Materials and methods for improved single-chain variable fragments

Abstract

Materials and methods for improved single-chain variable fragments are disclosed.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application is a compilation of U.S. Provisional Patent Applications No. 62 / 946,897, No. 62 / 946,886, No. 62 / 946,882, No. 62 / 946,877, No. 62 / 946,865, filed on December 11, 2019, and No. 62 / 946,865, filed on August 15, 2019. Claiming the interests of U.S. Provisional Patent Application No. 62 / 887,529, U.S. Provisional Patent Application No. 62 / 887,527 filed on August 15, 2019, U.S. Provisional Patent Application No. 62 / 887,524 filed on August 15, 2019, U.S. Provisional Patent Application No. 62 / 887,519 filed on August 15, 2019, and U.S. Provisional Patent Application No. 62 / 887,514 filed on August 15, 2019, each of which is incorporated herein by reference in whole.

[0002] (Sequence Listing) This application is incorporated by referencing the sequence listing submitted with this application, which is a text file titled "14620-227-228_SL.txt" created on August 5, 2020, with a size of 258,724 bytes.

[0003] (Field of invention) Materials and methods for improved single-chain variable fragments are disclosed. [Background technology]

[0004] Antigen-binding single-chain variable fragments (scFvs) are modules that can be widely used as therapeutic agents, imaging agents, diagnostic agents, or as part of heterogeneous molecules such as multispecific molecules. One of the challenges of scFvs is their low stability and tendency to aggregate (re-examined in Worn and Pluckthun (2001) J Mol Biol 305:989-1010; Rothlisberger et al., (2005) J Mol Biol 347:773-789; Gross et al., (1989) Transplant Proc 21(1 Pt 1):127-130; Porter et al., (2011) J Cancer 2:331-332; Porter et al., (2011) N Engl J Med 365:725-733).

[0005] Therefore, an improved scFv design is needed that can be arbitrarily incorporated into multispecific molecules and heterogeneous molecules.

[0006] (overview) In one embodiment, the present disclosure relates to an isolated single-chain variable fragment (scFv) comprising a heavy chain variable region (VH), a linker (L), and a light chain variable region (VL), wherein the scFv is The first disulfide bond between the structurally conserved surface-exposed VH cysteine ​​(Cys) and the first L Cys, A second disulfide bond between a structurally preserved surface-exposed VLCys and a second L-Cys, or The present invention provides an scFv comprising a first disulfide bond between a structurally preserved surface-exposed VH Cys and a first L Cys, and a second disulfide bond between a structurally preserved surface-exposed VL Cys and a second L Cys.

[0007] This disclosure also relates to isolated scFv comprising VH, L, and VL, VH contains VH Cys at structurally conserved surface-exposed VH framework residue positions, and L contains a first L Cy, VL contains VL Cys at structurally conserved surface-exposed VL framework residue positions, and L contains a second L Cy, ​​or The present invention provides an scFv in which VH contains VH Cys at structurally conserved surface-exposed VH framework residue positions, VL contains VL Cys at structurally conserved surface-exposed VL framework residue positions, and L contains a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys can form a disulfide bond, and the VL Cys and the second L Cys can form a disulfide bond.

[0008] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H105, VL includes Cys in L42, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which the scFv is in a VL-L-VH orientation.

[0009] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H105, VL includes Cys in L45, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which the scFv is in a VL-L-VH orientation.

[0010] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H105, VL includes Cys in L39, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which the scFv is in a VL-L-VH orientation.

[0011] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H5, VL includes Cys in L42, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which the scFv is in a VL-L-VH orientation.

[0012] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H5, VL includes Cys in L45, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which the scFv is in a VL-L-VH orientation.

[0013] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H5, VL includes Cys in L39, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which the scFv is in a VL-L-VH orientation.

[0014] This disclosure also includes scFv including VH, L and VL, VH contains Cys in H3, VL includes Cys in L42, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which the scFv is in a VL-L-VH orientation.

[0015] This disclosure also includes scFv including VH, L and VL, VH contains Cys in H3, VL includes Cys in L45, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which the scFv is in a VL-L-VH orientation.

[0016] This disclosure also includes scFv including VH, L and VL, VH contains Cys in H3, VL includes Cys in L39, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which the scFv is in a VL-L-VH orientation.

[0017] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H43, VL includes Cys in L100, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which scFv is in a VH-L-VL orientation.

[0018] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H43, VL includes Cys in L102, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which scFv is in a VH-L-VL orientation.

[0019] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H43, VL includes Cys in L5, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which scFv is in a VH-L-VL orientation.

[0020] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H43, VL includes Cys in L3, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which scFv is in a VH-L-VL orientation.

[0021] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H40, VL includes Cys in L100, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which scFv is in a VH-L-VL orientation.

[0022] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H40, VL includes Cys in L102, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which scFv is in a VH-L-VL orientation.

[0023] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H40, VL includes Cys in L5, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which scFv is in a VH-L-VL orientation.

[0024] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H40, VL includes Cys in L3, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which scFv is in a VH-L-VL orientation.

[0025] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H46, VL includes Cys in L100, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which scFv is in a VH-L-VL orientation.

[0026] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H46, VL includes Cys in L102, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which scFv is in a VH-L-VL orientation.

[0027] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H46, VL includes Cys in L5, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which scFv is in a VH-L-VL orientation.

[0028] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H46, VL includes Cys in L3, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which scFv is in a VH-L-VL orientation.

[0029] This disclosure also provides a pharmaceutical composition comprising the spFv of this disclosure and a pharmaceutically acceptable carrier.

[0030] This disclosure also provides polynucleotides comprising the spFv of this disclosure.

[0031] This disclosure also provides vectors comprising the polynucleotides of this disclosure.

[0032] This disclosure also provides host cells containing the vector of this disclosure.

[0033] The disclosure also provides a method for producing spFv of the disclosure, comprising culturing host cells of the disclosure under conditions in which spFv is produced, and purifying the spFv.

[0034] This disclosure also provides an anti-idiotype antibody that binds to the spFv of this disclosure.

[0035] This disclosure also provides a kit containing the spFv of this disclosure.

[0036] In another embodiment, the present disclosure is a multispecific molecule comprising a single-chain variable fragment (scFv) including a heavy-chain variable region (VH), a linker (L), and a light-chain variable region (VL), wherein the scFv is The first disulfide bond between the structurally conserved surface-exposed VH cysteine ​​(Cys) and the first L Cys, A second disulfide bond between a structurally preserved surface-exposed VL Cys and a second L Cys, or The present invention provides a multispecific molecule comprising a first disulfide bond between a structurally conserved surface-exposed VH Cys and a first L Cys, and a second disulfide bond between a structurally conserved surface-exposed VL Cys and a second L Cys.

[0037] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH contains VH Cys at structurally conserved surface-exposed VH framework residue positions, and L contains a first L Cy. VL contains VL Cys at structurally conserved surface-exposed VL framework residue positions, L contains a second L Cy, ​​or The present invention provides a multispecific molecule in which VH contains VH Cys at structurally conserved surface-exposed VH framework residue positions, VL contains VL Cys at structurally conserved surface-exposed VL framework residue positions, and L contains a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys can form disulfide bonds, and the VL Cys and the second L Cys can also form disulfide bonds.

[0038] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H105, VL includes Cys in L42, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VL-L-VH orientation.

[0039] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H105, VL includes Cys in L45, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which the scFv is in a VL-L-VH orientation.

[0040] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H105, VL includes Cys in L39, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VL-L-VH orientation.

[0041] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H5, VL includes Cys in L42, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VL-L-VH orientation.

[0042] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H5, VL includes Cys in L45, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VL-L-VH orientation.

[0043] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H5, VL includes Cys in L39, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VL-L-VH orientation.

[0044] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH contains Cys in H3, VL includes Cys in L42, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VL-L-VH orientation.

[0045] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH contains Cys in H3, VL includes Cys in L45, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VL-L-VH orientation.

[0046] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH contains Cys in H3, VL includes Cys in L39, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VL-L-VH orientation.

[0047] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H43, VL includes Cys in L100, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VH-L-VL orientation.

[0048] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H43, VL includes Cys in L102, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VH-L-VL orientation.

[0049] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H43, VL includes Cys in L5, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VH-L-VL orientation.

[0050] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H43, VL includes Cys in L3, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VH-L-VL orientation.

[0051] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H40, VL includes Cys in L100, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VH-L-VL orientation.

[0052] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H40, VL includes Cys in L102, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VH-L-VL orientation.

[0053] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H40, VL includes Cys in L5, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VH-L-VL orientation.

[0054] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H40, VL includes Cys in L3, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VH-L-VL orientation.

[0055] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H46, VL includes Cys in L100, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VH-L-VL orientation.

[0056] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H46, VL includes Cys in L102, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VH-L-VL orientation.

[0057] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H46, VL includes Cys in L5, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VH-L-VL orientation.

[0058] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H46, VL includes Cys in L3, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VH-L-VL orientation.

[0059] This disclosure also provides pharmaceutical compositions comprising multispecific molecules provided herein and pharmaceutically acceptable carriers.

[0060] In yet another embodiment, the present disclosure relates to a heterogeneous molecule comprising a single-chain variable fragment (scFv) including a heavy-chain variable region (VH), a linker (L), and a light-chain variable region (VL), wherein the scFv is The first disulfide bond between the structurally conserved surface-exposed VH cysteine ​​(Cys) and the first L Cys, A second disulfide bond between a structurally preserved surface-exposed VL Cys and a second L Cys, or The present invention provides heterogeneous molecules comprising a first disulfide bond between a structurally preserved surface-exposed VH Cys and a first L Cys, and a second disulfide bond between a structurally preserved surface-exposed VL Cys and a second L Cys.

[0061] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH contains VH Cys at structurally conserved surface-exposed VH framework residue positions, and L contains a first L Cy, VL contains VL Cys at structurally conserved surface-exposed VL framework residue positions, and L contains a second L Cy, ​​or The present invention provides heterologous molecules in which VH contains VH Cys at structurally conserved surface-exposed VH framework residue positions, VL contains VL Cys at structurally conserved surface-exposed VL framework residue positions, and L contains a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys can form disulfide bonds, and the VL Cys and the second L Cys can form disulfide bonds.

[0062] This disclosure also relates to heterogeneous molecules comprising scFv including VH, L, and VL, VH includes Cys in H105, VL includes Cys in L42, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VL-L-VH orientation.

[0063] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH includes Cys in H105, VL includes Cys in L45, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VL-L-VH orientation.

[0064] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH includes Cys in H105, VL includes Cys in L39, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VL-L-VH orientation.

[0065] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH includes Cys in H5, VL includes Cys in L42, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in the VL-L-VH orientation.

[0066] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH includes Cys in H5, VL includes Cys in L45, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in the VL-L-VH orientation.

[0067] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH includes Cys in H5, VL includes Cys in L39, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VL-L-VH orientation.

[0068] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH contains Cys in H3, VL includes Cys in L42, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VL-L-VH orientation.

[0069] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH contains Cys in H3, VL includes Cys in L45, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in the VL-L-VH orientation.

[0070] This disclosure also relates to heterogeneous molecules comprising scFv including VH, L, and VL, VH contains Cys in H3, VL includes Cys in L39, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VL-L-VH orientation.

[0071] This disclosure also relates to heterogeneous molecules comprising scFv including VH, L, and VL, VH includes Cys in H43, VL includes Cys in L100, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VH-L-VL orientation.

[0072] This disclosure also relates to heterogeneous molecules comprising scFv including VH, L, and VL, VH includes Cys in H43, VL includes Cys in L102, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VH-L-VL orientation.

[0073] This disclosure also relates to heterogeneous molecules comprising scFv including VH, L, and VL, VH includes Cys in H43, VL includes Cys in L5, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VH-L-VL orientation.

[0074] This disclosure also relates to heterogeneous molecules comprising scFv including VH, L, and VL, VH includes Cys in H43, VL includes Cys in L3, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VH-L-VL orientation.

[0075] This disclosure also relates to heterogeneous molecules comprising scFv including VH, L, and VL, VH includes Cys in H40, VL includes Cys in L100, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VH-L-VL orientation.

[0076] This disclosure also relates to heterogeneous molecules comprising scFv including VH, L, and VL, VH includes Cys in H40, VL includes Cys in L102, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VH-L-VL orientation.

[0077] This disclosure also relates to heterogeneous molecules comprising scFv including VH, L, and VL, VH includes Cys in H40, VL includes Cys in L5, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VH-L-VL orientation.

[0078] This disclosure also relates to heterogeneous molecules comprising scFv including VH, L, and VL, VH includes Cys in H40, VL includes Cys in L3, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VH-L-VL orientation.

[0079] This disclosure also relates to heterogeneous molecules comprising scFv including VH, L, and VL, VH includes Cys in H46, VL includes Cys in L100, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VH-L-VL orientation.

[0080] This disclosure also relates to heterogeneous molecules comprising scFv including VH, L, and VL, VH includes Cys in H46, VL includes Cys in L102, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VH-L-VL orientation.

[0081] This disclosure also relates to heterogeneous molecules comprising scFv including VH, L, and VL, VH includes Cys in H46, VL includes Cys in L5, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0082] This disclosure also relates to heterogeneous molecules comprising scFv including VH, L, and VL, VH includes Cys in H46, VL includes Cys in L3, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VH-L-VL orientation.

[0083] This disclosure also provides pharmaceutical compositions comprising heterologous molecules of this disclosure and pharmaceutically acceptable carriers.

[0084] In yet another aspect, the present disclosure relates to a process for preparing a stabilized scFv, To provide a heavy chain variable region (VH) and a light chain variable region (VL) that form an antigen-binding domain, To provide a linker (L) that includes or is manipulated to include a first L Cy, Manipulating VH to include VH Cys at structurally conserved surface-exposed VH framework residue positions, The present invention provides a process that includes forming a disulfide bond between VH Cys and a first L Cys to prepare a stabilized scFv.

[0085] This disclosure also relates to a process for preparing a stabilized scFv, To provide VH and VL that form antigen-binding domains, To provide L containing a second L Cys, or L manipulated to contain a second L Cys, Manipulating the VL to include VL Cys at structurally conserved surface-exposed VL framework residue positions, The present invention provides a process that includes forming a disulfide bond between a VL Cys and a second L Cys to prepare a stabilized scFv.

[0086] This disclosure also relates to a process for preparing a stabilized scFv, To provide VH and VL that form antigen-binding domains, To provide L which includes a first L Cys and a second L Cys, or L which has been manipulated to include them, Manipulating VH to include VH Cys at structurally conserved surface-exposed VH framework residue positions, Manipulating the VL to include VL Cys at structurally conserved surface-exposed VL framework residue positions, The present invention provides a process that includes forming a disulfide bond between VH Cys and a first L Cys, and forming a disulfide bond between VL Cys and a second L Cys to prepare a stabilized scFv.

[0087] This disclosure also relates to a process for preparing a stabilized scFv, To provide polynucleotides encoding VH, L, and VL, Does VH include Cys in H105, and does VL include Cys in L42? Does VH include Cys in H43, and does VL include Cys in L100? Does VH contain Cys in H3, and does VL contain Cys in L3? Does VH contain Cys in H3, and does VL contain Cys in L5? Does VH contain Cys in H3, and does VL contain Cys in L39? Does VH contain Cys in H3, and does VL contain Cys in L42? Does VH include Cys in H3, and does VL include Cys in L45? Does VH include Cys in H3, and does VL include Cys in L100? Does VH contain Cys in H3, and does VL contain Cys in L102? Does VH contain Cys in H5, and does VL contain Cys in L3? Does VH contain Cys in H5, and does VL contain Cys in L5? Does VH include Cys in H5, and does VL include Cys in L39? Does VH include Cys in H5, and does VL include Cys in L42? Does VH include Cys in H5, and does VL include Cys in L45? Does VH include Cys in H5, and does VL include Cys in L100? Does VH contain Cys in H5, and does VL contain Cys in L102? Does VH include Cys in H40, and does VL include Cys in L3? Does VH include Cys in H40, and does VL include Cys in L5? Does VH include Cys in H40, and does VL include Cys in L39? Does VH include Cys in H40, and does VL include Cys in L42? Does VH include Cys in H40, and does VL include Cys in L45? Does VH include Cys in H40, and does VL include Cys in L100? Does VH include Cys in H40, and does VL include Cys in L102? Does VH include Cys in H43, and does VL include Cys in L3? Does VH include Cys in H43, and does VL include Cys in L5? Does VH include Cys in H43, and does VL include Cys in L39? Does VH include Cys in H43, and does VL include Cys in L42? Does VH include Cys in H43, and does VL include Cys in L45? Does VH include Cys in H43, and does VL include Cys in L102? Does VH include Cys in H46, and does VL include Cys in L3? Does VH include Cys in H46, and does VL include Cys in L5? Does VH include Cys in H46, and does VL include Cys in L39? Does VH include Cys in H46, and does VL include Cys in L42? Does VH include Cys in H46, and does VL include Cys in L45? Does VH include Cys in H46, and does VL include Cys in L100? Does VH include Cys in H46, and does VL include Cys in L102? Does VH include Cys in H105, and does VL include Cys in L3? Does VH include Cys in H105, and does VL include Cys in L5? Does VH include Cys in H105, and does VL include Cys in L39? Does VH include Cys in H105, and does VL include Cys in L45? VH contains Cys in H105, VL contains Cys in L100, or VH contains Cys at H105, VL contains Cys at L102, and residue numbering follows Chothia. L provides a polynucleotide containing the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. The present invention provides a process that includes expressing polynucleotides in host cells to produce stabilized scFv.

[0088] In yet another aspect, the present disclosure provides an isolated single-chain variable fragment (scFv) comprising a heavy-chain variable region (VH), means for linking (L), and a light-chain variable region (VL), wherein the scFv is The first disulfide bond between the structurally conserved surface-exposed VH cysteine ​​(Cys) and the first L Cys, A second disulfide bond between a structurally preserved surface-exposed VL Cys and a second L Cys, or The present invention provides an scFv comprising a first disulfide bond between a structurally preserved surface-exposed VH Cys and a first L Cys, and a second disulfide bond between a structurally preserved surface-exposed VL Cys and a second L Cys.

[0089] This disclosure also relates to an isolated single-chain variable fragment (scFv) comprising means for antigen binding, a linker (L), and a light chain variable region (VL), wherein the scFv is Structurally conserved surface-exposed antigen-binding means: First disulfide bond between cysteine ​​(Cys) and first L-Cys, A second disulfide bond between a structurally preserved surface-exposed VL Cys and a second L Cys, or The present invention provides an scFv comprising a structurally conserved surface-exposed antigen-binding means, a first disulfide bond between cysteine ​​(Cys) and a first L-Cys, and a second disulfide bond between a structurally conserved surface-exposed VL-Cys and a second L-Cys.

[0090] This disclosure also relates to an isolated single-chain variable fragment (scFv) comprising a heavy chain variable region (VH), a linker (L), and means for antigen binding, wherein the scFv is The first disulfide bond between the structurally conserved surface-exposed VH cysteine ​​(Cys) and the first L Cys, A structurally conserved surface-exposed antigen-binding means Cys and a second disulfide bond between the second L Cys, or The present invention provides an scFv comprising a first disulfide bond between a structurally preserved surface-exposed VH Cys and a first L Cys, and a second disulfide bond between a structurally preserved surface-exposed antigen-binding means Cys and a second L Cys.

[0091] This disclosure also relates to a multispecific molecule comprising a single-chain variable fragment (scFv) including a heavy-chain variable region (VH), a linkage (L), and a light-chain variable region (VL), wherein the scFv is The first disulfide bond between the structurally conserved surface-exposed VH cysteine ​​(Cys) and the first L Cys, A second disulfide bond between a structurally preserved surface-exposed VL Cys and a second L Cys, or The present invention provides a multispecific molecule comprising a first disulfide bond between a structurally conserved surface-exposed VH Cys and a first L Cys, and a second disulfide bond between a structurally conserved surface-exposed VL Cys and a second L Cys.

[0092] This disclosure also relates to a multispecific molecule comprising means for antigen binding, a linker (L), and a single-chain variable fragment (scFv) comprising a light chain variable region (VL), wherein the scFv is Structurally conserved surface-exposed antigen-binding means: First disulfide bond between cysteine ​​(Cys) and first L-Cys, A second disulfide bond between a structurally preserved surface-exposed VL Cys and a second L Cys, or The present invention provides a multispecific molecule comprising a structurally conserved surface-exposed antigen-binding means, a first disulfide bond between cysteine ​​(Cys) and a first L-Cys, and a second disulfide bond between a structurally conserved surface-exposed VL-Cys and a second L-Cys.

[0093] This disclosure also relates to a multispecific molecule comprising a single-chain variable fragment (scFv) including a heavy-chain variable region (VH), a linker (L), and means for antigen binding (VL), wherein the scFv is The first disulfide bond between the structurally conserved surface-exposed VH cysteine ​​(Cys) and the first L Cys, A structurally conserved surface-exposed antigen-binding means Cys and a second disulfide bond between the second L Cys, or The present invention provides a multispecific molecule comprising a first disulfide bond between a structurally conserved surface-exposed VH Cys and a first L Cys, and a second disulfide bond between a structurally conserved surface-exposed antigen-binding means Cys and a second L Cys.

[0094] This disclosure also relates to heterogeneous molecules comprising a single-chain variable fragment (scFv) having a heavy-chain variable region (VH), a linkage (L), and a light-chain variable region (VL), wherein the scFv is The first disulfide bond between the structurally conserved surface-exposed VH cysteine ​​(Cys) and the first L Cys, A second disulfide bond between a structurally preserved surface-exposed VL Cys and a second L Cys, or The present invention provides heterogeneous molecules comprising a first disulfide bond between a structurally preserved surface-exposed VH Cys and a first L Cys, and a second disulfide bond between a structurally preserved surface-exposed VL Cys and a second L Cys.

[0095] This disclosure also relates to a heterogeneous molecule comprising means for antigen binding, a linker (L), and a single-chain variable fragment (scFv) comprising a light chain variable region (VL), wherein the scFv is Structurally conserved surface-exposed antigen-binding means: First disulfide bond between cysteine ​​(Cys) and first L-Cys, A second disulfide bond between a structurally preserved surface-exposed VL Cys and a second L Cys, or The present invention provides heterologous molecules comprising a structurally conserved surface-exposed antigen-binding means, a first disulfide bond between cysteine ​​(Cys) and a first L-Cys, and a second disulfide bond between a structurally conserved surface-exposed VL-Cys and a second L-Cys.

[0096] This disclosure also relates to a heterogeneous molecule comprising a single-chain variable fragment (scFv) including a heavy-chain variable region (VH), a linker (L), and means for antigen binding, wherein the scFv is The first disulfide bond between the structurally conserved surface-exposed VH cysteine ​​(Cys) and the first L Cys, A structurally conserved surface-exposed antigen-binding means Cys and a second disulfide bond between the second L Cys, or The present invention provides a heterologous molecule comprising a first disulfide bond between a structurally conserved surface-exposed VH Cys and a first L Cys, and a second disulfide bond between a structurally conserved surface-exposed antigen-binding means Cys and a second L Cys.

[0097] This disclosure also provides means for encoding the scFv provided herein.

[0098] This disclosure also provides means for replicating the vectors provided herein.

[0099] This disclosure also provides compositions comprising means for stabilizing scFv.

[0100] This disclosure also provides compositions comprising means for increasing the thermal stability of scFv.

[0101] This disclosure also provides a multispecific molecule including means for stabilizing scFv.

[0102] This disclosure also provides a multispecific molecule including means for increasing the thermal stability of scFv.

[0103] This disclosure also provides heterogeneous molecules including means for stabilizing scFv.

[0104] This disclosure also provides heterogeneous molecules including means for increasing the thermal stability of scFv. [Brief explanation of the drawing]

[0105] [Figure 1] An exemplary design of a stabilized scFv (spFv) is shown. VL and VH are connected by a movable linker, shown as a dashed line in the figure, which contains the staple sequence CPPC (SEQ ID NO: 1), and "SS" indicates disulfide bonds between the staple sequences of the linker and anchor points. [Figure 2] A graph of anchor point selection for spFv in VL-linker-VH orientation is shown. The germline human antibody Fv (pMESdb id 5I19, GLk1) was used for drafting and illustrative distance measurements. The dashed lines represent the distance between Cβ atoms of Å residues. Structurally conserved framework positions with desired distances were selected as anchor points for mutations to Cys. The anchor points for VL-linker-VH orientation were Chothia position 42 for VL (K42 in the figure) and position 105 for VH (Q105 in the figure). The C-terminal VL residue (K107) and N-terminal VH residue (Q1) are also shown. [Figure 3]A graph of anchor point selection for spFv in VH-linker-VL orientation is shown. Fv (pdb id 5I19, GLk1) of germline human antibody was used for drafting and illustrative distance measurements. The dashed lines represent the distance between Cβ atoms of Å residues. Structurally conserved framework positions with desired distances were selected as anchor points for mutations to Cys. The anchor points for VH-linker-VL orientation were Chothia position 43 for VH (K43 in the figure) and position 100 for VL (Q100 in the figure). The C-terminal VH residue (S114) and N-terminal VL residue (D1) are also shown. [Figure 4] This graph shows the Cβ(Cys1)-Cβ(Cys2) distance between two Cys residues in the mouse double-chain IgG2a (pdb id 1igt) hinge CPPC (SEQ ID NO: 1). In the graph, the distance is shown in angstroms. [Figure 5] This graph shows the Cβ(Cys1)-Cβ(Cys2) distance between two Cys residues in the two heavy chains of human IgG (pdb id 5dk3) hinge CPPC (SEQ ID NO: 1). In the graph, the distance is shown in angstroms. [Figure 6] Selected VL anchor points are highlighted in gray and numbered as 1 and 2 under the amino acid alignment. VL sequences are numbered according to the Chothia numbering scheme. VL anchor point 1 (Chothia position 42) is used for spFv with VL-linker-VH orientation, and VL anchor point 2 (Chothia position 100) is used for spFv with VH-linker-VL orientation. GLk1VL: SEQ ID NO: 56, GLk2VL: SEQ ID NO: 57, CAT2200VL: SEQ ID NO: 58; CAT2200bVL: SEQ ID NO: 59. [Figure 7]Selected VH anchor points are highlighted in gray and numbered as 1 and 2 under the amino acid alignment. VH sequences are numbered according to the Chothia numbering scheme. VH anchor point 1 (Chothia position 105) is used for spFv with VL-linker-VH orientation, and VH anchor point 2 (Chothia position 43) is used for spFv with VH-linker-VL orientation. Glk1VH: SEQ ID NO: 60; GLk2VH: SEQ ID NO: 61, CAT2200aVH: SEQ ID NO: 62. [Figure 8] The structure of GLk1 spFv VL-VH is shown. The formation of staples between the VH and VL anchor points and the linker is evident from the structure. [Figure 9] The structure of GLk1 spFv VH-VL is shown. The formation of staples between the VH and VL anchor points and the linker is evident from the structure. [Figure 10] The structure of GLk2 spFv VH-VL is shown. The formation of staples between the VH and VL anchor points and the linker is evident from the structure. [Figure 11] The structure of CAT2200b spFv VH-VL is shown. The formation of staples between the VH and VL anchor points and the linker is evident from the structure. [Figure 12] This shows a comparison of unbound CAT2200b spFv VH-VL (upper part) with CAT2200a scFv VL-VH (lower part) coupled to IL-17A. [Figure 13] This shows a comparison of the front views of the uncoupled CAT2200b spFv VH-VL (upper part) compared to the CAT2200a spFv VL-VH (lower part) coupled to IL-17A. [Figure 14] This shows a comparison of the rear view structures of an uncoupled CAT2200b spFv VH-VL (upper part) compared to a CAT2200a scFv VL-VH (lower part) coupled to IL-17A. [Figure 15A]A schematic diagram of the antibody fused to a stapled scFv is shown. A 2:1 heterodimer and isotype control antibody fused to a stapled scFv derived from LTBRmAb1 are also shown. [Figure 15B] A schematic diagram of the antibody fused to a stapled scFv is shown. A 2:1 heterodimer and isotype control antibody fused to a stapled scFv derived from LTBRmAb1 are also shown. [Figure 15C] A schematic diagram of the antibody fused to a stapled scFv is shown. A 2:1 heterodimer and isotype control antibody fused to a stapled scFv derived from LTBRmAb1 are also shown. [Figure 15D] A schematic diagram of the antibody fused to a stapled scFv is shown. A 2:1 heterodimer and isotype control antibody fused to a stapled scFv derived from LTBRmAb1 are also shown. [Figure 15E] A schematic diagram of the antibody fused to a stapled scFv is shown. The 2:1 heterodimer EDBmAb1, fused to a stapled scFv derived from LTBRmAb1, is also shown. [Figure 15F] A schematic diagram of the antibody fused to a stapled scFv is shown. The 2:1 heterodimer EDBmAb1, fused to a stapled scFv derived from LTBRmAb1, is also shown. [Figure 15G] A schematic diagram of the antibody fused to a stapled scFv is shown. The 2:1 heterodimer EDBmAb1, fused to a stapled scFv derived from LTBRmAb1, is also shown. [Figure 15H] A schematic diagram of the antibody fused to a stapled scFv is shown. The 2:1 heterodimer EDBmAb1, fused to a stapled scFv derived from LTBRmAb1, is also shown. [Figure 15I] A schematic diagram of the antibody fused to a stapled scFv is shown. EDBmAb1, a 2:1 heterodimer fused to a stapled scFv derived from a lower affinity variant of LTBRmAb1, is also shown. [Figure 15J]A schematic diagram of the antibody fused to a stapled scFv is shown. EDBmAb1, a 2:1 heterodimer fused to a stapled scFv derived from a lower affinity variant of LTBRmAb1, is also shown. [Figure 15K] A schematic diagram of an antibody fused to a stapled scFv is shown. It shows a 2:1 heterodimer, EDBmAb1, or B21M fused to a stapled scFv derived from LTBRmAb1, which does not contain the protein A mutation in the Fc region. [Figure 15L] A schematic diagram of an antibody fused to a stapled scFv is shown. It shows a 2:1 heterodimer, EDBmAb1, or B21M fused to a stapled scFv derived from LTBRmAb1, which does not contain the protein A mutation in the Fc region. [Figure 15M] A schematic diagram of the antibody fused to a stapled scFv is shown. The 2:1 heterodimer MSLNmAb1, fused to a stapled scFv derived from LTBRmAb1, is also shown. [Figure 16A] The graph illustrates the results of an A549 NF-κB reporter assay using 2:1 bispecific antibodies. It compares TAA-dependent LTBR activation by COVA1456 with COVA1482, their respective control molecules COVA1462 and COVA1486, and recombinant human LIGHT. [Figure 16B] The graph illustrates the results of the A549 NF-κB reporter assay using 2:1 bispecific antibodies. It compares TAA-dependent LTBR activation by COVA1482, as well as bispecific antibodies COVA14107 and COVA14108 containing lower affinity variants of LTBRmAb1, and COVA1486. [Figure 16C] The graph illustrates the results of an A549 NF-κB reporter assay using 2:1 bispecific antibodies. It compares TAA-dependent LTBR activation by COVA1482 and COVA14133 (constructs without protein A mutations), as well as their respective control molecules, COVA1486 and COVA14136. [Figure 17] Shows the results of low cytometry staining of ICAM-1 on A375 cells after co-culture experiment. Compare COVA1482 and its control molecule COVA1486 with recombinant human LIGHT. [Figure 18A] Shows a graph illustrating the measurement of cytokines in the supernatant of co-cultures treated with the anti-EDB / anti-LTBR bispecific antibody COVA14133 compared to COVA14136 and COVA1440. The assay is performed using the MSD platform. It is the concentration of human RANTES. [Figure 18B] Shows a graph illustrating the measurement of cytokines in the supernatant of co-cultures treated with the anti-EDB / anti-LTBR bispecific antibody COVA14133 compared to COVA14136 and COVA1440. The assay is performed using the MSD platform. It is the concentration of human IL-6. [Figure 18C] Shows a graph illustrating the measurement of cytokines in the supernatant of co-cultures treated with the anti-EDB / anti-LTBR bispecific antibody COVA14133 compared to COVA14136 and COVA1440. The assay is performed using the MSD platform. It is the concentration of human IL-8. [Figure 18D] Shows a graph illustrating the measurement of cytokines in the supernatant of co-cultures treated with the anti-EDB / anti-LTBR bispecific antibody COVA14133 compared to COVA14136 and COVA1440. The assay is performed using the MSD platform. It is the concentration of human MIP-3b. [Figure 19A] Shows LTBR activation by MSLN / LTBR bispecificity in A549 NF-κB reporter / CHOK1MSLN or A549 NF-κB reporter / H226 co-culture cell assays. It is the activation of LTBR in the A549 NF-κB reporter / H226 co-culture assay. Compare COVA14146 (2:1 MSLNmAb1×LTBRmAb1) with LIGHT and the isotype control 2:1 construct COVA1486. [Figure 19B]This shows LTBR activation due to MSLN / LTBR bispecificity in A549 NF-κB reporter / CHOK1MSLN or A549 NF-κB reporter / H226 co-culture cell assays. The concentration of RANTES secreted upon LTBR activation in the A549 NF-κB reporter / H226 co-culture assay is also shown. COVA14146 (2:1 MSLNmAb1×LTBRmAb1) is compared to the LIGHT and isotype control 2:1 construct COVA1486.

[0106] (Detailed description) The methods disclosed can be more readily understood by referring to the following detailed description made in relation to the accompanying drawings, which form part of this disclosure. It should be understood that the methods disclosed are not limited to any particular methods described and / or shown herein, and furthermore, that the terms used herein are intended solely to illustrate and not to limit any particular embodiment.

[0107] All patents, published patent applications, and publications referenced herein are incorporated by reference in the same manner as if they were included herein in their entirety.

[0108] Where a list is presented, it should be understood that, unless otherwise specified, each individual element of that list and all combinations of that list represent distinct embodiments. For example, a list of embodiments presented as "A, B, or C" should be interpreted as including embodiments "A", "B", "C", "A or B", "A or C", "B or C", or "A, B, or C".

[0109] When used herein and in the attached "Claims," ​​the singular forms "a," "an," and "the" include plural references unless specifically indicated otherwise. For example, a reference to "a cell" includes combinations of two or more cells, and similar combinations.

[0110] The transitional phrases “comprising,” “consisting essentially of,” and “consisting” are intended to imply their generally accepted meanings in patent terminology, namely, (i) “comprising” is synonymous with “containing,” “containing,” or “characterizing,” and is comprehensive or non-restrictive, not excluding other unlisted elements or process steps; (ii) “consisting of” excludes any elements, processes, or components not specified in the claims; and (iii) “consisting essentially of” limits the scope of the claims to specified materials or processes, as well as those that “do not substantially affect the basic and novel features” of the claimed invention. Embodiments described with respect to the phrase “comprising” (or its equivalent) also provide embodiments that are described independently with respect to “consisting” and “consisting essentially of.”

[0111] "Approximately" means that a particular value is within the acceptable margin of error as determined by those skilled in the art, which depends to some extent on the method by which the value is measured or determined, i.e., on the limitations of the measurement system. Unless otherwise expressly stated in the examples or elsewhere in the specification in the context of a particular assay, result, or embodiment, "approximately" means that a value is within the greater of one standard deviation or a range of up to 5% in accordance with the practices of the art.

[0112] An "alternative scaffold" refers to a single-chain protein framework containing a structured core that associates with a highly conformable variable domain. Because the variable domain allows for polymorphisms to be introduced without compromising the integrity of the scaffold, the variable domain can be genetically engineered and selected to bind to a specific antigen.

[0113] "Antibody-dependent cell-mediated cytotoxicity," or "ADCC," is a mechanism by which antibody-coated target cells induce cell death through the interaction of Fc gamma receptors (FcγR) expressed in effector cells with effector cells that possess lytic activity, such as natural killer cells (NK), monocytes, macrophages, and neutrophils.

[0114] "Antibody-dependent phagocytosis" or "ADCP" refers to a mechanism by which antibody-coated target cells are eliminated through uptake by phagocytic cells such as macrophages or dendritic cells.

[0115] An "antigen" refers to any molecule that can mediate an immune response (e.g., a protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, a portion thereof, or a combination thereof). Exemplary immune responses include antibody production and activation of immune cells such as T cells, B cells, or NK cells.

[0116] An "antigen-binding fragment" or "antigen-binding domain" means a portion of a protein that binds to an antigen. The antigen-binding fragment may be a synthetic polypeptide, an enzyme-available polypeptide, or a genetically modified polypeptide, and may include multispecific proteins containing VH, VL, VH and VL, Fab, F(ab')2, Fd and Fv fragments, a domain antibody (dAb) consisting of one VH domain or one VL domain, the smallest recognition unit consisting of amino acid residues that mimic the CDR of an antibody, such as a camelid VH domain, a VHH domain, or an FR3-CDR3-FR4 moiety, HCDR1, HCDR2, and / or HCDR3, as well as a portion of immunoglobulin bound to LCDR1, LCDR2, and / or LCDR3, an antigen-bound surrogate scaffold, and an antigen bound to the fragment. Antigen-binding fragments (such as VH and VL) can be linked together via synthetic linkers to form various types of single-chain antibody designs that form monovalent antigen-binding domains such as single-chain Fv (scFv) or antigens, or bispecific antibodies, by forming intramolecular or intermolecular pairing of VH / VL domains, or intermolecular pairing when the VH and VL domains are expressed on separate single chains. Antigen-binding fragments may also be conjugated to other antibodies, proteins, antigen-binding fragments, or alternative scaffolds, which may be monospecific or multispecific, in order to manipulate bispecific and multispecific proteins.

[0117] "Antibody" is intended in a broad sense and includes immunoglobulin molecules, including monoclonal antibodies such as mouse, human, humanized, and chimeric monoclonal antibodies; antigen-binding fragments; multispecific antibodies such as bispecific, tripspecific, and quadrupspecific antibodies; dimeric, tetrameric, or multimeric antibodies; single-chain antibodies; antibody domains; and any other modified configurations of immunoglobulin molecules including antigen-binding sites of the required specificity. "Full-length antibody" consists of two heavy chains (HC) and two light chains (LC), interconnected by disulfide bonds, and a multimer thereof (e.g., IgM). Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (consisting of domains CH1, hinge, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further classified into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with framework regions (FRs). Each VH and VL consists of three CDR and four FR segments arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG, and IgM, depending on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The antibody light chain of any vertebrate species can be assigned to one of two distinct types, namely kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0118] "Bispecificity" refers to a molecule (such as an antibody) that specifically binds to two different antigens or two different epitopes within the same antigen. Bispecific molecules may cross-react to the same antigen in other related antigens, such as humans or monkeys, e.g., Macaca cynomolgus (cyno) or Pan troglodytes, or they may bind to epitopes shared among two or more different antigens.

[0119] A “chimeric antigen receptor,” or “CAR,” refers to an engineered T cell receptor (e.g., naive T cells, central memory T cells, effector memory T cells, or a combination thereof) onto which ligand or antigen specificity is transplanted onto T cells. CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immunoreceptors. A CAR comprises an antigen, a transmembrane domain, and an extracellular domain capable of binding to at least one intracellular domain. The intracellular domain of the CAR contains a polypeptide known to function as a signaling domain that causes activation or inhibition of intracellular biological processes. The transmembrane domain is known to extend to the cell membrane and contains any peptide or polypeptide capable of functioning to bind the extracellular domain and the signaling domain. A chimeric antigen receptor may optionally contain a hinge domain that functions as a linker between the extracellular domain and the transmembrane domain.

[0120] Complement-dependent cell-mediated cytotoxicity, or CDC, refers to a mechanism by which the Fc effector domain of a target-binding protein binds to and activates complement component C1q, which in turn activates the complement cascade, leading to the death of target cells. Complement activation can also result in the deposition of complement components on the surface of target cells, facilitating CDC through the binding of complement receptors (e.g., CR3) to leukocytes.

[0121] The "complementarity-determining region" (CDR) is the antibody region that binds to the antigen. There are three CDRs in the VH (Very High Hormone) (HCDR1, HCDR2, HCDR3) and three CDRs in the VL (Very Low Hormone) (LCDR1, LCDR2, LCDR3). CDR can be defined using various descriptions, such as those by Kabat (Wu et al. (1970) J Exp Med 132:211-250, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al., (1987) J Mol Biol 196:901-17), IMGT (Lefranc et al., (2003) Dev Comp Immunol 27:55-77), and AbM (Martin and Thornton (1996) J Bmol Biol 263:800-815). Correspondence between various descriptions and variable region numbering is described (see, for example, Lefranc et al. (2003) Dev Comp Immunol 27:55-77; Honegger and Pluckthun, J Mol Biol (2001) 309:657-670; International ImMunoGeneTics (IMGT) database; web resource, http: / / www_imgt_org). CDRs can be depicted using available programs such as abYsis by UCL Business PLC. As used herein, the terms “CDR”, “HCDR1”, “HCDR2”, “HCDR3”, “LCDR1”, “LCDR2”, and “LCDR3” include CDRs as defined by any of the Kabat, Chothia, IMGT, or AbM methods described above, unless otherwise expressly stated in the specification.

[0122] "Decrease", "reduction", or "lowering" generally refers to the ability of a test molecule (i.e., downstream effect) to mediate a reduced response when compared to a response mediated by a control or vehicle. Exemplary responses include binding of a protein to its antigen or receptor, enhanced binding to FcγR such as enhanced ADCC, CDC, and / or ADCP, or enhanced Fc effector function. A decrease can be a statistically significant difference in the measured response between the test molecule and the control (or vehicle), or a decrease of about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30-fold or more, e.g., 500, 600, 700, 800, 900, or 1000-fold or more.

[0123] "Enhancement", "promotion", or "increase" generally refers to the ability of a test molecule (i.e., downstream effect) to mediate a greater response when compared to a response mediated by a control or vehicle. Exemplary responses are binding of a protein to its antigen or receptor, enhanced binding to FcγR, or enhanced Fc effector function such as enhanced ADCC, CDC, and / or ADCP. An enhancement can be a statistically significant difference in the measured response between the test molecule and the control (or vehicle), or an increase of about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30-fold or more, e.g., 500, 600, 700, 800, 900, or 1000-fold or more.

[0124] "Expression vector" refers to a vector that can be used in a biological system or a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0125] "Heterologous" refers to two or more polypeptides or two or more polynucleotides that are not found in the same relationship to each other in nature.

[0126] A "heterogeneic polynucleotide" refers to a polynucleotide that contains two or more polynucleotides that are not found in nature in the same relationship to each other.

[0127] A "heterogeneous peptide" refers to a polypeptide that contains two or more polypeptides that are not found in the same relationship to each other in nature.

[0128] A “human antibody” refers to an antibody optimized to minimize the immune response when administered to a human subject. The variable region of a human antibody is derived from a human immunoglobulin sequence. If a human antibody contains a constant region or a portion of a constant region, that constant region is also derived from a human immunoglobulin sequence. If the variable region of a human antibody is obtained from a system using human germline immunoglobulin or a rearranged immunoglobulin gene, the human antibody includes heavy-chain and light-chain variable regions that “derive” from a human-derived sequence. Such exemplary systems include human immunoglobulin gene libraries displayed on phages, and transgenic non-human animals possessing human immunoglobulin loci, such as mice or rats. A “human antibody” typically contains amino acid differences when compared to immunoglobulin expressed in humans, due to differences in the human antibody and the system used to obtain the human immunoglobulin locus, intentional introduction of somatic mutations or substitutions into the framework or CDR, or both. Typically, a “human antibody” is at least approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by a human germline immunoglobulin or rearranged immunoglobulin gene. In some cases, a “human antibody” may contain, for example, a consensus framework sequence obtained from human framework sequence analysis described in Knappik et al., (2000) J Mol Biol 296:57-86, or synthetic HCDR3 incorporated into a human immunoglobulin gene library presented on phages, for example, Shi et al., (2010) J Mol Biol 397:385-396 and International Publication No. 2009 / 085462. Antibodies in which at least one CDR originates from a non-human species are not included in the definition of "human antibodies."

[0129] A "humanized antibody" refers to an antibody in which at least one CDR is derived from a non-human species and at least one framework is derived from a human immunoglobulin sequence. Because humanized antibodies can contain substitutions in their framework, the framework may not be an exact copy of the expressed human immunoglobulin or human immunoglobulin germline gene sequence.

[0130] "Isolated" means a homogeneous population of molecules (scFv or heterologous proteins containing scFv of the Disclosure) that have been substantially separated and / or purified from other components of a system in which the molecules are produced, such as recombinant cells, as well as proteins subjected to at least one purification or isolation step. "Isolated" means molecules that are substantially free from other cellular material and / or chemicals, and includes molecules isolated to a higher purity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% purity.

[0131] "Modification" refers to either an enhancement or reduction in the ability of the test molecule to mediate a control-enhanced or reduced response compared to a response mediated by the control or vehicle (i.e., a downstream effect).

[0132] A "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibody molecules, i.e., an individual antibody that constitutes a population that is identical except for possible known modifications, such as removal of the C-terminal lysine from the antibody heavy chain, or isomerization or deamidation of amino acids, oxidation of methionine, or deamidation of asparagine or glutamine. Monoclonal antibodies typically bind to one antigenic epitope. Bispecific monoclonal antibodies bind to two different antigenic epitopes. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibodies may be monospecific or multispecific, such as bispecific, and may be monovalent, bivalent, or polyvalent.

[0133] "Multispecificity" refers to a molecule that binds to two or more different antigens, or to two or more different epitopes within the same antigen. Multispecific molecules may cross-react to other related antigens, such as the same antigen (homolog) from other species, such as humans or monkeys, such as cynomolgus monkeys (cyno) or chimpanzees, or they may bind to epitopes shared among two or more different antigens.

[0134] A "polynucleotide" refers to a molecule containing nucleotide chains covalently bonded by a sugar-phosphate backbone or other equivalent covalent chemistry. cDNA is a typical example of a polynucleotide.

[0135] In this specification, the terms “protein” and “polypeptide” as used interchangeably refer to molecules comprising one or more polypeptides, each consisting of at least two amino acid residues linked by peptide bonds. Proteins may be monomers or protein complexes of two or more subunits, which may be identical or distinct. Small polypeptide molecules consisting of fewer than 50 amino acids may be referred to as “peptides.” Proteins may be heterofusion proteins, glycoproteins, or proteins modified by post-translational modifications such as phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, citrullination, polyglutamylation, ADP-ribosylation, pegylation, or biotinylation.

[0136] "Recombinant organisms" refer to polynucleotides, polypeptides, vectors, viruses, and other macromolecules prepared, expressed, produced, or isolated by recombinant means.

[0137] "Single-chain Fv" or "scFv" refers to a single-chain protein comprising VH, VL, and a linker between VH and VL. The scFv can have VL and VH variable regions in either orientation, for example, with respect to the order of the N-terminus to the C-terminus of VH and VL. Thus, the scFv can be in the VL-linker-VH orientation or the VH-linker-VL orientation. The scFv may be engineered to include disulfide bonds between VH, VL, and the linker.

[0138] "Specifically binds", "specific binding", "specifically binding", or "binds" refers to a protein such as an scFv in which an antibody binds to an antigen or an epitope within an antigen with a higher affinity than to other antigens. Typically, a protein such as an scFv binds to an antigen or an epitope within an antigen with an equilibrium dissociation constant (K -6 ) of about 1×10 -7 M or less, about 1×10 -8 M or less, about 5×10 -8 M or less, about 1×10 -9 M or less, about 1×10 -10 M or less, about 1×10 -11 M or less, or about 1×10 -12 M or less, and typically, K D is at least 100-fold less than its K D for binding to non-specific antigens (e.g., BSA, casein). D

[0139] "Stapled single-chain Fv" or "spFv" refers to an scFv that includes one or more disulfide bonds between VH and the linker or between VL and the linker. Typically, the spFv can include one disulfide bond between VH and the linker, one disulfide bond between VL and the linker, or two disulfide bonds between VH and the linker and between VL and the linker. An scFv molecule that includes a disulfide bond between VH and VL is excluded from "spFv".

[0140] "Subject" includes any human or non-human animal. "Non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, and reptiles. The terms "subject" and "patient" may be used interchangeably herein.

[0141] The "therapeutic effective dose" refers to the effective amount required to obtain the desired therapeutic outcome with the necessary dosage and duration. The therapeutically effective dose may vary depending on factors such as the individual's condition, age, sex, and weight, as well as the ability of the drug or combination of drugs to elicit the desired response in the individual.

[0142] "To treat," "treating," or "treating" a disease or disability means achieving one or more of the following: reducing the severity and / or duration of the disability; inhibiting the exacerbation of symptoms characteristic of the disability being treated; limiting or preventing recurrence of the disability in a person who previously had the disability; or limiting or preventing recurrence of symptoms in a person who previously had the disability.

[0143] "Triple specificity" refers to a molecule (such as an antibody) that specifically binds to three different antigens within the same antigen, or to three different epitopes. A triple-specific molecule may cross-react to the same antigen in other related antigens, such as humans or monkeys, for example, Macaca cynomolgus (cyno) or Pan troglodytes, or it may bind to epitopes shared among three or more different antigens.

[0144] "Variant," "mutant," or "change" refers to a polypeptide or polynucleotide that is different from a reference polypeptide or reference polynucleotide by one or more modifications, such as one or more substitutions, insertions, or deletions.

[0145] Throughout this specification, unless otherwise explicitly stated herein, the numbering of amino acid residues in the antibody constant region follows the EU index described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991).

[0146] Mutations in the Ig constant region are referred to as follows: L351Y_F405A_Y407V refers to L351Y, F405A, and Y407V mutations in one immunoglobulin constant region. L351Y_F405A_Y407V / T394W refers to L351Y, F405A, and Y407V mutations in the first Ig constant region, and the T394W mutation in the second Ig constant region.

[0147] The numbering of variable regions follows Chothia unless otherwise explicitly stated.

[0148] "VH cysteine," or "VH Cys," refers to Cys residues present in the VH framework.

[0149] "VL cysteine," or "VL Cys," refers to Cys residues present in the VL framework.

[0150] "Stabilized" refers to scFv that maintains a bond equivalent to hK2 when compared to an unheated scFv sample, which refers to thermal stability.

[0151] "Improved stability" refers to the spFv of this disclosure having an elevated melting point (Tm) compared to a parent scFv lacking the disulfide bond and Cys residue introduced into the spFv. The elevated Tm may be 2°C or more, for example, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, or 15°C.

[0152] An "anchor point" refers to an scFv Vh or VL framework Cys residue that can be mutated to Cys without adversely affecting the overall scFv structure and can form a disulfide bond with Cys present in the scFv linker.

[0153] A "staple" refers to an scFv linker containing one or two Cys residues that can form a disulfide bond with an anchor Cys.

[0154] "Surface exposure" refers to amino acid residues that are at least partially exposed on the surface of a protein and are accessible to a solvent, for example, accessible for deuteration. Algorithms for predicting the surface accessibility of residues based on primary sequence or protein are well known in the art. Alternatively, surface-exposed residues can be identified from the crystalline structure of the protein.

[0155] "LTBR" refers to a polypeptide that is a cell surface receptor for lymphotoxins involved in apoptosis and cytokine release, and is a member of the tumor necrosis factor receptor superfamily. LTBR may also be referred to as "tumor necrosis factor receptor superfamily member 3 (TNFRSF3)". LTBR is expressed on the surface of many cell types, including epithelial and myeloid cells. LTBR can specifically bind to lymphotoxin membrane forms (lymphotoxin-alpha and lymphotoxin-beta complexes). Activation of LTBR can induce apoptosis via TRAF3 and TRAF5, which may lead to the release of interleukin-8. Unless otherwise specified, LTBR is preferably human LTBR. The amino acid sequence of human LTBR is provided by UniProt number P36941.

[0156] "EDB" or "Extra Domain B" refers to a fibronectin domain that may be included in the fibronectin molecule based on the splicing pattern of fibronectin premRNA. Extra Domain B is a complete fibronectin (FN) type III repeat containing 91 amino acid residues. Generally, EDBs are undetectable in normal adult tissues but show greater expression in fetal and tumor tissues in the extracellular matrix and accumulate around new vascular systems during the angiogenesis process, making EDBs a potential marker and target of angiogenesis. Unless otherwise stated, EDB is preferably human EDB. Human EDBs containing fibronectin isoform amino acid sequences are available under UniProt number P02751.

[0157] Fibronectin refers to a polypeptide, a high molecular weight glycoprotein of the extracellular matrix. Fibronectin can bind to transmembrane receptor proteins called integrins. Fibronectin can also bind to other extracellular matrix proteins such as collagen, fibrin, and heparan sulfate proteoglycans. Fibronectin can exist as a protein dimer consisting of two nearly identical monomers linked by a pair of disulfide bonds. Although fibronectin is produced from a single gene, alternative splicing of the fibronectin premRNA molecule creates several isoforms of fibronectin, one of which is EDB fibronectin. Fibronectin can play a role in cell adhesion, growth, migration, and differentiation, and may be important for processes such as wound healing and embryonic development. The amino acid sequence of human fibronectin is provided by UniProt number P02751, which contains extradomain B, and NCBI accession numbers NP_001263337 (isoform B), NP_001263338 (isoform c), NP_001263339 (isoform d), NP_001263340 (isoform e), and NP_001263341 (isoform f), NP_001293058 (isoform 8), NP_001293059 (isoform 9), NP_001293060 (isoform 10), NP_001293061 (isoform 11), and NP_002017 (isoform 3).

[0158] 5.1 Composition This disclosure provides stabilized scFv molecules (hereinafter referred to as spFv (staple-treated Fv)), heterogeneous and multispecific molecules containing spFv, encoding polynucleotides, vectors, host cells, and methods for constructing and using them. This disclosure is at least in part based on the identification of VH and / or VL (hereinafter referred to as VH anchor points or VL anchor points) and flexible linkers (hereinafter referred to as staples), which can be manipulated to cysteine ​​residues resulting in the formation of disulfide bonds between the linkers and variable domains in scFv. The “stapling” strategies described herein are broadly applicable to all VH / VL domains and existing scFv molecules that provide structural identity with scFv with improved stability. The spFv described herein may be conjugated to any heterogeneous protein, including chimeric antigen receptors (CARs), T cell redirecting molecules, and bispecific and multispecific molecules, in bispecific or multispecific formats, and may be used as therapeutic, diagnostic, and detection molecules.

[0159] spFv This disclosure relates to an isolated single-chain variable fragment (scFv) comprising a heavy-chain variable region (VH), a linker (L), and a light-chain variable region (VL), wherein the scFv is The first disulfide bond between the structurally conserved surface-exposed VH cysteine ​​(Cys) and the first L Cys, A second disulfide bond between a structurally preserved surface-exposed VL Cys and a second L Cys, or The present invention provides an scFv comprising a first disulfide bond between a structurally preserved surface-exposed VH Cys and a first L Cys, and a second disulfide bond between a structurally preserved surface-exposed VL Cys and a second L Cys.

[0160] This disclosure also relates to isolated scFv comprising VH, L, and VL, VH contains VH Cys at structurally conserved surface-exposed VH framework residue positions, and L contains a first L Cy. VL contains VL Cys at structurally conserved surface-exposed VL framework residue positions, L contains a second L Cy, ​​or The present invention provides an scFv in which VH comprises VH Cys at structurally conserved surface-exposed VH framework residue positions, VL comprises VL Cys at structurally conserved surface-exposed VL framework residue positions, and L comprises a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys can form a disulfide bond, and the VL Cys and the second L Cys can form a disulfide bond. The disulfide bond is typically formed during the expression of the scFv of this disclosure.

[0161] While certain examples disclose spFv having two disulfide bonds, it is readily conceivable that spFv having one disulfide bond formed between the linker Cys and either VH Cys or VL Cys can be fabricated and utilized to generate "half-anchored" molecules. The anchor position is the same for spFv having one or two disulfide bonds. The linker Cys position can vary in half-anchored molecules, as long as the distance and geometric requirements for disulfide bond formation by the anchor point are met. Half-anchored spFv are expected to suppress VL / VH relative movement similar to VL / VH pairs stabilized by two disulfide bonds, and are therefore stabilized.

[0162] The spFv of this disclosure showed increased thermal stability compared to the parent scFv lacking disulfide bonds. Generally, the Tm of the spFv was approximately 10°C higher than that of the parent scFv lacking disulfide bonds, regardless of the Tm of the parent scFv. Stability can generally be thermal stability or mechanical stability. Thermal stability can be assessed by differential thermal calorimetry (DSC) scans performed using a heated protein sample (e.g., a sample heated to 60°C), followed by a thermal melt profile obtained using two-state or non-two-state transitions. In the case of non-two-state transitions, two transitions (Tm1 and Tm2) corresponding to the melting Tm of the VL and VH domains are recorded, respectively.

[0163] In some embodiments, the distance between VH Cys and VL Cys is approximately 7 Å to approximately 9 Å. In some embodiments, the distance between VH Cys and VL Cys is approximately 7 Å. In some embodiments, the distance between VH Cys and VL Cys is approximately 8 Å. In some embodiments, the distance between VH Cys and VL Cys is approximately 9 Å.

[0164] In some embodiments, VH Cys are located at H3, H5, H40, H43, H46, or H105, and the residue numbering follows Chothia.

[0165] In some embodiments, VH Cys is located at H3.

[0166] In some embodiments, VH Cys is located at H5.

[0167] In some embodiments, VH Cys is located at H40.

[0168] In some embodiments, VH Cys is located at H43.

[0169] In some embodiments, VH Cys is located at H46.

[0170] In some embodiments, VH Cys is located at H105.

[0171] In some embodiments, the VL Cys are located at L3, L5, L39, L42, L45, L100, or L102, and the residue numbering follows Chothia.

[0172] In some embodiments, VL Cys is located at L3.

[0173] In some embodiments, VL Cys is located at L5.

[0174] In some embodiments, VL Cys is located at L39.

[0175] In some embodiments, VL Cys is located at L42.

[0176] In some embodiments, VL Cys is located at L45.

[0177] In some embodiments, VL Cys is located at L100.

[0178] In some embodiments, VL Cys is located at L102.

[0179] In some embodiments, VH Cys is located at H105 and VL Cys is located at L42.

[0180] In some embodiments, VH Cys is located at H43 and VL Cys is located at L100.

[0181] In some embodiments, VH Cys is located at H3 and VL Cys is located at L3.

[0182] In some embodiments, VH Cys is located at H3 and VL Cys is located at L5.

[0183] In some embodiments, VH Cys is located at H3 and VL Cys is located at L39.

[0184] In some embodiments, VH Cys is located at H3 and VL Cys is located at L42.

[0185] In some embodiments, VH Cys is located at H3 and VL Cys is located at L45.

[0186] In some embodiments, VH Cys is located at H3 and VL Cys is located at L100.

[0187] In some embodiments, VH Cys is located at H3 and VL Cys is located at L102.

[0188] In some embodiments, VH Cys is located at H5 and VL Cys is located at L3.

[0189] In some embodiments, VH Cys is located at H5 and VL Cys is located at L5.

[0190] In some embodiments, VH Cys is located at H5 and VL Cys is located at L39.

[0191] In some embodiments, VH Cys is located at H5 and VL Cys is located at L42.

[0192] In some embodiments, VH Cys is located at H5 and VL Cys is located at L45.

[0193] In some embodiments, VH Cys is located at H5 and VL Cys is located at L100.

[0194] In some embodiments, VH Cys is located at H5 and VL Cys is located at L102.

[0195] In some embodiments, VH Cys is located at H40 and VL Cys is located at L3.

[0196] In some embodiments, VH Cys is located at H40 and VL Cys is located at L5.

[0197] In some embodiments, VH Cys is located at H40 and VL Cys is located at L39.

[0198] In some embodiments, VH Cys is located at H40 and VL Cys is located at L42.

[0199] In some embodiments, VH Cys is located at H40 and VL Cys is located at L45.

[0200] In some embodiments, VH Cys is located at H40 and VL Cys is located at L100.

[0201] In some embodiments, VH Cys is located at H40 and VL Cys is located at L102.

[0202] In some embodiments, VH Cys is located at H43 and VL Cys is located at L3.

[0203] In some embodiments, VH Cys is located at H43 and VL Cys is located at L5.

[0204] In some embodiments, VH Cys is located at H43 and VL Cys is located at L39.

[0205] In some embodiments, VH Cys is located at H43 and VL Cys is located at L42.

[0206] In some embodiments, VH Cys is located at H43 and VL Cys is located at L45.

[0207] In some embodiments, VH Cys is located at H43 and VL Cys is located at L102.

[0208] In some embodiments, VH Cys is located at H46 and VL Cys is located at L3.

[0209] In some embodiments, VH Cys is located at H46 and VL Cys is located at L5.

[0210] In some embodiments, VH Cys is located at H46 and VL Cys is located at L39.

[0211] In some embodiments, VH Cys is located at H46 and VL Cys is located at L42.

[0212] In some embodiments, VH Cys is located at H46 and VL Cys is located at L45.

[0213] In some embodiments, VH Cys is located at H46 and VL Cys is located at L100.

[0214] In some embodiments, VH Cys is located at H46 and VL Cys is located at L102.

[0215] In some embodiments, VH Cys is located at H105 and VL Cys is located at L3.

[0216] In some embodiments, VH Cys is located at H105 and VL Cys is located at L5.

[0217] In some embodiments, VH Cys is located at H105 and VL Cys is located at L39.

[0218] In some embodiments, VH Cys is located at H105 and VL Cys is located at L45.

[0219] In some embodiments, VH Cys is located at H105 and VL Cys is located at L100.

[0220] In some embodiments, VH Cys is located at H105 and VL Cys is located at L102.

[0221] The residue numbering in the VH and VL regions follows Chothia.

[0222] Chothia numbering is well known. The positions of VH and VL residues can be numbered using other numbering systems such as Kabat or IMGT numbering, or sequential numbering. Table 1 shows the correspondence between Chothia, Kabat, and sequential numbering for exemplary VH and GLk1 VH (SEQ ID NO: 60). Table 2 shows the correspondence between Chothia, Kabat, and sequential numbering for exemplary VL and GLk1 VL (SEQ ID NO: 56).

[0223] [Table 1-1]

[0224] [Table 1-2]

[0225] [Table 2]

[0226] In some embodiments, L comprises a sequence of amino acids derived from an immunoglobulin (Ig) hinge region.

[0227] In some embodiments, the Ig hinge region is derived from a human or non-human Ig hinge region. Exemplary non-human Ig hinge regions are derived from non-human primates such as mice, rats, dogs, chickens, and monkeys.

[0228] In some embodiments, the Ig hinge region is derived from the human Ig hinge region.

[0229] In some embodiments, the human Ig hinge region is an IgG1, IgG2, IgG3, IgG4, IgM, IgA, or IgE isotype.

[0230] The Ig hinge region is generally defined as containing residue 216 and terminating at residue 230 in human IgG, with residue numbering following the EU index. In some cases, the lower hinge region from approximately residues 231 to 237 may also be included in the hinge. Therefore, the IgG1 hinge region may contain the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 63), or, if the lower hinge is included, the amino acid sequence EPKSCDKTHTCPPCPAPELLGG (SEQ ID NO: 64). Hinge regions of other Ig isotypes are well known, and their amino acid sequences can be obtained, for example, from the ImMunoGeneTics website. For example, the IgG2 hinge contains the amino acid sequence ERKCCVECPPCP (SEQ ID NO: 65).

[0231] L includes a sequence of amino acids "derived from" the Ig hinge region in those examples, if it includes at least a portion of the Ig hinge region amino acid sequence or at least a portion of the manipulated Ig hinge region. The manipulated Ig hinge region includes one or more mutations compared to the wild-type Ig hinge. Exemplary mutations that may be introduced are any conservative modifications such as substitutions of Cys residues that reduce the number of Cys in L to one or two, substitutions of Pro residues, or conservative substitutions.

[0232] "Conservative modification" refers to amino acid modifications that do not significantly affect or alter the binding properties of an antibody, including amino acid modifications. Conservative modifications include amino acid substitutions, additions, and deletions. A conservative amino acid substitution is a substitution in which an amino acid is replaced by an amino acid residue with a similar side chain. The families of amino acid residues with similar side chains are clearly defined and include amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), non-charged side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amides (e.g., asparagine, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine), and sulfur-containing side chains (cysteine, methionine). Furthermore, as previously described regarding alanine scanning mutagenesis ((MacLennan et al., (1988) Acta Physiol Scand Suppl 643:55-67, Sasaki et al., (1988) Adv Biophys 35:1-24), any native residue within the polypeptide may also be substituted with alanine. Amino acid substitution can be performed by known methods, e.g., PCR mutagenesis (U.S. Patent No. 4,683,195). The resulting mutant hinges can be incorporated into the spFv constructs of this disclosure and tested for their properties, such as stability and antigen binding, using known assays and assays described herein.

[0233] In some embodiments, L is the amino acid sequence C(X) yThe formula includes C (SEQ ID NO: 23), where X is glycine (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), threonine (Thr), tryptophan (Trp), or tyrosine (Tyr), and y is an integer from 1 to 3. Pro may be included in the linker that provides rigidity. Gly may be included in the linker that allows for maximum flexibility. Any other amino acid may also be used in L, except for Cys and Met.

[0234] In some embodiments, L is the amino acid sequence C(X) y The formula includes C (sequence number 24), where X is Gly, Ser, or Pro, and y is an integer between 1 and 3.

[0235] In some embodiments, L includes amino acid sequences CPC, CGC, CSC, CPPC (SEQ ID NO: 1), CGPC (SEQ ID NO: 28), CPGC (SEQ ID NO: 29), CGGC (SEQ ID NO: 30), CSPG (SEQ ID NO: 31), CPSC (SEQ ID NO: 32), CSSC (SEQ ID NO: 33), CGSC (SEQ ID NO: 34), CSGC (SEQ ID NO: 35), CPPPC (SEQ ID NO: 36), CGPPC (SEQ ID NO: 37), CPGPC (SEQ ID NO: 38), CPPGC (SEQ ID NO: 39), CGGPC (SEQ ID NO: 40), CPGGC (SEQ ID NO: 41), CGGGC (SEQ ID NO: 42), CSPPC (SEQ ID NO: 43), CPSPC (SEQ ID NO: 44), CPPSC (SEQ ID NO: 45), CSSPC (SEQ ID NO: 46), CPSSC (SEQ ID NO: 47), CSSSC (SEQ ID NO: 48), CGSPC (SEQ ID NO: 49), CPGSC (SEQ ID NO: 50), CSGPC (SEQ ID NO: 51), or CPSGC (SEQ ID NO: 52).

[0236] In some embodiments, L includes the amino acid sequence CPC.

[0237] In some embodiments, L comprises the amino acid sequence CGC.

[0238] In some embodiments, L includes the amino acid sequence CSC.

[0239] In some embodiments, L comprises the amino acid sequence CPPC (SEQ ID NO: 1).

[0240] In some embodiments, L comprises the amino acid sequence CGPC (SEQ ID NO: 28).

[0241] In some embodiments, L comprises the amino acid sequence CPGC (SEQ ID NO: 29).

[0242] In some embodiments, L comprises the amino acid sequence CGGC (SEQ ID NO: 30).

[0243] In some embodiments, L includes the amino acid sequence CSPG (SEQ ID NO: 31).

[0244] In some embodiments, L comprises the amino acid sequence CPSC (SEQ ID NO: 32).

[0245] In some embodiments, L comprises the amino acid sequence CSSC (SEQ ID NO: 33).

[0246] In some embodiments, L comprises the amino acid sequence CGSC (SEQ ID NO: 34).

[0247] In some embodiments, L comprises the amino acid sequence CSGC (SEQ ID NO: 35).

[0248] In some embodiments, L comprises the amino acid sequence CPPPC (SEQ ID NO: 36).

[0249] In some embodiments, L comprises the amino acid sequence CGPPC (SEQ ID NO: 37).

[0250] In some embodiments, L comprises the amino acid sequence CPGPC (SEQ ID NO: 38).

[0251] In some embodiments, L comprises the amino acid sequence CPPGC (SEQ ID NO: 39).

[0252] In some embodiments, L comprises the amino acid sequence CGGPC (SEQ ID NO: 40).

[0253] In some embodiments, L comprises the amino acid sequence CPGGC (SEQ ID NO: 41).

[0254] In some embodiments, L comprises the amino acid sequence CGGGC (SEQ ID NO: 42).

[0255] In some embodiments, L comprises the amino acid sequence CSPPC (SEQ ID NO: 43).

[0256] In some embodiments, L comprises the amino acid sequence CPSPC (SEQ ID NO: 44).

[0257] In some embodiments, L includes the amino acid sequence CPPSC (SEQ ID NO: 45).

[0258] In some embodiments, L comprises the amino acid sequence CSSPC (SEQ ID NO: 46).

[0259] In some embodiments, L comprises the amino acid sequence CPSSC (SEQ ID NO: 47).

[0260] In some embodiments, L comprises the amino acid sequence CSSSC (SEQ ID NO: 48).

[0261] In some embodiments, L comprises the amino acid sequence CGSPC (SEQ ID NO: 49).

[0262] In some embodiments, L comprises the amino acid sequence CPGSC (SEQ ID NO: 50).

[0263] In some embodiments, L comprises the amino acid sequence CSGPC (SEQ ID NO: 51).

[0264] In some embodiments, L comprises the amino acid sequence CPSGC (SEQ ID NO: 52).

[0265] In some embodiments, L contains approximately 14 to 19 amino acids.

[0266] In some embodiments, L contains approximately 14 amino acids.

[0267] In some embodiments, L contains approximately 15 amino acids.

[0268] In some embodiments, L contains approximately 16 amino acids.

[0269] In some embodiments, L contains approximately 17 amino acids.

[0270] In some embodiments, L contains approximately 18 amino acids.

[0271] In some embodiments, L contains approximately 19 amino acids.

[0272] In some embodiments, L is the amino acid sequence (X) m C(X) y C(X) n (Sequence ID 25) is included in the formula, where X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gln, His, Ile, leu, Lys, Phe Thr, Trp, or Tyr, m is an integer from 6 to 9, y is an integer from 1 to 3, and n is an integer from 4 to 6.

[0273] In some embodiments, L is the amino acid sequence (X) m C(X) y C(X) n(Sequence ID 26) is included in the formula, where X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gln, His, Ile, Leu, Lys, Thr, or Tyr, m is an integer from 6 to 9, y is an integer from 1 to 3, and n is an integer from 4 to 6.

[0274] In some embodiments, L is the amino acid sequence (X) m C(X) y C(X) n (Sequence code 27) is included in the formula, where X is Gly or Pro, m is an integer from 6 to 9, y is an integer from 1 to 3, and n is an integer from 4 to 6.

[0275] In some embodiments, L comprises the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7.

[0276] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 2.

[0277] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 3.

[0278] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 4.

[0279] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 5.

[0280] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 6.

[0281] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 7.

[0282] In some embodiments, the spFv of the present disclosure is in a VL-L-VH orientation.

[0283] In some embodiments, the spFv of the present disclosure is in a VH-L-VL orientation.

[0284] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H105, VL includes Cys in L42, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which the scFv is in a VL-L-VH orientation.

[0285] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H105, VL includes Cys in L45, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which the scFv is in a VL-L-VH orientation.

[0286] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H105, VL includes Cys in L39, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which the scFv is in a VL-L-VH orientation.

[0287] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H5, VL includes Cys in L42, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which the scFv is in a VL-L-VH orientation.

[0288] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H5, VL includes Cys in L45, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which the scFv is in a VL-L-VH orientation.

[0289] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H5, VL includes Cys in L39, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which the scFv is in a VL-L-VH orientation.

[0290] This disclosure also includes scFv including VH, L and VL, VH contains Cys in H3, VL includes Cys in L42, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which the scFv is in a VL-L-VH orientation.

[0291] This disclosure also includes scFv including VH, L and VL, VH contains Cys in H3, VL includes Cys in L45, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which the scFv is in a VL-L-VH orientation.

[0292] This disclosure also includes scFv including VH, L and VL, VH contains Cys in H3, VL includes Cys in L39, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which the scFv is in a VL-L-VH orientation.

[0293] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H43, VL includes Cys in L100, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which scFv is in a VH-L-VL orientation.

[0294] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H43, VL includes Cys in L102, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which scFv is in a VH-L-VL orientation.

[0295] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H43, VL includes Cys in L5, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which scFv is in a VH-L-VL orientation.

[0296] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H43, VL includes Cys in L3, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which scFv is in a VH-L-VL orientation.

[0297] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H40, VL includes Cys in L100, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which scFv is in a VH-L-VL orientation.

[0298] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H40, VL includes Cys in L102, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which scFv is in a VH-L-VL orientation.

[0299] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H40, VL includes Cys in L5, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which scFv is in a VH-L-VL orientation.

[0300] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H40, VL includes Cys in L3, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which scFv is in a VH-L-VL orientation.

[0301] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H46, VL includes Cys in L100, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which scFv is in a VH-L-VL orientation.

[0302] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H46, VL includes Cys in L102, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which scFv is in a VH-L-VL orientation.

[0303] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H46, VL includes Cys in L5, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which scFv is in a VH-L-VL orientation.

[0304] This disclosure also includes scFv including VH, L and VL, VH includes Cys in H46, VL includes Cys in L3, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. This provides scFv in which scFv is in a VH-L-VL orientation.

[0305] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 3.

[0306] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 4.

[0307] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 5.

[0308] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 6.

[0309] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 7.

[0310] heteromorphic molecules including spFv of this disclosure The spFvs of this disclosure, as well as unstabilized scFvs lacking disulfide bonds, can be conjugated to a second molecule, as is well known in the art. Exemplary second molecules disclosed herein include half-life extension moieties, contrast agents, therapeutic agents, various antibody forms and fragments thereof, antigen-binding domains, Fc regions, immunoglobulin heavy / light chains or fragments thereof, multispecific molecules, and chimeric antigen receptors (CARs).

[0311] This disclosure also relates to heterogeneous molecules comprising a single-chain variable fragment (scFv) including a heavy-chain variable region (VH), a linker (L), and a light-chain variable region (VL), wherein the scFv is The first disulfide bond between the structurally conserved surface-exposed VH cysteine ​​(Cys) and the first L Cys, A second disulfide bond between a structurally preserved surface-exposed VL Cys and a second L Cys, or The present invention provides heterogeneous molecules comprising a first disulfide bond between a structurally preserved surface-exposed VH Cys and a first L Cys, and a second disulfide bond between a structurally preserved surface-exposed VL Cys and a second L Cys.

[0312] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH contains VH Cys at structurally conserved surface-exposed VH framework residue positions, and L contains a first L Cy. VL contains VL Cys at structurally conserved surface-exposed VL framework residue positions, L contains a second L Cy, ​​or The present invention provides heterologous molecules in which VH contains VH Cys at structurally conserved surface-exposed VH framework residue positions, VL contains VL Cys at structurally conserved surface-exposed VL framework residue positions, and L contains a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys can form disulfide bonds, and the VL Cys and the second L Cys can form disulfide bonds.

[0313] In some embodiments, the distance between VH Cys and VL Cys is approximately 7 Å to approximately 9 Å.

[0314] In some embodiments, VH Cys are located at H3, H5, H40, H43, H46, or H105, and the residue numbering follows Chothia.

[0315] In some embodiments, the VL Cys are located at L3, L5, L39, L42, L45, L100, or L102, and the residue numbering follows Chothia.

[0316] In some embodiments, VH Cys is located at H105 and VL Cys is located at L42.

[0317] In some embodiments, VH Cys is located at H43 and VL Cys is located at L100.

[0318] In some embodiments, VH Cys is located at H3 and VL Cys is located at L3.

[0319] In some embodiments, VH Cys is located at H3 and VL Cys is located at L5.

[0320] In some embodiments, VH Cys is located at H3 and VL Cys is located at L39.

[0321] In some embodiments, VH Cys is located at H3 and VL Cys is located at L42.

[0322] In some embodiments, VH Cys is located at H3 and VL Cys is located at L45.

[0323] In some embodiments, VH Cys is located at H3 and VL Cys is located at L100.

[0324] In some embodiments, VH Cys is located at H3 and VL Cys is located at L102.

[0325] In some embodiments, VH Cys is located at H5 and VL Cys is located at L3.

[0326] In some embodiments, VH Cys is located at H5 and VL Cys is located at L5.

[0327] In some embodiments, VH Cys is located at H5 and VL Cys is located at L39.

[0328] In some embodiments, VH Cys is located at H5 and VL Cys is located at L42.

[0329] In some embodiments, VH Cys is located at H5 and VL Cys is located at L45.

[0330] In some embodiments, VH Cys is located at H5 and VL Cys is located at L100.

[0331] In some embodiments, VH Cys is located at H5 and VL Cys is located at L102.

[0332] In some embodiments, VH Cys is located at H40 and VL Cys is located at L3.

[0333] In some embodiments, VH Cys is located at H40 and VL Cys is located at L5.

[0334] In some embodiments, VH Cys is located at H40 and VL Cys is located at L39.

[0335] In some embodiments, VH Cys is located at H40 and VL Cys is located at L42.

[0336] In some embodiments, VH Cys is located at H40 and VL Cys is located at L45.

[0337] In some embodiments, VH Cys is located at H40 and VL Cys is located at L100.

[0338] In some embodiments, VH Cys is located at H40 and VL Cys is located at L102.

[0339] In some embodiments, VH Cys is located at H43 and VL Cys is located at L3.

[0340] In some embodiments, VH Cys is located at H43 and VL Cys is located at L5.

[0341] In some embodiments, VH Cys is located at H43 and VL Cys is located at L39.

[0342] In some embodiments, VH Cys is located at H43 and VL Cys is located at L42.

[0343] In some embodiments, VH Cys is located at H43 and VL Cys is located at L45.

[0344] In some embodiments, VH Cys is located at H43 and VL Cys is located at L100.

[0345] In some embodiments, VH Cys is located at H43 and VL Cys is located at L102.

[0346] In some embodiments, VH Cys is located at H46 and VL Cys is located at L3.

[0347] In some embodiments, VH Cys is located at H46 and VL Cys is located at L5.

[0348] In some embodiments, VH Cys is located at H46 and VL Cys is located at L39.

[0349] In some embodiments, VH Cys is located at H46 and VL Cys is located at L42.

[0350] In some embodiments, VH Cys is located at H46 and VL Cys is located at L45.

[0351] In some embodiments, VH Cys is located at H46 and VL Cys is located at L100.

[0352] In some embodiments, VH Cys is located at H46 and VL Cys is located at L102.

[0353] In some embodiments, VH Cys is located at H105 and VL Cys is located at L3.

[0354] In some embodiments, VH Cys is located at H105 and VL Cys is located at L5.

[0355] In some embodiments, VH Cys is located at H105 and VL Cys is located at L39.

[0356] In some embodiments, VH Cys is located at H105 and VL Cys is located at L42.

[0357] In some embodiments, VH Cys is located at H105 and VL Cys is located at L45.

[0358] In some embodiments, VH Cys is located at H105 and VL Cys is located at L100.

[0359] In some embodiments, VH Cys is located at H105 and VL Cys is located at L102.

[0360] The residue numbering in the VH and VL regions follows Chothia.

[0361] In some embodiments, L comprises a sequence of amino acids derived from an immunoglobulin (Ig) hinge region.

[0362] In some embodiments, the Ig hinge region is derived from a human or non-human Ig hinge region. Exemplary non-human Ig hinge regions are derived from non-human primates such as mice, rats, dogs, chickens, and monkeys.

[0363] In some embodiments, the Ig hinge region is derived from the human Ig hinge region.

[0364] In some embodiments, the human Ig hinge region is an IgG1, IgG2, IgG3, IgG4, IgM, IgA, or IgE isotype.

[0365] In some embodiments, L is the amino acid sequence C(X) y The formula includes C (SEQ ID NO: 23), where X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gln, His, Ile, Leu, Lys, Phe, Thr, Trp, or Tyr, and y is an integer from 1 to 3. Pro may be included in the linker that provides rigidity. Gly may be included in the linker that allows for maximum flexibility. Any other amino acids, except Cys and Met, may also be used in L.

[0366] In some embodiments, L is the amino acid sequence C(X) y The formula includes C (sequence number 24), where X is Gly, Ser, or Pro, and y is an integer between 1 and 3.

[0367] In some embodiments, L includes amino acid sequences CPC, CGC, CSC, CPPC (SEQ ID NO: 1), CGPC (SEQ ID NO: 28), CPGC (SEQ ID NO: 29), CGGC (SEQ ID NO: 30), CSPG (SEQ ID NO: 31), CPSC (SEQ ID NO: 32), CSSC (SEQ ID NO: 33), CGSC (SEQ ID NO: 34), CSGC (SEQ ID NO: 35), CPPPC (SEQ ID NO: 36), CGPPC (SEQ ID NO: 37), CPGPC (SEQ ID NO: 38), CPPGC (SEQ ID NO: 39), CGGPC (SEQ ID NO: 40), CPGGC (SEQ ID NO: 41), CGGGC (SEQ ID NO: 42), CSPPC (SEQ ID NO: 43), CPSPC (SEQ ID NO: 44), CPPSC (SEQ ID NO: 45), CSSPC (SEQ ID NO: 46), CPSSC (SEQ ID NO: 47), CSSSC (SEQ ID NO: 48), CGSPC (SEQ ID NO: 49), CPGSC (SEQ ID NO: 50), CSGPC (SEQ ID NO: 51), or CPSGC (SEQ ID NO: 52).

[0368] In some embodiments, L contains approximately 14 to 19 amino acids.

[0369] In some embodiments, L contains approximately 14 amino acids.

[0370] In some embodiments, L contains approximately 15 amino acids.

[0371] In some embodiments, L contains approximately 16 amino acids.

[0372] In some embodiments, L contains approximately 17 amino acids.

[0373] In some embodiments, L contains approximately 18 amino acids.

[0374] In some embodiments, L contains approximately 19 amino acids.

[0375] In some embodiments, L is the amino acid sequence (X) m C(X) y C(X) n (Sequence ID 25) is included in the formula, where X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gln, His, Ile, leu, Lys, Phe, Thr, Trp, or Tyr, m is an integer from 6 to 9, y is an integer from 1 to 3, and n is an integer from 4 to 6.

[0376] In some embodiments, L is the amino acid sequence (X) m C(X) y C(X) n (Sequence ID 26) is included in the formula, where X is Gly, Ser or Pro, Ala, Arg, Asn, Asp, Glu, Gln, His, Ile, Leu, Lys, Thr or Tyr, m is an integer from 6 to 9, y is an integer from 1 to 3, and n is an integer from 4 to 6.

[0377] In some embodiments, L is the amino acid sequence (X)m C(X) y C(X) n (Sequence code 27) is included in the formula, where X is Gly or Pro, m is an integer from 6 to 9, y is an integer from 1 to 3, and n is an integer from 4 to 6.

[0378] In some embodiments, L comprises the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7.

[0379] In some embodiments, the spFv of the present disclosure is in a VL-L-VH orientation.

[0380] In some embodiments, the spFv of the present disclosure is in a VH-L-VL orientation.

[0381] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH includes Cys in H105, VL includes Cys in L42, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VL-L-VH orientation.

[0382] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH includes Cys in H105, VL includes Cys in L45, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VL-L-VH orientation.

[0383] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH includes Cys in H105, VL includes Cys in L39, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VL-L-VH orientation.

[0384] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH includes Cys in H5, VL includes Cys in L42, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VL-L-VH orientation.

[0385] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH includes Cys in H5, VL includes Cys in L45, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VL-L-VH orientation.

[0386] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH includes Cys in H5, VL includes Cys in L39, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VL-L-VH orientation.

[0387] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH contains Cys in H3, VL includes Cys in L42, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VL-L-VH orientation.

[0388] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH contains Cys in H3, VL includes Cys in L45, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VL-L-VH orientation.

[0389] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH contains Cys in H3, VL includes Cys in L39, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides heterogeneous molecules in a VL-L-VH orientation.

[0390] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH includes Cys in H43, VL includes Cys in L100, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0391] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH includes Cys in H43, VL includes Cys in L102, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0392] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH includes Cys in H43, VL includes Cys in L5, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0393] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH includes Cys in H43, VL includes Cys in L3, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0394] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH includes Cys in H40, VL includes Cys in L100, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0395] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH includes Cys in H40, VL includes Cys in L102, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0396] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH includes Cys in H40, VL includes Cys in L5, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0397] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH includes Cys in H40, VL includes Cys in L3, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0398] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH includes Cys in H46, VL includes Cys in L100, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0399] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH includes Cys in H46, VL includes Cys in L102, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0400] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH includes Cys in H46, VL includes Cys in L5, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0401] This disclosure also relates to heterogeneous molecules including scFv containing VH, L, and VL, VH includes Cys in H46, VL includes Cys in L3, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0402] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 3.

[0403] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 4.

[0404] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 5.

[0405] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 6.

[0406] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 7.

[0407] In some embodiments, the scFv of this disclosure is conjugated to a second protein, polynucleotide, therapeutic agent, cytotoxic agent, or detectable label.

[0408] In some embodiments, the second protein is a half-life extension portion.

[0409] In some embodiments, the second protein is an antibody or a fragment thereof.

[0410] In some embodiments, the second protein is an antigen-binding fragment.

[0411] In some embodiments, the second protein is a therapeutic molecule.

[0412] heterogeneous molecules including spFv and half-life extension portion of the present disclosure In some embodiments, the spFv of this disclosure is conjugated to the half-life extension portion.

[0413] The half-life extension portion consists of immunoglobulin (Ig), Ig fragments, Ig constant region, Ig constant region fragments, Fc region, transferrin, albumin, albumin variant, albumin-binding domain, or polyethylene glycol. The amino acid sequence of human Ig is well known and includes IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE.

[0414] In some embodiments, the spFv of this disclosure is conjugated to Ig or a fragment of Ig.

[0415] In some embodiments, the spFv of this disclosure is conjugated to the Fc region.

[0416] In some embodiments, the spFv of this disclosure is conjugated to transferrin.

[0417] In some embodiments, the spFv of this disclosure is conjugated to albumin.

[0418] In some embodiments, the spFv of this disclosure is coupled to an albumin-binding protein.

[0419] In some embodiments, the spFv of this disclosure is conjugated to polyethylene glycol (PEG). Exemplary PEG molecules are PEG5000 or PEG20000.

[0420] In some embodiments, the spFv of the present disclosure is conjugated to a fatty acid or fatty acid ester. Exemplary fatty acids and fatty acid esters are polylysine, octane, carbohydrates (dextran, cellulose, oligo or polysaccharides), such as laurate, myristicate, stearate, arachidinate, behenate, oleate, arachidoneate, octanodioic acid, tetradecanediic acid, octadecanediic acid, docosanedioic acid, etc., for desired properties.

[0421] The half-life extension portion may be a direct fusion with the spFv of the present disclosure and can be generated by standard cloning and expression techniques. Alternatively, the portion can be conjugated to the spFv of the present disclosure produced by recombinant techniques using well-known chemical coupling methods.

[0422] The spFv and heterogeneous molecules including cytotoxic agents or detectable labels of the present disclosure The Disclosure also provides heterologous molecules comprising the spFv of the Disclosure, wherein the spFv of the Disclosure is conjugated to a second protein, polynucleotide, therapeutic agent, cytotoxic agent, or detectable label.

[0423] Heterogeneous molecules containing the spFv of this disclosure may be used in vitro or in vivo to direct therapeutic agents, mediate the killing of cells expressing antigens to which the spFv binds, visualize, identify, or purify them.

[0424] In some embodiments, the detectable label is also a cytotoxic agent.

[0425] The detectable label includes a composition that, when conjugated with the spFv of this disclosure, makes the spFv detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.

[0426] Examples of detectable labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron density reagents, enzymes (e.g., commonly used in ELISA), biotin, digoxigenin, haptens, luminescent molecules, chemiluminescent molecules, fluorescent dyes, fluorophores, fluorescent quenchers, colored molecules, radioisotopes, scintillates, avidin, streptavidin, protein A, protein G, antibodies or their fragments, polyhistidine, Ni 2+ Examples include Flag tags, myc tags, heavy metals, enzymes, alkaline phosphatases, peroxidases, luciferases, electron donors / receptors, acridinium esters, and colorimetric substrates.

[0427] A detectable label may spontaneously emit a signal, for example, when the detectable label is a radioactive isotope. In other cases, a detectable label emits a signal as a result of being stimulated by an external field.

[0428] Exemplary radioactive isotopes may be γ-emitting, Auger-emitting, β-emitting, alpha-emitting, or positron-emitting radioactive isotopes. Exemplary radioactive isotopes include: 3 H, 11 C, 13 C, 15 N, 18 F, 19 F, 55 Co, 57Co, 60 Co, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 68 Ga, 72 As, 75 Br, 86 Y, 89 Zr, 90 Sr, 94m Tc, 99m Tc, 115 In, 123 1. 124 1. 125 I, 131 1. 211 At, 212 Bi, 213 Bi, 223 Ra, 226 Ra, 225 Ac, and 227 Ac is one example.

[0429] Exemplary metal atoms include calcium, scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, bromine, krypton, rubidium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, indium, tin, antimony, tellurium, iodine, xenon, cesium, barium, lanthanum, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, mercury, and tali. These are metals with atomic numbers greater than 20, such as um, lead, bismuth, francium, radium, actinium, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, thorium, protactinium, uranium, neptunium, plutonium, americium, curium, bercklium, californium, einsteinium, fermium, mendelevium, nobelium, or lawrencium atoms.

[0430] In some embodiments, the metal atom may be an alkaline earth metal having an atomic number greater than 20.

[0431] In some embodiments, the metal atom may be a lanthanide.

[0432] In some embodiments, the metal atom may be an actinide.

[0433] In some embodiments, the metal atom may be a transition metal.

[0434] In some embodiments, the metal atom may be a base metal.

[0435] In some embodiments, the metal atoms may be gold atoms, bismuth atoms, tantalum atoms, and gadolinium atoms.

[0436] In some embodiments, the metal atoms may be metals having atomic numbers from 53 (i.e., iodine) to 83 (i.e., bismuth).

[0437] In some embodiments, metal atoms may be atoms suitable for magnetic resonance imaging.

[0438] Metal atoms are metal ions in the form of +1, +2, or +3 oxidation states, for example, Ba 2+ , Bi 3+ , Cs + Ca 2+ , Cr 2+ , Cr 3+ , Cr 6+ Co 2+ Co 3+ Cu + Cu 2+ Cu 3+ , Ga 3+ , Gd 3+ Au + Au 3+ Fe 2+ Fe 3+ F 3+ Pb 2+ Mn 2+ Mn3+ , Mn 4+ , Mn 7+ , Hg 2+ , Ni 2+ , Ni 3+ , Ag + , Sr 2+ , Sn 2+ , Sn 4+ , and Zn 2+ may be. The metal atom may include a metal oxide, for example, iron oxide, manganese oxide, or gadolinium oxide.

[0439] Suitable dyes include, for example, any commercially available dyes such as 5(6)-carboxyfluorescein, IRDye 680RD maleimide, or IRDye 800CW, ruthenium polypyridyl dyes.

[0440] Suitable fluorophores are fluorescein isothiocyanate (fluorescein isothiocyanate, FITC), fluorescein thiosemicarbazide, rhodamine, Texas Red, CyDye (e.g., Cy3, Cy5, Cy5.5), Alexa Fluors (e.g., Alexa488, Alexa555, Alexa594, Alexa647), near infrared (near infrared, NIR) (700~900nm) fluorescent dyes, as well as carbocyanine and aminostyryl dyes.

[0441] Heteromolecules comprising the scFv of the present disclosure conjugated to a detectable label can be used as a contrast agent.

[0442] In some embodiments, the cytotoxic agent is a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., a toxin having enzymatic activity derived from bacteria, fungi, plants, or animals, or a fragment thereof), or a radioisotope (i.e., a radioactive complex).

[0443] In some embodiments, the cytotoxic agent is a bacterial toxin such as daunomycin, doxorubicin, methotrexate, vindesine, diphtheria toxin, lysine, geldanamycin, mytansinoid, or calicheamicin. The cytotoxic agent may exert its cytotoxic or cell proliferation inhibitory effect through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.

[0444] In some embodiments, cytotoxic agents include diphtheria A chain, unbound active fragments of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modesine A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitors, curcin, crotin, sapaonaria officinalis inhibitors, geronin, mitogenin, restrictosin, phenomycin, enomycin, and toxins with enzymatic activity such as trichothecenes.

[0445] In some embodiments, the cytotoxic agent is 212 Bi, 131 I, 131 In, 90 Y, and 186 These are radioactive nuclides such as Re.

[0446] In some embodiments, the cytotoxic agent is drastatin or a peptide analog and derivative of drastatin, auristatin, or monomethyl auristatin phenylalanine. Exemplary molecules are disclosed in U.S. Patents 5,635,483 and 5,780,588. Drastatin and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division, and to possess anticancer and antifungal activity. The drastatin or auristatin drug site may be conjugated to the antibody of the present invention via the N (amino) or C (carboxyl) terminus of the peptide drug site (see International Publication No. 02 / 088172), or via any cysteine ​​manipulated within the antibody.

[0447] Conjugation to detectable markers can be performed using known methods.

[0448] In some embodiments, the detectable label forms a complex with a chelating agent.

[0449] In some embodiments, a detectable marker is conjugated to the spFv of this disclosure via a linker.

[0450] Detectable labels or cytotoxic agents can be directly or indirectly linked to the spFv of this disclosure using known methods. Suitable linkers are known in the art and include, for example, prosthetic groups, non-phenol linkers (derivatives of N-succimidyl benzoate, dodecaborate), chelate moieties of both macrocyclic and acyclic chelating agents, for example, derivatives of 1,4,7,10-tetraazacyclododecane-1,4,7,10,tetraacetic acid (DOTA), derivatives of diethylenetriaminepentaacetic acid (DTPA), derivatives of S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and derivatives of 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), N-succinimidyl-3-(2-p Examples include lysyl dithiol propionate (SPDP), iminothiolane (IT), difunctional derivatives of imide esters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl sverat), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene), as well as other chelate moieties. Suitable peptide linkers are well known.

[0451] heteromorphic molecules comprising spFv and immunoglobulin (Ig) constant regions or fragments thereof as disclosed herein. The spFvs of this disclosure can be conjugated to an Ig constant region or fragment of an Ig constant region to confer antibody-like properties including Fc effector function, C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, or downregulation of cell surface receptors (e.g., B cell receptors, BCRs). The Ig constant region or fragment of an Ig constant region also functions as a half-life extension portion as described herein. The spFvs of this disclosure can also be manipulated into full-length antibodies using standard methods. Full-length antibodies containing the spFvs of this disclosure can be further manipulated as described herein.

[0452] The immunoglobulin heavy chain constant region consists of subdomains CH1, CH2, and CH3. The CH1 domain extends from residues 118–215, CH2 domain residues 231–340, and CH3 domain residues 341–447 on the heavy chain, according to the EU index. In some cases, residue 341 is referred to as the CH2 domain residue. The hinge is generally defined as containing residue 216 and ending at 230 in human IgG1, but may include a lower hinge region of approximately residues 231–237, as described herein. The Ig Fc region includes at least the CH2 and CH3 domains of the Ig constant region and therefore includes at least the region of approximately 231–447 of the Ig heavy chain constant region.

[0453] The present invention also provides spFv of the present disclosure conjugated to an immunoglobulin (Ig) constant region or a fragment of an Ig constant region.

[0454] In some embodiments, the Ig steady-state region is the heavy chain steady-state region.

[0455] In some embodiments, the Ig steady-state region is the light chain steady-state region.

[0456] In some embodiments, the Ig steady-state region fragment includes an Fc region.

[0457] In some embodiments, the Ig constant region fragment includes a CH2 domain.

[0458] In some embodiments, the Ig constant region fragment includes a CH3 domain.

[0459] In some embodiments, the Ig constant region fragment includes a CH2 domain and a CH3 domain.

[0460] In some embodiments, the Ig constant region fragment includes at least a portion of the hinge, the CH2 domain, and the CH3 domain. The portion of the hinge refers to one or more amino acid residues of the Ig hinge.

[0461] In some embodiments, the Ig constant region fragment includes a hinge, a CH2 domain, and a CH3 domain.

[0462] In some embodiments, the spFv of this disclosure is conjugated to the N-terminus of the Ig steady region or to a fragment of the Ig steady region.

[0463] In some embodiments, the spFv of this disclosure is conjugated to the C-terminus of the Ig constant region or to a fragment of the Ig constant region.

[0464] The spFvs of this disclosure conjugated to the Ig constant region or a fragment of the Ig constant region may be evaluated for their functionality using several known assays. Binding to a target antigen may be evaluated using the methods described herein. Modified properties conferred by the Ig constant domain or a fragment of the Ig constant region, such as the Fc region, may be assayed in an Fc receptor binding assay using a soluble form of the receptor, such as FcγRI, FcγRII, FcγRIII, or FcRn, or using a cell-based assay that measures ADCC, CDC, or ADCP, for example.

[0465] ADCC activity can be evaluated using an in vitro assay that uses cells expressing the antigen to which the spFv of this disclosure binds as target cells and NK cells as effector cells. Cell lysis can be detected by the release of a label (e.g., a radioactive substrate, a fluorescent dye, or a native intracellular protein) from the lysed cells. In an exemplary assay, target cells are used in a ratio of one target cell to four effector cells. Target cells are pre-labeled with BATDA and combined with effector cells and a test antibody. Cell lysis was measured by incubating the sample for 2 hours and measuring the BATDA released into the supernatant. Data were normalized to the maximum cytotoxicity with 0.67% Triton X-100 (Sigma Aldrich), and a minimum control was determined by the spontaneous release of BATDA from target cells in the absence of any antibody.

[0466] ADCP can be evaluated by using monocyte-derived macrophages as effector cells, and any cell expressing the antigen to which the spFv of this disclosure binds as a target cell, and monocyte-derived macrophages engineered to express GFP or another labeled molecule. In an exemplary assay, the effector:target cell ratio may be, for example, 4:1. Effector cells may be incubated with target cells for 4 hours with or without the antibody of the present invention. After incubation, the cells can be detached using actase. Macrophages can be identified by fluorescently labeled anti-CD11b and anti-CD14 antibodies, but the rate of phagocytosis can be evaluated using standard methods for CD11 + and CD14 + This can be determined based on the percentage of GFP fluorescence in macrophages.

[0467] For example, the CDC of cells is 1 × 10⁶ Daudi cells in RPMI-B (RPMI supplemented with 1% BSA). 5The reaction can be measured by plating cells / well (50 μL / well), adding 50 μL of test protein to the well at a final concentration of 0–100 μg / mL, incubating the reaction at room temperature for 15 minutes, adding 11 μL of pooled human serum to the well, and incubating the reaction at 37°C for 45 minutes. The percentage of lysed cells (%) can be detected as the percentage of propidium iodide-stained cells in the FACS assay using a standard method.

[0468] heteromorphic molecules comprising spFv and chimeric antigen receptors (CARs) or fragments thereof as disclosed herein. The spFvs of this disclosure may be coupled to a chimeric antigen receptor (CAR) or a fragment of a CAR. Accordingly, a CAR containing the spFvs of this disclosure may be monospecific or multispecific, with its extracellular domain containing one or more scFv molecules of this disclosure.

[0469] Chimeric antigen receptors (CARs) are genetically modified receptors. These modified receptors can be readily inserted into immune cells, including T cells, and expressed by them, according to techniques known in the art. A single CAR can recognize a specific antigen and, upon binding to that antigen, activate immune cells to attack and destroy the cells carrying that antigen. If these antigens are present on tumor cells, immune cells expressing CARs can target and kill the tumor cells.

[0470] A CAR typically comprises an antigen ad, an optional linker, a transmembrane domain, and a cytoplasmic domain including a costimulatory domain and / or a signaling domain.

[0471] The extracellular domain of the CAR may contain any polypeptide that binds to a desired antigen, such as the scFv of this disclosure. The CAR may also be engineered to bind to two or more desired antigens that are arranged in tandem and can be separated by a linker sequence. For example, one or more scFv, domain antibody, rama VHH antibody, or other VH-only antibody fragments of this disclosure may be arranged in tandem via a linker to generate a bispecific or multispecific CAR.

[0472] The transmembrane domains of CARs include the transmembrane domain of CD8, the alpha, beta, or zeta chains of T cell receptors, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CDI la, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2R beta, IL2R gamma, and IL7R. a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDI Id, ITGAE, CD103, ITGAL, CDI la, LFA-1, ITGAM, CDI lb, ITGAX, CDI lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRT It may originate from AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0473] The intracellular costimulatory domain of a CAR may be derived from the intracellular domain of one or more costimulatory molecules. Costimulatory molecules are well-known cell surface molecules other than antigen receptors or Fc receptors that provide a second signal necessary for the efficient activation and function of T lymphocytes upon binding to an antigen. Exemplary costimulatory domains that may be used in a CAR are the intracellular domains of 4-1BB, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70.

[0474] The intracellular signaling domain of CARs may be derived from, for example, the signaling domains of CD3ζ, CD3ε, CD22, CD79a, CD66d, or CD39. The “intracellular signaling domain” refers to a portion of the CAR polypeptide involved in the transduction of the message of effective CAR binding to a target antigen into immune effector cells in order to induce effector cell function, such as activation, cytokine production, proliferation, and cytotoxic activity (including the release of cytotoxic factors to CAR-bound target cells, or other cellular responses induced after antigen binding to the extracellular CAR domain).

[0475] Any linker within a CAR, positioned between the extracellular domain and the transmembrane domain, can be a polypeptide approximately 2 to 100 amino acids long. The linker may contain or be composed of flexible residues such as glycine and serine, allowing adjacent protein domains to move freely relative to each other. Longer linkers can be used if it is desirable to ensure that two adjacent domains do not sterically interfere with each other. The linker may be cleavable or incleavable. An example of a cleavable linker is 2A.

[0476] An exemplary CAR comprises the scFv, CD8 transmembrane domain, and CD3ζ signaling domain of the present disclosure. Another exemplary CAR comprises the scFv, CD8 or CD28 transmembrane domain, CD28, 41BB or OX40 costimulatory domain, and CD3ζ signaling domain of the present disclosure.

[0477] CARs are produced using standard molecular biological techniques.

[0478] The spFv of this disclosure may be conjugated to a second molecule directly or via a linker. Exemplary linkers include immunoglobulin hinge regions, CL, or CH1 portions derived from immunoglobulin heavy or light chain isotypes, glycy-rich linkers, glycy and ser-containing linkers, glycy and ala-containing linkers, ala and ser-containing linkers, and pro-containing linkers. Exemplary amino acids that may be included in the linkers are glycy, ser, pro, thr, gluc, lys, arg, ile, leu, and his. Alternatively, various non-proteinoid polymers, including polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol, may be found to be used as linkers. Exemplary linkers are described, for example, in International Publication No. 2019 / 060695.

[0479] In some embodiments, the heterologous molecules are single-specific.

[0480] In some embodiments, heterologous molecules exhibit multiple specificities.

[0481] In some embodiments, heterologous molecules exhibit bispecificity.

[0482] In some embodiments, the heterologous molecules exhibit triple specificity.

[0483] In some embodiments, heterologous molecules exhibit quadruple specificity.

[0484] Multiple specific molecules including spFv in this disclosure This disclosure also relates to a multispecific molecule comprising a single-chain variable fragment (scFv) including a heavy-chain variable region (VH), a linker (L), and a light-chain variable region (VL), wherein the scFv is The first disulfide bond between the structurally conserved surface-exposed VH cysteine ​​(Cys) and the first L Cys, A second disulfide bond between a structurally preserved surface-exposed VL Cys and a second L Cys, or The present invention provides a multispecific molecule comprising a first disulfide bond between a structurally conserved surface-exposed VH Cys and a first L Cys, and a second disulfide bond between a structurally conserved surface-exposed VL Cys and a second L Cys.

[0485] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH contains VH Cys at structurally conserved surface-exposed VH framework residue positions, and L contains a first L Cy, VL contains VL Cys at structurally conserved surface-exposed VL framework residue positions, and L contains a second L Cy, ​​or The present invention provides a multispecific molecule in which VH contains VH Cys at structurally conserved surface-exposed VH framework residue positions, VL contains VL Cys at structurally conserved surface-exposed VL framework residue positions, and L contains a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys can form disulfide bonds, and the VL Cys and the second L Cys can also form disulfide bonds.

[0486] In some embodiments, the distance between VH Cys and VL Cys is approximately 7 Å to approximately 9 Å.

[0487] In some embodiments, VH Cys are located at H3, H5, H40, H43, H46, or H105, and the residue numbering follows Chothia.

[0488] In some embodiments, the VL Cys are located at L3, L5, L39, L42, L45, L100, or L102, and the residue numbering follows Chothia.

[0489] In some embodiments, VH Cys is at H105, and VL Cys is at L42, or VH Cys is located at H43, and VL Cys is located at L100, or VH Cys are located in H3, and VL Cys are located in L3, or VH Cys is located at H3, and VL Cys is located at L5, or VH Cys is located on H3, and VL Cys is located on L39, or VH Cys is located on H3, and VL Cys is located on L42, or VH Cys are located on H3, and VL Cys are located on L45, or VH Cys is H3, and VL Cys is at L100, VH Cys is located at H3, and VL Cys is located at L102, or VH Cys is at H5, and VL Cys is at L3, VH Cys is located at H5, and VL Cys is located at L5, or VH Cys is at H5, and VL Cys is at L39, VH Cys is located at H5, and VL Cys is located at L42, or VH Cys is at H5, and VL Cys is at L45, VH Cys is at H5, and VL Cys is at L100, VH Cys is located at H5, and VL Cys is located at L102, or VH Cys is at H40, and VL Cys is at L3, or VH Cys is at H40, and VL Cys is at L5, or VH Cys is at H40, and VL Cys is at L39, or VH Cys are at H40, and VL Cys are at L42, or VH Cys are at H40, and VL Cys are at L45, or VH Cys is at H40, and VL Cys is at L100, or VH Cys is at H40, and VL Cys is at L102, or VH Cys is located at H43, and VL Cys is located at L3, or VH Cys is located at H43, and VL Cys is located at L5, or VH Cys is located at H43, and VL Cys is located at L39, or VH Cys is located at H43, and VL Cys is located at L42, or VH Cys is located at H43, and VL Cys is located at L45, or VH Cys is located at H43, and VL Cys is located at L102, or VH Cys is located at H46, and VL Cys is located at L3, or VH Cys is located at H46, and VL Cys is located at L5, or VH Cys is located at H46, and VL Cys is located at L39, or VH Cys is located at H46, and VL Cys is located at L42, or VH Cys is located at H46, and VL Cys is located at L45, or VH Cys is located at H46, and VL Cys is located at L100, or VH Cys is located at H46, and VL Cys is located at L102, or VH Cys is located at H105, and VL Cys is located at L3, or VH Cys is located at H105, and VL Cys is located at L5, or VH Cys is at H105, and VL Cys is at L39, or VH Cys is at H105, and VL Cys is at L45, or VH Cys is at H105, VL Cys is at L100, or VH Cys is located at H105, and VL Cys is located at L102. In this specification, residue numbering follows Chothia.

[0490] In some embodiments, L comprises a sequence of amino acids derived from an immunoglobulin (Ig) hinge region.

[0491] In some embodiments, the Ig hinge region is derived from a human or non-human Ig hinge region.

[0492] In some embodiments, the Ig hinge region is derived from the human Ig hinge region.

[0493] In some embodiments, the human Ig hinge region is an IgG1, IgG2, IgG3, or IgG4 isotype.

[0494] In some embodiments, L is the amino acid sequence C(X) y The formula includes C (Sequence ID 23), where X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gln, His, Ile, Leu, Lys, Phe, Thr, Trp, or Tyr, and y is an integer from 1 to 3.

[0495] In some embodiments, L is the amino acid sequence C(X) y The formula includes C (sequence number 24), where X is Gly, Ser, or Pro, and y is an integer between 1 and 3.

[0496] In some embodiments, L includes amino acid sequences CPC, CGC, CSC, CPPC (SEQ ID NO: 1), CGPC (SEQ ID NO: 28), CPGC (SEQ ID NO: 29), CGGC (SEQ ID NO: 30), CSPG (SEQ ID NO: 31), CPSC (SEQ ID NO: 32), CSSC (SEQ ID NO: 33), CGSC (SEQ ID NO: 34), CSGC (SEQ ID NO: 35), CPPPC (SEQ ID NO: 36), CGPPC (SEQ ID NO: 37), CPGPC (SEQ ID NO: 38), CPPGC (SEQ ID NO: 39), CGGPC (SEQ ID NO: 40), CPGGC (SEQ ID NO: 41), CGGGC (SEQ ID NO: 42), CSPPC (SEQ ID NO: 43), CPSPC (SEQ ID NO: 44), CPPSC (SEQ ID NO: 45), CSSPC (SEQ ID NO: 46), CPSSC (SEQ ID NO: 47), CSSSC (SEQ ID NO: 48), CGSPC (SEQ ID NO: 49), CPGSC (SEQ ID NO: 50), CSGPC (SEQ ID NO: 51), or CPSGC (SEQ ID NO: 52).

[0497] In some embodiments, L comprises about 14 to about 19 amino acids, for example, about 14, about 15, about 16, about 17, about 18, or about 19 amino acids.

[0498] In some embodiments, L is the amino acid sequence (X) m C(X) y C(X) n (Sequence ID 25) is included in the formula, where X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gln, His, Ile, leu, Lys, Phe, Thr, Trp, or Tyr, m is an integer from 6 to 9, y is an integer from 1 to 3, and n is an integer from 4 to 6.

[0499] In some embodiments, L is the amino acid sequence (X) m C(X) y C(X) n (Sequence ID 26) is included in the formula, where X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gln, His, Ile, Leu, Lys, Thr, or Tyr, m is an integer from 6 to 9, y is an integer from 1 to 3, and n is an integer from 4 to 6.

[0500] In some embodiments, L is the amino acid sequence (X) m C(X) y C(X) n (Sequence code 27) is included in the formula, where X is Gly or Pro, m is an integer from 6 to 9, y is an integer from 1 to 3, and n is an integer from 4 to 6.

[0501] In some embodiments, L comprises the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7.

[0502] In some embodiments, the spFv of the present disclosure is in a VL-L-VH orientation.

[0503] In some embodiments, the spFv of the present disclosure is in a VH-L-VL orientation.

[0504] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H105, VL includes Cys in L42, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VL-L-VH orientation.

[0505] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H105, VL includes Cys in L45, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VL-L-VH orientation.

[0506] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H105, VL includes Cys in L39, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VL-L-VH orientation.

[0507] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H5, VL includes Cys in L42, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VL-L-VH orientation.

[0508] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H5, VL includes Cys in L45, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VL-L-VH orientation.

[0509] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H5, VL includes Cys in L39, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VL-L-VH orientation.

[0510] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH contains Cys in H3, VL includes Cys in L42, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VL-L-VH orientation.

[0511] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH contains Cys in H3, VL includes Cys in L45, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VL-L-VH orientation.

[0512] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH contains Cys in H3, VL includes Cys in L39, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv provides a multispecific molecule with a VL-L-VH orientation.

[0513] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H43, VL includes Cys in L100, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0514] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H43, VL includes Cys in L102, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0515] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H43, VL includes Cys in L5, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0516] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H43, VL includes Cys in L3, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0517] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H40, VL includes Cys in L100, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0518] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H40, VL includes Cys in L102, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0519] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H40, VL includes Cys in L5, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0520] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H40, VL includes Cys in L3, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0521] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H46, VL includes Cys in L100, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0522] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H46, VL includes Cys in L102, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0523] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H46, VL includes Cys in L5, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0524] This disclosure also relates to a multispecific molecule comprising scFv containing VH, L, and VL, VH includes Cys in H46, VL includes Cys in L3, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0525] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 3.

[0526] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 6.

[0527] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 7.

[0528] In some embodiments, the multispecific molecule includes an antibody or an antibody fragment.

[0529] In some embodiments, the multispecific protein comprises an Ig constant region or a fragment of an Ig constant region.

[0530] In some embodiments, the Ig steady-state region includes the Fc region.

[0531] In some embodiments, the Ig constant region includes a CH2 domain.

[0532] In some embodiments, the Ig constant region fragment includes a CH3 domain.

[0533] In some embodiments, the Ig constant region fragment includes a CH2 domain and a CH3 domain.

[0534] In some embodiments, the Ig steady-state region fragment includes a hinge, a CH2 domain, and at least a portion of the CH3 domain.

[0535] In some embodiments, the Ig constant region fragment includes a hinge, a CH2 domain, and a CH3 domain.

[0536] In some embodiments, the spFv of this disclosure is conjugated to the N-terminus of the Ig steady region or the N-terminus of a fragment of the Ig steady region.

[0537] In some embodiments, the spFv of this disclosure is conjugated to the C-terminus of the Ig steady region or the C-terminus of a fragment of the Ig steady region.

[0538] In some embodiments, the Ig steady-state region or fragments of the Ig steady-state region are IgG1, IgG2, and IgG3 or IgG4 isotypes.

[0539] In some embodiments, the Ig constant region or a fragment of the Ig constant region contains at least one mutation that reduces the binding of multispecific molecules to FcγR.

[0540] In some embodiments, at least one mutation that reduces the binding of multispecific molecules to FcγR is F234A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G236 deletion / A327G The residues are selected from the group consisting of / P331A / D365E / L358M, H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S and S228P / F234A / L235A / G236 deletion / G237A / P238S, and residue numbering follows the EU index.

[0541] In some embodiments, the Ig constant region or a fragment of the Ig constant region contains at least one mutation that enhances the binding of multispecific molecules to FcγR.

[0542] In some embodiments, at least one mutation that enhances the binding of multispecific molecules to FcγR is selected from the group consisting of S239D / I332E, S298A / E333A / K334A, F243L / R292P / Y300L, F243L / R292P / Y300L / P396L, F243L / R292P / Y300L / V305I / P396L, and G236A / S239D / I332E, with residue numbering following the EU index.

[0543] In some embodiments, FcγR is FcγRI, FcγRIIA, FcγRIIB, or FcγRIII.

[0544] In some embodiments, the Ig constant region or a fragment of the Ig constant region contains at least one mutation that modulates the half-life of the multispecific molecule.

[0545] In some embodiments, at least one mutation that modulates the half-life of the multispecific molecule is selected from the group consisting of H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A, and H435R, and the residue numbering follows the EU index.

[0546] In some embodiments, the Ig constant region or a fragment of the Ig constant region contains at least one mutation in the CH3 domain.

[0547] In some embodiments, at least one mutation in the CH3 domain is T350V, L351Y, F405A, Y407V, T366Y, T366W, F405W, T394W, T394S, Y407T, Y407A, T366S / L368A / Y407V, L351Y / F405A / Y407V, T366I / K392M / T394W, F405A / Y40 The residues are selected from the group consisting of 7V, T366L / K392M / T394W, L351Y / Y407A, T366A / K409F, L351Y / Y407A, T366V / K409F, T366A / K409F, T350V / L351Y / F405A / Y407V, and T350V / T366L / K392L / T394W, and the residue numbering follows the EU index.

[0548] In some embodiments, the multispecific molecules are bispecific.

[0549] In some embodiments, the multispecific molecules are triple specific.

[0550] In some embodiments, the multispecific molecules are quadruple specific.

[0551] 5.2 Generation of multispecific proteins including spFv of the Disclosure The spFvs of this disclosure can be manipulated into any known form of multispecific molecule using known recombinant techniques, expression, and purification protocols.

[0552] The spFv of this disclosure can be engineered into full-length multispecific antibodies having one or more mutations in the CH3 domain that promote the stability of the two halves of the molecule. These multispecific antibodies can be generated in vitro using Fab arm exchange or by co-expression of various chains. In the case of in vitro Fab arm exchange, two monospecific bivalent antibodies are engineered to have one or more substitutions in the CH3 domain, and the antibodies are incubated together under reducing conditions sufficient to induce disulfide isomerization of the cysteine ​​in the hinge region, thereby generating a bispecific antibody by Fab arm exchange. The incubation conditions can be optimally returned to non-reducing conditions. Typical reducing agents that can be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. For example, incubation can be performed at a temperature of at least 20°C in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreitol, at a pH of 5 to 8, for example, pH 7.0 or pH 7.4, for at least 90 minutes.

[0553] Possible CH3 mutations include techniques such as knob-in-hole mutations (Genentech), electrostatic match mutations (Chugai, Amgen, NovoNordisk, Oncomed), strand exchange manipulation domain bodies (SEEDbody) (EMD Serono), Duobody® mutations (Genmab), and other asymmetric mutations (e.g., Zymeworks).

[0554] Knob-in-hole mutations, disclosed for example in International Publication No. 1996 / 027011, include interfacial mutations of the CH3 region in which an amino acid with a small side chain (hole) is introduced into the first CH3 region and an amino acid with a large side chain (knob) is introduced into the second CH3 region, resulting in a preferential interaction between the first and second CH3 regions. Exemplary CH3 region mutations that form knobs and holes are T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V.

[0555] Heavy chain heterodimer formation can be facilitated by using electrostatic interactions by substituting a positively charged residue on the first CH3 region and a negatively charged residue on the second CH3 region, as described in U.S. Patent Application Publications 2010 / 0015133, 2009 / 0182127, 2010 / 028637, or 2011 / 0123532.

[0556] Other asymmetric mutations that can be used to promote heavy chain heterodimerization include L351Y_F405A_Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T3 described in U.S. Patent Application Publication No. 2012 / 0149876 or 2013 / 0195849 (Zymeworks). These are 66L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W.

[0557] SEED body mutations, as described in U.S. Patent Application Publication No. 2007 / 0287170, involve the substitution of selected IgG residues with IgA residues to promote heavy chain heterodimerization.

[0558] Other exemplary variants that may be used are R409D_K370E / D399K_E357K, S354C_T366W / Y349C_T366S_L368A_Y407V, Y349C_T366W / S354C_T366W described in International Publication No. 2007 / 147901, International Publication No. 2011 / 143545, International Publication No. 2013 / 157954, International Publication No. 2013 / 096291 and U.S. Patent Application Publication No. 2018 / 0118849. S_L368A_Y407V, T366K / L351D, L351K / Y349E, L351K / Y349D, L351K / L368E, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, K392D / D399K, K392D / E356K, K253E_D282K_K322D / D239K_E240K_K292D, K392D_K409D / D356K_D399K.

[0559] Duobody® variants (Genmab) are disclosed, for example, in U.S. Patent Application Publication No. 2014 / 0303356 and include variants such as F405L / K409R, wild-type / F405L_R409K, T350I_K370T_F405L / K409R, K370W / K409R, D399AFGHILMNRSTVWY / K409R, T366ADEFGHILMQVY / K409R, L368ADEGHNRSTVQ / K409AGRH, D399FHKRQ / K409AGRH, F405IKLSTVW / K409AGRH, and Y407LWQ / K409AGRH.

[0560] Additional bispecific or multispecific structures into which the spFv of this disclosure may be incorporated include: Dual Variable Domain immunoglobulin (DVD) (International Publication No. 2009 / 134776, DVD is a full-length antibody comprising a heavy chain having a VH1-linker-VH2-CH structure and a light chain having a VL1-linker-VL2-CL structure, with the linker being optional); structures containing various dimerization domains for conjugating two antibody arms with different specificities, such as a leucine zipper or collagen dimerization domain (International Publication No. 2012 / 022811, U.S. Patent No. 5,932,448, U.S. Patent No. 6,833,441); two or more domain antibodies (dAb) conjugated together; diabodies; heavy chain-only antibodies such as camelid antibodies and engineered camelid antibodies; dual-target (DT)-Ig (GSK / Domantis); 2-in-1 antibodies (Genentech); cross-linked Mab (Karmanos Cancer This includes mAb2 (F-Star), CovX body (CovX / Pfizer), IgG-like bispecificity (InnClone / Eli Lilly), Ts2Ab (MedImmune / AZ), BsAb (Zymogenetics), HERCULES (Biogen Idec), TvAb (Roche), ScFv / Fc fusion (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), biaffinity retargeting technology (Fc-DART) (MacroGenics), and bivalent (ScFv)2-Fab (National Research Center for Antibody Medicine--China), biactive or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), divalent bispecificity (Biotecnol), and Fab-Fv (UCB-Celltech).ScFv antibodies, diabody-based antibodies, and domain antibodies include, but are not limited to, bispecific T cell engagers (BiTE) (Micromet), tandem diabodies (Tandab) (Affimed), biaffinity retargeting technology (DART) (MacroGenics), single-chain diabodies (Academic), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack), and COMBODY (Epigen Biotech), bitarget nanobodies (Ablynx), and bitarget heavy chain-only domain antibodies.

[0561] The scFv of this disclosure may also be engineered into a multispecific protein comprising three polypeptide chains. In such a design, at least one antigen-binding domain is present in the form of the scFv of this disclosure. An exemplary design includes (where "1" represents the first antigen-binding domain, "2" represents the second antigen-binding domain, and "3" represents the third antigen-binding domain): Design 1: Chain A) scFv1-CH2-CH3, Chain B) VL2-CL, Chain C) VH2-CH1-hinge-CH2-CH3 Design 2: Chain A) scFv1-hinge-CH2-CH3, Chain B) VL2-CL, Chain C) VH2-CH1-hinge-CH2-CH3 Design 3: Chain A) scFv1-CH1-hinge-CH2-CH3, Chain B) VL2-CL, Chain C) VH2-CH1-hinge-CH2-CH3 Design 4: Chain A) CH2-CH3-scFv1, Chain B) VL2-CL, Chain C) VH2-CH1-hinge-CH2-CH3

[0562] CH3 operation is described in U.S. Patent Application Publication No. 2012 / 0149876 or U.S. Patent Application Publication No. 2013 / 0195849 (Zymeworks) as L351Y_F405A_Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F40 Variations such as 5A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W can be incorporated into designs 1-4.

[0563] 5.3 Isotype, Allotype, and Fc Operation The Ig constant region or fragment of the Ig constant region, such as the Fc region, present in the multispecific molecule or the heterogeneous molecule of this disclosure may be of any allotype or isotype.

[0564] In some embodiments, the Ig steady-state region or a fragment of the Ig steady-state region is an IgG1 isotype.

[0565] In some embodiments, the Ig steady-state region or a fragment of the Ig steady-state region is an IgG2 isotype.

[0566] In some embodiments, the Ig steady-state region or a fragment of the Ig steady-state region is an IgG3 isotype.

[0567] In some embodiments, the Ig steady-state region or a fragment of the Ig steady-state region is an IgG4 isotype.

[0568] The Ig constant region or fragments of the Ig constant region can be any allotype. The allotype is not expected to affect the properties of the Ig constant region, such as binding or Fc-mediated effector function. The immunogenicity of therapeutic proteins containing the Ig constant region fragment is associated with an increased risk of injection reaction and a shortened therapeutic response (Baert et al., (2003) N Engl J Med 348:602-608). The extent to which therapeutic proteins containing the Ig constant region fragment induce an immune response in the host may be partially determined by the Ig constant region allotype (Stickler et al., (2011) Genes and Immunity 12:213-221). The Ig constant region allotype is related to mutations in the amino acid sequence at specific positions in the antibody's constant region sequence. Table 3 shows the selected IgG1, IgG2, and IgG4 allotypes.

[0569] [Table 3]

[0570] C-terminal lysine (CTL) can be removed from the Ig constant region by endogenous circulating carboxypeptidase in the bloodstream (Cai et al., (2011) Biotechnol Bioeng 108:404-412). During manufacturing, extracellular Zn is used as described in U.S. Patent Application Publication No. 2014 / 0273092. 2+ , EDTA or EDTA-Fe 3+ By controlling the concentration of [the substance], CTL removal can be controlled to below the maximum level. The CTL content of the protein can be measured using known methods.

[0571] In some embodiments, the spFv of the Disclosure conjugated to the Ig constant region has a C-terminal lysine content of about 10% to about 90%. In some embodiments, the C-terminal lysine content is about 20% to about 80%. In some embodiments, the C-terminal lysine content is about 40% to about 70%. In some embodiments, the C-terminal lysine content is about 55% to about 70%. In some embodiments, the C-terminal lysine content is about 60%.

[0572] Fc region mutations may be made to multispecific or heterologous molecules of this disclosure, including the Ig constant region or fragments of the Ig constant region, in order to modulate their effector functions, such as ADCC, ADCP, and / or ADCP and / or pharmacokinetic properties. This can be achieved by introducing mutations into Fc to control the binding of the mutant Fc to activated FcγRs (FcγRI, FcγRIIa, FcγRIII), inhibitory FcγRIIb, and / or FcRn.

[0573] In some embodiments, the multispecific molecules or heterologous molecules of this disclosure include at least one mutation in the Ig constant region or a fragment of the Ig constant region.

[0574] In some embodiments, at least one mutation is located in the Fc region.

[0575] In some embodiments, the multispecific molecules or heterologous molecules of this disclosure contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations in the Fc region.

[0576] In some embodiments, the multispecific or heterologous molecules of this disclosure include at least one mutation in the Fc region that modulates antibody binding to FcRn.

[0577] Fc locations that can be mutated to regulate the half-life (e.g., binding to FcRn) include positions 250, 252, 253, 254, 256, 257, 307, 376, 380, 428, 434, and 435. Exemplary mutations that can be performed individually or in combination are the mutations T250Q, M252Y, I253A, S254T, T256E, P257I, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A, and H435R. Exemplary mutations, either alone or in combination, that can be performed to increase the half-life of an antibody are the mutations M428L / N434S, M252Y / S254T / T256E, T250Q / M428L, N434A, and T307A / E380A / N434A. Exemplary mutations, either alone or in combination, that can be performed to decrease the half-life are the mutations H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A, and H435R.

[0578] In some embodiments, the multispecific molecules or heterologous molecules of this disclosure contain the M252Y / S254T / T256E mutation in the Fc region.

[0579] In some embodiments, the multispecific or heterologous molecules of this disclosure include at least one mutation in the Fc region that reduces the binding of a protein to an activated Fcγ receptor (FcγR) and / or reduces Fc effector functions such as C1q binding, complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), or phagocytosis (ADCP).

[0580] The fc positions that can be mutated to reduce the effector function after reducing the binding of the multispecific molecules or heterologous molecules of this disclosure to activated FcγR include positions 214, 233, 234, 235, 236, 237, 238, 265, 267, 268, 270, 295, 297, 309, 327, 328, 329, 330, 331, and 365. Exemplary mutations that may occur individually or in combination include mutations in IgG1, IgG2, IgG3, or IgG4: K214T, E233P, L234V, L234A, deletion of G236, V234A, F234A, L235A, G237A, P238A, P238S, D265A, D265S, S267E, H268A, H268Q, Q268A, N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F, A330S, and P331S. Exemplary mutation combinations resulting in reduced ADCC for the multispecific or heterologous molecules of this disclosure include: L234A / L235A in IgG1, L234A / L235A / D265S in IgG1, V234A / G237A / P238S / H268A / V309L / A330S / P331S in IgG2, F234A / L235A in IgG4, S228P / F234A / L235A in IgG4, N297A in all Ig isotypes, V234A / G237A in IgG2, and K214T / E233P / L234V / L2 in IgG1. These include 35A / G236 deletion / A327G / P331A / D365E / L358M, H268Q / V309L / A330S / P331S in IgG2, S267E / L328F in IgG1, L234F / L235E / D265A in IgG1, L234A / L235A / G237A / P238S / H268A / A330S / P331S in IgG1, S228P / F234A / L235A / G237A / P238S in IgG4, and S228P / F234A / L235A / G236-deleted / G237A / P238S in IgG4. Hybrid IgG2 / 4 Fc domains, such as Fc having residues 117-260 derived from IgG2 and residues 261-447 derived from IgG4, may also be used.

[0581] An exemplary mutation resulting in a reduced CDC of the multispecific or heterogeneous molecules of this disclosure is the K322A mutation.

[0582] The well-known S228P mutation can be added to an IgG4 antibody to enhance the stability of IgG4.

[0583] In some embodiments, the multispecific molecules or heterologous molecules of this disclosure include at least one mutation in an Fc region selected from the group consisting of deletions of K214T, E233P, L234V, L234A, G236, V234A, F234A, L235A, G237A, P238A, P238S, D265A, S267E, H268A, H268Q, Q268A, N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F, A330S, and P331S.

[0584] In some embodiments, the multispecific molecules or heterologous molecules of this disclosure contain the L234A / L235A / D265S mutation in the Fc region.

[0585] In some embodiments, the multispecific molecules or heterologous molecules of this disclosure contain the L234A / L235A mutation in the Fc region.

[0586] In some embodiments, the multispecific molecules or heterologous molecules of the Disclosure include at least one mutation in an Fc region that enhances the binding of the multispecific molecules or heterologous molecules of the Disclosure to FcγR and / or enhances Fc effector functions such as C1q binding, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), or phagocytosis (ADCP).

[0587] Fc positions that can be mutated to increase the binding of the multispecific or heterologous molecules of this disclosure to activated FcγR and / or enhance Fc effector function include positions 236, 239, 243, 256, 290, 292, 298, 300, 305, 312, 326, 330, 332, 333, 334, 345, 360, 339, 378, 396, or 430 (residue numbering according to the EU index). Exemplary mutations that can be performed individually or in combination include G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305L, K326A, A330K, I332E, E333A, K334A, A339T, and P396L. Exemplary mutation combinations that result in increased ADCC or ADCP proteins include S239D / I332E, S298A / E333A / K334A, F243L / R292P / Y300L, F243L / R292P / Y300L / P396L, F243L / R292P / Y300L / V305I / P396L, and G236A / S239D / I332E.

[0588] Fc positions that can be mutated to enhance CDC include positions 267, 268, 324, 326, 333, 345, and 430. Exemplary mutations that can occur individually or in combination are S267E, F1268F, S324T, K326A, K326W, E333A, E345K, E345Q, E345R, E345Y, E430S, E430F, and E430T. Exemplary combinations of mutations resulting in multispecific or heterogeneous molecules of this disclosure with increased CDC are K326A / E333A, K326W / E333A, H268F / S324T, S267E / H268F, S267E / S324T, and S267E / H268F / S324T.

[0589] The specific mutations described herein are those compared to the wild-type amino acid sequences of IgG1, IgG2, and IgG4, respectively, of SEQ ID NOs. 66, 67, and 68.

[0590] Sequence ID 66, wild-type IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0591] Sequence ID 67; wild-type IgG2 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKDTLMISRTPEVTCVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0592] Sequence ID 68; wild-type IgG4 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0593] The binding of the multispecific or heterologous molecules of this disclosure to FcγR or FcRn can be evaluated using flow cytometry in cells engineered to express each receptor. In the exemplary binding assay, 2 x 10⁶ wells were used in a 96-well plate. 5Cells were seeded and blocked in BSA Stain Buffer (BD Biosciences, San Jose, USA) at 4°C for 30 minutes. Cells were incubated with the test multispecificity molecule or test heterogeneity molecule of this disclosure on ice at 4°C for 1.5 hours. After washing twice with BSA stain buffer, cells were incubated with R-PE labeled anti-human IgG secondary antibody (Jackson Immunoresearch Laboratories) at 4°C for 45 minutes. Cells were washed twice with stain buffer and then resuspended in 150 μL of Stain Buffer containing 1:200 diluted DRAQ7 live / dead cell staining reagent (Cell Signaling Technology, Danvers, USA). PE and DRAQ7 signals from stained cells were detected using a Miltenyi MACSQuant flow cytometer (Miltenyi Biotec, Auburn, USA) using channels B2 and B4, respectively. Live cells were gated out by DRAQ7 exclusion, and the geometric mean fluorescence signal was determined for at least 10,000 live cell events collected. FlowJo software (Tree Star) was used for the analysis. The data was plotted as the logarithm of antibody concentration against the mean fluorescence signal. Nonlinear regression analysis was performed.

[0594] 5.4 Glycotechnology The ability to mediate ADCC of multispecific or heterologous molecules of the Disclosure conjugated to the Ig constant region or fragments of the Ig constant region can be enhanced by manipulating the Ig constant region or fragments of the Ig constant region oligosaccharide component. Human IgG1 or IgG3 is N-glycosylated at Asn297, where the majority of the glycan is in the known bibranched G0, G0F, G1, G1F, G2, or G2F form. Ig constant region-containing proteins that can be produced by unmanipulated CHO cells typically have a glycan fucose content of at least about 85%. Removal of core fucose from bibranched complex oligosaccharides conjugated to the Ig constant region or fragments of the Ig constant region enhances ADCC of multispecific or heterologous molecules of the Disclosure via improved FcγRIIIa binding without altering antigen binding or CDC activity. Such multispecific or heterologous molecules have been used to control the osmolality of the culture medium (Konno et al., Cytotechnology 64:249-265, 2012), to use mutant CHO strain Lec13 as a host cell line (Shields et al., (2002) J Biol Chem 277:26733-26740), to use mutant CHO strain EB66 as a host cell line (Olivier et al., (2010) MAbs;2:405-415), to use rat hybridoma cell line YB2 / 0 as a host cell line (Shinkawa et al., (2003) J Biol Chem 278:3466-3473), and to specifically introduce small interfering RNAs into the 1,6-fucosyltransferase (FUT8) gene (Mori et al., (2004) Biotechnol Bioeng This can be achieved using different methods that have been reported to lead to the successful expression of relatively high fucose-unmodified immunoglobulins with branched complexes of Fc oligosaccharides, such as co-expression of β-1,4-N-acetylglucosaminyltransferase III and kifunensin, a Golgi α-mannosidase II or potent α-mannosidase I inhibitor (Ferrara et al., (2006) J Biol Chem 281:5032-5036).

[0595] In some embodiments, the multispecific molecules or heterologous molecules of the Disclosure, including an Ig constant region or a fragment of an Ig constant region, have a branched glycan structure having a fucose content of about 1% to about 15%, for example, about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the multispecific molecules or heterologous molecules of the Disclosure, including an Ig constant region or a fragment of an Ig constant region, have a glycan structure having a fucose content of about 50%, 40%, 45%, 40%, 35%, 30%, 25%, or 20%.

[0596] "Fucose content" refers to the amount of fucose monosaccharides within the sugar chain in Asn297. The relative amount of fucose is the ratio of the fucose-containing structure to the total sugar structure. These include multiple methods, e.g., 1) the use of MALDI-TOF (e.g., complex, hybrid, oligo, and high-mannose structures) of N-glycosidase F-treated samples, as described in International Publication No. 2008 / 077546; 2) enzymatic release of Asn297 glycan, subsequent derivatization, and detection / quantification by HPLC (UPLC) and / or HPLC-MS (UPLC-MS) with fluorescence detection; 3) intact protein analysis of natural or reduced mAbs with or without treatment of Asn297 glycan with Endo S or other enzymes that cleave between the first and second GlcNAc monosaccharides, leaving fucose attached to the first GlcNAc; 4) digestion of mAbs into constituent peptides by enzymatic digestion (e.g., trypsin or endopeptidase Lys-C), followed by separation, detection, and quantification by HPLC-MS (UPLC-MS); 5) Asn The mAb oligosaccharides can be characterized and quantified by separating them from the mAb protein via specific enzymatic deglycosylation using PNGase F at position 297. The oligosaccharides thus released can be labeled with fluorophores and separated and identified by various supplementary techniques that enable detailed characterization of the glycan structure by matrix-assisted laser desorption / ionization (MALDI) mass spectrometry by comparing the measured mass with the theoretical mass, determination of the degree of sialylation by ion-exchange HPLC (GlycoSep C), separation and quantification of oligosaccharide types according to hydrophilicity criteria by normal-phase HPLC (GlycoSep N), and separation and quantification of oligosaccharides by high-performance capillary electrophoresis-laser-induced fluorescence (HPCE-LIF).

[0597] "Low fucose" or "low fucose content" refers to the multispecific molecules or heterogeneous molecules of this disclosure that include an Ig constant region or a fragment of an Ig constant region having a fucose content of approximately 1% to 15%.

[0598] "Normal fucose" or "normal fucose content" refers to the multispecific or heterogeneous molecules of this disclosure that include an Ig constant region or fragment of an Ig constant region having a fucose content of more than approximately 50%, typically more than approximately 80%, or more than 85%.

[0599] 5.5 Anti-idiotype antibodies The anti-idiotype antibody is an antibody that specifically binds to the spFv of this disclosure.

[0600] The present invention also provides an anti-idiotype antibody that specifically binds to the spFv of the present disclosure.

[0601] In some embodiments, an anti-idiotype antibody binds to the disulfide bond in the spFv of this disclosure.

[0602] In some embodiments, an anti-idiotype antibody binds to the antigen-binding domain of the spFv of this disclosure.

[0603] 5.6 Polynucleotides, vectors, and host cells This disclosure also provides isolated polynucleotides encoding the spFv of this disclosure.

[0604] This disclosure also provides vectors comprising the polynucleotides of this disclosure.

[0605] In some embodiments, the vector is an expression vector. The expression vector may be a plasmid vector, a viral vector, a vector for baculovirus expression, a vector for prokaryotic expression, a vector for eukaryotic expression, a transposon vector, or any other vector suitable for introducing the polynucleotide of the Disclosure into a given cell or organism. The polynucleotide encoding the spFv of the Disclosure may be operably ligated to a regulatory sequence of the expression vector that promotes spFv expression. Such regulatory elements may include a transcription promoter, a sequence encoding a suitable mRNA-ribosome binding site, and sequences that control the termination of transcription and translation. The expression vector may also contain one or more non-transcription elements, such as an origin of replication, other 5' or 3' adjacent non-transcription sequences, 5' or 3' untranslated sequences (such as a required ribosome binding site), splice donor and acceptor sites, or a selection marker. The polynucleotide may be cDNA. The promoter driving spFv expression may be a strong, weak, tissue-specific, inducible, or developmentally specific promoter. Exemplary promoters that may be used include hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin, human myosin, human hemoglobin, and human muscle creatine. In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for use in the embodiments described.Examples of such viral promoters include, but are not limited to, the early promoter of cytomegalovirus (CMV), the early and late promoters of SV40, the promoter of mouse mammary tumor virus (MMTV), Moloney's leukemia virus, human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), Rous sarcoma virus (RSV), and long terminal repeats (LTRs) of other retroviruses, as well as the thymidine kinase promoter of herpes simplex virus. Inducible promoters include metallothionein promoters, tetracycline-inducible promoters, doxycycline-inducible promoters, protein kinase R 2',5'-oligoadenylate synthetase, and one or more interferon-stimulated response elements (ISREs) such as the Mx gene and ADAR1. The vectors of this disclosure may also contain one or more internal ribosome entry sites (IRESs). Inclusion of IRES sequences in fusion vectors may be beneficial for enhancing the expression of certain proteins. The vectors of this disclosure may be circular or linear. They may be prepared to contain a functional replication system in prokaryotic or eukaryotic host cells. The replication system may be derived from, for example, ColE1, SV40, 2μ plasmid, λ, bovine papillomavirus, etc. Expression vectors may be designed for transient expression, stable expression, or both. Expression vectors may be prepared for constitutive or inducible expression.

[0606] Exemplary vectors that may be used include: Bacteria: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotes: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia), pEE6.4 (Lonza), and pEE12.4 (Lonza). Additional vectors include the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors such as λGT10, λGT11, λEMBL4, and λNM1149, λZapII (Stratagene) can also be used. Exemplary plant expression vectors include pBI01, pBI01.2, pBI121, pBI101.3, and pBIN19 (Clontech). Exemplary animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The expression vector may be a viral vector, such as a retroviral vector, such as a gamma retroviral vector.

[0607] This disclosure also provides host cells containing the vector of this disclosure.

[0608] "Host cell" refers to the cell into which the vector has been introduced. The term "host cell" is understood to refer not only to a specific target cell, but also to the progeny of such cells, and stable cell lines generated from a specific target cell. Such progeny may not be identical to the parent cell, as certain modifications may occur in subsequent generations due to either mutation or environmental influences, but they remain within the scope of the term "host cell" as used herein. Such host cells may be eukaryotic, prokaryotic, plant, or archaeal cells. Examples of prokaryotic host cells include rod-shaped bacteria such as Escherichia coli and Bacillus subtilis, as well as other Enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species. Other microorganisms, such as yeast, are also useful for expression. Examples of suitable yeast host cells include the genera Saccharomyces (e.g., S. cerevisiae) and Pichia. Exemplary eukaryotic cells may be derived from mammals, insects, birds, or other animals. Examples of mammalian eukaryotic cells include immortalized cell lines (e.g., hybridomas) or myeloma cell lines (e.g., SP2 / 0 (American Type Culture Collection (ATCC), Manassas, VA, CRL-1581), NS0 (European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646), and Ag653 (ATCC CRL-1580) mouse cell lines). An exemplary human myeloma cell line is U266 (ATCC CRL-TIB-196). Other useful cell lines include those derived from Chinese hamster ovary (CHO) cells, such as CHO-K1SV (Lonza Biologics (Walkersville, MD)), CHO-K1 (ATCC CRL-61), or DG44.

[0609] The Disclosure also provides a method for producing the scFv of the Disclosure, comprising culturing the host cells of the Disclosure under conditions that produce the spFv, and recovering the spFv produced by the host cells. Methods for preparing and purifying scFv are known. Once synthesized (chemically or recombinantly), the scFv of the Disclosure can be purified by standard procedures including ammonium sulfate precipitation, affinity columns, column chromatography, high-performance liquid chromatography (HPLC) purification, gel electrophoresis, etc. (see, in general, Scopes, Protein Purification (Springer-Verlag, NY, (1982)). The scFv of the Disclosure may be substantially pure and free from contaminants such as cell debris, macromolecules other than the target protein, for example, with a purity of at least about 80% to 85%, at least about 85% to 90%, at least about 90% to 95%, or at least about 98% to 99%, or higher.

[0610] The polynucleotide encoding the scFv of this disclosure can be incorporated into a vector using standard molecular biological methods. Transformation, culture, antibody expression, and purification of host cells are performed using well-known methods.

[0611] 5.7 Pharmaceutical Compositions and Administration This disclosure also provides pharmaceutical compositions comprising the spFv, a heterogeneous molecule containing spFv, or a multispecific molecule containing spFv, and a pharmaceutically acceptable carrier. For therapeutic use, the spFv, a heterogeneous molecule containing spFv, or a multispecific molecule containing spFv may be prepared as a pharmaceutical composition containing an effective amount of the spFv, a heterogeneous molecule containing spFv, or a multispecific molecule containing spFv as the active ingredient in a pharmaceutically acceptable carrier. "Carrier" means a diluent, adjuvant, excipient, or vehicle on which the spFv, a heterogeneous molecule containing spFv, or a multispecific molecule containing spFv is administered. Such a vehicle may be water and liquids including oils derived from petroleum, animal, plant, or synthetic sources, such as peanut oil, soybean oil, mineral oil, or sesame oil. For example, 0.4% saline and 0.3% glycine may be used. These solutions are sterile and generally free of particulate matter. These can be sterilized by conventionally known sterilization techniques (e.g., filtration). The composition may contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters and buffers, stabilizers, thickeners, lubricants, and colorants. The concentration of spFv, spFv-containing heterogeneous molecules, or spFv-containing multispecific molecules of the present disclosure in such pharmaceutical formulations may vary from less than about 0.5% by weight to typically at least about 1% by weight, and up to 15 or 20% by weight, and may be selected mainly based on the required dose, fluid volume, viscosity, etc., depending on the chosen method of administration. Suitable vehicles and formulations (including other human proteins, e.g., human serum albumin) are described, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, DB ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing, pp. 691-1092, and in particular, see pp. 958-989.

[0612] The spFv, heterogeneous molecules containing spFv, or multispecific molecules containing spFv described herein may be administered via any preferred route, such as parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, transmucosal (oral, intranasal, vaginal, rectal)) or other means as well known in the art.

[0613] 5.8 Process for preparing the spFv of this disclosure This disclosure also relates to a process for preparing a stabilized scFv, To provide heavy chain variable regions (VH) and light chain variable regions (VL) that form antigen-binding sites, To provide a linker (L) that includes or is manipulated to include a first L Cys, Manipulating VH to include VH Cys at structurally conserved surface-exposed VH framework residue positions, The present invention provides a process that includes forming a disulfide bond between VH Cys and a first L Cys to prepare a stabilized scFv.

[0614] This disclosure also relates to a process for preparing a stabilized scFv, To provide VH and VL that form antigen-binding sites, To provide L containing a second L Cys, or L manipulated to contain a second L Cys, Manipulating the VL to include VL Cys at structurally conserved surface-exposed VL framework residue positions, The present invention provides a process that includes forming a disulfide bond between a VL Cys and a second L Cys to prepare a stabilized scFv.

[0615] This disclosure also relates to a process for preparing a stabilized scFv, To provide heavy chain variable regions (VH) and light chain variable regions (VL) that form antigen-binding sites, To provide a linker (L) that includes or is operated to include a first L Cys and a second L Cys, Manipulating VH to include VH Cys at structurally conserved surface-exposed VH framework residue positions, Manipulating the VL to include VL Cys at structurally conserved surface-exposed VL framework residue positions, The present invention provides a process for preparing stabilized scFv by forming disulfide bonds between VH Cys and a first L Cys, and between VL Cys and a second L Cys.

[0616] Disulfide bonds are typically formed during scFv expression.

[0617] Any known VH / VL pair of an scFv forming an antigen-binding domain can be manipulated into the stabilized scFv of this disclosure. Alternatively, the antigen-binding VH / VL pair of interest may be novelly identified using known methods, and the resulting VH / VL pair may be manipulated into spFv form.

[0618] For example, the Kohler-Milstein hybridoma method can be used to identify VH / VL pairs that bind to the target antigen and whose resulting VH / VL pairs can be manipulated as spFv. Alternatively, antigen-binding fragments can be generated using transgenic animals such as mice, rats, or chickens that carry human immunoglobulin (Ig) loci in their own genomes, as described, for example, in U.S. Patent No. 6,150,584, International Publication Nos. 1999 / 45962, 2002 / 066630, 2002 / 43478, 2002 / 043478, and 1990 / 04036. The endogenous immunoglobulin loci of such animals may be disrupted or deleted, and at least one complete or partial human immunoglobulin locus may be inserted into the animal genome using homologous or non-homologous recombination, using transchromosomes, or using minigenes. Companies such as Regeneron (http: / / _www.regeneron_com), Harbour Antibodies (http: / / _www.harbourantibodies_com), Open Monoclonal Technology, Inc. (OMT) (http: / / _www.omtinc_net), KyMab (http: / / _www.kymab_com), Trianni (http: / / _www.trianni_com), and Ablexis (http: / / _www.ablexis_com) may be working to provide human antibodies targeting selected antigens using the above technologies. Phage display can also be used to generate antigen-binding fragments that can be manipulated as spFvs.

[0619] In some embodiments, the spFv of the Disclosure is humanized. In some embodiments, the spFv of the Disclosure is human. In some embodiments, the spFv of the Disclosure is non-human.

[0620] In some embodiments, the distance between VH Cys and VL Cys is about 7 Å to about 9 Å in the stabilized scFv.

[0621] In some embodiments, VH Cys are located at H3, H5, H40, H43, H46, or H105, and the residue numbering follows Chothia.

[0622] In some embodiments, the VL Cys are located at L3, L5, L39, L42, L45, L100, or L102, and the residue numbering follows Chothia.

[0623] In some embodiments, VH Cys is at H105, and VL Cys is at L42, or VH Cys is located at H43, and VL Cys is located at L100, or VH Cys are located in H3, and VL Cys are located in L3, or VH Cys is located at H3, and VL Cys is located at L5, or VH Cys is located on H3, and VL Cys is located on L39, or VH Cys is located on H3, and VL Cys is located on L42, or VH Cys are located on H3, and VL Cys are located on L45, or VH Cys is located at H3, and VL Cys is located at L100, or VH Cys is located at H3, and VL Cys is located at L102, or VH Cys is located at H5, and VL Cys is located at L3, or VH Cys is located at H5, and VL Cys is located at L5, or VH Cys is located at H5, and VL Cys is located at L39, or VH Cys is located at H5, and VL Cys is located at L42, or VH Cys is located at H5, and VL Cys is located at L45, or VH Cys is at H5, and VL Cys is at L100, or VH Cys is located at H5, and VL Cys is located at L102, or VH Cys is at H40, and VL Cys is at L3, or VH Cys is at H40, and VL Cys is at L5, or VH Cys is at H40, and VL Cys is at L39, or VH Cys are at H40, and VL Cys are at L42, or VH Cys are at H40, and VL Cys are at L45, or VH Cys is at H40, and VL Cys is at L100, or VH Cys is at H40, and VL Cys is at L102, or VH Cys is located at H43, and VL Cys is located at L3, or VH Cys is located at H43, and VL Cys is located at L5, or VH Cys is located at H43, and VL Cys is located at L39, or VH Cys is located at H43, and VL Cys is located at L42, or VH Cys is located at H43, and VL Cys is located at L45, or VH Cys is located at H43, and VL Cys is located at L102, or VH Cys is located at H46, and VL Cys is located at L3, or VH Cys is located at H46, and VL Cys is located at L5, or VH Cys is located at H46, and VL Cys is located at L39, or VH Cys is located at H46, and VL Cys is located at L42, or VH Cys is located at H46, and VL Cys is located at L45, or VH Cys is located at H46, and VL Cys is located at L100, or VH Cys is located at H46, and VL Cys is located at L102, or VH Cys is located at H105, and VL Cys is located at L3, or VH Cys is located at H105, and VL Cys is located at L5, or VH Cys is at H105, and VL Cys is at L39, or VH Cys is at H105, and VL Cys is at L45, or VH Cys is at H105, VL Cys is at L100, or The VH Cys is located at H105, the VL Cys is at L102, and the residue numbering follows Chothia.

[0624] In some embodiments, L comprises a sequence of amino acids derived from an immunoglobulin (Ig) hinge region.

[0625] In some embodiments, the Ig hinge region is derived from a human or non-human Ig hinge region.

[0626] In some embodiments, the Ig hinge region is derived from the human Ig hinge region.

[0627] In some embodiments, the human Ig hinge region is an IgG1, IgG2, IgG3, or IgG4 isotype.

[0628] In some embodiments, L is the amino acid sequence C(X) y The formula includes C (Sequence ID 23), where X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gln, His, Ile, Leu, Lys, Phe, Thr, Trp, or Tyr, and y is an integer from 1 to 3.

[0629] In some embodiments, L is the amino acid sequence C(X) y The formula includes C (sequence number 24), where X is Gly, Ser, or Pro, and y is an integer between 1 and 3.

[0630] In some embodiments, L includes amino acid sequences CPC, CGC, CSC, CPPC (SEQ ID NO: 1), CGPC (SEQ ID NO: 28), CPGC (SEQ ID NO: 29), CGGC (SEQ ID NO: 30), CSPG (SEQ ID NO: 31), CPSC (SEQ ID NO: 32), CSSC (SEQ ID NO: 33), CGSC (SEQ ID NO: 34), CSGC (SEQ ID NO: 35), CPPPC (SEQ ID NO: 36), CGPPC (SEQ ID NO: 37), CPGPC (SEQ ID NO: 38), CPPGC (SEQ ID NO: 39), CGGPC (SEQ ID NO: 40), CPGGC (SEQ ID NO: 41), CGGGC (SEQ ID NO: 42), CSPPC (SEQ ID NO: 43), CPSPC (SEQ ID NO: 44), CPPSC (SEQ ID NO: 45), CSSPC (SEQ ID NO: 46), CPSSC (SEQ ID NO: 47), CSSSC (SEQ ID NO: 48), CGSPC (SEQ ID NO: 49), CPGSC (SEQ ID NO: 50), CSGPC (SEQ ID NO: 51), or CPSGC (SEQ ID NO: 52).

[0631] In some embodiments, L comprises about 14 to about 19 amino acids, for example, about 14, about 15, about 16, about 17, about 18, or about 19 amino acids.

[0632] In some embodiments, L is the amino acid sequence (X) m C(X) y C(X) n (Sequence ID 25) is included in the formula, where X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gln, His, Ile, leu, Lys, Phe, Thr, Trp, or Tyr, m is an integer from 6 to 9, y is an integer from 1 to 3, and n is an integer from 4 to 6.

[0633] In some embodiments, L is the amino acid sequence (X) m C(X) y C(X) n (Sequence ID 26) is included in the formula, where X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gln, His, Ile, Leu, Lys, Thr, or Tyr, m is an integer from 6 to 9, y is an integer from 1 to 3, and n is an integer from 4 to 6.

[0634] In some embodiments, L is the amino acid sequence (X) m C(X) y C(X) n (Sequence code 27) is included in the formula, where X is Gly or Pro, m is an integer from 6 to 9, y is an integer from 1 to 3, and n is an integer from 4 to 6.

[0635] In some embodiments, L comprises the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7.

[0636] In some embodiments, the stabilized spFv of the present disclosure is in a VL-L-VH orientation.

[0637] In some embodiments, the stabilized spFv of the present disclosure is in a VH-L-VL orientation.

[0638] In some embodiments, VH includes Cys in H105, VL includes Cys in L42, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in the VL-L-VH orientation.

[0639] In some embodiments, VH includes Cys in H105, VL includes Cys in L45, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in the VL-L-VH orientation.

[0640] In some embodiments, VH includes Cys in H105, VL includes Cys in L39, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in the VL-L-VH orientation.

[0641] In some embodiments, VH includes Cys in H5, VL includes Cys in L42, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in the VL-L-VH orientation.

[0642] In some embodiments, VH includes Cys in H5, VL includes Cys in L45, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in the VL-L-VH orientation.

[0643] In some embodiments, VH includes Cys in H5, VL includes Cys in L39, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in the VL-L-VH orientation.

[0644] In some embodiments, VH contains Cys in H3, VL includes Cys in L42, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in the VL-L-VH orientation.

[0645] In some embodiments, VH contains Cys in H3, VL includes Cys in L45, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in the VL-L-VH orientation.

[0646] In some embodiments, VH contains Cys in H3, VL includes Cys in L39, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in the VL-L-VH orientation.

[0647] In some embodiments, VH includes Cys in H43, VL includes Cys in L100, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0648] In some embodiments, VH includes Cys in H43, VL includes Cys in L102, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0649] In some embodiments, VH includes Cys in H43, VL includes Cys in L5, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0650] In some embodiments, VH includes Cys in H43, VL includes Cys in L3, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0651] In some embodiments, VH includes Cys in H40, VL includes Cys in L100, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0652] In some embodiments, VH includes Cys in H40, VL includes Cys in L102, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0653] In some embodiments, VH includes Cys in H40, VL includes Cys in L5, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0654] In some embodiments, VH includes Cys in H40, VL includes Cys in L3, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0655] In some embodiments, VH includes Cys in H46, VL includes Cys in L100, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0656] In some embodiments, VH includes Cys in H46, VL includes Cys in L102, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0657] In some embodiments, VH includes Cys in H46, VL includes Cys in L5, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0658] In some embodiments, VH includes Cys in H46, VL includes Cys in L3, L contains the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, or 7. scFv is in a VH-L-VL orientation.

[0659] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 3.

[0660] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 6.

[0661] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 7.

[0662] In some embodiments, the stabilized spFv of this disclosure binds to the antigen with comparable affinity to a parental scFv lacking a disulfide bond.

[0663] This disclosure also relates to a process for preparing a stabilized scFv, To provide polynucleotides encoding VH, L, and VL, Does VH include Cys in H105, and does VL include Cys in L42? Does VH include Cys in H43, and does VL include Cys in L100? Does VH contain Cys in H3, and does VL contain Cys in L3? Does VH contain Cys in H3, and does VL contain Cys in L5? Does VH contain Cys in H3, and does VL contain Cys in L39? Does VH contain Cys in H3, and does VL contain Cys in L42? Does VH include Cys in H3, and does VL include Cys in L45? Does VH include Cys in H3, and does VL include Cys in L100? Does VH contain Cys in H3, and does VL contain Cys in L102? Does VH contain Cys in H5, and does VL contain Cys in L3? Does VH contain Cys in H5, and does VL contain Cys in L5? Does VH include Cys in H5, and does VL include Cys in L39? Does VH include Cys in H5, and does VL include Cys in L42? Does VH include Cys in H5, and does VL include Cys in L45? Does VH include Cys in H5, and does VL include Cys in L100? Does VH contain Cys in H5, and does VL contain Cys in L102? Does VH include Cys in H40, and does VL include Cys in L3? Does VH include Cys in H40, and does VL include Cys in L5? Does VH include Cys in H40, and does VL include Cys in L39? Does VH include Cys in H40, and does VL include Cys in L42? Does VH include Cys in H40, and does VL include Cys in L45? Does VH include Cys in H40, and does VL include Cys in L100? Does VH include Cys in H40, and does VL include Cys in L102? Does VH include Cys in H43, and does VL include Cys in L3? Does VH include Cys in H43, and does VL include Cys in L5? Does VH include Cys in H43, and does VL include Cys in L39? Does VH include Cys in H43, and does VL include Cys in L42? Does VH include Cys in H43, and does VL include Cys in L45? Does VH include Cys in H43, and does VL include Cys in L102? Does VH include Cys in H46, and does VL include Cys in L3? Does VH include Cys in H46, and does VL include Cys in L5? Does VH include Cys in H46, and does VL include Cys in L39? Does VH include Cys in H46, and does VL include Cys in L42? Does VH include Cys in H46, and does VL include Cys in L45? Does VH include Cys in H46, and does VL include Cys in L100? Does VH include Cys in H46, and does VL include Cys in L102? Does VH include Cys in H105, and does VL include Cys in L3? Does VH include Cys in H105, and does VL include Cys in L5? Does VH include Cys in H105, and does VL include Cys in L39? Does VH include Cys in H105, and does VL include Cys in L45? VH contains Cys in H105, VL contains Cys in L100, or VH contains Cys at H105, VL contains Cys at L102, and residue numbering follows Chothia. L contains the amino acid sequence of SEQ ID NOs. 2, 3, 4, 5, 6, or 7, and is provided as such. The present invention provides a process that includes expressing polynucleotides in host cells to produce stabilized scFv.

[0664] In some embodiments, the host cell is a prokaryotic cell.

[0665] In some embodiments, the host cell is a eukaryotic cell.

[0666] To further illustrate some of the embodiments disclosed herein, the following examples are provided. These examples are illustrative and not limiting to the embodiments of the present disclosure. [Examples]

[0667] 6.1 Example 1: Design of a stabilized scFv Monoclonal antibodies (mAbs) recognize their target antigens via two variable domains, VL and VH. Single-chain Fvs (scFvs) were first designed by Bird et al. (1988) Science 242:423-426 (1988) as gene fusions of VL and VH with a flexible linker in either a VL-linker-VH or VH-linker-VL orientation. The flexible linker is typically three or four repeats of a glycine-serine linker, such as (GGGGS)n;n=1-4 (SEQ ID NO: 2, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55). scFvs replicate antigen-binding specificity and, primarily, affinity for their parent mAb. These scFv molecules have found a wide range of applications, either as detection / diagnostic reagents or as building blocks for creating more sophisticated molecules such as bipolar and multispecific therapeutics (Brinkmann and Kontermann (2017) MAb 9:182-212) or CAR-T therapy (Gross et al., (1989), Transplant Proc 21 (1 Pt 1):127-130, Porter et al., (2011) J Cancer 2:331-332, Porter et al., (2011) N Engl J Med 365:725-733).

[0668] One of the challenges with scFv molecules is their low stability and tendency to aggregate (as outlined in Worn and Pluckthun (2001) J Mol Biol 305:989-1010 and Rothlisberger et al., (2005) J Mol Biol 347:773-789). Many strategies have been attempted to improve these characteristics (Arnd et al., (2001) J Mol Biol 312:221-228, Monsellier et al., (2006) J Mol Biol 362:580-593, Zhao et al., (2010) Int J Mol Sci 12:1-11, Perchiacca and Tessier (2012) Annu Rev Chem Biomol Eng 3:263-286, Asial et al., (2013) Nat Commmun 4:2901, Gil and Schrum (2013) Adv Biosci Biteccchnol 4:73-84, Tiller and Tessier (2015) Annu rev ​​Biomed Eng 17:191-216). These strategies include introducing disulfide bonds between VL / VH domains, using additional dimerization motifs, and improving the stability and / or interfacial interactions of the VL / VH domains using different experimental methods. A significant difficulty is that most of these strategies are often specific to VH / VL pairs and cannot be readily transferred to other VH / VL pairs. In some cases, the manipulation can adversely affect the VL / VH structure and scFv properties. Recently, Zhang et al. successfully achieved significant stabilization of scFv while maintaining its binding affinity by introducing a disulfide between position 44 of VH and position 100 of VL in anti-aflatoxin B1scFv(H4) (Zhao et al., (2010) Int J Mol Sci 12:1-11). However, due to the distance and angle constraints between the two selected positions, when applied to other VL / VH pairs, the VL / VH inter-disulfide can restrict / distort the relative orientation between the two domains that are often required for bonding.

[0669] The interface between the heavy and light chains of the Fab fragment includes VH / VL and CH1 / CL interactions. These two independent sets of interactions result in a synergistic stabilization effect. Furthermore, the V / C junction also contributes to some stabilization. In comparison, in scFv, the VH / VL interface is maintained solely by VH / VL interactions. The linker is designed to be flexible and non-restrictive, except when its length is designed to be short to facilitate scFv-scFv interactions for dimer and oligomer formation. It is known that the length and properties of the linker, when sufficiently long, contribute little to the stability of scFv.

[0670] 6.1.1 "Staple Processing" Design The objective of this specification was to design and generate a stabilized scFv without adversely affecting the relative movement between the VH and VL that form the scFv. This was achieved by stabilizing the scFv by manipulating the disulfide bonds between the VH and the linker and between the VL and the linker. The constraints (i.e., disulfide bonds), when properly positioned, play a role in the synergistic effects brought about by the CH1 / CL and V / C interactions described above. For this purpose, two structurally conserved surface-exposed framework sites (anchor points), one each in the VH and VL, were identified, which did not overlap with typical predicted antigen-binding sites and were mutated to cysteine ​​(Cys) residues. Subsequently, the two sites were selected in a flexible linker relative to the Cys sites. If the distance and position between linker Cys residues are designed in a manner that promotes the formation of disulfide bonds between the linker Cys and each anchor point, the VH and VL can be anchored more rigidly compared to anchoring in the absence of disulfide bonds. This scheme is shown in Figure 1, which has an exemplary linker containing the CPPC sequence (SEQ ID NO: 1). The concept of forming a disulfide bond between the flexible linker and the anchor point is referred to herein as “stapling.” The resulting “stapled” scFv molecule is referred herein as spFv (“stapled Fv”).

[0671] 6.1.2 Anchor point selection, staple arrangement design, and linker In widely applicable stapling schemes, it is important that anchor points are structurally conserved, exposed on the surfaces of both VL and VH, and that mutations in Cys residues do not affect the folding or antigen binding of VL and VH. The anchor points of the VL and VH domains, as well as the distance and geometric shape of the N and C termini, are also important considerations for proper disulfide formation.

[0672] Anchor points were selected separately for spFv in VL-linker-VH and VH-linker-VL orientations. In the case of VL-linker-VH orientation, Chothia position 42 in VL and Chothia position 105 in VH were selected as anchor points. A graph of the selected anchor points for spFv in VL-linker-VH orientation is shown in Figure 2 within the Fv of the human germline antibody (hereinafter, pdb ID 5I19, GLk1). In GLk1, VL Chothia position 42 is lysine (K) and VH Chothia position 105 is glutamine (Q). In the case of VH-linker-VL orientation, Chothia position 100 in VL and Chothia position 43 in VH were selected as anchor points. Figure 3 shows a graph of selected anchor points for spFv in the VH-linker-VL orientation within Fv of a human germline antibody (pdb ID 5I19, GLk1). In GLk1, VL Chothia position 100 is glutamine (Q), and VH Chothia position 43 is lysine (K). The selected anchor points were structurally conserved, and the geometry was very similar in antibodies containing either kappa or lambda light chains. The distance between pairs of anchor points ranged from approximately 7 Å (for VL-linker-VH orientation) to approximately 9 Å (for VH-linker-VL orientation).

[0673] The staple sequence embedded within the linker connecting VH and VL was designed to have a length similar to the distance between anchor points in spFv. As an early example of a staple sequence, CPPC (SEQ ID NO: 1) was selected as a possible staple sequence because this sequence occurs naturally in the human IgG1 hinge and in some rodent IgGs. The hinge structures of human and mouse IgG molecules showed that the Cβ(cys1)-Cβ(cys2) distance in the mouse IgG hinge (Figure 4) and human IgG (Figure 5) is in the range of approximately 7 Å to 9 Å. Since this range is very similar to the distance between two anchor points in both VL-linker-VH and VH-linker-VL orientations, the CPPC (SEQ ID NO: 1) staple sequence has the potential to provide the correct geometric shape for stapling, i.e., to efficiently and correctly form appropriate disulfide bonds at the anchor points. In general, staple sequences were designed to have two Cys residues. For proper stapling, the N-terminal Cys of the staple sequence formed disulfide bonds with the spFv N-terminal domain anchor point, and the C-terminal Cys of the staple sequence formed disulfide bonds with the spFv C-terminal domain anchor point.

[0674] Therefore, the linker connecting VH and VL is designed to include staple arrays as well as connecting arrays at both the N-terminus and C-terminus, extending the linker to provide sufficient linker length to enable endoskin folding of VH and VL and to facilitate proper positioning of the staple arrays.

[0675] In the VL-linker-VH design, the distances between the VL anchor point (K42), the VH anchor point (Q105), the C-terminus of VL (K107), and the N-terminus of VH (Q1) are shown in Figure 2. In the VH-linker-VL design, the distances between the VL anchor point (Q100), the VH anchor point (K43), the C-terminus of VH (S114), and the N-terminus of VL (D1) are shown in Figure 3. Modeling suggested that these distances could be extended by a linker length of approximately 14-19 residues, and that a 4-residue staple sequence was adjacent to an N-terminal linker extension of approximately 6-9 residues and a C-terminal linker extension of approximately 4-6 residues. Therefore, the designed linker length could be expressed as n+4+m, where n=6-9 residues and m=4-6 residues, and 4 represents the length of the CPPC (SEQ ID NO: 1) staple sequence. n and m residues may be glycine, serine, or other amino acid residues. The length of these linkers is expected to be too short to allow crumble, but long enough to allow stapling and sufficiently flexible.

[0676] 6.2 Example 2: Generation and Characterization of spFv To evaluate the stapling design, three human antibodies were selected to generate scFv and corresponding spFv: two antibodies (GLk1 and GLk2) with kappa light chains from the synthetic phage antibody library Shi et al., (2010) J Mol Biol 397:385-396) and a lambda-containing antibody (CAT2200) obtained from the paper (Gerhardt et al. (2009) J Mol Biol 394:905-921). For CAT2200, the T28G mutation was introduced into the parental VH to generate a mutant (CAT2200a) which reduced some of its interactions with its target IL-17. Furthermore, the S42Q mutation (Chothia) was manipulated into the parental CAT2200 VL and paired with the T28G VH to generate CAT2200b. The amino acid sequences of the VL and VH domains of GLk1, GLk2, CAT2200a, and CAT2200b are shown in Figures 6 and 7, respectively. The VH domain amino acid sequence is identical between CAT2200a and CAT2200b. GLk1VH is derived from human IGHV2-23. * 01 GLk2VH vs. human IGHV5-51, CAT2200VH vs. human IGHV2-23 * It is closest to 01. GLk1VL is human IGKV1-39 * 01, GLk2VL vs. Human IGKV3-20 * 01, and CAT2200VL vs. human IGLV 6-57 * It is closest to 01.

[0677] All scFv and spFv molecules were generated and expressed in both VL-linker-VH and VH-linker-VL orientations. For scFv constructs, the standard (GGGGS)4 (SEQ ID NO: 2) linker was used. For spFv, different linker lengths within the n and m ranges described above were used. For GLk1 spFv, the 9-4-5 linker was used for both orientations. For GLk2 spFv, the 9-4-5 and 6-4-6 linker lengths were used for VL-VH and VH-VL orientations, respectively. For CAT2200a spFv, the VL-VH molecules were prepared with 8-4-4 and 9-4-4 linkers, respectively, and the CAT2200b spFv VH-VL was prepared with the 9-4-4 linker. Table 4 shows the generated molecules and their linker sequences. Table 5 shows the amino acid sequences of the generated molecules.

[0678] [Table 4]

[0679] [Table 5-1]

[0680] [Table 5-2]

[0681] All scFv and spFv molecules except CAT2200a scFv VL-VH were cloned into CMV promoter-driven mammalian expression vectors. These constructs were transfected into Expi293 cells using the manufacturer's protocol, and the cells were cultured for 5 days. Each protein was purified from the supernatant clarified on a 1 ml His-TRAPHP column (GE Healthcare) using an AKTAXPRESS system (GE Healthcare). The column was prepared with elution buffers of 0–100% gradient (wash buffer: 50 mM Tris, pH 7.5, 500 mM NaCl, 20 mM imidazole; elution buffer: 50 mM Tris, pH 7.5, 500 mM NaCl, 500 mM imidazole) to remove loosely bound nickel, and then re-equilibriumated with DPBS. The cleared supernatant was first fitted with 50 mM Tris, pH 7.5, and 20 mM imidazole, and then loaded onto a 1 mL HisTRAP HP column at 4°C and 0.8 mL / min. The column was then washed with PBS until a stable baseline was obtained. The column was then further washed with 20 CV of wash buffer, eluted into a single injection loop with elution buffer, desalted in 1x DPBS on a 26 / 10 HiPrep desalted column, and the fraction was collected. The fractions containing the purified protein were then pooled and concentrated. Glk2 scFv and spFv proteins were dialyzed in DPBS for thermal stability measurements (DSC and NanoDSF) and in 25 mM Tris, pH 7.5, and 100 mM NaCl for other studies. Other scFv and spFv proteins were dialyzed in 25 mM MES, pH 6.0, and 100 mM NaCl.

[0682] CAT2200a scFv VL-VH was purchased from a vendor. The concentration was 0.77 mg / mL in DPBS, pH 7.2. A variant of IL-17 (12-132 with the K70Q A132Q C106S mutation, hereafter referred to as IL-17 for simplification) (SEQ ID NO: 22) was purchased from Accelagen (CA). The protein was refolded from E. coli inclusion bodies after their proprietary refolding protocol and supplied at 1.50 mg / mL in 20 mM NaCl, 20 mM MES, pH 6.0.

[0683] Sequence ID 22 (IL-17A variant) MNSEDKNFPRTVMVNLNIHNRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAQCRHLGCINADGNVDYHMNSVPIQQEILVLRREPPHSPNSFRLEKILVSVGCTCVTPIVHHVQ

[0684] 6.2.1 Thermal stability of generated scFv and spFv molecules The thermal stability of scFv and spFv molecules was investigated by differential thermocalorimetry (DSC). scFv and spFv proteins were dialyzed overnight in 1x DPBS (Gibco) for GLk1 and in CAT2200a / CAT2200b or MES (25 mM MES, pH 6.0, 100 mM NaCl) for GLk2. The dialyzed buffer was then filtered through a 0.22 micrometer sieve and used as a reference solution and as a buffer buffer blank in the DSC experiments. The proteins were diluted to approximately 0.5 mg / mL in the filtered buffer, and 400 μL of each protein or buffer sample was loaded into a 96-deep-well plate (MicroLiter Analytical Supplies, 07-2100) and maintained at 4°C in an autosampler drawer throughout the experiment. DSC experiments were performed using MicroCal capillary DSC with an autosampler (Malvern). DSC scanning was performed at 25–95°C with a scan rate of 60°C / h and no sample rescanning. Feedback was not selected, and the filtering period was set to 15 seconds. After each sample, cells were washed with 10% Contrad-70 solution, and a buffer-buffer blank was performed. Data analysis was performed using Origin 7.0 with the MicroCal VP-Capillary DSC Automated Analysis Add-on (Malvern). Baseline range and type were manually selected and then subtracted. Concentration-dependent normalization was performed after subtracting the previous buffer blank from the sample curve. Thermal fusion profiles were analyzed using both two-state and non-two-state transitions. The two-state fit (one transition) did not agree well with the experimental curve. Therefore, two transitions (Tm1 and Tm2) were calculated by manually performing a non-two-state fit. Tm data are reported in Table 6. The DSC profiles for all scFv and spFv proteins showed skewness that could only be fitted by non-two-state transitions. Therefore, two transitions (Tm1 and Tm2) were reported for each scFv or spFv (Table 6). Almost certainly, these two transitions correspond to the melting Tm of the VL and VH domains, respectively.In general, when comparing the difference between scFv and spFv for either Tm1 or Tm2, ​​stapling increases the stability of the initial scFv by approximately 10°C, regardless of the Tm of the initial scFv. There is only one exception, namely the difference between GLk2 scFv and spFv (VH-VL orientation), which is approximately 7°C. This is likely due to the shorter 6+4+6 linker, which may cause slight strain on the geometric shape of the stapling treatment. The fact that ΔTm1(VL) and ΔTm2(VH) were nearly identical suggests that stapling leads to the stabilization of its own domain, in addition to enhancing the VL / VH interaction. Alternatively, stronger VH / VL interaction would transfer the stabilizing effect to the stabilization of the VL / VH domain. In summary, the stapling treatment described herein significantly increases the stability of the scFv.

[0685] [Table 6]

[0686] The CAT2200 spFv was tested for its coupling to IL-17. The coupling was equivalent to that of the CAT2200 scFvs.

[0687] 6.3 Example 3: Verification of appropriate stapling treatment by crystallization of generated scFv and spFv molecules The proteins were concentrated in their respective buffers: GLk1 spFv VL-VH was concentrated at 8.67 mg / ml in 25 mM MES, pH 6.0, and 100 mM NaCl; GLk1 spFv VH-VL was concentrated at 5 mg / ml in 25 mM MES, pH 6.0, and 100 mM NaCl; GLk2 spFv VH-VL was concentrated at 8.66 mg / ml in 25 mM Tris, pH 7.5, and 100 mM NaCl; and cat2200b spFv VH-VL was concentrated at 25 mM MES, pH 6.0, and 100 mM NaCl. Crystallization was set up for each protein in a sitting drop format in a Corning 3550 crystal tray using a Mosquito robot. Each well contained 100 nl of protein and 100 nl of reservoir solution and was incubated against a 70 μl reservoir at 20°C. The reservoir solutions were IH1 and IH2 custom conditions, as well as PEG Ion Screen HT (Hampton Research). Several initial conditions were refined by varying the reservoir components in optimization trials. Diffraction-quality crystals were obtained for several scFv and spFv proteins. Table 7 summarizes the conditions used. Crystals were immersed for several seconds in a mother liquor supplemented with 20% glycerol and flash-frozen in liquid nitrogen. X-ray data were collected at Argonne National Lab using IMCA-CAT Beamline 17ID.

[0688] [Table 7]

[0689] 6.3.1 Crystallization of CAT2200a scFv VL-VH and CAT2200a spFv VL-VH combined with IL-17 The IL-17 / CAT2200a scFv VL-VH complex was prepared by mixing 333 μL of IL17 (SEQ ID NO: 22) (1.5 mg / mL) with 1.74 ml of Cat2200a scFv (0.69 mg / mL) and incubating at 4°C for 3 hours. The mixture was concentrated to approximately 400 μL using a 10 kDa cutoff Amicon Ultra concentrator and loaded onto a Superdex75 column equilibrated with 250 mM NaCl, 20 mM HEPES, and pH 7.5. The fraction corresponding to the complex was pooled and concentrated to a volume of 150 μL. The sample was diluted and concentrated four times (350 μL with 50 mM NaCl, 20 mM HEPES, and pH 7.5 added to a concentration of just under 150 μL). The volume was adjusted to approximately 105 μL, and the concentration was determined to be 2.69 mg / mL. Crystallization was set up using a sitting-drop method with a Corning 3550 plate containing 150 nL of protein + 150 nL of reservoir in an 80 μL reservoir, using a pre-designed buffer and precipitate condition set in-house. The plate was incubated at 20°C. Very small crystals were produced under one of the conditions (sodium acetate, pH 4.5, 25% PEG3K, 0.2 M ammonium acetate). These were collected and converted into crystallization seeds using Hampton seed beads in a Hampton seed bead tube containing 100 μL of 27% PEG3350, 200 mM ammonium acetate, 100 mM sodium acetate, pH 4.5.

[0690] Diffraction-quality crystals were obtained using the same procedure as described above, except for the addition of the seed (150 nL protein + 100 nL reservoir + 50 μL seed). Crystals were grown from 0.1 M Tris 8.5, 18% PEG3K, and 0.2 M LiSO4, transferred to the synthetic mother liquor (0.1 M Tris, pH 8.5, 10% PEG3350, 0.2 M LiSO4, and 20% glycerol), and rapidly frozen in liquid nitrogen. X-ray diffraction data were collected using IMCA-CAT ID17 at Argonne National Laboratory.

[0691] The IL-17-CAT2200a spFv VL-VH complex was prepared by mixing 167 μl of IL-17 (250 μg) with 154 μl of MSCW274 (467 μg at 250 mM NaCl, 20 mM MES, pH 6.5) and incubating overnight at 4°C. The mixture was concentrated to approximately 100 μL in a 10 kDa MWCO Amicon Ultra 0.5 mL concentrator, then diluted and concentrated five times (to approximately 150 μL), and 350 μL of 50 mM NaCl, 20 mM HEPES, pH 7.5 was added. The final volume was 100 μL, and the concentration of the complex was determined to be 6.0 mg / ml. Crystallization was set up using a Mosquito robot, similar to the case of the scFv / IL-17 complex in a sitting drop. Sitting drops consisted of 150 nL of protein + 120 nL of reservoir + 30 nL of seed (scFv / IL-17 as described above). The reservoir solutions were a set of various PEG3350 concentrations and salt conditions. Crystallization plates were incubated at 20°C. Small crystals were obtained from 15.5% PEG3350 and 0.4 M NaH2PO4. The crystals were transferred to LN2 with 16% PEG3350, 0.2 M NaH2PO4, 20% glycerol, and rapid freezing. X-ray diffraction data were collected using IMCA-CAT ID17 at Argonne National Laboratory.

[0692] All X-ray diffraction data were processed using XDS (Kabsch et al. (2010) Acta Crystallogr D Biol Crystallogr 66 (Pt.2):125-132, Monsellier and Bedouelle (2006) J Mol Biol 362:580-593) and CCP4 (Collaborative Computational Project, N. (1994) Acta Crystallogr D Biol Crystallogr 53:240-255). All crystal structures, with the exception of the scFv CAT2200a scFv VL-VH / IL-17 complex, were analyzed by molecular substitution (MR) using a phaser (Read (2001) Acta Crystallogr D Biol Crystallogr 57 (Pt 10):1373-1382) with a homology model generated at MOE (Montreal, Canada). In contrast, the structure of pdb id 2vxs (Gerhardt) (Gerhardt et al. (2009) J Mol Biol 394:905-921) was used as a search model. The structural model was refined using PHENIX (Adams et al. (2004) J Synchrotron Radiat 11 (Pt 1):53-55) and manually adjusted using Coot (Emsley et al. (2010) Acta Crystallogr D Biol Crystallogr 66 (Pt 4):486-501). Molecular graphics were generated using PyMol (www.schrodinger.com).

[0693] 6.3.2 Structure The structures using unbound scFv and spFv molecules are shown in Figures 8, 9, 10, and 11. Figure 8 shows the structure of GLk1 spFv VL-VH. Figure 9 shows the structure of GLk1 spFv VH-VL. Figure 10 shows the structure of GLk2 spFv VH-VL. Figure 11 shows the structure of CAT2200b spFv VH-VL. The structures were consistent with typical Fv structures where both the VL and VH domains pack each other. In general, most linker residues were ordered and resolved in electron density maps. Disulfide bonds between staples and anchor points were generally well ordered in both VL-VH orientations. In addition to unbound scFv and spFv structures, we also attempted to elucidate any structural effects on antigen binding. CAT2200 scFv and spFv variant molecules were crystallized in complex with their congener target IL-17. In the case of crystallized CAT2200 variants, scFv and spFv, the structures are nearly identical regardless of the presence or absence of a bound target (Figures 12, 13, and 14). Figure 12 shows a comparison of unbound CAT2200b spFv VH-VL with CAT2200a scFv VL-VH bound to IL-17. Figure 13 shows a comparison of the front view structures of unbound CAT2200b spFv VH-VL with CAT2200a spFv VL-VH bound to IL-17. Figure 14 shows a comparison of the back view structures of unbound CAT2200b spFv VH-VL with CAT2200a spFv VL-VH bound to IL-17. The structures were identical regardless of the orientation or presence or absence of staples. The rmsd for all matching Cα atoms between structural pairs is very small (0.41 Å between unbound spFv-VH-VL and antigen-bound scFv-VL-VH (Figure 12), 0.46 Å between unbound spFv-VH-VL and spFv-VL-VH (Figures 13 and 14, respectively), and 0.37 Å between bound scFv and bound spFv). Structural evidence indicates that stapling functions as designed. Furthermore, stapling does not affect the VL and VH domain structure or relative VL / VH packing.

[0694] 6.4 Example 4: Design of Additional Anchor Points Using the approach described in Example 1, any additional anchor points for stapling were identified. The following anchor points were identified:

[0695] For VL-linker-VH orientation: VL Chothia positions 42, 45, and 39, and VH Chothia positions 105, 5, and 3. In Figure 6, the VL residues on GLk1VL are K42, K45, and K39, and the VH residues on GLk1 are Q105, L5, and Q3. Staples are formed between any of the indicated positions.

[0696] In the case of VH-linker-VL orientation (VH Chothia positions 43, 40 and 46, VL Chothia positions 102, 5 and 3), staples are formed between any of those positions.

[0697] The spFv having the anchor points described in this example are cloned, expressed, and tested for staple formation and their thermal stability using the assays and Example 2 described herein.

[0698] 6.5 Example 5: Partial stapling The construct is generated and expressed to contain one staple either between VH and the linker or between VL and the linker. The generated construct is expressed, purified and analyzed using the method described herein.

[0699] 6.6 Example 6: Multiple specificity construct containing spFv Exemplary multispecific binding molecules incorporating the spFv structures provided herein were constructed and tested in this example. Specifically, the bispecific antibodies and control molecules were derived from the target binding molecules shown in Table 8, transiently expressed in serum-free / animal component-free medium in CHO suspension culture medium, and purified using AKTA PURE instrumentation (GE Healthcare) by protein A affinity chromatography followed by preparative size exclusion chromatography (SEC) on a Superdex200 10 / 300GL column (GE Healthcare). The heavy chain contained a knob-into-hole (KiH) mutation that promotes heterodimerization (Ridgway et al., Protein Eng. 9(7):617-21 (1996), Atwell et al., J.Mol.Biol. 270(1):26-35 (1997), Merchant et al., Nat.Biotechnol. 16(7):677-81 (1998)). The antibody contained IgG1 sigma Fc, including a set of seven Fc mutations—L234A, L235A, G237A, P238S, H268A, A330S, and P331S—compared to wild-type IgG1 which reduces Fc receptor interaction (Tam et al., Antibodies (2017)).

[0700] Bispecific antibodies were generated by IgG1 sigma mutations and KiH mutations.

[0701] [Table 8] * The EDBmAb1 used herein (International Publication No. 9745544) is a clinically tested anti-ED-B antibody, while other antibodies that bind to ED-B or its adjacent domain have been previously described (Carnemolla et al. Int. J. Cancer 68:397-405 (1996)).

[0702] The arrangement in Table 8 is as follows: Sequence ID 69 (VH BHA10) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSS

[0703] Sequence ID 70 (VL BHA10) DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIK

[0704] Sequence ID 71 (VH L19) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSS

[0705] Sequence ID 72 (VL L19) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIK

[0706] Sequence ID 73 (VH B21M) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSS

[0707] Sequence ID 74 (VL B21M) DIVMTQSPDSLAVSLGERATINCRASQSVDYNGISYMHWYQQKPGQPPKLLIYAASNPESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQIIEDPWTFGQGTKVEIK

[0708] Sequence ID 75 (VH MSLNmAb1) QVQLQQSGPELEKPGASVKISKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSS

[0709] Sequence ID 76 (VL MSLNmAb1) DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK

[0710] Protein concentration was determined by absorbance measurement at 280 nm (OD280), and the purification yield was determined. Analytical SEC was performed using a Bio SEC-5 column (Agilent, 5 μm particle size, 300 Å) in a Thermo VANQUISH HPLC system. 10 μl of purified protein was packed into the column, and elution was recorded by OD280.

[0711] Table 9 outlines the structural properties of the bispecific antibodies and control molecules described in this embodiment. The molecules in bold are exemplary molecules according to the present invention, while the others are controls of different embodiments. Table 10 shows the structural properties of another comparable bispecific antibody targeting LTBR and mesothelin (tumor-associated antigens not present in the extracellular matrix), as discussed in Example 9.

[0712] [Table 9] *Mutations in the :Fc region inhibit binding to protein A and promote the purification of the heterodimer described in International Publication No. 2010 / 151792.

[0713] [Table 10]

[0714] Asymmetric antibodies with the 2:1 stoichiometry listed above (all IgG1 sigma, all KiH mutations) were generated as follows. i.COVA1484 was generated by co-expression of an anti-RSV B21M antibody heavy chain carrying an N-terminally stapled scFv BHA10 (VH-VL oriented SEQ ID NO: 77) fusion (including SEQ ID NOs: 78 and 79) with the heavy chain (HC, SEQ ID NO: 80) and light chain (LC, SEQ ID NO: 81) of an anti-RSV B21M antibody (Figure 15A).

[0715] Sequence ID 77 [Staple processing scFv BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0716] Sequence ID 78 (HC B21M N-terminal stapled BHA10 (VH-VL), IgG1s, knob, pA mutation present) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPK SLISSASYRYSGVPSRFSGGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGGSQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDT ATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSA EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0717] Sequence ID 79 (HC B21M(RSV)IgG1s knob with pA mutation) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAEAPEAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0718] SEQ ID NO:80(HC B21M(RSV)IgG1sホール) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAPEAAGGASSVFLFPPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0719] Sequence ID 81 [LC B21M(RSV)] DIVMTQSPDSLAVSLGERATINCRASQSVDYNGISYMHWYQQKPGQPPKLLIYAASNPESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQIIEDPWTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0720] ii. COVA1485 was generated by co-expression of an anti-RSV B21M antibody heavy chain carrying an N-terminally stapled scFv BHA10 (VL-VH oriented SEQ ID NO: 82) fusion (including SEQ ID NO: 83 and SEQ ID NO: 79) with the heavy chain (HC, SEQ ID NO: 80) and light chain (LC, SEQ ID NO: 81) of the anti-RSV B21M antibody (Figure 15B).

[0721] Sequence ID 82 [Staple processing scFv BHA10(VL-VH)] DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPC GSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSS

[0722] Sequence ID 83 (HCB 21M N-terminal stapled BHA10 (VL-VH), IgG1s, knob, pA mutation present) DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPCGSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGN VHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSSGGGGSGGGGSGGGGGSGGGGSGGGGSQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTA TYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSA EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0723] Sequence ID 79 (HC B21M(RSV)IgG1s knob with pA mutation) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAEAPEAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0724] SEQ ID NO:80(HC B21M(RSV)IgG1sホール) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAPEAAGGASSVFLFPPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0725] Sequence ID 81 [LC B21M(RSV)] DIVMTQSPDSLAVSLGERATINCRASQSVDYNGISYMHWYQQKPGQPPKLLIYAASNPESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQIIEDPWTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0726] iii. COVA1486 was generated by co-expression of an anti-RSV B21M antibody heavy chain carrying a C-terminally stapled scFv BHA10 (VH-VL oriented SEQ ID NO: 77) fusion (including SEQ ID NOs: 84 and 79) with the heavy chain (HC, SEQ ID NO: 80) and light chain (LC, SEQ ID NO: 81) of an anti-RSV B21M antibody (Figure 15C).

[0727] Sequence ID 77 [Staple processing scFv BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0728] Sequence ID No. 84 (HC B21M C-terminal stapled BHA (VH-VL), IgG1s, knob, pA mutation present) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVY TLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEK FKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGP

[0729] Sequence ID 79 (HC B21M(RSV)IgG1s knob with pA mutation) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAEAPEAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0730] SEQ ID NO:80(HC B21M(RSV)IgG1sホール) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAPEAAGGASSVFLFPPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0731] Sequence ID 81 [LC B21M(RSV)] DIVMTQSPDSLAVSLGERATINCRASQSVDYNGISYMHWYQQKPGQPPKLLIYAASNPESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQIIEDPWTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0732] iv.COVA1487 was generated by co-expression of an anti-RSV B21M antibody heavy chain carrying a C-terminally stapled scFv BHA10 (VL-VH oriented SEQ ID NO: 82) fusion (including SEQ ID NOs: 85 and 79) with the heavy chain (HC, SEQ ID NO: 80) and light chain (LC, SEQ ID NO: 81) of an anti-RSV B21M antibody (Figure 15D).

[0733] Sequence ID 82 [Staple processing scFv BHA10(VL-VH)] DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPC GSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSS

[0734] Sequence ID 85 (HC B21M C-staple treated BHA (VL-VH), IgG1s, knob, pA mutation present) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVY TLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFS GSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPCGSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSS

[0735] Sequence ID 79 (HC B21M(RSV)IgG1s knob with pA mutation) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAEAPEAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0736] SEQ ID NO:80(HC B21M(RSV)IgG1sホール) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAPEAAGGASSVFLFPPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0737] Sequence ID 81 [LC B21M(RSV)] DIVMTQSPDSLAVSLGERATINCRASQSVDYNGISYMHWYQQKPGQPPKLLIYAASNPESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQIIEDPWTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0738] v.COVA1480 was generated by co-expression of the heavy chain (HC, SEQ ID NO: 88) and light chain (LC, SEQ ID NO: 89) of the anti-EDB antibody EDBmAb1, which carries an N-terminal stapled scFv BHA10 (VH-VL oriented SEQ ID NO: 77) fusion (including SEQ ID NOs: 86 and 87) (Figure 15E).

[0739] Sequence ID 77 [Staple processing scFv BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0740] Sequence ID No. 86 (HC L19 N-staple treated BHA10 (VH-VL), IgG1s, knob, pA mutation present) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAP KSLISSASYRYSGVPSRFSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0741] Sequence ID 87 (HC L19 IgG1s knob with pA mutation) VQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0742] Sequence ID 88 (HC L19 IgG1s hole) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVCTLPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0743] Sequence ID 89 (LC L19) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0744] vi.COVA1481 was generated by co-expression of the heavy chain (HC, SEQ ID NO: 88) and light chain (LC, SEQ ID NO: 89) of the anti-EDB antibody EDBmAb1, which carries an N-terminal staple-treated scFv BHA10 (VL-VH oriented SEQ ID NO: 82) fusion (including SEQ ID NOs: 90 and 87) (Figure 15F).

[0745] Sequence ID 82 [Staple processing scFv BHA10(VL-VH)] DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPC GSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSS

[0746] Sequence ID 90 (HC L19 N-staple treated BHA10 (VL-VH), IgG1s, knob, pA mutation present) DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPCGSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPG NVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGP AVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0747] Sequence ID 87 (HC L19 IgG1s knob with pA mutation) VQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0748] Sequence ID 88 (HC L19 IgG1s hole) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVCTLPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0749] Sequence ID 89 (LC L19) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0750] vii.COVA1482 was generated by co-expression of the heavy chain (HC, SEQ ID NO: 88) and light chain (LC, SEQ ID NO: 89) of the anti-EDB antibody EDBmAb1, which carries a C-terminal stapled scFv BHA10 (VH-VL oriented SEQ ID NO: 77) fusion (including SEQ ID NO: 91 and SEQ ID NO: 87) (Figure 15G).

[0751] Sequence ID 77 [Staple processing scFv BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0752] Sequence ID 91 (HC L19 C-staple treated BHA10 (VH-VL), IgG1s, knob, pA mutation present) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTL PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0753] Sequence ID 87 (HC L19 IgG1s knob with pA mutation) VQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0754] Sequence ID 88 (HC L19 IgG1s hole) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVCTLPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0755] Sequence ID 89 (LC L19) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0756] viii.COVA1483 was generated by co-expression of the heavy chain (HC, SEQ ID NO: 88) and light chain (LC, SEQ ID NO: 89) of the anti-EDB antibody EDBmAb1, which carries a C-terminal stapled scFv BHA10 (VL-VH oriented SEQ ID NO: 82) fusion (including SEQ ID NOs: 92 and 87) (Figure 15H).

[0757] Sequence ID 82 [Staple processing scFv BHA10(VL-VH)] DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPC GSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSS

[0758] Sequence ID 92 (HC L19 C-staple treated BHA10 (VL-VH), IgG1s, knob, pA mutation present) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTL PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFSG SGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPCGSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSS

[0759] Sequence ID 87 (HC L19 IgG1s knob with pA mutation) VQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0760] Sequence ID 88 (HC L19 IgG1s hole) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVCTLPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0761] Sequence ID 89 (LC L19) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0762] ix.COVA14107 was generated by co-expression of the heavy chain (HC, SEQ ID NO: 88) and light chain (LC, SEQ ID NO: 89) of the anti-EDB antibody EDBmAb1, which carries a C-terminal stapled scFv BHA10 (VH-VL orientation, VL3 Y36F_S49Y_F87Y SEQ ID NO: 93) fusion (including SEQ ID NO: 94 and SEQ ID NO: 87) of the anti-EDB antibody EDBmAb1 (Figure 15I).

[0763] Sequence ID 93 [Staple processing scFv(VL3_Y36F_S49Y_F87Y)BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWFQQKPGKAPKSLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDTYPFTFGCGTKVEIK

[0764] Sequence ID 94 (HC L19 C-staple treated (VL3_Y36F S49Y_F87Y) BHA (VH-VL), IgG1s, knob, pA mutation present) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTL PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWFQQKPGKAPKSLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDTYPFTFGCGTKVEIK

[0765] Sequence ID 87 (HC L19 IgG1s knob with pA mutation) VQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0766] Sequence ID 88 (HC L19 IgG1s hole) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVCTLPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0767] Sequence ID 89 (LC L19) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0768] x.COVA14108 was generated by co-expression of the heavy chain (HC, SEQ ID NO: 88) and light chain (LC, SEQ ID NO: 89) of the anti-EDB antibody EDBmAb1, which carries a C-terminal stapled scFv BHA10 (VH-VL orientation, VH_CDR1_Y33A SEQ ID NO: 95) fusion (including SEQ ID NO: 96 and SEQ ID NO: 87) with the heavy chain (HC, SEQ ID NO: 88) and light chain (LC, SEQ ID NO: 89) of the anti-EDB antibody EDBmAb1 (Figure 15J).

[0769] Sequence ID 95 [Staple processing scFv(VH_CDR1_Y33A)BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYALHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0770] Sequence ID 96 (HC L19 C-staple treated (VH_CDR1_Y33A) BHA10 (VH-VL), IgG 1s, knob, pA mutation present) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTL PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYALHWVRQAPGCGLEWMGWIYPGNVHAQYNEKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0771] Sequence ID 87 (HC L19 IgG1s knob with pA mutation) VQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0772] Sequence ID 88 (HC L19 IgG1s hole) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVCTLPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0773] Sequence ID 89 (LC L19) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0774] xi.COVA14133 was generated by co-expression of the heavy chain (HC, SEQ ID NO: 88) and light chain (LC, SEQ ID NO: 89) of the anti-EDB antibody EDBmAb1, which carries a C-terminal stapled scFv BHA10 (VH-VL oriented SEQ ID NO: 77) fusion (including SEQ ID NOs: 97 and 98) (Figure 15K).

[0775] Sequence ID 77 [Staple processing scFv BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0776] Sequence ID 97 (HC L19 C-staple treated BHA10 (VH-VL), IgG1s, knob, pA mutation present) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTL PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0777] Sequence ID 98 (HC L19 IgG1s knob) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0778] Sequence ID 88 (HC L19 IgG1s hole) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVCTLPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0779] Sequence ID 89 (LC L19) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0780] xii.COVA14136 was generated by co-expression of the heavy chain (HC, SEQ ID NO: 88) and light chain (LC, SEQ ID NO: 81) of the anti-EDB antibody EDBmAb1, which carries a C-terminal stapled scFv BHA10 (VH-VL oriented SEQ ID NO: 77) fusion (including SEQ ID NO: 99 and SEQ ID NO: 100) of the anti-RSV B21M antibody (Figure 15L).

[0781] Sequence ID 77 [Staple processing scFv BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0782] Sequence ID 99 (HC B21M C-staple treated BHA10 (VH-VL), IgG1s, knob, no pA mutation) QITLKESGPTLVKPTQTLLTTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVY TLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEK FKGRVTITADKSTSTAYMELSSLRSEDTAVYCARSWEGFPYWGQGTTVTVSSGGGSGGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0783] SEQ ID NO:100(HC B21M(RSV)IgG1sノブ) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAPEAAGGASSVFLFPPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0784] SEQ ID NO:80(HC B21M(RSV)IgG1sホール) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAPEAAGGASSVFLFPPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0785] Sequence ID 81 [LC B21M(RSV)] DIVMTQSPDSLAVSLGERATINCRASQSVDYNGISYMHWYQQKPGQPPKLLIYAASNPESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQIIEDPWTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0786] xiii.COVA14146 was generated by co-expression of the heavy chain (HC, SEQ ID NO: 88) and light chain (LC, SEQ ID NO: 104) of the anti-mesothelin antibody MSLNmAb1, carrying a C-terminal stapled scFv BHA10 (VH-VL oriented SEQ ID NO: 77) fusion (including SEQ ID NOs: 101 and 102) (Figure 15M).

[0787] Sequence ID 77 [Staple processing scFv BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0788] Sequence ID 101 (MSLNmAb1 HC C-staple treated BHA10 (VH-VL), IgG1s, knob, pA mutation present) QVQLQQSGPELEKPGASVKISKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYT LPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0789] Sequence ID No. 102 (MSLNmAb1 HC, IgG1s, knob, pA mutation present) QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSG TPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEK TISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0790] Sequence ID 103 (HC MSLNmAb1 IgG1s hole) QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSG TPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEK TISKAKGQPREPQVCTLPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0791] Sequence ID 104 (LC MLSNmAb1) DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0792] All of the above constructs can be expressed and purified in high yield and high purity (see Table 11 below), and the bispecific constructs incorporating the spFv provided herein have been shown to have good biophysical properties.

[0793] [Table 11]

[0794] 6.7 Example 7: EDB-dependent in vitro LTBR-activated NF-κB luciferase reporter assay To demonstrate that the EDB / LTBR bispecific compound can activate LTBR in an EDB-dependent manner, the compound's activity was tested in an A549 cell NF-κB luciferase reporter assay in the presence or absence of EDB-containing fibronectin (EDB + fibronectin). NF-κB signaling plays a central role in regulating cell development and immunohomeostasis. Activation of NF-κB via tumor necrosis factor receptor (TNFR) or TNFR superfamily members (e.g., LTBR) occurs in conjunction with their respective ligands. The A549 lung epithelial cell line spontaneously expresses LTBR, ​​and the NF-κB luciferase reporter construct is stably integrated into the genome of the A549 lung epithelial cell line. Following activation by the stimulant, the endogenous NF-κB transcription factor binds to the DNA response element to induce transcription of the luciferase gene.

[0795] To demonstrate EDB-dependent activation of LTBR, ​​high-binding 96-well μClear flat-bottom plates (Greiner;Monroe, NC) were coated overnight with 150 ng / well of human recombinant EDB fibronectin domain 7-B-8-9 (EDB, SEQ ID NO: 105) or 150 ng / well of human recombinant fibronectin domain 7-8-9 (EDB-, SEQ ID NO: 106) (these sequences are listed below).

[0796] Sequence ID 105 (fibronectin domain 7B89) PLSPPTNLHLEANPDTGVLTVSWERSTTPDITGYRITTTPTNGQQGNSLEEVVHADQSSCTFDNLSPGLEYNVSVYTVKDDKESVPISDTIIPEVPQLTDLSFVDITDSSIGLRWTPLNSSTIIGYRITVVAAGEGIPIFEDFVDSSVGYYTVTGLEPGIDYDISVITLINGGESAPTTLTQQTA VPPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLIGQQSTHHHHHH

[0797] Sequence ID 106 (fibronectin domain 789) PLSPPTNLHLEANPDTGVLTVSWERSTTPDITGYRITTTPTNGQQGNSLEEVVHADQSSCTFDNLSPGLEYNVSVYTVKDDKESVPISDTIIPAVPPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVA ELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREEESPLLIGQQSTHHHHHH

[0798] After incubation overnight, the coated plates were washed with PBS and blocked in assay medium (DMEM + 10% thermally inactivated FBS) at 37°C for 2 hours. A 1:5 dilution series of the compound to be tested was prepared in assay medium as 2x concentration stocks (the final concentrations tested ranged from 200 nM to 2.6 pM). After removing the blocking solution by aspirate, 50 μl of the diluted compound was added to the pre-blocked plates. 50 μl of A549 cell suspension (cell suspension concentration = 0.4 MiO cells / ml assay medium) was added to each well (20,000 cells / well). A549 cells were pre-isolated from cell culture flasks using Accutase / EDTA and then transplanted into assay medium. The cells were incubated with the compound at 37°C / 5% CO2 for 18–20 hours.

[0799] After incubation for 18 hours, luciferase activity was detected using the BIO-GLO luciferase assay system (Promega, Madison, WI). Luminescence was measured using the TECAN M 1000Pro instrument with an integration time of 500 milliseconds. From the obtained relative luminous units (RLU), the induction factor of LTBR signaling was calculated as follows: Induction Factor = RLU 刺激済み cells / average RLU 未刺激 Cells (unstimulated cells were included as a control in each plate tested).

[0800] Dose-response curves, including standard deviation, were plotted using GRAPHPAD Prism, and nonlinear fitting was applied where applicable (logarithmic (agonist) versus response (variable gradient - 3 parameters)). To fit the data, x values ​​(compound concentration) were transformed using the x=Log(x) function in GRAPHPAD Prism.

[0801] COVA1482 was compared to COVA1456 using the same A549 NF-κB reporter assay. COVA1482 differs from COVA1456 only in the stabilization method used for the scFv. The scFv in COVA1482, also derived from LTBRmAb1, is stabilized using the stapled platform described herein (i.e., stabilized via VH / linker and VL linker disulfide bonds), while in COVA1456 it is a disulfide stabilized between VH and VL (i.e., stabilized via VH / VL disulfide bonds). Figure 16A shows that both COVA1482 and COVA1456 potently activated LTBR in an EDB-dependent manner. The corresponding isotype controls COVA1486 and COVA1462 did not activate LTBR (Figure 16A). These results indicate that the incorporation of spFv into the multispecific molecule did not adversely affect the activity of the multispecific molecule. 2:1 bispecific EDB / LTBR antibodies (COVA1482 or COVA1456) showed increased potency in inducing NF-κB signaling in this reporter assay. The mean EC25 was calculated for COVA1482 across several assays with the same experimental setup. 50 The EC2 was approximately 30 pM ± 10 pM, while the control 1:1 heterodimer construct showed an EC2 of approximately 3 nM (data not shown here) in the assay. 50 This demonstrates that 2:1 bispecificity can be 100 times stronger than 1:1 bispecificity. This can be explained by the increased clustering of LTBR binding sites achieved by the two binding sites to TAA.

[0802] To study the effect of affinity to LTBR on the ability of such bispecific antibodies to activate LTBR in a TAA-dependent manner, lower affinity variants of scFv fragments derived from LTBRmAb1 (SEQ ID NO: 107, KD ≈ 60 nM and SEQ ID NO: 108, KD ≈ 600 nM) were generated and used to construct 2:1 bispecific COVA14107 and COVA14108 (see Table 9).

[0803] Sequence ID 107 [Staple processing scFv(VL3_Y36F_S49Y_F87Y)BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWFQQKPGKAPKSLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDTYPFTFGCGTKVEIK

[0804] Sequence ID 108 [Staple Processing scFv(VH_CDR 1_Y33A)BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYALHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0805] The generated bispecific compounds were tested using the A549 NF-κB reporter assay to confirm the effect of their affinity on LTBR activation. Figure 16B shows that lower affinity for LTBR corresponded to a reduced ability of the bispecific compounds to activate LTBR in a TAA-dependent manner in this assay. The data also demonstrated that the incorporation of spFv into the multispecific molecule did not adversely affect the molecule's activity.

[0806] As described in Example 6, mutations that suppress binding to protein A (used for antibody purification) (International Publication No. 2010 / 151792) were introduced into the Fc region of several constructs to facilitate the purification of the desired heterodimer. COVA14133 was generated without these mutations, and its activity, compared to COVA 1482, showed that the mutations in the Fc region did not affect the activity of the bispecific strain. COVA14133 and COVA1482, as well as their respective isotype controls COVA14136 and COVA1486, were compared in the A549 NF-κB reporter assay. Figure 16C shows that COVA14133 activated LTBR in a TAA-dependent manner with similar efficiency to COVA1482, demonstrating that the mutations in Fc did not affect the bispecific ability to activate LTBR and did not affect the function of spFv.

[0807] In conclusion, COVA14133 demonstrated excellent ability to activate LTBR in a TAA-dependent manner.

[0808] 6.8 Example 8: EDB-dependent in vitro LTBR activation-A375 / WI38VA sub-lineage 2RA co-culture cell assay To investigate whether activation of LTBR in the presence of EDB + fibronectin (produced and deposited in the extracellular matrix by WI38VA cells (Zardi, L., et al., EMBO J, 6, 2337-42 (1987)) does not adversely affect the activity of cytokine and chemokine release, upregulation of the adhesion molecule ICAM-1 on A375 cells, and spFv incorporation, an A375 / WI38VA sublineage 2RA co-culture assay was performed. WI38VA sublineage 2RA (ATCC® CCL75.1®) cells were seeded at a density of 5000 cells / well in 96-well plates and grown in growth medium (MEM) at 37°C / 5%CO2. The cells were incubated for 48 hours in (w / o glutamine + 10% heat-inactivated FBS + 0.1 mM NEAA + 2 mM L-Gln + 1 mM sodium pyruvate). A 1:5 dilution series of the compounds to be tested was prepared as 2-fold stocks in assay medium (DMEM + 10% heat-inactivated FBS) (the final concentrations tested ranged from 40 nM to 0.5 pM). Prior to incubation in co-culture with WI38VA sublineage 2RA cells, A375 cells (ATCC® CRL-1619®) were labeled with CELLTRACE violet (CTV, Invitrogen, Carlsbad, CA). For labeling, 10 × 10⁶ cells were used in 5% FBS in PBS. 6 Cell suspensions with a concentration of cells / ml and 2.5 μMCTV were incubated at room temperature for 5 minutes while protected from light. Then the cells were washed and 0.4 × 10⁶ cells were removed. 6 The cells were resuspended in assay medium at a density of cells / ml. Following careful removal of the culture medium from the plate containing a 48-hour WI38VA sub-lineage 2RA culture, 50 μl of A375 cell suspension (20,000 cells / well, CTV+ or CTV-) was added to each well. 50 μl of serially diluted compound (final volume 100 μl per well) was added to the cells and incubated at 37°C / 5% CO2 for 24 hours.

[0809] After incubation for 24 hours, the supernatant was cleared by centrifugation and stored for cytokine and chemokine measurement using the MSD assay. The cells were further processed by flow cytometry for ICAM-1 measurement.

[0810] 6.8.1 Detection of ICAM-1 by flow cytometry All remaining culture medium in the 96-well plate was carefully removed, the cells were detached with Accutase and transferred to a DeepWell 96-well plate (pooled in one well in three different ways), washed, resuspended in 100 μl of FACS buffer (PBS + 1% FBS + 0.1% NaN3), and transferred to a round-bottom 96-well plate. Antibodies, i.e., labeled anti-human ICAM-1 PE (clone 1H4, Thermo, Waltham, MA) or labeled isotype control antibody PE (MPC-11, BioLegend, San Diego, CA) and LIVE / DEAD fixable near-IR staining (Invitrogen), single staining or combination staining were diluted as shown in Table 12.

[0811] [Table 12]

[0812] Cells were centrifuged at 400×g for 4 minutes at 4°C, the supernatant was discarded, and 50 μl of antibody solution was prepared as shown in Table 12. Cells and antibody were incubated in the dark at 4°C for 30 minutes. After incubation, 120 μl was added to each well, and the cells were then centrifuged at 400×g for 4 minutes at 4°C. The cells were washed once with FACS buffer, centrifuged, and resuspended in 90 μL of FACS buffer. The cells were then fixed by adding 90 μl of 3.7% formalin solution in PBS and incubated on ice in the dark for 15 minutes. After fixation, the cells were centrifuged at 400×g for 4 minutes at 4°C and resuspended in 100 μL of FACS buffer. Cells were measured using a MACS Quant instrument at high flow rate in screen mode, and 49 μl / well was obtained. Data were analyzed using Flowlogics Software (version 700.2A) and plotted in Graphpad Prism.

[0813] 6.8.2 Cytokine measurement in supernatant of treated cells using the MSD platform Several cytokines known to be regulated by NF-κB signaling were measured using the MSD platform and multiplex MSD plates. Some examples of the measured cytokines are listed here. ■RANTES: R-Plex antibody set using human RANTES (MSD) ■Using I-TAC, IP-10, and MIP-3b:3-PLEX cytokine release assay (MSD), ■IL-8, IP-10, MIP-3b:3-PLEX cytokine release assay (MSD) was used, and ■IL-12p70, IL-6, TNF-α, MIP-3α, SDF-1α: 5-PLEX cytokine release assay (MSD) was used.

[0814] The cytokine concentrations in the supernatant of treated cells were measured using the MSD platform according to the manufacturer's instructions. Briefly, the protocol included the following steps: (1) Plate preparation involved coating the provided plates with linker-bound capture antibodies. The plates were incubated overnight at 2–8°C with shaking. The following day, the plates were washed with PBST (PBS + 0.05% Tween-20) using a plate washer (Biotek; Winooski, VT). (2) Calibration standards and detection antibody solutions were prepared. (3) Depending on the availability of the material, the supernatant was diluted to a ratio of 1:3 or 1:5.

[0815] 6.8.3 Assay Protocol: Step 1: Add the sample or calibration standard to the plate and incubate at room temperature for 1 hour while shaking the plate. Step 2: Wash the plate and add the detection antibody. Incubate the plate at room temperature for 1 hour with shaking. Step 3: The plate was washed and 2x read buffer T was added. The plate was analyzed using an MSD instrument.

[0816] The data was analyzed using MESOSCALE software (MSD Exploration Workbench Program v 4.0.12.1) and plotted using GRAPHPAD Prism.

[0817] 6.8.4 Results - Detection of ICAM-1 by flow cytometry It has been previously shown that NF-κB signaling may lead to the upregulation of ICAM-1 on the cell surface (da Silva Antunes, et al. Front Immunol, 9:576, (2018)). Therefore, we measured the level of ICAM-1 expression on the surface of A375 cells after co-culture incubation with an EDB / LTBR bispecific molecule. As an example, Figure 17 shows the upregulation of ICAM-1 after incubation with the EDB / LTBR bispecific molecule COVA 1482, demonstrating the functionality of LTBR-binding spFv. The isotype control molecule COVA1486 did not induce upregulation of ICAM-1. These findings indicate that the ability to cluster LTBR scFv via binding to EDB is a prerequisite for LTBR activation and, consequently, the upregulation of ICAM-1.

[0818] 6.8.5 Results - Measurement of cytokines in the supernatant of treated cells Several cytokines and chemokines expressed as a result of LTBR activation were measured in the supernatant of co-cultures treated with EDB / LTBR bispecific and control molecules, as described above. Figures 18A–18D show four representative examples of cytokines upregulated by LTBR activation with COVA14133 (Figure 18A: RANTES, Figure 18B: IL-6, Figure 18C: IL-8, and Figure 18D: MIP-3b). Untargeted LTBRmAb1-derived scFv in COVA14136 did not activate LTBR, ​​and consequently, cytokine concentrations in the supernatant did not increase beyond the background. The background is represented by the level achieved with B21M antibody or COVA1440 (2(mAb) B21M isotype control mAb) (shown as a single concentration in the plot). The results indicate that LTBR-bound spFv is functional in vitro.

[0819] In summary, the upregulation of ICAM-1 and cytokine secretion during LTBR activation confirmed the expected effects that LTBR activation may have on cells.

[0820] In this example, it was demonstrated that the molecules provided herein achieved efficient tumor-associated antigen (in this case, EDB-containing fibronectin)-dependent activation of LTBR.

[0821] 6.9 Example 9: Co-culture cell assay using mesothelin-dependent in vitro LTBR-activated A549 NF-κB reporter cells and CHOK1-huMSLN or H226 In Examples 7 and 8, it was demonstrated that bispecific antibodies comprising the spFv structure, targeted EDB (tumor-associated antigen in the extracellular matrix), and LTBR provided herein highly efficiently activated LTBR in a tumor antigen-dependent manner. To confirm whether this finding applies to any tumor antigen, despite its location (deposited in the extracellular matrix or on the cell surface of tumor cells), bispecific 2:1 antibody-targeted mesothelin (MSLN), tumor-associated antigen expressed in different types of tumors (Hassan and Ho, Eur. J. Cancer, 44:46-53 (2008)), and LTBR were designed and produced as described in Example 6. COVA14146 is a 2:1 MSLN / LTBR bispecific antibody consisting of an anti-mesothelin antibody (MSLNmAb1) fused to an spFv fragment derived from LTBRmAb1. To demonstrate whether LTBR bispecific antibodies and tumor-associated antigens (e.g., mesothelin) present on the surface of tumor cells can efficiently activate LTBR in a tumor-dependent manner, co-culture cell assays were used. The co-culture assays used were the A549 cell NF-κB luciferase reporter cell assay (described in Example 7) and H226 cells (mesothelioma cell line, ATCC® CRL-5826) (known to express mesothelin (Fan et al. Mol. Canc. Ther. 1:595-600 (2002))) and LTBR.

[0822] 6.9.1 Preparation of H226 cells 10,000 cells per well of a suspension of H226 cells (expressing approximately 200,000 copies of mesothelin and 10,000 copies of LTBR) were seeded in 75 μl of assay medium (DMEM + 10% FBS-HI) into a 96-well tissue culture plate. These cells were incubated for 6 hours at 37°C / 5% CO2 in growth medium (MEM + 2 mM glutamine + 10% FBS-HI + 10 μg / ml puromycin and RPMI-1640 + 10% FBS + 1 mM Na-pyruvate, respectively) to allow the cells to adhere to the plate.

[0823] 6.9.2 Preparation of Compounds The compounds were tested at concentrations ranging from 100 nM to 1.3 pM. Four-fold 1:5 serial dilutions of the compounds were prepared in assay medium (DMEM + 10% FBS-HI) and stored at 4°C until use.

[0824] 6.9.3A549 Preparation and addition of reporter cells A549 reporter cells were isolated from cell culture flasks using Accutase / EDTA and transferred to assay medium (DMEM + 10% FBS-HI). A total of 20,000 A549 reporter cells per well were added to plates containing H226 cells, and then 50 μL of pre-diluted compound was added to each well. The mixture was incubated at 37°C / 5% CO2 for 20 hours.

[0825] 6.9.4 Measurement of luminescence in treated co-cultures After a 20-hour incubation, luciferase activity was detected using a BIO-GLO luciferase assay system (Promega, Madison, WI) according to the manufacturer's instructions. Luminescence was measured using a TECAN M 1000Pro instrument with an integration time of 500 milliseconds. From the obtained relative light units (RLU), the induction factor of LTBR signaling was calculated as follows: Induction Factor = RLU 刺激済み cells / average RLU 未刺激 Cells (unstimulated cells were included as a control in each plate tested).

[0826] Dose-response curves, including standard deviation, were plotted using GRAPHPAD Prism, and nonlinear fitting was applied where applicable (logarithmic (agonist) versus response (variable gradient - 3 parameters)). To fit the data, x values ​​(compound concentration) were transformed using the x=Log(x) function in GRAPHPAD Prism.

[0827] 6.9.5 Cytokine Measurement in Supernatant of Treated Cells Using the MSD Platform Several cytokines known to be regulated by NF-κB signaling can be measured using the MSD platform and multiplexed MSD plates. As an example, a method for measuring RANTES using the R-Plex antibody set Human RANTES (MSD) is described herein.

[0828] The concentration of RANTES in the supernatant of treated cells was measured using the MSD platform according to the manufacturer's instructions. Briefly, the protocol included the following steps: (1) Plate preparation involved coating the provided plates with linker-bound capture antibodies. The plates were incubated overnight at 2–8°C with shaking. The following day, the plates were washed with PBST (PBS + 0.05% Tween-20) using a plate washer (Biotek; Winooski, VT). (2) Calibration standards and detection antibody solutions were prepared. (3) Depending on the availability of the material, the supernatant was diluted to a ratio of 1:3 or 1:5.

[0829] 6.9.6 Assay Protocol: Step 1: Add the sample or calibration standard to the plate and incubate at room temperature for 1 hour while shaking the plate. Step 2: Wash the plate and add the detection antibody. Incubate the plate at room temperature for 1 hour with shaking. Step 3: The plate was washed and 2x read buffer T was added. The plate was analyzed using an MSD instrument.

[0830] The data was analyzed using MESOSCALE software (MSD Exploration Workbench Program v 4.0.12.1) and plotted using GRAPHPAD Prism.

[0831] 6.9.7 Results - Mesothelin-dependent activation of LTBR in A549 reporter cell / H226 co-culture assay Co-culture assays using A549 reporter cells and H226 cells were performed to investigate whether COVA14146 could activate LTBR in a more physiological system when LTBR and mesothelin (another tumor-associated antigen on the surface of tumor cells, e.g., EGFR) are expected to be co-expressed on the surface of tumor cells due to their broad expression (Lukashev, et al. Cancer Res., 66(19):9617-24 (2006)). Figure 19A shows that COVA14146 did not efficiently activate LTBR under these conditions. The concentration of RANTES secreted into the supernatant of treated cells was measured to confirm that COVA14146 could not efficiently activate LTBR. As expected, Figure 19B shows that RANTES was secreted by cells treated with COVA14146 to the same extent as by cells treated with the isotype control molecule COVA1486, confirming that LTBR could not be activated under these conditions.

[0832] 6.10 Example 10: Bispecific antibody that specifically binds to LTBR and other TAAs present in the extracellular matrix The above example demonstrates that bispecific antibodies targeting LTBR and TAA are expressed in the extracellular matrix, in which case the EDB of fibronectin can selectively activate LTBR in tumor tissue expressing the EDB. This example demonstrates that this is generally applicable by providing two or more examples of such TAAs, namely domain A2 of tenascin C and extradomain A of fibronectin, and thus bispecific antibodies targeting LTBR and other TAAs present in the extracellular matrix.

[0833] Generally, further bispecific antibodies that bind to LTBR and other TAAs present in the extracellular matrix, namely domain A2 of tenascin C (TnCA2) or extra domain A (EDA) of fibronectin, were prepared according to the methods described in the examples above. The sequences of these TAAs are, for example, -TnCA2:UniProt accession number P24821.3, -EDA: UniProt accession number P02751 is listed.

[0834] Furthermore, antibodies against these TAAs are, for example, previously described below: -TnCA2: International Publication No. 2011 / 020783, -EDA: European Patent No. 2142567.

[0835] Exemplary sequences of the binding domains to these targets used in this embodiment (by combining them with LTBR scFv to create the multispecific binding molecules of the present invention) are provided as follows: -TnCA2: VH of sequence number 109 and VL of sequence number 110, -EDA: VH of sequence number 111 and VLV of sequence number 112.

[0836] These sequences are as follows: Sequence ID 109 (VH of anti-TnCA2 Ab 2B10) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLYGYAYYGAFDYWGQGTTVTVSS

[0837] Sequence ID 110 (VL of anti-TnCA2 Ab 2B10) DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGGGSGTEFTLTISSLQPEDFATYYCLQNGLQPATFGQGTKVEIK

[0838] Sequence ID 111 (VH of anti-EDA Ab F8) EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS

[0839] Sequence ID 112 (VL of anti-EDA Ab F8) EIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK

[0840] Bispecific antibodies binding to LTBR and their TAAs were prepared and tested for TAA-specific activation of LTBR in a TAA-dependent in vitro LTBR activation-NF-κB luciferase reporter assay following the same procedure as described in Example 7.

[0841] Asymmetric antibodies with a 2:1 stoichiometry were generated as follows: COVA14198 was generated by co-expression of an anti-EDA antibody heavy chain (HC, SEQ ID NO: 115) and light chain (LC; SEQ ID NO: 116) of an anti-EDA antibody, carrying a C-terminally stapled scFv BHA10 (VH-VL oriented SEQ ID NO: 77) fusion (including SEQ ID NOs: 113 and 114).

[0842] The arrays are listed below. Sequence ID 77 [Staple processing scFv BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0843] Sequence ID 113 (BHA10 stapled (VH-VL) scFv C-terminal fusion, IgG1 sigma, F8 HC with knob mutation) EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYT LPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0844] Sequence ID 114 (F8 HC, IgG1 sigma, knob mutation) EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPPAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0845] Sequence ID 115 (F8 HC, IgG1 sigma and hole mutation) EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPPAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVCTLPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0846] Sequence ID 116 (F8 LC) EIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0847] COVA14202 was generated by co-expression of the antidomain A2 of the tenascin C antibody heavy chain, which carries a C-terminal stapled scFv BHA10 (VH-VL oriented SEQ ID NO: 77) fusion (including SEQ ID NOs: 117 and 118), with the heavy chain (HC, SEQ ID NO: 119) and light chain (LC, SEQ ID NO: 120) of the antidomain A2 of the tenascin C antibody. The sequences are listed below.

[0848] Sequence ID 77 [Staple processing scFv BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0849] Sequence ID 117 (BHA10 stapled (VH-VL) scFv C-terminal fusion, IgG1 sigma, 2B10 HC with knob mutation) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLYGYAYYGAFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV...

Claims

1. 1. An isolated single chain variable fragment (scFv) comprising: comprising a heavy chain variable region (VH), a linker (L) and a light chain variable region (VL), the VH comprises a structurally conserved, surface-exposed VH cysteine ​​(Cys); the VL comprises a structurally conserved, surface-exposed VL Cys; wherein the scFv is (i) a first disulfide bond between the structurally conserved, surface-exposed VH Cys and the first L Cys; (ii) a second disulfide bond between the structurally conserved, surface-exposed VL Cys and a second L Cys; or (iii) the first disulfide bond between the structurally conserved, surface-exposed VH Cys and the first L Cys, and the second disulfide bond between the structurally conserved, surface-exposed VL Cys and the second L Cys; a) the VH Cys is at H3, H5, H40, H43, H46, or H105, residue numbering according to Chothia; and b) the VL Cys is at L3, L5, L39, L42, L45, L100, or L102, and residue numbering is according to Chothia; The L is a) Amino acid sequence C(X) ｙ C (SEQ ID NO:23), wherein X is glycine (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), threonine (Thr), tryptophan (Trp), or tyrosine (Tyr), and y is an integer from 1 to 3. b) the amino acid sequences CPC, CGC, CSC, CPPC (SEQ ID NO: 1), CGPC (SEQ ID NO: 28), CPGC (SEQ ID NO: 29), CGGC (SEQ ID NO: 30), CSPG (SEQ ID NO: 31), CPSC (SEQ ID NO: 32), CSSC (SEQ ID NO: 33), CGSC (SEQ ID NO: 34), CSGC (SEQ ID NO: 35), CPPPC (SEQ ID NO: 36), CGPPC (SEQ ID NO: 37), CPGPC (SEQ ID NO: 38), CPPGC (SEQ ID NO: 39), 39), CGGPC (SEQ ID NO: 40), CPGGC (SEQ ID NO: 41), CGGGC (SEQ ID NO: 42), CSPPC (SEQ ID NO: 43), CPSPC (SEQ ID NO: 44), CPPSC (SEQ ID NO: 45), CSSPC (SEQ ID NO: 46), CPSSC (SEQ ID NO: 47), CSSSC (SEQ ID NO: 48), CGSPC (SEQ ID NO: 49), CPGSC (SEQ ID NO: 50), CSGPC (SEQ ID NO: 51) or CPSGC (SEQ ID NO: 52), c) Amino acid sequence (X) ｍ C(X) ｙ C(X) ｎ (SEQ ID NO:25), wherein X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gln, His, Ile, leu, Lys, Phe, Thr, Trp, or Tyr; m is an integer from 6 to 9; y is an integer from 1 to 3; and n is an integer from 4 to 6; or d) comprising the amino acid sequence of SEQ ID NO: 3, 4, 5, 6 or 7; the distance between the VH Cys and the VL Cys is about 7 Å to about 9 Å; The amino acid sequence contained in the L includes a first L Cys and a second L Cys; scFv.

2. (i) the VH Cys is at H105 and the VL Cys is at L42; (ii) the VH Cys is at H43 and the VL Cys is at L100; (iii) the VH Cys is in H3 and the VL Cys is in L3; (iv) the VH Cys is in H3 and the VL Cys is in L5; (v) the VH Cys is in H3 and the VL Cys is in L39; (vi) the VH Cys is in H3 and the VL Cys is in L42; (vii) the VH Cys is in H3 and the VL Cys is in L45; (viii) the VH Cys is in H3 and the VL Cys is in L100; (ix) the VH Cys is at H3 and the VL Cys is at L102; (x) the VH Cys is in H5 and the VL Cys is in L3; (xi) the VH Cys is in H5 and the VL Cys is in L5; (xii) the VH Cys is at H5 and the VL Cys is at L39; (xiii) the VH Cys is at H5 and the VL Cys is at L42; (xiv) the VH Cys is in H5 and the VL Cys is in L45; (xv) the VH Cys is at H5 and the VL Cys is at L100; (xvi) the VH Cys is at H5 and the VL Cys is at L102; (xvii) the VH Cys is in H40 and the VL Cys is in L3; (xviii) the VH Cys is in H40 and the VL Cys is in L5; (xix) the VH Cys is at H40 and the VL Cys is at L39; (xx) the VH Cys is at H40 and the VL Cys is at L42; (xxi) the VH Cys is at H40 and the VL Cys is at L45; (xxii) the VH Cys is at H40 and the VL Cys is at L100; (xxiii) the VH Cys is at H40 and the VL Cys is at L102; (xxiv) the VH Cys is in H43 and the VL Cys is in L3; (xxv) the VH Cys is in H43 and the VL Cys is in L5; (xxvi) the VH Cys is at H43 and the VL Cys is at L39; (xxvii) the VH Cys is at H43 and the VL Cys is at L42; (xxviii) the VH Cys is at H43 and the VL Cys is at L45; (xxix) the VH Cys is at H43 and the VL Cys is at L102; or (xxx) the VH Cys is in H46 and the VL Cys is in L3; (xxxi) the VH Cys is at H46 and the VL Cys is at L5; (xxxii) the VH Cys is at H46 and the VL Cys is at L39; or (xxxiii) the VH Cys is at H46 and the VL Cys is at L42; or (xxxiv) the VH Cys is at H46 and the VL Cys is at L45; or (xxxv) the VH Cys is at H46 and the VL Cys is at L100; or (xxxvi) the VH Cys is at H46 and the VL Cys is at L102; (xxxvii) the VH Cys is at H105 and the VL Cys is at L3; (xxxviii) the VH Cys is at H105 and the VL Cys is at L5; (xxxix) the VH Cys is at H105 and the VL Cys is at L39; or (xl) the VH Cys is at H105 and the VL Cys is at L45; or (xli) the VH Cys is at H105 and the VL Cys is at L100; or (xlii) the VH Cys is at H105 and the VL Cys is at L102, wherein residue numbering is according to Chothia; The scFv of claim 1.

3. The L is an amino acid sequence C(X) ｙ C (SEQ ID NO:24), wherein X is Gly, Ser, or Pro, and y is an integer from 1 to 3; 3. The scFv of claim 1 or 2.

4. The L is an amino acid sequence (X): ｍ C(X) ｙ C(X) ｎ (SEQ ID NO:26), wherein X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gln, His, He, Leu, Lys, Thr, or Tyr; m is an integer from 6 to 9; y is an integer from 1 to 3; and n is an integer from 4 to 6; or The L is an amino acid sequence (X): ｍ C(X) ｙ C(X) ｎ (SEQ ID NO:27), wherein X is Gly or Pro, m is an integer from 6 to 9, y is an integer from 1 to 3, and n is an integer from 4 to 6.

3. The scFv of claim 1 or 2.

5. the scFv is in a VL-L-VH orientation; The scFv according to any one of claims 1 to 4.

6. 2. The scFv of claim 1, a) comprising a VH, L, and VL, wherein: (i) the VH comprises a Cys at H105; (ii) the VL comprises a Cys at L42; (iii) L comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, or 7; (iv) the scFv is in a VL-L-VH orientation; b) comprising a VH, L, and VL, wherein: (i) the VH comprises a Cys at H105; (ii) the VL comprises a Cys at L45; (iii) L comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, or 7; (iv) the scFv is in a VL-L-VH orientation; c) comprising a VH, L, and VL, wherein: (i) the VH comprises a Cys at H105; (ii) the VL comprises a Cys at L39; (iii) L comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, or 7; (iv) the scFv is in a VL-L-VH orientation; d) comprising a VH, L, and VL, wherein: (i) the VH comprises Cys at H5; (ii) the VL comprises a Cys at L42; (iii) L comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, or 7; (iv) the scFv is in a VL-L-VH orientation; e) VH, L, and VL, wherein: (i) the VH comprises Cys at H5; (ii) the VL comprises a Cys at L45; (iii) L comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, or 7; (iv) the scFv is in a VL-L-VH orientation; f) VH, L, and VL, wherein: (i) the VH comprises Cys at H5; (ii) the VL comprises a Cys at L39; (iii) L comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, or 7; (iv) the scFv is in a VL-L-VH orientation; g) VH, L, and VL, wherein: (i) the VH comprises Cys in H3; (ii) the VL comprises a Cys at L42; (iii) L comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, or 7; (iv) the scFv is in a VL-L-VH orientation; h) VH, L, and VL, wherein: (i) the VH comprises Cys in H3; (ii) the VL comprises a Cys at L45; (iii) L comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, or 7; (iv) the scFv is in a VL-L-VH orientation; i) comprising a VH, L, and VL, wherein: (i) the VH comprises Cys in H3; (ii) the VL comprises a Cys at L39; (iii) L comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, or 7; (iv) the scFv is in a VL-L-VH orientation; j) VH, L, and VL, wherein: (i) the VH comprises Cys at H43; (ii) the VL comprises Cys at L100; (iii) L comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, or 7; (iv) the scFv is in a VH-L-VL orientation; k) comprises a VH, L, and VL, wherein: (i) the VH comprises Cys at H43; (ii) the VL comprises Cys at L102; (iii) L comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, or 7; (iv) the scFv is in a VH-L-VL orientation; l) VH, L, and VL, wherein: (i) the VH comprises Cys at H43; (ii) the VL comprises Cys at L5; (iii) L comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, or 7; (iv) the scFv is in a VH-L-VL orientation; m) VH, L, and VL, wherein: (i) the VH comprises Cys at H43; (ii) the VL comprises Cys at L3; (iii) L comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, or 7; (iv) the scFv is in a VH-L-VL orientation; n) VH, L, and VL, wherein: (i) the VH comprises Cys at H40; (ii) the VL comprises Cys at L100; (iii) L comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, or 7; (iv) the scFv is in a VH-L-VL orientation; o) comprising a VH, L, and VL, wherein: (i) the VH comprises Cys at H40; (ii) the VL comprises Cys at L102; (iii) L comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, or 7; (iv) the scFv is in a VH-L-VL orientation; p) VH, L, and VL, wherein: (i) the VH comprises Cys at H40; (ii) the VL comprises Cys at L5; (iii) L comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, or 7; (iv) the scFv is in a VH-L-VL orientation; q) comprises a VH, L, and VL, wherein: (i) the VH comprises Cys at H40; (ii) the VL comprises Cys at L3; (iii) L comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, or 7; (iv) the scFv is in a VH-L-VL orientation; r) VH, L, and VL, wherein: (i) the VH comprises Cys at H46; (ii) the VL comprises Cys at L100; (iii) L comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, or 7; (iv) the scFv is in a VH-L-VL orientation; s) VH, L, and VL, wherein: (i) the VH comprises Cys at H46; (ii) the VL comprises Cys at L102; (iii) L comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, or 7; (iv) the scFv is in a VH-L-VL orientation; t) VH, L, and VL, wherein: (i) the VH comprises Cys at H46; (ii) the VL comprises Cys at L5; (iii) L comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, or 7; (iv) the scFv is in a VH-L-VL orientation; or u) comprising a VH, L, and VL, wherein: (i) the VH comprises Cys at H46; (ii) the VL comprises Cys at L3; (iii) L comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, or 7; (iv) the scFv is in a VH-L-VL orientation; scFv.

7. a) said L is (i) SEQ ID NO: 3; (ii) SEQ ID NO: 6, or (iii) SEQ ID NO: 7 and / or comprising the amino acid sequence b) the scFv is conjugated to a second molecule; An scFv according to any one of claims 1 to 6.

8. (i) the second molecule is a half-life extending moiety; or (ii) the second molecule is a cytotoxic agent or a detectable label; or (iii) the second molecule is a chimeric antigen receptor (CAR). The scFv of claim 7.

9. the half-life extending moiety is an immunoglobulin (Ig), a fragment of the Ig, an Ig constant region, a fragment of the Ig constant region, an Fc region, transferrin, albumin, an albumin binding domain, or polyethylene glycol; The scFv of claim 8.

10. The scFv of claim 8 , wherein the second molecule is an antibody or a fragment thereof.

11. The scFv of claim 10, wherein the scFv and the antibody or fragment thereof bind to different antigens.

12. A pharmaceutical composition comprising the scFv of any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

13. A polynucleotide encoding the scFv of any one of claims 1 to 11.

14. A vector comprising the polynucleotide of claim 13.

15. A host cell comprising the vector of claim 14.

16. A method for producing the scFv of any one of claims 1 to 11, comprising culturing the host cell of claim 15 under conditions in which the scFv is produced, and purifying the scFv.

17. 17. The method of claim 16, wherein the host cell is a prokaryotic or eukaryotic cell.

18. A kit comprising the scFv of any one of claims 1 to 11.

19. A multispecific molecule comprising an scFv according to any one of claims 1 to 11.

20. a) the multispecific molecule comprises an antibody or an antibody fragment; b) the multispecific molecule is a multispecific protein, wherein the multispecific protein comprises an Ig constant region or a fragment of an Ig constant region; c) the scFv is (i) conjugated to the N-terminus of the Ig constant region or the N-terminus of the fragment of the Ig constant region; or (ii) conjugated to the C-terminus of the Ig constant region or the N-terminus of the fragment of the Ig constant region; d) the Ig constant region or the fragment of the Ig constant region is an IgG1, IgG2, and IgG3 or IgG4 isotype; e) the Ig constant region or the fragment of an Ig constant region comprises at least one mutation that reduces binding of the multispecific molecule to an FcγR; f) the Ig constant region or the fragment of an Ig constant region comprises at least one mutation that enhances binding of the multispecific molecule to an FcγR; g) the Ig constant region or the fragment of the Ig constant region comprises at least one mutation that modulates the half-life of the multispecific molecule, or h) the Ig constant region or fragment of the Ig constant region comprises at least one mutation in the CH3 domain; 20. The multispecific molecule of claim 19.

21. (i) the fragment of the Ig constant region comprises an Fc region; (ii) the fragment of the Ig constant region comprises a CH2 domain; or (iii) the fragment of the Ig constant region comprises a CH3 domain; or (iv) the fragment of the Ig constant region comprises the CH2 domain and the CH3 domain; or (v) the fragment of the Ig constant region comprises at least a portion of a hinge, the CH2 domain, and the CH3 domain; or (vi) the fragment of the Ig constant region comprises the hinge, the CH2 domain, and the CH3 domain.

21. The multispecific molecule of claim 20.

22. 21. The multispecific molecule of claim 20, wherein the FcγR is FcγRI, FcγRIIA, FcγRIIB, or FcγRIII, or any combination thereof.

23. the at least one mutation that reduces binding of the multispecific molecule to an FcγR is selected from the group consisting of F234A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G236-deletion / A327G / P331A / D365E / L358M, 21. The multispecific molecule of claim 20, wherein the residues are selected from the group consisting of H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S and S228P / F234A / L235A / G236-deletion / G237A / P238S, and wherein residue numbering is according to the EU index.

24. 21. The multispecific molecule of claim 20, wherein the at least one mutation that enhances binding of the multispecific molecule to an FcγR is selected from the group consisting of S239D / I332E, S298A / E333A / K334A, F243L / R292P / Y300L, F243L / R292P / Y300L / P396L, F243L / R292P / Y300L / V305I / P396L and G236A / S239D / I332E, wherein residue numbering is according to the EU index.

25. 21. The multispecific molecule of claim 20, wherein the at least one mutation that modulates the half-life of the multispecific molecule is selected from the group consisting of H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A and H435R, wherein residue numbering is according to the EU index.

26. The at least one mutation in the CH3 domain is selected from the group consisting of T350V, L351Y, F405A, Y407V, T366Y, T366W, F405W, T394W, T394S, Y407T, Y407A, T366S / L368A / Y407V, L351Y / F405A / Y407V, T366I / K392M / T394W, F405A / Y4 21. The multispecific molecule of claim 20, wherein the residue numbering is according to the EU index.

27. the multispecific molecule comprising: a) It is bispecific; b) is trispecific, or c) is tetraspecific; A multispecific molecule according to any one of claims 19 to 26.

28. 28. A pharmaceutical composition comprising the multispecific molecule of any one of claims 19 to 27 and a pharmaceutically acceptable carrier.

29. A heterologous molecule comprising an scFv according to any one of claims 1 to 11.

30. 30. The heterologous molecule of claim 29, wherein the scFv is conjugated to a second protein, polynucleotide, therapeutic agent, cytotoxic agent, or detectable label.

31. (i) the second protein is an antibody or a fragment thereof; or (ii) the second protein is a chimeric antigen receptor (CAR) or a fragment thereof; 31. The heterologous molecule of claim 30.

32. the heterologous molecule is (i) is monospecific, or (ii) is multispecific; A heterologous molecule according to any one of claims 29 to 31.

33. the heterologous molecule is A) It is bispecific, B) trispecific, or C) is tetraspecific; A heterologous molecule according to any one of claims 29 to 31.

34. A pharmaceutical composition comprising the heterologous molecule of any one of claims 29 to 33 and a pharmaceutically acceptable carrier.

35. 10. A process for preparing the stabilized scFv of claim 1, comprising: a) (i) providing a heavy chain variable region (VH) and a light chain variable region (VL) that form an antigen-binding domain; (ii) providing a linker (L) that includes or is engineered to include a first L Cys; (iii) engineering said VH to contain a VH Cys at a structurally conserved, surface-exposed VH framework residue position; (iv) forming a disulfide bond between the VH Cys and the first L Cys to prepare the stabilized scFv; b) (i) providing a VH and a VL that form an antigen-binding domain; (ii) providing an L that includes or is engineered to include a second L Cys; (iii) engineering the VL to include a VL Cys at a structurally conserved, surface-exposed VL framework residue position; (iv) forming a disulfide bond between the VL Cys and the second L Cys to prepare the stabilized scFv; or c) (i) providing a VH and a VL that form an antigen-binding domain; (ii) providing L comprising or engineered to comprise a first L Cys and a second L Cys; (iii) engineering said VH to contain a VH Cys at a structurally conserved, surface-exposed VH framework residue position; (iv) engineering the VL to include a VL Cys at a structurally conserved, surface-exposed VL framework residue position; (v) forming a disulfide bond between the VH Cys and the first L Cys, and forming a disulfide bond between the VL Cys and the second L Cys to prepare the stabilized scFv; process.

36. A) The stabilized scFv according to any one of a) to c) of claim 35 is an scFv according to any one of claims 1 to 11, and / or B) The stabilized scFv of any one of claims 35 a) to c) binds to an antigen with comparable affinity when compared to a control scFv lacking the disulfide bond.

36. The process of claim 35.

37. 37. A process for preparing the stabilized scFv of claim 36, comprising: a) providing polynucleotides encoding VH, L and VL, i. the VH comprises a Cys at H105 and the VL comprises a Cys at L42; ii. the VH comprises a Cys at H43 and the VL comprises a Cys at L100; iii. The VH comprises Cys in H3 and the VL comprises Cys in L3; iv. the VH comprises a Cys in H3 and the VL comprises a Cys in L5; v. the VH comprises a Cys at H3 and the VL comprises a Cys at L39; vi. The VH comprises a Cys at H3 and the VL comprises a Cys at L42; vii. The VH comprises a Cys at H3 and the VL comprises a Cys at L45; viii. the VH comprises a Cys at H3 and the VL comprises a Cys at L100; ix. the VH comprises a Cys at H3 and the VL comprises a Cys at L102; x. The VH comprises a Cys in H5 and the VL comprises a Cys in L3; xi. The VH comprises Cys at H5 and the VL comprises Cys at L5; xii. The VH comprises a Cys at H5 and the VL comprises a Cys at L39; xiii. The VH comprises Cys at H5 and the VL comprises Cys at L42; xiv. the VH comprises Cys at H5 and the VL comprises Cys at L45; xv. the VH comprises a Cys at H5 and the VL comprises a Cys at L100; xvi. The VH comprises Cys at H5 and the VL comprises Cys at L102; xvii. The VH comprises Cys at H40 and the VL comprises Cys at L3; xviii. The VH comprises Cys at H40 and the VL comprises Cys at L5; xix. The VH comprises Cys at H40 and the VL comprises Cys at L39; xx. The VH comprises Cys at H40 and the VL comprises Cys at L42; xxi. The VH comprises Cys at H40 and the VL comprises Cys at L45; xxii. the VH comprises Cys at H40 and the VL comprises Cys at L100; xxiii. the VH comprises Cys at H40 and the VL comprises Cys at L102; xxiv. the VH comprises Cys at H43 and the VL comprises Cys at L3; xxv. the VH comprises Cys at H43 and the VL comprises Cys at L5; xxvi. The VH comprises a Cys at H43 and the VL comprises a Cys at L39; xxvii. the VH comprises Cys at H43 and the VL comprises Cys at L42; xxviii. The VH comprises Cys at H43 and the VL comprises Cys at L45; xxix. the VH comprises Cys at H43 and the VL comprises Cys at L102; xxx. The VH comprises Cys at H46 and the VL comprises Cys at L3; xxxi. The VH comprises Cys at H46 and the VL comprises Cys at L5; xxxii. the VH comprises Cys at H46 and the VL comprises Cys at L39; xxxiii. The VH comprises Cys at H46 and the VL comprises Cys at L42; xxxiv. the VH comprises Cys at H46 and the VL comprises Cys at L45; xxxv. the VH comprises a Cys at H46 and the VL comprises a Cys at L100; xxxvi. The VH comprises a Cys at H46 and the VL comprises a Cys at L102; xxxvii. The VH comprises Cys at H105 and the VL comprises Cys at L3; xxxviii. The VH comprises Cys at H105 and the VL comprises Cys at L5; xxxix. The VH comprises Cys at H105 and the VL comprises Cys at L39; xl. the VH comprises a Cys at H105 and the VL comprises a Cys at L45; xli. The VH comprises a Cys at H105 and the VL comprises a Cys at L100; or xlii. the VH comprises a Cys at H105 and the VL comprises a Cys at L102; providing that residue numbering is according to Chothia; b) L comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, or 7; c) expressing the polynucleotide in a host cell to produce the stabilized scFv.

38. 38. The process of claim 37, wherein the host cell is a prokaryotic or eukaryotic cell.

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