Multispecific transthyretin immunoglobulin fusion

Abstract

The present invention relates to multispecific transthyretin (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 for treating diseases using the TTR complexes of the invention are described herein.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority and benefit to U.S. Provisional Patent Application No. 62 / 871,247, filed Jul. 8, 2019, which is hereby incorporated by reference in its entirety.

[0002] Reference to Sequence Listing This application includes a Sequence Listing in computer - readable form. The Sequence Listing is provided as a text file named A - 2414 - WO - PCT_SeqList_ST25.txt, created on Jul. 1, 2020, and is 101,660 bytes in size. The information in the electronic format of this Sequence Listing is hereby incorporated by reference in its entirety.

[0003] The present invention relates to multispecific transthyretin (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 present invention are described herein.

Background Art

[0004] Monospecific antibodies, i.e., antibodies that bind to a single antigen, are a well - established class of compounds approved in a variety of therapeutic fields. In fact, over the past decade, many monospecific antibody - based pharmaceuticals have been approved in various countries.

[0005] Multispecific proteins, such as multispecific antibodies, are increasingly the subject of research. Multispecific proteins can bind to two or more different antigens on the same or different proteins. This enables the possibility of two different biological pathways, or the ability to execute those pathways simultaneously.

[0006] Transthyretin (TTR) is a non-covalently bound tetrameric human serum and cerebrospinal fluid protein that plays a role in the transport of a portion of circulating thyroxine and the serum half-life of retinol-binding protein. TTR typically exists as a tetramer (about 56 kDa) serum protein, and each monomer unit has a molecular weight of about 14 kDa.

[0007] Attempts made prior to multimerizing the protein 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 / barstar complexes (Deyev et al., Nature Biotechnology, 21(12):1486-1492(2003)), and Dock N Lock technology (protein kinase and A-kinase anchor protein anchor domain interaction) (Goldenberg et al., Journal of Nuclear Medicine, 49(1):158-163(2008)).

[0008] However, there is still a need for an efficient means of generating multispecific proteins (e.g., whole antibodies and antibody fragments) that can bind to multiple epitopes on the same or different proteins.

Prior Art Documents

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

Means for Solving the Problems

[0010] The present invention relates to a TTR protein complex, where the TTR protein complex includes TTR subunits A, B, C, and D; TTR subunits A and B dimerize to form a TTR dimer AB; TTR subunits C and D dimerize to form a TTR dimer CD; TTR dimers AB and CD further dimerize to form a TTR tetramer ABCD; Each of A, B, C, and D contains the amino acid sequence of SEQ ID NO: 1, except that at least one amino acid at the interface between the TTR dimer AB and the TTR dimer CD is mutated such that the formation of the ABCD tetramer is more favorable than the formation of any other tetramer (e.g., ABAB tetramer or CDCD tetramer).

[0011] Each of the A, B, C, and D subunits of the TTR protein complex can contain the amino acid sequence of SEQ ID NO: 1 having the following mutations: C10A, K15A, or both C10A and K15A.

[0012] Thus, in one embodiment, the present invention relates to a TTR protein complex, where both A and B, both C and D, or all four of A, B, C, and D contain mutations at one or more amino acid positions selected from the list comprising positions 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.

[0013] In another embodiment, the present invention relates to a TTR protein complex, where both A and B, both C and D, or all four of A, B, C, and D contain mutations at one or more amino acid positions selected from the list comprising positions 15, 17, 20, 21, 22, 23, 24, 51, 52, 84, 106, 108, 112, 114, 115, 119, 121, and 123 of SEQ ID NO: 1.

[0014] In another embodiment, the present invention relates to a TTR protein complex, where both A and B, both C and D, or all four of A, B, C, and D contain mutations at one or more amino acid positions selected from the list comprising positions 15, 17, 20, 21, 22, 23, 24, 51, 52, 84, 106, 108, 112, 114, 115, 119, 121, and 123 of SEQ ID NO: 1, where the amino acids are mutated to aspartate, glutamate, arginine, lysine, or histidine.

[0015] In another embodiment, the present invention relates to a TTR protein complex, where A and B contain mutations at one or more amino acid positions selected from the list comprising positions 15, 17, 20, 21, 22, 23, 24, 51, 52, 84, 106, 108, 112, 114, 115, 119, 121, and 123 of SEQ ID NO: 1, where the amino acids are mutated to aspartate or glutamate.

[0016] In yet another embodiment, the invention relates to a TTR protein complex, where C and D comprise mutations at one or more amino acid positions selected from the list comprising positions 15, 17, 20, 21, 22, 23, 24, 51, 52, 84, 106, 108, 112, 114, 115, 119, 121, and 123 of SEQ ID NO: 1, where the amino acids are mutated to arginine, lysine, or histidine.

[0017] In certain embodiments, A and B comprise mutations at one or more amino acid positions selected from the list comprising positions 15, 17, 20, 21, 22, 23, 24, 51, 52, 84, 106, 108, 112, 114, 115, 119, 121, and 123 of SEQ ID NO: 1, where the amino acids are mutated to aspartate or glutamate; C and D comprise mutations at one or more amino acid positions selected from the list comprising positions 15, 17, 20, 21, 22, 23, 24, 51, 52, 84, 106, 108, 112, 114, 115, 119, 121, and 123 of SEQ ID NO: 1, where the amino acids are mutated to arginine, lysine, or histidine.

[0018] In some embodiments, A and B comprise at least one mutation in SEQ ID NO: 1, and said mutation is selected from the list comprising 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, and the mutation is selected from the list comprising L17D, L17E, V20D, V20E, G22D, G22E, S112D, S112E, T119D, T119E, V121D, and V121E.

[0019] In some embodiments, C and D comprise at least one mutation in SEQ ID NO: 1, and said mutation is selected from the list comprising K15R, L17R, V20R, G22R, S23R, P24R, D51R, S52R, I84R, T106R, A108R, S112R, Y114R, S115R, T119R, V121R, S123R, L17K, V20K, R21K, G22K, S23K, P24K, D51K, S52K, I84K, T106K, A108K, S112K, Y114K, S115K, T119K, V121K, S123K, K15H, L17H, V20H, R21H, G22H, S23H, P24H, D51H, S52H, I84H, T106H, 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, and the mutation is selected from the list comprising L17R, L17K, L17H, V20R, V20K, V20H, G22R, G22K, G22H, S112R, S112K, S112H, T119R, T119K, T119H, V121R, V121K, and V121H.

[0020] In another embodiment, both A and B, both C and D, or all four of A, B, C, and D independently contain one of the above-described mutations. In yet another embodiment, both A and B, both C and D, or all four of A, B, C, and D independently contain two of the above-described mutations.

[0021] In certain embodiments, the invention relates to a TTR protein, wherein each of A, B, C, and D independently contains the amino acid sequence of SEQ ID NO: 1 having the following mutations: A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / L17R (or vice versa); A and B contain C10A / K15A / L17E, and C and D contain C10A / K15A / L17R (or vice versa); A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / L17R (or vice versa); A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / L17R (or vice versa); A and B contain C10A / K15A / G22D, and C and D contain C10A / K15A / L17R (or vice versa); A and B contain C10A / K15A / G22E, and C and D contain C10A / K15A / L17R (or vice versa); A and B contain C10A / K15A / S112D, and C and D contain C10A / K15A / L17R (or vice versa); A and B contain C10A / K15A / S112E, and C and D contain C10A / K15A / L17R (or vice versa); A and B contain C10A / K15A / T119D, and C and D contain C10A / K15A / L17R (or vice versa); A and B contain C10A / K15A / T119E, and C and D contain C10A / K15A / L17R (or vice versa); A and B contain C10A / K15A / V121D, and C and D contain C10A / K15A / L17R (or vice versa); A and B contain C10A / K15A / V121E, and C and D contain C10A / K15A / L17R (or vice versa); A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / L17K (or vice versa); A and B contain C10A / K15A / L17E, and C and D contain C10A / K15A / L17K (or vice versa); A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / L17K (or vice versa); A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / L17K (or vice versa); A and B contain C10A / K15A / G22D, and C and D contain C10A / K15A / L17K (or vice versa); A and B contain C10A / K15A / G22E, and C and D contain C10A / K15A / L17K (or vice versa); A and B contain C10A / K15A / S112D, and C and D contain C10A / K15A / L17K (or vice versa); A and B contain C10A / K15A / S112E, and C and D contain C10A / K15A / L17K (or vice versa); A and B contain C10A / K15A / T119D, and C and D contain C10A / K15A / L17K (or vice versa); A and B contain C10A / K15A / T119E, and C and D contain C10A / K15A / L17K (or vice versa); A and B contain C10A / K15A / V121D, and C and D contain C10A / K15A / L17K (or vice versa); A and B contain C10A / K15A / V121E, and C and D contain C10A / K15A / L17K (or vice versa); A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / V20R (or vice versa); A and B contain C10A / K15A / L17E, and C and D contain C10A / K15A / V20R (or vice versa); A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / V20R (or vice versa); A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / V20R (or vice versa); A and B contain C10A / K15A / G22D, and C and D contain C10A / K15A / V20R (or vice versa); A and B contain C10A / K15A / G22E, and C and D contain C10A / K15A / V20R (or vice versa); A and B contain C10A / K15A / S112D, and C and D contain C10A / K15A / V20R (or vice versa); A and B contain C10A / K15A / S112E, and C and D contain C10A / K15A / V20R (or vice versa); A and B contain C10A / K15A / T119D, and C and D contain C10A / K15A / V20R (or vice versa); A and B contain C10A / K15A / T119E, and C and D contain C10A / K15A / V20R (or vice versa); A and B contain C10A / K15A / V121D, and C and D contain C10A / K15A / V20R (or vice versa); A and B contain C10A / K15A / V121E, and C and D contain C10A / K15A / V20R (or vice versa); A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / V20K (or vice versa); A and B contain C10A / K15A / L17E, and C and D contain C10A / K15A / V20K (or vice versa); A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / V20K (or vice versa); A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / V20K (or vice versa); A and B contain C10A / K15A / G22D, and C and D contain C10A / K15A / V20K (or vice versa); A and B contain C10A / K15A / G22E, and C and D contain C10A / K15A / V20K (or vice versa); A and B contain C10A / K15A / S112D, and C and D contain C10A / K15A / V20K (or vice versa); A and B contain C10A / K15A / S112E, and C and D contain C10A / K15A / V20K (or vice versa); A and B contain C10A / K15A / T119D, and C and D contain C10A / K15A / V20K (or vice versa); A and B contain C10A / K15A / T119E, and C and D contain C10A / K15A / V20K (or vice versa); A and B contain C10A / K15A / V121D, and C and D contain C10A / K15A / V20K (or vice versa); A and B contain C10A / K15A / V121E, and C and D contain C10A / K15A / V20K (or vice versa); A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / G22R (or vice versa); A and B contain C10A / K15A / L17E, and C and D contain C10A / K15A / G22R (or vice versa); A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / G22R (or vice versa); A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / G22R (or vice versa); A and B contain C10A / K15A / G22D, and C and D contain C10A / K15A / G22R (or vice versa); A and B contain C10A / K15A / G22E, and C and D contain C10A / K15A / G22R (or vice versa); A and B contain C10A / K15A / S112D, and C and D contain C10A / K15A / G22R (or vice versa); A and B contain C10A / K15A / S112E, and C and D contain C10A / K15A / G22R (or vice versa); A and B contain C10A / K15A / T119D, and C and D contain C10A / K15A / G22R (or vice versa); A and B contain C10A / K15A / T119E, and C and D contain C10A / K15A / G22R (or vice versa); A and B contain C10A / K15A / V121D, and C and D contain C10A / K15A / G22R (or vice versa); A and B contain C10A / K15A / V121E, and C and D contain C10A / K15A / G22R (or vice versa); A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / G22K (or vice versa); A and B contain C10A / K15A / L17E, and C and D contain C10A / K15A / G22K (or vice versa); A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / G22K (or vice versa); A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / G22K (or vice versa); A and B contain C10A / K15A / G22D, and C and D contain C10A / K15A / G22K (or vice versa); A and B contain C10A / K15A / G22E, and C and D contain C10A / K15A / G22K (or vice versa); A and B contain C10A / K15A / S112D, and C and D contain C10A / K15A / G22K (or vice versa); A and B contain C10A / K15A / S112E, and C and D contain C10A / K15A / G22K (or vice versa); A and B contain C10A / K15A / T119D, and C and D contain C10A / K15A / G22K (or vice versa); A and B contain C10A / K15A / T119E, and C and D contain C10A / K15A / G22K (or vice versa); A and B contain C10A / K15A / V121D, and C and D contain C10A / K15A / G22K (or vice versa); A and B contain C10A / K15A / V121E, and C and D contain C10A / K15A / G22K (or vice versa); A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / S112R (or vice versa); A and B contain C10A / K15A / L17E, and C and D contain C10A / K15A / S112R (or vice versa); A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / S112R (or vice versa); A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / S112R (or vice versa); A and B contain C10A / K15A / G22D, and C and D contain C10A / K15A / S112R (or vice versa); A and B contain C10A / K15A / G22E, and C and D contain C10A / K15A / S112R (or vice versa); A and B contain C10A / K15A / S112D, and C and D contain C10A / K15A / S112R (or vice versa); A and B contain C10A / K15A / S112E, and C and D contain C10A / K15A / S112R (or vice versa); A and B contain C10A / K15A / T119D, and C and D contain C10A / K15A / S112R (or vice versa); A and B contain C10A / K15A / T119E, and C and D contain C10A / K15A / S112R (or vice versa); A and B contain C10A / K15A / V121D, and C and D contain C10A / K15A / S112R (or vice versa); A and B contain C10A / K15A / V121E, and C and D contain C10A / K15A / S112R (or vice versa); A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / S112K (or vice versa); A and B contain C10A / K15A / L17E, and C and D contain C10A / K15A / S112K (or vice versa); A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / S112K (or vice versa); A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / S112K (or vice versa); A and B contain C10A / K15A / G22D, and C and D contain C10A / K15A / S112K (or vice versa); A and B contain C10A / K15A / G22E, and C and D contain C10A / K15A / S112K (or vice versa); A and B contain C10A / K15A / S112D, and C and D contain C10A / K15A / S112K (or vice versa); A and B contain C10A / K15A / S112E, and C and D contain C10A / K15A / S112K (or vice versa); A and B contain C10A / K15A / T119D, and C and D contain C10A / K15A / S112K (or vice versa); A and B contain C10A / K15A / T119E, and C and D contain C10A / K15A / S112K (or vice versa); A and B contain C10A / K15A / V121D, and C and D contain C10A / K15A / S112K (or vice versa); A and B contain C10A / K15A / V121E, and C and D contain C10A / K15A / S112K (or vice versa); A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / T119R (or vice versa); A and B contain C10A / K15A / L17E, and C and D contain C10A / K15A / T119R (or vice versa); A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / T119R (or vice versa); A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / T119R (or vice versa); A and B contain C10A / K15A / G22D, and C and D contain C10A / K15A / T119R (or vice versa); A and B contain C10A / K15A / G22E, and C and D contain C10A / K15A / T119R (or vice versa); A and B contain C10A / K15A / S112D, and C and D contain C10A / K15A / T119R (or vice versa); A and B contain C10A / K15A / S112E, and C and D contain C10A / K15A / T119R (or vice versa); A and B contain C10A / K15A / T119D, and C and D contain C10A / K15A / T119R (or vice versa); A and B contain C10A / K15A / T119E, and C and D contain C10A / K15A / T119R (or vice versa); A and B contain C10A / K15A / V121D, and C and D contain C10A / K15A / T119R (or vice versa); A and B contain C10A / K15A / V121E, and C and D contain C10A / K15A / T119R (or vice versa); A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / T119K (or vice versa); A and B contain C10A / K15A / L17E, and C and D contain C10A / K15A / T119K (or vice versa); A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / T119K (or vice versa); A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / T119K (or vice versa); A and B contain C10A / K15A / G22D, and C and D contain C10A / K15A / T119K (or vice versa); A and B contain C10A / K15A / G22E, and C and D contain C10A / K15A / T119K (or vice versa); A and B contain C10A / K15A / S112D, and C and D contain C10A / K15A / T119K (or vice versa); A and B contain C10A / K15A / S112E, and C and D contain C10A / K15A / T119K (or vice versa); A and B contain C10A / K15A / T119D, and C and D contain C10A / K15A / T119K (or vice versa); A and B contain C10A / K15A / T119E, and C and D contain C10A / K15A / T119K (or vice versa); A and B contain C10A / K15A / V121D, and C and D contain C10A / K15A / T119K (or vice versa); A and B contain C10A / K15A / V121E, and C and D contain C10A / K15A / T119K (or vice versa); A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / V121R (or vice versa); A and B contain C10A / K15A / L17E, and C and D contain C10A / K15A / V121R (or vice versa); A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / V121R (or vice versa); A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / V121R (or vice versa); A and B contain C10A / K15A / G22D, and C and D contain C10A / K15A / V121R (or vice versa); A and B contain C10A / K15A / G22E, and C and D contain C10A / K15A / V121R (or vice versa); A and B contain C10A / K15A / S112D, and C and D contain C10A / K15A / V121R (or vice versa); A and B contain C10A / K15A / S112E, and C and D contain C10A / K15A / V121R (or vice versa); A and B contain C10A / K15A / T119D, and C and D contain C10A / K15A / V121R (or vice versa); A and B contain C10A / K15A / T119E, and C and D contain C10A / K15A / V121R (or vice versa); A and B contain C10A / K15A / V121D, and C and D contain C10A / K15A / V121R (or vice versa); A and B contain C10A / K15A / V121E, and C and D contain C10A / K15A / V121R (or vice versa); A and B contain C10A / K15A / L17D, and C and D contain C10A / K15A / V121K (or vice versa); A and B contain C10A / K15A / L17E, and C and D contain C10A / K15A / V121K (or vice versa); A and B contain C10A / K15A / V20D, and C and D contain C10A / K15A / V121K (or vice versa); A and B contain C10A / K15A / V20E, and C and D contain C10A / K15A / V121K (or vice versa); A and B contain C10A / K15A / G22D, and C and D contain C10A / K15A / V121K (or vice versa); A and B contain C10A / K15A / G22E, and C and D contain C10A / K15A / V121K (or vice versa); A and B contain C10A / K15A / S112D, and C and D contain C10A / K15A / V121K (or vice versa); A and B contain C10A / K15A / S112E, and C and D contain C10A / K15A / V121K (or vice versa); A and B contain C10A / K15A / T119D, and C and D contain C10A / K15A / V121K (or vice versa); A and B contain C10A / K15A / T119E, and C and D contain C10A / K15A / V121K (or vice versa); A and B contain C10A / K15A / V121D, and C and D contain C10A / K15A / V121K (or vice versa); or A and B comprise C10A / K15A / V121E, and C and D comprise C10A / K15A / V121K (or vice versa).

[0022] 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 having the following mutations: A and B comprise C10A / K15A / L17D, and C and D comprise C10A / K15A / V121R (or vice versa); A and B comprise C10A / K15A / L17D, and C and D comprise C10A / K15A / V121K (or vice versa); A and B comprise C10A / K15A / L17E, and C and D comprise C10A / K15A / V121R (or vice versa); A and B comprise C10A / K15A / V20D, and C and D comprise C10A / K15A / V20R (or vice versa); A and B comprise C10A / K15A / V20D, and C and D comprise C10A / K15A / V20K (or vice versa); A and B comprise C10A / K15A / V20E, and C and D comprise C10A / K15A / V20R (or vice versa); A and B comprise C10A / K15A / V20E, and C and D comprise C10A / K15A / V20K (or vice versa); A and B comprise C10A / K15A / T119D, and C and D comprise C10A / K15A / L17R (or vice versa); A and B comprise C10A / K15A / T119D, and C and D comprise C10A / K15A / L17K (or vice versa); or A and B comprise C10A / K15A / V121E, and C and D comprise C10A / K15A / L17K (or vice versa).

[0023] In some embodiments, A and B contain two mutations in SEQ ID NO: 1, and the mutations are selected from the list including 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.

[0024] In some embodiments, C and D contain two mutations in SEQ ID NO: 1, and the mutations are selected from the list including 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.

[0025] The present invention also includes embodiments in which each of A, B, C, and D in the TTR protein complex contains the amino acid sequence of SEQ ID NO: 1 having the following mutations: A and B contain C10A / K15A / L17D / V20D, and C and D contain C10A / K15A / L17K / V20K (or vice versa); A and B contain C10A / K15A / L17D / V20E, and C and D contain C10A / K15A / L17K / V20R (or vice versa); A and B contain C10A / K15A / L17E / V20D, and C and D contain C10A / K15A / L17R / V20K (or vice versa); A and B contain C10A / K15A / L17E / V20E, and C and D contain C10A / K15A / L17R / V20R (or vice versa); A and B contain C10A / K15A / L17D / T119D, and C and D contain C10A / K15A / L17K / V121K (or vice versa); A and B contain C10A / K15A / L17D / V121E, and C and D contain C10A / K15A / L17K / V121R (or vice versa); A and B contain C10A / K15A / L17E / T119D, and C and D contain C10A / K15A / L17R / V121K (or vice versa); A and B contain C10A / K15A / L17E / V121E, and C and D contain C10A / K15A / L17R / V121R (or vice versa); A and B contain C10A / K15A / V20D / T119D, and C and D contain C10A / K15A / V20K / V121K (or vice versa); A and B contain C10A / K15A / V20D / V121E, and C and D contain C10A / K15A / V20K / V121R (or vice versa); A and B contain C10A / K15A / V20E / T119D, and C and D contain C10A / K15A / V20R / V121K (or vice versa); or A and B contain C10A / K15A / V20E / V121E, and C and D contain C10A / K15A / V20R / V121R (or vice versa).

[0026] In some embodiments, the TTR protein complex attaches to 1, 2, 3, 4, 5, 6, 7, or 8 bioactive proteins, peptides, or small molecules. In some embodiments, the TTR protein complex attaches to 1, 2, 3, 4, 5, 6, 7, or 8 antigen-binding proteins or peptides. In other embodiments, the TTR protein complex attaches to 1, 2, 3, or 4 antigen-binding proteins or peptides. The antigen-binding protein or peptide can attach to the TTR protein complex at the C-terminus of the TTR subunit, or at the N-terminus of the TTR subunit. Additionally, the TTR protein complex can attach directly to 1, 2, 3, 4, 5, 6, 7, or 8 antigen-binding proteins or peptides; or can attach to 1, 2, 3, 4, 5, 6, 7, or 8 antigen-binding proteins or peptides via a linker. In certain embodiments, the TTR protein complex attaches directly to 1, 2, 3, or 4 antigen-binding proteins or peptides; or attaches to 1, 2, 3, or 4 antigen-binding proteins or peptides via a linker.

[0027] 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 yet another embodiment, the linker is an amino acid-based linker comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments, the linker is G, GG, GGG, GGGG, GGGGG, GGGGGG, GGGGGGG, GGGGGGGG, GGGGGGGGG, or GGGGGGGGGG. In other certain embodiments, the linker is selected from the list comprising GG, GGGG, GGGSGG, GGGSGGGG, and GGAGGGAGGG.

[0028] Other suitable linkers are G(G x B y ) r G z comprising 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 is a linker, wherein B = Q, S, A, E, P, T, K, R, D or N; x = 4; y = 1; z = 4; and r = 1.

[0029] In some embodiments of the present invention, the TTR protein complex is attached to two antigen-binding proteins, and each antigen-binding protein binds to a different antigen. In another embodiment of the present invention, the TTR protein complex is attached to four antigen-binding proteins, and the antigen-binding proteins bind to at least two different antigens (e.g., one antigen-binding protein binds to a first antigen and three antigen-binding proteins bind to a second antigen; or two antigen-binding proteins bind to a first antigen and two antigen-binding proteins bind to a second antigen).

[0030] The antigen-binding protein may be an antibody. In other embodiments, the antigen-binding protein is a Fab or scFv. In certain embodiments, the antigen-binding protein is a Fab. In other embodiments, the antigen-binding protein is a mixture of an antibody and a Fab.

[0031] The present invention also includes a pharmaceutical composition comprising any of the TTR protein complexes described above.

[0032] In addition, the present invention includes a method for treating cancer using any of the TTR protein complexes discussed herein. The TTR protein complexes of the present invention can be used for the treatment of cancer. The present invention also includes any of the TTR protein complexes discussed herein for use in the treatment of cancer.

[0033] In another embodiment, the present invention includes one or more isolated nucleic acids encoding any of the TTR protein complexes discussed herein. In addition, the present invention includes an expression vector comprising a nucleic acid encoding any of the TTR protein complexes discussed herein. The present invention further includes a recombinant host cell comprising such a nucleic acid or vector. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell, an E5 cell, a baby hamster kidney (BHK) cell, a simian kidney (COS) cell, a human hepatocellular carcinoma cell, or a human embryonic kidney 293 (HEK293) cell.

[0034] In some embodiments, the present invention relates to a method for producing a TTR protein complex described herein, the method comprising: a) culturing a recombinant host cell; and b) isolating the TTR protein complex from the culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0035]

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DETAILED DESCRIPTION OF THE INVENTION

[0036] The section headings used in this specification are for structural purposes only and should not be construed as limiting the subject matter described.

[0037] In this specification, unless otherwise defined, scientific and technical terms used in connection with this application have the meanings commonly understood by those skilled in the art. Further, unless the context requires a different interpretation, singular terms shall include the plural, and plural terms shall include the singular.

[0038] In general, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and the chemistry and hybridization of the proteins and nucleic acids described herein are well known and commonly used in the art. Unless otherwise noted, the methods and procedures of the present application are generally carried out according to conventional methods well known in the art, and such methods and procedures are described in various general and specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001), Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990) (these references are incorporated herein by reference). Enzyme reactions and purification procedures are performed according to the manufacturer's instructions, as commonly accomplished in the art, or as described herein. The specialized terms used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as their laboratory procedures and techniques, are well known and commonly used in the art. Standard techniques may be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and for the treatment of patients.

[0039] The present invention is not limited to the specific methodologies, protocols, reagents, etc. described herein, and thus should be understood to be variable. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the disclosure, which is defined only by the claims.

[0040] Unless otherwise noted in examples or described in another form, all numbers indicating amounts of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." The term "about" when used in connection with percentages can mean ±1%.

[0041] All embodiments within a narrower range, specifically all ranges narrower than the variations defined by specific paragraphs herein, should be considered to be included in this disclosure. For example, a particular aspect is described as a genus concept, and it should be understood that all components of the genus concept can be, individually, embodiments. Also, an aspect of selecting an aspect or component described as a genus concept should be understood to include combinations of two or more components of the genus concept. Also, while the language "comprising" is presented in various embodiments under various circumstances, related embodiments can also be described using the language "consisting of" or "consisting essentially of."

[0042] In this application, the use of "or" means "and / or" unless otherwise specified. Further, the use of the term "comprising" and other forms such as "comprises" and "comprised of" is not limiting. Also, terms such as "element" or "component" include both elements and components that include one unit and elements and components that include two or more sub-units, unless otherwise specified.

[0043] Definitions "Amino acid" includes its standard meaning in the art. The 20 naturally occurring amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis, 2nd Edition, (E.S. Golub and D.R. Green, eds.), Sinauer Associates: Sunderland, Mass. (1991) (this reference is incorporated herein by reference for any purpose). Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids), unnatural amino acids such as [α]-, [α]-disubstituted amino acids, N-alkyl amino acids, and other non-conventional amino acids may also be suitable components of polypeptides and are included in the term "amino acid". Examples of non-conventional amino acids include the following: 4-hydroxyproline, [γ]-carboxyglutamic acid, [ε]-N,N,N-trimethyllysine, [ε]-N-acetyllysine, 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, according to standard usage and convention, the left hand direction is the amino terminal direction and the right hand direction is the carboxyl terminal direction.

[0044] As used herein, "antagonist" generally refers to a molecule, e.g., an antigen-binding protein as presented herein, which can bind to an antigen and inhibit, reduce, or eliminate the biological signal associated with the antigen.

[0045] As used herein, the term "antibody" refers to a protein having the conventional immunoglobulin structure, including heavy and light chains, and variable and constant regions. For example, an antibody can be an IgG having a "Y-shaped" structure with two pairs of identical polypeptide chains, each pair having one "light" chain (usually with a molecular weight of about 25 kDa) and one "heavy" chain (usually with a molecular weight of about 50-70 kDa). Antibodies have variable and constant regions. In the IgG structure, the variable region generally consists of about 100-110 or more amino acids, contains three complementarity-determining regions (CDRs), is mainly involved in antigen recognition, and is substantially different among other antibodies that bind to different antigens. The constant region enables the antibody to mobilize cells and molecules of the immune system. The variable region is made up of the N-terminal regions of each light and heavy chain, while the constant region is made up of the respective C-terminal portions of the heavy and light chains. (Janeway et al., "Structure of the Antibody Molecule and the Immunoglobulin Genes", Immunobiology: The Immune System in Health and Disease, 4 th ed. Elsevier Science Ltd. / Garland Publishing, (1999)).

[0046] An antibody can include any constant region known in the art. Human light chains are classified into human kappa light chains and lambda light chains. Heavy chains are classified into mu, delta, gamma, alpha or epsilon, defining the isotype of the antibody 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. Embodiments of the present disclosure include all such classes or isotypes of antibodies. The light chain constant region can be, for example, a kappa-type or lambda-type light chain constant region, such as a human kappa-type or lambda-type light chain constant region. The heavy chain constant region can be, for example, an alpha-type, delta-type, epsilon-type, gamma-type or mu-type heavy chain constant region, such as a human alpha-type, delta-type, epsilon-type, gamma-type or mu-type heavy chain constant region. Thus, in an exemplary embodiment, the antibody is an antibody of isotype IgA, IgD, IgE, IgG or IgM that includes any one of IgG1, IgG2, IgG3 or IgG4.

[0047] The term "antigen" refers to a molecule or part of a molecule that has the ability to be bound by a binder such as an antigen-binding protein (e.g., including an antibody), and further can be used in an animal to generate an antibody that has the ability to bind to that antigen. An antigen can have one or more epitopes that have the ability to interact with different antigen-binding proteins (e.g., antibodies).

[0048] As used herein, the term "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 antibodies that include at least two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments and mutants thereof. Antigen-binding proteins also include domain antibodies such as Fab, Fab’, F(ab’)2, Fv fragments, Nanobodies® and scFv, as described in more detail below.

[0049] "Antigen-binding region" or "antigen-binding domain" means a portion of a protein such as 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, that portion of an antigen-binding protein that contains the amino acid residues that interact with the antigen and confer its specificity and affinity for the antigen to the antigen-binding protein is referred to as the "antigen-binding region". The antigen-binding region may contain one or more "complementary determining regions" ("CDRs"). A particular antigen-binding region also includes one or more "framework" regions. The "framework" regions may contribute directly to the specific binding of the antigen-binding protein, but typically serve to maintain the proper three-dimensional structure of the CDRs, thereby facilitating the binding between the antigen-binding region and the antigen.

[0050] The terms "cancer", "tumor", "cancerous", and "malignant" typically relate to or describe a mammalian physiological condition characterized by unregulated cell growth. Examples of cancers include, but are not limited to, carcinomas such as adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, and leukemia. Further specific examples of such cancers include melanoma, lung cancer, head and neck cancer, renal cell cancer, colon cancer, colorectal cancer, squamous cell cancer, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin and non-Hodgkin lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer such as hepatocellular carcinoma and hepatoma, bladder cancer, breast cancer, endometrial cancer, myeloma (such as multiple myeloma), salivary gland cancer, kidney cancer such as renal cell cancer and Wilms tumor, basal cell cancer, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, and esophageal cancer.

[0051] The term "CDR" and its plural form "CDRs" (also referred to as "hypervariable regions") refer to the complementarity-determining regions of proteins such as antibodies or fragments, derivatives, or variants 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 responsible for the specific interaction between the antibody and the antigen and thus contribute to the functional activity of the antibody molecule. CDRs are the major determinants of antigen specificity.

[0052] The precise definition of the CDR boundaries and lengths follows various classification and numbering schemes. Thus, CDRs can be represented by Kabat, Chothia, contact, or any other boundary definition, including the numbering scheme described herein. The Kabat numbering scheme is a widely adopted standard for numbering the amino acid residues of antibody variable domains in a consistent manner and is the preferred scheme for application in the present invention as mentioned elsewhere in this specification. Additional structural considerations may be used to determine the canonical structure of the antibody. For example, differences not fully reflected by the Kabat numbering method can be described by the Chothia et al. numbering scheme and / or revealed by other techniques such as crystallography and two-dimensional or three-dimensional computer modeling. Even though the boundaries are different, each of these methods has some overlap in the portions that constitute the CDRs within the variable sequences. Thus, the definitions of CDRs by these methods may differ in the boundary regions with respect to length and adjacent framework regions. See, for example, Kabat (a method based on sequence variability between species), Chothia (a method based on crystallographic studies of antigen-antibody complexes), and / or MacCallum (Kabat et al., supra; 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 the antigen-binding site is the AbM definition used by Oxford Molecular's AbM antibody modeling software. See, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). For an overview of antibody structure, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, eds. Harlow et al., 1988.

[0053] Typically, a CDR forms a loop structure that can be classified as a canonical structure. The term "canonical structure" refers to the conformation of the backbone taken by the antigen-binding (CDR) loop. From comparative structural studies, it has been found that 5 out of 6 antigen-binding loops have only a limited repertoire of available conformations. Each canonical structure can be characterized by the torsion angles of the polypeptide backbone. Thus, corresponding loops between antibodies can have very similar three-dimensional structures, even though there is high amino acid sequence variability in most of the loop (Chothia and Lesk, J. Mol. Biol., 1987, 196:901; Chothia et al., Nature, 1989, 342:877; Martin and Thornton, J. Mol. Biol, 1996, 263:800). Furthermore, there is a relationship between the adopted loop structure and the amino acid sequence around it. The conformation of a particular canonical class is determined by the loop length and by amino acid residues present at important positions within the loop and within the conserved framework (i.e., outside the loop). Thus, assignment to a particular canonical class can be made based on the presence of these important amino acid residues.

[0054] When used in the context of antigen-binding proteins (e.g., antibodies or fragments thereof) that compete for the same epitope, the term "competing" means competition between the antigen-binding proteins, as determined by an assay in which the specific binding of a reference antigen-binding protein to a common antigen is blocked or inhibited by the antigen-binding protein (e.g., antibody or fragment thereof) being tested. Many types of competitive binding assays can be used, e.g., solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (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 labeled assay, solid-phase direct labeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct labeled RIA using I-125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid-phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, in such assays, a purified antigen bound to a solid surface or cells expressing such antigen, an unlabeled test antigen-binding protein, and a labeled reference antigen-binding protein are used. 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. Usually, 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 protein and antigen-binding proteins that bind to an adjacent epitope that is sufficiently close to the epitope to which the reference antigen-binding protein binds such that steric hindrance occurs.Further details regarding methods for determining competitive binding are provided herein. For example, in one embodiment, competition is determined according to a BiaCore assay. Typically, when competing antigen-binding proteins are present in excess, the competing antigen-binding proteins will inhibit the specific binding of the reference antigen-binding protein to the common antigen by at least 20%, 25%, 30%, 35%, 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%. In some cases, the binding is inhibited by at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% or more.

[0055] The term "control sequence" refers to a polynucleotide sequence that can affect the expression and processing of the coding sequence to which it is ligated. The nature of such control sequences may depend on the host organism. In certain embodiments, prokaryotic control sequences may include a promoter, ribosome binding site, and transcription termination sequence. For example, eukaryotic control sequences may include a promoter that contains one or more recognition sites for transcription factors, a transcription enhancer sequence, and a transcription termination sequence. A "control sequence" may include a leader sequence and / or a fusion partner sequence.

[0056] A "derivative" of a polypeptide is a polypeptide that has been modified (e.g., chemically) in some manner different from an insertion, deletion, or substitution variant, for example, by conjugation to another chemical moiety.

[0057] A "domain antibody" is an immunologically functional immunoglobulin fragment that contains only the variable region of the heavy chain or only the variable region of the light chain. Examples of domain antibodies include Nanobodies®. In some cases, two or more V H regions are covalently linked via a peptide linker to create a bivalent domain antibody. The two V H regions of the bivalent domain antibody may target the same or different antigens.

[0058] An "effective amount" generally is an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate symptoms and / or the underlying cause, prevent the occurrence of symptoms and / or their underlying cause, and / or improve or repair damage caused by or associated with cancer. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" is an amount sufficient for the treatment of a medical condition (e.g., cancer) or symptoms, specifically, a condition or symptom associated with the medical condition, or for the prevention, prevention, delay, or improvement of the progression of any other undesirable symptom associated with the medical condition or disease, regardless of the method. A "prophylactically effective amount" is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, e.g., prevention or delay of the onset (or recurrence) of cancer, or reduction in the likelihood of the onset (or recurrence) of cancer or cancer symptoms. A complete therapeutic or prophylactic effect does not necessarily occur upon administration of a single dose and may occur only after administration of a series of doses. Thus, a therapeutically effective amount or a prophylactically effective amount may be administered in one or multiple administrations.

[0059] The term "epitope" refers to the portion of an antigen that can be recognized and specifically bound by an antigen-binding protein (e.g., an antibody). In the context of a polypeptide, an epitope can be formed from contiguous or non-contiguous amino acids juxtaposed by the tertiary folding of the protein. Epitopes formed from adjacent amino acids are typically retained upon protein denaturation, whereas epitopes formed by tertiary folding are typically lost upon protein denaturation. Epitopes typically contain at least 3, more typically at least 5 or 8 - 10 amino acids in a unique spatial higher-order structure. A "linear epitope" or "continuous epitope" is an epitope that is recognized by an antigen-binding protein (e.g., an antibody) in its linear sequence of amino acids or primary structure. A "conformational epitope" or "discontinuous epitope" is an epitope that is recognized by an antigen-binding protein (e.g., an antibody) in its tertiary structure. The residues that make up these epitopes may not be adjacent in the primary amino acid sequence but are close to each other in the tertiary structure of the molecule. Linear and conformational epitopes generally behave differently when the protein is denatured, fragmented, or reduced.

[0060] The term "expression vector" or "expression construct" refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that (in cooperation with the host cell) induce and / or control the expression of one or more heterologous coding regions that are operably linked thereto. An expression construct can include, but is not limited to, sequences that affect or control transcription, translation, and, if introns are present, RNA splicing of the coding region that is operably linked thereto.

[0061] 「Fab fragment」 or 「Fab」 consists of one light chain and the C H region and variable region of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0062] A 「Fab’ fragment」 or 「Fab’」 consists of one light chain and V HDomain and C H In addition to 1 domain, C H 1 domain and C H It includes a part of one heavy chain including the region between 2 domains, and as a result, an F(ab’)2 molecule can be formed by forming an interchain disulfide bond between the two heavy chains of the two Fab’ fragments.

[0063] The term “F(ab’)2 fragment” or “F(ab’)2” refers to two light chains and C H 1 domain and C H It includes two heavy chains including a part of the constant region between 1 domain and 2 domains. Therefore, the F(ab’)2 fragment is composed of two Fab’ fragments joined together by a disulfide bond between the two heavy chains.

[0064] The “Fc region” refers to the C of the antibody H 2 domains and C H It includes two heavy chain fragments including 3 domains. The two heavy chain fragments are joined together by two or more disulfide bonds and the hydrophobic interaction of the C H 3 domains.

[0065] The “Fv region” includes variable regions derived from both the heavy chain and the light chain, but lacks the constant region.

[0066] The term “heavy chain” as used with respect to an antigen-binding protein, antibody, or fragment thereof includes the full-length heavy chain. The full-length heavy chain includes a variable region domain (V H ) and three constant region domains (C H 1, C H 2, and C H 3). The V H domain is at the amino terminus of the polypeptide, the C H domain is at the carboxyl terminus, and the C H3 is closest to the carboxy terminus of the polypeptide. The heavy chain can be of any isotype, such as IgG (including IgG1 subtype, IgG2 subtype, IgG3 subtype and IgG4 subtype), IgA (including IgA1 subtype and IgA2 subtype), IgM, and IgE. The fragment of the heavy chain has a variable region sequence sufficient to confer binding specificity.

[0067] "Blood cancer" is a cancer that occurs in blood-forming tissues such as the bone marrow, or cells of the immune system. Examples of blood cancers are leukemia, lymphoma, and multiple myeloma.

[0068] The term "heterodimeric fusion protein" or "heterodimeric protein complex" refers to a fusion protein that includes two different proteins (e.g., an antigen-binding protein; a peptide such as an agonist peptide; and an agonist protein domain). In certain examples, the heterodimer can be a TTR heterodimeric fusion protein that includes two different antigen-binding proteins (e.g., two different antibodies) linked via a TTR protein, as described herein. In another example, the heterodimer can be a TTR heterodimeric fusion protein that includes one antibody and one Fab linked via a TTR protein, as described herein. Exemplary heterodimeric fusion proteins are shown in FIGS. 1b and 2b.

[0069] The term "heterotrimeric fusion protein" or "heterotrimeric protein complex" refers to a fusion protein that includes three different proteins (e.g., an antigen-binding protein; a peptide such as an agonist peptide; and an agonist protein domain). In certain examples, the heterotrimer can be a TTR heterotrimeric fusion protein that includes one antibody and two Fabs linked via a TTR protein, as described herein (see, e.g., FIG. 2c).

[0070] 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 certain examples, the heterotetramer is a TTR heterotetrameric fusion protein in which, as described herein, for example, an antibody, Fab, or a mixture thereof is linked via the TTR protein. Examples include those in which the antigen-binding protein is an antibody (see, e.g., FIG. 2a) or a Fab (see, e.g., FIGS. 1d and 2a). In certain examples, the heterotetrameric fusion protein is a TTR heterotetrameric fusion protein in which, as described herein, for example, an antibody, Fab, or a mixture thereof is linked via the TTR protein.

[0071] The term "host cell" means a cell that has been transformed with a nucleic acid sequence and thereby expresses the gene of interest. This term includes the progeny of the parental cell, whether or not the progeny's form or genetic makeup is identical to that of the original parental cell, as long as the gene of interest is present.

[0072] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, which is determined by aligning and comparing the sequences. "Percent identity" means the percentage of residues that are identical between amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps (if any) in the alignment must be addressed by a particular 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, A.M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D.W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A.M., and Griffin, H.G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[0073] When calculating the percent identity, the arrays to be compared are aligned in such a way as to give the greatest match between the arrays. The computer program used to determine the percent identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI). The computer algorithm GAP is used to take an alignment of two polypeptides or polynucleotides for which the percent sequence identity is to be determined. The sequences are arranged so that their respective amino acids or nucleotides are optimally matched (the "match span" determined by the algorithm). A gap start penalty (calculated as 3× the average diagonal, where the "average diagonal" is the average of the diagonals of the comparison matrix used and the "diagonal" is the score or number assigned to each perfect amino acid match by a particular comparison matrix) and a gap extension penalty (usually 1 / 10 times the gap start penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62, are used with the algorithm. In certain embodiments, standard comparison matrices (see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; see Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM 62 comparison matrix) are also used by the algorithm.

[0074] The recommended parameters for determining the percent identity of a polypeptide or nucleotide sequence using the GAP program are as follows. Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453; Comparison matrix: BLOSUM 62 of Henikoff et al., 1992 (supra); Gap penalty: 12 (no penalty for end gaps) Gap length penalty: 4 Similarity threshold: 0

[0075] A specific alignment scheme for aligning two amino acid sequences can result in a match of only a short region of the two sequences, and this small aligned region can have a very high sequence identity even though there is no significant relationship between the two full-length sequences. Thus, the selected alignment method (GAP program) can be adjusted, if desired, to yield an alignment over at least 50 contiguous amino acids of the target polypeptide.

[0076] The term "immunomodulator" refers to a molecule that causes, enhances, or suppresses an immune response. An immunostimulant is a molecule that causes or enhances an immune response. An immunosuppressant is a molecule that decreases or suppresses an immune response. Thus, activation immunotherapy is a therapy involving administration of a molecule to cause or enhance the immune system of a subject. Suppression immunotherapy is a therapy in which a subject is treated with a molecule to decrease or suppress the immune system of the subject.

[0077] As used herein, the term "fragment" of an antibody or an immunoglobulin chain (heavy or light chain) is an antigen-binding protein that lacks at least some of the amino acids present in the full-length chain but can specifically bind to an antigen, regardless of how that portion is obtained or synthesized. Such fragments are biologically active in that they specifically bind to the target antigen and can compete with other antigen-binding proteins, including intact antibodies, for binding to a given epitope. In one aspect, such fragments retain at least one CDR present in the full-length light or heavy chain, and in some embodiments, will include a single heavy and / or light chain or a portion thereof. Such biologically active fragments may be produced by recombinant DNA techniques or may be produced by enzymatic or chemical cleavage of antigen-binding proteins, including intact antibodies. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv, domain antibodies, and scFv and may be derived from any mammalian source, including, but not limited to, human, mouse, rat, camel, or rabbit. For example, functional portions of the antigen-binding proteins disclosed herein, such as one or more CDRs, are further contemplated to be covalently attached to a second protein or small molecule to create a therapeutic agent targeted to a specific target in the body or to extend the serum half-life.

[0078] An "isolated nucleic acid molecule" is DNA or RNA that is derived from genomic, mRNA, cDNA, or synthesis, or any combination thereof, and is a DNA or RNA in which the isolated polynucleotide is not accompanied by all or part of the polynucleotide in which it is naturally found, or is linked to a polynucleotide to which it is not naturally linked. For the purposes of this disclosure, a nucleic acid molecule "comprising" a particular nucleotide sequence should be understood not to include an intact chromosome. An isolated nucleic acid molecule "comprising" a particular nucleic acid sequence may, in addition to that particular sequence, include sequences encoding up to 10 or even up to 20 other proteins or portions thereof, or may include regulatory sequences operably linked to control the expression of the coding region of the recited nucleic acid sequence, and / or may include vector sequences.

[0079] As used herein, the terms "isolated polypeptide", "purified polypeptide", "isolated protein", or "purified protein" are intended to refer to a composition that is separable from other components, where the polypeptide is purified to any degree compared to its naturally available state. Thus, a purified polypeptide also refers to a polypeptide that is free from the environment in which it may naturally occur. Generally, "purified" refers to a polypeptide composition that has been fractionated to remove various other components, and this composition substantially maintains its expressed biological activity. When the term "substantially purified" is used, this notation refers to a polypeptide or peptide composition in which the polypeptide or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the protein in the composition.

[0080] The term "light chain" as used in reference to an antigen-binding protein, antibody or fragment thereof includes the full-length light chain. The full-length light chain has a variable region domain (V L ) and a constant region domain (C LIt includes (0). The variable region domain of the light chain is at the amino terminus of the polypeptide. The light chain includes a κ chain and a λ chain. The fragment of the light chain has a variable region sequence sufficient to confer binding specificity.

[0081] The term "naturally occurring" as used throughout this specification in connection with biological materials such as polypeptides, nucleic acids, host cells, etc. refers to materials found in nature.

[0082] The term "oligonucleotide" means a polynucleotide containing 200 or fewer nucleotides. In some embodiments, the oligonucleotide is 10 - 60 bases in length. In other embodiments, the oligonucleotide is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 - 40 nucleotides in length. The oligonucleotide can be, for example, single-stranded or double-stranded for use in constructing mutant genes. The oligonucleotide can be a sense oligonucleotide or an antisense oligonucleotide. The oligonucleotide can contain a label, including a radiolabel, a fluorescent label, a hapten, or an antigenic label for a detection assay. The oligonucleotide can be used, for example, as a PCR primer, a cloning primer, or a hybridization probe.

[0083] As used herein, "operably linked" means that the components to which the term applies are in a relationship such that they are capable of performing their inherent functions under appropriate conditions. For example, a control sequence in a vector "operably linked" to a protein coding sequence is linked to the protein coding sequence such that expression of the protein coding sequence occurs under conditions compatible with the transcriptional activity of the control sequence.

[0084] The terms "polynucleotide" or "nucleic acid" include both single-stranded nucleotide polymers and double-stranded nucleotide polymers. The nucleotides that make up a polynucleotide can be ribonucleotides or 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 modifications of the internucleotide linkages such as phosphorothioate, phosphorodithioate, phosphorothioate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, and phosphoroamidate.

[0085] Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence discussed herein is the 5' end, and the left-hand direction of a double-stranded polynucleotide sequence is called the 5' direction. The direction of addition of nascent RNA transcripts from 5' to 3' is called the transcription direction, the sequence region on the DNA strand that has the same sequence as the RNA transcript and is on the 5' side of the 5' end of the RNA transcript is called the "upstream sequence", and the sequence region on the DNA strand that has the same sequence as the RNA transcript and is on the 3' side of the 3' end of the RNA transcript is called the "downstream sequence".

[0086] The terms "polypeptide" or "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms also apply to amino acid polymers in which one or more amino acid residues are analogs or mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. These terms may also include, for example, amino acid polymers modified by the addition of carbohydrate residues (for forming glycoproteins), or by phosphorylation. Polypeptides and proteins can be produced by naturally occurring and non-recombinant cells, or by genetically engineered or recombinant cells, and include molecules having the amino acid sequence of a native protein, or molecules having one or more amino acid deletions, additions and / or substitutions from the native sequence. The term "polypeptide fragment" refers to a polypeptide having an amino-terminal deletion, a carboxyl-terminal deletion and / or an internal deletion as compared to the full-length protein. Such fragments may also include amino acids modified as compared to the full-length protein. In certain embodiments, the fragment is about 5 to 500 amino acids in length. For example, the 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.

[0087] A "recombinant protein", including a recombinant TTR protein, is a protein made through the use of recombinant techniques, i.e., via the expression of a recombinant nucleic acid as described herein. Methods and techniques for generating recombinant proteins are well known in the art.

[0088] A "single-chain Fv" (scFv) is an Fv molecule in which the heavy-chain variable region and the light-chain variable region are linked by a flexible linker to form a single polypeptide chain, and the antigen-binding region is formed by this single polypeptide chain. scFv has been discussed in detail in WO 88 / 01649 pamphlet, as well as in U.S. Patent Nos. 4,946,778 and 5,260,203.

[0089] A "solid tumor" generally refers to an abnormal growth or mass of tissue that does not typically contain cysts or fluid regions. Solid tumors can be benign (non-cancerous) or malignant (cancerous). Different types of solid tumors are named according to the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemia (cancer of the blood) generally does not form solid tumors.

[0090] When an antigen-binding protein shows little or no binding to molecules other than the antigen, the antigen-binding protein "specifically binds" to the antigen. However, an antigen-binding protein that specifically binds to an antigen can cross-react with antigens from different species. Typically, when measured by surface plasmon resonance techniques (e.g., BIACore, GE-Healthcare Uppsala, Sweden), when the dissociation constant (K D ) is ≤ 10 -7 M, the antigen-binding protein specifically binds to the antigen. The antigen-binding protein binds with "high affinity" when K D ≤ 5 × 10 -8 M and with "very high affinity" when K D ≤ 5 × 10 -9 M, and specifically binds to the antigen.

[0091] As used herein, "subject" or "patient" can be any mammal. In a typical embodiment, the subject or patient is a human.

[0092] As used herein, "substantially pure" means that the described molecular species is the predominant species present, i.e., is more abundant than any other individual species in the same mixture on a molar basis. In certain embodiments, a substantially pure molecule is a composition that contains at least 50% (on a molar basis) of all polymeric species in which the species of interest is present. In other embodiments, a substantially pure composition contains at least 80%, 85%, 90%, 95% or 99% of all polymeric species present in the composition. In other embodiments, the species of interest cannot be detected as a contaminating species in the composition by conventional detection methods, and thus the composition is purified to substantial homogeneity consisting of a single detectable polymeric species.

[0093] The term "treating" refers to any indication of success in the treatment or amelioration of an injury, condition, or disorder, and such indications include, for example, reduction of symptoms, remission, regression, or improvement in patient tolerance of an injury, condition, or disorder; blunting of the rate of worsening or decline; attenuation of the end-point of worsening; and improvement in the physical or mental health of the patient. Treatment or amelioration of symptoms can be based on objective or subjective parameters, including the results of physical examination, neuropsychiatric examination, and / or psychiatric evaluation. For example, certain methods presented herein are successful in treating cancer and tumors by, for example, reducing the progression or spread of cancer, inhibiting tumor growth, causing tumor regression, and / or improving symptoms associated with cancer or tumors. Similarly, other methods presented herein treat an infectious disease by reducing the progression or spread of the infectious disease, reducing the extent of the infectious disease, and / or improving symptoms associated with the infectious disease.

[0094] 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 for human TTR is also described in the UniProt Knowledgebase (www.uniprot.org / uniprot / P02766#sequences), and is cited herein as SEQ ID NO:1. Nucleic acids for human TTR are described at NCBI (www.ncbi.nlm.nih.gov / gene / 7276). See also GenBank deposit K02091.1. The nucleic acid sequence for human TTR is listed herein as SEQ ID NO:44. The amino acid and nucleic acid sequences for murine TTR are shown as 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.

[0095] 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 having truncations at the C or N terminus.

[0096] "Tumor" refers to a mass of tissue formed when cancerous cells grow and multiply, which may invade and destroy adjacent normal tissue. Cancer cells can break away from the malignant tumor and enter the bloodstream or lymphatic system, and the cancer cells may spread from the primary tumor to form new tumors in other organs.

[0097] A "variant" of a polypeptide includes an amino acid sequence in which one or more amino acid residues have been inserted, deleted, and / or substituted in the amino acid sequence as compared to another polypeptide sequence. Variants include fusion proteins.

[0098] The term "vector" is intended to refer, in the context of a nucleic acid molecule, to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. A "plasmid", which is a type of vector, refers to a circular double-stranded DNA loop to which additional DNA segments can be linked. Another type of vector is a viral vector, in which additional DNA segments can be linked to the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of the host cell upon introduction into the host cell and are thereby replicated along with the host genome. Furthermore, certain vectors can direct the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA technology are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably when the plasmid is the most commonly used form of the vector. However, the present invention is intended to include other forms of expression vectors such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses) that perform equivalent functions.

[0099] TTR variant As described above, human TTR is a non-covalently bound tetrameric protein. The TTR tetrameric protein is composed of two dimers of dimers (Figure 3). Interestingly, the interface between TTR monomers that form a TTR dimer (Figure 3, left side) and the interface between TTR dimers that form a TTR tetramer (Figure 3, right side) are different. Due to the difference between the two interfaces, it becomes possible to engineer TTR variants that modulate the interaction between TTR dimers without disrupting the interface between TTR monomers.

[0100] 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 - where 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 associate to form TTR tetramer ABCD. The TTR monomers of the present invention contain at least one amino acid mutation (relative to SEQ ID NO: 1) at the interface between TTR dimer AB and TTR dimer CD such that the formation of the ABCD tetramer is more favorable than the formation of any other tetramer (e.g., ABAB tetramer or CDCD tetramer).

[0101] Accordingly, the present invention relates to a TTR protein complex, where the TTR protein complex comprises TTR subunits A, B, C, and D; TTR subunits A and B dimerize to form TTR dimer AB; TTR subunits C and D dimerize to form TTR dimer CD; TTR dimers AB and CD further dimerize to form TTR tetramer ABCD; each of A, B, C, and D contains the amino acid sequence of SEQ ID NO: 1, except that at least one amino acid at the interface between TTR dimer AB and TTR dimer CD is mutated such that the formation of the ABCD tetramer is more favorable than the formation of any other tetramer (e.g., ABAB tetramer or CDCD tetramer).

[0102] Each of A, B, C, and D of the TTR protein complex can include the amino acid sequence of SEQ ID NO: 1 having the following mutations: C10A, K15A, or both C10A and K15A.

[0103] Thus, in one embodiment, the present 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 include mutations at one or more amino acid positions selected from the list including 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 mutations are in addition to C10A and K15A.

[0104] In another embodiment, the present 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 include mutations at one or more amino acid positions selected from the list including 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 mutations are in addition to C10A and K15A.

[0105] In another embodiment, the present 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 include mutations at one or more amino acid positions selected from the list including 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 acids are mutated to aspartate, glutamate, arginine, lysine, or histidine. In some embodiments, the mutations are in addition to C10A and K15A.

[0106] In another embodiment, the present invention relates to a TTR protein complex, where A and B include mutations at one or more amino acid positions selected from the list consisting of positions 15, 17, 20, 21, 22, 23, 24, 51, 52, 84, 106, 108, 112, 114, 115, 119, 121, and 123 of SEQ ID NO: 1, and where the amino acid is mutated to aspartate or glutamate. In some embodiments, the mutations are added to C10A and K15A.

[0107] In yet another embodiment, the present invention relates to a TTR protein complex, where C and D include mutations at one or more amino acid positions selected from the list consisting of positions 15, 17, 20, 21, 22, 23, 24, 51, 52, 84, 106, 108, 112, 114, 115, 119, 121, and 123 of SEQ ID NO: 1, and where the amino acid is mutated to arginine, lysine, or histidine. In some embodiments, the mutations are added to C10A and K15A.

[0108] In certain embodiments, A and B include mutations at one or more amino acid positions selected from the list consisting of positions 15, 17, 20, 21, 22, 23, 24, 51, 52, 84, 106, 108, 112, 114, 115, 119, 121, and 123 of SEQ ID NO: 1, and where the amino acid is mutated to aspartate or glutamate; C and D include mutations at one or more amino acid positions selected from the list consisting of positions 15, 17, 20, 21, 22, 23, 24, 51, 52, 84, 106, 108, 112, 114, 115, 119, 121, and 123 of SEQ ID NO: 1, and where the amino acid is mutated to arginine, lysine, or histidine. In some embodiments, the mutations are added to C10A and K15A.

[0109] In some embodiments, A and B comprise at least one mutation in SEQ ID NO: 1, said mutation being selected from the list comprising 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 invention also relates to a TTR protein complex, wherein A and B comprise at least one mutation in SEQ ID NO: 1, the mutation being selected from the list comprising L17D, L17E, V20D, V20E, G22D, G22E, S112D, S112E, T119D, T119E, V121D, and V121E. In some embodiments, the mutation is additionally C10A and K15A.

[0110] In some embodiments, C and D comprise at least one mutation in SEQ ID NO:1, said mutation being selected from the list comprising K15R, L17R, V20R, G22R, S23R, P24R, D51R, S52R, I84R, T106R, A108R, S112R, Y114R, S115R, T119R, V121R, S123R, L17K, V20K, R21K, G22K, S23K, P24K, D51K, S52K, I84K, T106K, A108K, S112K, Y114K, S115K, T119K, V121K, S123K, K15H, L17H, V20H, R21H, G22H, S23H, P24H, D51H, S52H, I84H, T106H, 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, the mutation being selected from the list comprising 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 added to C10A and K15A.

[0111] The TTR protein complex of the invention can comprise a TTR subunit, wherein both A and B, both C and D, or all four of A, B, C, and D are independently, include one or two mutations discussed herein. In some embodiments, the TTR protein complex of the invention can comprise a TTR subunit, wherein both A and B, both C and D, or all four of A, B, C, and D are independently, include one mutation discussed herein. In some embodiments, the mutation is added to C10A and K15A.

[0112] In certain embodiments, the TTR protein complex of the invention comprises a TTR subunit, wherein each of A, B, C, and D comprises an amino acid sequence of SEQ ID NO:1 having the mutations (and their reverse) of Table 1 below:

[0113]

Table 1

[0114]

Table 2

[0115]

Table 3

[0116] Any of the TTR variants and variant pairings in Table 1 are suitable for use in the present invention. Table 2 shows the amount of TTR tetramer formation observed for specific variants and pairings (see Example 2 and Figure 7).

[0117]

Table 4

[0118]

Table 5

[0119] The TTR protein complex of the present invention can include TTR subunits, where both A and B, both C and D, or all four of A, B, C, and D independently include the two mutations discussed herein. In some embodiments, A and B include the two mutations in SEQ ID NO: 1, the mutations being selected from the list including 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 additionally C10A and K15A.

[0120] In some embodiments, C and D contain two mutations in SEQ ID NO:1, said mutations being selected from the list including 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 added to C10A and K15A.

[0121] In certain embodiments, the TTR protein complex of the present invention comprises a TTR subunit, wherein each of A, B, C, and D comprises an amino acid sequence of SEQ ID NO:1 having the mutations (and their reverse) in Table 3 below:

[0122]

Table 6

[0123] In some embodiments, the TTR protein complex of the present invention comprises a TTR subunit, wherein A and B, or C and D, comprise an amino acid sequence of SEQ ID NO:1 having 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.

[0124] 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. A TTR variant comprises a protein having an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a TTR protein having a displacement mutation in relation to SEQ ID NO:1.

[0125] The cysteines present in human TTR (SEQ ID NO: 1) can be used as sites for conjugation to bioactive 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, in the present invention, TTR variants that enable site-specific conjugation, such as TTR variants having genetically engineered cysteines, may be used. See, for example, U.S. Patent No. 8,633,153, which is incorporated herein by reference. For example, the TTR variant may comprise one or more of the following cysteine mutations: A37C, D38C, A81C, or G83C.

[0126] Further variants useful in the present invention include, for example, TTR proteins having a truncation at the C 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 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 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.

[0127] Further TTR variants that can be used in the present invention include those that reduce or block the binding of TTR to thyroxine. Each TTR tetramer containing two thyroxine-binding sites is located in the central channel of the TTR tetramer. Such variants can, for example, avoid interfering with the patient's thyroxine biology and may avoid the TTR fusion acting in the thyroxine metabolic pathway. Still other TTR variants that can be used in the present invention include those that reduce or eliminate the proteolytic activity of TTR.

[0128] In addition, a TTR-His tag fusion may be used in the present invention. For example, the TTR-His tag fusion can be used in the purification of a TTR Fab construct where the Fab lacks the Fc, or in the purification of a TTR Ab construct where it is beneficial 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 may also be present in the final therapeutic molecule (i.e., the tag may 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 certain embodiments, 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 contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 glycine amino acids as a linker. In a particular embodiment, the His tag contains two glycines (e.g., GGHHHHHH).

[0129] In some embodiments, the two glycine amino acid linker can be inserted between the TTR variant and the heavy or light chain.

[0130] Furthermore, the TTR variants of the present invention can include variants incorporating glycosylation sites that can help regulate the PK or solubility properties of the TTR fusion. In addition, the TTR variants or TTR fusion proteins of the present invention can be modified to include moieties that confer beneficial PK properties, such as a triazine-containing moiety (contained within a construct having a terminal group capable of reacting with the protein; see, for example, International Publication No. WO 2017 / 083604, which is hereby incorporated by reference in its entirety).

[0131] In some embodiments, the TTR protein complex attaches to 1, 2, 3, 4, 5, 6, 7, or 8 antigen-binding proteins or peptides. In other embodiments, the TTR protein complex attaches to 1, 2, 3, or 4 antigen-binding proteins or peptides. The antigen-binding protein or peptide can attach to the TTR protein complex at the C-terminus of the TTR subunit, or at the N-terminus of the TTR subunit. Additionally, the TTR protein complex can attach directly to 1, 2, 3, 4, 5, 6, 7, or 8 antigen-binding proteins or peptides; or can attach to 1, 2, 3, 4, 5, 6, 7, or 8 antigen-binding proteins or peptides via a linker. In certain embodiments, the TTR protein complex attaches directly to 1, 2, 3, or 4 antigen-binding proteins or peptides; or attaches to 1, 2, 3, or 4 antigen-binding proteins via a linker or peptide.

[0132] Heterodimeric fusion protein (complex) As described herein, the invention relates in part to the use of TTR in the multimerization of antigen-binding proteins such as antibodies. Since TTR is a human extracellular protein found in human serum, it is present in relatively large amounts throughout the human body, and thus TTR is less likely to induce an immune response when present in the multimerization constructs of the invention (e.g., when compared to non-human proteins, intracellular proteins, and rare proteins). Therefore, its use in the multimerization technology of the invention is advantageous.

[0133] For example, TTR can be used for dimerization of antibodies that bind to different epitopes, where the epitopes are present, for example, on the same or different proteins. In such heterodimeric fusion proteins, TTR (SEQ ID NO: 1) or its variant exists as a tetramer, where the TTR subunit is linked to the C-terminus of the antibody heavy chain to form a TTR-antibody heterodimer. For example, the C-terminus of each antibody heavy chain (each antibody contains two such C-termini) may be linked to the N-terminus of each TTR subunit (see FIGS. 1 and 2). Thus, each antibody is linked to two TTR subunits in the TTR tetramer to obtain a TTR-antibody heterodimer.

[0134] Accordingly, the present invention relates to a heterodimeric fusion protein comprising two antigen-binding proteins, where each antigen-binding protein binds to a different epitope, and the epitopes are 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, where the TTR protein complex is a TTR tetramer. In some embodiments, the antigen-binding protein is an antibody.

[0135] In certain embodiments, the present invention relates to a heterodimeric fusion protein comprising two antibodies linked to a TTR tetramer, where each antibody-binding protein binds to a different epitope, and the epitopes are present, for example, on the same or different proteins. The antibodies may be linked to the TTR tetramer without a linker (i.e., the antibodies are directly linked to the TTR).

[0136] In other embodiments, the antibody is linked to the TTR tetramer via a linker. For example, an amino acid linker can be used to link the C-terminus of the heavy chain of the antibody to the N-terminus of the TTR subunit. In some embodiments, the linker is 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1 to 35, or 1 to 40 amino acids in length. In some embodiments, 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 length. In other embodiments, the linker is 0, 1, 5, 10, 15, 20, 25, 30, 35, or 40 amino acids in length. In other embodiments, the linker is up to 5, 10, 15, 20, 25, 30, 35, or 40 amino acids in length. In some embodiments, the linker is up to 5, 10, 15, or 20 amino acids in length. In certain embodiments, the linker is 0, 5, 10, 15, or 20 amino acids in length.

[0137] 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, the linker is GGGGS, GGGGSGGGGS (i.e., (GGGGS)2), GGGGSGGGGSGGGGS (i.e., (GGGGS)3), or GGGGSGGGGSGGGGSGGGGS (i.e., (GGGGS)4).

[0138] 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).

[0139] In some embodiments, the antibody is linked to the truncated TTR subunit, with or without a linker. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids may be removed from the N - terminus of one or more TTR subunits, and the antibody may be attached to the N - terminus of the truncated TTR subunit.

[0140] The present invention also relates to nucleic acid molecules encoding the heterodimeric fusion proteins described herein. Details regarding exemplary methods for generating heterodimeric fusion proteins can be found in the Examples.

[0141] Heterotrimeric and heterotetrameric fusion proteins (complexes) The present invention also relates, in part, to the use of TTR in the trimerization or tetramerization of antigen - binding proteins such as antibodies.

[0142] In such heterotetrameric fusion proteins, TTR (SEQ ID NO: 1) or its variant is still present as a tetramer. However, in the context of the TTR - antibody heterotetramer, a single antibody heavy chain (i.e., only one of the two heavy chains present in a single antibody) is linked to each TTR subunit, enabling the linkage of the four antibodies to the TTR tetramer (see Figure 2e). One of the two heavy chains at the C - terminus of the antibody may be linked to the N - terminus of each TTR subunit (see Figure 2e). Thus, each antibody is linked to one TTR subunit in the TTR tetramer, resulting in a TTR - antibody heterotetramer.

[0143] In such heterotetrameric fusion proteins, the formation of the Fc heterodimer (described above) is inhibited by mutations in the Fc. Such modifications include Fc mutations such as knobs-into-holes, DuoBody, Azymetric, charge pairs, HA-TF, SEEDbody, and modifications with different protein A affinities. See, for example, Spiess et al., Molecular Immunology, 67(2, Part A), 2015, pp. 95-106. Knobs-into-holes mutations include T366W in the first heavy chain, and T366S, L368A and / or Y407V in the second heavy chain. See, for example, 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, for example, Labrijn et al., Proc. Natl. Acad. Sci. U.S.A., 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, for example, 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, for example, 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, for example, Davis et al., Protein Eng. Des. Sel., 23(2010), pp. 195-202. Mutations with different protein A affinities include H435R in one heavy chain and no mutation in the other heavy chain. See, for example, U.S. Patent No. 8,586,713. Each of these documents is incorporated by reference in its entirety.

[0144] In certain embodiments, the use of Fc charge pairs that inhibit antibody heavy chain dimerization can promote antibody heterotetramerization and thus support heavy chain dimerization between one antibody heavy chain linked to a TTR subunit and one antibody heavy chain not linked to TTR (see Figure 1c). For example, a set of charge mutations can have either a negative charge on one heavy chain and a positive charge on the corresponding heavy chain, or a mixture of negative and positive charges on one heavy chain that pair with the corresponding positive and negative charges on the corresponding heavy chain, incorporated into the C H 3 domain. Exemplary negative charges include K392D & K409D, and exemplary positive charges include E356K & D399K. While different charges are attracted, the charges at the C H 3 interface repel, so homodimerization is inhibited while heterodimerization is favored. TTR is fused to a heavy chain of only one charge type (either positive or negative, but not both); thus, a complete antibody composed of four chains (two light chains, one unfused heavy chain, and one TTR-fused heavy chain) results in a TTR subunit. Additionally, charge pair mutations are described, for example, in U.S. Patent No. 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., 420 (2012), pp. 204-219. Each of these references is incorporated herein by reference in its entirety.

[0145] In the heterotrimeric fusion protein, TTR (SEQ ID NO: 1) or its variant also exists as a tetramer. Examples of heterotrimeric fusion proteins include those containing one antibody and two Fabs. In the context of the 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, and the C-terminus of each of the two Fabs can be linked to the N-terminus of each of the two TTR subunits to form a TTR Ab / Fab heterotrimer (see FIGS. 2c and 2d). Thus, the antibody links to two TTR subunits in the TTR tetramer, and each Fab links to a TTR subunit to obtain a TTR Ab / Fab heterotrimer containing a TTR tetramer, one antibody, and two Fabs.

[0146] The present invention also relates, in part, to the use of TTR in the tetramerization of Fabs. In such a heterotetrameric fusion protein, TTR (SEQ ID NO: 1), or its variant, also exists as a tetramer, and each TTR subunit links to the C-terminus of each Fab to form a TTR Fab heterotetramer (see FIG. 2a). Thus, each Fab links to a single TTR subunit in the TTR tetramer to obtain a TTR Fab heterotetramer.

[0147] Accordingly, the present invention relates to heterotrimeric and heterotetrameric fusion proteins comprising three or four antigen-binding proteins (e.g., Ab / Fab trimers, Fab tetramers, or Ab tetramers). In some embodiments, the heterotrimeric and heterotetrameric fusion proteins comprise antigen-binding proteins linked to a protein complex. In some embodiments, the protein complex is a TTR protein complex, where 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 heterotetrameric fusion protein comprises a mixture of an antibody and a Fab.

[0148] In certain embodiments, the invention relates to a heterotetrameric fusion protein comprising four antibodies linked to a TTR tetramer. In other embodiments, the invention relates to a heterotetrameric fusion protein comprising four Fabs linked to a TTR tetramer via a linker. In other embodiments, the invention relates to a heterotrimeric fusion protein comprising one Ab and two Fabs linked to a TTR tetramer via a linker. In some embodiments, the antibody or Fab is linked to the TTR tetramer without a linker (i.e., the antibody or Fab is directly linked to the TTR).

[0149] 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, or 20 amino acids. In other embodiments, the linker is an amino acid-based linker comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In yet other embodiments, the linker is an amino acid-based linker comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments, the linker is G, GG, GGG, GGGG, GGGGG, GGGGGG, GGGGGGGG, GGGGGGGGG, GGGGGGGGGG, or GGGGGGGGGG. In other specific embodiments, the linker is selected from the list comprising GG, GGGG, GGGSGG, GGGSGGGG, and GGAGGGAGGG.

[0150] 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, the linker is GGGGS, GGGGSGGGGS (i.e., (GGGGS)2), GGGGSGGGGSGGGGS (i.e., (GGGGS)3), or GGGGSGGGGSGGGGSGGGGS (i.e., (GGGGS)4).

[0151] Other suitable linkers are G(G x B y ) r G z linker, where G = glycine; B = any amino acid; x = 1 to 15; y = 1 to 5; z = 1 to 15; and r = 1 to 20. In another embodiment, the linker is G(G x B y ) r G z linker, where B = Q, S, A, E, P, T, K, R, D or N; x = 4; y = 1; z = 4; and r = 1.

[0152] Additional suitable amino acid linkers include, for example, disulfide bonds, (Gly) n (n = 1 to 10), (EAAAK) n (n = 1 to 5), A(EAAAK)4ALEA(EAAAK)4A, PAPAP, AEAAAKEAAAKA, (Ala - Pro) n(n = 1 to 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 within constructs having end - groups capable of reacting with proteins; see, for example, WO 2017 / 083604 pamphlet, which is hereby incorporated by reference in its entirety).

[0153] 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 may be removed from the N - terminus of one or more TTR subunits, and the antibody or Fab may be attached to the N - terminus of the truncated TTR subunit.

[0154] The invention also relates to nucleic acid molecules encoding the heterotrimeric and heterotetrameric fusion proteins described herein. Details regarding exemplary methods for generating heterotrimeric and heterotetrameric fusion proteins can be found in the Examples.

[0155] Antigen - binding protein Any antigen - binding protein (e.g., Fab, antibody, scFv, scFab) can be used in the TTR fusion proteins of the invention. In addition, proteins such as enzymes can be combined with the antigen - binding protein and used in the TTR fusion proteins of the invention.

[0156] The fusion proteins of the present invention enable the binding of different epitopes (e.g., on the same or different proteins), so the fusion proteins are useful in situations where it is beneficial to bring different targets into proximity. Examples of successful implementation of such techniques include emicizumab, which acts to bring together activated factor IX and factor X, thereby allowing the coagulation process to continue without the need to replace factor VIII for the treatment of hemophilia.

[0157] The fusion proteins of the present invention may also be useful in the field of oncology. For example, depending on the mechanism of action related to an oncology target, cross-linking of target cells (e.g., cancer cells) with effector cells (e.g., T cells) may be desirable. Such an approach has been shown to be successful in the context of BiTE® (bispecific T cell engager) antibody constructs. Other examples include trispecifics that can bind to two different tumor markers (e.g., via the Ab and / or Fab of the TTR fusion protein of the present invention) and CD3 (e.g., via an anti-CD3 scFv, Ab, or Fab).

[0158] The fusion proteins of the present invention can also address the complexities associated with the regulatory evaluation / approval of combination therapies. Clinical trials of combination therapies may require more complex clinical trial strategies to assess safety and efficacy, particularly when none of the individual components have been previously evaluated. The fusion proteins of the present invention address such complexities by combining multiple components into a single construct.

[0159] Method for producing TTR heterodimer, heterotrimer, and heterotetramer fusion proteins The method for producing the TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) fusions of the present invention is discussed in the Examples.

[0160] Generally, the TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) fusions of the present invention can be generated using recombinant methods. Accordingly, the present invention includes polynucleotides encoding TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) fusions. In another aspect, the present invention includes an expression vector comprising a polynucleotide encoding a TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) fusion. In certain embodiments, the expression vector includes control sequences (e.g., promoter, enhancer) operably linked to the polynucleotide encoding the TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) fusion to facilitate expression in a suitable host cell. In certain embodiments, the expression vector also includes a polynucleotide sequence that enables chromosomal-independent replication in the host cell. Exemplary vectors include, but are not limited to, plasmids, cosmids, and YACs. In certain embodiments, the vector is pTT5.

[0161] Generally, mammalian host cells are utilized 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 prokaryotes (bacteria) and non-mammalian (e.g., yeast) host cells can also be used.

[0162] In yet another embodiment, the invention includes a host cell comprising the expression vector of the invention. Methods for transfecting a host cell with the expression vector and culturing the transfected host cell under conditions suitable for the expression of TTR hetero-oligomer (e.g., hetero-dimer, hetero-trimer, and hetero-tetramer) fusions are known in the art. The transfection procedure used may depend on the host being transformed. Particular methods for introducing heterologous polynucleotides into mammalian cells are known in the art and include, but are not limited to, dextran-mediated gene transfer, calcium phosphate precipitation, polybrene-mediated gene transfer, protoplast fusion, electroporation, encapsulation of polynucleotides into liposomes, and direct microinjection of DNA into the nucleus. Particular mammalian cell lines available as hosts for expression are 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, simian kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human fetal kidney cells (HEK293), and many other cell lines. In certain embodiments, the cell line may be selected by determining which cell line expresses and produces high levels of the TTR hetero-dimer, hetero-trimer, and hetero-tetramer fusions.

[0163] Accordingly, the invention also relates to a method for producing the TTR hetero-oligomer (e.g., hetero-dimer, hetero-trimer, and hetero-tetramer) fusion proteins described herein. For example, the TTR hetero-oligomer (e.g., hetero-dimer, hetero-trimer, and hetero-tetramer) fusion proteins are: a) culturing a recombinant host cell comprising a polynucleotide encoding a TTR hetero-oligomer (e.g., hetero-dimer, hetero-trimer, and hetero-tetramer) fusion; b) isolating a TTR hetero-oligomer (e.g., hetero-dimer, hetero-trimer, and hetero-tetramer) fusion protein from said culture; can be produced by

[0164] Pharmaceutical composition In some embodiments, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of one or more of the TTR hetero-oligomer (e.g., hetero-dimer, hetero-trimer, and hetero-tetramer) fusion proteins of the invention, along with a pharmaceutically effective diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant. The pharmaceutical compositions of the invention include, but are not limited to, liquid, frozen, and lyophilized compositions.

[0165] Preferably, the formulation materials are non-toxic to the recipient at the dosages and concentrations employed. In specific embodiments, there is provided a pharmaceutical composition comprising a therapeutically effective amount of a TTR hetero-oligomer (e.g., hetero-dimer, hetero-trimer, and hetero-tetramer) fusion protein.

[0166] In certain embodiments, the pharmaceutical composition may include formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution rate or release rate, absorbability, or permeability of the composition. In such embodiments, suitable formulation materials include amino acids (such as glycine, glutamine, asparagine, arginine, proline, or lysine); antibacterial agents; antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite); buffers (such as boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid or other organic acids); fillers (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); injectables; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrin); proteins (such as serum albumin, gelatin or immunoglobulins); coloring agents, flavoring agents and diluents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancers (such as sucrose or sorbitol); isotonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants, but are not limited thereto. See REMINGTON’S PHARMACEUTICAL SCIENCES, 18” Edition, (A.R. Genrmo, ed.), 1990, Mack Publishing Company.

[0167] In certain embodiments, the optimal pharmaceutical composition will be determined by one of ordinary skill in the art, for example, according to the intended route of administration, delivery format, and desired dosage. See, e.g., REMINGTON’S PHARMACEUTICAL SCIENCES (supra). In certain embodiments, such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the antigen-binding protein of the invention. In certain embodiments, the major vehicle or carrier in a pharmaceutical composition can have either aqueous or non-aqueous properties. For example, suitable vehicles or carriers can be water for injection, aqueous saline solution, or artificial cerebrospinal fluid, optionally supplemented with other materials common in parenteral compositions. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In a specific embodiment, the pharmaceutical composition can include a Tris buffer at about pH 7.0 - 8.5, or an acetate buffer at about pH 4.0 - 5.5, and can further include sorbitol or a suitable alternative thereof. In certain embodiments of the invention, TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) compositions can be prepared for storage by mixing a selected composition having a desired degree of purity, in the form of a lyophilized cake or aqueous solution, with optional formulation agents (REMINGTON’S PHARMACEUTICAL SCIENCES, supra). Further, in certain embodiments, TTR heteromultimers (e.g., heterodimers, heterotrimers, and heterotetramers) can be formulated as lyophilized products using suitable excipients such as sucrose.

[0168] The pharmaceutical compositions of the invention can be selected for parenteral delivery. Alternatively, the compositions can be selected for delivery via the digestive tract, such as by inhalation or orally. The preparation of such pharmaceutically acceptable compositions is within the scope of the art. The formulation ingredients are preferably present at concentrations acceptable to the site of administration. In certain embodiments, buffers are used to maintain the composition within a physiological pH or slightly lower pH, typically in the pH range of about 5 to about 8.

[0169] When parenteral administration is contemplated, the therapeutic composition for use in the present invention can be provided in the form of a pyrogen-free parenterally acceptable aqueous solution containing the desired TTR hetero-oligomers (e.g., hetero-dimers, hetero-trimers, and hetero-tetramers) in a pharmaceutically acceptable vehicle. A vehicle particularly suitable for parenteral injection is sterile distilled water, and the TTR hetero-oligomers (e.g., hetero-dimers, hetero-trimers, and hetero-tetramers) are formulated as a sterile isotonic solution properly stored therein. In certain embodiments, the formulation can include an agent that can provide controlled or sustained release of the product deliverable via depot injection, such as injectable microspheres, biodegradable particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, in formulation with the desired molecule. In certain embodiments, hyaluronic acid having the effect of enhancing the duration in circulation can also be used. In certain embodiments, an implantable drug delivery device may be used to introduce the desired antigen-binding protein.

[0170] The pharmaceutical compositions of the present invention can be formulated for the purpose of inhalation. In these embodiments, the TTR hetero-oligomers (e.g., hetero-dimers, hetero-trimers, and hetero-tetramers) are advantageously formulated as a dry, inhalable powder. In a specific embodiment, the TTR hetero-oligomer (e.g., hetero-dimer, hetero-trimer, and hetero-tetramer) inhalation solution may be formulated with a propellant for aerosol delivery. In certain embodiments, the solution may be nebulized. Transpulmonary administration, and thus the formulation method, is further described in International Patent Application PCT / US Patent Application Publication No. 94 / 001875 (incorporated by reference), which describes the transpulmonary delivery of chemically modified proteins.

[0171] The formulation may also be considered suitable for oral administration. TTR hetero-oligomers (e.g., hetero-dimers, hetero-trimers, and hetero-tetramers) administered in this manner can be formulated with carriers customarily used in the formulation of solid dosage forms such as tablets and capsules, or without such carriers. In certain embodiments, the capsule may be designed such that the active portion of the formulation is released at a point where bioavailability in the gastrointestinal tract is maximized and pre-systemic degradation is minimized. Additional agents can be included to facilitate the absorption of TTR hetero-oligomers (e.g., hetero-dimers, hetero-trimers, and hetero-tetramers). Diluents, flavoring agents, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and binders may be used.

[0172] In sustained or controlled delivery formulations, additional pharmaceutical compositions comprising formulations with TTR heteromultimers (e.g., heterodimers, heterotrimers, and heterotetramers) will be apparent to those skilled in the art. Formulation techniques for various other sustained or controlled delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art. See, for example, International Patent Application No. PCT / US93 / 00829 (incorporated by reference), which describes the controlled release of porous polymer microparticles for delivering pharmaceutical compositions. Sustained release preparations can include, for example, a semipermeable polymer matrix in the form of a shaped article such as a film or microcapsule. Sustained release matrices can include polyesters, hydrogels, polylactides (disclosed in U.S. Patent No. 3,773,919 and European Patent Application Publication No. 058481, each incorporated by reference), copolymers of L-glutamic acid and γ-ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinyl acetate (Langer et al., 1981, supra), or poly-D(-)-3-hydroxybutyric acid (European Patent Application Publication No. 133,988). Sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art. See, for example, Eppstein et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:3688-3692; European Patent Application Publication No. 036,676; No. 088,046; and No. 143,949, each incorporated by reference.

[0173] Pharmaceutical compositions for in vivo administration are typically provided as sterile preparations. Sterilization can be achieved by filtration through a sterile filtration membrane. When the composition is lyophilized, sterilization using this method may be carried out either before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or as a solution. Parenteral compositions are generally filled into containers having a sterile access port, such containers being, for example, intravenous solution bags or vials having a stopper penetrable by a hypodermic needle.

[0174] Aspects of the invention include self-buffering TTR hetero-oligomers (e.g., hetero-dimers, hetero-trimers, and hetero-tetramers), which can be used as pharmaceutical compositions as described in International Publication No. WO 2006 / 138181 A2 (PCT / US2006 / 022599), which is hereby incorporated by reference in its entirety.

[0175] As noted above, certain embodiments provide TTR hetero-oligomer (e.g., hetero-dimer, hetero-trimer, and hetero-tetramer) compositions, particularly pharmaceutical TTR hetero-oligomer (e.g., hetero-dimer, hetero-trimer, and hetero-tetramer) compositions, that include, in addition to the hetero-oligomer, one or more excipients, such as those exemplified elsewhere in this section and in this specification. Excipients can be used in the present invention in view of a wide range of purposes, such as adjusting the physical, chemical, or biological properties of the formulation, such as viscosity, and / or improving efficacy and / or stabilizing such a formulation, as well as methods for degradation and damage due to stress occurring during, for example, manufacture, transportation, storage, preparation prior to use, administration, and after these.

[0176] A variety of explanations are available for the stabilization of proteins and pharmaceutical materials and methods useful in this regard, for example, 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": 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 protein pharmaceuticals and processes for veterinary and / or human medical use, and in particular, reference is made to the parts regarding the same excipients and processes as the self-buffering protein formulations according to the present invention.

[0177] Salts can be used, according to certain embodiments of the present invention, for example, to adjust the ionic strength and / or isotonicity of a formulation, and / or to improve the solubility and / or physical stability of the protein or other components of the composition according to the present invention.

[0178] As is well known, ions can stabilize native proteins by binding to charged residues on the surface of the protein and by shielding the charged and polar groups in the protein, thereby reducing the strength of its electrostatic interactions, attractive and repulsive interactions. Ions can also stabilize denatured proteins, particularly by binding to the denatured peptide bonds (--CONH) of the protein. Furthermore, ionic interactions with charged and polar groups in the protein can reduce intermolecular electrostatic interactions, thereby preventing or reducing protein aggregation and insolubilization.

[0179] Their effects on proteins vary significantly depending on the ionic species. Several classifications of the effects on ions and proteins have been proposed and can be used for formulating the pharmaceutical compositions according to the present invention. One example is the Hofmeister series that ranks ionic and polar non-ionic solutes according to their effects on the conformational stability of proteins in solution. Solutes that stabilize are termed "cosmotropic." Solutes that destabilize are termed "chaotropic." Cosmotropes are generally used at high concentrations (e.g., >1 molar ammonium sulfate) to precipitate proteins from solution ("salting out"). Chaotropes are generally used to denature and / or solubilize proteins ("salting in"). The relative effects of ions on "salting in" and "salting out" define the position of the ions in the Hofmeister series.

[0180] Free amino acids can be used in formulations of TTR heteromultimers (e.g., heterodimers, heterotrimers, and heterotetramers) according to various embodiments of the present invention as fillers, stabilizers, and antioxidants, as well as for other standard uses. Lysine, proline, serine, and alanine can be used to stabilize the proteins in the formulation. Glycine is useful for ensuring an appropriate cake structure and properties in lyophilization. Arginine can be useful for inhibiting protein aggregation in both liquid and lyophilized formulations. Methionine is useful as an antioxidant.

[0181] Polyols include sugars such as mannitol, sucrose, and sorbitol, as well as polyhydric alcohols such as glycerol and propylene glycol, and polyethylene glycol (PEG) and related substances for the purposes of discussion herein. Polyols are cosmotropic. They are useful stabilizers for protecting proteins from physical and chemical degradation processes in both liquid and lyophilized formulations. Polyols are also useful for adjusting the isotonicity of the formulation.

[0182] Among the polyols, useful in selected embodiments of the present invention is mannitol, which is commonly used to ensure the structural stability of the cake in a lyophilized formulation. Mannitol ensures the structural stability of the cake. Generally, this is used together with a lyoprotectant such as sucrose. Sorbitol and sucrose are included among those preferred as agents for adjusting isotonicity and stabilizers for protecting against freeze-thaw stress during transportation or during bulk preparation in the manufacturing process. Reducing sugars (containing free aldehyde or ketone groups), such as glucose and lactose, can glycate surface lysine and arginine residues. Thus, they are generally not included among the preferred polyols for use according to the present invention. In addition, sugars that form such reactive species, such as sucrose, are also not included among the preferred polyols of the present invention in that they are hydrolyzed to fructose and glucose under acidic conditions, resulting in fructose glycation. PEG is useful for stabilizing proteins and as a cryoprotective substance and can be used in the present invention in this regard.

[0183] Embodiments of TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) formulations further comprise a surfactant. Protein molecules can be prone to causing adsorption to surfaces and denaturation and consequent aggregation at gas-liquid interfaces, solid-liquid interfaces, and liquid-liquid interfaces. These effects generally vary inversely with protein concentration. These detrimental interactions generally vary inversely with protein concentration and are typically exacerbated by physical agitation that occurs, for example, during product transportation and handling.

[0184] Surfactants are conventionally used to prevent, minimize, or reduce surface adsorption. Surfactants useful in the present invention in this regard include polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan polyethoxylate, and poloxamer 188.

[0185] Surfactants are also commonly used to control the conformational stability of proteins. In this regard, the use of surfactants is protein-specific, as any given surfactant will typically stabilize some proteins and destabilize others.

[0186] Polysorbates are prone to oxidative degradation and often contain sufficient amounts of peroxide to cause oxidation of the side chains of protein residues, particularly methionine, when supplied. Therefore, polysorbates should be used with caution and at the minimum effective concentration during use. In this regard, polysorbates exemplify the general rule that excipients should be used at the minimum effective concentration.

[0187] Embodiments of TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) formulations further comprise one or more antioxidants. By maintaining appropriate levels of ambient oxygen and ambient temperature and by avoiding exposure to light, harmful oxidation of proteins in pharmaceutical formulations can be prevented to some extent. Antioxidant excipients can be used similarly to prevent oxidative degradation of proteins. Antioxidants that are particularly useful in this regard include reducing agents, oxygen / free radical scavengers, and chelating agents. Antioxidants for use in therapeutic protein formulations according to the present invention are preferably water-soluble and maintain their activity over the shelf life of the product. In this regard, EDTA is a preferred antioxidant according to the present invention.

[0188] Antioxidants can damage proteins. For example, reducing agents such as glutathione can break intramolecular disulfide bonds in particular. Therefore, antioxidants for use in the present invention are selected, inter alia, to rule out or sufficiently reduce the possibility that they themselves can damage the proteins in the formulation.

[0189] The formulations according to the present invention are cofactors of proteins and may contain metal ions required to form protein coordination compounds, such as zinc required to form certain insulin suspensions. Metal ions can also inhibit some of the processes that degrade proteins. However, metal ions also catalyze the physical and chemical processes that degrade proteins.

[0190] Magnesium ions (10 - 120 mM) can be used to inhibit the isomerization of aspartic acid to isoaspartic acid. Ca +2 ions (up to 100 mM) can increase the stability of human deoxyribonuclease. However, Mg +2 , Mn +2 and Zn +2 can destabilize rhDNase. Similarly, Ca +2 and Sr +2 can stabilize factor VIII, which can be destabilized by Mg +2 , Mn +2 and Zn +2 , Cu +2 and Fe +2 and its aggregation can be increased by Al +3 ions.

[0191] Embodiments of TTR hetero-oligomer (e.g., hetero-dimer, hetero-trimer, and hetero-tetramer) formulations further include one or more preservatives. Preservatives are required when developing multi-dose parenteral formulations involving more than two withdrawals from the same container. Its main function is to inhibit the growth of microorganisms over the storage life or shelf life of the formulation and ensure the sterility of the product. Commonly used preservatives include benzyl alcohol, phenol, and m-cresol. Preservatives have a long history of use with low molecular weight parenteral drugs, but the development of protein formulations containing preservatives can be difficult. Preservatives almost always have a destabilizing effect (aggregation) on proteins, which is a major factor limiting their use in multi-dose protein formulations. To date, most protein drugs have been formulated only for single use. However, when multi-dose formulations are possible, there are advantages in terms of patient convenience and high marketability. The development of a preserved formulation led to the commercialization of a more convenient multi-use injection pen. Human growth hormone (hGH) is a good example. 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.

[0192] During the formulation and development of preserved dosage forms, several aspects need to be considered. The effective preservative concentration in the formulation must be optimized. This requires testing a given preservative in the dosage form within a concentration range that confers an antimicrobial effect without compromising protein stability.

[0193] As can be expected, the development of liquid formulations containing preservatives is more difficult than that of lyophilized formulations. Freeze-dried products are lyophilized without preservatives and can be reconstituted with a preservative-containing diluent at the time of use. This shortens the time the preservative is in contact with the protein and significantly minimizes the associated stability risks. In the case of liquid formulations, the effectiveness and stability of the preservative should be maintained throughout the product shelf life (e.g., about 18 - 24 months). An important point to note is that the effectiveness of the preservative needs to be demonstrated in the final formulation containing the active drug and all excipient components.

[0194] Formulations of TTR heteromultimers (e.g., heterodimers, heterotrimers, and heterotetramers) will generally be designed according to specific routes and methods of administration, specific dosages and dosing frequencies, and specific treatments for specific diseases, especially in terms of the range of bioavailability and persistence. Thus, the formulations can be designed according to the present invention for delivery by any suitable route including, but not limited to, oral, auditory, ocular, rectal, and vaginal, as well as by parenteral routes including intravenous and intra-arterial injection, intramuscular injection, and subcutaneous injection.

[0195] Once the pharmaceutical composition is formulated, it may be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, crystal, or dehydrated or lyophilized powder. Such formulations may be stored either in a form ready for immediate use or in a form that needs to be reconstituted prior to administration (e.g., lyophilized product). The present invention also provides a kit for generating single-dose administration units. The kit of the present invention includes both a first container having a dry protein and a second container having an aqueous formulation. In certain embodiments of the present invention, kits are provided that include single and multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes).

[0196] The therapeutically effective amount of a pharmaceutical composition containing TTR hetero-oligomers (e.g., hetero-dimers, hetero-trimers, and hetero-tetramers) to be used will depend, for example, on the nature and purpose of the treatment. The dosage levels suitable for treatment will vary somewhat depending on the molecule being delivered, the TTR hetero-oligomers (e.g., hetero-dimers, hetero-trimers, and hetero-tetramers), the indication for which they are used, the route of administration, and the size (weight, body surface, or organ size) and / or condition (age and general health) of the patient, as will be understood by those skilled in the art. In certain embodiments, the clinician may determine the potency of the dosage and modify the route of administration to obtain the optimal therapeutic effect. Typical dosages can range from about 0.1 μg / kg to up to about 30 mg / kg or more, depending on the above factors. In specific embodiments, the dosage can range from 1.0 μg / kg to up 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.

[0197] A therapeutically effective amount of a TTR hetero-oligomer (e.g., hetero-dimer, hetero-trimer, and hetero-tetramer) preferably results in a decrease in the severity of disease symptoms, an increase in the frequency or duration of disease symptom-free periods, or the prevention of disease-related disability or incapacity due to pain.

[0198] The pharmaceutical composition may be administered using a medical device. Examples of medical devices for administering the pharmaceutical composition are described in U.S. Patent 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).

[0199] Therapeutic uses of TTR hetero-dimer, hetero-trimer, and hetero-tetramer fusion proteins As shown in the examples, it has been discovered that the multispecific TTR fusion proteins of the present invention can bind to two or more epitopes on one or more proteins. Such multispecific TTR fusions are particularly useful in that they can participate in multiple biological pathways and enable more effective treatment of disease states (e.g., cancer) compared to traditional treatment modalities.

[0200] The multispecific TTR fusion proteins of the present invention have advantages over many known bispecific / multispecific approaches. For example, the present invention provides a bivalent bispecific presentation of antigen-binding domains that reduces or eliminates affinity loss, for example, compared to hetero-IgG constructs. Further advantages over hetero-IgG constructs include that the multispecific TTR fusion proteins of the present invention can be generated without the need for Fc charge pair mutations (CPMs) that are required to drive heavy chain heterodimerization in hetero-IgG constructs, and the reduction or elimination of unwanted by-products such as half-antibodies and light chain mismatches (present in hetero-IgG and IgG-Fab constructs). Indeed, the TTR fusion proteins of the present invention reduce many (optionally, all) of the Ab or Fab manipulations required for other constructs.

[0201] 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 regions are exposed and sterically induced affinity loss is reduced or eliminated.

[0202] Another advantage of the TTR fusion proteins of the present invention stems from the use of the naive IgG format, which helps to reduce affinity loss and the increased tendency to aggregate observed when converting mAbs to scFv constructs. Gil and Schrum, Advances in Bioscience and Biotechnology, 4:73-84 (2013).

[0203] In addition, the TTR fusion proteins of the present invention enable efficient incorporation of diverse antigen-binding domains, allowing for rapid scanning of bispecific (or multispecific) combinations.

[0204] TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) fusion proteins also exhibit improved antigen clustering compared to individual antibodies and / or Fabs. When an antibody (e.g., IgG antibody) binds to an antigen on a target cell (e.g., a tumor cell), the resulting clustered Fc domains bind to FcγRs found on immune effector cells such as NK cells and macrophages. This clustering aids in signal transduction via FcγRs, leading to the initiation of cell-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP). Thus, TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) fusion proteins are particularly useful for targeting ligands where high antibody or Fab affinity / avidity activities lead to enhanced biological effects. Enhancement of cell-mediated effector functions by the TTR heteromultimer (e.g., heterodimer, heterotrimer, and heterotetramer) constructs of the present invention results in an increased ability to kill cells, which is useful, for example, in the treatment of cancer.

[0205] Accordingly, the present invention also relates to methods of treating cancer using the heterodimeric fusion proteins and heterotetrameric fusion proteins described herein.

[0206] 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.

[0207] In yet other embodiments, the present invention relates to the heterodimeric fusion proteins and heterotetrameric fusion proteins described herein for use in the treatment of cancer.

Examples

[0208] The following examples are provided for the purpose of exemplifying specific embodiments or features of the present invention and are not intended to limit its scope.

[0209] Example 1: General Techniques Example 1 describes the general techniques used to generate and characterize the TTR negative and positive constructs discussed in the remainder of the examples.

[0210] Using the following techniques, TTR negative and positive constructs containing TTR variants with one TTR dimer / dimer interface mutation per TTR subunit ( "C10A / K15A / XX") were generated.

[0211] Cloning of TTR Negative and Positive Variants in Escherichia coli (E. coli) Amgen Large Molecule Registry Construct C37979 (pAMG21: huTTR (opt-C10A, K15A)) was used as a template for all TTR negative and positive variants (containing 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.

[0212] These procedures were generally carried out according to methods found in Molecular Cloning: A Laboratory Manuel, 3rd ed., Sambrook et al., 2001, Cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y.

[0213] Expression of TTR Negative and Positive Variants in Escherichia coli (E. coli) BL21 cells containing the pAMG21 vector encoding TTR negative and positive variants were grown overnight at 30 - 37°C in 50 ml volume of Terrific Broth (Teknova T7060) containing 20 μg / ml kanamycin in a 250 ml baffled shake flask. 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 antifoaming agent and incubated at 33°C until the OD at 600 nm reached 0.4. 1 ml of Sigma K - 3255 N-(β-ketocaproyl)-DL-homoserine lactone autoinducer (stock solution dissolved in ethanol) was added to the culture and expressed at 30 - 33°C for 4 hours.

[0214] Purification of TTR negative and positive variants from Escherichia coli (E. coli) Frozen Escherichia coli (E. coli) cell paste was homogenized using an Omni TH (Omni International, Kennesaw, Georgia, USA) handheld homogenizer in a 1:10 (weight to volume) solution of 50 mM sodium phosphate, 300 mM NaCl, pH 8.0. The resulting suspension was then processed twice at 13,800 PSI through an M - 110S Microfluidizer (Microfluidics Corporation, Irvine, California, USA). The lysate was then centrifuged at 22,000 RCF for 1 hour at 4°C. The soluble fraction was filtered through a 0.45 μm cellulose acetate filter (Corning Life Sciences, Tewksbury, Massachusetts, USA) and retained as the starting material for FPLC purification; the insoluble fraction was discarded as waste.

[0215] A 5 mL Ni-NTA SuperFlow column (Qiagen, Hilden, Germany) connected to an ÄKTA purifier (GE Healthcare Bio-Sciences, Marlborough, Massachusetts, USA) was equilibrated with 5 column volumes (CV) of 50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 8.0 prior to sample application. The filtered soluble lysate was injected onto the column and 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.

[0216] The purified pool was concentrated using a VivaSpin 10 kDa MWCO (Sartorius AG, Göttingen, Germany) centrifugal filter and centrifuged at 3,000 RCF until the desired volume was reached.

[0217] The concentrated sample was dialyzed against 10 mM Tris-HCl, pH 8.0, 150 mM NaCl using a Slide-a-lyzer 10 kDa MWCO (Thermo Fisher Scientific, Waltham, Massachusetts, USA) dialysis cartridge until the starting buffer was less than 1% by calculation.

[0218] PC analysis of TTR negative and positive variants (E. coli) Protein quantification was performed by measuring the UV absorbance at 280 nm using a Nanodrop 2000c (Thermo Fisher Scientific).

[0219] Non-reducing SDS-PAGE analysis was performed with and without heating the samples. In both cases, the samples were treated with SDS-PAGE sample buffer and run on a 4–20% Tris-Gly SDS-PAGE (Thermo Fisher Scientific) according to the manufacturer's protocol. In the heating experiment, the samples and sample buffer were heated at 85 °C for 5 min and then loaded onto the gel; non-heated samples were loaded directly onto the gel after addition of the sample buffer. The gels were stained using SimplyBlue SafeStain (Thermo Fisher Scientific) according to the manufacturer's microwave protocol.

[0220] HPLC SEC analysis was performed on a SEC-3000, 7.8 × 300 mm column (Phenomenex, Torrance, California, USA) connected to an Agilent 1290 Infinity HPLC system (Agilent Technologies, Santa Clara, California, USA), with an isocratic mobile phase of 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 flowing at 1 mL / min, and UV absorbance observed at 280 nm.

[0221] TTR negative and positive variant dimerization The purified TTR samples were normalized to the lowest common molar concentration of the experimental cohort by diluting with 10 mM Tris-HCl, pH 8.0, 150 mM NaCl. The samples were combined in equal volumes and incubated overnight at 4 °C.

[0222] A portion of the mixed sample was processed by caspase cleavage as follows. The purified protein sample concentration was adjusted to 2.5 mg / mL by diluting with 10 mM Tris-HCl, pH 8.0, 150 mM NaCl. A 5× digestion buffer consisting of 250 mM NaCl, 15 mM 2-mercaptoethanol, pH 8.0 was prepared, brought to 25 °C in a water bath, and a 1× digestion buffer was also prepared by diluting the 5× buffer with water. A stock aliquot of caspase-3 (Amgen Inc., Thousand Oaks, CA, USA) was diluted to 0.1 mg / mL using the 1× digestion buffer. Four parts of protein, four parts of diluted caspase-3, eight parts of 5× digestion buffer and twenty parts of water were combined and incubated at 25 °C for 2 hours in a water bath. The digestion solution was removed and twenty parts of SDS-PAGE sample buffer (the same reagent as previously specified for SDS-PAGE analysis) was added. The cleavage reaction on SDS-PAGE was carried out using the same protocol as previously specified.

[0223] The resulting molecular mixtures, caspase-treated and untreated, were analyzed by non-reducing, non-heating SDS-PAGE and HPLC SEC.

[0224] (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) cloning of molecules (without linker) Using standard molecular biology techniques including polymerase chain reaction (PCR), site-directed PCR mutagenesis, restriction endonuclease digestion, and enzymatic ligation into mammalian expression plasmids, TTR was fused to several engineered variants of hybridoma-derived anti-CB1, anti-GITR, and anti-TR2 antibody heavy chains (HC). His-tagged Fab-TTR molecules were also generated. These cloned TTR fusion variant heavy chains and Fab DNAs were used in combination with their respective cloned anti-CB1, anti-GITR, and anti-TR2 antibody light chain (LC) DNAs to transfect mammalian cells for the expression of 2X Ab-TTR, 4X Ab-TTR, and 4X Fab-TTR. This technique was generally performed according to methods rd described in Moletular Cloning: A Laboratory Manuel, 3

[0225] ed., Sambrook et al., 2001, Cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y. TTR antibody and Fab fusion sequences were generated by GENEART® Seamless Cloning (GSC) or Golden Gate Assembly (GGA). The combined DNA fragments were generated by splicing overlap extension PCR (SOE-PCR) or ordered synthetically from an external vendor. The SOE-PCR products used for GSC cloning were made using mutagenic palindromic primers paired with flanking primers that created two PCR products sharing a 15 bp overlap region surrounding the codons for the desired amino acid change sites. The SOE-PCR products used for GGA were designed to contain a directional unique 4 base pair overhang generated by BsmBI digestion.

[0226] In summary, GGA uses type II restriction enzymes and T4 DNA ligase to cleave and seamlessly ligate multiple DNA fragments (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 full antibody gene, a linker, and a TTR sequence (GeneByte, Gen9, Cambridge, MA); and (ii) an expression vector backbone. The GGA reaction consisted of 50 ng of GeneByte, 20 ng of the expression vector, 1 μl of 10× Fast Digest Reaction Buffer + 0.5 mM ATP (Thermo Fisher, Waltham, MA), 0.5 μl of FastDigest Esp3I (Thermo Fisher, Waltham, MA), 1 μl of T4 DNA ligase (5 U / μl, Thermo Fisher, Waltham, MA), and water to 10 μl. The reaction was carried out over 15 cycles consisting of a 2-minute digestion step at 37°C and a 3-minute ligation step at 16°C. After 15 cycles, a final 5-minute digestion step at 37°C and a 5-minute enzyme inactivation step at 80°C followed.

[0227] [Ab "A"] = [negative TTR]2: [positive TTR]2 = expression of [Ab "B"] molecule (without linker) HEK 293-6E cells were maintained at 36°C in a shaking flask in an incubator with 5% CO2 and 80% - 90% humidity in FreeStyle F17 Medium (Thermo Fisher Scientific) supplemented with 0.1% (w / v) Poloxamer 188 (Sigma-Aldrich), 6 mM L-glutamine (Thermo Fisher Scientific), and 25 μg / ml G418 (Thermo Fisher Scientific), and stirred at 120 rpm on a shaker with a 25 mm shaking diameter. Two days before transfection, the 293-6E cells were seeded at 0.4×10 6 cells / ml. On the day of transfection, the cells were in the exponential growth phase (about 1.5×10 6cells / ml, >95% viability). Transient transfection was performed at 20% gene dosage by adding a mixture of 0.5 mg / L DNA (0.1 mg / L of the target gene construct DNA + 0.4 mg / L of vector DNA) and 2 mg / L of PEI Max (Polyethylenimine Max, Polysciences, Cat# 24765-2) to the cell culture. Four hours after transfection, a proprietary feed {Yeastolate (0.5% w / v) and glucose (3 g / L)} was added. The product was recovered 6 days after transfection by centrifuging the cells at 4000 rpm (3485 xg) for 40 minutes. The supernatant was filtered through a 0.45 μM PES (polyethersulfone) filter.

[0228] [Ab "A"] = [negative TTR]2: [positive TTR]2 = purification of [Ab "B"] molecule (without linker) An rProtein A Fast Flow column (GE Healthcare Bio-Sciences, Marlborough, Massachusetts, USA) connected to an AeKTApurifier (GE Healthcare Bio-Sciences) FPLC was equilibrated with Dulbecco's PBS (DPBS) prior to sample application. The filtered cell culture medium was injected onto the column, washed with 5 column volumes (CV) of DPBS, and eluted stepwise with 8 CV of 50 mM HOAc, pH 3.2. The eluate 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, NY, USA). The titrated and filtered rProtein A pool was split into two separate pools for further purification.

[0229] Half of the rProtein A pool was diluted 1:5 volume with 20 mM MES, pH 5.0 and then injected onto an SP Sepharose High Performance column (GE Healthcare Bio-Sciences) connected to an ÄKTA purifier (GE Healthcare Bio-Sciences) FPLC previously 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.

[0230] SP Sepharose fractions were analyzed on a Caliper LabChip GXII microcapillary electrophoresis system according to the manufacturer's protocol using a Protein Express Assay LabChip (Perkin Elmer, Waltham, MA, USA). Fractions were selected and pooled for enrichment of bands at the approximate molecular weights of monomeric Ab-TTR vs. non-conforming MW species.

[0231] The SP Sepharose pool was dialyzed against 10 mM MES, 150 mM NaCl, pH 6.5 using a Slide-a-lyzer 10 kDa MWCO (Thermo Fisher Scientific, Waltham, Massachusetts, USA) dialysis cartridge until the starting buffer was <1% by calculation.

[0232] The other half of the rProtein A pool was injected onto a Sephadex G-25 column (GE Healthcare Bio-Sciences) connected to an ÄKTA purifier (GE Healthcare Bio-Sciences) FPLC previously equilibrated with 20 mM MES, pH 6.5. The column was eluted isocratically in 10 mM MES, 150 mM NaCl, pH 6.5.

[0233] [Ab "A"] = [negative TTR]2 : [positive TTR]2 = PC analysis of [Ab "B"] molecule (without linker) Protein quantification was performed by measuring the UV absorbance at 280 nm using a Nanodrop 2000c (Thermo Fisher Scientific).

[0234] After treating the samples with SDS-PAGE sample buffer (Thermo Fisher Scientific) containing 100 mM iodoacetamide (Sigma-Aldrich, St. Louis, MO, USA), they were directly loaded onto a 10% Tris-Gly gel and electrophoresed according to the manufacturer's protocol to perform non-reducing SDS-PAGE analysis. Reducing SDS-PAGE analysis was performed by treating the samples with SDS-PAGE sample buffer and a sample reducing agent (Thermo Fisher Scientific). After incubating the samples at 85 °C for 5 minutes, they were loaded onto a 10% Tris-Gly gel and electrophoresed according to the manufacturer's protocol. The gels were stained using SimplyBlue SafeStain (Thermo Fisher Scientific) according to the manufacturer's microwave protocol.

[0235] HPLC SEC analysis was performed on a Zenix-C SEC-300, 7.8 × 300 mm column (Sepax Technologies Inc., Newark, DE, USA) connected to an Agilent 1290 Infinity HPLC system (Agilent Technologies, Santa Clara, California, USA). A constant composition mobile phase of 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 was flowed at 1 mL / min, and the UV absorbance was observed at 280 nm.

[0236] [Ab "A"] = [negative TTR]2 : [positive TTR]2 = DAS analysis of [Ab "B"] molecule (without linker) Denaturing LC-MS: All LC-MS data were acquired on an Agilent 6230 TOF LC / MS system with a 1290 Infinity LC system. Chromatographic separation was achieved using a Zorbax SB300-C8 3.5 μm 2.1×50 mm column operated at a temperature of 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 - 1.0 min; 20 - 70% mobile phase B from 1.0 - 9.0 min; 70 - 100% mobile phase B from 9.0 - 10.0 min, and held at 100% for an additional 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 obtained over the m / z range 1000 - 7000. The source fragmentor, skimmer, and octapole 1 RF values were: 460 V, 95 V, and 800 V (peak-to-peak), respectively. The ESI capillary voltage was 5.9 kV. The gas temperature was 340 °C. The drying gas was 13 L / min. The nebulizer was 25 psig. Oa-ToF calibration was performed using an Agilent Tune Mix with an automated calibration procedure implemented by MassHunter Data Acquisition version B.06.01, Build 6.01.6157.

[0237] [655 - 341 Ab]=[[LX]-[negative TTR]]2:[[positive TTR]-[LX]]2 = expression of [655 - 341 Ab] molecule Expression was performed as described for the [Ab "A"] = [negative TTR]2:[positive TTR]2 = [Ab "B"] molecule.

[0238] [655 - 341 Ab]=[[LX]-[negative TTR]]2:[[positive TTR]-[LX]]2 = purification of [655 - 341 Ab] molecule The filtered cell culture medium was injected into an in-line tandem purification system consisting of an rProtein A Fast Flow HiTrap column (GE Healthcare Bio-Sciences) and a Desalting HiTrap column (GE Healthcare Bio-Sciences), each equilibrated with DPBS and 10 mM MES 150 mM NaCl, pH 6.5, connected to an AeKTA purifier (GE Healthcare Bio-Sciences). 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 to 10 mM MES, 150 mM NaCl, pH 6.5 on the Desalting HiTrap column.

[0239] [655 - 341 Ab]=[[LX]-[negative TTR]]2:[[positive TTR]-[LX]]2 = DAS analysis of the [655 - 341 Ab] molecule Denaturing LC-MS was performed as described for the [Ab "A"]=[negative TTR]2:[positive TTR]2=[Ab "B"] molecule.

[0240] (1) [[Fab "A"]-[negative TTR]]2:[[positive TTR]-[Fab "B"]]2, (2) [Ab "A"]=[negative TTR]2:[positive TTR]2=[Ab "B"], and (3) expression of the [Ab "A"]=[negative TTR]2:[[positive TTR]-[Fab "B"]]2 molecule (co-expression) The CHO-K1 growth medium consists of 50% CS9 Media (non-selective, Amgen proprietary) + 50% ExCell302 (SAFC Biosciences #14324C) + 2 mM L-glutamine (Gibco #25030-081). The selection medium consists of the growth medium + 10 μg / ml puromycin (Gibco #A11138-03) + 500 μg / ml hygromycin (Invitrogen #10687-010). The production medium consists of CHO-K1 6DCD (ATO Media Lab, Amgen proprietary).

[0241] The transfection reagent consists of Lipofectamine LTX (Gibco #15338-100 (p / n 94756)) and Opti-MEM I Reduced Serum Medium (Gibco #31985-070). The growth conditions were suspension growth at 36°C + 5% CO2 in a humidified incubator shaken at 120 RPM using a vented shake flask. The transfection procedure was as follows. One day prior to transfection, the host culture was split to 7 - 10e 5 VCD / ml. The DNA / Lipofectamine LTX complex was prepared as follows. 4 μg of non-linearized DNA was diluted in 0.5 ml of Opti-MEM medium in a 24DWB (2.0 μg of GOI (gene of interest) and 2.0 μg of PB200 (hyperactive transposase)). For 4-strand transfection, 0.5 μg of each strand and 2.0 μg of PB200 (hyperactive transposase) were used for a total of 4.0 μg / transfection. For 3-strand transfection, 0.66 μg of each strand and 2.0 μg of PB200 (hyperactive transposase) were used for a total of 4.0 μg / transfection. 10 μl of Lipofectamine LTX was diluted in 0.5 ml of Opti-MEM medium in a 15 ml polypropylene tube and left for 5 minutes. Then, the diluted DNA was combined with Lipofectamine LTX and thoroughly mixed by pipetting. The mixture was incubated at room temperature for 15 - 20 minutes and mixed occasionally. Then, 2e 6Transfer the viable cells / transfection to a 15 - 50 ml polypropylene tube, rotate at 1200 rpm for 5 minutes, and aspirate the medium. Then, wash the cells with 1xPBS by complete resuspension and rotate at 1200 rpm for 5 minutes. Next, aspirate the 1xPBS and resuspend the cells in 1 ml of Opti - MEM (per transfection). Then, add 1 ml of the cells to each well, and then add the DNA / LTX complex dropwise to each well. Incubate the cells with shaking at 235 rpm, 36 °C + 5% CO2 for 5 - 6 hours. Next, add 2.0 ml of non - selective growth medium (CHO - K1 medium) to the cells. Selection 72 hours after transfection was performed by placing the cells in 4 ml of selective medium by complete resuspension. The growth scale - up on day 6 was performed by directly adding 1.6 ml from the DWB culture to 12 ml in a 50 ml vented spin tube. The production on day 10 was performed by resuspending approximately 13 ml of the N - 1 culture in production medium and inoculating a 40 ml batch product. The harvest on day 17 was performed by centrifuging the cells and then sterile - filtering the conditioned medium.

[0242] Purification of [[Fab "A"] - [negative TTR]]2:[[positive TTR] - [Fab "B"]]2 molecules (co - expression) The Fab - TTR fusion protein contains a C - terminal 6x his - tag and was captured from the CM via IMAC affinity chromatography (1 ml HisTrap Excel, GE Healthcare; 17 - 3712 - 05) at a flow rate of 2 ml / min. Then, wash the IMAC column at a flow rate of 4 ml / min with 5 CV of 20 mM sodium phosphate, 250 mM sodium chloride, pH 7.4, and elute the protein step - wise at 2 ml / min with 20 mM sodium phosphate, 250 mM sodium chloride, 0.5 M imidazole, pH 7.4. Strip the IMAC column with 6 M guanidine - HCl, 50 mM Tris, pH 8, equilibrate it with 20 mM sodium phosphate, 250 mM sodium chloride, pH 7.4, and then load the next sample.

[0243] All purified proteins were finally buffer-exchanged into 10 mM MES, 150 mM sodium chloride, pH 6 by passing them through a 5 ml HiTrap Desalting column (GE Healthcare; 17-1408-01) at a flow rate of 2 ml / min. All preparative chromatography procedures were performed using an AeKTA Purifiers (GE Healthcare). Subsequently, the quantity and quality of the generated proteins were characterized using a combination of analytical methods including A280 protein quantification, size exclusion chromatography (SEC), microcapillary electrophoresis (MCE), and SDS-PAGE.

[0244] [Ab "A"] = [negative TTR]2: [positive TTR]2 = [Ab "B"] and [Ab "A"] = [negative TTR]2: [[positive TTR] - [Fab "B"]]2 Purification of molecules (co-expression) The antibody-TTR fusion protein was supplemented from CM via protein A affinity chromatography (1 ml MabSelect SuRe HiTrap, GE Healthcare, Bio-Sciences, Marlborough, Massachusetts, USA; 11-0034-93) at a flow rate of 2 ml / min. Subsequently, the protein A column was washed with 5 CV of 25 mM Tris, 100 mM sodium chloride, pH 7.4 at a flow rate of 4 ml / min by stepwise elution of the protein at 2 ml / min with 100 mM acetic acid, pH 3.6. The column was stripped with 6 M guanidine-HCl, 50 mM Tris, pH 8 and equilibrated with 25 mM Tris, 100 mM sodium chloride, pH 7.4 before loading the next sample.

[0245] All purified proteins were finally buffer-exchanged into 10 mM MES, 150 mM sodium chloride, pH 6 by passing them through a 5 ml HiTrap Desalting column (GE Healthcare; 17-1408-01) at a flow rate of 2 ml / min. All preparative chromatography procedures were performed using an AeKTA Purifiers (GE Healthcare).

[0246] (1) [[Fab“A”]-[Negative TTR]]2:[[Positive TTR]-[Fab“B”]]2, (2) [Ab“A”]=[Negative TTR]2:[Positive TTR]2=[Ab“B”], and (3) PC analysis of [Ab“A”]=[Negative TTR]2:[[Positive TTR]-[Fab“B”]]2 molecules (co-expression) A280 quantification - Protein quantification was performed by measuring the UV absorbance at 280 nm using a Multiskan Go (Thermo Fisher Scientific, Waltham, Massachusetts, USA).

[0247] SEC - The TTR-fusion protein samples were applied to an ACQUITY UPLC BEH 200Å, 1.7 μm, 4.6 × 300 mm SEC column (Waters, Milford, Massachusetts, USA; 186005226) at a flow rate of 0.4 ml / min in a mobile phase of 100 mM sodium phosphate, 50 mM sodium chloride, 7.5% ethanol, pH 6.9, and the UV absorbance at 280 nm was observed. Analytical SEC was performed using a 1290 Infinity HPLC (Agilent Technologies, Santa Clara, California, USA). Due to the large MW (249 - 347 kDa) of these TTR-fusion molecules and manufacturing-related impurities, the approximate SEC retention times of specific fusion molecules were evaluated using MW benchmark molecules. These benchmark molecules were molecules manufactured by Amgen, Inc., and included two different antibodies (145 kDa each; protein lots BR4214-1 and PL41591), an antibody-TTR heterotetramer (635 kDa; protein lot PL38002), an antibody-TTR heterodimer (265 kDa; protein lot PL46796), and a Fab-TTR heterotetramer (248 kDa; protein lot PL38000).

[0248] The characteristics of the TTR-fusion protein samples were evaluated by microcapillary electrophoresis using an MCE-LabChip GXII (Caliper LifeSciences, Mountainview, California, USA). Samples were prepared both reductively and non-reductively according to the manufacturer's guidelines. The microfluidic chip technology automatically stains, destains, electrophoretically separates, and analyzes protein samples.

[0249] SDS-PAGE - TTR-fusion protein samples were electrophoresed on a variety of Tris-glycine, one-dimensional gels containing 8%, 10%, and 4–20% (Invitrogen, Carlsbad, California, USA; Wedge Well: XP00080, XP00100, XP04200, respectively). Samples were prepared non-reduced either without heating or by heating at 85 °C for 10 min. Gels were stained using SimpyBlue SafeStain (Invitrogen; LC6060) and compared to MW reference standards for identification of the desired product bands.

[0250] (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 molecule (co-expression) DAS analysis Denaturing LC-MS was performed as described for the [Ab "A"]=[negative TTR]2:[positive TTR]2=[Ab "B"] molecule.

[0251] SEC-Native-MS: All QToF experiments were performed on a Synapt G1 HDMS instrument operated in positive ESI mode. This instrument was converted to an RF-confined drift tube instrument similar to that described by Bush et al., Anal Chem 2010, 82:9557-9565. All important 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; transfer 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; transfer inlet 1.0 V; transfer outlet 1.0 V; transfer velocity 248 m / s; transfer wave amplitude 3.0 V; source RF amplitude (peak-to-peak) 450 V; triwave RF amplitude (peak-to-peak) trap 380 V, IMS 250 V, transfer 380 V; source backing pressure 6.0 mbar; trap / transfer pressure cC4F8, 2.00e-2 mbar (Pirani gauge reading: flow rate 4.0 mL / min). Instrument control and data acquisition were performed using MassLynx 4.1 SCN 872.

[0252] SEC was performed using an Agilent 1200 pump system operating at a flow rate of 75 μL / min at ambient temperature and a 2.1×50 mm, 300 Å, Waters BEH. The mobile phase was 200 mM ammonium acetate. The SEC separation was performed using a constant composition 6-minute method. 25 - 50 μg of material was injected for analysis. 200 mM ammonium acetate was used because it is a volatile buffer and thus compatible with the mass spectrometer. Instrument control was performed using ChemStation.

[0253] The following techniques were used to generate TTR negative and positive constructs containing a TTR variant with two TTR dimer / dimer interface mutations (「C10A / K15A / XX / YY」) per TTR subunit.

[0254] Cloning of Fab TTR dimers with TTR variants containing two TTR dimer / dimer interface mutations Cloning of Fab TTR dimers having a TTR variant containing two TTR dimer / dimer interface mutations was achieved using a method similar to that described in the section describing the cloning of molecules (without linker) 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).

[0255] Expression of Fab TTR dimers having a TTR variant containing two TTR dimer / dimer interface mutations Transfection was performed on a 50 ml scale. HEK 293-6E cells were maintained 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 shaking flask in an incubator at 36 °C with 5% CO2 and 80% - 90% humidity and stirred at 120 rpm on a shaker with a 25 mm shaking diameter. Two days before transfection, 293-6E cells were seeded at 0.4 × 10 6 cells / ml. On the day of transfection, the cells were in the exponential growth phase (about 1.5 × 10 6cells / ml, >95% viability). Transient transfection was performed by adding a mixture of 0.5 mg / L DNA and 2 mg / L PEI Max (Polyethylenimine Max, Polysciences, Cat# 24765-2) to the cell culture. Four hours after transfection, a proprietary feed {Yeastolate (0.5% w / v) and glucose (3 g / L)} was added. The product was recovered 6 days after transfection by centrifuging the cells at 4000 rpm (3485 xg) for 40 minutes. The supernatant was filtered through a 0.45 μM PES (polyethersulfone) filter.

[0256] Purification of Fab TTR dimers with TTR variants containing two TTR dimer / dimer interface mutations The filtered cell culture medium was injected into an in-line tandem purification system consisting of a HisTrap Excel column (GE Healthcare Bio-Sciences) and a Desalting HiTrap column (GE Healthcare Bio-Sciences) equilibrated with 20 mM sodium phosphate, 500 mM NaCl, pH 7.4 and 10 mM MES 150 mM NaCl, pH 6.0, respectively, connected to an AeKTApurifier (GE Healthcare Bio-Sciences). The HisTrap Excel column was washed with 20 mM sodium phosphate, 500 mM NaCl, pH 7.4 and eluted stepwise with 20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4. The HisTrap Excel eluate was buffer-exchanged to 10 mM MES, 150 mM NaCl, pH 6.0 on the Desalting HiTrap column.

[0257] PC of Fab TTR dimers with TTR variants containing two TTR dimer / dimer interface mutations Protein quantification was performed by measuring the UV absorbance at 280 nm using a MultiSkan FC Microplate Photometer (Thermo Fisher Scientific).

[0258] Non-reducing and reducing microcapillary electrophoresis analyses were performed on a Caliper LabChip GXII system using a Protein Express Assay LabChip (Perkin Elmer) according to the manufacturer's protocol.

[0259] HPLC-SEC analysis was performed on an ACQUITY UPLC BEH450 SEC 2.5 μm 7.8×300 mm column (Waters Corp., Milford, MA, USA) connected to an Agilent 1290 Infinity HPLC system (Agilent Technologies). A mobile phase of constant composition 100 mM NaH2PO4, 50 mM NaCl, 7.5% EtOH, pH 6.9 was flowed at 0.4 mL / min, and UV absorbance was observed at 280 nm.

[0260] Heterodimerization of [[Fab "A"]-[double negative TTR]] and [[double positive TTR]-[Fab "B"]]: Mixing and analysis of separately generated constructs 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.

[0261] Co-expression of [[Fab "A"]-[double negative TTR]] and [[double positive TTR]-[Fab "B"]] Transfections were performed individually and after completion (1 - 4 hours later), the [[Fab "A"]-[double negative TTR]] and [[double positive TTR]-[Fab "B"]] constructs were pooled and generated together at a 4 ml scale. HEK 293-6E cells were maintained at 36 °C in a shaker flask in an incubator with 5% CO2 and 80% - 90% humidity in FreeStyle F17 Medium (Thermo Fisher Scientific) supplemented with 0.1% (w / v) Poloxamer 188 (Sigma-Aldrich), 6 mM L-glutamine (Thermo Fisher Scientific), and 25 μg / ml G418 (Thermo Fisher Scientific), and stirred at 120 rpm on a shaker with a 25 mm shaking diameter. Two days before transfection, 293-6E cells were seeded at 0.4×10 6 cells / ml. On the day of transfection, the cells were in the exponential growth phase (about 1.5×10 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 (Polyethylenimine Max, Polysciences, Cat# 24765-2) to the cell culture. Four hours after transfection, a proprietary feed {Yeastolate (0.5% w / v) and glucose (3 g / L)} was added. The product was harvested 6 days after transfection by centrifuging the cells at 4000 rpm (3485 xg) for 40 minutes. The supernatant was filtered through a 0.45 μM PES (polyethersulfone) filter.

[0262] Purification of co-expressed [[Fab "A"]-[double negative TTR]] and [[double positive TTR]-[Fab "B"]] The filtered cell culture medium was injected into an in-line tandem purification system consisting of a HisTrap Excel column (GE Healthcare Bio-Sciences) and a Desalting HiTrap column (GE Healthcare Bio-Sciences), equilibrated with 20 mM sodium phosphate, 500 mM NaCl, pH 7.4 and 10 mM MES, 150 mM NaCl, pH 6.0, respectively, connected to an AeKTA purifier (GE Healthcare Bio-Sciences). The HisTrap Excel column was washed with 20 mM sodium phosphate, 500 mM NaCl, pH 7.4 and eluted stepwise with 20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4. The HisTrap Excel eluate was buffer-exchanged to 10 mM MES, 150 mM NaCl, pH 6.0 on the Desalting HiTrap column.

[0263] PC analysis of co-expressed [[Fab "A"]-[double negative TTR]] and [[double positive TTR]-[Fab "B"]] Protein quantification was performed by measuring the UV absorbance at 280 nm using a MultiSkan FC Microplate Photometer (Thermo Fisher Scientific). Non-reducing and reducing microcapillary electrophoresis analyses were performed on a Caliper LabChip GXII system using a Protein Express Assay LabChip (Perkin Elmer) according to the manufacturer's protocol. HPLC-SEC analysis was performed on an ACQUITY UPLC BEH450 SEC 2.5 μm 7.8×150 mm column (Waters Corp., Milford, MA, USA) connected to an Agilent 1290 Infinity HPLC system (Agilent Technologies), flowing a constant composition mobile phase of 100 mM NaH2PO4, 50 mM NaCl, 7.5% EtOH, pH 6.9 at 0.4 mL / min and observing the UV absorbance at 280 nm.

[0264] Example 2: Evaluation of TTR hetero-tetramers containing TTR variants produced in E. coli and having one TTR dimer / dimer interface mutation per TTR subunit (「C10A / K15A / XX」) Eighteen TTR charge variants (C10A / K15A / XX) of TTR (SEQ ID NO: 1) were generated to determine which charge mutations result in substantial repulsion at the TTR dimer / dimer interface (see Figure 4). Each of the TTR variants contained the C10A and K15A mutations and a third mutation designated as 「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.

[0265] Nine of the TTR variants (P24R, I84R, L17R, V121R, V20R, G22R, S112R, T119R, Y114R, and S115R) showed substantial weakening of the TTR dimer / dimer interface, as evidenced by the fact that the TTR tetramer disassembled into the corresponding TTR dimer in the presence of SDS (chaotrope) without heating (see Figure 5, 「non-heated」 gel). Six of the variants (V20R, G22R, S112R, T119R, Y114R, and S115R) also interfere with dimer / dimer interactions under non-denaturing (native) conditions, as evaluated by SEC (see Figure 6). In addition, four of the variants (P24R, I84R, L17R, and V121R) were observed to primarily form TTR tetramers under non-denaturing SEC conditions, showed a lower melting temperature, and again indicated that the dimer / dimer interface was weakened in these variants.

[0266] The six most preferred dimer / dimer interface mutation sites (Figure 6, red) were selected to generate additional TTR variants and to evaluate the formation of TTR hetero - tetramers containing two different TTR monomer sequences. In these experiments, the desired TTR hetero - tetramers consisted of [1] one TTR dimer consisting of two TTR monomers itself (each monomer being a "negative" TTR variant); and [2] one TTR dimer consisting of two TTR monomers itself (each monomer being a "positive" TTR variant). The six preferred dimer / dimer interface mutation sites were selected based on their ability to form hetero - tetramers under SEC and SDS - PAGE conditions. Mutation sites that predominantly led to dimer formation under SDS - PAGE conditions were selected as an initial cut - off (i.e., L17R, V121R, V20R, G22R, S112R, T119R, Y114R, and S115R). Y114R and S115R were excluded from further consideration because the protein yields were significantly low.

[0267] The negative TTR variants contained the C10A / K15A / XX mutation, where each XX was L17D, L17E, V20D, V20E, G22D, G22E, S112D, S112E, T119D, T119E, V121D, or V121E. Similarly, the positive TTR variants contained the C10A / K15A / XX mutation, where 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 negative and 12 positive) were generated (Figure 7).

[0268] The positive variants were mixed with the negative variants (in a pairwise fashion), and TTR hetero - tetramer formation was evaluated by both SDS - PAGE and SEC. Many of the variant pairings showed some tendency to form the desired hetero - tetramers, as indicated by the non - zero SEC values in Figure 7 (the values represent the % of hetero - tetramer formation). In fact, some pairings showed a very high tendency to form hetero - tetramers, with tetramer formation being 40 - 100%.

[0269] Many of these TTR hetero-tetramers were resistant to dissociation by chaotropic SDS, as shown by the SDS-PAGE results also shown in Figure 7. Positive / negative pairings that showed a high tendency to form stable hetero-tetramers (see cross-reference of SEC and SDS-PAGE data): include L17R / T119D, L17K / T119D, L17K / V121E, V20R / V20D, V20R / V20E, V20K / V20D, V20K / V20E, V121R / L17D, V121R / L17E, and V121K / L17D.

[0270] Positive / negative pairings that showed a high tendency to form stable TTR tetramers were further evaluated by SDS-PAGE. In this experiment, for each pairing, [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 a tetramer); and [4] the combination of the negative and positive variants exposed to caspase were evaluated (Figure 8). As shown in Figure 8, the separate negative and positive variants migrated to the bottom of the gel, while the combination of the negative and positive variants migrated only partway down the gel. This further demonstrates that when the negative and positive variants are combined, they form a high molecular weight species (HMW) (presumably the desired hetero-tetramer). Treating the combination of the negative and basic variant with caspase (which only cleaves the polyhistidine tag from the positive variant due to the inclusion of the DEVD sequence) produced a nearly uniform band that ran slightly lower than the uncleaved tetramer, indicating the presence of the positive component and that the tetramer is nearly uniform and presumably a hetero-tetramer.

[0271] Next, heterotetramers containing the L17R / T119D, L17K / T119D, L17K / V121E, V20R / V20D, V20R / V20E, V20K / V20D, V20K / V20E, V121R / L17D, V121R / L17E, and V121K / L17D pairings were exposed to pH 5.0 conditions to determine whether they could maintain their tetrameric state under conditions similar to those found in pharmaceutical formulations (by SEC) (Figure 9). Indeed, the single peak in Figure 9 indicates that the heterotetramers were able to maintain their tetrameric state.

[0272] The melting temperatures of three heterotetramers were evaluated. In each case, the heterotetramers were stable up to at least 92 °C, indicating that the heterotetramers are very thermally stable (Figure 10).

[0273] Example 3: Evaluation of TTR heterotetrameric Fab, Ab, and mixed Fab / Ab constructs containing TTR variants having one TTR dimer / dimer interface mutation per TTR subunit (''C10A / K15A / XX'') produced in mammalian cells The ability to form TTR heterotetrameric Fab, Ab, and mixed Fab / Ab constructs was evaluated. In these constructs, TTR tetramers containing two positive and two negative TTR variants (as described above) were used to generate TTR heterotetramers attached to four Fabs, two Abs, or one Ab and two Fabs. See Figures 2a, 2b, and 2c, respectively. Charge pair mutations in the Fab constructs can be used to drive proper HC / LC Fab pairing. One advantage of such TTR heterotetrameric constructs is that the fusion of TTR monomer units to the C-terminus of the Ab and / or Fab allows for the assembly of multiple antigen-targeting moieties (e.g., Ab and / or Fab). This orients the Ab and / or Fab such that their antigen-binding domains are less subject to steric hindrance seen in other bivalent bispecific platforms (e.g., IgG-Fab and IgG scFv constructs) that typically result from the fusion of a Fab or scFv N-terminus to the IgG C-terminus.

[0274] TTR heterotetramer Ab construct Bispecific TTR heterotetramer Ab constructs were generated. In these constructs, one Ab (655-341 Ab) was 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) was specific for DNP. An exemplary bispecific TTR heterotetramer Ab construct is shown in FIG. 11, where each heavy chain of the 655-341 Ab (hatched) is attached to the N-terminus of the negative TTR monomer (forming a negative TTR dimer together), and each heavy chain of the DNP-3B1 Ab (solid) is attached to the N-terminus of the positive TTR monomer (forming a positive TTR dimer together).

[0275] Four negative TTR variants were fused to the 655-341 Ab, and four positive TTR variants were fused to the DNP-3B1 Ab (FIG. 11). All of the Ab-TTR fusions were made without a linker between the Ab and the TTR monomer. These variants were generated in mammalian cells (293-6E HEK cells) for secretion into the medium. Two sets of transformed cells were generated: one to produce the 655-341 Ab / negative TTR fusion ([655-341 Ab]=[negative TTR]2), and the other to produce the DNP-3B1 Ab / positive TTR fusion ([positive TTR]2=[DNP-3B1 Ab]).

[0276] Interestingly, in contrast to the TTR tetramers produced in E. coli (Example 2), significant amounts of the secreted constructs were: the self-associating 655-341 Ab / negative TTR fusions (i.e., [655-341 Ab]=[negative TTR]2:[negative TTR]2=[655-341 Ab]), the self-associating DNP-3B1 Ab / positive TTR fusions (i.e., [DNP-3B1 Ab]=[positive TTR]2:[positive TTR]2=[DNP-3B1 Ab]), or other HMW species. The free 655-341 Ab / negative TTR fusions ([655-341 Ab]=[negative TTR]2) and DNP-3B1 Ab / positive TTR fusions ([positive TTR]2=[DNP-3B1 Ab]) accounted for 11-32% of the secreted constructs (Figure 12). Rapid buffer exchange decreased the amount of the HMW species, which mainly resulted in an increase in the self-associating species.

[0277] The effect of adding a linker between the Ab heavy chain and the TTR monomer was examined using the same eight charge variants. Five linkers in the range of 2 - 10 amino acids in size were evaluated (Figure 13). The linker-containing Ab / TTR fusions that were mainly secreted were again self-associates (e.g., [655-341 Ab]=[[LX]:[negative TTR]]2:[[negative TTR]:[LX]]2=[655-341 Ab]) and HMW species. The free 655-341 Ab / negative TTR fusion and the DNP-3B1 Ab / positive TTR fusion ([655-341 Ab]=[[LX]:[negative TTR]]2 and [[positive TTR]:[LX]]2=[DNP-3B1 Ab]) accounted for up to 31% of the secreted constructs (Figures 14 and 15). In particular, shorter linkers generally resulted in higher titers and yields (i.e., more protein production) and lower levels of HMW species (Figure 16). Additionally, the negative TTR fusions resulted in moderately high yields and lower HMW species levels. None of the linker-containing Ab-TTR fusions formed the 655-341 Ab / negative TTR fusion or the DNP-3B1 Ab / positive TTR fusion as the main product. Other observations included that shorter linkers produced higher levels of SDS-resistant self-associating Ab / TTR fusions; that V20K, V20R, and V121K produced higher levels of SDS-sensitive self-associating Ab / TTR fusions; that T119D, V20D, V121E, and L17D produced higher titers / yields and reduced amounts of free Ab / TTR fusions; and that the negative variants appeared to produce less amounts of HMW species.

[0278] In light of these observations, the hypothesis was put forward that mammalian cells cannot efficiently produce "non-tetramerized" TTR. That is, mammalian cells may have difficulty producing [655-341 Ab]=[negative TTR]2 or [positive TTR]2=[DNP-3B1 Ab] (with or without a linker), because such constructs contain only two TTR monomers (compared to native TTR having four TTR monomers). To investigate this, five combinations of 655-341 Fab / negative TTR fusions and DNP-3B1 Fab / positive TTR fusions were co-produced in a mammalian cell line (CHO K1). In these constructs, the Fab HC was attached to TTR via a GG linker (Figure 17).

[0279] Co-production of [655-341 Fab]-[GG]-[negative TTR] and [positive TTR]-[GG]-[DNP-3B1 Fab] in CHO K1 resulted in significant formation of the desired [[655-341 Fab]-[GG]-[negative TTR]]2:[[positive TTR]-[GG]-[DNP-3B1 Fab]]2 (78 - 88%) and reduced amounts of HMW species (less than 5% in each case) (Figure 18). Fab formation was found to be very efficient for all mutant pairs, although only a slight advantage seemed to exist for the L17D / V121R pair. In addition, it seemed as if the V20R / V20D pair could form weak heterotetramers that could be disrupted by SDS.

[0280] When Ab- and Fab-TTR fusions and non-fused Ab were used as SEC standards, it was found that molecule 15524 ([655-341 Fab]-[GG]-[TTR(C10A / K15A / L17D)] and [TTR(C10A / K15A / V121R)]-[GG]-[DNP-3B1 Fab]) had a retention time similar to that of the 4X-Fab homomultimer and was substantially shorter than the retention time expected for the 2X-Fab-TTR fusion (which should elute after the control Ab) (Figure 19). Furthermore, when 15524 was evaluated by SEC-coupled MS, the molecular mass of the eluted species was consistent with what was expected (Figure 20).

[0281] The same procedure was carried out to determine whether co-expression of the Ab-containing TTR fusion would result in the production of the desired TTR heterotetramer [655-341 Ab]=[[LX]-[negative TTR]]2:[[positive 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 (Figure 21). Significant amounts of the desired TTR heterotetramer were formed up to 70% for many of the combinations (Figure 22). In addition, the 4-amino acid linker resulted in higher titers and yields compared to the 0-amino acid linker. When Ab- and Fab-TTR fusions and unfused Ab were used as SEC standards, it was found that molecule 15539 ([655-341 Ab]=[[GGAGGGAGGG]-[TTR(C10A / K15A / L17D]]2:[[TTR(C10A / K15A / V121K)]-[GGAGGGAGGG]]2=[DNP-3B1 Ab]) had an elution profile with a major peak retention time nearly identical to that of the 2X-Ab-TTR homomultimer (Figure 23). Furthermore, when the constructs were evaluated by SEC-coupled MS, the molecular masses of the eluting species were consistent with those of the desired TTR heterotetramer (Figure 24). On average, the L4 linker was observed to result in good yields associated with good preferential production of the desired product. In addition, combinations of the L17K / T119D, V20K / V20D, and V20R / V20D mutations appeared to result in high expression and yields. Finally, combinations of the L17K / V121E, V121K / L17D, and V20K / V20D mutations appeared to be most likely to drive the desired assembly of the TTR heterotetramer. See Figure 25.

[0282] Next, the inventors sought to determine whether co-expression of negative and positive TTR variants (four of each mutation) fused to Ab and Fab in the same cell line would result in the generation of an Ab-Fab-TTR construct (i.e., the [Ab "A"] = [negative TTR]2:[[positive TTR]=[Fab "B"]]2 construct). Five TTR variants, combining three linker lengths (X = 0, 4, and 10 amino acids) and two Ab / Fabs, were tested for a total of 30 combinations (Figure 26). For many combinations, based on SEC, the amount of Ab fusion that formed the desired Ab-Fab-TTR construct was up to 45.6% (Figure 27). Additionally, the 4- and 10-amino acid linkers appear to produce more of the desired Ab-Fab-TTR construct compared to the 0-amino acid linker (based on titer and purification yield). When Ab- and Fab-TTR fusions and unfused Ab were used as SEC standards, molecule 15545 ([655-341 Ab]=[[GGGG]-[TTR(C10A / K15A / V20D)]]2:[[TTR(C10A / K15A / V20R)]-[GG]-[DNP-3B1-Fab]]2) was observed to generate a peak with retention between the 2X-Ab-TTR homotetramer and the 4X-Fab-TTR homotetramer, as expected (Figure 28). Furthermore, when this molecule was evaluated by SEC-coupled MS, the molecular mass of the eluted species was consistent with the desired construct (Figure 29). On average, the L10 linker was observed to be preferred in the Ab-Fab-TTR construct. Additionally, the L17K / T119D TTR mutation appears to be preferred in the Ab-Fab-TTR construct. See Figure 30.

[0283] Example 4: Evaluation of TTR Heterotetrameric Fab, Ab, and Mixed Fab / Ab Constructs Containing TTR Variants with Two TTR Dimer / Dimer Interface Mutations ("C10A / K15A / XX / YY") per TTR Subunit in Mammalian Cells Ab- and Fab-TTR fusions with two charged interface mutations were generated separately in mammalian cells as previously described for single charged interface mutations. Titers, yields after affinity chromatography 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 a matrix format and the mixture was evaluated by SEC as previously described for single charge variants.

[0284] Ab- and Fab-TTR fusions with single and double charged interface mutations were generated by co-culturing mammalian cells with oppositely charged TTR variants as previously described for single charged interface mutations. Yields after affinity chromatography and SEC performance were evaluated as previously described for single charged interface mutations.

[0285] The results of these experiments can be found in FIGS. 31 - 40. As can be appreciated in FIGS. 35 and 36, certain combinations of variants resulted in a significant increase in 4X-Fab formation compared to that produced for a simple mixture of the molecules produced individually prior to mixing (determined by taking the average of the premix 4X-Fab levels and subtracting that value from the observed 4X-Fab). FIG. 37 illustrates this by showing that the 4X-Fab SEC peak of the mixture is much larger than the premix 4X-Fab peak. In addition, certain co-culture conditions resulted in a greater amount of 4X-Fab formation (FIGS. 38 and 39). FIG. 40 shows that the co-cultured 4X-Fab peak is significantly larger than the 4X-Fab peak of the individually cultured molecules, indicating a possible increase in 4X-Fab formation in the presence of charge counter variants.

[0286]

Table 7

[0287]

Table 8

[0288]

Table 9

[0289]

Table 10

[0290]

Table 11

[0291]

Table 12

[0292]

Table 13

[0293]

Table 14

[0294]

Table 15

[0295]

Table 16

Claims

1. A transthyretin (TTR) protein complex, wherein the TTR protein complex comprises TTR subunits A, B, C, and D; TTR subunits A and B dimerize to form a TTR dimer AB; TTR subunits C and D dimerize to form a TTR dimer CD; TTR dimer AB and TTR dimer CD further dimerize to form a TTR tetramer ABCD; Each of A, B, C, and D comprises the amino acid sequence of SEQ ID NO: 1, except for the following mutations, Each of A, B, C, and D comprises the mutations C10A and K15A, Each of A and B comprises at least one mutation selected from L17D / E, V20D / E, G22D / E, S112D / E, T119D / E, and V121D / E, Each of C and D comprises at least one mutation selected from L17R / K, V20R / K, G22R / K, S112R / K, T119R / K, and V121R / K, A transthyretin (TTR) protein complex.

2. Each of A and B comprises at least two mutations selected from L17D / E, V20D / E, G22D / E, S112D / E, T119D / E, and V121D / E, Each of C and D comprises at least two mutations selected from L17R / K, V20R / K, G22R / K, S112R / K, T119R / K, and V121R / K, The TTR protein complex according to claim 1.

3. Each of A, B, C, and D Both A and B comprise L17D, and both C and D comprise L17R, Both A and B comprise L17E, and both C and D comprise L17R, Both A and B comprise V20D, and both C and D comprise L17R, Both A and B contain V20E, and both C and D contain L17R. Both A and B contain G22D, and both C and D contain L17R. Both A and B contain G22E, and both C and D contain L17R. Both A and B contain S112D, and both C and D contain L17R. Both A and B contain S112E, and both C and D contain L17R. Both A and B contain T119D, and both C and D contain L17R. Both A and B contain T119E, and both C and D contain L17R. Both A and B contain V121D, and both C and D contain L17R. Both A and B contain V121E, and both C and D contain L17R. Both A and B contain L17D, and both C and D contain L17K. Both A and B contain L17E, and both C and D contain L17K. Both A and B contain V20D, and both C and D contain L17K. Both A and B contain V20E, and both C and D contain L17K. Both A and B contain G22D, and both C and D contain L17K. Both A and B contain G22E, and both C and D contain L17K. Both A and B contain S112D, and both C and D contain L17K. Both A and B contain S112E, and both C and D contain L17K. Both A and B contain T119D, and both C and D contain L17K. Both A and B contain T119E, and both C and D contain L17K. Both A and B contain V121D, and both C and D contain L17K. Both A and B contain V121E, and both C and D contain L17K. Both A and B contain L17D, and both C and D contain V20R. Both A and B contain L17E, and both C and D contain V20R. Both A and B contain V20D, and both C and D contain V20R. Both A and B contain V20E, and both C and D contain V20R. Both A and B contain G22D, and both C and D contain V20R. Both A and B contain G22E, and both C and D contain V20R. Both A and B contain S112D, and both C and D contain V20R. Both A and B contain S112E, and both C and D contain V20R. Both A and B contain T119D, and both C and D contain V20R. Both A and B contain T119E, and both C and D contain V20R. Both A and B contain V121D, and both C and D contain V20R. Both A and B contain V121E, and both C and D contain V20R. Both A and B contain L17D, and both C and D contain V20K. Both A and B contain L17E, and both C and D contain V20K. Both A and B contain V20D, and both C and D contain V20K. Both A and B contain V20E, and both C and D contain V20K. Both A and B contain G22D, and both C and D contain V20K. Both A and B contain G22E, and both C and D contain V20K. Both A and B contain S112D, and both C and D contain V20K. Both A and B contain S112E, and both C and D contain V20K. Both A and B contain T119D, and both C and D contain V20K. Both A and B contain T119E, and both C and D contain V20K. Both A and B contain V121D, and both C and D contain V20K. Both A and B contain V121E, and both C and D contain V20K. Both A and B contain L17D, and both C and D contain G22R. Both A and B contain L17E, and both C and D contain G22R. Both A and B contain G22D, and both C and D contain G22R. Both A and B contain G22E, and both C and D contain G22R. Both A and B contain S112D, and both C and D contain G22R. Both A and B contain S112E, and both C and D contain G22R. Both A and B contain T119D, and both C and D contain G22R. Both A and B contain T119E, and both C and D contain G22R. Both A and B contain V121E, and both C and D contain G22R. Both A and B contain L17D, and both C and D contain G22K. Both A and B contain L17E, and both C and D contain G22K. Both A and B contain G22D, and both C and D contain G22K. Both A and B contain G22E, and both C and D contain G22K. Both A and B contain S112D, and both C and D contain G22K. Both A and B contain S112E, and both C and D contain G22K. Both A and B contain T119D, and both C and D contain G22K. Both A and B contain T119E, and both C and D contain G22K. Both A and B contain V121E, and both C and D contain G22K. Both A and B contain L17D, and both C and D contain S112R. Both A and B contain L17E, and both C and D contain S112R. Both A and B contain V20D, and both C and D contain S112R. Both A and B contain V20E, and both C and D contain S112R. Both A and B contain G22D, and both C and D contain S112R. Both A and B contain G22E, and both C and D contain S112R. Both A and B contain S112D, and both C and D contain S112R. Both A and B contain S112E, and both C and D contain S112R. Both A and B contain T119D, and both C and D contain S112R. Both A and B contain T119E, and both C and D contain S112R. Both A and B contain V121D, and both C and D contain S112R. Both A and B contain V121E, and both C and D contain S112R. Both A and B contain V20D, and both C and D contain S112K. Both A and B contain V20E, and both C and D contain S112K. Both A and B contain G22D, and both C and D contain S112K. Both A and B contain G22E, and both C and D contain S112K. Both A and B contain S112D, and both C and D contain S112K. Both A and B contain S112E, and both C and D contain S112K. Both A and B contain T119E, and both C and D contain S112K. Both A and B contain V121D, and both C and D contain S112K. Both A and B contain L17D, and both C and D contain T119R. Both A and B contain L17E, and both C and D contain T119R. Both A and B contain V20E, and both C and D contain T119R. Both A and B contain G22D, and both C and D contain T119R. Both A and B contain G22E, and both C and D contain T119R. Both A and B contain S112D, and both C and D contain T119R. Both A and B contain S112E, and both C and D contain T119R. Both A and B contain T119D, and both C and D contain T119R. Both A and B contain T119E, and both C and D contain T119R. Both A and B contain V121D, and both C and D contain T119R. Both A and B contain V121E, and both C and D contain T119R. Both A and B contain L17D, and both C and D contain T119K. Both A and B contain L17E, and both C and D contain T119K. Both A and B contain V20D, and both C and D contain T119K. Both A and B contain V20E, and both C and D contain T119K. Both A and B contain G22D, and both C and D contain T119K. Both A and B contain G22E, and both C and D contain T119K. Both A and B contain S112D, and both C and D contain T119K. Both A and B contain S112E, and both C and D contain T119K. Both A and B contain T119D, and both C and D contain T119K. Both A and B contain T119E, and both C and D contain T119K. Both A and B contain V121D, and both C and D contain T119K. Both A and B contain V121E, and both C and D contain T119K. Both A and B contain L17D, and both C and D contain V121R. Both A and B contain L17E, and both C and D contain V121R. Both A and B contain G22D, and both C and D contain V121R. Both A and B contain G22E, and both C and D contain V121R. Both A and B contain T119D, and both C and D contain V121R. Both A and B contain T119E, and both C and D contain V121R. Both A and B contain V121D, and both C and D contain V121R. Both A and B contain V121E, and both C and D contain V121R. Both A and B contain L17D, and both C and D contain V121K. Both A and B contain L17E, and both C and D contain V121K. Both A and B contain G22D, and both C and D contain V121K. Both A and B contain G22E, and both C and D contain V121K. Both A and B contain T119D, and both C and D contain V121K. Both A and B contain T119E, and both C and D contain V121K. Both A and B contain V121D, and both C and D contain V121K, or Both A and B contain V121E, and both C and D contain V121K. The TTR protein complex according to claim 1 or 2, comprising the amino acid sequence of SEQ ID NO: 1 having a mutation.

4. Both A and B contain L17D, and both C and D contain V121K. Both A and B contain L17E, and both C and D contain V121R. Both A and B contain V20D, and both C and D contain V20R. Both A and B contain V20D, and both C and D contain V20K. Both A and B contain V20E, and both C and D contain V20R. Both A and B contain V20E, and both C and D contain V20K. Both A and B contain T119D, and both C and D contain L17R. Both A and B contain T119D, and both C and D contain L17K, or Both A and B contain V121E, and both C and D contain L17K. The TTR protein complex according to claim 1 or 2.

5. Each of A, B, C, and D Both A and B contain L17D / V20D, and both C and D contain L17K / V20K. Both A and B contain L17D / V20E, and both C and D contain L17K / V20R. Both A and B contain L17E / V20D, and both C and D contain L17R / V20K. Both A and B contain L17E / V20E, and both C and D contain L17R / V20R. Both A and B contain L17D / T119D, and both C and D contain L17K / V121K. Both A and B contain L17D / V121E, and both C and D contain L17K / V121R. Both A and B contain L17E / T119D, and both C and D contain L17R / V121K. Both A and B contain L17E / V121E, and both C and D contain L17R / V121R. Both A and B contain V20D / T119D, and both C and D contain V20K / V121K. Both A and B contain V20D / V121E, and both C and D contain V20K / V121R. Both A and B contain V20E / T119D, and both C and D contain V20R / V121K, or Both A and B contain V20E / V121E, and both C and D contain V20R / V121R. The TTR protein complex according to claim 1 or 2, comprising the amino acid sequence of SEQ ID NO: 1 having a mutation.

6. Both A and B contain V20E / T119D, and both C and D contain V20R / V121K. Both A and B contain L17D / T119D, and both C and D contain L17K / V121K. Both A and B contain L17E / T119D, and both C and D contain L17R / V121K. Both A and B contain L17E / V20D, and both C and D contain L17R / V20K. Both A and B contain L17D / V20D, and both C and D contain L17K / V20K, or Both A and B contain L17E / V121E, and both C and D contain L17R / V121R. The TTR protein complex according to claim 1 or 2, comprising the amino acid sequence of SEQ ID NO: 1 having a mutation.

7. The TTR protein complex according to any one of claims 1 to 6, wherein the TTR protein complex is bound to an antigen-binding protein or peptide of 1, 2, 3, 4, 5, 6, 7, or 8.

8. The TTR protein complex according to claim 7, wherein the TTR protein complex is bound to an antigen-binding protein or peptide of 1, 2, 3, or 4.

9. The TTR protein complex according to claim 7 or 8, wherein the antigen-binding protein or peptide is bound to the TTR protein complex at the C-terminus of the TTR subunit.

10. The TTR protein complex according to claim 7 or 8, wherein the antigen-binding protein or peptide is bound to the TTR protein complex at the N-terminus of the TTR subunit.

11. The TTR protein complex according to any one of claims 7 to 10, wherein the TTR protein complex is directly bound to the antigen-binding protein.

12. The TTR protein complex according to any one of claims 7 to 10, wherein the TTR protein complex is bound to the antigen-binding protein via a linker.

13. The linker is an amino acid-based linker containing 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, and the TTR protein complex according to claim 12.

14. The linker is an amino acid-based linker containing 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, and the TTR protein complex according to claim 12.

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

16. The linker is an amino acid-based linker containing 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, and the TTR protein complex according to claim 12.

17. The linker is G, GG, GGG, GGGG, GGGGG, GGGGGG, GGGGGGG, GGGGGGGG, GGGGGGGGG, or GGGGGGGGGG, and the TTR protein complex according to claim 12.

18. The linker is G(GxB y)rG z, where: G = glycine; B = amino acid x = 1 to 15; y = 1 to 5; z = 1 to 15; and The TTR protein complex according to claim 12, wherein r = 1 to 20.

19. B = Q, S, A, E, P, T, K, R, D or N; x = 4; y = 1; z = 4; and The TTR protein complex according to claim 18, wherein r = 1.

20. The TTR protein complex according to claim 12, wherein the linker is selected from the list comprising: GG, GGGG, GGGSGG, GGGGSGGG, and GGAGGGAGGGG.

21. The TTR protein complex according to any one of claims 7 to 20, wherein the TTR protein complex is bound to two antigen-binding proteins, and the antigen-binding proteins bind to different antigens.

22. The TTR protein complex according to any one of claims 7 to 20, wherein the TTR protein complex is bound to four antigen-binding proteins, and the antigen-binding proteins bind to at least two different antigens.

23. The TTR protein complex according to any one of claims 7 to 22, wherein the antigen-binding protein is an antibody.

24. The TTR protein complex according to any one of claims 7 to 22, wherein the antigen-binding protein is Fab or scFv.

25. The TTR protein complex according to claim 24, wherein the antigen-binding protein is Fab.

26. The TTR protein complex according to any one of claims 7 to 22, wherein the antigen-binding protein is a mixture of an antibody and Fab.

27. One or more isolated nucleic acids encoding the TTR protein complex according to any one of claims 1 to 26.

28. An expression vector comprising the nucleic acid according to Claim 27.

29. A recombinant host cell comprising the nucleic acid according to Claim 27 or the vector according to Claim 28.

30. The recombinant host cell according to Claim 29, wherein the host cell is a Chinese hamster ovary (CHO) cell, an E5 cell, a baby hamster kidney (BHK) cell, a simian kidney (COS) cell, a human hepatocellular carcinoma cell, or a human embryonic kidney 293 (HEK293) cell.

31. A method for producing the TTR protein complex according to any one of Claims 1 to 26, the method comprising: a) culturing the recombinant host cell according to Claim 29 or 30; and b) isolating the TTR protein complex from the culture. A method comprising the above steps.

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