cys Gly Thr Cys Gly Thr Thr Ala Cys Ala Cys Gly 1055 1060 1065 Ala Thr Cys Gly Cys Thr Gly Cys Thr Cys Thr Gly Cys Thr Gly 1070 1075 1080 Thr Cys Cys Cys Cys Gly Thr Ala Cys Thr Cys Cys Thr Ala Cys 1085 1090 1095 Thr Cys Cys Ala Cys Cys Ala Cys Cys Gly Cys Thr Gly Thr Thr 1100 1105 1110 Gly Thr Thr Ala Cys Cys Ala Ala Cys Cys Cys Gly Ala Ala Ala 1115 1120 1125 Gly Ala Ala Gly Gly Cys Gly Gly Thr Cys Ala Cys Cys Ala Thr 1130 1135 1140 Cys Ala Cys Cys Ala Thr Cys Ala Cys Cys Ala Cys 1145 1150 1155 <210> 43 <211> 385 <212> PRT <213> Artificial Sequence <220> <223> DNP Fab HC AA (TTR construct) <400> 43 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Gln Glu Ser Gly Pro Gly 20 25 30 Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly 35 40 45 Gly Ser Ile Ser Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly 50 55 60 Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Asn Thr Asn 65 70 75 80 Ser Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser 85 90 95 Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Arg Thr Tyr Tyr Asp Ser Ser Gly Tyr Tyr 115 120 125 Tyr Arg Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser 130 135 140 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 145 150 155 160 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 165 170 175 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 180 185 190 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 195 200 205 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 210 215 220 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 225 230 235 240 Lys Lys Val Glu Pro Lys Ser Cys Gly Gly Gly Pro Thr Gly Thr Gly 245 250 255 Glu Ser Lys Ala Pro Leu Met Val Ala Val Lys Asp Ala Val Arg Gly 260 265 270 Ser Pro Ala Ile Asn Val Ala Val His Val Phe Arg Lys Ala Ala Asp 275 280 285 Asp Thr Trp Glu Pro Phe Ala Ser Gly Lys Thr Ser Glu Ser Gly Glu 290 295 300 Leu His Gly Leu Thr Thr Glu Glu Glu Phe Val Glu Gly Ile Tyr Lys 305 310 315 320 Val Glu Ile Asp Thr Lys Ser Tyr Trp Lys Ala Leu Gly Ile Ser Pro 325 330 335 Phe His Glu His Ala Glu Val Val Phe Thr Ala Asn Asp Ser Gly Pro 340 345 350 Arg Arg Tyr Thr Ile Ala Ala Leu Leu Ser Pro Tyr Ser Tyr Ser Thr 355 360 365 Thr Ala Val Val Thr Asn Pro Lys Glu Gly Gly His His His His His 370 375 380 His 385 <210> 44 <211> 381 <212> DNA <213> Artificial Sequence <220> <223> Human TTR NA <400> 44 60. ggtccaactg gtaccggtga atccaagtgt cctctgatgg tcaaagttct agatgctgtc 120. cgaggcagtc ctgccatcaa tgtggccgtg catgtgttca gaaaggctgc tgatgacacc tgggagccat ttgcctctgg gaaaaccagt gagtctggag agctgcatgg gctcacaact gaggaggat ttgtagaagg throwcaaa gtggaatag acaccaaatc ttactggaag gcacttggca tctccccatt ccatgagcat gcagaggtgg tattcacagc caacgactcc ggccccgcc gctacaccat tgccgccctg ctgagcccct actcctattc caccacggct 360 gtcgtcacca atcccaagga a

Claims

1. A thyroxine transporter (TTR) protein complex, wherein the TTR protein complex comprises TTR subunits A, B, C and D; Among them, TTR subunits A and B dimerize to form TTR dimer AB; Among them, TTR subunits C and D dimerize to form TTR dimer CD; Wherein TTR dimers AB and CD further dimerize to form TTR tetramer ABCD; and wherein each of A, B, C, and D comprises the amino acid sequence of SEQ ID NO: 1, except that: at least one amino acid in the interface between TTR dimer AB and TTR dimer CD is mutated so as to favor the formation of an ABCD tetramer over the formation of an ABAB tetramer or a CDCD tetramer; Where each of A, B, C, and D contains the following mutations: A and B both contain C10A / K15A / L17D, and C and D both contain C10A / K15A / V121R; A and B both contain C10A / K15A / L17D, and C and D both contain C10A / K15A / V121K; A and B both contain C10A / K15A / L17E, and C and D both contain C10A / K15A / V121R; A and B both contain C10A / K15A / V20D, and C and D both contain C10A / K15A / V20R; A and B both contain C10A / K15A / V20D, and C and D both contain C10A / K15A / V20K; A and B both contain C10A / K15A / V20E, and C and D both contain C10A / K15A / V20R; A and B both contain C10A / K15A / V20E, and C and D both contain C10A / K15A / V20K; A and B both contain C10A / K15A / T119D, and C and D both contain C10A / K15A / L17R; A and B both contain C10A / K15A / T119D, and C and D both contain C10A / K15A / L17K; or A and B both contained C10A / K15A / V121E, and C and D both contained C10A / K15A / L17K.

2. The TTR protein complex of claim 1, wherein each of A, B, C and D comprises the amino acid sequence of SEQ ID NO: 1 with the following mutations: A and B both contain C10A / K15A / L17D / V20D, and C and D both contain C10A / K15A / L17K / V20K; A and B both contain C10A / K15A / L17D / V20E, and C and D both contain C10A / K15A / L17K / V20R; A and B both contain C10A / K15A / L17E / V20D, and C and D both contain C10A / K15A / L17R / V20K; A and B both contain C10A / K15A / L17E / V20E, and C and D both contain C10A / K15A / L17R / V20R; A and B both contain C10A / K15A / L17D / T119D, and C and D both contain C10A / K15A / L17K / V121K; A and B both contain C10A / K15A / L17D / V121E, and C and D both contain C10A / K15A / L17K / V121R; A and B both contain C10A / K15A / L17E / T119D, and C and D both contain C10A / K15A / L17R / V121K; A and B both contain C10A / K15A / L17E / V121E, and C and D both contain C10A / K15A / L17R / V121R; A and B both contain C10A / K15A / V20D / T119D, and C and D both contain C10A / K15A / V20K / V121K; A and B both contain C10A / K15A / V20D / V121E, and C and D both contain C10A / K15A / V20K / V121R; A and B both contain C10A / K15A / V20E / T119D, and C and D both contain C10A / K15A / V20R / V121K; or A and B both contain C10A / K15A / V20E / V121E, and C and D both contain C10A / K15A / V20R / V121R.

3. The TTR protein complex according to claim 1 or 2, wherein each of A, B, C and D comprises the amino acid sequence of SEQ ID NO: 1 with the following mutations: A and B both contain C10A / K15A / V20E / T119D, and C and D both contain C10A / K15A / V20R / V121K; A and B both contain C10A / K15A / L17D / T119D, and C and D both contain C10A / K15A / L17K / V121K; A and B both contain C10A / K15A / L17E / T119D, and C and D both contain C10A / K15A / L17R / V121K; A and B both contain C10A / K15A / L17E / V20D, and C and D both contain C10A / K15A / L17R / V20K; A and B both contain C10A / K15A / L17D / V20D, and C and D both contain C10A / K15A / L17K / V20K; or Both A and B contained C10A / K15A / L17E / V121E, and both C and D contained C10A / K15A / L17R / V121R.

4. The TTR protein complex of any one of claims 1-3, wherein the TTR protein complex is attached to 1, 2, 3 or 4 antigen binding proteins or peptides, and wherein the antigen binding proteins are antibodies, Fabs, scFvs, or a mixture of antibodies and Fabs.

5. The TTR protein complex of claim 4, wherein the antigen binding protein or peptide is attached to the TTR protein complex at the C-terminus of the TTR subunit.

6. The TTR protein complex of claim 4, wherein the antigen binding protein or peptide is attached to the TTR protein complex at the N-terminus of the TTR subunit.

7. The TTR protein complex of any one of claims 4-6, wherein the TTR protein complex: directly attached to the antigen binding protein; or Attached to the antigen binding protein via a linker.

8. The TTR protein complex of claim 7, wherein the linker is an amino acid based linker comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids.

9. The TTR protein complex of claim 8, wherein the linker is an amino acid based linker comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids.

10. The TTR protein complex of claim 9, wherein the linker is an amino acid based linker comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.

11. The TTR protein complex of claim 10, wherein the linker is an amino acid based linker comprising 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

12. The TTR protein complex of claim 8, wherein the linker is G, GG, GGG, GGGG, GGGGG, GGGGGG, GGGGGGG, GGGGGGGG, GGGGGGGGG, or GGGGGGGGGGGG.

13. The TTR protein complex of claim 8, wherein the linker is G(G x B y ) r G z And among them: G = glycine; B = any amino acid; x＝1-15； y＝1-5； z = 1-15; and r＝1-20。 14. The TTR protein complex according to claim 13, wherein: B = Q, S, A, E, P, T, K, R, D or N; x＝4； y＝1； z = 4; and r＝1。 15. The TTR protein complex of claim 8, wherein the linker is selected from the list consisting of GG, GGGG, GGGSGG, GGGSGGGG, and GGAGGGAGGG.

16. The TTR protein complex of any one of claims 4-15, wherein the TTR protein complex is attached to two antigen binding proteins, wherein the antigen binding proteins bind different antigens.

17. The TTR protein complex of any one of claims 4-15, wherein the TTR protein complex is attached to four antigen binding proteins, wherein the antigen binding proteins bind at least two different antigens.

18. A pharmaceutical composition comprising the TTR protein complex according to any one of claims 1-17.

19. Use of the TTR protein complex according to any one of claims 4 to 17 in the preparation of a medicament for treating cancer.

20. One or more isolated nucleic acids encoding the TTR protein complex of any one of claims 1-17.

21. An expression vector comprising the nucleic acid according to claim 20.

22. A recombinant host cell comprising the nucleic acid according to claim 20 or the vector according to claim 21.

23. The recombinant host cell of claim 22, wherein the host cell is a Chinese hamster ovary (CHO) cell, an E5 cell, a baby hamster kidney (BHK) cell, a monkey kidney (COS) cell, a human hepatocellular carcinoma cell, or a human embryonic kidney 293 (HEK 293) cell.

24. A method for preparing the TTR protein complex according to any one of claims 1-17, the method comprising: a) culturing the recombinant host cell according to claim 22 or 23; and b) isolating the TTR protein complex from said culture.

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