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 TO RELATED APPLICATIONS) This application is a continuation of U.S. Provisional Patent Application No. 62 / 946,897 filed December 11, 2019, U.S. Provisional Patent Application No. 62 / 946,886 filed December 11, 2019, U.S. Provisional Patent Application No. 62 / 946,882 filed December 11, 2019, U.S. Provisional Patent Application No. 62 / 946,877 filed December 11, 2019, U.S. Provisional Patent Application No. 62 / 946,865 filed December 11, 2019, U.S. Provisional Patent Application No. 62 / 946,866 filed December 11, 2019, U.S. Provisional Patent Application No. 62 / 946,877 filed December 11, 2019, U.S. Provisional Patent Application No. 62 / 946,865 filed August 15, 2019, This application claims the benefit of U.S. Provisional Patent Application No. 62 / 887,529, filed August 15, 2019, U.S. Provisional Patent Application No. 62 / 887,527, filed August 15, 2019, U.S. Provisional Patent Application No. 62 / 887,524, filed August 15, 2019, U.S. Provisional Patent Application No. 62 / 887,519, filed August 15, 2019, and U.S. Provisional Patent Application No. 62 / 887,514, filed August 15, 2019, each of which is incorporated herein by reference in its entirety.

[0002] (Sequence Listing) This application incorporates by reference the Sequence Listing submitted herewith in text format entitled "14620-227-228_SL.txt", created on August 5, 2020, having a size of 258,724 bytes.

[0003] FIELD OF THE INVENTION Materials and methods for improved single-chain variable fragments are disclosed. [Background technology]

[0004] Antigen-binding single-chain variable fragments (scFv) are modules that can be widely used as therapeutic agents, imaging agents, diagnostic agents, or as part of heterologous molecules such as multispecific molecules. One of the challenges of scFv is its low stability and tendency to aggregate (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, there is a need for improved scFv designs that can be optionally incorporated into multispecific and heterologous molecules.

[0006] (overview) In one aspect, the present disclosure provides 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 comprises: 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 conserved surface-exposed VLCys and a second L Cys, or The present invention provides an scFv 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.

[0007] The present disclosure also provides an isolated scFv comprising a VH, L and VL, VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position and L comprises a first L Cy; or VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position and L comprises a second L Cy; or The scFv includes: VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position; VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position; L comprises a first L Cys and a second L Cys; the VH Cys and the first L Cys are capable of forming a disulfide bond; and the VL Cys and the second L Cys are capable of forming a disulfide bond.

[0008] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H105, VL contains Cys at L42, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VL-L-VH orientation.

[0009] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H105, VL contains Cys at L45, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VL-L-VH orientation.

[0010] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H105, VL contains Cys at L39, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VL-L-VH orientation.

[0011] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H5, VL contains Cys at L42, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VL-L-VH orientation.

[0012] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H5, VL contains Cys at L45, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VL-L-VH orientation.

[0013] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H5, VL contains Cys at L39, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VL-L-VH orientation.

[0014] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H3, VL contains Cys at L42, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VL-L-VH orientation.

[0015] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H3, VL contains Cys at L45, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VL-L-VH orientation.

[0016] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H3, VL contains Cys at L39, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VL-L-VH orientation.

[0017] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H43, VL contains Cys at L100, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VH-L-VL orientation.

[0018] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H43, VL contains Cys at L102, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VH-L-VL orientation.

[0019] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H43, VL contains Cys at L5, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VH-L-VL orientation.

[0020] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H43, VL contains Cys at L3, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VH-L-VL orientation.

[0021] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H40, VL contains Cys at L100, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VH-L-VL orientation.

[0022] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H40, VL contains Cys at L102, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VH-L-VL orientation.

[0023] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H40, VL contains Cys at L5, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VH-L-VL orientation.

[0024] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H40, VL contains Cys at L3, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VH-L-VL orientation.

[0025] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H46, VL contains Cys at L100, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VH-L-VL orientation.

[0026] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H46, VL contains Cys at L102, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VH-L-VL orientation.

[0027] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H46, VL contains Cys at L5, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VH-L-VL orientation.

[0028] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H46, VL contains Cys at L3, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VH-L-VL orientation.

[0029] The present disclosure also provides a pharmaceutical composition comprising an spFv of the present disclosure and a pharmaceutically acceptable carrier.

[0030] The present disclosure also provides polynucleotides comprising the spFv of the present disclosure.

[0031] The present disclosure also provides a vector comprising a polynucleotide of the present disclosure.

[0032] The present disclosure also provides a host cell comprising a vector of the present disclosure.

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

[0034] The present disclosure also provides anti-idiotypic antibodies that bind to spFvs of the present disclosure.

[0035] The present disclosure also provides kits comprising the spFvs of the present disclosure.

[0036] In another aspect, the present disclosure provides a multispecific molecule comprising a 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 comprises: 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 conserved, 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] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position, and L comprises a first L Cy; VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position and L comprises a second L Cy; or The present invention provides a multispecific molecule, wherein VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position, VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position, L comprises a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys are capable of forming a disulfide bond, and the VL Cys and the second L Cys are capable of forming a disulfide bond.

[0038] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H105, VL contains Cys at L42, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VL-L-VH orientation to provide multispecific molecules.

[0039] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H105, VL contains Cys at L45, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VL-L-VH orientation.

[0040] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H105, VL contains Cys at L39, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VL-L-VH orientation to provide multispecific molecules.

[0041] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H5, VL contains Cys at L42, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VL-L-VH orientation to provide multispecific molecules.

[0042] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H5, VL contains Cys at L45, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VL-L-VH orientation to provide multispecific molecules.

[0043] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H5, VL contains Cys at L39, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VL-L-VH orientation to provide multispecific molecules.

[0044] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H3, VL contains Cys at L42, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VL-L-VH orientation to provide multispecific molecules.

[0045] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H3, VL contains Cys at L45, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VL-L-VH orientation to provide multispecific molecules.

[0046] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H3, VL contains Cys at L39, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VL-L-VH orientation to provide multispecific molecules.

[0047] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H43, VL contains Cys at L100, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VH-L-VL orientation to provide multispecific molecules.

[0048] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H43, VL contains Cys at L102, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VH-L-VL orientation to provide multispecific molecules.

[0049] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H43, VL contains Cys at L5, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VH-L-VL orientation to provide multispecific molecules.

[0050] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H43, VL contains Cys at L3, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VH-L-VL orientation to provide multispecific molecules.

[0051] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H40, VL contains Cys at L100, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VH-L-VL orientation to provide multispecific molecules.

[0052] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H40, VL contains Cys at L102, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VH-L-VL orientation to provide multispecific molecules.

[0053] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H40, VL contains Cys at L5, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VH-L-VL orientation to provide multispecific molecules.

[0054] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H40, VL contains Cys at L3, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VH-L-VL orientation to provide multispecific molecules.

[0055] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H46, VL contains Cys at L100, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VH-L-VL orientation to provide multispecific molecules.

[0056] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H46, VL contains Cys at L102, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VH-L-VL orientation to provide multispecific molecules.

[0057] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H46, VL contains Cys at L5, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VH-L-VL orientation to provide multispecific molecules.

[0058] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H46, VL contains Cys at L3, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VH-L-VL orientation to provide multispecific molecules.

[0059] The present disclosure also provides pharmaceutical compositions comprising the multispecific molecules provided herein and a pharmaceutically acceptable carrier.

[0060] In yet another aspect, the present disclosure provides a heterologous molecule comprising a 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 comprises: 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 conserved, surface-exposed VL Cys and a second L Cys; or The heterologous molecule includes 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.

[0061] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position and L comprises a first L Cy; or VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position and L comprises a second L Cy; or The heterologous molecule is provided, wherein VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position, VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position, L comprises a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys are capable of forming a disulfide bond, and the VL Cys and the second L Cys are capable of forming a disulfide bond.

[0062] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H105, VL contains Cys at L42, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VL-L-VH orientation.

[0063] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H105, VL contains Cys at L45, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VL-L-VH orientation.

[0064] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H105, VL contains Cys at L39, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VL-L-VH orientation.

[0065] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H5, VL contains Cys at L42, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VL-L-VH orientation.

[0066] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H5, VL contains Cys at L45, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VL-L-VH orientation.

[0067] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H5, VL contains Cys at L39, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VL-L-VH orientation.

[0068] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H3, VL contains Cys at L42, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VL-L-VH orientation.

[0069] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H3, VL contains Cys at L45, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VL-L-VH orientation.

[0070] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H3, VL contains Cys at L39, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VL-L-VH orientation.

[0071] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H43, VL contains Cys at L100, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VH-L-VL orientation.

[0072] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H43, VL contains Cys at L102, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VH-L-VL orientation.

[0073] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H43, VL contains Cys at L5, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VH-L-VL orientation.

[0074] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H43, VL contains Cys at L3, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VH-L-VL orientation.

[0075] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H40, VL contains Cys at L100, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VH-L-VL orientation.

[0076] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H40, VL contains Cys at L102, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VH-L-VL orientation.

[0077] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H40, VL contains Cys at L5, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VH-L-VL orientation.

[0078] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H40, VL contains Cys at L3, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VH-L-VL orientation.

[0079] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H46, VL contains Cys at L100, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VH-L-VL orientation.

[0080] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H46, VL contains Cys at L102, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VH-L-VL orientation.

[0081] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H46, VL contains Cys at L5, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0082] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L, and VL, VH contains Cys at H46, VL contains Cys at L3, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VH-L-VL orientation.

[0083] The present disclosure also provides a pharmaceutical composition comprising a heterologous molecule of the present disclosure and a pharmaceutically acceptable carrier.

[0084] In yet another aspect, the present disclosure provides a process for preparing a stabilized scFv, comprising: providing a heavy chain variable region (VH) and a light chain variable region (VL) that form an antigen-binding domain; providing a linker (L) that includes or is engineered to include a first L Cy; engineering a VH to contain a VH Cys at a structurally conserved, surface-exposed VH framework residue position; forming a disulfide bond between the VH Cys and the first L Cys to prepare a stabilized scFv.

[0085] The present disclosure also provides a process for preparing a stabilized scFv, comprising: providing VH and VL that form an antigen-binding domain; providing an L that includes or is engineered to include a second L Cys; engineering VL to contain a VL Cys at a structurally conserved, surface-exposed VL framework residue position; forming a disulfide bond between the VL Cys and the second L Cys to prepare a stabilized scFv.

[0086] The present disclosure also provides a process for preparing a stabilized scFv, comprising: providing VH and VL that form an antigen-binding domain; providing L comprising or engineered to comprise a first L Cys and a second L Cys; engineering a VH to contain a VH Cys at a structurally conserved, surface-exposed VH framework residue position; engineering VL to contain a VL Cys at a structurally conserved, surface-exposed VL framework residue position; 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 a stabilized scFv.

[0087] The present disclosure also provides a process for preparing a stabilized scFv, comprising: providing polynucleotides encoding VH, L and VL, VH contains Cys at H105 and VL contains Cys at L42, or VH contains Cys at H43 and VL contains Cys at L100, or VH contains Cys in H3 and VL contains Cys in L3, or VH contains Cys in H3 and VL contains Cys in L5, or VH contains Cys at H3 and VL contains Cys at L39, or VH contains Cys at H3 and VL contains Cys at L42, or VH contains Cys at H3 and VL contains Cys at L45, or VH contains Cys at H3 and VL contains Cys at L100, or VH contains Cys at H3 and VL contains Cys at L102, or VH contains Cys at H5 and VL contains Cys at L3, or VH contains Cys at H5 and VL contains Cys at L5, or VH contains Cys at H5 and VL contains Cys at L39, or VH contains Cys at H5 and VL contains Cys at L42, or VH contains Cys at H5 and VL contains Cys at L45, or VH contains Cys at H5 and VL contains Cys at L100, or VH contains Cys at H5 and VL contains Cys at L102, or VH contains Cys at H40 and VL contains Cys at L3, or VH contains Cys at H40 and VL contains Cys at L5, or VH contains Cys at H40 and VL contains Cys at L39, or VH contains Cys at H40 and VL contains Cys at L42, or VH contains Cys at H40 and VL contains Cys at L45, or VH contains Cys at H40 and VL contains Cys at L100, or VH contains Cys at H40 and VL contains Cys at L102, or VH contains Cys at H43 and VL contains Cys at L3, or VH contains Cys at H43 and VL contains Cys at L5, or VH contains Cys at H43 and VL contains Cys at L39, or VH contains Cys at H43 and VL contains Cys at L42, or VH contains Cys at H43 and VL contains Cys at L45, or VH contains Cys at H43 and VL contains Cys at L102, or VH contains Cys at H46 and VL contains Cys at L3, or VH contains Cys at H46 and VL contains Cys at L5, or VH contains Cys at H46 and VL contains Cys at L39, or VH contains Cys at H46 and VL contains Cys at L42, or VH contains Cys at H46 and VL contains Cys at L45, or VH contains Cys at H46 and VL contains Cys at L100, or VH contains Cys at H46 and VL contains Cys at L102, or VH contains Cys at H105 and VL contains Cys at L3, or VH contains Cys at H105 and VL contains Cys at L5, or VH contains Cys at H105 and VL contains Cys at L39, or VH contains Cys at H105 and VL contains Cys at L45, or VH contains a Cys at H105 and VL contains a Cys at L100, or VH contains a Cys at H105 and VL contains a Cys at L102, residue numbering is according to Chothia, providing a polynucleotide wherein L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; and expressing the polynucleotide in a host cell to produce the stabilized scFv.

[0088] In yet another aspect, the disclosure provides an isolated single chain variable fragment (scFv) comprising a heavy chain variable region (VH), a means for linking (L), and a light chain variable region (VL), wherein the scFv comprises: 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 conserved, surface-exposed VL Cys and a second L Cys; or The present invention provides an scFv 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.

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

[0091] The present disclosure also provides a multispecific molecule comprising a single chain variable fragment (scFv) comprising a heavy chain variable region (VH), a joining (L), and a light chain variable region (VL), wherein the scFv comprises: 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 conserved, 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] The present disclosure also provides a multispecific molecule comprising a single chain variable fragment (scFv) comprising a means for antigen binding, a linker (L), and a light chain variable region (VL), wherein the scFv comprises: a first disulfide bond between a structurally conserved surface-exposed antigen-binding means cysteine ​​(Cys) and a first L Cys; a second disulfide bond between a structurally conserved, 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 antigen binding means 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] The present disclosure also provides a multispecific molecule comprising a single chain variable fragment (scFv) comprising a heavy chain variable region (VH), a linker (L) and means for antigen binding (VL), wherein the scFv comprises: the first disulfide bond between the structurally conserved surface-exposed VH cysteine ​​(Cys) and the first L Cys; a second disulfide bond between the structurally conserved surface-exposed antigen-binding means 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 antigen binding means Cys and a second L Cys.

[0094] The present disclosure also provides a heterologous molecule comprising a single chain variable fragment (scFv) comprising a heavy chain variable region (VH), a linking (L), and a light chain variable region (VL), wherein the scFv comprises: 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 conserved, surface-exposed VL Cys and a second L Cys; or The heterologous molecule includes 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.

[0095] The present disclosure also provides a heterologous molecule comprising a single chain variable fragment (scFv) comprising a means for antigen binding, a linker (L), and a light chain variable region (VL), wherein the scFv comprises: a first disulfide bond between a structurally conserved surface-exposed antigen-binding means cysteine ​​(Cys) and a first L Cys; a second disulfide bond between a structurally conserved, surface-exposed VL Cys and a second L Cys; or The heterologous molecule includes a first disulfide bond between a structurally conserved surface-exposed antigen binding means 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] The present disclosure also provides a heterologous molecule comprising a single chain variable fragment (scFv) comprising a heavy chain variable region (VH), a linker (L), and a means for antigen binding, wherein the scFv comprises: the first disulfide bond between the structurally conserved surface-exposed VH cysteine ​​(Cys) and the first L Cys; a second disulfide bond between the structurally conserved surface-exposed antigen-binding means Cys and a second L Cys; or The heterologous molecule comprises 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] The present disclosure also provides means for encoding the scFvs provided herein.

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

[0099] The present disclosure also provides compositions comprising means for stabilizing scFvs.

[0100] The present disclosure also provides compositions comprising means for increasing the thermal stability of scFvs.

[0101] The present disclosure also provides multispecific molecules comprising means for stabilizing scFvs.

[0102] The present disclosure also provides multispecific molecules that include means for increasing the thermal stability of the scFv.

[0103] The present disclosure also provides heterologous molecules that include means for stabilizing scFvs.

[0104] The present disclosure also provides heterologous molecules that include means for increasing the thermal stability of scFvs. [Brief explanation of the drawings]

[0105] [Figure 1] 1 shows an exemplary design of a stabilized scFv (spFv), where the VL and VH are connected by a flexible linker, shown as a dashed line in the figure, that contains the staple sequence CPPC (SEQ ID NO: 1), and "SS" indicates a disulfide bond between the linker and the staple sequence at the anchor point. [Figure 2] Figure 1 shows a graphical representation of anchor point selection for spFv in a VL-linker-VH orientation. A germline human antibody Fv (pMESdb id 5I19, GLk1) was used for drawing and exemplary distance measurements. The distances indicated by dashed lines are between the Cβ atoms of residues in Å. Structurally conserved framework positions with the desired distances were selected as anchor points for mutation to Cys. The anchor points for the 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]Figure 1 shows a graphical representation of anchor point selection for spFv in a VH-linker-VL orientation. A germline human antibody Fv (pdb id 5I19, GLk1) was used for drawing and exemplary distance measurements. Distances indicated by dashed lines are between Cβ atoms of residues in Å. Structurally conserved framework positions with the desired distance were selected as anchor points for mutation to Cys. The anchor points for the 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] 1 shows a graphical representation of the Cβ(Cys1)-Cβ(Cys2) distance between two Cys residues in the mouse two-chain IgG2a (pdb id 1igt) hinge CPPC (SEQ ID NO: 1), where distances are shown in Angstroms. [Figure 5] 1 shows a graphical representation of 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), where distances are shown in Angstroms. [Figure 6] Selected VL anchor points are shown highlighted in gray and numbered as 1 and 2 below the amino acid alignment. VL sequences are numbered according to the Chothia numbering scheme. VL anchor point 1 (Chothia position 42) was used for spFv in a VL-linker-VH orientation, and VL anchor point 2 (Chothia position 100) was used for spFv in a 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 shown highlighted in gray and numbered as 1 and 2 below the amino acid alignment. VH sequences are numbered according to the Chothia numbering scheme. VH anchor point 1 (Chothia position 105) was used for spFv in a VL-linker-VH orientation, and VH anchor point 2 (Chothia position 43) was used for spFv in a 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] Figure 1 shows the structure of CAT2200b spFv VH-VL. Staple formation between the VH and VL anchor points and the linker is evident from the structure. [Figure 12] A comparison of non-binding CAT2200b spFv VH-VL (top) compared to CAT2200a scFv VL-VH bound to IL-17A (bottom) is shown. [Figure 13] A comparison of the front view structures of unbound CAT2200b spFv VH-VL (top) compared to IL-17A bound CAT2200a spFv VL-VH (bottom) is shown. [Figure 14] A comparison of the back view of the structure of unbound CAT2200b spFv VH-VL (top) compared to IL-17A bound CAT2200a scFv VL-VH (bottom) is shown. [Figure 15A]Schematic diagram of antibody fused to stapled scFv. 2:1 heterodimer, isotype control antibody fused to stapled scFv derived from LTBRmAb1. [Figure 15B] Schematic diagram of antibody fused to stapled scFv. 2:1 heterodimer, isotype control antibody fused to stapled scFv derived from LTBRmAb1. [Figure 15C] Schematic diagram of antibody fused to stapled scFv. 2:1 heterodimer, isotype control antibody fused to stapled scFv derived from LTBRmAb1. [Figure 15D] Schematic diagram of antibody fused to stapled scFv. 2:1 heterodimer, isotype control antibody fused to stapled scFv derived from LTBRmAb1. [Figure 15E] 1 shows a schematic diagram of an antibody fused to a stapled scFv. 2:1 heterodimer, EDBmAb1, fused to a stapled scFv derived from LTBRmAb1 is shown. [Figure 15F] 1 shows a schematic diagram of an antibody fused to a stapled scFv. 2:1 heterodimer, EDBmAb1, fused to a stapled scFv derived from LTBRmAb1 is shown. [Figure 15G] 1 shows a schematic diagram of an antibody fused to a stapled scFv. 2:1 heterodimer, EDBmAb1, fused to a stapled scFv derived from LTBRmAb1 is shown. [Figure 15H] 1 shows a schematic diagram of an antibody fused to a stapled scFv. 2:1 heterodimer, EDBmAb1, fused to a stapled scFv derived from LTBRmAb1 is shown. [Figure 15I] 1 shows a schematic diagram of an antibody fused to a stapled scFv. 2:1 heterodimer, EDBmAb1, fused to a stapled scFv derived from a lower affinity variant of LTBRmAb1 is shown. [Figure 15J]1 shows a schematic diagram of an antibody fused to a stapled scFv. 2:1 heterodimer, EDBmAb1, fused to a stapled scFv derived from a lower affinity variant of LTBRmAb1 is shown. [Figure 15K] Schematic diagram of antibodies fused to stapled scFvs. Shown are 2:1 heterodimers, EDBmAb1 or B21M fused to stapled scFvs derived from LTBRmAb1, which does not contain Protein A mutations in the Fc region. [Figure 15L] Schematic diagram of antibodies fused to stapled scFvs. Shown are 2:1 heterodimers, EDBmAb1 or B21M fused to stapled scFvs derived from LTBRmAb1, which does not contain Protein A mutations in the Fc region. [Figure 15M] 1 shows a schematic diagram of an antibody fused to a stapled scFv. 2:1 heterodimer, MSLNmAb1, fused to a stapled scFv derived from LTBRmAb1 is shown. [Figure 16A] 1 shows a graph illustrating the results of an A549 NF-κB reporter assay using a 2:1 bispecific antibody: TAA-dependent LTBR activation by COVA1456 compared with COVA1482, their respective control molecules COVA1462 and COVA1486, and recombinant human LIGHT. [Figure 16B] 1 shows a graph illustrating the results of an A549 NF-κB reporter assay using a 2:1 bispecific antibody. Comparison of TAA-dependent LTBR activation by COVA1482, and bispecific antibodies COVA14107 and COVA14108 containing lower affinity variants of LTBRmAb1, and COVA1486. [Figure 16C] Graphs illustrating the results of an A549 NF-κB reporter assay using a 2:1 bispecific antibody. Comparison of TAA-dependent LTBR activation by COVA1482 and COVA14133 (constructs without protein A mutations) and their respective control molecules COVA1486 and COVA14136. [Figure 17] Figure 1 shows the results of hypocytometric staining of ICAM-1 on A375 cells after co-culture experiments, comparing COVA1482 and its control molecule COVA1486 with recombinant human LIGHT. [Figure 18A] Graphs illustrating the measurement of cytokines in the supernatants of co-cultures treated with the anti-EDB / anti-LTBR bispecific antibody COVA14133 compared to COVA14136 and COVA1440. Assays are performed using the MSD platform. Concentration of human RANTES. [Figure 18B] Graphs illustrating the measurement of cytokines in the supernatants of co-cultures treated with the anti-EDB / anti-LTBR bispecific antibody COVA14133 compared to COVA14136 and COVA1440. Assays are performed using the MSD platform. Human IL-6 concentration. [Figure 18C] Figure 1 shows a graph illustrating the measurement of cytokines in the supernatants of co-cultures treated with the anti-EDB / anti-LTBR bispecific antibody COVA14133 compared to COVA14136 and COVA1440. Assays are performed using the MSD platform. Concentration of human IL-8. [Figure 18D] Figure 1 shows a graph illustrating the measurement of cytokines in the supernatants 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. The concentration of human MIP-3b. [Figure 19A] Figure 1 shows LTBR activation by MSLN / LTBR bispecifics in A549 NF-κB reporter / CHOK1MSLN or A549 NF-κB reporter / H226 co-culture cell assays. Activation of LTBR in A549 NF-κB reporter / H226 co-culture assays. COVA14146 (2:1 MSLNmAb1 x LTBRmAb1) is compared to LIGHT and the isotype control 2:1 construct COVA1486. [Figure 19B]Figure 1 shows LTBR activation by MSLN / LTBR bispecifics in A549 NF-κB reporter / CHOK1MSLN or A549 NF-κB reporter / H226 co-culture cell assays. Concentrations of RANTES secreted upon LTBR activation in A549 NF-κB reporter / H226 co-culture assays. COVA14146 (2:1 MSLNmAb1 x LTBRmAb1) is compared to LIGHT and the isotype control 2:1 construct COVA1486.

[0106] (Detailed description) The disclosed methods may be understood more readily by reference to the following detailed description taken in conjunction with the accompanying drawings, which form a part of this disclosure: It is to be understood that the disclosed methods are not limited to the specific methods described and / or illustrated herein, and further, the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting.

[0107] All patents, published patent applications and publications cited herein are incorporated by reference as if set forth in their entireties.

[0108] Where lists are presented, it is to be understood that each individual element of that list and every combination of that list is a separate embodiment, unless otherwise specified. For example, a list of embodiments presented as "A, B, or C" should be interpreted to include the embodiments "A," "B," "C," "A or B," "A or C," "B or C," or "A, B, or C."

[0109] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

[0110] The transitional phrases "comprising," "consisting essentially of," and "consisting" are intended to connote their generally accepted meanings in patent terminology, i.e., (i) "comprising" is synonymous with "comprising," "containing," or "characterized by" and is inclusive or open-ended and does not exclude other unrecited elements or method steps; (ii) "consisting of" excludes any element, step, or ingredient not specified in the claim; and (iii) "consisting essentially of" limits the scope of the claim to the specified materials or steps and those that do not materially affect the "basic and novel characteristics" of the claimed invention. Embodiments described with the phrase "comprising" (or its equivalents) also provide those described independently with the phrases "consisting of" and "consisting essentially of," as embodiments.

[0111] "About" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on the limitations of the method, i.e., measurement system, by which the value is measured or determined. Unless expressly stated otherwise in the examples or elsewhere in the specification in the context of a particular assay, result, or embodiment, "about" means within one standard deviation or a range of up to 5%, whichever is greater, according to practice in the art.

[0112] "Alternative scaffold" refers to a single-chain protein framework containing a structured core associated with conformationally tolerant variable domains that can be engineered and selected to bind specific antigens, allowing polymorphism to be introduced without compromising the integrity of the scaffold.

[0113] "Antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" is a mechanism of cell death induction that relies on the interaction of antibody-coated target cells with lytic effector cells, such as natural killer (NK) cells, monocytes, macrophages, and neutrophils, via Fc gamma receptors (FcγR) expressed on the effector cells.

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

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

[0116] "Antigen-binding fragment" or "antigen-binding domain" refers to a portion of a protein that binds to an antigen. Antigen-binding fragments may be synthetic, enzymatically obtainable, or recombinant polypeptides, and include VH, VL, VH and VL, Fab, F(ab')2, Fd, and Fv fragments, domain antibodies (dAbs) consisting of one VH domain or one VL domain, camelized VH domains, VHH domains, minimal recognition units consisting of amino acid residues mimicking the CDRs of an antibody, such as FR3-CDR3-FR4 portions, portions of immunoglobulins bound to HCDR1, HCDR2, and / or HCDR3, and LCDR1, LCDR2, and / or LCDR3, alternative scaffolds bound to antigens, and multispecific proteins containing antigen-bound fragments. Antigen-binding fragments (such as VH and VL) can be linked together via synthetic linkers to form various types of single chain antibody designs, where the VH / VL domains pair intramolecularly, or intermolecularly when the VH and VL domains are expressed as separate single chains, to form monovalent antigen-binding domains such as single chain Fvs (scFvs) or antigens, or bispecific antibodies. Antigen-binding fragments can also be linked to other antibodies, proteins, antigen-binding fragments, or alternative scaffolds, which can be monospecific or multispecific, to engineer bispecific and multispecific proteins. Conjugates This may be done.

[0117] The term "antibody" is intended in a broad sense and includes immunoglobulin molecules, including monoclonal antibodies, including murine, human, humanized, and chimeric monoclonal antibodies; antigen-binding fragments; multispecific antibodies, such as bispecific, trispecific, and tetraspecific antibodies; dimeric, tetrameric, or multimeric antibodies; single-chain antibodies; antibody domains; and any other modified configuration of an immunoglobulin molecule containing an antigen-binding site of the required specificity. A "full-length antibody" consists of two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (consisting of domains CH1, hinge, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions are further divided into regions of hypervariability called complementarity determining regions (CDRs), which are interspersed with framework regions (FRs). Each VH and VL is composed of three CDR and four FR segments, arranged from amino- to carboxy-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. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0118] "Bispecific" 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 be cross-reactive with other related antigens, e.g., the same antigen in other species (homologues), such as humans or monkeys, e.g., Macaca cynomolgus (cynomolgus monkey, cyno) or Pan troglodytes, or may bind to an epitope shared between two or more different antigens.

[0119] "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) that transfers ligand or antigen specificity onto a T cell. CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors. CARs comprise an extracellular domain capable of binding to an antigen, a transmembrane domain, and at least one intracellular domain. The CAR intracellular domain comprises a polypeptide known to function as a domain that transmits a signal that causes activation or inhibition of a biological process within the cell. The transmembrane domain comprises any peptide or polypeptide known to span the cell membrane and that can function to connect the extracellular domain and the signaling domain. Chimeric antigen receptors may optionally comprise a hinge domain that functions as a linker between the extracellular domain and the transmembrane domain.

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

[0121] The "complementarity-determining region" (CDR) is the region of an antibody that binds to an antigen. VH has three CDRs (HCDR1, HCDR2, and HCDR3), and VL has three CDRs (LCDR1, LCDR2, and LCDR3). CDRs can be defined using various descriptions, such as 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)). The correspondence between various descriptions and the numbering of variable regions has been described (see, e.g., Lefranc et al. (2003) Dev Comp Immunol 27:55-77; Honegger and Pluckthun, J Mol Biol (2001) 309:657-670; the International ImMunoGeneTics (IMGT) database; web resource, http: / / www_imgt_org). CDRs can be delineated 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 defined by any of the Kabat, Chothia, IMGT, or AbM methods described above, unless otherwise expressly stated in the specification.

[0122] "Decrease," "reduction," or "reduction" generally refers to the ability of a test molecule to mediate a diminished response (i.e., a downstream effect) 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 FcγRs or enhanced Fc effector function such as enhanced ADCC, CDC, and / or ADCP. A decrease can be a statistically significant difference in the measured response between the test molecule and the control (or vehicle), or about a 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30-fold or greater decrease, e.g., a 500-, 600-, 700-, 800-, 900-, or 1000-fold or greater decrease.

[0123] "Enhancement," "promotion," or "increase" generally refers to the ability of a test molecule to mediate a greater response (i.e., a downstream effect) 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. 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., a 500, 600, 700, 800, 900, or 1000 fold or more.

[0124] An "expression vector" refers to a vector that can be utilized in a biological system or 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 "heterologous polynucleotide" refers to a polynucleotide that comprises two or more polynucleotides that are not found in the same relationship to each other in nature.

[0127] A "heterologous peptide" refers to a polypeptide that comprises 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 an immune response when administered to a human subject. The variable regions of a human antibody are derived from human immunoglobulin sequences. If a human antibody contains a constant region or a portion of a constant region, the constant region is also derived from a human immunoglobulin sequence. A human antibody contains heavy and light chain variable regions "derived" from sequences of human origin when the variable regions of the human antibody are obtained from a system using human germline immunoglobulins or rearranged immunoglobulin genes. Exemplary such systems include phage-displayed human immunoglobulin gene libraries and transgenic non-human animals, such as mice or rats, carrying human immunoglobulin loci. A "human antibody" typically contains amino acid differences compared to immunoglobulins expressed in humans due to differences in the systems used to obtain human antibodies and human immunoglobulin loci, the introduction of somatic mutations or intentional substitutions into frameworks or CDRs, or both. Typically, a "human antibody" is at least about 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. Optionally, a "human antibody" may contain a consensus framework sequence obtained from human framework sequence analysis, e.g., as described in Knappik et al., (2000) J Mol Biol 296:57-86, or a synthetic HCDR3 incorporated into a phage-displayed human immunoglobulin gene library, e.g., as described in Shi et al., (2010) J Mol Biol 397:385-396 and WO 2009 / 085462. Antibodies in which at least one CDR is derived from a non-human species are not included in the definition of "human antibody."

[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. Humanized antibodies can contain substitutions in the framework, so that the framework may not be an exact copy of an expressed human immunoglobulin or human immunoglobulin germline gene sequence.

[0130] "Isolated" refers to a homogenous population of molecules (scFvs of the disclosure or heterologous proteins comprising scFvs of the disclosure) that have been substantially separated and / or purified from other components of the system in which they are produced, such as recombinant cells, and to proteins that have been subjected to at least one purification or isolation step. "Isolated" refers to molecules that are substantially free of other cellular material and / or chemicals and includes molecules isolated to greater degrees of 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] "Modulation" refers to either an enhancement or a decrease in the ability of a test molecule to mediate a control-enhanced or reduced response (i.e., a downstream effect) when compared to a control or vehicle-mediated response.

[0132] A "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibody molecules, i.e., the individual antibodies comprising the population are identical except for possible, well-known alterations, such as removal of the C-terminal lysine from the antibody heavy chain, or post-translational modifications such as 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 multivalent.

[0133] "Multispecific" 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 be cross-reactive to other related antigens, e.g., the same antigen (homologues) from other species such as humans or monkeys, e.g., cynomolgus monkeys (cyno) or chimpanzees, or may bind to epitopes shared between two or more different antigens.

[0134] "Polynucleotide" refers to a molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. cDNA is a typical example of a polynucleotide.

[0135] As used interchangeably herein, "protein" or "polypeptide" refers to a molecule comprising one or more polypeptides, each consisting of at least two amino acid residues linked by a peptide bond. A protein may be a monomer or a protein complex of two or more subunits, which may be identical or different. Small polypeptides consisting of fewer than 50 amino acids may be referred to as "peptides." A protein may be a heterologous fusion protein, a glycoprotein, or a protein modified by post-translational modifications such as phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, citrullination, polyglutamylation, ADP-ribosylation, pegylation, or biotinylation.

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

[0137] A "single-chain Fv" or "scFv" refers to a single-chain protein comprising a VH, a VL, and a linker between the VH and VL. An scFv can have the VL and VH variable regions in either orientation, e.g., with respect to the N-terminal to C-terminal order of the VH and VL. Thus, an scFv can be in a VL-linker-VH or VH-linker-VL orientation. An scFv can also be engineered to contain disulfide bonds between the VH, VL, and linker.

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

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

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

[0141] A "therapeutically effective amount" refers to an amount effective to obtain a desired therapeutic result, at dosages and for periods of time necessary. A therapeutically effective amount may vary depending on factors such as the individual's condition, age, sex, and weight, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual.

[0142] "Treating," "treating," or "treatment" of a disease or disorder refers to achieving one or more of the following: reducing the severity and / or duration of the disorder, inhibiting the worsening of symptoms characteristic of the disorder being treated, limiting or preventing the recurrence of the disorder in a subject who previously had the disorder, or limiting or preventing the recurrence of symptoms in a subject who was previously symptomatic for the disorder.

[0143] "Trispecific" refers to a molecule (such as an antibody) that specifically binds to three different antigens, or three different epitopes, within the same antigen. Trispecific molecules may have cross-reactivity to other related antigens, e.g., the same antigen in other species (homologues), such as humans or monkeys, e.g., Macaca cynomolgus (cynomolgus monkey, cyno) or Pan troglodytes, or may bind to an epitope shared among three or more different antigens.

[0144] A "variant," "mutant," or "alteration" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide by one or more modifications, e.g., one or more substitutions, insertions, or deletions.

[0145] Throughout this specification, the numbering of amino acid residues in antibody constant regions is according to the EU index as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), unless otherwise expressly stated herein.

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

[0147] Numbering of variable regions is according to Chothia unless explicitly stated otherwise.

[0148] "VH cysteine" or "VH Cys" refers to a Cys residue present in the VH framework.

[0149] "VL cysteine" or "VL Cys" refers to a Cys residue present in the VL framework.

[0150] "Stabilized" refers to scFv that retains equivalent binding to hK2 when compared to an unheated scFv sample, which refers to it being heat stable.

[0151] "Improved stability" refers to an spFv of the disclosure having an increased melting temperature (Tm) when compared to a parent scFv lacking the disulfide bond and Cys residues introduced into the SpFv. The increased Tm can be an increase of 2°C or more, e.g., 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] "Anchor point" refers to an scFv Vh or VL framework Cys residue that can be mutated to a Cys without adversely affecting the overall scFv structure and that can form a disulfide bond with a Cys present in the scFv linker.

[0153] "Staple" refers to an scFv linker containing one or two Cys residues capable of forming a disulfide bond with the anchor point Cys.

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

[0155] "LTBR" refers to a polypeptide that is a cell surface receptor for lymphotoxins, which are members of the tumor necrosis factor receptor superfamily and are involved in apoptosis and cytokine release. 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 lineage 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 and can lead to the release of interleukin-8. Unless otherwise specified, the LTBR is preferably human LTBR. The human LTBR amino acid sequence is provided by UniProt number P36941.

[0156] "EDB" or "extra domain B" refers to a domain of fibronectin that can be contained in a fibronectin molecule based on the splicing pattern of fibronectin pre-mRNA. Extra domain B is a complete fibronectin (FN) type III repeat containing 91 amino acid residues. Generally, EDB is undetectable in normal adult tissues, but shows greater expression in fetal and tumor tissues in the extracellular matrix and accumulates around neovasculature during the angiogenesis process, making EDB a potential marker and target for angiogenesis. Unless otherwise specified, EDB is preferably human EDB. Human EDB containing fibronectin isoform amino acid sequences is provided by UniProt number P02751.

[0157] "Fibronectin" refers to a polypeptide that is 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 pre-mRNA 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, including extra domain B, is provided by UniProt number P02751, 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 The present disclosure provides stabilized scFv molecules (referred to herein as spFvs (stapled Fvs)), heterologous and multispecific molecules comprising spFvs, polynucleotides encoding them, vectors, host cells, and methods of making and using them. The present disclosure is based, at least in part, on the identification of VH and / or VL (referred to herein as VH anchor points or VL anchor points) and flexible linkers (referred to herein as staples), which can be engineered into cysteine ​​residues that result in sulfide bond formation between the linker and variable domains in scFvs. The "stapling" strategy described herein is broadly applicable to all VH / VL domains and existing scFv molecules that provide structural identity with scFvs with improved stability. The spFvs described herein can be incorporated into any heterologous protein, bispecific, or multispecific format, including chimeric antigen receptors (CARs), T cell redirecting molecules, and bi- and multispecific molecules. Conjugates These can be used as therapeutic, diagnostic and detection molecules.

[0159] spFv of the present disclosure The present disclosure provides 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 comprises: 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 conserved, surface-exposed VL Cys and a second L Cys; or The present invention provides an scFv 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.

[0160] The present disclosure also provides an isolated scFv comprising a VH, L and VL, VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position, and L comprises a first L Cy; VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position and L comprises a second L Cy; or The present disclosure provides an scFv in which VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position, VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position, and L comprises a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys are capable of forming a disulfide bond, and the VL Cys and the second L Cys are capable of forming a disulfide bond. The disulfide bonds are typically formed during expression of the scFvs of the present disclosure.

[0161] Although specific examples disclose spFvs with two disulfide bonds, it is readily envisioned that spFvs with one disulfide bond formed between the linker Cys and either the VH Cys or the VL Cys can be constructed and utilized to generate "half-anchored" molecules. The anchor position is the same for spFvs with one or two disulfide bonds. The linker Cys position can vary in half-anchored molecules as long as it satisfies the distance and geometric requirements for disulfide bond formation by the anchor point. Half-anchored spFvs are expected to inhibit VL / VH relative movement similar to that of a VL / VH pair stabilized by two disulfide bonds, and are therefore stabilized.

[0162] The spFvs of the present disclosure exhibited increased thermal stability when compared to parent scFvs lacking disulfide bonds. Generally, the Tm of the spFvs was approximately 10°C higher when compared to 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 using differential thermal calorimetry (DSC), in which DSC scans are performed using a heated protein sample (such as a sample heated to 60°C), followed by a thermal melting profile obtained using a two-state or non-two-state transition. In the case of a non-two-state transition, two transitions (Tm1 and Tm2) corresponding to the melting Tms of the VL and VH domains, respectively, are recorded.

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

[0164] In some embodiments, the VH Cys is at H3, H5, H40, H43, H46, or H105, where residue numbering is according to Chothia.

[0165] In some embodiments, the VH Cys is in H3.

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

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

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

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

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

[0171] In some embodiments, the VL Cys is at L3, L5, L39, L42, L45, L100, or L102, where residue numbering is according to Chothia.

[0172] In some embodiments, the VL Cys is in L3.

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

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

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

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

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

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

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

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

[0181] In some embodiments, the VH Cys is in H3 and the VL Cys is in L3.

[0182] In some embodiments, the VH Cys is in H3 and the VL Cys is in L5.

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

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

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

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

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

[0188] In some embodiments, the VH Cys is in H5 and the VL Cys is in L3.

[0189] In some embodiments, the VH Cys is in H5 and the VL Cys is in L5.

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

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

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

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

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

[0195] In some embodiments, the VH Cys is in H40 and the VL Cys is in L3.

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

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

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

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

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

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

[0202] In some embodiments, the VH Cys is in H43 and the VL Cys is in L3.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0221] Residue numbering for the VH and VL regions is according to Chothia.

[0222] Chothia numbering is well known. Other numbering systems, such as Kabat or IMGT numbering, or consecutive numbering, can be used to number the VH and VL residue positions. Table 1 shows the correspondence between Chothia, Kabat, and consecutive numbering for an exemplary VH, GLk1 VH (SEQ ID NO: 60). Table 2 shows the correspondence between Chothia, Kabat, and consecutive numbering for an exemplary VL, GLk1 VL (SEQ ID NO: 56).

[0223] [Table 1-1]

[0224] [Table 1-2]

[0225] [Table 2]

[0226] In some embodiments, L comprises a contiguous amino acid sequence 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 from mouse, rat, dog, chicken, and non-human primates such as monkeys.

[0228] In some embodiments, the Ig hinge region is derived from a 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 including residue 216 and ending at residue 230 for human IgG, with residue numbering according to the EU index. In some cases, the lower hinge region from about residue 231 to about residue 237 may also be included in the hinge. Thus, an IgG1 hinge region may include 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 for other Ig isotypes are well known, and their amino acid sequences can be obtained, for example, from the ImMunoGeneTics website. For example, an IgG2 hinge includes the amino acid sequence ERKCCVECPPCP (SEQ ID NO: 65).

[0231] When L comprises at least a portion of an Ig hinge region amino acid sequence or at least a portion of an engineered Ig hinge region, it comprises a contiguous amino acid sequence "derived from" an Ig hinge region in those examples. An engineered Ig hinge region comprises one or more mutations compared to a wild-type Ig hinge. Exemplary mutations that can be introduced are: and can be any conservative modification, such as substitution of a Cys residue to reduce the number of Cys in L to one or two, substitution of a Pro residue, or a conservative substitution.

[0232] "Conservative modifications" refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid modification. Conservative modifications include amino acid substitutions, additions, and deletions. A conservative amino acid substitution is one in which an amino acid is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are well-defined and include amino acids with 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), uncharged polar 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). Additionally, any naturally occurring residue in the polypeptide can be substituted with alanine, as previously described for alanine scanning mutagenesis (MacLennan et al., (1988) Acta Physiol Scand Suppl 643:55-67; ​​Sasaki et al., (1988) Adv Biophys 35:1-24). Amino acid substitutions can be made by known methods, such as PCR mutagenesis (U.S. Pat. No. 4,683,195). The resulting mutant hinges can be incorporated into spFv constructs of the present disclosure and tested for their properties, such as stability and binding to antigen, using known assays and those described herein.

[0233] In some embodiments, L is the amino acid sequence C(X) yC (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 can be included in the linker to provide rigidity. Gly can be included in the linker to allow maximum flexibility. Any other amino acid, except Cys and Met, can also be used in L.

[0234] In some embodiments, L is the 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.

[0235] In some embodiments, L comprises the amino acid sequence 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 comprises the amino acid sequence CPC.

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

[0238] In some embodiments, L comprises 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 comprises 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 comprises 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 comprises about 14-19 amino acids.

[0266] In some embodiments, L comprises about 14 amino acids.

[0267] In some embodiments, L comprises about 15 amino acids.

[0268] In some embodiments, L comprises about 16 amino acids.

[0269] In some embodiments, L comprises about 17 amino acids.

[0270] In some embodiments, L comprises about 18 amino acids.

[0271] In some embodiments, L comprises about 19 amino acids.

[0272] In some embodiments, L is the 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 of 6 to 9, y is an integer of 1 to 3, and n is an integer of 4 to 6.

[0273] In some embodiments, L is the 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, 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 (SEQ ID NO: 27), wherein X is Gly or Pro, m is an integer of 6 to 9, y is an integer of 1 to 3, and n is an integer of 4 to 6.

[0275] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 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 disclosure is in a VL-L-VH orientation.

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

[0284] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H105, VL contains Cys at L42, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VL-L-VH orientation.

[0285] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H105, VL contains Cys at L45, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VL-L-VH orientation.

[0286] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H105, VL contains Cys at L39, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VL-L-VH orientation.

[0287] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H5, VL contains Cys at L42, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VL-L-VH orientation.

[0288] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H5, VL contains Cys at L45, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VL-L-VH orientation.

[0289] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H5, VL contains Cys at L39, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VL-L-VH orientation.

[0290] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H3, VL contains Cys at L42, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VL-L-VH orientation.

[0291] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H3, VL contains Cys at L45, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VL-L-VH orientation.

[0292] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H3, VL contains Cys at L39, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VL-L-VH orientation.

[0293] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H43, VL contains Cys at L100, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VH-L-VL orientation.

[0294] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H43, VL contains Cys at L102, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VH-L-VL orientation.

[0295] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H43, VL contains Cys at L5, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VH-L-VL orientation.

[0296] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H43, VL contains Cys at L3, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VH-L-VL orientation.

[0297] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H40, VL contains Cys at L100, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VH-L-VL orientation.

[0298] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H40, VL contains Cys at L102, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VH-L-VL orientation.

[0299] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H40, VL contains Cys at L5, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VH-L-VL orientation.

[0300] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H40, VL contains Cys at L3, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VH-L-VL orientation.

[0301] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H46, VL contains Cys at L100, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VH-L-VL orientation.

[0302] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H46, VL contains Cys at L102, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VH-L-VL orientation.

[0303] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H46, VL contains Cys at L5, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the scFv is in a VH-L-VL orientation.

[0304] The present disclosure also provides an scFv comprising a VH, L and VL, VH contains Cys at H46, VL contains Cys at L3, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; An scFv is provided, wherein the 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] Heterologous molecules comprising spFv of the present disclosure The spFvs of the present disclosure, like unstabilized scFvs lacking disulfide bonds, can be bound to a second molecule, as is well known in the art. Conjugates Exemplary second molecules are disclosed herein and include half-life extending moieties, imaging agents, therapeutic agents, various antibody formats and fragments thereof, antigen binding domains, Fc regions, immunoglobulin heavy / light chains or fragments thereof, multispecific molecules, and chimeric antigen receptors (CARs).

[0311] The present disclosure also provides a heterologous molecule comprising a 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 comprises: 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 conserved, surface-exposed VL Cys and a second L Cys; or The heterologous molecule includes 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.

[0312] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position, and L comprises a first L Cy; VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position and L comprises a second L Cy; or The heterologous molecule is provided, wherein VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position, VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position, L comprises a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys are capable of forming a disulfide bond, and the VL Cys and the second L Cys are capable of forming a disulfide bond.

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

[0314] In some embodiments, the VH Cys is at H3, H5, H40, H43, H46, or H105, where residue numbering is according to Chothia.

[0315] In some embodiments, the VL Cys is at L3, L5, L39, L42, L45, L100, or L102, where residue numbering is according to Chothia.

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

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

[0318] In some embodiments, the VH Cys is in H3 and the VL Cys is in L3.

[0319] In some embodiments, the VH Cys is in H3 and the VL Cys is in L5.

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

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

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

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

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

[0325] In some embodiments, the VH Cys is in H5 and the VL Cys is in L3.

[0326] In some embodiments, the VH Cys is in H5 and the VL Cys is in L5.

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

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

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

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

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

[0332] In some embodiments, the VH Cys is in H40 and the VL Cys is in L3.

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

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

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

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

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

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

[0339] In some embodiments, the VH Cys is in H43 and the VL Cys is in L3.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0360] Residue numbering for the VH and VL regions is according to Chothia.

[0361] In some embodiments, L comprises a contiguous amino acid sequence 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 from mouse, rat, dog, chicken, and non-human primates such as monkeys.

[0363] In some embodiments, the Ig hinge region is derived from a 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 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 can be included in the linker to provide rigidity. Gly can be included in the linker to allow for maximum flexibility. Any other amino acid, except Cys and Met, can also be used in L.

[0366] In some embodiments, L is the 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.

[0367] In some embodiments, L comprises the amino acid sequence 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 comprises about 14-19 amino acids.

[0369] In some embodiments, L comprises about 14 amino acids.

[0370] In some embodiments, L comprises about 15 amino acids.

[0371] In some embodiments, L comprises about 16 amino acids.

[0372] In some embodiments, L comprises about 17 amino acids.

[0373] In some embodiments, L comprises about 18 amino acids.

[0374] In some embodiments, L comprises about 19 amino acids.

[0375] In some embodiments, L is the 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 of 6 to 9; y is an integer of 1 to 3; and n is an integer of 4 to 6.

[0376] In some embodiments, L is the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 26), wherein 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 (SEQ ID NO: 27), wherein X is Gly or Pro, m is an integer of 6 to 9, y is an integer of 1 to 3, and n is an integer of 4 to 6.

[0378] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, or 7.

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

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

[0381] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H105, VL contains Cys at L42, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VL-L-VH orientation.

[0382] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H105, VL contains Cys at L45, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VL-L-VH orientation.

[0383] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H105, VL contains Cys at L39, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VL-L-VH orientation.

[0384] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H5, VL contains Cys at L42, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VL-L-VH orientation.

[0385] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H5, VL contains Cys at L45, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VL-L-VH orientation.

[0386] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H5, VL contains Cys at L39, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VL-L-VH orientation.

[0387] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H3, VL contains Cys at L42, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VL-L-VH orientation.

[0388] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H3, VL contains Cys at L45, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VL-L-VH orientation.

[0389] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H3, VL contains Cys at L39, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv provides a heterologous molecule in a VL-L-VH orientation.

[0390] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H43, VL contains Cys at L100, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0391] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H43, VL contains Cys at L102, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0392] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H43, VL contains Cys at L5, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0393] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H43, VL contains Cys at L3, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0394] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H40, VL contains Cys at L100, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0395] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H40, VL contains Cys at L102, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0396] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H40, VL contains Cys at L5, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0397] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H40, VL contains Cys at L3, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0398] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H46, VL contains Cys at L100, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0399] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H46, VL contains Cys at L102, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0400] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H46, VL contains Cys at L5, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0401] The present disclosure also provides a heterologous molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H46, VL contains Cys at L3, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The 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 the disclosure is linked to a second protein, polynucleotide, therapeutic agent, cytotoxic agent, or detectable label. Conjugates will be done.

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

[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] Heterologous molecules comprising spFv and half-life extending moieties of the present disclosure In some embodiments, the spFv of the present disclosure comprises a half-life extending moiety. Conjugates will be done.

[0413] The half-life extending moiety is an immunoglobulin (Ig), a fragment of Ig, an Ig constant region, a fragment of an Ig constant region, an Fc region, transferrin, albumin, an albumin variant, an albumin binding domain, or polyethylene glycol. The amino acid sequences of human Igs are well known and include IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE.

[0414] In some embodiments, the spFv of the disclosure binds to an Ig or a fragment of an Ig. Conjugates will be done.

[0415] In some embodiments, the spFv of the present disclosure comprises in the Fc region Conjugates will be done.

[0416] In some embodiments, the spFv of the disclosure binds to transferrin. Conjugates will be done.

[0417] In some embodiments, the spFv of the present disclosure binds to albumin. Conjugates will be done.

[0418] In some embodiments, the spFv of the present disclosure binds to an albumin binding protein. Conjugates will be done.

[0419] In some embodiments, the spFv of the disclosure is attached to polyethylene glycol (PEG). Conjugates Exemplary PEG molecules are PEG 5000 or PEG 20,000.

[0420] In some embodiments, the spFv of the disclosure is linked to a fatty acid or fatty acid ester. Conjugates Exemplary fatty acids and fatty acid esters are laurate, myristate, stearate, arachidate, behenate, oleate, arachidonate, octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, etc., polylysine, octane, carbohydrates (dextran, cellulose, oligo- or polysaccharides) for desired properties.

[0421] The half-life extending moiety can be a direct fusion to the spFv of the present disclosure and can be produced by standard cloning and expression techniques. Alternatively, the moiety can be attached to a recombinantly produced spFv of the present disclosure using well-known chemical coupling methods.

[0422] Heterologous molecule comprising an spFv of the disclosure and a cytotoxic agent or detectable label The present disclosure also provides a heterologous molecule comprising an spFv of the present disclosure, wherein the spFv of the present disclosure is linked to a second protein, polynucleotide, therapeutic agent, cytotoxic agent, or detectable label. Conjugates The heterologous molecule is provided.

[0423] Heterologous molecules comprising the spFv of the present disclosure may be used to direct therapeutic agents, mediate killing of cells expressing the antigen to which the spFv binds, visualize, identify, or purify in vitro or in vivo.

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

[0425] The detectable label may be used in conjunction with the spFv of the present disclosure. Conjugates The term "spFv" encompasses compositions that, when activated, render the spFv detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.

[0426] Exemplary detectable labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., commonly used in ELISAs), biotin, digoxigenin, haptens, luminescent molecules, chemiluminescent molecules, fluorescent dyes, fluorophores, fluorescence quenchers, colored molecules, radioisotopes, scintillates, avidin, streptavidin, protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni 2+ , Flag tags, myc tags, heavy metals, enzymes, alkaline phosphatase, peroxidase, luciferase, electron donors / acceptors, acridinium esters, and colorimetric substrates.

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

[0428] Exemplary radioisotopes can be gamma-, Auger-, beta-, alpha-, or positron-emitting radioisotopes. 3 H, 11 C. 13 C. 15 N, 18F, 19 F, 55 Co, 57 Co, 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 an 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, thallium and metals having an atomic number greater than 20, such as uranium, 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, berkelium, californium, einsteinium, fermium, mendelevium, nobelium, or lawrencium atoms.

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

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

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

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

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

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

[0436] In some embodiments, the metal atom can be a metal having an atomic number between 53 (ie, iodine) and 83 (ie, bismuth).

[0437] In some embodiments, the metal atom can be an atom suitable for magnetic resonance imaging.

[0438] The metal atom may be in the form of a metal ion in the +1, +2, or +3 oxidation state, e.g., 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+ , Mn 3+ , Mn 4+ , Mn 7+ , Hg 2+ , Ni 2+ , Ni 3+ , Ag + , Sr 2+ , Sn 2+ , Sn 4+ , and Zn 2+ The metal atom may comprise a metal oxide, such as iron oxide, manganese oxide, or gadolinium oxide.

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

[0440] Suitable fluorophores include 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 (NIR) (700-900 nm) fluorescent dyes, and carbocyanine and aminostyryl dyes.

[0441] Detectable Label Conjugates Heterologous molecules comprising the scFv of the present disclosure can be used as imaging agents.

[0442] In some embodiments, the cytotoxic agent is a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioactive isotope (i.e., a radioconjugate).

[0443] In some embodiments, the cytotoxic agent is daunomycin, doxorubicin, methotrexate, vindesine, a bacterial toxin such as diphtheria toxin, ricin, geldanamycin, a maytansinoid, or calicheamicin. A cytotoxic agent may cause its cytotoxic or cytostatic effect by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.

[0444] In some embodiments, the cytotoxic agent is an enzymatically active toxin such as diphtheria A chain, nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes.

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

[0446] In some embodiments, the cytotoxic agent is a dolastatin or a peptide analog or derivative of dolastatin, an auristatin, or monomethylauristatin phenylalanine. Exemplary molecules are disclosed in U.S. Patent Nos. 5,635,483 and 5,780,588. Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division, and to have anticancer and antifungal activity. The dolastatin or auristatin drug moiety can be attached to the antibody of the invention via the N-terminus or C-terminus of the peptide drug moiety (see WO 02 / 088172), or via any cysteine ​​engineered into the antibody.

[0447] Conjugation to a detectable label can be carried out using known methods.

[0448] In some embodiments, the detectable label is complexed to a chelator.

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

[0450] The detectable label or cytotoxic agent can be linked directly or indirectly to the spFv of the present disclosure using known methods. Suitable linkers are known in the art and include, for example, prosthetic groups, non-phenolic linkers (e.g., derivatives of N-succinimidyl-benzoate, dodecaborate), chelating moieties of both macrocyclic and acyclic chelators, such as 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-pyridyl) ... Examples of suitable peptide linkers include lysyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), activated esters (e.g., disuccinimidyl suberate), 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 chelating moieties. Suitable peptide linkers are well known.

[0451] Heterologous molecules comprising spFv and immunoglobulin (Ig) constant regions or fragments thereof of the present disclosure The spFv of the present disclosure binds to an Ig constant region or a fragment of an Ig constant region. ConjugatesThe spFvs of the present disclosure can be engineered into full-length antibodies using standard methods. Full-length antibodies, including spFvs of the present disclosure, can be further engineered as described herein.

[0452] The immunoglobulin heavy chain constant region consists of the subdomains CH1, CH2, and CH3. The CH1 domain spans residues 118-215 on the heavy chain, CH2 domain residues 231-340, and CH3 domain residues 341-447 according to the EU index. Residue 341 is sometimes referred to as the CH2 domain residue. The hinge is generally defined as including residue 216 and ending at residue 230 in human IgG1, but may include the lower hinge region from about residue 231 to about residue 237 as described herein. The Ig Fc region contains at least the CH2 and CH3 domains of the Ig constant region and thus contains at least the region from about residue 231 to about residue 447 of the Ig heavy chain constant region.

[0453] The present invention also relates to immunoglobulin (Ig) constant regions or fragments of Ig constant regions. Conjugates The spFv of the present disclosure is provided.

[0454] In some embodiments, the Ig constant region is a heavy chain constant region.

[0455] In some embodiments, the Ig constant region is a light chain constant region.

[0456] In some embodiments, the fragment of an Ig constant region comprises an Fc region.

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

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

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

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

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

[0462] In some embodiments, the spFv of the disclosure is directed to the N-terminus of an Ig constant region or a fragment of an Ig constant region. Conjugates will be done.

[0463] In some embodiments, the spFv of the disclosure is linked to the C-terminus of an Ig constant region or a fragment of an Ig constant region. Conjugates will be done.

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

[0465] ADCC activity can be assessed using an in vitro assay using cells expressing the antigen bound by the spFv of the present disclosure as target cells and NK cells as effector cells. Cytolysis can be detected by the release of a label (e.g., a radioactive substrate, a fluorescent dye, or a natural intracellular protein) from lysed cells. In an exemplary assay, target cells are used at a ratio of 1 target cell to 4 effector cells. Target cells are pre-labeled with BATDA and combined with effector cells and test antibodies. Cytolysis was measured by incubating samples for 2 hours and measuring BATDA released into the supernatant. Data were normalized to maximal 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 assessed by using monocyte-derived macrophages as effector cells and any cells expressing the antigen to which the spFv of the present disclosure binds as target cells, and monocyte-derived macrophages as target cells engineered to express GFP or another marker molecule. In an exemplary assay, the effector:target cell ratio can be, for example, 4:1. Effector cells can be incubated with target cells for 4 hours, with or without the addition of an antibody of the present invention. After incubation, cells can be detached using actase. Macrophages can be identified with anti-CD11b and anti-CD14 antibodies conjugated to fluorescent labels, while the rate of phagocytosis can be measured using standard methods to measure CD11b and CD14 phagocytosis. + and CD14 + It can be determined based on the percentage of GFP fluorescence in macrophages.

[0467] For example, Daudi cells were cultured at 1 × 10 in RPMI-B (RPMI supplemented with 1% BSA) for CDC. 5Lysate can be measured by plating 50 μL / well of cells, 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 % of propidium iodide-stained cells in a FACS assay using standard methods.

[0468] Heterologous molecules comprising spFv of the present disclosure and chimeric antigen receptor (CAR) or fragments thereof The spFv of the present disclosure can be used to target a chimeric antigen receptor (CAR) or a fragment of a CAR. Conjugates Thus, a CAR comprising an spFv of the present disclosure can be monospecific or multispecific, comprising one or more scFv molecules of the present disclosure as its extracellular domain.

[0469] Chimeric antigen receptors (CARs) are genetically engineered receptors. These engineered receptors can be easily inserted into and expressed by immune cells, including T cells, according to techniques known in the art. CARs allow a single receptor to recognize a specific antigen, and upon binding to that antigen, activate immune cells to attack and destroy cells bearing that antigen. If these antigens are present on tumor cells, immune cells expressing the CAR 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 containing a costimulatory domain and / or a signaling domain.

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

[0472] The transmembrane domain of CARs is the transmembrane domain of CD8, the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, 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 can be derived from AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), 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 can 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 efficient activation and function of T lymphocytes upon binding to an antigen. Exemplary costimulatory domains that can 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 a CAR can be derived, for example, from the signaling domain of CD3ζ, CD3ε, CD22, CD79a, CD66d, or CD39. "Intracellular signaling domain" refers to the portion of a CAR polypeptide that is involved in transducing the message of effective CAR binding to a target antigen inside an immune effector cell to induce effector cell function, such as activation, cytokine production, proliferation, and cytotoxic activity (including release of cytotoxic factors into the CAR-bound target cell, or other cellular responses elicited after antigen binding to the extracellular CAR domain).

[0475] The optional linker within the CAR, located between the extracellular domain and the transmembrane domain, can be a polypeptide of approximately 2 to 100 amino acids in length. The linker can 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 can be cleavable or non-cleavable. An exemplary cleavable linker includes 2A.

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

[0477] CARs are produced by standard molecular biology techniques.

[0478] The spFv of the present disclosure can be linked to a second molecule directly or via a linker. Conjugates Exemplary linkers include portions of immunoglobulin hinge regions, CL, or CH1 derived from immunoglobulin heavy or light chain isotypes, Gly-rich linkers, Gly and Ser-containing linkers, Gly and Ala-containing linkers, Ala and Ser-containing linkers, and Pro-containing linkers. Exemplary amino acids that can be included in the linker are Gly, Ser, Pro, Thr, Glu, Lys, Arg, Ile, Leu, and His. Alternatively, various non-proteinaceous polymers, including polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, can find use as linkers. Exemplary linkers are described, for example, in WO 2019 / 060695.

[0479] In some embodiments, the heterologous molecule is monospecific.

[0480] In some embodiments, the heterologous molecule is multispecific.

[0481] In some embodiments, the heterologous molecule is bispecific.

[0482] In some embodiments, the heterologous molecule is trispecific.

[0483] In some embodiments, the heterologous molecule is tetraspecific.

[0484] Multispecific molecules comprising spFv of the present disclosure The present disclosure also provides a multispecific molecule comprising a 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 comprises: 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 conserved, 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] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position and L comprises a first L Cy; or VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position and L comprises a second L Cy; or The present invention provides a multispecific molecule, wherein VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position, VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position, L comprises a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys are capable of forming a disulfide bond, and the VL Cys and the second L Cys are capable of forming a disulfide bond.

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

[0487] In some embodiments, the VH Cys is at H3, H5, H40, H43, H46, or H105, where residue numbering is according to Chothia.

[0488] In some embodiments, the VL Cys is at L3, L5, L39, L42, L45, L100, or L102, where residue numbering is according to Chothia.

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

[0490] In some embodiments, L comprises a contiguous amino acid sequence 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 a 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 C (SEQ ID NO: 23), wherein 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 C (SEQ ID NO: 24), wherein X is Gly, Ser or Pro, and y is an integer from 1 to 3.

[0496] In some embodiments, L comprises the amino acid sequence 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, eg, 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 (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 of 6 to 9; y is an integer of 1 to 3; and n is an integer of 4 to 6.

[0499] In some embodiments, L is the 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, 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 (SEQ ID NO: 27), wherein X is Gly or Pro, m is an integer of 6 to 9, y is an integer of 1 to 3, and n is an integer of 4 to 6.

[0501] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, or 7.

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

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

[0504] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H105, VL contains Cys at L42, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VL-L-VH orientation to provide multispecific molecules.

[0505] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H105, VL contains Cys at L45, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VL-L-VH orientation to provide multispecific molecules.

[0506] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H105, VL contains Cys at L39, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VL-L-VH orientation to provide multispecific molecules.

[0507] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H5, VL contains Cys at L42, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VL-L-VH orientation to provide multispecific molecules.

[0508] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H5, VL contains Cys at L45, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VL-L-VH orientation to provide multispecific molecules.

[0509] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H5, VL contains Cys at L39, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VL-L-VH orientation to provide multispecific molecules.

[0510] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H3, VL contains Cys at L42, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VL-L-VH orientation to provide multispecific molecules.

[0511] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H3, VL contains Cys at L45, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VL-L-VH orientation to provide multispecific molecules.

[0512] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H3, VL contains Cys at L39, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFvs are in a VL-L-VH orientation to provide multispecific molecules.

[0513] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H43, VL contains Cys at L100, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0514] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H43, VL contains Cys at L102, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0515] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H43, VL contains Cys at L5, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0516] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H43, VL contains Cys at L3, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0517] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H40, VL contains Cys at L100, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0518] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H40, VL contains Cys at L102, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0519] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H40, VL contains Cys at L5, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0520] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H40, VL contains Cys at L3, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0521] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H46, VL contains Cys at L100, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0522] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H46, VL contains Cys at L102, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0523] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H46, VL contains Cys at L5, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0524] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains Cys at H46, VL contains Cys at L3, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The 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 comprises an antibody or 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 constant region comprises an Fc region.

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

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

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

[0534] In some embodiments, the fragment of an Ig constant region comprises at least a portion of a hinge, a CH2 domain, and a CH3 domain.

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

[0536] In some embodiments, the spFv of the disclosure comprises an Ig constant region N-terminal to the Ig constant region or a fragment of the Ig constant region. Conjugates will be done.

[0537] In some embodiments, the spFv of the disclosure comprises a nucleotide sequence at the C-terminus of an Ig constant region or a fragment of an Ig constant region. Conjugates will be done.

[0538] In some embodiments, the Ig constant region or fragment of an Ig constant region is an IgG1, IgG2, and IgG3 or IgG4 isotype.

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

[0540] In some embodiments, 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, F234A / L235A, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G236 deletion / A327G / 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, where residue numbering is according to the EU index.

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

[0542] In some embodiments, 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, where residue numbering is according to the EU index.

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

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

[0545] In some embodiments, 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, where residue numbering is according to the EU index.

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

[0547] In some embodiments, 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 / Y40 7V, T366L / K392M / T394W, L351Y / Y407A, T366A / K409F, L351Y / Y407A, T366V / K409F, T366A / K409F, T350V / L351Y / F405A / Y407V and T350V / T366L / K392L / T394W, wherein residue numbering is according to the EU index.

[0548] In some embodiments, the multispecific molecule is bispecific.

[0549] In some embodiments, the multispecific molecule is trispecific.

[0550] In some embodiments, the multispecific molecule is tetraspecific.

[0551] 5.2 Generation of multispecific proteins containing spFvs of the present disclosure The spFvs of the present disclosure can be engineered into multispecific molecules of any known format using known recombinant techniques, expression and purification protocols.

[0552] The spFvs of the present disclosure can be engineered into full-length multispecific antibodies with one or more mutations in the CH3 domain that promote the stability of the two half molecules. These multispecific antibodies can be generated in vitro using Fab arm exchange or by coexpression of the various chains. For in vitro Fab arm exchange, two monospecific bivalent antibodies are engineered with one or more substitutions in the CH3 domain, and the antibodies are incubated together under reducing conditions sufficient to cause disulfide bond isomerization at the cysteines in the hinge region, thereby generating bispecific antibodies via Fab arm exchange. The incubation conditions can optimally be returned to non-reducing conditions. Representative reducing agents that can be used include 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol, and 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, e.g., pH 7.0 or 7.4, for at least 90 minutes.

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

[0554] Knob-in-hole mutations, for example, are disclosed in WO 1996 / 027011 and include mutations at the interface of the CH3 domains in which an amino acid with a small side chain (hole) is introduced into the first CH3 domain and an amino acid with a large side chain (knob) is introduced into the second CH3 domain, resulting in preferential interactions between the first and second CH3 domains. Exemplary CH3 domain 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 promoted using electrostatic interactions by substituting positively charged residues on the first CH3 region with negatively charged residues on the second CH3 region, as described in U.S. Patent Application Publication Nos. 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 ... 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 involve replacing selected IgG residues with IgA residues to promote heavy chain heterodimerization, as described in US Patent Application Publication No. 2007 / 0287170.

[0558] Other exemplary mutations that may be used include R409D_K370E / D399K_E357K, S354C_T366W / Y349C_T366S_L368A_Y407V, Y349C_T366W / S354C_T366S_L368A_Y407V, and R409D_K370E / D399K_E357K, as described in WO 2007 / 147901, WO 2011 / 143545, WO 2013 / 157954, WO 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® mutations (Genmab) are disclosed, for example, in U.S. Patent Application Publication No. 2014 / 0303356 and include the following mutations: 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 spFvs of the present disclosure can be incorporated include Dual Variable Domain immunoglobulins (DVDs) (WO 2009 / 134776, where a DVD is a full-length antibody comprising a heavy chain with a VH1-linker-VH2-CH structure and a light chain with a VL1-linker-VL2-CL structure, where the linker is optional), structures comprising various dimerization domains for linking two antibody arms with different specificities, such as leucine zipper or collagen dimerization domains (WO 2012 / 022811, U.S. Pat. No. 5,932,448, U.S. Pat. No. 6,833,441), together ConjugatesHeavy chain-only antibodies, such as engineered two or more domain antibodies (dAbs), diabodies, camelid antibodies and engineered camelid antibodies, dual targeting (DT)-Ig (GSK / Domantis), 2in1 antibodies (Genentech), cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star), and CovX bodies (CovX / Pfizer), IgG-like bispecifics (InnClone / Eli Lilly), Ts2Ab (MedImmune / AZ), and BsAb (Zymogenetics), HERCULES (Biogen Idec), and TvAb (Roche), ScFv / Fc fusions (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), dual affinity retargeting technology (Fc-DART) (MacroGenics), and dual (ScFv)2-Fab (National Research Center for Antibody Medicine--China), biactive or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), bivalent bispecific (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), dual affinity retargeting technology (DART) (MacroGenics), single-chain diabodies (Academic), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack), and COMBODY (Epigen Biotech), dual-targeting nanobodies (Ablynx), and dual-targeting heavy chain-only domain antibodies.

[0561] The scFvs of the present disclosure can also be engineered into multispecific proteins comprising three polypeptide chains. In such designs, at least one antigen-binding domain is in the form of an scFv of the present disclosure. Exemplary designs include (where "1" indicates the first antigen-binding domain, "2" indicates the second antigen-binding domain, and "3" indicates 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 was performed using the L351Y_F405A_Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, and T366L_K392M_T394W / F405A_Y407V methods described in U.S. Patent Application Publication No. 2012 / 0149876 or U.S. Patent Application Publication No. 2013 / 0195849 (Zymeworks). Mutations 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 engineering The Ig constant region or fragment of an Ig constant region, such as an Fc region, present in the multispecific molecule or heterologous molecule of the present disclosure can be of any allotype or isotype.

[0564] In some embodiments, the Ig constant region or fragment of an Ig constant region is of the IgG1 isotype.

[0565] In some embodiments, the Ig constant region or fragment of an Ig constant region is of the IgG2 isotype.

[0566] In some embodiments, the Ig constant region or fragment of an Ig constant region is of the IgG3 isotype.

[0567] In some embodiments, the Ig constant region or fragment of an Ig constant region is of the IgG4 isotype.

[0568] The Ig constant region or a fragment of the Ig constant region can be of 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 fragments of the Ig constant region is associated with an increased risk of infusion reactions and a shorter duration of therapeutic response (Baert et al., (2003) N Engl J Med 348:602-608). The extent to which therapeutic proteins containing fragments of the Ig constant region induce an immune response in the host may be determined, in part, by the allotype of the Ig constant region (Stickler et al., (2011) Genes and Immunity 12:213-221). The allotype of the Ig constant region is related to variations in the amino acid sequence at specific positions in the antibody constant region sequence. Table 3 shows selected IgG1, IgG2, and IgG4 allotypes.

[0569] [Table 3]

[0570] The C-terminal lysine (CTL) can be removed from the Ig constant region by endogenous circulating carboxypeptidases in the bloodstream (Cai et al., (2011) Biotechnol Bioeng 108:404-412). During production, extracellular Zn was removed as described in U.S. Patent Application Publication No. 2014 / 0273092. 2+ , EDTA or EDTA-Fe3+ By controlling the concentration of the CTL, the removal of CTL can be controlled to less than the maximum level. The CTL content of the protein can be measured using known methods.

[0571] In some embodiments, the Ig constant region Conjugates The spFv of the present disclosure 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 can be made to the multispecific or heterologous molecules of the present disclosure comprising an Ig constant region or a fragment of an Ig constant region to modulate its effector functions, such as ADCC, ADCP, and / or pharmacokinetic properties. This can be achieved by introducing mutations into the Fc to control binding of the mutant Fc to activating FcγRs (FcγRI, FcγRIIa, FcγRIII), inhibitory FcγRIIb, and / or FcRn.

[0573] In some embodiments, the multispecific or heterologous molecule of the present disclosure comprises at least one mutation in an Ig constant region or a fragment of an Ig constant region.

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

[0575] In some embodiments, the multispecific or heterologous molecule of the disclosure comprises 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 the present disclosure comprise at least one mutation in the Fc region that modulates binding of the antibody to FcRn.

[0577] Positions in Fc that can be mutated to modulate 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 made alone 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 that may be made, alone or in combination, 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 that may be made, alone or in combination, 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 or heterologous molecule of the present disclosure comprises M252Y / S254T / T256E mutations in the Fc region.

[0579] In some embodiments, the multispecific or heterologous molecules of the present disclosure comprise at least one mutation in the Fc region that reduces binding of the protein to activating Fcγ receptors (FcγRs) and / or reduces an Fc effector function such as C1q binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) or phagocytosis (ADCP).

[0580] F c positions that can be mutated to reduce binding of a multispecific molecule or heterologous molecule of the disclosure to activating FcγR and subsequently reduce effector function 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 be made alone or in combination are the mutations 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 in IgG1, IgG2, IgG3 or IgG4. Exemplary mutation combinations that result in multispecific or heterologous molecules of the disclosure with reduced ADCC are 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, K214T / E233P / L234V / L231S in IgG1 ... 35A / G236 deletion / A327G / P331A / D365E / L358M in IgG2, 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. A hybrid IgG2 / 4 Fc domain may also be used, such as an Fc having residues 117-260 from IgG2 and residues 261-447 from IgG4.

[0581] An exemplary mutation that results in a multispecific or heterologous molecule of the disclosure having reduced CDC is the K322A mutation.

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

[0583] In some embodiments, the multispecific or heterologous molecule of this disclosure comprises at least one mutation in the Fc region selected from the group consisting of K214T, E233P, L234V, L234A, deletion of 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 or heterologous molecule of the present disclosure comprises L234A / L235A / D265S mutations in the Fc region.

[0585] In some embodiments, the multispecific or heterologous molecule of the present disclosure comprises L234A / L235A mutations in the Fc region.

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

[0587] Fc positions that may be mutated to increase binding of the multispecific or heterologous molecules of the disclosure to activating FcγRs 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 may be made singly or in combination are G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305L, K326A, A330K, I332E, E333A, K334A, A339T, and P396L. Exemplary combinations of mutations that result in proteins with increased ADCC or ADCP are 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 be made singly or in combination are S267E, F1268F, S324T, K326A, K326W, E333A, E345K, E345Q, E345R, E345Y, E430S, E430F, and E430T. Exemplary combinations of mutations that result in multispecific or heterologous molecules of the 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 mutations relative to the IgG1, IgG2 and IgG4 wild-type amino acid sequences of SEQ ID NOs: 66, 67 and 68, respectively.

[0590] SEQ ID NO: 66, wild-type IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0591] SEQ ID NO: 67; wild type IgG2 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKDTLMISRTPEVTCVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0592] SEQ ID NO: 68; wild type IgG4 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0593] Binding of the multispecific or heterologous molecules of the present disclosure to FcγR or FcRn can be assessed in cells engineered to express each receptor using flow cytometry. In an exemplary binding assay, 2x10 cells per well in a 96-well plate are used. 5Cells were seeded and blocked in BSA Stain Buffer (BD Biosciences, San Jose, USA) for 30 minutes at 4°C. The cells were incubated with the test multispecific molecules or heterologous molecules of the present disclosure for 1.5 hours on ice at 4°C. After washing twice with BSA stain buffer, the cells were incubated with R-PE-labeled anti-human IgG secondary antibody (Jackson Immunoresearch Laboratories) for 45 minutes at 4°C. The cells were washed twice with stain buffer and then resuspended in 150 μL of Stain Buffer containing a 1:200 dilution of DRAQ7 Live / Dead Cell Stain Reagent (Cell Signaling Technology, Danvers, USA). The PE and DRAQ7 signals of the stained cells were detected using the B2 and B4 channels, respectively, on a Miltenyi MACSQuant flow cytometer (Miltenyi Biotec, Auburn, USA). Live cells are gated by DRAQ7 exclusion and the geometric mean fluorescence signal is determined for at least 10,000 live cell events collected. Analysis is performed using FlowJo software (Tree Star). Data are plotted as the logarithm of the antibody concentration versus the mean fluorescence signal. Nonlinear regression analysis is performed.

[0594] 5.4 Glycotechnology Ig constant region or fragment of Ig constant region ConjugatesThe ability of engineered multispecific or heterologous molecules of the present disclosure to mediate ADCC can be enhanced by engineering fragments of the Ig constant region or Ig constant region oligosaccharide moieties. Human IgG1 or IgG3 is N-glycosylated at Asn297, where the majority of glycans are in the known biantennary G0, G0F, G1, G1F, G2, or G2F forms. Ig constant region-containing proteins that can be produced by unengineered CHO cells typically have a glycan fucose content of at least about 85%. Removal of core fucose from biantennary complex-type oligosaccharides attached to Ig constant regions or fragments of Ig constant regions enhances ADCC of multispecific or heterologous molecules of the present disclosure through improved FcγRIIIa binding without altering antigen binding or CDC activity. Such multispecific or heterologous molecules can be used for a variety of purposes, including controlling the osmolality of the culture medium (Konno et al., Cytotechnology 64:249-265, 2012), using the mutant CHO line Lec13 as a host cell line (Shields et al., (2002) J Biol Chem 277:26733-26740), using the mutant CHO line EB66 as a host cell line (Olivier et al., (2010) MAbs;2:405-415), using the rat hybridoma cell line YB2 / 0 as a host cell line (Shinkawa et al., (2003) J Biol Chem 278:3466-3473), and introducing small interfering RNA specifically against the 1,6-fucosyltransferase (FUT8) gene (Mori et al., (2004) Biotechnol Bioeng 88:901-908), or co-expression of β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II or the potent α-mannosidase I inhibitor kifunensine (Ferrara et al., (2006) J Biol Chem 281:5032-5036), which have been reported to lead to the successful expression of relatively high levels of fucose-unmodified immunoglobulins with biantennary complex types of Fc oligosaccharides.

[0595] In some embodiments, multispecific or heterologous molecules of the present disclosure comprising an Ig constant region or a fragment of an Ig constant region have biantennary glycan structures with a fucose content of about 1% to about 15%, e.g., about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, multispecific or heterologous molecules of the present disclosure comprising an Ig constant region or a fragment of an Ig constant region have glycan structures with a fucose content of about 50%, 40%, 45%, 40%, 35%, 30%, 25%, or 20%.

[0596] "Fucose content" refers to the amount of fucose monosaccharide in the glycan at Asn 297. The relative amount of fucose is the ratio of fucose-containing structures to total glycan structures. These include several methods, such as: 1) using MALDI-TOF of N-glycosidase F treated samples (e.g., complex, hybrid, and oligo- and high mannose structures) as described in WO 2008 / 077546; 2) enzymatic release of the Asn297 glycan followed by derivatization and detection / quantification by HPLC with fluorescence detection (UPLC) and / or HPLC-MS (UPLC-MS); 3) intact protein analysis of native or reduced mAbs, with or without treatment of the Asn297 glycan with Endo S or other enzymes that cleave between the first and second GlcNAc monosaccharides, leaving the fucose attached to the first GlcNAc; 4) enzymatic digestion (e.g., trypsin or endopeptidase Lys-C) of the mAb into its constituent peptides, followed by separation, detection, and quantification by HPLC-MS (UPLC-MS); 5) derivatization of the Asn297 glycan by enzymatic digestion (e.g., trypsin or endopeptidase Lys-C) followed by separation, detection, and quantification by HPLC-MS (UPLC-MS); mAb oligosaccharides can be characterized and quantified by specific enzymatic deglycosylation with PNGase F at position 297, separating them from the mAb protein. The oligosaccharides thus released can be labeled with fluorophores and separated and identified by a variety of complementary techniques, including detailed characterization of glycan structures by matrix-assisted laser desorption / ionization (MALDI) mass spectrometry by comparison of observed masses with theoretical masses, 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 a multispecific molecule or heterologous molecule of the present disclosure comprising an Ig constant region or a fragment of an Ig constant region having a fucose content of between about 1% and 15%.

[0598] "Normal fucose" or "normal fucose content" refers to a multispecific molecule or heterologous molecule of the present disclosure comprising an Ig constant region or a fragment of an Ig constant region having a fucose content of greater than about 50%, typically greater than about 80% or 85%.

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

[0600] The present invention also provides anti-idiotypic antibodies that specifically bind to the spFv of the present disclosure.

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

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

[0603] 5.6 Polynucleotides, Vectors and Host Cells The present disclosure also provides isolated polynucleotides encoding the spFvs of the present disclosure.

[0604] The present disclosure also provides a vector comprising a polynucleotide of the present disclosure.

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

[0606] Exemplary vectors that can be used are: bacterial: 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); eukaryotic: 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.), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors such as λGT10, λGT11, λEMBL4, and λNM1149, λZapII (Stratagene) can 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 can be a viral vector, eg, a retroviral vector, eg, a gamma retroviral vector.

[0607] The present disclosure also provides a host cell comprising a vector of the present disclosure.

[0608] A "host cell" refers to a cell into which a vector has been introduced. It is understood that the term host cell is intended to refer not only to the particular subject cell, but also to the progeny of such a cell, as well as to stable cell lines generated from the particular subject cell. Because certain modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included 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 are bacilli such as Escherichia coli, Bacillus subtilis, and 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 are Saccharomyces (e.g., S. cerevisiae) and Pichia. Exemplary eukaryotic cells may be of mammalian, insect, avian, or other animal origin. 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) murine 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 present disclosure also provides a method for producing an spFv of the present disclosure, comprising culturing a host cell of the present disclosure under conditions in which the spFv is produced, and recovering the spFv produced by the host cell. Methods for making scFvs and purifying them are known. Once synthesized (chemically or recombinantly), the scFv of the present disclosure can be purified according to standard procedures, including ammonium sulfate precipitation, affinity columns, column chromatography, high-performance liquid chromatography (HPLC) purification, gel electrophoresis, and the like (see generally, Scopes, Protein Purification (Springer-Verlag, NY, (1982))). The scFv of the present disclosure may be substantially pure, e.g., free from contaminants such as cellular debris, macromolecules other than the protein of interest, and the like, e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or at least about 98% to 99% pure, or even more pure.

[0610] Polynucleotides encoding the scFv of the present disclosure can be incorporated into vectors using standard molecular biology techniques, and host cell transformation, culture, antibody expression, and purification are carried out using well-known methods.

[0611] 5.7 Pharmaceutical Compositions and Administration The present disclosure also provides pharmaceutical compositions comprising an spFv, a heterologous molecule comprising an spFv, or a multispecific molecule comprising an spFv of the present disclosure and a pharmaceutically acceptable carrier. For therapeutic use, the spFv, a heterologous molecule comprising an spFv, or a multispecific molecule comprising an spFv of the present disclosure can be prepared as a pharmaceutical composition containing an effective amount of the spFv, a heterologous molecule comprising an spFv, or a multispecific molecule comprising an spFv of the present disclosure as an active ingredient in a pharmaceutically acceptable carrier. "Carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the spFv, a heterologous molecule comprising an spFv, or a multispecific molecule comprising an spFv of the present disclosure is administered. Such vehicles can be liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, stabilizers, thickeners, lubricants, and coloring agents. The concentration of the spFv, spFv-containing heterologous molecule, or spFv-containing multispecific molecule of the present disclosure in such pharmaceutical formulations may vary from less than about 0.5% by weight, usually to at least about 1% by weight, up to 15 or 20% by weight, and may be selected primarily based on the required dose, fluid volume, viscosity, etc., according to the selected 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, see in particular pp. 958-989.

[0612] The method of administration of the spFv, heterologous molecule comprising an spFv, or multispecific molecule comprising an spFv of the present disclosure can be by any suitable route, such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, transmucosal (oral, intranasal, intravaginal, rectal), or other means understood by one of skill in the art, as is well known in the art.

[0613] 5.8 Processes for preparing spFv of the present disclosure The present disclosure also provides a process for preparing a stabilized scFv, comprising: providing a heavy chain variable region (VH) and a light chain variable region (VL) that form an antigen-binding site; providing a linker (L) that includes or is engineered to include a first L Cys; engineering a VH to contain a VH Cys at a structurally conserved, surface-exposed VH framework residue position; forming a disulfide bond between the VH Cys and the first L Cys to prepare a stabilized scFv.

[0614] The present disclosure also provides a process for preparing a stabilized scFv, comprising: providing VH and VL that form an antigen-binding site; providing an L that includes or is engineered to include a second L Cys; engineering VL to contain a VL Cys at a structurally conserved, surface-exposed VL framework residue position; forming a disulfide bond between the VL Cys and the second L Cys to prepare a stabilized scFv.

[0615] The present disclosure also provides a process for preparing a stabilized scFv, comprising: providing a heavy chain variable region (VH) and a light chain variable region (VL) that form an antigen-binding site; providing a linker (L) comprising or engineered to comprise a first L Cys and a second L Cys; engineering a VH to contain a VH Cys at a structurally conserved, surface-exposed VH framework residue position; engineering VL to contain a VL Cys at a structurally conserved, surface-exposed VL framework residue position; forming a disulfide bond between the VH Cys and the first L Cys and a disulfide bond between the VL Cys and the second L Cys to prepare a stabilized scFv.

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

[0617] Any known VH / VL pair of scFvs that form antigen-binding domains can be engineered into stabilized scFvs of the present disclosure. Alternatively, antigen-binding VH / VL pairs of interest can be identified de novo using known methods, and the resulting VH / VL pairs can be engineered into an spFv format.

[0618] For example, the hybridoma method of Kohler and Milstein can be used to identify VH / VL pairs that bind to the antigen of interest, and the resulting VH / VL pairs can be engineered as spFvs. Alternatively, transgenic animals, such as mice, rats, or chickens, carrying human immunoglobulin (Ig) loci in their genomes can be used to produce antigen-binding fragments; these are described, for example, in U.S. Pat. No. 6,150,584, WO 1999 / 45962, WO 2002 / 066630, WO 2002 / 43478, WO 2002 / 043478, and WO 1990 / 04036. The endogenous immunoglobulin loci of such animals can be disrupted or deleted, and at least one complete or partial human immunoglobulin locus can be inserted into the animal's 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 technology. Phage display may also be used to generate antigen-binding fragments that can be engineered as spFvs.

[0619] In some embodiments, the spFv of the present disclosure is humanized. In some embodiments, the spFv of the present disclosure is human. In some embodiments, the spFv of the present 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, the VH Cys is at H3, H5, H40, H43, H46, or H105, where residue numbering is according to Chothia.

[0622] In some embodiments, the VL Cys is at L3, L5, L39, L42, L45, L100, or L102, where residue numbering is according to Chothia.

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

[0624] In some embodiments, L comprises a contiguous amino acid sequence 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 a 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 C (SEQ ID NO: 23), wherein 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 C (SEQ ID NO: 24), wherein X is Gly, Ser or Pro, and y is an integer from 1 to 3.

[0630] In some embodiments, L comprises the amino acid sequence 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, eg, 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 (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 of 6 to 9; y is an integer of 1 to 3; and n is an integer of 4 to 6.

[0633] In some embodiments, L is the 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, 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 (SEQ ID NO: 27), wherein X is Gly or Pro, m is an integer of 6 to 9, y is an integer of 1 to 3, and n is an integer of 4 to 6.

[0635] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 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 disclosure is in a VH-L-VL orientation.

[0638] In some embodiments, VH contains Cys at H105, VL contains Cys at L42, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VL-L-VH orientation.

[0639] In some embodiments, VH contains Cys at H105, VL contains Cys at L45, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VL-L-VH orientation.

[0640] In some embodiments, VH contains Cys at H105, VL contains Cys at L39, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VL-L-VH orientation.

[0641] In some embodiments, VH contains Cys at H5, VL contains Cys at L42, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VL-L-VH orientation.

[0642] In some embodiments, VH contains Cys at H5, VL contains Cys at L45, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VL-L-VH orientation.

[0643] In some embodiments, VH contains Cys at H5, VL contains Cys at L39, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VL-L-VH orientation.

[0644] In some embodiments, VH contains Cys at H3, VL contains Cys at L42, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VL-L-VH orientation.

[0645] In some embodiments, VH contains Cys at H3, VL contains Cys at L45, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VL-L-VH orientation.

[0646] In some embodiments, VH contains Cys at H3, VL contains Cys at L39, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VL-L-VH orientation.

[0647] In some embodiments, VH contains Cys at H43, VL contains Cys at L100, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0648] In some embodiments, VH contains Cys at H43, VL contains Cys at L102, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0649] In some embodiments, VH contains Cys at H43, VL contains Cys at L5, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0650] In some embodiments, VH contains Cys at H43, VL contains Cys in L3, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0651] In some embodiments, VH contains Cys at H40, VL contains Cys at L100, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0652] In some embodiments, VH contains Cys at H40, VL contains Cys at L102, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0653] In some embodiments, VH contains Cys at H40, VL contains Cys at L5, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0654] In some embodiments, VH contains Cys at H40, VL contains Cys in L3, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0655] In some embodiments, VH contains Cys at H46, VL contains Cys at L100, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0656] In some embodiments, VH contains Cys at H46, VL contains Cys at L102, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0657] In some embodiments, VH contains Cys at H46, VL contains Cys at L5, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The scFv is in a VH-L-VL orientation.

[0658] In some embodiments, VH contains Cys at H46, VL contains Cys in L3, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; The 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 the present disclosure binds to antigen with comparable affinity when compared to the parent scFv lacking disulfide bonds.

[0663] The present disclosure also provides a process for preparing a stabilized scFv, comprising: providing polynucleotides encoding VH, L, and VL, VH contains Cys at H105 and VL contains Cys at L42, or VH contains Cys at H43 and VL contains Cys at L100, or VH contains Cys in H3 and VL contains Cys in L3, or VH contains Cys in H3 and VL contains Cys in L5, or VH contains Cys at H3 and VL contains Cys at L39, or VH contains Cys at H3 and VL contains Cys at L42, or VH contains Cys at H3 and VL contains Cys at L45, or VH contains Cys at H3 and VL contains Cys at L100, or VH contains Cys at H3 and VL contains Cys at L102, or VH contains Cys at H5 and VL contains Cys at L3, or VH contains Cys at H5 and VL contains Cys at L5, or VH contains Cys at H5 and VL contains Cys at L39, or VH contains Cys at H5 and VL contains Cys at L42, or VH contains Cys at H5 and VL contains Cys at L45, or VH contains Cys at H5 and VL contains Cys at L100, or VH contains Cys at H5 and VL contains Cys at L102, or VH contains Cys at H40 and VL contains Cys at L3, or VH contains Cys at H40 and VL contains Cys at L5, or VH contains Cys at H40 and VL contains Cys at L39, or VH contains Cys at H40 and VL contains Cys at L42, or VH contains Cys at H40 and VL contains Cys at L45, or VH contains Cys at H40 and VL contains Cys at L100, or VH contains Cys at H40 and VL contains Cys at L102, or VH contains Cys at H43 and VL contains Cys at L3, or VH contains Cys at H43 and VL contains Cys at L5, or VH contains Cys at H43 and VL contains Cys at L39, or VH contains Cys at H43 and VL contains Cys at L42, or VH contains Cys at H43 and VL contains Cys at L45, or VH contains Cys at H43 and VL contains Cys at L102, or VH contains Cys at H46 and VL contains Cys at L3, or VH contains Cys at H46 and VL contains Cys at L5, or VH contains Cys at H46 and VL contains Cys at L39, or VH contains Cys at H46 and VL contains Cys at L42, or VH contains Cys at H46 and VL contains Cys at L45, or VH contains Cys at H46 and VL contains Cys at L100, or VH contains Cys at H46 and VL contains Cys at L102, or VH contains Cys at H105 and VL contains Cys at L3, or VH contains Cys at H105 and VL contains Cys at L5, or VH contains Cys at H105 and VL contains Cys at L39, or VH contains Cys at H105 and VL contains Cys at L45, or VH contains a Cys at H105 and VL contains a Cys at L100, or VH contains a Cys at H105 and VL contains a Cys at L102, residue numbering is according to Chothia, providing L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; and expressing the polynucleotide in a host cell to produce the 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] The following examples are provided to further describe some of the embodiments disclosed herein and are intended to illustrate, but not limit, the embodiments of the present disclosure. [Example]

[0667] 6.1 Example 1: Design of stabilized scFvs 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 genetic fusions of VL and VH with a flexible linker in either the VL-linker-VH or VH-linker-VL orientation. The flexible linker is typically a triplet or quadruple repeat 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 reproduce the antigen-binding specificity and largely the affinity of their parent mAbs. These scFv molecules find wide application as detection / diagnostic reagents or as building blocks for creating more sophisticated molecules such as bispecific, multispecific therapeutics (Brinkmann and Kontermann (2017) MAb 9:182-212) or CAR-T therapeutics (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 of scFv molecules is their low stability and tendency to aggregate (reviewed in Worn and Pluckthun (2001) J Mol Biol 305:989-1010; Rothlisberger et al., (2005) J Mol Biol 347:773-789). Many strategies have been attempted to improve their properties (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 Commun 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, improving the stability and / or interfacial interactions of the VL / VH domains using different experimental methods, such as using additional dimerization motifs. A key difficulty is that most of these strategies are often specific to a VH / VL pair and cannot be easily transferred to other VH / VL pairs. In some cases, the manipulations can adversely affect the VL / VH structure and scFv properties. Recently, Zhang et al. successfully introduced a disulfide between position 44 of the VH and position 100 of the VL of the anti-aflatoxin B1 scFv (H4) and significantly stabilized the scFv while maintaining its binding affinity (Zhao et al., (2010) Int J Mol Sci 12:1-11). However, due to the restriction of the distance and angle between the two selected positions, when applied to other VL / VH pairs, inter-VL / VH disulfides may restrict / distort the relative orientation between the two domains that is often required for binding.

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

[0670] 6.1.1 "Staple Processing" Design The goal here was to design and generate stabilized scFvs without adversely affecting the relative movement between the VH and VL that form the scFv. This was achieved by stabilizing the scFvs by engineering disulfide bonds between the VH and linker and between the VL and linker. When appropriately positioned, the constraints (i.e., disulfide bonds) play a role in the synergistic effects provided by the CH1 / CL and V / C interactions described above. To this end, two structurally conserved, surface-exposed framework positions (anchor points), one in the VH and one in the VL, that do not overlap with typical predicted antigen-binding sites were identified and mutated to cysteine ​​(Cys) residues. Two positions were then selected in the flexible linker for the Cys positions. When the distance and position between the linker Cys residues are designed in a manner that promotes disulfide bond formation between the linker Cys and each anchor point, the VH and VL can be tethered more tightly compared to tethering in the absence of disulfide bonds. This scheme is shown in Figure 1 with an exemplary linker containing the CPPC sequence (SEQ ID NO: 1). The concept of forming a disulfide bond between a flexible linker and an anchor point is referred to herein as "stapling." The resulting "stapled" scFv molecule is referred to herein as an spFv ("stapled Fv").

[0671] 6.1.2 Selection of anchor points, staple sequence design and linker For a broadly applicable stapling scheme, it is important that the anchor points are structurally conserved and exposed on the surface of both VL and VH, and that mutations to Cys residues do not affect VL and VH folding or antigen binding. The distance and geometry of the anchor points and the N- and C-termini of the VL and VH domains are also important considerations for proper disulfide formation.

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

[0673] The staple sequence embedded within the linker connecting the VH and VL was designed to be of a length similar to the distance between anchor points in spFv. As an initial example of a staple sequence, CPPC (SEQ ID NO: 1) was selected as a possible staple sequence because this sequence naturally occurs in the human IgG1 hinge and in some rodent IgGs. The structures of the hinges 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) ranges from approximately 7 Å to 9 Å. Because this range is very similar to the distance between the two anchor points in both the VL-linker-VH and VH-linker-VL orientations, the CPPC (SEQ ID NO: 1) staple sequence has the potential to provide the correct geometry for stapling, i.e., to efficiently and correctly form the appropriate disulfide bond at the anchor point. In general, staple sequences were designed to have two Cys residues. For proper stapling, the N-terminal Cys of the staple sequence formed a disulfide bond with the spFv N-terminal domain anchor point, and the C-terminal Cys of the staple sequence formed a disulfide bond with the spFv C-terminal domain anchor point.

[0674] Therefore, the linker connecting VH and VL is designed to include staple sequences and connecting sequences at both the N- and C-terminus to extend the linker and provide a linker length sufficient to allow endothelial folding of VH and VL and to facilitate proper positioning of the staple sequences.

[0675] For 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. For 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 spanned by a linker length of approximately 14-19 residues, with a four-residue staple sequence flanked by an N-terminal linker extension of approximately 6-9 residues and a C-terminal linker extension of approximately 4-6 residues. Thus, 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. The n and m residues can be glycine or serine, or other amino acid residues. The length of these linkers is expected to be too short to allow for crambling, yet long enough and flexible enough to allow stapling.

[0676] 6.2 Example 2: Generation and characterization of spFv To evaluate the stapling design, three human antibodies were selected to generate scFvs and corresponding spFvs: two antibodies with kappa light chains (GLk1 and GLk2) from a synthetic phage antibody library (Shi et al., (2010) J Mol Biol 397:385-396) and a lambda-containing antibody (CAT2200) derived from a paper (Gerhardt et al., (2009) J Mol Biol 394:905-921). For CAT2200, a T28G mutation was introduced into the parent VH to generate a variant (CAT2200a) to reduce some of its interactions with its target IL-17. Additionally, a S42Q mutation (Chothia) was engineered into the parent 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 sequences are identical between BAT2200a and CAT2200b. GLk1VH is a human IGHV2-23 * 01 GLk2VH vs. human IGHV5-51, CAT2200VH vs. human IGHV2-23 * 01. GLk1VL is human IGKV1-39 * 01. GLk2VL vs. human IGKV3-20 * 01, and CAT2200VL versus human IGLV 6-57 * 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 spFvs, different linker lengths within the n and m ranges described above were used. For GLk1 spFv, a 9-4-5 linker was used for both orientations. For GLk2 spFv, 9-4-5 and 6-4-6 linker lengths were used for the VL-VH and VH-VL orientations, respectively. For CAT2200a spFv, VL-VH molecules were generated with 8-4-4 and 9-4-4 linkers, respectively, and CAT2200b spFv VH-VL was generated with a 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 for CAT2200a scFv VL-VH, were cloned into a CMV promoter-driven mammalian expression vector. 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 clarified supernatant on a 1 ml His-TRAPHP column (GE Healthcare) using the AKTAXPRESS system (GE Healthcare). The column was prepared with a 0-100% gradient of elution buffer (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-equilibrated with DPBS. The clarified supernatant was first adjusted against 50 mM Tris, pH 7.5, and 20 mM imidazole and then loaded onto a 1 mL HisTRAP HP column at 0.8 mL / min at 4°C. 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 and eluted with elution buffer into a single injection loop. The column was then desalted on a 26 / 10 HiPrep desalting column into 1x DPBS, and fractions were collected. Fractions containing 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. IL-17 mutant (12-132 with K70Q A132Q C106S mutations, hereafter referred to as IL-17 for brevity) (SEQ ID NO: 22) was purchased from Accelagen (CA). The protein was refolded from E. coli inclusion bodies following their proprietary refolding protocol and provided at 1.50 mg / mL in 20 mM NaCl, 20 mM MES, pH 6.0.

[0683] SEQ ID NO: 22 (IL-17A variant) MNSEDKNFPRTVMVNLNIHNRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAQCRHLGCINADGNVDYHMNSVPIQQEILVLRREPPHSPNSFRLEKILVSVGCTCVTPIVHHVQ

[0684] 6.2.1 Thermal Stability of Produced scFv and spFv Molecules The thermal stability of scFv and spFv molecules was investigated by differential scanning calorimetry (DSC). The scFv and spFv proteins were dialyzed overnight against 1x DPBS (Gibco) for GLk1 and CAT2200a / CAT2200b or MES (25 mM MES, pH 6.0, 100 mM NaCl) for GLk2. The dialysis buffer was then 0.22 micrometer filtered and used as a reference solution and for buffer blanks in DSC experiments. 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 kept at 4 °C in an autosampler drawer throughout the experiment. DSC experiments were performed using a MicroCal capillary DSC with an autosampler (Malvern). DSC scans were performed from 25 to 95 °C at a scan rate of 60 °C / h without sample rescans. 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 run. Data analysis was performed using Origin 7.0 with the MicroCal VP-Capillary DSC Autoanalysis Add-on (Malvern). The baseline range and type were manually selected and then subtracted. After subtracting the previous buffer blank from the sample curve, concentration-dependent normalization was performed. Thermal melting profiles were analyzed using both two-state and non-two-state transitions. A two-state fit (one transition) did not adequately match 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. DSC profiles of all scFv and spFv proteins showed skewness that could only be fitted by non-two-state transitions. Thus, for each scFv or spFv, two transitions (Tm1 and Tm2) were reported (Table 6). Arguably, these two transitions correspond to the melting Tms of the VL and VH domains, respectively.Generally, when comparing the difference between scFv and spFv for either Tm1 or Tm2, ​​stapling results in an increase of approximately 10°C, regardless of the Tm of the starting scFv. There is only one exception: the difference between GLk2 scFv and spFv (VH-VL orientation) is approximately 7°C. This is likely due to the shorter 6+4+6 linker, which may cause slight distortion in the stapling geometry. The fact that ΔTm1(VL) and ΔTm2(VH) were nearly identical suggests that stapling leads to stabilization of its own domain in addition to strengthening the VL / VH interaction. Alternatively, stronger VH / VL interactions may transfer a stabilizing effect to the stabilization of the VL / VH domain. In summary, the stapling process described herein significantly increases the stability of scFvs.

[0685] [Table 6]

[0686] CAT2200 spFv was tested for its binding to IL-17, and binding was comparable when compared to CAT2200 scFvs.

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

[0688] [Table 7]

[0689] 6.3.1 Crystallization of CAT2200a scFv VL-VH and CAT2200a spFv VL-VH in Complex with IL-17 The IL-17 / CAT2200a scFv VL-VH complex was generated 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 Superdex 75 column equilibrated with 250 mM NaCl, 20 mM HEPES, pH 7.5. Fractions corresponding to the complex were pooled and concentrated to a volume of 150 μL. The sample was diluted and concentrated four times (350 μL of 50 mM NaCl, 20 mM HEPES, pH 7.5 was added to a concentration of just under 150 μL). The volume was brought to approximately 105 μL, and the concentration was determined to be 2.69 mg / mL. Crystallizations were set up in a sitting drop format using a Mosquito crystallization robot with 150 nL of protein + 150 nL of reservoir in a Corning 3550 plate against an 80 μL reservoir, a set of buffer and precipitation conditions previously designed in-house. Plates were incubated at 20°C. One of the conditions (sodium acetate, pH 4.5, 25% PEG3K, 0.2 M Am-acetate) produced very small crystals. These were collected and converted into crystallization seeds using Hampton seed beads in 100 μL of 27% PEG3350, 200 mM ammonium acetate, 100 mM sodium acetate, pH 4.5 in a Hampton Seed Bead tube.

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

[0691] The IL-17-CAT2200a spFv VL-VH complex was produced by mixing 167 μl of IL-17 (250 μg) with 154 μl of MSCW274 (467 μg in 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 repeatedly diluted and concentrated five times (to approximately 150 μL) before adding 350 μL of 50 mM NaCl, 20 mM HEPES, pH 7.5. The final volume was 100 μL, and the concentration of the complex was determined to be 6.0 mg / mL. Crystallization was set up similarly to the scFv / IL-17 complex in sitting drops using a Mosquito robot. The sitting drop consisted of 150 nL of protein, 120 nL of reservoir, and 30 nL of seed (scFv / IL-17 as described above). The reservoir solution was a set of conditions with various PEG3350 concentrations and salts. The crystallization plate was incubated at 20°C. Small crystals were obtained from 15.5% PEG3350, 0.4 M NaH2PO4. Crystals were transferred to 16% PEG3350, 0.2 M NaH2PO4, 20% glycerol, and flash-frozen LN2. X-ray diffraction data were collected on an IMCA-CAT ID17 at Argonne National Laboratory.

[0692] All X-ray diffraction data were processed with 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 were solved by molecular replacement (MR) using Phaser (Read (2001) Acta Crystallogr D Biol Crystallogr 57(Pt 10):1373-1382) with a homology model generated in MOE (Montreal, Canada), except for the scFv CAT2200a scFv VL-VH / IL-17 complex, for which the structure with 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 in PHENIX (Adams et al. (2004) J Synchrotron Radiat 11(Pt 1):53-55) and manually adjusted in Coot (Emsley et al. (2010) Acta Crystallogr D Biol Crystallogr 66(Pt 4):486-501). Molecular graphics were generated with PyMol (www_schrodinger_com).

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

[0694] 6.4 Example 4: Design of additional anchor points Any additional anchor points for stapling were identified using the approach described in Example 1. The following anchor points were identified:

[0695] For the 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, K39, and the VH residues on GLk1 are Q105, L5, and Q3. Staples are formed between any of the positions shown.

[0696] In the case of the 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] spFvs with the anchor points described in this example are cloned, expressed, and tested for staple formation and their thermal stability using the assays described herein and in Example 2.

[0698] 6.5 Example 5: Partial Stapling Constructs are generated and expressed to contain one staple either between the VH and the linker or between the VL and the linker, and the generated constructs are expressed, purified, and analyzed using the methods described herein.

[0699] 6.6 Example 6: Multispecific constructs containing spFvs Exemplary multispecific binding molecules incorporating the spFv structures provided herein were constructed and tested in this example. Specifically, bispecific antibodies and control molecules were derived from the target-binding molecules shown in Table 8 and were transiently expressed in CHO suspension cultures in serum-free / animal component-free medium and purified by Protein A affinity chromatography using an AKTA PURE instrument (GE Healthcare), 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 to promote 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 an IgG1 sigma Fc with a set of seven Fc mutations—L234A, L235A, G237A, P238S, H268A, A330S, and P331S—compared to wild-type IgG1, which reduced Fc-receptor interaction (Tam et al., Antibodies (2017)).

[0700] The bispecific antibody was generated by IgG1 sigma and KiH mutations.

[0701] [Table 8] * : EDBmAb1 (WO 9745544) used herein is an anti-ED-B antibody that has been tested in the clinic; other antibodies that bind to ED-B or adjacent domains have been previously described (Carnemolla et al. Int. J. Cancer 68:397-405 (1996)).

[0702] The sequences in Table 8 are as follows: SEQ ID NO: 69 (VH BHA10) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSS

[0703] SEQ ID NO: 70 (VL BHA10) DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIK

[0704] SEQ ID NO: 71 (VH L19) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSS

[0705] SEQ ID NO: 72 (VL L19) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIK

[0706] SEQ ID NO: 73 (VH B21M) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSS

[0707] SEQ ID NO: 74 (VL B21M) DIVMTQSPDSLAVSLGERATINCRASQSVDYNGISYMHWYQQKPGQPPKLLIYAASNPESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQIIEDPWTFGQGTKVEIK

[0708] SEQ ID NO: 75 (VH MSLNmAb1) QVQLQQSGPELEKPGASVKISKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSS

[0709] SEQ ID NO: 76 (VL MSLNmAb1) DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK

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

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

[0712] [Table 9] *: Mutations in the Fc portion abrogate binding to Protein A and facilitate purification of the heterodimer described in WO 2010 / 151792.

[0713] [Table 10]

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

[0715] SEQ ID NO: 77 [stapled scFv BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0716] SEQ ID NO: 78 (HC B21M N-terminally stapled BHA10 (VH-VL), IgG1s, knob, with pA mutation) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPK SLISSASYRYSGVPSRFSGGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGGSQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDT ATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSA EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0717] SEQ ID NO: 79 (HC B21M (RSV) IgG1s knob with pA mutation) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAEAPEAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

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

[0719] SEQ ID NO: 81 [LC B21M (RSV)] DIVMTQSPDSLAVSLGERATINCRASQSVDYNGISYMHWYQQKPGQPPKLLIYAASNPESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQIIEDPWTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

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

[0721] SEQ ID NO: 82 [stapled scFv BHA10(VL-VH)] DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPC GSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSS

[0722] SEQ ID NO: 83 (HCB 21M N-terminally stapled BHA10 (VL-VH), IgG1s, knob, with pA mutation) DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPCGSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGN VHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSSGGGGSGGGGSGGGGGSGGGGSGGGGSQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTA TYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSA EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0723] SEQ ID NO: 79 (HC B21M (RSV) IgG1s knob with pA mutation) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAEAPEAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

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

[0725] SEQ ID NO: 81 [LC B21M (RSV)] DIVMTQSPDSLAVSLGERATINCRASQSVDYNGISYMHWYQQKPGQPPKLLIYAASNPESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQIIEDPWTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

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

[0727] SEQ ID NO: 77 [stapled scFv BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0728] SEQ ID NO: 84 (HC B21M C-terminally stapled BHA (VH-VL), IgG1s, knob, with pA mutation) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVY TLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEK FKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGP

[0729] SEQ ID NO: 79 (HC B21M (RSV) IgG1s knob with pA mutation) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAEAPEAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

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

[0731] SEQ ID NO: 81 [LC B21M (RSV)] DIVMTQSPDSLAVSLGERATINCRASQSVDYNGISYMHWYQQKPGQPPKLLIYAASNPESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQIIEDPWTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

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

[0733] SEQ ID NO: 82 [stapled scFv BHA10(VL-VH)] DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPC GSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSS

[0734] SEQ ID NO: 85 (HC B21M C-stapled BHA (VL-VH), IgG1s, knob, with pA mutation) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVY TLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFS GSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPCGSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSS

[0735] SEQ ID NO: 79 (HC B21M (RSV) IgG1s knob with pA mutation) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAEAPEAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

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

[0737] SEQ ID NO: 81 [LC B21M (RSV)] DIVMTQSPDSLAVSLGERATINCRASQSVDYNGISYMHWYQQKPGQPPKLLIYAASNPESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQIIEDPWTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0738] v.COVA1480 was generated by co-expression of the anti-EDB antibody EDBmAb1 heavy chain carrying the N-terminally stapled scFv BHA10 (VH-VL orientation SEQ ID NO: 77) fusion (SEQ ID NO: 86, including 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 15E).

[0739] SEQ ID NO: 77 [stapled scFv BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0740] SEQ ID NO: 86 (HC L19 N-stapled BHA10 (VH-VL), IgG1s, knob, with pA mutation) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAP KSLISSASYRYSGVPSRFSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0741] SEQ ID NO: 87 (HC L19 IgG1s knob with pA mutation) VQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0742] SEQ ID NO: 88 (HC L19 IgG1s Whole) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVCTLPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0743] SEQ ID NO: 89 (LC L19) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0744] vi. COVA1481 was generated by co-expression of the anti-EDB antibody EDBmAb1 heavy chain carrying the N-terminally stapled scFv BHA10 (VL-VH orientation SEQ ID NO: 82) fusion (SEQ ID NO: 90, including 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 15F).

[0745] SEQ ID NO: 82 [stapled scFv BHA10(VL-VH)] DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPC GSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSS

[0746] SEQ ID NO: 90 (HC L19 N-stapled BHA10 (VL-VH), IgG1s, knob, with pA mutation) DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPCGSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPG NVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGP AVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0747] SEQ ID NO: 87 (HC L19 IgG1s knob with pA mutation) VQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0748] SEQ ID NO: 88 (HC L19 IgG1s Whole) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVCTLPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0749] SEQ ID NO: 89 (LC L19) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0750] vii. COVA1482 was generated by co-expression of the anti-EDB antibody EDBmAb1 heavy chain carrying a C-terminally stapled scFv BHA10 (VH-VL orientation SEQ ID NO: 77) fusion (SEQ ID NO: 91, including 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 15G).

[0751] SEQ ID NO: 77 [stapled scFv BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0752] SEQ ID NO: 91 (HC L19 C-stapled BHA10 (VH-VL), IgG1s, knob, with pA mutation) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTL PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0753] SEQ ID NO: 87 (HC L19 IgG1s knob with pA mutation) VQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0754] SEQ ID NO: 88 (HC L19 IgG1s Whole) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVCTLPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0755] SEQ ID NO: 89 (LC L19) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0756] viii. COVA1483 was generated by co-expression of the anti-EDB antibody EDBmAb1 heavy chain carrying a C-terminally stapled scFv BHA10 (VL-VH orientation SEQ ID NO: 82) fusion (SEQ ID NO: 92, including 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 15H).

[0757] SEQ ID NO: 82 [stapled scFv BHA10(VL-VH)] DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPC GSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSS

[0758] SEQ ID NO: 92 (HC L19 C-stapled BHA10 (VL-VH), IgG1s, knob, with pA mutation) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTL PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFSG SGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPCGSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSS

[0759] SEQ ID NO: 87 (HC L19 IgG1s knob with pA mutation) VQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0760] SEQ ID NO: 88 (HC L19 IgG1s Whole) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVCTLPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0761] SEQ ID NO: 89 (LC L19) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0762] ix.COVA14107 was generated by co-expression of the anti-EDB antibody EDBmAb1 heavy chain carrying the C-terminally stapled scFv BHA10 (VH-VL orientation, VL3 Y36F_S49Y_F87Y SEQ ID NO: 93) fusion (SEQ ID NO: 94, containing 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 15I).

[0763] SEQ ID NO: 93 [stapled scFv(VL3_Y36F_S49Y_F87Y)BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWFQQKPGKAPKSLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDTYPFTFGCGTKVEIK

[0764] SEQ ID NO: 94 (HC L19 C-stapled (VL3_Y36F S49Y_F87Y) BHA (VH-VL), IgG1s, knob, with pA mutation) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTL PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWFQQKPGKAPKSLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDTYPFTFGCGTKVEIK

[0765] SEQ ID NO: 87 (HC L19 IgG1s knob with pA mutation) VQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0766] SEQ ID NO: 88 (HC L19 IgG1s Whole) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVCTLPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0767] SEQ ID NO: 89 (LC L19) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0768] x.COVA14108 was generated by co-expression of the anti-EDB antibody EDBmAb1 heavy chain carrying a C-terminally stapled scFv BHA10 (VH-VL orientation, VH_CDR1_Y33A SEQ ID NO: 95) fusion (SEQ ID NO: 96, including 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] SEQ ID NO: 95 [stapled scFv(VH_CDR1_Y33A)BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYALHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0770] SEQ ID NO: 96 (HC L19 C-stapled (VH_CDR1_Y33A) BHA10 (VH-VL), IgG 1s, knob, with pA mutation) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTL PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYALHWVRQAPGCGLEWMGWIYPGNVHAQYNEKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0771] SEQ ID NO: 87 (HC L19 IgG1s knob with pA mutation) VQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0772] SEQ ID NO: 88 (HC L19 IgG1s Whole) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVCTLPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0773] SEQ ID NO: 89 (LC L19) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

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

[0775] SEQ ID NO: 77 [stapled scFv BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0776] SEQ ID NO: 97 (HC L19 C-stapled BHA10 (VH-VL), IgG1s, knob, with pA mutation) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTL PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0777] SEQ ID NO: 98 (HC L19 IgG1s knob) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0778] SEQ ID NO: 88 (HC L19 IgG1s Whole) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVCTLPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0779] SEQ ID NO: 89 (LC L19) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

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

[0781] SEQ ID NO: 77 [stapled scFv BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0782] SEQ ID NO: 99 (HC B21M C-stapled BHA10 (VH-VL), IgG1s, knob, no pA mutations) QITLKESGPTLVKPTQTLLTTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVY TLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEK FKGRVTITADKSTSTAYMELSSLRSEDTAVYCARSWEGFPYWGQGTTVTVSSGGGSGGGSGCPPCGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

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

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

[0785] SEQ ID NO: 81 [LC B21M (RSV)] DIVMTQSPDSLAVSLGERATINCRASQSVDYNGISYMHWYQQKPGQPPKLLIYAASNPESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQIIEDPWTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

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

[0787] SEQ ID NO: 77 [stapled scFv BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0788] SEQ ID NO: 101 (MSLNmAb1 HC C-stapled BHA10 (VH-VL), IgG1s, knob, with pA mutation) QVQLQQSGPELEKPGASVKISKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYT LPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0789] SEQ ID NO: 102 (MSLNmAb1 HC, IgG1s, knob, with pA mutation) QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSG TPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEK TISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0790] SEQ ID NO: 103 (HC MSLNmAb1 IgG1s Whole) QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSG TPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEK TISKAKGQPREPQVCTLPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0791] SEQ ID NO: 104 (LC MLSNmAb1) DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0792] All of the above constructs can be expressed and purified with high yield and purity (see Table 11 below), demonstrating that the bispecific constructs incorporating spFvs provided herein have good biophysical properties.

[0793] [Table 11]

[0794] 6.7 Example 7: EDB-Dependent In Vitro LTBR Activation—NF-κB Luciferase Reporter Assay To demonstrate that EDB / LTBR bispecifics can activate LTBR in an EDB-dependent manner, the activity of the compounds 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 cellular development and immune homeostasis. Activation of NF-κB via tumor necrosis factor receptors (TNFRs) or TNFR superfamily members (e.g., LTBR) occurs upon engagement with their respective ligands. The A549 lung epithelial cell line naturally expresses LTBR, ​​and an NF-κB luciferase reporter construct was stably integrated into the genome of the A549 lung epithelial cell line. Following activation by a stimulant, the endogenous NF-κB transcription factor binds to a DNA response element and induces 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] SEQ ID NO: 105 (fibronectin domain 7B89) PLSPPTNLHLEANPDTGVLTVSWERSTTPDITGYRITTTPTNGQQGNSLEEVVHADQSSCTFDNLSPGLEYNVSVYTVKDDKESVPISDTIIPEVPQLTDLSFVDITDSSIGLRWTPLNSSTIIGYRITVVAAGEGIPIFEDFVDSSVGYYTVTGLEPGIDYDISVITLINGGESAPTTLTQQTA VPPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLIGQQSTHHHHHH

[0797] SEQ ID NO: 106 (fibronectin domain 789) PLSPPTNLHLEANPDTGVLTVSWERSTTPDITGYRITTTPTNGQQGNSLEEVVHADQSSCTFDNLSPGLEYNVSVYTVKDDKESVPISDTIIPAVPPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVA ELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREEESPLLIGQQSTHHHHHH

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

[0799] After 18 hours of incubation, luciferase activity was detected using the BIO-GLO Luciferase Assay System (Promega, Madison, WI). Luminescence was measured using a TECAN M 1000Pro instrument with an integration time of 500 ms. From the resulting relative light units (RLU), the fold induction of LTBR signaling was calculated as follows: fold induction = 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 deviations, were plotted using GRAPHPAD Prism, and where applicable, a nonlinear fit was applied (log(agonist) vs. response (variable slope - 3 parameters)). To fit the data, x values ​​(compound concentrations) were transformed using the x = Log(x) function in GRAPHPAD Prism.

[0801] COVA1482 was compared with COVA1456 in 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, was stabilized using the stapled platform described herein (i.e., stabilized via VH / linker and VL linker disulfide bonds), while COVA1456 is disulfide stabilized between VH and VL (i.e., stabilized via VH / VL disulfide bonds). Figure 16A shows that both COVA1482 and COVA1456 potently activated the LTBR in an EDB-dependent manner. The corresponding isotype controls, COVA1486 and COVA1462, did not activate the LTBR (Figure 16A). These results indicate that the incorporation of spFvs into multispecific molecules did not adversely affect the activity of the multispecific molecules. The 2:1 bispecific EDB / LTBR antibodies (COVA1482 or COVA1456) showed increased potency in inducing NF-κB signaling in this reporter assay. The average EC calculated for COVA1482 across several assays with the same experimental setup 50 is approximately 30 pM ± 10 pM, and the control 1:1 heterodimer construct has an EC of approximately 3 nM in the assay (data not shown). 50 This shows that 2:1 bispecificity can be 100-fold more potent than 1:1 bispecificity, which can be explained by the increased clustering of LTBR binding sites achieved with two binding sites to the TAA.

[0802] To study the effect of affinity for 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 the 2:1 bispecifics COVA14107 and COVA14108 (see Table 9).

[0803] SEQ ID NO: 107 [stapled scFv(VL3_Y36F_S49Y_F87Y)BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWFQQKPGKAPKSLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDTYPFTFGCGTKVEIK

[0804] SEQ ID NO: 108 [stapled scFv(VH_CDR1_Y33A)BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYALHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0805] The resulting bispecifics were tested in an A549 NF-κB reporter assay to confirm the effect of affinity on LTBR activation. Figure 16B shows that lower affinity for LTBR corresponds to a reduced ability of the bispecifics to activate LTBR in a TAA-dependent manner in this assay. The data also demonstrated that incorporating spFv into multispecific molecules did not adversely affect the activity of the molecules.

[0806] As described in Example 6, mutations (WO 2010 / 151792) that abrogate binding to Protein A (used for antibody purification) were introduced into the Fc region of some constructs to facilitate purification of the desired heterodimer. COVA14133 was generated without these mutations, and its activity was compared to that of COVA1482, indicating that the mutations in the Fc region did not affect the activity of the bispecific. COVA14133 and COVA1482, as well as their respective isotype controls, COVA14136 and COVA1486, were compared in an A549 NF-κB reporter assay. Figure 16C shows that COVA14133 activated the LTBR in a TAA-dependent manner with similar efficiency to COVA1482, demonstrating that the mutations in the Fc did not affect the ability of the bispecific to activate the LTBR or affect spFv function.

[0807] In conclusion, COVA14133 was shown to have a potent ability to activate LTBR in a TAA-dependent manner.

[0808] 6.8 Example 8: EDB-Dependent In Vitro LTBR Activation—A375 / WI38VA Subline 2RA Co-Culture Cell Assay To verify 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)) leads to the release of cytokines and chemokines and upregulation of the adhesion molecule ICAM-1 on A375 cells, and whether incorporation of spFv does not adversely affect activity, an A375 / WI38VA sublineage 2RA coculture assay was performed. WI38VA sublineage 2RA (ATCC® CCL75.1™) cells were seeded into 96-well plates at a density of 5000 cells / well and cultured in growth medium (MEM) at 37°C / 5% CO. The cells were incubated for 48 hours in a 1:5 dilution series of the compounds to be tested as 2x stocks in assay medium (DMEM + 10% heat-inactivated FBS) (with or without 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 in assay medium (DMEM + 10% heat-inactivated FBS) (final concentrations tested ranged from 40 nM to 0.5 pM). Prior to incubation in coculture with WI38VA subline 2RA cells, A375 cells (ATCC® CRL-1619™) were labeled with CELLTRACE Violet (CTV, Invitrogen, Carlsbad, CA). For labeling, 10 x 10 cells were diluted in 5% FBS in PBS. 6 The cell suspension, with a concentration of 0.4 × 10 cells / ml and 2.5 μM CTV, was incubated for 5 minutes at room temperature, protected from light. The cells were then washed and diluted to 0.4 × 10 cells / ml. 6 Cells were resuspended in assay medium at a density of 1000 cells / ml. Following careful removal of culture medium from plates containing 48-hour WI38VA sublineage 2RA cultures, 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 for 24 hours at 37°C / 5% CO2.

[0809] After 24 hours of incubation, the supernatant was clarified by centrifugation and saved for cytokine and chemokine measurement using the MSD assay. Cells were further processed for ICAM-1 measurement by flow cytometry.

[0810] 6.8.1 Detection of ICAM-1 by Flow Cytometry Any remaining medium in the 96-well plate was carefully removed, and cells were detached with Accutase, transferred to a DeepWell 96-well plate (pooled in triplicate), 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 stain (Invitrogen), single stain, or combined stain, were diluted as shown in Table 12.

[0811] [Table 12]

[0812] The cells were centrifuged at 400 x g for 4 minutes at 4°C, the supernatant was discarded, and 50 μl of antibody solution was prepared as described in Table 12. The cells and antibodies were incubated in the dark for 30 minutes at 4°C. After incubation, 120 μl was added to each well, and the cells were then centrifuged at 400 x 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 x g for 4 minutes at 4°C and resuspended in 100 μL of FACS buffer. The cells were measured using a MACS Quant instrument in screen mode at high flow rate, acquiring 49 μl per well. Data were analyzed using Flowlogics Software (version 700.2A) and plotted using Graphpad Prism.

[0813] 6.8.2 Cytokine Measurement in Supernatants of Treated Cells Using the MSD Platform Several cytokines known to be under the control of NF-κB signaling have been measured using the MSD platform and multiplex MSD plates. Some examples of cytokines measured are listed here. ■RANTES: R-Plex antibody set using human RANTES (MSD) ■ I-TAC, IP-10, MIP-3b: Using 3-PLEX cytokine release assay (MSD) IL-8, IP-10, MIP-3b: using the 3-PLEX cytokine release assay (MSD), and IL-12p70, IL-6, TNF-α, MIP-3α, SDF-1α: Using 5-PLEX cytokine release assay (MSD)

[0814] Cytokine concentrations in the supernatants 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-conjugated capture antibodies. The plates were incubated overnight at 2-8°C with shaking. The next day, the plates were washed with PBST (PBS + 0.05% Tween-20) using a plate washer (Biotek; Winooski, VT). (2) Calibrator standards and detection antibody solutions were prepared. (3) The supernatant was diluted 1:3 or 1:5 depending on the availability of material.

[0815] 6.8.3 Assay Protocol: Step 1: Samples or calibrator standards were added to the plate and the plate was incubated for 1 hour at room temperature with shaking. Step 2: The plate was washed and the detection antibody was added. The plate was incubated at room temperature for 1 hour with shaking. Step 3: Plates were washed and 2x Read Buffer T was added. Plates were analyzed on an MSD instrument.

[0816] Data were analyzed using MESOSCALE software (MSD Discovery 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 previously been shown that NF-κB signaling can lead to the upregulation of ICAM-1 on the surface of cells (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 coculture incubation with the EDB / LTBR bispecific. As an example, Figure 17 shows the upregulation of ICAM-1 after incubation with the EDB / LTBR bispecific COVA 1482, demonstrating the functionality of the LTBR-binding spFv. The isotype control molecule COVA1486 did not cause ICAM-1 upregulation. These findings indicated that the ability to cluster the LTBR scFv via binding to EDB is a prerequisite for LTBR activation and, consequently, ICAM-1 upregulation.

[0818] 6.8.5 Results - Cytokine Measurement in Supernatants of Treated Cells Several cytokines and chemokines expressed as a result of LTBR activation were measured in the supernatants of cocultures treated with the 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). The non-targeted LTBR mAb1-derived scFv in COVA14136 did not activate the LTBR, ​​and as a result, cytokine concentrations in the supernatants did not increase above background. Background was represented by the levels achieved with the B21M antibody or COVA1440 (the B21M isotype control mAb) (shown as a single concentration in the plot). The results demonstrate that the LTBR-binding spFv is functional in vitro.

[0819] Taken together, the upregulation of ICAM-1 and cytokine secretion upon LTBR activation confirmed the expected effects on cells that LTBR activation may have.

[0820] In this example, it was demonstrated that the molecules provided herein achieved efficient tumor-associated antigen (here, EDB-containing fibronectin)-dependent activation of LTBR.

[0821] 6.9 Example 9: Mesothelin-Dependent In Vitro LTBR Activation—Co-Culture Cell Assay Using A549 NF-κB Reporter Cells and CHOK1-huMSLN or H226 Examples 7 and 8 demonstrate that a bispecific antibody comprising the spFv construct provided herein, targeting EDB (a tumor-associated antigen in the extracellular matrix) and LTBR, ​​highly efficiently activated LTBR in a tumor antigen-dependent manner. To determine whether this finding applies to any tumor antigen, regardless of its location (deposited in the extracellular matrix or on the cell surface of tumor cells), a bispecific 2:1 antibody targeting mesothelin (MSLN), a tumor-associated antigen expressed in different types of tumors (Hassan and Ho, Eur. J. Cancer, 44:46-53 (2008)), and LTBR was 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 targeting LTBR and tumor-associated antigens (e.g., mesothelin) present on the cell surface of tumor cells can efficiently activate LTBR in a tumor-dependent manner, a co-culture cell assay was used. The co-culture assays used were 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 H226 cells (expressing approximately 200,000 copies of mesothelin and 10,000 copies of LTBR) were seeded at 10,000 cells per well (75 μl of assay medium, DMEM + 10% FBS-HI) in 96-well tissue culture plates and incubated in their growth medium (MEM + 2 mM glutamine + 10% FBS-HI + 10 μg / ml puromycin and RPMI-1640 + 10% FBS + 1 mM Na-pyruvate, respectively) at 37°C / 5% CO2 for 6 hours to allow the cells to adhere to the plates.

[0823] 6.9.2 Preparation of Compounds Compounds were tested at concentrations ranging from 100 nM to 1.3 pM. Four-fold 1:5 serial dilutions of compounds were prepared in assay medium (DMEM + 10% FBS-HI) and stored at 4°C until use.

[0824] 6.9.3 Preparation and Addition of A549 Reporter Cells A549 reporter cells were detached from cell culture flasks with Accutase / EDTA and transferred to assay medium (DMEM + 10% FBS-HI). A total of 20,000 A549 reporter cells per well were added to the plate containing H226 cells, and 50 μL of pre-diluted compound was added to each well and incubated at 37°C / 5% CO for 20 hours.

[0825] 6.9.4 Measurement of luminescence in treated co-cultures After 20 hours of incubation, luciferase activity was detected using the 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 ms. From the resulting relative light units (RLU), the fold induction of LTBR signaling was calculated as follows: fold induction = 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 deviations, were plotted using GRAPHPAD Prism, and where applicable, a nonlinear fit was applied (log(agonist) vs. response (variable slope - 3 parameters)). To fit the data, x values ​​(compound concentrations) were transformed using the x = Log(x) function in GRAPHPAD Prism.

[0827] 6.9.5 Cytokine Measurement in Supernatants of Treated Cells Using the MSD Platform Several cytokines known to be under the control of NF-κB signaling can be measured using the MSD platform and multiplex 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-conjugated capture antibodies. The plates were incubated overnight at 2-8°C with shaking. The next day, the plates were washed with PBST (PBS + 0.05% Tween-20) using a plate washer (Biotek; Winooski, VT). (2) Calibrator standards and detection antibody solutions were prepared. (3) The supernatant was diluted 1:3 or 1:5 depending on the availability of material.

[0829] 6.9.6 Assay Protocol: Step 1: Samples or calibrator standards were added to the plate and the plate was incubated for 1 hour at room temperature with shaking. Step 2: The plate was washed and the detection antibody was added. The plate was incubated at room temperature for 1 hour with shaking. Step 3: Plates were washed and 2x Read Buffer T was added. Plates were analyzed on an MSD instrument.

[0830] Data were analyzed using MESOSCALE software (MSD Discovery 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 Assays A coculture assay using A549 reporter cells and H226 cells was performed to verify whether COVA14146 could activate LTBR in a more physiological system where LTBR and mesothelin (as well as other tumor-associated antigens, such as EGFR) are expected to be coexpressed on the cell surface of tumor cells due to its widespread 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 was unable to efficiently activate LTBR. As expected, Figure 19B shows that RANTES was secreted by cells treated with COVA14146 to the same extent as cells treated with the isotype control molecule COVA1486, confirming that LTBR was unable to be activated under these conditions.

[0832] 6.10 Example 10: Bispecific antibodies that specifically bind to LTBR and other TAAs present in the extracellular matrix The above example demonstrated that a bispecific antibody targeting LTBR and a TAA expressed in the extracellular matrix, where the EDB of fibronectin can selectively activate LTBR in tumor tissue expressing the EDB, is more generally applicable to bispecific antibodies targeting LTBR and another TAA present in the extracellular matrix by providing two or more examples of such a TAA, namely, domain A2 of tenascin-C and extradomain A of fibronectin.

[0833] Generally, following the methods described in the above examples, further bispecific antibodies were prepared that bind to LTBR and other TAAs present in the extracellular matrix, namely domain A2 of tenascin-C (TnCA2) or extra domain A of fibronectin (EDA). The sequences of these TAAs are, for example: -TnCA2: UniProt accession number P24821.3; -EDA: Described in UniProt accession number P02751.

[0834] Additionally, antibodies against these TAAs have been previously described, for example, in: -TnCA2: International Publication No. 2011 / 020783, -EDA: European Patent No. 2142567.

[0835] Exemplary sequences of binding domains for these targets used in this example (by combining with LTBR scFv to generate multispecific binding molecules of the invention) are provided as follows: TnCA2: VH of SEQ ID NO: 109 and VL of SEQ ID NO: 110, -EDA: VH of SEQ ID NO: 111 and VLV of SEQ ID NO: 112.

[0836] These sequences are as follows: SEQ ID NO: 109 (VH of anti-TnCA2 Ab 2B10) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLYGYAYYGAFDYWGQGTTVTVSS

[0837] SEQ ID NO: 110 (VL of anti-TnCA2 Ab 2B10) DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGGGSGTEFTLTISSLQPEDFATYYCLQNGLQPATFGQGTKVEIK

[0838] SEQ ID NO: 111 (VH of anti-EDA Ab F8) EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS

[0839] SEQ ID NO: 112 (VL of anti-EDA Ab F8) EIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK

[0840] Bispecific antibodies binding to LTBR and these TAAs were prepared and tested for TAA-specific activation of LTBR in a TAA-dependent in vitro LTBR activation-NF-κB luciferase reporter assay according to a procedure similar to that described in Example 7.

[0841] Asymmetric antibodies with a 2:1 stoichiometry were generated as follows: COVA14198 was produced by co-expression of an anti-EDA antibody heavy chain carrying a C-terminally stapled scFv BHA10 (VH-VL orientation SEQ ID NO:77) fusion (SEQ ID NO:113, including SEQ ID NO:114) with an anti-EDA antibody heavy chain (HC, SEQ ID NO:115) and light chain (LC; SEQ ID NO:116).

[0842] The sequences are listed below. SEQ ID NO: 77 [stapled scFv BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0843] SEQ ID NO: 113 (BHA10 stapled (VH-VL) scFv C-terminal fusion, IgG1 sigma, F8 HC with knob mutation) EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYT LPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0844] SEQ ID NO: 114 (F8 HC, IgG1 Sigma, Knob Mutation) EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPPAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0845] SEQ ID NO: 115 (F8 HC, IgG1 sigma and hole mutations) EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPPAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVCTLPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0846] SEQ ID NO: 116 (F8 LC) EIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0847] COVA14202 was generated by co-expression of the anti-domain A2 of the tenascin-C antibody heavy chain carrying the C-terminally stapled scFv BHA10 (VH-VL orientation SEQ ID NO: 77) fusion (including SEQ ID NO: 117 and SEQ ID NO: 118) with the anti-domain A2 heavy chain (HC, SEQ ID NO: 119) and light chain (LC, SEQ ID NO: 120) of the tenascin-C antibody. The sequences are listed below.

[0848] SEQ ID NO: 77 [stapled scFv BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0849] SEQ ID NO: 117 (BHA10 stapled (VH-VL) scFv C-terminal fusion, IgG1 sigma, 2B10 HC with knob mutation) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLYGYAYYGAFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVY TLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEK FKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGP

[0850] SEQ ID NO: 118 (2B10 HC, IgG1 Sigma, Knob Mutation) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLYGYAYYGAFDYWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPRE...

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; or (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; or (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 Cys at H3 and the VL comprises 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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