Materials and methods for improved single chain variable fragments

Abstract

To provide antigen-binding single-chain variable fragments (scFv) that can be optionally incorporated into multispecific and heterologous molecules in which low stability and aggregation tendency have been improved.SOLUTION: Provided is an isolated single chain variable fragment (scFv) comprising a heavy chain variable region (VH), a linker (L) and a light chain variable region (VL), the scFv comprising: a) a first disulfide bond between a structurally conserved surface exposed VH cysteine (Cys) and a first L Cys; b) a second disulfide bond between a structurally conserved surface exposed VL Cys and a second L Cys; or c) 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.SELECTED DRAWING: None

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of U.S. Provisional Patent Application No. 62 / 946,89 7, filed on December 11, 2019, U.S. Provisional Patent Application No. 62 / 946,886, filed on December 11, 2019, U.S. Provisional Patent Application No. 62 / 946,882, 201 9, filed on December 11, 2019, U.S. Provisional Patent Application No. 62 / 946,877, 2019, 1 filed on December 11, 2019, U.S. Provisional Patent Application No. 62 / 946,865, August 15, 2019 filed on August 15, 2019, U.S. Provisional Patent Application No. 62 / 887,529, filed on August 15, 2019 U.S. Provisional Patent Application No. 62 / 887,527, filed on August 15, 2019, U.S. Provisional Patent Application No. 62 / 887,524, filed on August 15, 2019 U.S. Provisional Patent Application No. 62 / 887,519, and U.S. Provisional Patent Application No. 62 / 887,514, filed on August 15, 2019, and each of them is hereby incorporated by reference in its entirety into this specification.

[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 Art

[0004] ​​​​​An antigen-binding single-chain variable fragment (scFv) is a therapeutic agent that is widely used as an imaging agent, a diagnostic agent, or as part of a heterologous molecule such as a bispecific molecule and is a module that can be obtained. One of the problems with scFv is its low stability and tendency to aggregate (W orn and Pluckthun (2001) J Mol Biol 305:98 9-1010; Rothlisberger et al., (2005) J Mol Biol 347:773-789; Gross et al., (1989) Tran splant 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) and has been reinvestigated igated.

[0005] Therefore, there is a need for an improved scFv design that can be optionally incorporated into bispecific and heterologous molecules igated.

[0006] (Summary) 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 a first disulfide bond between a structurally conserved surface-exposed VH 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 a third disulfide bond between a structurally conserved surface-exposed VH Cys and a structurally conserved surface-exposed VL Cys , and the scFv has improved stability and reduced aggregation tendency compared to a wild-type scFv . bond, or 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, are included, to provide an scFv. 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, are included, to provide an scFv. 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, are included, to provide an scFv.

[0007] The present disclosure also provides an isolated scFv comprising VH, L, and VL, wherein VH comprises VH Cys at a structurally conserved surface-exposed VH framework residue position, L comprises a first L Cy, or VL comprises VL Cys at a structurally conserved surface-exposed VL framework residue position, L comprises a second L Cy, or VH comprises VH Cys at a structurally conserved surface-exposed VH framework residue position, VL comprises VL Cys at a structurally conserved surface-exposed VL framework residue position, L comprises a first L Cys and a second L Cys, and VH Cys and the first L Cys can form a disulfide bond, and VL Cys and the second L Cy s can form a disulfide bond, to provide an scFv.

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

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

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

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

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

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

[0014] The present disclosure also provides an scFv comprising VH, L, and VL, wherein 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, providing an scFv wherein the scFv is in the VL-L-VH orientation.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0035] The present disclosure also provides a kit comprising the spFv 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 a first disulfide bond between a structurally conserved surface-exposed VH 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 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. (VL), wherein the scFv comprises a first disulfide bond between a structurally conserved surface-exposed VH cysteine (Cys) and a first L Cys, A first disulfide bond between a structurally conserved surface-exposed VH cysteine (Cys) and a first L Cys, A first disulfide bond, A second disulfide bond between a structurally conserved surface-exposed VL Cys and a second L Cys, or A second disulfide bond, or 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, A second disulfide bond between a structurally conserved surface-exposed VL Cys and a second L Cys, Provided is 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 VH, L, and VL, wherein VH comprises a VH Cys at a structurally conserved surface-exposed VH framework residue position and L comprises a first L Cy, and VL comprises a VL Cys at a structurally conserved surface-exposed VL framework residue position and L comprises a second L Cy, or VH comprises a VH Cys at a structurally conserved surface-exposed VH framework residue position and 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, and VH Cys and the first L Cys can form a disulfide bond, and VL Cys and the second L Cy s can form a disulfide bond, providing a multispecific molecule.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0054] The present disclosure also relates to a multispecific molecule comprising an scFv comprising VH, L, and VL, wherein VH contains Cys at H40, VL contains Cys at L3, L contains the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7, Provided is a multispecific molecule in which the scFv is in the VH-L-VL orientation.

[0055] The present disclosure also relates to a multispecific molecule comprising an scFv comprising VH, L, and VL, wherein VH contains Cys at H46, VL contains Cys at L100, L contains the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7, Provided is a multispecific molecule in which the scFv is in the VH-L-VL orientation.

[0056] The present disclosure also relates to a multispecific molecule comprising an scFv comprising VH, L, and VL, wherein VH contains Cys at H46, VL contains Cys at L102, L contains the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7, Provided is a multispecific molecule in which the scFv is in the VH-L-VL orientation.

[0057] The present disclosure also relates to a multispecific molecule comprising an scFv comprising VH, L, and VL, wherein VH contains Cys at H46, VL contains Cys at L5, L contains the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7, Provided is a multispecific molecule in which the scFv is in the VH-L-VL orientation.

[0058] The present disclosure also provides a multispecific molecule comprising a single-chain variable fragment (scFv) comprising VH, L, and VL, wherein VH comprises Cys at H46, VL comprises Cys at L3, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, or 7, and the scFv is in the VH-L-VL orientation.

[0059] The present disclosure also provides a pharmaceutical composition comprising the multispecific molecule 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 a first disulfide bond between a structurally conserved surface-exposed VH 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 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 a single-chain variable fragment (scFv) comprising VH, L, and VL, wherein VH comprises VH Cys at a structurally conserved surface-exposed VH framework residue position, L comprises a first L Cy, or VL comprises VL Cys at a structurally conserved surface-exposed VL framework residue position, L comprises a second L Cy, or VH comprises VH Cys at a structurally conserved surface-exposed VH framework residue position, wherein VL contains a VL Cys at a structurally conserved surface-exposed VL framework residue position, L contains a first L Cys and a second L Cys, and VH Cys and the first L Cys can form a disulfide bond, and VL Cys and the second L Cy s can form a disulfide bond, providing a heteromolecule.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0084] In yet another aspect, the present disclosure is 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, and providing a linker (L) that comprises a first L Cy or is engineered to comprise the same. and engineering VH to include VH Cys at a structurally conserved surface-exposed VH framework residue position and forming a disulfide bond between VH Cys and a first L Cys to prepare a stabilized scF v, and providing a process comprising the same.

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

[0086] The present disclosure also provides a process for preparing a stabilized scFv, comprising providing VH and VL that form an antigen-binding domain and providing an L that includes or is engineered to include a first L Cys and a second L Cys and engineering VH to include VH Cys at a structurally conserved surface-exposed VH framework residue position and engineering VL to include VL Cys at a structurally conserved surface-exposed VL framework residue position and forming a disulfide bond between VH Cys and the first L Cys and forming a disulfide bond between VL Cys and the second L Cys to prepare a stabilized scFv, and providing a process comprising the same. and

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

[0088] In yet another aspect, the present disclosure provides a method for the preparation of a heavy chain variable region (VH), a means for linking (L) and a light chain variable region (L). An isolated single chain variable fragment (scFv) comprising a chain variable region (VL), wherein the scFv comprises: The first amino acid sequence between the structurally conserved surface-exposed VH cysteine ​​(Cys) and the first L Cys 1 disulfide bond, A second disulfide between the structurally conserved surface-exposed VL Cys and a second L Cys bond, or The first disulfide between the structurally conserved surface-exposed VH Cys and the first L Cys The second bond between the structurally conserved surface-exposed VL Cys and the second L Cys is The present invention provides an scFv comprising:

[0089] The present disclosure also provides a method for antigen binding, a linker (L), a light chain variable region (VL), and 1. An isolated single chain variable fragment (scFv) comprising: A structurally conserved surface-exposed antigen-binding site cysteine ​​(Cys) and the first L Cys A first disulfide bond between A second disulfide between the structurally conserved surface-exposed VL Cys and a second L Cys bond, or Between the structurally conserved surface-exposed antigen-binding cysteine ​​Cys and the first L Cys The first disulfide bond and the structurally conserved surface-exposed VL Cys and the second LC a second disulfide bond between the srcDNA and srcDNA, and a second disulfide bond between the srcDNA and srcDNA.

[0090] The present disclosure also provides a single-chain variable fragment (scFv) comprising a heavy-chain variable region (VH), a linker (L), and means for antigen binding, wherein the scFv comprises a first disulfide bond between a structurally conserved surface-exposed VH cysteine (Cys) and a first L Cys, a second disulfide bond between a structurally conserved surface-exposed antigen-binding means Cys and a second L Cys, or 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. 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 a first disulfide bond between a structurally conserved surface-exposed VH 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 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. The present disclosure also provides a multispecific molecule comprising a single-chain variable fragment (scFv) comprising means for antigen binding, a linker (L), and a light-chain variable region (VL), wherein the scFv

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

[0092] The present disclosure also provides a multispecific molecule comprising a single-chain variable fragment (scFv) comprising 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 VH cysteine (Cys) and a first L Cys, Structurally conserved surface-exposed antigen-binding means cysteine (Cys) and the first L Cys and a first disulfide bond between Structurally conserved surface-exposed VL Cys and a second L Cys and a second disulf ide bond, or Structurally conserved surface-exposed antigen-binding means cysteine Cys and the first L Cys and a first disulfide bond between and a second disulfide bond between the structurally conserved surface-exposed VL Cys and the second L C ys, to provide a multispecific molecule.

[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 is 、 a 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 the second L Cys 、or a first disulfide bond between the structurally conserved surface-exposed VH Cys and the first L Cys and a second disulf ide bond between the structurally conserved surface-exposed antigen-binding means Cys and the second L Cys 、to provide a multispecific molecule.

[0094] 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 is a 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 VL Cys and the second L Cys An ID bond, or a first disulfide between a structurally conserved surface-exposed VH Cys and a first L Cys an ID bond and a second disulfide between a structurally conserved surface-exposed VL Cys and a second L Cys are provided, a heterologous molecule comprising.

[0095] The present disclosure also provides a heterologous molecule comprising a means for antigen binding, a linker (L), and a variable light chain region (VL) and a single-chain variable fragment (scFv), 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 an ID bond, or 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 C ys, are provided, a heterologous molecule comprising.

[0096] The present disclosure also provides a heterologous molecule comprising a variable heavy chain region (VH), a linker (L), and a means for antigen binding and a single-chain variable fragment (scFv), wherein the scFv comprises a first disulfide bond between a structurally conserved surface-exposed VH cysteine (Cys) and a first L Cys ; a second disulfide bond between a structurally conserved surface-exposed antigen-binding means Cys and a second L Cys an ID bond, or 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 are provided, a heterologous molecule comprising.

[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 a composition comprising means for stabilizing an scFv.

[0100] The present disclosure also provides a composition comprising means for increasing the thermal stability of an scFv.

[0101] The present disclosure also provides a multispecific molecule comprising means for stabilizing an scFv.

[0102] The present disclosure also provides a multispecific molecule comprising means for increasing the thermal stability of an scFv. to provide.

[0103] The present disclosure also provides a heterologous molecule comprising means for stabilizing an scFv.

[0104] The present disclosure also provides a heterologous molecule comprising means for increasing the thermal stability of an scFv. . BRIEF DESCRIPTION OF THE DRAWINGS

[0105]

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[0106] (Detailed description) The disclosed methods are made in connection with the accompanying drawings, which form a part of this disclosure, and the following detailed description. They can be more readily understood by reference to the following detailed description. The disclosed methods are not limited to the specific methods described and / or shown herein. Further, the terms used herein are for the purpose of illustrating particular embodiments by way of example only and are not intended to be limiting. It should be understood that.

[0107] All patents, published patent applications, and publications cited herein are hereby incorporated by reference in their As if the whole of it were incorporated in the same manner as described in this specification.

[0108] When a list is presented, unless otherwise specified, it should be understood that each individual element of the list and all combinations of the list are separate embodiments. For example, a list of embodiments presented as "A, B, or C" should be interpreted as including 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 context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes combinations of two or more cells, and the like.

[0110] The transitional phrases "comprising", "consisting essentially of", and "consisting of" are intended to imply their generally accepted meaning in patent terminology, i.e., (i) "comprising" is synonymous with "including", "containing", or "characterized by", is inclusive or open-ended and does not exclude additional, unrecited elements or method steps, (ii) "consisting of" excludes any element, step, or ingredient not specified in the claims, and (iii) "consisting essentially of" includes the specified materials or steps, as well as those that do not materially affect the "basic and novel characteristics of the claimed invention. ​​ which does not substantially affect it」restricts the scope of the claims. Embodiments described with respect to the phrase「comprising」(or its equivalent) also provide those independently described with respect to「consisting of」and「consisting essentially of」. For embodiments described with respect to the phrase「comprising」(or its equivalent), they also provide those independently described with respect to「consisting of」and「consisting essentially of」. 「consisting of」and「consisting essentially of」.

[0111] 「about」means within the allowable error range for a specific value determined by a person skilled in the art, which depends to some extent on the method by which the value is measured or determined, i.e., the limitations of the measurement system. Unless otherwise explicitly stated elsewhere in the examples or the specification in the context of a particular assay, result, or embodiment,「about」means within the larger of the range of one standard deviation or 5% in accordance with the practice in the relevant technical field. which depends to some extent on the limitations of the measurement system, i.e., the method by which the value is measured or determined. In the context of a particular assay, result, or embodiment, unless otherwise explicitly stated elsewhere in the examples or the specification,「about」means within the larger of the range of one standard deviation or 5% in accordance with the practice in the relevant technical field. In the context of a particular assay, result, or embodiment, unless otherwise explicitly stated elsewhere in the examples or the specification,「about」means within the larger of the range of one standard deviation or 5% in accordance with the practice in the relevant technical field. In the context of a particular assay, result, or embodiment, unless otherwise explicitly stated elsewhere in the examples or the specification,「about」means within the larger of the range of one standard deviation or 5% in accordance with the practice in the relevant technical field. In the context of a particular assay, result, or embodiment, unless otherwise explicitly stated elsewhere in the examples or the specification,「about」means within the larger of the range of one standard deviation or 5% in accordance with the practice in the relevant technical field.

[0112] 「Alternative scaffold」refers to a single-chain protein framework containing a structured cofactor that associates with a variable domain with high conformational tolerance. The variable domain can be genetically engineered and selected to bind to a specific antigen in order to allow polymorphisms to be introduced without compromising the integrity of the scaffold. The variable domain can be genetically engineered and selected to bind to a specific antigen in order to allow polymorphisms to be introduced without compromising the integrity of the scaffold. The variable domain can be genetically engineered and selected to bind to a specific antigen in order to allow polymorphisms to be introduced without compromising the integrity of the scaffold. The variable domain can be genetically engineered and selected to bind to a specific antigen in order to allow polymorphisms to be introduced without compromising the integrity of the scaffold.

[0113] 「Antibody-dependent cell-mediated cytotoxicity」,「antibody-dependent cell-mediated cytotoxicity」, or「ADCC」is a mechanism that induces cell death, which depends on the interaction between antibody-coated target cells and effector cells with lytic activity, such as natural killer (NK) cells, monocytes, macrophages, and neutrophils, mediated by Fc gamma receptors (FcγR) expressed on the effector cells. 「Antibody-dependent cell-mediated cytotoxicity」,「antibody-dependent cell-mediated cytotoxicity」, or「ADCC」is a mechanism that induces cell death, which depends on the interaction between antibody-coated target cells and effector cells with lytic activity, such as natural killer (NK) cells, monocytes, macrophages, and neutrophils, mediated by Fc gamma receptors (FcγR) expressed on the effector cells. 「Antibody-dependent cell-mediated cytotoxicity」,「antibody-dependent cell-mediated cytotoxicity」, or「ADCC」is a mechanism that induces cell death, which depends on the interaction between antibody-coated target cells and effector cells with lytic activity, such as natural killer (NK) cells, monocytes, macrophages, and neutrophils, mediated by Fc gamma receptors (FcγR) expressed on the effector cells. 「Antibody-dependent cell-mediated cytotoxicity」,「antibody-dependent cell-mediated cytotoxicity」, or「ADCC」is a mechanism that induces cell death, which depends on the interaction between antibody-coated target cells and effector cells with lytic activity, such as natural killer (NK) cells, monocytes, macrophages, and neutrophils, mediated by Fc gamma receptors (FcγR) expressed on the effector cells. 「Antibody-dependent cell-mediated cytotoxicity」,「antibody-dependent cell-mediated cytotoxicity」, or「ADCC」is a mechanism that induces cell death, which depends on the interaction between antibody-coated target cells and effector cells with lytic activity, such as natural killer (NK) cells, monocytes, macrophages, and neutrophils, mediated by Fc gamma receptors (FcγR) expressed on the effector cells.

[0114] "Antibody-dependent cell phagocytosis" or "ADCP" refers to a 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., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, portions thereof, or combinations 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 part of a protein that binds to an antigen. Antigen-binding fragments may be synthetic polypeptides, polypeptides obtainable by enzymes, or recombinantly engineered polypeptides, and may include VH, VL, VH and VL, Fab, F(ab’)2, Fd and Fv fragments, domain antibodies (dAb) 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 moieties, H CDR1, HCDR2, and / or HCDR3, and LCDR1, LCDR2, and / or LCDR3, a portion of an immunoglobulin that binds to them, an alternative scaffold that binds to an antigen, and multispecific proteins that include an antigen bound to a fragment. Antigen-binding fragments (such as VH and VL) are linked to each other via a synthetic linker such that the VH / VL domains pair intramolecularly or intermolecularly when the VH and VL domains are expressed by separate single chains, to form a monovalent antigen-binding domain such as a single chain Fv (scFv) or an antigen. or can form the design of various types of single-chain antibodies that form bispecific antibodies. Anti- genic binding fragments can also be monospecific or multispecific for engineering bispecific and multispecific proteins, and can be conjugated to other antibodies, proteins, antigen-binding fragments, or alternative scaffolds, whether monospecific or multispecific.

[0117] "Antibody" is intended in a broad sense and includes monoclonal antibodies, antigen-binding fragments, bispecific, trispecific, tetra specific, etc. multispecific antibodies, dimeric, tetrameric, or multimeric antibodies, single-chain antibodies, antibody domains, and any other modified form of immunoglobulin molecules containing antigen-binding sites of the required specificity, including murine, human, humanized, and chimeric monoclonal antibodies. "Full-length antibody" consists of two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain consists of a heavy chain variable region (VH), as well as a heavy chain constant region (consisting of domains CH1, hinge, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH region and VL region can be further classified into hypervariable regions called framework regions (FR) interspersed with complementarity determining regions (CDR). Each VH and VL consists of three CDRs and four FR segments arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, from the amino terminus towards the carboxy terminus. ​​​​​​​​​. Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG, and IgM, according to the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subclassified as the isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and Ig G4. Antibody light chains of any vertebrate species can be assigned to one of two distinct types, namely, kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domain.

[0118] "Bispecificity" means a molecule (such as an antibody) that specifically binds to two different antigens or two different epitopes within the same antigen. Bispecific molecules can have cross-reactivity with the same antigen of other species (homologs), such as humans or monkeys, such as Macaca cynomolgus (cynomolgus monkey, cyno ) or Pan troglodytes, or can bind to epitopes shared between two or more different antigens.

[0119] A "chimeric antigen receptor", or "CAR", refers to an engineered T cell receptor that transfers ligand or antigen specificity onto T cells (e.g., naive T cells, central memory T cells , effector memory T cells or combinations thereof). CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immunoreceptors. A CAR comprises an extracellular domain that can bind to an antigen, a transmembrane domain, and at least one intracellular domain. The CAR intracellular domain is known to function as a domain that transmits signals that activate or inhibit intracellular biological processes. domains that are known to function as domains that transmit signals that activate or inhibit intracellular biological processes. It contains a peptide. The transmembrane domain is known to span the cell membrane and can function to bind an extracellular domain and a signaling domain, any peptide or polypeptide including it. The chimeric antigen receptor may optionally include a hinge domain that functions as a linker between the extracellular domain and the transmembrane domain.

[0120] "Complement-dependent cytotoxicity", i.e., "CDC", refers to a mechanism of inducing cell death in which the Fc effector domain of a target-binding protein binds to and activates complement component C1q, and such complement component C1q then activates the complement cascade to cause the death of target cells. Activation of the complement can also result in the deposition of complement components on the surface of target cells, facilitating CDC by binding of complement receptors (e.g., CR3) on white blood cells.

[0121] "Complementary determining region" (CDR) is the antibody region that binds to an antigen. There are three CDRs (HCDR1, HCDR2, HCDR3) in VH and three CDRs (LCD R1, LCDR2, LCDR3) in VL. CDRs are defined by Kabat (Wu et al .(1970) J Exp Med 132:211 - 250, Kabat et al ., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, N ational Institutes of Health, Bethesda, Md ., 1991), Chothia (Chothia et al., (1987) J M ol 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)) and can be defined using a variety of descriptions. A variety of descriptions and the correspondence with the numbering of the variable regions are described (e.g., Lefranc et al . (2003) Dev Comp Immunol 27:55-77;, Honegg er and Pluckthun, J Mol Biol (2001) 309:657 -670; International ImMunoGeneTics (IMGT) database - see web resource, http: / / www_imgt_org). Available programs such as abYsis by UCL Business PLC can be used to depict the CDRs. As used herein, the terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2", and "LCDR3" include CDRs defined by any of the Kabat, C hothia, IMGT, or AbM methods described above, unless otherwise explicitly stated in the specification.

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

[0123] "Enhancement", "promotion" or "increase" generally refers to the ability of a test molecule (i.e., downstream effect) to mediate a greater response when compared to a response mediated by a control or vehicle. Exemplary responses are enhanced binding of a protein to its antigen or receptor, enhanced binding to FcγR, or enhanced Fc effector functions such as enhanced ADCC, CDC and / or ADCP. Enhancement is 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, for example, 500, 600, 700 , 800, 900 or 1000-fold or more may be possible. effector function.

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

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

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

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

[0128] "Human antibody" refers to an antibody that has been optimized to minimize the immune response when administered to a human subject. The variable regions of a human antibody are derived from human immunoglobulin sequences. When a human antibody contains a constant region or a part of the constant region, the constant region is also derived from human immunoglobulin sequences. A human antibody contains "derived from" human-origin sequences in the heavy chain variable region and the light chain variable region when the variable regions of the human antibody are obtained from a system using human germline immunoglobulin or rearranged immunoglobulin genes. Such exemplary systems include human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals carrying the human immunoglobulin locus, such as mice or rats. "Human antibody" typically contains differences in amino acids when compared to immunoglobulins expressed in humans due to, for example, differences in the systems used to obtain the human antibody and the human immunoglobulin locus, intentional introduction or substitution of somatic mutations into the framework or CDRs, or both. Typically, a "human antibody" has an amino acid sequence that 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 to the amino acid sequence encoded by a human germline immunoglobulin or rearranged immunoglobulin gene. In some cases, a "human antibody" is, for example, Knappik displayed human immunoglobulin gene libraries, and transgenic non-human animals carrying the human immunoglobulin locus, such as mice or rats. "Human antibody" typically contains differences in amino acids when compared to immunoglobulins expressed in humans due to, for example, differences in the systems used to obtain the human antibody and the human immunoglobulin locus, intentional introduction or substitution of somatic mutations into the framework or CDRs, or both. Typically, a "human antibody" has an amino acid sequence that 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 to the amino acid sequence encoded by a human germline immunoglobulin or rearranged immunoglobulin gene. In some cases, a "human antibody" is, for example, Knappik displayed human immunoglobulin gene libraries, and transgenic non-human animals carrying the human immunoglobulin locus, such as mice or rats. "Human antibody" typically contains differences in amino acids when compared to immunoglobulins expressed in humans due to, for example, differences in the systems used to obtain the human antibody and the human immunoglobulin locus, intentional introduction or substitution of somatic mutations into the framework or CDRs, or both. Typically, a "human antibody" has an amino acid sequence that 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 to the amino acid sequence encoded by a human germline immunoglobulin or rearranged immunoglobulin gene. In some cases, a "human antibody" is, for example, Knappik %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, or 99% identical to the amino acid sequence encoded by a human germline immunoglobulin or rearranged immunoglobulin gene. In some cases, a "human antibody" is, for example, Knappik %, or 99% identical to the amino acid sequence encoded by a human germline immunoglobulin or rearranged immunoglobulin gene. In some cases, a "human antibody" is, for example, Knappik et al., (2000) J Mol Biol 296:57-86, as described in the human consensus framework sequence obtained from framework sequence analysis, or, for example , Shi et al., (2010) J Mol Biol 397:385-396 and International Publication No. WO 2009 / 085462, may contain synthetic HCDR3 incorporated into a human immunoglobulin gene library displayed on a phage. An antibody in which at least one CDR is derived from a non-human species is not included in the definition of "human antibody". An "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. Since a humanized antibody can include substitutions in the framework, the framework may not be an exact copy of the expressed human immunoglobulin

[0129] or the human immunoglobulin germline gene sequence.

[0130] "Isolated" refers to a homogeneous population of molecules (such as the scFv of the present disclosure or a heterologous protein containing the scFv of the present disclosure) that are substantially separated and / or purified from other components of the system in which the molecule is produced, such as a recombinant cell, and also refers to a protein that has been subjected to at least one purification or isolation step. "Isolated" refers to a molecule that is substantially free of other cellular materials and / or chemical substances, and includes molecules isolated to a higher purity, such as 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 increase or a decrease in the ability of a test molecule to mediate a response that is enhanced or reduced compared to a response mediated by a control or vehicle (i.e., a downstream effect). That is, it refers to either an increase or a decrease in the ability of a test molecule to mediate a response that is enhanced or reduced compared to a response mediated by a control or vehicle (i.e., a downstream effect). That is, it refers to either an increase or a decrease in the ability of a test molecule to mediate a response that is enhanced or reduced compared to a response mediated by a control or vehicle (i.e., a downstream effect).

[0132] "Monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibody molecules, i.e., individual antibodies that constitute a population that is identical except for possible well-known variations 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. That is, it refers to individual antibodies that constitute a population that is identical except for possible well-known variations 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. That is, it refers to individual antibodies that constitute a population that is identical except for possible well-known variations 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. That is, it refers to individual antibodies that constitute a population that is identical except for possible well-known variations 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 can have heterogeneous glycosylation within the antibody population. Monoclonal antibodies can be monospecific, or they can be multispecific such as bispecific, and can be monovalent, bivalent, or multivalent. Monoclonal antibodies typically bind to one antigenic epitope. Bispecific monoclonal antibodies bind to two different antigenic epitopes. Monoclonal antibodies can have heterogeneous glycosylation within the antibody population. Monoclonal antibodies can be monospecific, or they can be multispecific such as bispecific, and can be monovalent, bivalent, or multivalent. Monoclonal antibodies typically bind to one antigenic epitope. Bispecific monoclonal antibodies bind to two different antigenic epitopes. Monoclonal antibodies can have heterogeneous glycosylation within the antibody population. Monoclonal antibodies can be monospecific, or they can be multispecific such as bispecific, and can be monovalent, bivalent, or multivalent. Monoclonal antibodies typically bind to one antigenic epitope. Bispecific monoclonal antibodies bind to two different antigenic epitopes. Monoclonal antibodies can have heterogeneous glycosylation within the antibody population. Monoclonal antibodies can be monospecific, or they can be multispecific such as bispecific, and can be monovalent, bivalent, or multivalent. Monoclonal antibodies typically bind to one antigenic epitope. Bispecific monoclonal antibodies bind to two different antigenic epitopes. Monoclonal antibodies can have heterogeneous glycosylation within the antibody population. Monoclonal antibodies can be monospecific, or they can be multispecific such as bispecific, and can be monovalent, bivalent, or multivalent.

[0133] "Multispecificity" refers to a molecule that binds to two or more different antigens, or two or more different epitopes within the same antigen. Multispecific molecules can have cross-reactivity with other related antigens, e.g., the same antigen (homolog) from other species such as human or monkey, e.g., cynomolgus or chimpanzee, or they can bind to epitopes shared between two or more different antigens. Multispecific molecules can have cross-reactivity with other related antigens, e.g., the same antigen (homolog) from other species such as human or monkey, e.g., cynomolgus or chimpanzee, or they can bind to epitopes shared between two or more different antigens. Multispecific molecules can have cross-reactivity with other related antigens, e.g., the same antigen (homolog) from other species such as human or monkey, e.g., cynomolgus or chimpanzee, or they can bind to epitopes shared between two or more different antigens. Multispecific molecules can have cross-reactivity with other related antigens, e.g., the same antigen (homolog) from other species such as human or monkey, e.g., cynomolgus or chimpanzee, or they can bind to epitopes shared between two or more different antigens.

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

[0135] As used interchangeably herein, "protein" or "polypeptide" each refers to a molecule comprising one or more polypeptides consisting of at least two amino acid residues linked by peptide bonds. 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 less than 50 amino acids may be referred to as "peptides". A protein may be a heterologous fusion protein, 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. A "recombinant" refers to a polynucleotide, polypeptide, vector, virus, and other macromolecules prepared, expressed, produced, or isolated by recombinant means. "Single-chain Fv" or "scFv" refers to a single-chain protein comprising VH, VL, and a linker between VH and VL. The scFv may have VL and VH variable regions in either orientation, for example, with respect to the order from the N-terminus to the C-terminus of VH and VL. Thus, the scFv may be in the VL-linker-VH orientation or the VH-linker-VL orientation. The scFv may be engineered to include disulfide bonds between VH, VL, and the linker.

[0136]

[0137]

[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 the antigen with a higher affinity than to other antigens . Typically, a protein such as an scFv binds to an antigen or an epitope within the antigen with an equilibrium dissociation constant (K -6 d ) of about 1×10 -7 M or less, about 1×10 -8 M or less, about 5×10 -8 M or less, about 1×10 -1 -9 M or less, about 1×10 -10 M or less, about 1×10 -11 M or less, or about 1×10 -1 2 M or less, and typically, D K d D is at least 100-fold less than its K D for binding to non-specific antigens (e.g., BSA, casein).

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

[0140] A "subject" includes any human or non-human animal. A "non-human animal" includes any spinal cord or spinal cord injury. Vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cats, cows, etc. The terms "subject" and "patient" refer to animals, including, but not limited to, mammals, cattle, chickens, amphibians, reptiles, etc. may be used interchangeably herein.

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

[0142] "Treating," "treating" or "treatment" of a disease or disorder means one or more of the following: refers to achieving two or more of the following: reducing the severity and / or duration of the disorder; Inhibits the worsening of symptoms characteristic of the disorder in a subject who previously had the disorder. Limiting or preventing recurrence or symptoms in a subject who was previously symptomatic for the disorder Limit or prevent the recurrence of

[0143] "Trispecific" refers to a compound that is specific for three different antigens or three different epitopes within the same antigen. A trispecific molecule is a molecule (such as an antibody) that binds specifically to another related antigen, e.g. Humans or monkeys, e.g., Macaca cynomolgus ) or against the same antigen in other species (homologs), such as Pan troglodytes It may have cross-reactivity or may have epitopes shared among three or more different antigens. The peptide can be bound to the peptide.

[0144] "Variant", "mutant" or "variation" refers to a polypeptide or polynucleotide that is different from a reference polypeptide or reference 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 follows the EU index as described in Kabat et al., Sequences of Pro teins of Immunological Interest, 5th Ed. P ublic Health Service, National Institutes of Health, Bethesda, MD. (1991), unless otherwise explicitly indicated herein.

[0146] Mutations in the Ig constant region 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 the first Ig constant region, as well as the T394W mutation in the second Ig constant region.

[0147] Unless otherwise explicitly indicated, the numbering of variable regions follows Chothia.

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

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

[0150] ​​​​​​​ "Stabilized" refers to an scFv that retains binding equivalent to hK2 when compared to a non-heated scFv sample that is thermally stable.

[0151] "Improved stability" refers to an spFv of the present disclosure having an elevated melting temperature (Tm) when compared to a parental scFv lacking the disulfide bonds and Cys residues introduced into the SpFv. The elevated Tm can be an increase of 2°C or more, such as 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 a Cys residue in the scFv Vh or VL framework that can be mutagenized to 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 that can form a disulfide bond with an anchor point Cys.

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

[0155] "LTBR" is a polypeptide that is a cell surface receptor for lymphotoxin that is involved in apoptosis and cytokine release and is a member of the tumor necrosis factor receptor superfamily. refers to. LTBR can also be referred to as "tumor necrosis factor receptor superfamily member 3 (tumo r necrosis factor receptor superfamily member 3, TNFRSF3)". . LTBR is expressed on the surface of many cell types, including epithelial and myeloid lineage cells. LT BR can specifically bind to the lymphotoxin membrane form (a complex of lymphotoxin-alpha and lymphotoxin-beta). Activation of LTBR can induce apoptosis via TRAF3 and TRAF5 and can lead to the release of interleukin 8. Unless stated otherwise, preferably, LTBR is human LTBR. The human LTBR amino acid sequence is provided by UniProt accession number P36941. "EDB" or "extra domain B" refers to a domain of fibronectin that can be included in fibronectin molecules based on alternative splicing patterns of fibronectin pre-mRNA. Extra domain B is a complete fibronectin (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 the new vasculature during the angiogenesis process, making EDB a potential

[0156] marker and target for angiogenesis. Unless stated otherwise, preferably, EDB is human E DB. Human EDB containing the fibronectin isoform amino acid sequence is provided by U niProt accession number P02751. "Fibronectin" is a high molecular weight glycoprotein of the extracellular matrix, a polypeptide that is generally undetectable in normal adult tissues, but shows greater expression in fetal and tumor tissues in the extracellular matrix and accumulates around the new vasculature during the angiogenesis process, making EDB a potential marker and target for angiogenesis. Unless stated otherwise, preferably, EDB is human E DB. Human EDB containing the fibronectin isoform amino acid sequence is provided by U niProt accession number P02751.

[0157] "Fibronectin" is a high molecular weight glycoprotein of the extracellular matrix, a polypeptide Fibronectin binds to transmembrane receptor proteins called integrins. Fibronectin also binds to collagen, fibrin, and heparan sulfate proteins. Fibronectin can bind to other extracellular matrix proteins such as glycoglycans. , a protein consisting of two nearly identical monomers linked by a pair of disulfide bonds Fibronectin is produced by a single gene, but can exist as a fibronectin dimer. Alternative splicing of the fibronectin pre-mRNA molecule results in the formation of some Fibronectin isoforms have been created, one of which is EDB fibronectin. Lonectin may play a role in cell adhesion, growth, migration, and differentiation, and is involved in wound healing. It may be important for processes such as healing and embryonic development. The sequence is UniProt number P02751, which contains the extra domain B, as well as NC BI accession numbers NP_001263337 (isoform B), NP_001 263338 (isoform c), NP_001263339 (isoform d), NP_001263340 (isoform e) and NP_001263341 (isoform i) isoform f), NP_001293058 (isoform 8), NP_001293 059 (isoform 9), NP_001293060 (isoform 10), NP _001293061 (isoform 11) and NP_002017 (isoform This information is provided by the following company:

[0158] 5.1 Composition The present disclosure relates to stabilized scFv molecules (referred to herein as spFv (stapled Fv) , heterologous and multispecific molecules comprising spFv, polynucleotides encoding them, vectors, host cells, and methods for making and using them are provided. 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 to cysteine residues that result in the formation of disulfide bonds between the linker and the variable domains in scFv. The "staple treatment" strategy described herein is widely applicable to all VH / VL domains and existing scFv molecules that provide structural identity with scFv with improved stability. The spFv described herein can be conjugated to any heterologous protein, bispecific or multispecific format, including chimeric antigen receptors (CARs), T cell receptor molecules, bispecific and multispecific molecules, and can be used as therapeutic, diagnostic, and detection molecules. The present disclosure provides heterologous and multispecific molecules comprising spFv, polynucleotides encoding them, vectors, host cells, and methods for 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 to cysteine residues that result in the formation of disulfide bonds between the linker and the variable domains in scFv. The "staple treatment" strategy described herein is widely applicable to all VH / VL domains and existing scFv molecules that provide structural identity with scFv with improved stability. The spFv described herein can be conjugated to any heterologous protein, bispecific or multispecific format, including chimeric antigen receptors (CARs), T cell receptor molecules, bispecific and multispecific molecules, and can be used as therapeutic, diagnostic, and detection molecules. 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 to cysteine residues that result in the formation of disulfide bonds between the linker and the variable domains in scFv. The "staple treatment" strategy described herein is widely applicable to all VH / VL domains and existing scFv molecules that provide structural identity with scFv with improved stability. The spFv described herein can be conjugated to any heterologous protein, bispecific or multispecific format, including chimeric antigen receptors (CARs), T cell receptor molecules, bispecific and multispecific molecules, and can be used as therapeutic, diagnostic, and detection molecules. 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 to cysteine residues that result in the formation of disulfide bonds between the linker and the variable domains in scFv. The "staple treatment" strategy described herein is widely applicable to all VH / VL domains and existing scFv molecules that provide structural identity with scFv with improved stability. The spFv described herein can be conjugated to any heterologous protein, bispecific or multispecific format, including chimeric antigen receptors (CARs), T cell receptor molecules, bispecific and multispecific molecules, and can be used as therapeutic, diagnostic, and detection molecules. 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 to cysteine residues that result in the formation of disulfide bonds between the linker and the variable domains in scFv. The "staple treatment" strategy described herein is widely applicable to all VH / VL domains and existing scFv molecules that provide structural identity with scFv with improved stability. The spFv described herein can be conjugated to any heterologous protein, bispecific or multispecific format, including chimeric antigen receptors (CARs), T cell receptor molecules, bispecific and multispecific molecules, and can be used as therapeutic, diagnostic, and detection molecules. 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 to cysteine residues that result in the formation of disulfide bonds between the linker and the variable domains in scFv. The "staple treatment" strategy described herein is widely applicable to all VH / VL domains and existing scFv molecules that provide structural identity with scFv with improved stability. The spFv described herein can be conjugated to any heterologous protein, bispecific or multispecific format, including chimeric antigen receptors (CARs), T cell receptor molecules, bispecific and multispecific molecules, and can be used as therapeutic, diagnostic, and detection molecules. 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 to cysteine residues that result in the formation of disulfide bonds between the linker and the variable domains in scFv. The "staple treatment" strategy described herein is widely applicable to all VH / VL domains and existing scFv molecules that provide structural identity with scFv with improved stability. The spFv described herein can be conjugated to any heterologous protein, bispecific or multispecific format, including chimeric antigen receptors (CARs), T cell receptor molecules, bispecific and multispecific molecules, and can be used as therapeutic, diagnostic, and detection molecules. 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 to cysteine residues that result in the formation of disulfide bonds between the linker and the variable domains in scFv. The "staple treatment" strategy described herein is widely applicable to all VH / VL domains and existing scFv molecules that provide structural identity with scFv with improved stability. The spFv described herein can be conjugated to any heterologous protein, bispecific or multispecific format, including chimeric antigen receptors (CARs), T cell receptor molecules, bispecific and multispecific molecules, and can be used as therapeutic, diagnostic, and detection molecules. The spFv described herein can be conjugated to any heterologous protein, bispecific or multispecific format, including chimeric antigen receptors (CARs), T cell receptor molecules, bispecific and multispecific molecules, and can be used as therapeutic, diagnostic, and detection molecules. The spFv described herein can be conjugated to any heterologous protein, bispecific or multispecific format, including chimeric antigen receptors (CARs), T cell receptor molecules, bispecific and multispecific molecules, and can be used as therapeutic, diagnostic, and detection molecules. The spFv described herein can be conjugated to any heterologous protein, bispecific or multispecific format, including chimeric antigen receptors (CARs), T cell receptor molecules, bispecific and multispecific molecules, and can be used as therapeutic, diagnostic, and detection molecules.

[0159] The 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 is a first disulfide bond between a structurally conserved surface-exposed VH 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 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, or 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, or 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, or 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, or Provided is an scFv comprising a disulfide bond.

[0160] The present disclosure also provides an isolated scFv comprising VH, L, and VL, wherein VH comprises VH Cys at a structurally conserved surface-exposed VH framework residue position, L comprises a first L Cy, VL comprises VL Cys at a structurally conserved surface-exposed VL framework residue position, L comprises a second L Cy, or VH comprises VH Cys at a structurally conserved surface-exposed VH framework residue position, VL comprises VL Cys at a structurally conserved surface-exposed VL framework residue position, L comprises a first L Cys and a second L Cys, and VH Cys and the first L Cys can form a disulfide bond, and VL Cys and the second L Cy s can form a disulfide bond, thereby providing an scFv. The disulfide bond is typically formed during the expression of the scFv of the present disclosure.

[0161] Certain embodiments disclose an spFv having two disulfide bonds, but it is readily envisioned that an spFv having one disulfide bond formed between a linker -Cys and either VH Cys or VL Cys can be made and utilized to generate a "half-anchored" molecule. The anchor position is the same in an spFv having one or two disulfide bonds. The linker Cys position can vary in the half-anchored molecule as long as it meets the distance and geometric requirements for disulfide bond formation by the anchor point. The half-anchored spFv is a V stabilized by two disulfide bonds bond. bond. bond. bond. bond. It is expected that the relative movement of VL / VH is suppressed in the same way as the L / VH pair, and thus stabilization is achieved.

[0162] The spFv of the present disclosure showed an increase in thermal stability when compared with the parental scFv lacking disulfide bonds. Generally, the Tm of the spFv was about 10 °C higher regardless of the Tm of the parental scFv when compared with the parental scFv lacking disulfide bonds. Stability can generally be either thermal stability or mechanical stability. Thermal stability can be evaluated by differential scanning calorimetry (DSC) using a DSC scan performed on a heated protein sample (any 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 Tm of the VL and VH domains are recorded respectively. In some embodiments, the distance between VH Cys and VL Cys is from 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. In some embodiments, VH Cys is at H3, H5, H40, H43, H46 or H105, and the residue numbering follows Chothia.

[0163] In some embodiments, VH Cys is at H3.

[0164]

[0165]

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

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

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

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

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

[0171] In some embodiments, VL Cys is at L3, L5, L39, L42, L45, L 100 or L102, and the residue numbering follows Chothia.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0219] In some embodiments, VH Cys is at H105 and VL Cys is at L10 0 and is present.

[0220] In some embodiments, VH Cys is at H105 and VL Cys is at L10 2 and is present.

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

[0222] Chothia numbering is well known. Kabat or IMGT numbering, or The positions of the VH and VL residues can be numbered using other numbering systems such as sequential numbering. Table 1 shows the correspondence between exemplary VH, Chothia for GLk1 VH, Kabat, and sequential numbering (SEQ ID NO: 60). Table 2 shows the correspondence between exemplary VL, Chothia for GLk1 VL, Kabat, and sequential numbering ( 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 those derived from non-human primates such as mouse, rat, dog, chicken, and monkey.

[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 of the IgG1, IgG2, IgG3, I gG4, IgM, IgA, or IgE isotype.

[0230] The Ig hinge region generally includes residue 216 and ends at residue 230 of human IgG and is defined, and the residue numbering follows 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, the 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 EPKSCDKTHTCPPCPAPELLG G (SEQ ID NO: 64). The hinge regions of other Ig isotypes are well known and their amino acid sequences can be obtained, for example, from the ImMunoGeneTics website . For example, the IgG2 hinge includes the amino acid sequence ERKCCVECPPCP (SEQ ID NO: 6 5).

[0231] L contains a contiguous amino acid sequence "derived from" the Ig hinge region in those examples when it includes at least a portion of the Ig hinge region amino acid sequence or at least a portion of the engineered Ig hinge region . The engineered Ig hinge region contains one or more mutations compared to the wild-type Ig hinge . Exemplary mutations that can be introduced include substitutions of Cys residues that reduce the number of Cys in L to one or two, substitutions of Pro residues, or any conservative modification such as a conservative substitution . "Conservative modification" refers to an amino acid modification that does not significantly affect or alter the binding properties of the antibody containing the amino acid modification. Conservative modifications include substitutions, additions, and deletions of amino acids. Conservative amino acid substitutions are substitutions in which one amino acid is replaced with an amino acid residue having a similar side chain

[0232] . Families of amino acid residues having similar side chains are clearly defined . and are substitutions in which one amino acid is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are clearly is defined and includes amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leu cine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains ( e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyr osine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, his tidine, 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 (cys teine, methionine). Further, as previously described with respect to alanine scanning mutagenesis ((MacLennan et al.,(1988 )Acta Physiol Scand Suppl 643:55-67, Sasa ki et al.,(1988)Adv Biophys 35:1-24), any of the native residues within a polype ptide can be replaced with alanine. Amino acid substitutions can be made by known methods , for example, by PCR mutagenesis (U.S. Patent No. 4,683,195). The resulting mutant hinge is incorporated into the spFv constructs of the present disclosure and tested for their properties such as stability and binding to antigen using known assays and the assays described herein. In some embodiments, L comprises the amino acid sequence C(X) C (SEQ ID NO: 23).

[0233] y C (SEQ ID NO: 23) and ​In the formula, 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 , leucine (Leu), lysine (Lys), phenylalanine (Phe), threonine (Thr), tryptophan (Trp) or tyrosine (Tyr), and y is , an integer from 1 to 3. Pro may be included in the linker that provides rigidity. Gly may be included in the linker that allows for maximum flexibility. Except for Cys and Met, any other amino acid may also be used in L.

[0234] In some embodiments, L includes the amino acid sequence C(X) y C (SEQ ID NO: 24), where X is Gly, Ser or Pro, and y is an integer from 1 to 3.

[0235] In some embodiments, L is 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), CPPS C (SEQ ID NO: 45), CSSPC (SEQ ID NO: 46), CPSSC (SEQ ID NO: 47), CS SSC (SEQ ID NO: 48), CGSPC (SEQ ID NO: 49), CPGSC (SEQ ID NO: 50), includes CSGPC (SEQ ID NO: 51) or CPSGC (SEQ ID NO: 52).

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

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

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

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

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

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

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

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

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

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

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

[0247] In some embodiments, L includes 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 to 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 has 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 Val. It is 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. It is an integer.

[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 from 6 to 9, y is an integer from 1 to 3, and n is an integer from 4 to 6.

[0275] In some embodiments, L comprises the amino acid sequence of SEQ ID 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 present disclosure is in the VL-L-VH orientation.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0304] The present disclosure also provides a scFv comprising VH, L, and VL, wherein VH comprises Cys at H46, VL comprises Cys at L3, L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, or 7, and the scFv is in the 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 the spFv of the present disclosure As is well known in the art, the spFv of the present disclosure is an unstable s lacking a disulfide bond cFv, and like cFv, can be conjugated to a second molecule. Exemplary second molecules are disclosed herein and include half-life extending moieties, contrast 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 a first disulfide bond between a structurally conserved surface-exposed VH 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 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. The present disclosure also provides a heterologous molecule comprising an scFv comprising VH, L and VL, wherein VH comprises VH Cys at a structurally conserved surface-exposed VH framework residue position, L comprises a first L Cy , VL comprises VL Cys at a structurally conserved surface-exposed VL framework residue position, L comprises a second L Cy, or VH comprises VH Cys at a structurally conserved surface-exposed VH framework residue position, VL comprises VL Cys at a structurally conserved surface-exposed VL framework residue position and L comprises a first L Cy and a second L Cy.

[0312] The present disclosure also provides a heterologous molecule comprising an scFv comprising VH, L and VL, wherein VH comprises VH Cys at a structurally conserved surface-exposed VH framework residue position, L comprises a first L Cy , VL comprises VL Cys at a structurally conserved surface-exposed VL framework residue position, L comprises a second L Cy, or VH comprises VH Cys at a structurally conserved surface-exposed VH framework residue position, VL comprises VL Cys at a structurally conserved surface-exposed VL framework residue position and L comprises a first L Cy and a second L Cy. VH comprises VH Cys at a structurally conserved surface-exposed VH framework residue position, VL comprises VL Cys at a structurally conserved surface-exposed VL framework residue position and L comprises a first L Cy and a second L Cy. comprising, L comprises a first L Cys and a second L Cys, and VH Cys and the first L Cys can form a disulfide bond, and VL Cys and the second L Cy s can form a disulfide bond, to provide a heteromolecule.

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

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

[0315] In some embodiments, VL Cys is at L3, L5, L39, L42, L45, L 100 or L102, and the residue numbering follows Chothia.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0358] In some embodiments, VH Cys is at H105 and VL Cys is at L10 0

[0359] In some embodiments, VH Cys is at H105 and VL Cys is at L10 2

[0360] Residue numbering of the VH and VL regions follows 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 non - human primates such as mouse, rat, dog, chicken and monkey .

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

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

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

[0366] In some embodiments, L comprises 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 is the amino acid sequence CPC, CGC, CSC, CPPC (arrangement 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 ( Array number 42), CSPPC (Array number 43), CPSPC (Array number 44), CPPS C (Array number 45), CSSPC (Array number 46), CPSSC (Array number 47), CS SSC (Array number 48), CGSPC (Array number 49), CPGSC (Array number 50), CSGPC (Array number 51) or CPSGC (Array number 52).

[0368] In some embodiments, L comprises about 14 to 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 (Array number 25), wherein X is Gly, Ser, Pro, Ala, Arg, Asn, As p, Glu, Gln, His, Ile, leu, Lys, Phe, Thr, Trp or T yr, m is an integer from 6 to 9, y is an integer from 1 to 3, and n is an integer from 4 to 6 .

[0376] In some embodiments, L comprises 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 comprises 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 from 6 to 9, y is an integer from 1 to 3, and n is an integer from 4 to 6.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0401] The present disclosure also provides a heterologous molecule comprising an scFv comprising VH, L and VL, wherein VH comprises Cys at H46, VL contains Cys in L3, L contains the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7, The scFv is in the VH-L-VL orientation.

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

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

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

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

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

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

[0408] In some embodiments, the second protein is a half-life extension 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] The heterologous molecule comprising the spFv and the half-life extension moiety of the present disclosure In some embodiments, the spFv of the present disclosure is conjugated to a half-life extension moiety.

[0413] The half-life extension 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. In some embodiments, the spFv of the present disclosure is conjugated to an Ig or a fragment of an Ig. In some embodiments, the spFv of the present disclosure is conjugated to an Fc region. In some embodiments, the spFv of the present disclosure is conjugated to transferrin.

[0414] In some embodiments, the spFv of the present disclosure is conjugated to albumin.

[0415] In some embodiments, the spFv of the present disclosure is conjugated to an albumin binding protein.

[0416] In some embodiments, the spFv of the present disclosure is conjugated to polyethylene glycol (PEG). Exemplary PEG molecules are PEG 5000 or PEG 20,000.

[0417] In some embodiments, the spFv of the present disclosure is conjugated to a fatty acid or a fatty acid ester. 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

[0418] In some embodiments, the spFv of the present disclosure is conjugated to an albumin binding protein.

[0419] In some embodiments, the spFv of the present disclosure is conjugated to polyethylene glycol (PEG). Exemplary PEG molecules are PEG 5000 or PEG 20,000. In some embodiments, the spFv of the present disclosure is conjugated to a fatty acid or a fatty acid ester. 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

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

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

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

[0423] Using the heterologous molecule comprising the spFv of the present disclosure, in vitro or in vivo, a therapeutic agent can be directed, or the spFv can mediate killing of cells expressing the antigen to which it binds, or it can be visualized, identified, or purified.

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

[0425] The detectable label, when conjugated to the spFv of the present disclosure, can include a composition that makes 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 density reagents, enzymes (e.g., those commonly used in ELISA ), biotin, digoxigenin, haptens, luminescent molecules, chemiluminescent molecules, fluorescent dyes, fluoro​​​​​​ Rophore, fluorescence quencher, colored molecule, radioisotope, scintillates, avidin biotin, streptavidin, protein A, protein G, antibody or fragment thereof, polyhistidine tag, Ni 2+ , Flag tag, myc tag, heavy metal, enzyme, alkaline phosphatase , peroxidase, luciferase, electron donor / acceptor, acridinium ester, and colorimetric substrate.

[0427] A detectable label can emit a signal spontaneously, for example, 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 γ-emitting, Auger-emitting, β-emitting, α-emitting, or positron-emitting radioisotopes. Exemplary radioisotopes include 3 H, 11 C, 13 C, 15 N, 18 F, 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, 21 ​1 At, 212 Bi, 213 Bi, 223 Ra, 226 Ra, 225 Ac, and 227 A c are included.

[0429] Exemplary metal atoms include calcium, scandium, titanium, vanadium, chromium, man ganese, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, brom ine, krypton, rubidium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, ind ium, tin, antimony, tellurium, iodine, xenon, cesium, barium, lanthanum , hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum , gold, mercury, thallium, 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, ferm ium, mendelevium, nobelium, or lawrencium atoms, etc., which are metals having an atomic number greater than 20 .

[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 atom can be a gold atom, a bismuth atom, a tantalum atom, and a gadolinium atom.

[0436] In some embodiments, the metal atom can be a metal having an atomic number from 53 (i.e., iodine) to 83 (i.e., bismuth).

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

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

[0439] Suitable dyes include, for example, 5(6)-carboxyfluorescein, IRDye 6 80RD maleimide, or any commercially available dyes such as IRDye 800CW, ruthenium polypyridyl dyes can be mentioned.

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

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

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

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

[0444] In some embodiments, the cytotoxic agent is diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crocin, saponaria officinalis inhibitor, gelonin, mitogelin, a toxin having enzymatic activity such as restrictocin, phenomycin, enomycin, and trichothecene.

[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 dolastatin or a peptide of dolastatin Is a somatostatin and derivatives, auristatin or monomethyl auristatin phenylalanine . Exemplary molecules are disclosed in U.S. Patent Nos. 5,635,483 and 5,780,588 . Dolastatin and auristatin have been shown to interfere with microtubule dynamics, hydrolysis of GTP, as well as nuclear and cell division, and have anti-cancer and anti-fungal activities. The dolastatin or auristatin drug moiety can be conjugated to the antibodies of the present invention via the N (amino) terminus or the C (carboxyl) terminus of the peptide drug moiety (see WO 02 / 088172) , or via any cysteine engineered into the antibody. Conjugation to a detectable label can be performed using known methods.

[0447] In some embodiments, the detectable label forms a complex with a chelating agent.

[0448] In some embodiments, the detectable label serves in concert with the spFv of the present disclosure via a linker.

[0449] In some embodiments, the detectable label or cytotoxic agent can be directly or indirectly linked to the spFv of the present disclosure using known methods. Suitable linkers are known in the art

[0450] and include, for example, families of bridging molecules, non-phenolic linkers (derivatives of N-succinimidyl-benzoate , dodecaborate), chelating moieties of both macrocyclic and acyclic chelating agents, such as derivatives of 1,4 ,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), derivatives of diethylenetriaminepentaacetic acid (DTPA), S-2-(4-isothiocyanato ​​​​(Tobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) derivatives, and 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA) derivatives, N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g. dimethyl adipimidate HCl), active esters (e.g. disuccinimidyl suberate), aldehydes (e.g. glutaraldehyde), bis-azide 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), and other chelating moieties. Suitable peptide linkers are well known.

[0451] The spFv of the present disclosure and heterodimers comprising the immunoglobulin (Ig) constant region or fragments thereof The spFv of the present disclosure is conjugated to an Ig constant region or a fragment of an Ig constant region to confer antibody-like properties including Fc effector function C1q binding, complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis or down-regulation of cell surface receptors (e.g. B cell receptor, BCR). The Ig constant region or a fragment of an Ig constant region also functions as a half-life extending moiety as described herein. The present disclosure l-mediated cytotoxicity, ADCC), phagocytosis or down-regulation of cell surface receptors (e.g. B cell receptor, BCR). The Ig constant region or a fragment of an Ig constant region also functions as a half-life extending moiety as described herein. The present disclosure ​ The spFv of can also be engineered into a full-length antibody using standard methods. The spFv of the present disclosure Full-length antibodies containing the spFv of can be further engineered as described herein.

[0452] The immunoglobulin heavy chain constant region consists of subdomains CH1, CH2, and CH3. C The H1 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. In some cases residue 341 is referred to as a CH2 domain residue. The hinge generally includes residue 216 and is defined as ending at 230 for human IgG1, but as described herein may include a lower hinge region of about residues 231 to about residues 237. The Ig Fc region includes at least the CH2 and CH3 domains of the Ig constant region and thus includes at least the region of about 231-447 of the Ig heavy chain constant region.

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

[0454] In some embodiments, the Ig 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 the Ig constant region includes an Fc region.

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

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

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

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

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

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

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

[0464] The spFv of the present disclosure conjugated to the Ig constant region or a fragment of the Ig constant region can be evaluated for their functionality using some known assays. Binding to the target antigen can be evaluated using the methods described in this specification. The altered properties conferred by Ig constant domains such as the Fc region or fragments of the Ig constant region can be assayed in Fc receptor binding assays using soluble forms of receptors such as FcγRI, FcγRII, Fc γRIII or FcRn, or using cell-based assays that measure, for example, AD CC, CDC or ADCP. γRIII or FcRn, or using cell-based assays that measure, for example, AD CC, CDC or ADCP. binding assays.

[0465] The ADCC activity is determined using cells expressing the antigen to which the spFv of the present disclosure binds as target cells and can be evaluated using in vitro assays that use 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 native cellular protein) from the lysed cells. In an exemplary assay, target cells are used at a ratio of one target cell to four effector cells. The target cells are pre-labeled with BATDA and combined with effector cells and test antibodies. The samples are incubated for 2 hours and cytolysis is measured by measuring the BATDA released into the supernatant. Data are normalized to the maximum cytotoxicity by 0.67% Triton X-100 (Sigma Aldrich), and the minimum control is determined by the spontaneous release of BATDA from the target cells in the absence of any antibody.

[0466] ADCP can be evaluated by using monocyte-derived macrophages as effector cells and any cell expressing an antigen to which the scFv of the present disclosure binds as a target cell and monocyte-derived macrophages as target cells engineered to express GFP or another labeled molecule. In an exemplary assay, the effector:target cell ratio can be, for example, 4:1. The effector cells can be incubated with the target cells for 4 hours with or without the addition of the antibody of the present invention. After incubation, the cells can be detached using an enzyme. Macrophages can be identified by anti-CD11b antibody and anti-CD14 antibody conjugated to a fluorescent label, and the rate of phagocytosis can be determined based on the percentage (%) of GFP fluorescence in CD11 and CD14 macrophages using standard methods. + and CD14 + macrophages. ​

[0467] The CDC of cells can be measured, for example, by plating Daudi cells in RPMI-B (RPMI supplemented with 1% BSA) at 1×10 cells / well (50 μL / well), adding 50 μ 5 L of the test protein to the wells 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 wells, and incubating the reaction at 3 7 °C for 45 minutes. The percentage of lysed cells (%) can be detected as the percentage of propidium iodide-stained cells in the FACS assay using standard methods. )

[0468] Heterologous molecules containing the spFv and chimeric antigen receptor (CAR) or fragments thereof of the present disclosure The spFv of the present disclosure can be conjugated to a chimeric antigen receptor (CAR) or a fragment of the CAR. Therefore, a CAR containing the spFv of the present disclosure can be monospecific or multispecific, containing one or two or more scFv molecules of the present disclosure as its extracellular domain.

[0469] A chimeric antigen receptor (CAR) is a genetically engineered receptor. These engineered receptors can be easily inserted into immune cells including T cells and expressed therein according to techniques known in the art. By means of the CAR, a single receptor can recognize a specific antigen and, when bound to that antigen, program immune cells to activate and attack and destroy cells carrying that antigen . When 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, any linker, a transmembrane domain, and a cytoplasmic domain containing a co-stimulatory domain and / or a signaling domain. The extracellular domain of the CAR may contain any polypeptide that binds to a desired antigen, such as the scFv of the present disclosure. The CAR may also be engineered to bind to two or more desired antigens that are arranged in tandem and separated by a linker sequence. For example,

[0471] one or more scFvs, domain antibodies, llama VHH antibodies or other VH-like antibody fragments of the present disclosure may be configured in tandem via a linker to generate a bispecific or multispecific CAR. The transmembrane domain of the CAR is the transmembrane domain of CD8, the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD 8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86 CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD1 37), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR),

[0472] SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, I L2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4 CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, IT GAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11 b, CD89, 2B4 (CD244), CD138 (syndecan-1), BTLA, CD 229 (Siglec-9), CD300lf, CLEC12A, CD123 (IL3R alpha), CD23 (Fc epsilon RII), CD233 (band 3), CD235a (glycophorin A), CD235b (glycophorin B), CD247 (zeta chain), CD279 (PD-1), CD282 (TREM2), CD288 (LILRB4), CD300a (p75-AIRM1), CD300c (CLM-1), CD300e (VSIG4), CD302 (LILRB2), CD303 (BDCA-2), b, ITGAX, CDI lc, ITGB1, CD29, ITGB2, CD18, LFA -1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244 , 2B4), CD84, CD96 (tactile), CEACAM1, CRT AM, Ly9 (C D229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SL AMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO- 3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C may be derived from them.

[0473] The intracellular co-stimulatory domain of the CAR may be derived from the intracellular domain of one or more co-stimulatory molecules. Co-stimulatory 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 co-stimulatory domains that can be used in the CAR are the intracellular domains of 4-1BB, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD13 4 (OX40), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD278 (ICOS), DAP10, LAT , NKD2C SLP76, TRIM, and the intracellular domains of ZAP70. The intracellular signaling domain of the CAR may be derived from the signaling domains of, for example, CD3ζ, CD3ε, CD22, C

[0474] D79a, CD66d or CD39. "Intracellular sig naling The "signal transduction domain" refers to a part of the CAR polypeptide that mediates the introduction of the message of effective CAR binding to the target antigen into immune effector cells in order to induce effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity (including release of cytotoxic factors to the CAR-binding target cells or other cellular responses induced after antigen binding to the extracellular CAR domain). Any linker within the CAR positioned between the extracellular domain and the transmembrane domain can be a polypeptide about 2 to 100 amino acids in length. The linker can contain flexible residues such as glycine and serine, or can be composed of residues, so that adjacent protein domains can move freely relative to each other. If it is desired to ensure that two adjacent domains do not sterically interfere with each other, a longer linker can be used. The linker can be cleavable or non-cleavable. Exemplary cleavable linkers include 2A. Exemplary CARs include the scFv, CD8 transmembrane domain, and CD3ζ signal transduction domain of the present disclosure. Another exemplary CAR includes the scFv, CD8 or CD28 transmembrane domain, CD28, 41BB or OX40 co-stimulatory domain, and CD3ζ signal transduction domain of the present disclosure. CARs are produced by standard molecular biology techniques. The spFv of the present disclosure can be conjugated directly to a second molecule or via a linker. Exemplary linkers include immunoglobulin heavy or light chain isotypes, Gly-rich linkers.

[0475]

[0476]

[0477]

[0478] ​​​​​​​​​​​​​​​ Immunoglobulin hinge regions, CL or CH1 moieties derived from Gly- and Ser-containing linkers, Gly- and Ala-containing linkers, Ala- and Ser-containing linkers, and Pro-containing linkers. Exemplary amino acids that may 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, polyoxyalkylene, or a copolymer of polyethylene glycol and polypropylene glycol, may find use as linkers. Exemplary linkers are described, for example, in International Publication No. WO 2019 / 060695. In some embodiments, the heteromolecule is monospecific. In some embodiments, the heteromolecule is multispecific. In some embodiments, the heteromolecule is bispecific. In some embodiments, the heteromolecule is trispecific. In some embodiments, the heteromolecule is tetravalent. Multispecific molecules comprising the spFv of the present disclosure

[0479] The present disclosure also provides multispecific molecules 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 has a first disulfide bond between a structurally conserved surface-exposed VH cysteine (Cys) and a first L Cys,

[0480]

[0481]

[0482]

[0483]

[0484] ​​​​​​​​​A second disulfide between the structurally conserved surface-exposed VL Cys and a second L Cys bond, or The first disulfide between the structurally conserved surface-exposed VH Cys and the first L Cys The second bond between the structurally conserved surface-exposed VL Cys and the second L Cys is The present invention provides a multispecific molecule comprising:

[0485] The present disclosure also provides a multispecific molecule comprising an scFv comprising a VH, L and VL, VH contains a VH Cys at a structurally conserved, surface-exposed VH framework residue position. L includes a first L Cy, VL contains a VL Cys at a structurally conserved, surface-exposed VL framework residue position. and L contains a second L Cy; or VH contains a VH Cys at a structurally conserved, surface-exposed VH framework residue position. and VL has a VL Cys at a structurally conserved, surface-exposed VL framework residue position. L comprises a first L Cys and a second L Cys; VH Cys and the first L Cys can form a disulfide bond, and VL Cys and a second L Cy The present invention provides a multispecific molecule in which s is 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. Å.

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

[0488] In some embodiments, the VL Cys is L3, L5, L39, L42, L45, L at 100 or L102, and residue numbering follows Chothia.

[0489] In some embodiments, the VH Cys is at H105 and the VL Cys is at L42, or the VH Cys is at H43 and the VL Cys is at L100, or the VH Cys is at H3 and the VL Cys is at L3, or the VH Cys is at H3 and the VL Cys is at L5, or the VH Cys is at H3 and the VL Cys is at L39, or the VH Cys is at H3 and the VL Cys is at L42, or the VH Cys is at H3 and the VL Cys is at L45, or the VH Cys is at H3 and the VL Cys is at L100, or the VH Cys is at H3 and the VL Cys is at L102, or the VH Cys is at H5 and the VL Cys is at L3, or the VH Cys is at H5 and the VL Cys is at L5, or the VH Cys is at H5 and the VL Cys is at L39, or the VH Cys is at H5 and the VL Cys is at L42, or the VH Cys is at H5 and the VL Cys is at L45, or the VH Cys is at H5 and the VL Cys is at L100, or the VH Cys is at H5 and the VL Cys is at L102, or the VH Cys is at H40 and the VL Cys is at L3, or the VH Cys is at H40 and the VL Cys is at L5, or the VH Cys is at H40 and the VL Cys is at L39, or the VH Cys is at H40 and the VL Cys is at L42, or The VH Cys is at H40, and the VL Cys is at L45, or The VH Cys is at H40, and the VL Cys is at L100, or The VH Cys is at H40, and the VL Cys is at L102, or The VH Cys is at H43, and the VL Cys is at L3, or The VH Cys is at H43, and the VL Cys is at L5, or The VH Cys is at H43, and the VL Cys is at L39, or The VH Cys is at H43, and the VL Cys is at L42, or The VH Cys is at H43, and the VL Cys is at L45, or The VH Cys is at H43, and the VL Cys is at L102, or The VH Cys is at H46, and the VL Cys is at L3, or The VH Cys is at H46, and the VL Cys is at L5, or The VH Cys is at H46, and the VL Cys is at L39, or The VH Cys is at H46, and the VL Cys is at L42, or The VH Cys is at H46, and the VL Cys is at L45, or The VH Cys is at H46, and the VL Cys is at L100, or The VH Cys is at H46, and the VL Cys is at L102, or The VH Cys is at H105, and the VL Cys is at L3, or The VH Cys is at H105, and the VL Cys is at L5, or The VH Cys is at H105, and the VL Cys is at L39, or The VH Cys is at H105, and the VL Cys is at L45, or The VH Cys is at H105, and the VL Cys is at L100, or The VH Cys is at H105, and the VL Cys is at L102, In this specification, residue numbering follows 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 of the IgG1, IgG2, IgG3 or IgG4 isotype.

[0494] In some embodiments, L comprises 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 comprises 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 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: 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), CPPS C (SEQ ID NO: 45), CSSPC (SEQ ID NO: 46), CPSSC (SEQ ID NO: 47), CS SSC (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 from about 14 to about 19 amino acids, such as 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, As p, Glu, Gln, His, Ile, leu, Lys, Phe, Thr, Trp or T yr, m is an integer from 6 to 9, y is an integer from 1 to 3, and n is an integer from 4 to 6 .

[0499] In some embodiments, L is the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 26), wherein X is Gly, Ser, Pro, Ala, Arg, Asn, As p, 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 comprises 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 from 6 to 9, y is an integer from 1 to 3, and n is an integer from 4 to 6.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0525] In some embodiments, L contains 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 an antibody fragment.

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

[0530] In some embodiments, the Ig 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 the Ig constant region comprises a CH3 domain.

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

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

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

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

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

[0538] In some embodiments, the Ig constant region or fragment of the Ig constant region is of the IgG1, IgG 2, and IgG3 or IgG4 isotype.

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

[0540] In some embodiments, at least one mutation that reduces the binding of the multispecific molecule to FcγR is F234A / L235A, L234A / L235A, L234A / L235 A / D265S, V234A / G237A / P238S / H268A / V309L / A3 30S / P331S, F234A / L235A, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235 A / G236 deletion / A327G / P331A / D365E / L358M, H268Q / V 309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A33 0S / P331S, S228P / F234A / L235A / G237A / P238S and S228P / F234A / L235A / G236 deletion / G237A / P238S, and is selected from the group consisting of residue numbering follows the EU index.

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

[0542] In some embodiments, at least one mutation that enhances the binding of the multispecific molecule to FcγR is S239D / I332E, S298A / E333A / K334A, F243 L / R292P / Y300L, F243L / R292P / Y300L / P396L, F2 43L / R292P / Y300L / V305I / P396L, and G236A / S239D / I332E, and the residue numbering follows the EU index.

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

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

[0545] In some embodiments, at least one mutation that regulates the half-life of the multispecific molecule is , H435A, P257I / N434H, D376V / N434H, M252Y / S25 4T / T256E / H433K / N434F, T308P / N434A, and H435R, and the residue numbering follows the EU index. selected from the group consisting of

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

[0547] In some embodiments, at least one mutation in the CH3 domain is T350V, L 351Y, F405A, Y407V, T366Y, T366W, F405W, T394W , T394S, Y407T, Y407A, T366S / L368A / Y407V, L35 1Y / F405A / Y407V, T366I / K392M / T394W, F405A / Y 407V, T366L / K392M / T394W, L351Y / Y407A, T366A / K409F, L351Y / Y407A, T366V / K409F, T366A / K40 9F, T350V / L351Y / F405A / Y407V and T350V / T366L / selected from the group consisting of K392L / T394W, with residue numbering according to the EU index followed.

[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 tetravalent.

[0551] 5.2 Generation of multispecific proteins comprising the spFv of the present disclosure The spFv of the present disclosure can be engineered into any known form of multispecific molecule using known recombinant techniques, expression, and purification protocols.

[0552] The spFv of the present disclosure can be engineered into full-length multispecific antibodies having one or two 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 co-expression of the various chains. In the case of in vitro Fab-arm exchange, two monospecific bivalent antibodies are engineered to have one or two or more substitutions in the C H3 domain, and the antibodies are in the hinge region of one or two substitutions, and the antibodies are in the hinge region of Incubated together under reducing conditions sufficient to effect disulfide bond isomerization of cysteine, thereby generating bispecific antibodies by Fab arm exchange. The incubation conditions can optimally be returned to non-reducing conditions. Representative reducing agents that can be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. For example, at a temperature of at least 20°C, in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol, at pH 5-8, such as pH 7.0 or pH 7.4, an incubation of at least 90 minutes can be used. Thereby, bispecific antibodies are generated by Fab arm exchange. The incubation conditions can optimally be returned to non-reducing conditions. Representative reducing agents that can be used are 2- mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. For example, at a temperature of at least 20°C, in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol, at pH 5-8, such as pH 7.0 or pH 7.4, an incubation of at least 90 minutes can be used. Thereby, bispecific antibodies are generated by Fab arm exchange. The incubation conditions can optimally be returned to non-reducing conditions. Representative reducing agents that can be used are 2- mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. For example, at a temperature of at least 20°C, in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol, at pH 5-8, such as pH 7.0 or pH 7.4, an incubation of at least 90 minutes can be used. Thereby, bispecific antibodies are generated by Fab arm exchange. The incubation conditions can optimally be returned to non-reducing conditions. Representative reducing agents that can be used are 2- mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. For example, at a temperature of at least 20°C, in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol, at pH 5-8, such as pH 7.0 or pH 7.4, an incubation of at least 90 minutes can be used. Thereby, bispecific antibodies are generated by Fab arm exchange. The incubation

[0553] CH3 mutations that can be used include the knob-in-hole mutation (Genentech), electrostatic match mutations (Chugai, Amgen, NovoNordisk, Oncomed), chain exchange operation domain body (SEEDbody) (EMD Serono), Duobody (registered trademark) mutation (Genmab), and other asymmetric mutations (e.g., Zymeworks) and other techniques. Thereby, bispecific antibodies are generated by Fab arm exchange. The incubation

[0554] The knob-in-hole mutation is disclosed, for example, in International Publication No. WO 1996 / 027011, where an amino acid with a small side chain (hole) is introduced into the first CH3 region, and an amino acid with a large side chain (knob) is introduced into the second CH3 region, and the first CH3 region and the second C (knob) is introduced into the second CH3 region, and the first CH3 region and the second C It includes mutations on the interface of the CH3 region that result in a preferential interaction with the H3 region. Exemplary CH3 region mutations that form knobs and holes are T366Y / F405A, T36 6W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y 407A, T366W / T394S, F405W / T394S, and T366W / T36 6S_L368A_Y407V.

[0555] Heavy chain heterodimer formation can be promoted by using electrostatic interactions by substituting positively charged residues on the first CH3 region and negatively charged residues on the second CH3 region, as described in U.S. Patent Application Publication Nos. 2010 / 0015133, 20 09 / 0182127, 2010 / 028637, or 2011 / 0123 532. Other asymmetric mutations that can be used to promote heavy chain heterodimerization are L351Y_F405A_Y407V / T394W, T366I described in U.S. Patent Application Publication Nos. 2012 / 0149876 or 2013 / 0195849 (Zym

[0556] eworks), T366L_K392M_T39 4W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L 351Y_Y407A / T366V_K409F, Y407A / T366A_K409F , or T350V_L351Y_F405A_Y407V / T350V_T366L_K 392L_T394W. 351Y_Y407A / T366V_K409F, Y407A / T366A_K409F , or T350V_L351Y_F405A_Y407V / T350V_T366L_K 392L_T394W.

[0557] SEED BODY mutations are as described in U.S. Patent Application Publication No. 2007 / 0287170 substituting the selected IgG residue with an IgA residue to promote heavy chain heterodimerization is involved.

[0558] Other exemplary mutations that can be used are described in WO 2007 / 147901, WO 2011 / 143545, WO 2013 / 157954, WO 2013 / 096291 and US Patent Application Publication No. 2018 / 0118849, including R409 D_K370E / D399K_E357K, S354C_T366W / Y349C_T3 66S_L368A_Y407V, Y349C_T366W / S354C_T366S_ L368A_Y407V, T366K / L351D, L351K / Y349E, L351 K / Y349D, L351K / L368E, L351Y_Y407A / T366A_K4 09F, L351Y_Y407A / T366V_K409F, K392D / D399K, K392D / E356K, K253E_D282K_K322D / D239K_E240 K_K292D, K392D_K409D / D356K_D399K.

[0559] Duobody® mutations (Genmab) are disclosed, for example, in US Patent Application Publication No. 2 014 / 0303356, including mutations of F405L / K409R, wild type / F40 5L_R409K, T350I_K370T_F405L / K409R, K370W / K 409R, D399AFGHILMNRSTVWY / K409R, T366ADEFGH ILMQVY / K409R, L368ADEGHNRSTVQ / K409AGRH, D3 99FHKRQ / K409AGRH, F405IKLSTVW / K409AGRH and Y 407LWQ / K409AGRH.

[0560] Additional bispecific or multispecific structures into which the spFv of the present disclosure can be incorporated include dual variable domain immunoglobulins (Dual Variable Domain, DVD) (International Publication No. 2009 / 1 34776, the DVD is a full-length antibody comprising a heavy chain having a VH1-linker-VH2-CH structure and a light chain having a VL1 -linker-VL2-CL structure, and the linker is optional ), structures comprising various dimerization domains for binding two antibody arms having different specificities, such as leucine zippers or collagen dimerization domains (International Publication No. 20 12 / 022811, U.S. Patent No. 5,932,448, U.S. Patent No. 6,833,44 1), two or more domain antibodies (dAb) conjugated together, diabodies, single-chain antibodies such as camelid antibodies and engineered camelid antibodies, dual target (DT)-Ig (GSK / Domantis), 2in1 antibody (Genentech), cross-linked Mab (K armanos Cancer Center), mAb2 (F-Star), and Co vX body (CovX / Pfizer), IgG-like bispecific (InnClone / El i Lilly), Ts2Ab (MedImmune / AZ), and BsAb (Zymo genetics), HERCULES (Biogen Idec), and TvAb (R oche), ScFv / Fc fusions (Academic Institution), S CORPION (Emergent BioSolutions / Trubion, Zy mogenetics / BMS), dual affinity retargeting technology (Fc-DART) (Macr oGenics), and bis(ScFv)2-Fab (National Resear oGenics), and the like. ch Center for Antibody Medicine--China), dual activity or Bis-Fab (Genentech), Dock-and-Lock (D NL) (ImmunoMedics), bivalent bispecificity (Biotecnol), and F ab-Fv (UCB-Celltech) are included. ScFv antibodies, antibodies based on diabodies and domain antibodies include bispecific T cell engagers (BiTE) (Mi cromet), tandem diabodies (Tandab) (Affimed), dual affinity retargeting technology (DART) (MacroGenics), single-chain diabodies (Acad emic), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack), and COMBODY (Epigen Bi otech), bispecific nanobodies (Ablynx), and bispecific heavy-chain-only domain antibodies are included, but are not limited to these.

[0561] The scFv of the present disclosure can also be engineered into multispecific proteins containing three polypeptide chains. In such designs, at least one antigen-binding domain is in the form of the scFv of the present disclosure. Exemplary designs include (where "1" represents the first antigen-binding domain, "2 " represents the second antigen-binding domain, and "3" represents the third antigen-binding domain): Design 1: Chain A) scFv1-CH2-CH3, Chain B) VL2-CL, Chain C) VH2-C H1-hinge-CH2-CH3 Design 2: Chain A) scFv1-hinge-CH2-CH3, Chain B) VL2-CL, Chain C) V H2-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) scFv1-CH2-CH3, Chain B) VL2-CL, Chain C) VH2-CH1-hinge-CH2-CH3 Chain C) VH2-CH1-Hinge-CH2-CH3 Design 4: Chain A) CH2-CH3-scFv1, Chain B) VL2-CL, Chain C) VH2-C H1-Hinge-CH2-CH3

[0562] CH3 engineering can be incorporated into Designs 1-4 such as the mutations of L351Y_F405A _Y407V / T394W, T366I_K392M_T394W / F405A_Y40 7V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y 407A / T366A_K409F, L351Y_Y407A / T366V_K409F , Y407A / T366A_K409F, or T350V_L351Y_F405A_Y 407V / T350V_T366L_K392L_T394W, etc. can be incorporated into Designs 1-4.

[0563] 5.3 Isotype, Allotype and Fc Engineering Ig constant regions or fragments of Ig constant regions, such as the Fc region present in a multispecific molecule or a heteromolecule of the present disclosure, can be of any allotype or isotype.

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

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

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

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

[0568] The Ig constant region or a fragment of the Ig constant region can be of any allotype. The allotype is expected not to affect the properties of the Ig constant region such as binding or Fc-mediated effector functions. The immunogenicity of a therapeutic protein containing the Ig constant region of the fragment is associated with an increased risk of infusion reactions and a shortened duration of the therapeutic response (Baert et al., (2003) N Engl J Med 348:602 - 608). The degree to which a therapeutic protein containing the Ig constant region of the fragment induces an immune response in the host can be partially determined 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 mutations in the amino acid sequence at specific positions in the constant region sequence of the antibody. 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 carboxypeptidase in the bloodstream (Cai et al., (2011) Biotechnol Bioeng 108:404 - 412). During production, as described in US Patent Application Publication No. 2014 / 0273092, extracellular Zn, EDTA or EDTA-Fe 2+ ​​​​​​​​​​​​​​​3+ By controlling the concentration of 3+ , the removal of CTL can be controlled to be below the maximum level. The CTL content of the protein can be measured using known methods. It is possible.

[0571] In some embodiments, the spFv of the present disclosure conjugated to the Ig constant region has a C-terminal lysine content of about 10% to about 90%. In some embodiments, the C-terminal lysine content is about 20% to about 80%. In some embodiments, the C-terminal lysine content is about 40 % to about 70%. In some embodiments, the C-terminal lysine content is about 55% to about 7 0%. 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 that include an Ig constant region or a fragment of an Ig constant region to modulate their effector functions such as ADCC, ADCP, and / or ADCP and / or pharmacokinetic properties. This can be achieved by introducing mutations into the Fc to control the binding of the mutant Fc to activating FcγRs (FcγRI, FcγRIIa, FcγRIII ), inhibitory FcγRIIb, and / or FcRn. It can be done. ), inhibitory FcγRIIb, and / or FcRn. ). It is achievable.

[0573] In some embodiments, the multispecific or heterologous molecules of the present disclosure include at least one mutation in an Ig constant region or a fragment of an Ig constant region. It contains 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 molecules of the present disclosure have at least comprising from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mutations.

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

[0577] Positions of the 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 include the mutations T250Q, M252Y, I253A, S254T, T256E , P257I, T307A, D376V, E380A, M428L, H433K, N43 4S, N434A, N434H, N434F, H435A, and H435R. Exemplary mutations that can be made alone or in combination to increase the half-life of an antibody include the mutations M428L / N434S, M252Y / S254T / T256E, T250Q / M428 L, N434A, and T307A / E380A / N434A. Exemplary mutations that can be made alone or in combination to decrease the half-life include the mutations H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H43 3K / N434F, T308P / N434A, and H435R.

[0578]

[0579] In some embodiments, the multispecific or heterologous molecules of the disclosure comprise the M25 2Y / S254T / T256E mutation in the Fc region.

[0579] In some embodiments, the multispecific or heterologous molecules of the present disclosure have the activity of a protein reduce binding to activating Fcγ receptors (FcγRs) and / or reduce C1q binding, complement-dependent cyto toxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) or phagocytosis (AD P), etc., and include at least one mutation in the Fc region to reduce Fc effector functions such as.

[0580] After reducing the binding of the multispecific or heterologous molecules of the present disclosure to activating FcγRs, fc positions that can be mutated to reduce effector functions include positions 214, 23 3, 234, 235, 236, 237, 238, 265, 267, 268, 270, 29 5, 297, 309, 327, 328, 329, 330, 331 and 365. Exemplary mutations that can be made alone or in combination are mutations K214T, E233P, L234V, L234A, G236 in IgG1, IgG2, IgG3 or IgG4 deletion, V234A, F234A, L235A, G237A, P238A, P238S, D 265A, D265S, S267E, H268A, H268Q, Q268A, N297A , A327Q, P329A, D270A, Q295A, V309L, A327S, L32 8F, A330S and P331S. Exemplary combinations of mutations that result in a multispecific or heterologous molecule of the present disclosure with reduced ADCC are L234A / L2 in IgG1 35A, L234A / L235A / D265S in IgG1, V2 in IgG2 34A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A in IgG4, S228P / F234A / in IgG4 L235A, N297A in all Ig isotypes, V234 in IgG2 A / G237A, K214T / E233P / L234V / L235A / in IgG1 G236 deletion / A327G / P331A / D365E / L358M, H in IgG2 268Q / V309L / A330S / P331S, S267E / L32 in IgG1 8F, L234F / L235E / D265A in IgG1, L23 in IgG1 4A / L235A / G237A / P238S / H268A / A330S / P331S, I gG4, S228P / F234A / L235A / G237A / P238S, and I gG4, S228P / F234A / L235A / G236 - deleted / G2 37A / P238S. Using a hybrid IgG2 / 4 Fc domain such as Fc having residues 117 - 260 from IgG2 and residues 261 - 447 from IgG4 may also be used. It may be good.

[0581] Exemplary mutations that result in the multispecific or heterologous molecules of the present disclosure with reduced CDC are the K322A mutation.

[0582] Adding the well - known S228P mutation to an IgG4 antibody can enhance the stability of IgG4. It can be done.

[0583] In some embodiments, the multispecific or heterologous molecules of the present disclosure are K214T, E2 33P, L234V, L234A, deletion of G236, V234A, F234A, L235 A, G237A, P238A, P238S, D265A, S267E, H268A, H2 68Q, Q268A, N297A, A327Q, P329A, D270A, Q295A, selected from the group consisting of V309L, A327S, L328F, A330S and P331S and comprises at least one mutation in the Fc region.

[0584] In some embodiments, the multispecific or heterologous molecule of the disclosure comprises L234A / L235A / D265S mutations in the Fc region.

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

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

[0587] Fc positions that can be mutated to increase the binding of the multispecific or heterologous molecule of the disclosure to activating FcγR and / or enhance Fc effector functions include positions 236, 239, 243, 256, 290, 292, 298, 300, 305, 312, 326, 330, 332, 333, 334, 345, 360, 339, 378, 396 or 430 (residue numbering according to the EU index). Exemplary mutations that can be made singly or in combination are G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305L, K326A, A330K, I332E, E333A, K334A, A339T and P396L. ADCC or An exemplary combination of mutations that results in a protein with increased ADCP is S239D / I 332E, S298A / E333A / K334A, F243L / R292P / Y300L , F243L / R292P / Y300L / P396L, F243L / R292P / Y30 0L / V305I / P396L and G236A / S239D / I332E.

[0588] Fc positions that can be mutated to enhance CDC include positions 267, 268, 32 4, 326, 333, 345 and 430. Exemplary mutations include S267E, F1268F, S324T, K326A, K326W, E333A, E345K, E345Q, E345R, E345Y, E430S, E430 The multispecific or heterologous molecules of the present disclosure that have increased CDC are Exemplary combinations of mutations that result in a nucleotide sequence similar to that of the nucleotide sequence ... A, H268F / S324T, S267E / H268F, S267E / S324T and S 267E / H268F / S324T.

[0589] The specific mutations described herein are SEQ ID NOs: 66, 67 and 68, respectively, IgG1, I These are mutations compared to the gG2 and IgG4 wild-type amino acid sequences.

[0590] SEQ ID NO: 66, wild type IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK

[0591] SEQ ID NO: 67; wild-type IgG2 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQ TYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYK CKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK

[0592] SEQ ID NO: 68; wild-type IgG4 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK TYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPSQEEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK

[0593] Binding of the multispecific or heterologous molecules of the present disclosure to FcγR or FcRn was determined using flow cytometry. Using chromatometry, the expression of each receptor can be evaluated in cells engineered to express the respective receptor. In the exemplary binding assay, 2x10 per well in a 96-well plate 5 Cells After seeding, the cells were stained with BSA Stain Buffer (BD Biosciences, San The cells were blocked in PBS (Bio-Rad, Jose, USA) for 30 min at 4°C. The cells were then incubated on ice for 1.5 h at 4°C. , and incubating with a test multispecific molecule of the present disclosure or a test heterologous molecule. After washing twice with staining buffer, the cells were stained with R-PE-conjugated anti-human IgG secondary antibody (Jack Son Immunoresearch Laboratories) at 4°C for 4 Incubate for 5 minutes. Wash cells twice with staining buffer and then resuspend in 1:200 dilution of Diluted DRAQ7 Live / Dead Cell Stain (Cell Signaling Technology) In 150 μL of Stain Buffer containing 100 mM Tris-HCl (pH 7.0) The PE and DRAQ7 signals of stained cells were analyzed according to the Miltenyi M ACSQuant flow cytometer (Miltenyi Biotec, Aubur n, USA) detect using B2 and B4 channels, respectively. Live cells are gated on DRAQ7 exclusion and the geometric mean fluorescence signal is determined for at least 10,000 live cell events . FlowJo software (Tree S tar) is used for analysis. The data is plotted as the logarithm of the antibody concentration against the mean fluorescence signal . Non-linear regression analysis is performed.

[0594] 5.4 Glycan engineering The ability of the multispecific molecules or heterologous molecules of the present disclosure conjugated to an Ig constant region or a fragment of an Ig constant region to mediate ADCC can be enhanced by manipulating the Ig constant region or a fragment of the oligosaccharide component of the Ig constant region. Human IgG1 or IgG3 is N-glycosylated at Asn297. Here, most of the glycans are in the form of the known bi-branched G0, G0F, G 1, G1F, G2, or G2F. Ig constant region-containing proteins that can be produced by unmanipulated CHO cells typically have a glycan fucose content of at least about 85%. Removal of core fucose from bi-branched complex-type oligosaccharides linked to an Ig constant region or a fragment of an Ig constant region enhances the ADCC of the multispecific molecules or heterologous molecules of the present disclosure via improved FcγRIIIa binding without changing antigen binding or CDC activity. Such multispecific molecules or heterologous molecules are controlled by the osmolality of the medium (K onno et al., Cytotechnology 64:249-265, 20 12), application as a host cell line of the mutant CHO strain Lec13 (Shields et al., (2002) J Biol Chem277:26733-26740), mutation onno et al., Cytotechnology 64:249-265, 20 12), application as a host cell line of the mutant CHO strain Lec13 (Shields et al., (2002) J Biol Chem277:26733-26740), mutation Application as a host cell line for the body CHO strain EB66 (Olivier et al., (20 10) MAbs; 2:405 - 415), application as a host cell line for the rat hybridoma cell line YB2 / 0 (Shinkawa et al., (2003) J Biol C hem 278:3466 - 3473), introduction of small interfering RNA against the specifically 1,6 - fucosyltransferase (FUT8) gene (Mori et al., (20 04) Biotechnol Bioeng 88:901 - 908), or co - expression of β - 1 ,4 - N - acetylglucosaminyltransferase III and Golgi α - mannosida se II or kifunensine, a potent α - mannosidase I inhibitor (Fe rrara et al., (2006) J Biol Chem 281:5032 - 5036), etc., can be achieved using different methods reported to lead to the successful expression of relatively high fucose - unmodified immunoglobulins having a bisected complex type of Fc oligosaccharide.

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

[0596] ​​​The "fucose content" refers to the amount of fucose monosaccharide in the sugar chain at Asn297. The relative amount of fucose is the ratio to the total sugar structure of the fucose-containing structure. These can be determined by a plurality of methods, for example, 1) use of MALDI-TOF of N-gly cosidase F-treated samples (e.g., complex, hybrid, and oligo structures and high-mannose structures), 2) enzymatic release of the Asn297 glycan, followed by derivatization and detection / quantification by HPLC (UPLC) and / or HPLC-MS ( UPLC-MS) with fluorescence detection, 3) cleavage between the first GlcNAc monosaccharide and the second GlcN Ac monosaccharide, leaving fucose attached to the first GlcNAc, with or without treatment of the Asn297 glycan with Endo S or other enzymes, for intact protein analysis of native or reduced mAb, 4) digestion of the mAb into constituent peptides by enzymatic digestion (e.g., trypsin or endopeptidase Lys-C), followed by separation, detection, and quantification by HPLC-MS (UPLC-MS), 5) separation of mAb oligosaccharides from the mAb protein by specific enzymatic deglycosylation with PNGase F at Asn 297, thereby enabling characterization and quantification. The oligosaccharides thus released are labeled with a fluorophore and subjected to detailed characterization of the glycan structure by matrix-assisted laser desorption ionization (MALDI) mass spectrometry by comparison with the theoretical mass of the measured mass, determination of the degree of sialylation by ion-exchange HPLC (GlycoSep C), separation and quantification of oligosaccharide types according to hydrophilic criteria by normal-phase HPLC (GlycoSep N), and high-performance capillary electrophoresis-laser-induced fluorescence (HPCE-LI ), etc. The oligosaccharides released in this way are labeled with a fluorophore and compared with the theoretical mass of the measured mass for detailed characterization of the glycan structure by matrix-assisted laser desorption ionization (MALDI) mass spectrometry, determination of the degree of sialylation by ion-exchange HPLC (GlycoSep C), separation and quantification of oligosaccharide types according to hydrophilic criteria by normal-phase HPLC (GlycoSep N), and high-performance capillary electrophoresis-laser-induced fluorescence (HPCE-LI Separation and quantification of oligosaccharides according to (F), and can be separated and characterized by various supplementary techniques to be able to.

[0597] "Low fucose" or "low fucose content" refers to the multispecific molecules or heterologous molecules of the present disclosure that contain an Ig constant region or a fragment of an Ig constant region having a fucose content of about 1% to 15% to. Refers to.

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

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

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

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

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

[0603] 5.6 Polynucleotides, vectors and host cells The present disclosure also provides an isolated polynucleotide encoding the spFv of the present disclosure.

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

[0605] In some embodiments, the vector is an expression vector. The expression vector is a plus mid vector, viral vector, vector for baculovirus expression, vector for prokaryotic expression, vector for eukaryotic expression, transposon-based vector, or any other vector suitable for introducing the polynucleotides of the present disclosure into a given cell or organism. The polynucleotide encoding the spFv of the present disclosure can be operably linked to a control sequence of an expression vector that promotes the expression of the spFv. Such regulatory elements include a transcription promoter, a sequence encoding a suitable mRNA ribosome binding site, and sequences that control the termination of transcription and translation. The expression vector can also include an origin of replication, other 5' or 3' flanking non-transcribed sequences, 5' or 3' untranslated sequences (such as the necessary ribosome binding site), splice donor and acceptor sites, or one or more non-transcribed elements such as a selectable marker. The polynucleotide can be cDNA. The promoter driving spFv expression can be a strong, weak, tissue-specific, inducible, or developmental-specific promoter. Exemplary promoters that can be used are hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin, human myosin, human hemoglobin globin, 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 the cytomegalovirus (CMV) immediate early promoter and the like. In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for use in the described embodiments. Such viral promoters include the cytomegalovirus (CMV) immediate early promoter Motor, SV40 early and late promoters, Mouse Mammary Tumor Virus (MMTV) promoter, Moloney leukemia virus, Human Immunodeficiency Virus (HIV), Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV), and long terminal repeats (LTRs) of other retroviruses, as well as the thymidine kinase promoter of herpes simplex virus, are included, but not limited to these. Inducible promoters such as metallothionein promoters, tetracycline-inducible promoters, doxycycline-inducible promoters, protein kinase R 2’,5’-oligoadenylate synthetase, Mx gene, and one or more interferon-stimulated response elements (ISREs) such as ADAR1. The vectors of the present disclosure may also contain one or more internal ribosome entry sites (IRES). Inclusion of the IRES sequence into the fusion vector may be beneficial for enhancing the expression of some proteins. The vectors of the present disclosure may be circular or linear. These can be prepared to contain a replication system functional in prokaryotic or eukaryotic host cells. The replication system may be derived from, for example, ColE1, SV40, 2μ plasmid, λ, bovine papillomavirus, etc. Expression vectors can be designed for transient expression, stable expression, or both. Expression vectors r Virus, MMTV) promoter, Moloney leukemia virus, Human Immunodeficiency Virus (Hum an Immunodeficiency Virus, HIV), Epstein Barr Virus (Epstein Barr Viru s, EBV), Rous Sarcoma Virus (Rous Sarcoma Virus, RSV), and other retroviruses long terminal repeat (LTR), and the thymidine kinase promoter of herpes simplex virus are included, but not limited to these. Metallothionein promoter, tetracycline-inducible promoter, doxycycline-inducible promoter, protein kinase R 2’,5’-oligoadenylate synthetase, Mx gene, and A DAR1 and other one or more interferon-stimulated response elements (interferon-stimula ted response element, ISRE). The vectors of the present disclosure may also contain one or more internal ribosome entry sites (Internal Ribosome Entry Site 、IRES). Inclusion of the IRES sequence into the fusion vector may be beneficial for enhancing the expression of some proteins. The vectors of the present disclosure may be circular or linear. These can be prepared to contain a replication system functional in prokaryotic or eukaryotic host cells. The replication system may be derived from, for example, ColE1, SV40, 2μ plasmid, λ, bovine papillomavirus, etc. Expression vectors can be designed for transient expression, stable expression, or both. Expression vectors can be designed for transient expression, stable expression, or both. Expression vectors can be designed for transient expression, stable expression, or both. Expression vectors can be made for constitutive or inducible expression.

[0606] Exemplary vectors that can be used include bacteria: pBs, phagescript, PsiX 174, pBluescript SK, pBs KS, pNH8a, pNH16a, p NH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotes: pWL neo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pS VK3, pBPV, pMSG and pSVL (Pharmacia), pEE6.4 (Lo nza) and pEE12.4 (Lonza). Additional vectors include pUC series (Fermentas Life Sciences, Glen Burnie , Md.), pBluescript series (Stratagene, LaJolla , Calif.), pET series (Novagen, Madison, Wis.), p GEX series (Pharmacia Biotech, Uppsala, Sweden ), and pEX series (Clontech, Palo Alto, Calif.) can be mentioned. Bacteriophage vectors such as λGT10, λGT11, λEMBL4, and λNM1149, λZapII (Stratagene) can be used. Exemplary plant expression vectors include pBI01, pBI01.2, pBI121, pB I101.3, and pBIN19 (Clontech). Exemplary animal expression Examples of vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech ). Expression vectors can be viral vectors, for example, retroviral vectors , such as gamma-retroviral vectors.

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

[0608] "Host cell" refers to a cell into which a vector has been introduced. The term "host cell" is intended to refer not only to a particular target cell, but also to the progeny of such a cell, as well as stable cell lines generated from a particular target cell. It is understood that due to either mutation or environmental influence, certain modifications may occur in subsequent generations, and thus such progeny may not be identical to the parental cell, but are still included within the scope of the term "host cell" as used herein. Such host cells may be eukaryotic cells, prokaryotic cells, plant cells, or archaebacterial cells. Examples of prokaryotic host cells include Escherichia coli, bacilli such as Bacillus subtilis, and other Enterobacteriaceae such as Salmonella, Serratia, and various species of Pseudomonas. Other microorganisms such as yeast are also useful for expression. Examples of suitable yeast host cells include Saccharomyces (e.g., S. cerevisiae) and Pichia. Exemplary eukaryotic cells may be derived from mammals, insects, birds, or other animals. Mammalian eukaryotic cells include immortalized cell lines (e.g., hybridomas) or ​​​​​​​​​​​​Myeloma cell lines (e.g., SP2 / 0 (American Type Culture Co llection (ATCC), Manassas, VA, CRL-1581), NS0 (European Collection of Cell Cultures (EC ACC), Salisbury, Wiltshire, UK, ECACC No. 851 10503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL -1580) mouse cell lines) can be mentioned. Exemplary human myeloma cell lines are U266 (A TTC CRL-TIB-196). Other useful cell lines include CHO-K1S V (Lonza Biologics (Walkersville, MD)), CHO- K1 (ATCC CRL-61), or those derived from Chinese hamster ovary (CHO) cells such as DG44.

[0609] The present disclosure also provides a method for producing the spFv of the present disclosure, which includes culturing the host cells of the present disclosure under conditions where the spFv is produced, and recovering the spFv produced by the host cells. Methods for making and purifying scFvs are known. Once synthesized (chemically or recombinantly), the scFvs 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, etc. (generally, see Sc opes, Protein Purification (Springer-Verla g, N.Y., (1982)). The scFvs of the present disclosure may be substantially pure, for example, free of contaminants such as cell debris, macromolecules other than the target protein chromatography, high performance liquid chromatography (HPLC) purification, gel electrophoresis, etc. can be purified according to standard procedures including ammonium sulfate precipitation, affinity columns, column opes, Protein Purification (Springer-Verla g, N.Y., (1982)). The scFvs of the present disclosure may be substantially pure and may, for example, contain no contaminants such as cell debris, macromolecules other than the target protein i. For example, a purity of at least about 80% - 85%, at least about 85% - 90% purity, at least about 90% - 95% purity, or at least about 98% - 99% purity, or even higher purity may be used.

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

[0611] 5.7 Pharmaceutical Compositions and Administration The present disclosure also provides a pharmaceutical composition comprising the spFv of the present disclosure, a heterologous molecule comprising the spFv, or a multispecific molecule comprising the spFv, and a pharmaceutically acceptable carrier. For therapeutic use, the spFv of the present disclosure, a heterologous molecule comprising the spFv, or a multispecific molecule comprising the spFv can be prepared as a pharmaceutical composition containing an effective amount of the spFv of the present disclosure, a heterologous molecule comprising the spFv, or a multispecific molecule comprising the spFv as an active ingredient in a pharmaceutically acceptable carrier. The "carrier" refers to a diluent, adjuvant, excipient, or vehicle in which the spFv of the present disclosure, a heterologous molecule comprising the spFv, or a multispecific molecule comprising the spFv is administered. Such vehicles may be liquids such as water and oils derived from petroleum, animals, plants, or synthetic sources, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc. For example, 0.4% saline and 0.3% glycine may be used. These solutions are sterilized and generally do not contain particulate matter. They can be sterilized by conventionally well-known sterilization techniques (e.g., filtration). The composition may contain pharmaceutically acceptable adjuvants required to approximate physiological conditions, for example, pH It may contain regulators, buffers, stabilizers, thickeners, lubricants, colorants, etc. Such pharmaceuticals The concentration of the spFv, heterologous molecule containing spFv or multispecific molecule containing spFv of the present disclosure in the pharmaceutical preparation is usually at least about 1% by weight to up to 15 or 20% by weight, from less than about 0.5% by weight, and may vary up to 6% by weight, and may be selected mainly based on the required dose, fluid volume , viscosity, etc. according to the administration method selected. Suitable vehicles and formulations (including other human proteins , for example, human serum albumin) are described, for example, in Remington: The Sc ience and Practice of Pharmacy, 21st Edit ion, Troy, D.B. ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Phar maceutical Manufacturing pp 691-1092, see especially pp. 958-989.

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

[0613] 5.8 Process for preparing the 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 and providing a linker (L) that contains a first L Cys or is engineered to contain it and engineering the VH to include a VH Cys at a structurally conserved surface-exposed VH framework residue position and forming a disulfide bond between the VH Cys and a first L Cys to prepare a stabilized scF v, comprising a process.

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

[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 and providing a linker (L) that includes, or is engineered to include, a first L Cys and a second L Cys and engineering the VH to include a VH Cys at a structurally conserved surface-exposed VH framework residue position and engineering the VL to include a VL Cys at a structurally conserved surface-exposed VL framework residue position and forming a disulfide bond between the VH Cys and the first L Cys and between the VL Cys and the second L Cys to prepare a stabilized scFv and forming a disulfide bond between the VH Cys and the first L Cys and between the VL Cys and the second The present invention provides a process including:

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

[0617] Any known VH / VL pair of scFv that forms an antigen-binding domain can be used in the stabilization of the present disclosure. Alternatively, the antigen-binding VH / VL pair of interest can be engineered into a scFv using known methods. As such, VH / VL pairs can be identified de novo and the resulting VH / VL pairs can be engineered into an spFv format.

[0618] For example, the hybridoma method of Kohler and Milstein may be used to identify the target antigen. Identify VH / VL pairs that can be combined with the original and the resulting VH / VL pair engineered as an spFv. Alternatively, they can carry human immunoglobulin (Ig) loci in their genome. Transgenic animals, such as mice, rats, or chickens, that express the antigen-binding fragment These can be produced by, for example, the methods described in U.S. Pat. No. 6,150,584 and International Publication No. No. 1999 / 45962, No. 2002 / 066630, No. 2002 / 43478 Nos. 2002 / 043478 and 1990 / 04036. The endogenous immunoglobulin loci of such animals may be disrupted or deleted, and homologous or by using non-homologous recombination, by using transchromosomes, or by using minigenes The gene is used to express at least one complete or partial human immunoglobulin locus. It can also be inserted into the genome of a living organism. neron_com), Harbor Antibodies(http: / / _ww w_harbourantibodies_com), Open Monoclonal Technology, Inc. (OMT) (http: / / _www_omtinc _net), KyMab (http: / / _www_kymab_com), Trian ni (http: / / _www.trianni_com) and Ablexis (htt p: / / _www_ablexis_com) and other companies may be working to use the above technologies to provide human antibodies targeting selected antigens. Phage display may also be used to generate antigen-binding fragments that can be engineered as spFvs. Some embodiments of the spFvs of the present disclosure are humanized. In some embodiments, the spFvs of the present disclosure are human. In some embodiments, the spFvs of the present disclosure are

[0619] non-human.

[0620]

[0621] In some embodiments, the distance between VH Cys and VL Cys is about 7 Å to about 9 Å in a stabilized scFv.

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

[0622] In some embodiments, VL Cys is at L3, L5, L39, L42, L45, L 100 or L102, and the residue numbering follows Chothia.

[0623] In some embodiments, VH Cys is at H105 and VL Cys is at L42, or VH Cys is at H43 and VL Cys is at L100, or VH Cys is at H3 and VL Cys is at L3, or The VH Cys is in H3, and the VL Cys is in L5, or The VH Cys is in H3, and the VL Cys is in L39, or The VH Cys is in H3, and the VL Cys is in L42, or The VH Cys is in H3, and the VL Cys is in L45, or The VH Cys is in H3, and the VL Cys is in L100, or The VH Cys is in H3, and the VL Cys is in L102, or The VH Cys is in H5, and the VL Cys is in L3, or The VH Cys is in H5, and the VL Cys is in L5, or The VH Cys is in H5, and the VL Cys is in L39, or The VH Cys is in H5, and the VL Cys is in L42, or The VH Cys is in H5, and the VL Cys is in L45, or The VH Cys is in H5, and the VL Cys is in L100, or The VH Cys is in H5, and the VL Cys is in L102, or The VH Cys is in H40, and the VL Cys is in L3, or The VH Cys is in H40, and the VL Cys is in L5, or The VH Cys is in H40, and the VL Cys is in L39, or The VH Cys is in H40, and the VL Cys is in L42, or The VH Cys is in H40, and the VL Cys is in L45, or The VH Cys is in H40, and the VL Cys is in L100, or The VH Cys is in H40, and the VL Cys is in L102, or The VH Cys is in H43, and the VL Cys is in L3, or The VH Cys is in H43, and the VL Cys is in L5, or The VH Cys is in H43, and the VL Cys is in L39, or The VH Cys is at H43, and the VL Cys is at L42, or The VH Cys is at H43, and the VL Cys is at L45, or The VH Cys is at H43, and the VL Cys is at L102, or The VH Cys is at H46, and the VL Cys is at L3, or The VH Cys is at H46, and the VL Cys is at L5, or The VH Cys is at H46, and the VL Cys is at L39, or The VH Cys is at H46, and the VL Cys is at L42, or The VH Cys is at H46, and the VL Cys is at L45, or The VH Cys is at H46, and the VL Cys is at L100, or The VH Cys is at H46, and the VL Cys is at L102, or The VH Cys is at H105, and the VL Cys is at L3, or The VH Cys is at H105, and the VL Cys is at L5, or The VH Cys is at H105, and the VL Cys is at L39, or The VH Cys is at H105, and the VL Cys is at L45, or The VH Cys is at H105, and the VL Cys is at L100, or The VH Cys is at H105, and the VL Cys is at L102, and the residue numbering follows 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 IgG1, IgG2, IgG3 or IgG4 isotype.

[0628] In some embodiments, L comprises the amino acid sequence C(X) y C (SEQ ID NO: 23), wherein X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gl n, His, Ile, Leu, Lys, Phe, Thr, Trp or Tyr, and y is , an integer from 1 to 3.

[0629] In some embodiments, L comprises 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 is the amino acid sequence CPC, CGC, CSC, CPPC (arrangement SEQ ID NO: 1), CGPC (SEQ ID NO: 28), CPGC (SEQ ID NO: 29), CGGC (arrangement SEQ ID NO: 30), CSPG (SEQ ID NO: 31), CPSC (SEQ ID NO: 32), CSSC (arrangement SEQ ID NO: 33), CGSC (SEQ ID NO: 34), CSGC (SEQ ID NO: 35), CPPPC (arrangement SEQ ID NO: 36), CGPPC (SEQ ID NO: 37), CPGPC (SEQ ID NO: 38), CPPGC (arrangement 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), CPPS C (SEQ ID NO: 45), CSSPC (SEQ ID NO: 46), CPSSC (SEQ ID NO: 47), CS SSC (SEQ ID NO: 48), CGSPC (SEQ ID NO: 49), CPGSC (SEQ ID NO: 50), It contains CSGPC (SEQ ID NO: 51) or CPSGC (SEQ ID NO: 52).

[0631] In some embodiments, L comprises about 14 to about 19 amino acids, such as 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 from 6 to 9, y is an integer from 1 to 3, and n is an integer from 4 to 6.

[0633] In some embodiments, L is the amino acid sequence (X) m C(X) y C(X) n (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 from 6 to 9, y is an integer from 1 to 3, and n is an integer from 4 to 6.

[0635] In some embodiments, L is the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, or 7​ includes.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0658] In some embodiments, VH contains Cys at H46, VL contains Cys at L3, L contains the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7, The scFv is in the 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 lacks disulfide bonds and binds to the antigen with an equivalent affinity when compared to the parental scFv.

[0663] The present disclosure also provides a process for preparing a stabilized scFv, comprising providing polynucleotides encoding VH, L, and VL, wherein VH comprises Cys at H105 and VL comprises Cys at L42, or VH comprises Cys at H43 and VL comprises Cys at L100, or VH comprises Cys at H3 and VL comprises Cys at L3, or VH comprises Cys at H3 and VL comprises Cys at L5, or VH comprises Cys at H3 and VL comprises Cys at L39, or VH comprises Cys at H3 and VL comprises Cys at L42, or VH comprises Cys at H3 and VL comprises Cys at L45, or VH comprises Cys at H3 and VL comprises Cys at L100, or VH comprises Cys at H3 and VL comprises Cys at L102, or VH comprises Cys at H5 and VL comprises Cys at L3, or VH comprises Cys at H5 and VL comprises Cys at L5, or VH comprises Cys at H5 and VL comprises Cys at L39, or Whether VH contains Cys at H5 and VL contains Cys at L42, or Whether VH contains Cys at H5 and VL contains Cys at L45, or Whether VH contains Cys at H5 and VL contains Cys at L100, or Whether VH contains Cys at H5 and VL contains Cys at L102, or Whether VH contains Cys at H40 and VL contains Cys at L3, or Whether VH contains Cys at H40 and VL contains Cys at L5, or Whether VH contains Cys at H40 and VL contains Cys at L39, or Whether VH contains Cys at H40 and VL contains Cys at L42, or Whether VH contains Cys at H40 and VL contains Cys at L45, or Whether VH contains Cys at H40 and VL contains Cys at L100, or Whether VH contains Cys at H40 and VL contains Cys at L102, or Whether VH contains Cys at H43 and VL contains Cys at L3, or Whether VH contains Cys at H43 and VL contains Cys at L5, or Whether VH contains Cys at H43 and VL contains Cys at L39, or Whether VH contains Cys at H43 and VL contains Cys at L42, or Whether VH contains Cys at H43 and VL contains Cys at L45, or Whether VH contains Cys at H43 and VL contains Cys at L102, or Whether VH contains Cys at H46 and VL contains Cys at L3, or Whether VH contains Cys at H46 and VL contains Cys at L5, or Whether VH contains Cys at H46 and VL contains Cys at L39, or Whether VH contains Cys at H46 and VL contains Cys at L42, or Whether VH contains Cys at H46 and VL contains Cys at L45, or Whether VH contains Cys at H46 and VL contains Cys at L100, or either 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 Cys at H105 and VL contains Cys at L100, or VH contains Cys at H105 and VL contains Cys at L102, with residue numbering in accordance with Chothia, 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 a stabilized scFv, and thereby providing a process.

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

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

[0666] To further illustrate some of the embodiments disclosed herein, the following examples are provided for purposes of illustration only and are not intended to limit the embodiments of the present disclosure.

Examples

[0667] 6.1 Example 1: Design of Stabilized scFv Monoclonal antibodies (mAbs) recognize their target antigens via two variable domains, VL and VH. Single-chain Fv (scFv) is a genetic fusion of VL and VH with a flexible linker in either the VL-linker-VH or VH-linker-VL orientation. ​ rd et al. (1988) Science 242:423-426 (1988) was first designed by. Flexible linkers are typically (GGGGS)n; n = 1 ~4 (SEQ ID NO: 2, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55), etc., three or four repeats of glycine-serine linkers. The scFv reproduces the antigen-binding specificity and mainly the affinity of its parent m Ab. These scFv molecules are used as detection / diagnostic reagents or as components for making more sophisticated molecules such as bispecific , multispecific therapeutic agents (Brinkmann and Kontermann (2017) MAb 9:182-212) or CAR-T therapeutic agents (Gross et al., (1 989), Transplant Proc 21(1 Pt 1):127-130, Porter et al., (2011) J Cancer 2:331-332, P orter et al., (2011) N Engl J Med 365:725- 733), etc., and have found wide applications.

[0668] One of the problems with scFv molecules is their low stability and tendency to aggregate (Worn an d Pluckthun (2001) J Mol Biol 305:989-1010 , Rothlisberger et al., (2005) J Mol Biol 3 47:773-789). 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 B iol 362:580-593, Zhao et al., (2010) Int J ​ Mol Sci 12:1-11, Perchiacca and Tessier (2 012) Annu Rev Chem Biomol Eng 3:263-286, A sial et al., (2013) Nat Commmun 4:2901, Gil and Schrum (2013) Adv Biosci Biteccchnol 4:73-84, Tiller and Tessier (2015) Annu rev Biomed Eng 17:191-216). These strategies include additional dimerization motifs, etc., and use different experimental methods to improve the stability and / or interface interactions of VL / VH domains, and introduce disulfide bonds between VL / VH domains to achieve this. An important difficulty is that most of these strategies are specific to VH / VL pairs and cannot be easily transferred to other VH / VL pairs. In some cases, the manipulation can adversely affect the VL / VH structure and scFv properties. Recently, Zhang et al. introduced a disulfide bond between position 44 of VH and position 100 of VL of the anti-aflatoxin B1 scFv (H4), and successfully achieved significant stabilization of the scFv while maintaining its binding affinity (Zhao et al., (2010) Int J Mol Sci 12:1- 11). However, due to the distance and angle limitations between the two selected positions, when applied to other VL / VH pairs, the VL / VH disulfide can limit / distort the relative orientation between the two domains that are often required for binding. The interface between the heavy and light chains of the Fab fragment includes VH / VL and CH1 / CL interactions including

[0669] ​. Two independent interaction sets result in a synergistic stabilization effect. Furthermore, the V / C junction also contributes to some stabilization effect. In comparison, in scFv, the VH / VL interface is maintained only by VH / VL interaction. The linker is designed to be flexible and non-limiting, except when its length is designed to be short to facilitate scFv-scFv interactions for dimer and oligomer formation. The length and nature of the linker are known to contribute little to the stability of scFv when it is sufficiently long.

[0670] 6.1.1 "Staple treatment" design The purpose of this specification was to design and generate a stabilized scFv without adversely affecting the relative movement between VH and VL that form the scFv. This was achieved by manipulating the disulfide bonds between VH and the linker and between VL and the linker to stabilize the scFv. The limitation (i.e., the disulfide bond), when properly positioned, serves the role of the synergistic effect brought about by the above-mentioned CH1 / CL and V / C interactions. For this purpose, one surface-exposed framework position (anchor point) conserved in two structures was identified in VH and VL respectively, which did not overlap with the typical predicted antigen-binding site and was mutated to a cysteine (Cys) residue. Then, two positions were selected in a flexible linker for the Cys positions. When the distance and position between the linker Cys residues were designed in a manner that promoted the formation of disulfide bonds between the linker Cys and each anchor point, VH and VL could be tethered more firmly compared to tethering in the absence of disulfide bonds. This scheme is C An exemplary linker containing the PPC array (SEQ ID NO: 1) is shown in FIG. 1. The flexible The concept of forming a disulfide bond between the linker and the anchor point is described herein as "stapling". The resulting "stapled" scFv molecule is referred to herein as spFv ("stapled Fv").

[0671] 6.1.2 Selection of Anchor Points, Design of Staple Arrays, and Linkers In a widely applicable stapling scheme, the anchor points are structurally conserved and exposed on the surfaces of both VL and VH, and it is important that mutations to Cys residues do not affect the folding of VL and VH or their binding to the antigen. The main anchor points of VL and VH, as well as the distances and geometric shapes of the N and C termini, are also important considerations for proper disulfide formation. The anchor points are selected separately for spFv in the VL-linker-VH and VH-linker-VL orientations. In the case of the VL-linker-VH orientation, Chothia position 42 in VL and Chothia position 105 in VH were selected as the anchor points. A graphical representation of the selected anchor points of spFv in the VL-linker-VH orientation is shown within the Fv of the human germline antibody (hereinafter, pdb ID 5I19, GLk1) in FIG. 2. In GLk1, VL Chothia position 42 is lysine (K) and VH Chothia position 105 is glutamine (Q). In the case of the VH-linker-VL orientation, Chothia position 100 in VL and Chothia position 43 in VH are the selected anchor points.

[0672] The anchor points were separately selected for spFv 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 in VH were selected as the anchor points. In the case of the VL-linker-VH orientation, Chothia position 42 in VL and Chothia position 105 in VH were selected as the anchor points. In the case of the VL-linker-VH orientation, Chothia position 42 in VL and Chothia position 105 in VH were selected as the anchor points. A graphical representation of the selected anchor points of spFv in the VL-linker-VH orientation is shown in FIG. 2 within the Fv of the human germline antibody (hereinafter, pdb ID 5I19, GLk1). In GLk1, VL Chothia position 42 is lysine (K) and VH Chothia position 105 is glutamine (Q). In the case of the VH-linker-VL orientation, Chothia position 100 in VL and Chothia position 43 in VH are the selected anchor points. In the case of the VH-linker-VL orientation, Chothia position 100 in VL and Chothia position 43 in VH are the Selected as the carp points. Figure 3 shows the selected anchor points for spFv in the VH-linker-VL orientation within the Fv of a human germline antibody (pdb ID 5I19 , GLk1). In GLk1, the VL Chothia position 100 is glutamine (Q), and the VH Chothia position 43 is lysine (K). The selected anchor points were structurally conserved, and the geometry was very similar in antibodies containing either kappa or lambda light chains. The distance between pairs of anchor points was in the range of about 7 Å (for the VL-linker-VH orientation) to about 9 Å (for the VH-linker-VL orientation). The staple sequences embedded within the linker connecting VH and VL were designed to be of a length similar to the distance between the anchor points in the spFv. As an initial example of a staple sequence, CPPC (SEQ ID NO: 1) was selected as a possible staple sequence because this sequence occurs naturally in the human IgG1 hinge as well as in

[0673] several murine IgGs. The structure of the hinge 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) was in the range of about 7 Å to 9 Å. This range is very similar to the distance between the two anchor points for both the VL-linker-VH and VH-linker-VL orientations, so the CPPC (SEQ ID NO: 1) staple sequence has the potential to provide the correct geometric shape for stapling, i.e., to form appropriate disulfide bonds efficiently and correctly to the anchor points. In general, the staple sequences were designed to have two Cys residues. ​​​​​​​ For proper stapling, the N-terminal Cys of the staple array forms a disulfide bond with the spFv N-terminal domain main anchor point, and the C-terminal Cys of the staple array forms a disulfide bond with the spFv C-terminal domain anchor point.

[0674] Therefore, the linker connecting VH and VL is designed to include the staple array as well as the connecting arrays at both the N-terminus and C-terminus to extend the linker to allow for the internal folding of VH and VL and to promote proper positioning of the staple array, providing a linker length sufficient for this purpose.

[0675] In the VL-linker-VH design, the distances between the VL anchor point (K42), VH anchor point (Q105), C-terminus of VL (K107), and N-terminus of VH (Q1) are shown in Figure 2. In the VH-linker-VL design, the distances between the VL anchor point (Q100), VH anchor point (K43), C-terminus of VH (S114), and N-terminus of VL (D1) are shown in Figure 3. Modeling suggests that these distances can be spread by a linker length of approximately 14 - 19 residues, and that the 4-residue staple array is 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 can be represented 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 array. The n and m residues can be glycine or serine, or other amino acid residues. These linker lengths are too short to allow for scrambling, but are sufficient for staple processing It is of sufficient length to enable and is expected to be sufficiently flexible.

[0676] 6.2 Example 2: Generation and Characterization of spFv To evaluate the staple processing design, three human antibodies were selected to generate scFv and the corresponding spFv: two antibodies (GL k1 and GLk2) with kappa light chains from the synthetic phage antibody library Shi et al., (2010) J M ol Biol 397:385-396) and a lambda-containing antibody (CAT2200) obtained from the paper (Gerhardt et al. (2009) J Mol Biol 394:905-921). For CAT2200, the T28G mutation was introduced into the parental VH to generate a mutant (CA T2200 a) that reduced some of its interaction with the target IL-17. Furthermore, the S42Q mutation (Chothia) was engineered into the parental CAT2200 VL and paired with T2 8G VH to generate CAT2200b. The amino acid sequences of the VL and VH domains of GLk1, GLk2, CAT220 0a and CAT2200b are shown in Figures 6 and 7, respectively. The VH domain amino acid sequences are identical between BAT2200a and CAT2200b. GLk1VH is closest to human IGHV2-23 01 GLk2VH to human IG * 01 GLk2VH to human IG HV5-51, CAT2200VH to human IGHV2-23 * 01. GLk1 VL is closest to human IGKV1-39 * 01, GLk2VL to human IGKV3-20 * 01, and CAT2200VL to human IGLV 6-57 * 01.

[0677] All scFv and spFv molecules were generated and expressed in both the VL-linker-VH and VH-linker-VL orientations. For the scFv constructs, the standard (GGGGS)4 (SEQ ID NO: 2) linker was used. For the spFv, different linker lengths within the above n and m ranges were used. For the GLk1 spFv, a 9-4-5 linker was used for both orientations. For the GLk2 spFv, 9-4-5 and 6-4-6 linker lengths were used for the VL-VH and VH-VL orientations, respectively. For the CAT2200a spFv, VL-VH molecules were made with 8-4-4 and 9-4-4 linkers, respectively, and the CAT2200b spFv VH-VL was made 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. -VL were expressed. For the scFv constructs, the standard (GGGGS)4 (SEQ ID NO: 2) linker was used. For the spFv, different linker lengths within the above n and m ranges were used. For the GLk1 spFv, a 9-4-5 linker was used for both orientations. For the GLk2 spFv, 9-4-5 and 6-4-6 linker lengths were used for the VL-VH and VH-VL orientations, respectively. For the CAT2200a spFv, VL- VH molecules were made with 8-4-4 and 9-4-4 linkers, respectively, and the CAT2200b spFv VH-VL was made 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 the 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 using an AKTAXPRESS system (GE Healthcare) e) Purified from the clarified supernatant on a 1 ml His-TRAP HP column (GE Healthcare). The column was prepared with a 0 - 100% gradient of elution buffer (washing 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 to 50 mM Tris, pH 7.5 and 20 mM imidazole and then loaded onto a 1 ml HisTRAP HP column at 4 °C at 0.8 mL / min. The column was then washed with PBS until a stable baseline was obtained. The column was further washed with 20 CV of washing buffer and eluted into a single injection loop with elution buffer, desalted in 1x DPBS on a 26 / 10 HiPrep desalting column, and fractions were collected. The fractions containing the purified protein were then pooled and concentrated. Glk2 scFv and spFv proteins were dialyzed into DPBS for thermal stability measurements (DSC and NanoDSF) and into 25 mM Tris, pH 7.5 and 100 mM NaCl for other studies. Other scFv and spFv proteins were dialyzed into 25 mM MES, pH 6.0 and 100 mM NaCl. CAT2200a scFv VL-VH was purchased from the vendor. The concentration was 0.77 mg / mL in DPBS, pH 7.2. A mutant of IL-17 (12 - 132 with K70Q A132Q C106S mutations, hereinafter abbreviated as IL-17 for simplicity) (SEQ ID NO: 22) was purchased from Accelagen (CA). The proteins were in their native form. form. form. form. form.

[0682] CAT2200a scFv VL-VH was purchased from the vendor. The concentration was 0.77 mg / mL in DPBS, pH 7.2. An IL-17 mutant (12 - 132 with K70Q A132Q C106S mutations, hereinafter abbreviated as IL-17 for simplicity) (SEQ ID NO: 22) was purchased from Accelagen (CA). The proteins were in their native form. refolded from E. coli inclusion bodies after the 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) MNSEDKNFPRTVMVNLNIHNRNTNTNPKRSSDYYNRSTS PWNLHRNEDPERYPSVIWEAQCRHLGCINADGNVDYHMNS VPIQQEILVLRREPPHSPNSFRLEKILVSVGCTCVTPIVH HVQ

[0684] 6.2.1 Thermal stability of the generated scFv and spFv molecules The thermal stability of the scFv and spFv molecules was investigated by differential scanning calorimetry (DSC). The scFv and spFv proteins were dialyzed overnight against 1× DPBS (Gibco) for GLk1 and against CAT2200a / CAT2200b or MES (25 mM MES, pH 6.0, 100 mM NaCl) for GLk2. The dialysis buffer was then filtered through 0.22 micrometers and used as the reference solution and the buffer blank in the DSC experiment. The proteins were diluted to approximately 0.5 mg / mL in the filtered buffer, and 400 μL of each protein or buffer sample was loaded into a 96-deep well plate (MicroLiter Analytical Supplies, 07-2100) and held at 4 °C in the autosampler drawer throughout the experiment. MicroCal capillary DSC using an autosampler (Malvern) was used to perform the DSC experiment. The DSC scan was from 25 to 95 °C without sample rescan at 60 °C. Performed at a scan rate of / h. Without selecting feedback, the filtering period was set to 15 seconds After each sample, the 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 DS C automatic analysis add-on (Malvern). The baseline range and type were selected manually and then subtracted. After subtracting the previous buffer blank from the sample curve, concentration-dependent normalization was performed. The thermal melting profiles were analyzed using both the 2 state and the non-2 state transitions. The 2-state fit (one transition) did not match the experimental curve well. Therefore, two transitions (Tm1 and Tm2) were calculated by performing a non- 2-state fit manually. The Tm data are reported in Table 6. All DSC profiles of scFv and spFv proteins showed skewness that could be fitted by non-2 state transitions. Therefore, for each scFv or spFv, two transitions (Tm1 and Tm2) were reported (Table 6). Almost certainly, these two transitions correspond to the melting Tm of the VL and VH domains, respectively. Generally, when comparing the differences between scFv and spFv for either Tm1 or Tm2, there is an increase of about 10 °C due to stapling, regardless of the Tm of the starting scFv. There is only one exception, namely that the difference between GLk2 scFv and spFv (VH-VL orientation) is about 7 °C. This is likely due to the shorter 6+4+6 linker that can cause a slight distortion in the geometry of the stapling process The fact that ΔTm1 (VL) and ΔTm2 (VH) were nearly identical indicates that in addition to strengthening the VL / VH interaction, the stapling process also​​​​​​​​ suggests that it leads to the stabilization of its own domain. Alternatively, a stronger VH / V L interaction transmits the stabilizing effect to the stabilization of the VL / VH domain. In summary, the staple treatment described in this specification significantly increases the stability of the scFv.

[0685]

Table 6

[0686] The CAT2200 spFv was tested for its binding to IL-17. The binding was equivalent when compared to the C AT2200 scFvs.

[0687] 6.3 Example 3: Verification of Appropriate Staple Treatment by Crystallization of the Generated scFv and spFv Molecules The proteins were concentrated in their respective buffers: GLk1 spFv VL-VH was 8.67 mg / ml in 25 mM MES, pH 6.0, 100 mM NaCl, GLk1 spFv VH-VL was 5 mg / ml in 25 mM MES, pH 6.0, 100 mM NaCl, GLk2 spFv VH-VL was 8.66 mg / ml in 25 mM Tris, pH 7.5, 1 00 mM NaCl, cat2200b spFv VH-VL was 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 Mosquit o robot. Each well contained 100 nl of protein and 1 00 nl of reservoir solution and was incubated at 20 °C against 70 μl of reservoir. The reservoir solution was IH1 and IH2 custom conditions, as well as PEG Ion Scr ed. ​ is HT (Hampton Research). Some initial conditions were refined by varying the reservoir components in the optimization trials. Crystals of diffraction quality were obtained for some of the scFv and spFv proteins. Table 7 shows a summary of the conditions used. The crystals were soaked in the mother liquor supplemented with 20% glycerol for several seconds and flash frozen in liquid nitrogen. X-ray data were collected at the IMCA-CAT Beamline 17ID at Argonne National Lab.

[0688]

Table 7

[0689] 6.3.1 Crystallization of CAT2200a scFv VL-VH and CAT 2200a spFv VL-VH complexed 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 cut-off Amicon Ultra concentrator and loaded onto a Superdex75 column equilibrated with 250 mM NaCl, 20 mM HEPES, pH 7.5. The fractions corresponding to the complex were pooled and concentrated to a volume of 150 μL. The sample was diluted and concentrated 4 times (350 μL of 50 mM NaCl, 20 mM HEPES, pH 7.5 was added to make a concentration of just less than 150 μL). The volume was brought to approximately 105 μL and the concentration was determined to be 2.69 mg / mL. A buffer devised in-house and ​​​​​​​​​​​​​For an 80 μL reservoir which is a set of precipitation conditions, Corning 3550 plate In the Mosquito crystallization robot with 150 nL of protein + 150 nL of reservoir, crystallization was set up in the sitting-drop method using the robot. The plate was incubated at 2 0 °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 to crystallization seeds using Hampton Seed beads in 100 μL of 27% PEG3350, 200 mM ammonium acetate, 100 mM sodium acetate, pH 4.5.

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

[0691] The IL-17-CAT2200a spFv VL-VH complex was generated 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 passed through a 10 kDa MWCO Amicon Ultra 0.5 Concentrate to about 100 μL in an mL concentrator, then repeat the dilution and concentrate 5 times (to about 150 μL), and add 350 μL of 50 mM NaCl, 20 mM HEPES, pH 7. 5. The final volume is 100 μL, and the concentration of the complex is determined to be 6.0 mg / ml as follows. Crystallization was set up in the sitting drop as in the case of the scFv / IL-17 complex using a Mosquito robot. The sitting drop consisted of 150 nL of protein + 120 nL of reservoir + 30 nL of seed (the above scF v / IL-17). The reservoir solution was a set of various PEG3350 concentrations and salt conditions . The crystallization plate was incubated at 20 °C. Small crystals were obtained from 15 .5% PEG3350, 0.4 M NaH2PO4. The crystals were transferred into 16% P EG3350, 0.2 M NaH2PO4, 20% glycerol, and liquid nitrogen (LN 2) for rapid freezing. X-ray diffraction data were collected at IMCA-CAT ID 17 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 (Collaborati ve Computational Project, N. (1994) Acta Cr ystallogr D Biol Crystallogr 53: 240 - 255) . All crystal structures were of scFv CAT2200a scFv VL-VH Except for the / IL-17 complex, generated in MOE (Montreal, Canada) Phaser using homology models (Read (2001) Acta Crystall ogr D Biol Crystallogr 57(Pt 10):1373-13 82) was used for analysis by molecular replacement (MR), for which the structure of pdb id 2v xs (Gerhardt) (Gerhardt et al. (2009) J M ol Biol 394:905-921) was used as the search model. The structure model was refined with PHENIX (Adams et al. (2004) J Synchrotro n Radiat 11(Pt 1):53-55) and manually adjusted with Coot (Emsl ey et al. (2010) Acta Crystallogr D Biol C rystallogr 66(Pt 4):486-501). Molecular graphics were generated with PyMol (www_schrodinger_com) .

[0693] 6.3.2 Structure Structures using unbound scFv and spFv molecules are shown in Figures 8, 9, 10, and 11 . Figure 8 shows the structure of GLk1 spFv VL-VH. Figure 9 shows the GLk1 spFv VH-VL structure. Figure 10 shows the GLk2 spFv VH-VL structure. Figure 11 shows the structure of CAT2200b spFv VH-VL. The structures are consistent with a typical Fv structure packing both the VL domain and the VH domain against each other . Generally, most of the linker residues were ordered and solved in the electron density map. The disulfide bond between the staple and the anchor point is generally in both VL-VH orientations Well-ordered in a square. In addition to the non-conjugated scFv and spFv structures, we also attempted to clarify any structural effects on antigen binding. The CAT2200 scFv and spF v variant molecules were crystallized in complex with their cognate target, IL-17. For the scFv and spFv of the crystallized CAT 2200 variants, the structures are nearly identical regardless of the presence or absence of the bound target (Figures 12, 13, 14). Figure 12 shows the non-binding CAT2200b spFv VH-VL compared to the IL-17-bound CAT2200a scFv VL-VH . Figure 13 shows the front view comparison of the non-binding CAT2200b spFv VH-VL compared to the IL-17-bound CAT2200a spF v VL-VH. Figure 14 shows the back view comparison of the non-binding CAT2200b spFv VH-VL compared to the IL-17-bound CAT2200a spFv VL -VH. The structures were identical regardless of the orientation or presence of the staples. The rmsd for all matching Cα atoms between pairs of structures was very small (0.41 Å between non-binding spFv-VH -VL and antigen-binding scFv-VL-VH (Figure 12), 0.46 Å between non-binding spFv -VH-VL and spFv-VL-VH (for each of Figures 13 and 14 ), and 0.37 Å between binding scFv and binding spFv). Structural evidence shows that the staple treatment functions as designed. Also, the staple treatment does not affect the VL and VH domain structures or the relative VL / VH packing. ). ). ). The structural evidence shows that the staple treatment functions as designed. Also, the staple treatment does not affect the VL and VH domain structures or the relative VL / VH packing.

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

[0695] For 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 Q 3. The staple is formed between any of the indicated positions.

[0696] For the VH - linker - VL orientation (VH Chothia positions 43, 40, and 46, VL C hothia positions 102, 5, and 3), the staple is formed between any of those positions.

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

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

[0699] 6.6 Example 6: Multispecific constructs containing spFv Exemplary multispecific binding molecules incorporating the spFv structures provided herein were constructed and tested in this example. Specifically, bispecific antibodies and control molecules were tested against the targets shown in Table 8. It is derived from a binding molecule and transiently expressed in CHO suspension culture in a serum-free / animal component-free medium and purified using an AKTA PURE instrument (GE Healthcare) by Protein A affinity chromatography, followed by size exclusion chromatography on a Superdex200 10 / 300GL column ( GE Healthcare), SEC). The heavy chain contained a kn ob-into-hole (KiH) mutation that promotes heterodimerization (Ridgway et al. , Protein Eng. 9(7):617-21(1996), Atwell et al., J. Mol. Biol. 270(1):26-35(1997), Merch ant et al., Nat. Biotechnol. 16(7):677-81(1 998)). The antibody contained an IgG1 sigma Fc with a set of 7 Fc mutations - L234A, L235A, G237A, P238S, H268A, A33 0S, and P331S when compared to wild-type IgG1 that reduces Fc receptor interaction (Tam et al., Antibodies(2017)).

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

[0701]

Table 8

[0702] The sequences in Table 8 are as follows. Accession No. 69 (VH BHA10) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQ APGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTA YMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSS

[0703] Accession No. 70 (VL BHA10) DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQK PGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQ PEDFATYFCQQYDTYPFTFGQGTKVEIK

[0704] Accession No. 71 (VH L19) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQ APGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTL YLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSS

[0705] Accession No. 72 (VL L19) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQ KPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRL EPEDFAVYYCQQTGRIPPTFGQGTKVEIK

[0706] Accession No. 73 (VH B21M) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWI RQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQ VVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVS S

[0707] Sequence number 74 (VL B21M) DIVMTQSPDSLAVSLGERATINCRASQSVDYNGISYMHW YQQKPGQPPKLLIYAASNPESGVPDRFSGSGSGTDFTLTI SSLQAEDVAVYYCQQIIEDPWTFGQGTKVEIK

[0708] Sequence number 75 (VH MSLNmAb1) QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQ SHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTA YMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSS

[0709] Sequence number 76 (VL MSLNmAb1) DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKS GTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEA EDDATYYCQQWSKHPLTFGSGTKVEIK

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

[0711] Table 9 shows an overview of the structural characteristics of the bispecific antibodies and control molecules described in this example. The molecules of the world body are exemplary molecules according to the present invention, and the others are controls in different aspects. 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).

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

[0713] [Table 10]

[0714] The asymmetric antibodies having the 2:1 stoichiometry listed above (all IgG1 sigma, all with KiH mutations) were generated as follows. i. COVA1484 is an anti-RSV B2 1M antibody heavy chain (HC, SEQ ID NO: 80) carrying an N-terminal stapled scFv BHA10 (VH-VL orientation SEQ ID NO: 77) fusion (including SEQ ID NO: 78, SEQ ID NO: 79) and the light chain (LC, SEQ ID NO: 81) of the anti-RSV B21M antibody were co-expressed (Figure 15A).

[0715] SEQ ID NO: 77 [Stapled scFv BHA10 (VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQ APGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTA YMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGG SGGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKAS QNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSG SGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEI K

[0716] SEQ ID NO: 78 (HC B21M N-terminal stapled BHA10 (VH-VL), I gG1s, knob, pA mutant) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQ APGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTA YMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGG SGGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKAS QNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSG SGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEI KGGGGSGGGGSGGGGSGGGGSGGGGSQITLKESGPTLVKP TQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIY WDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTAT YYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTL MISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSS IEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0717] Sequence number 79 (HC B21M(RSV)IgG1s knob with pA mutation) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWI RQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQ VVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAG ASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCRE EMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRF TQKSLSLSPGK

[0718] SEQ ID NO: 80 (HC B21M(RSV)IgG1s hole) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWI RQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQ VVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAG ASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSRE EMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK

[0719] SEQ ID NO: 81 [LC B21M(RSV)] DIVMTQSPDSLAVSLGERATINCRASQSVDYNGISYMHW YQQKPGQPPKLLIYAASNPESGVPDRFSGSGSGTDFTLTI SSLQAEDVAVYYCQQIIEDPWTFGQGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

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

[0721] SEQ ID NO: 82 [Stapled scFv BHA10 (VL-VH)] DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQK PGCAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQ PEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPC GSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHW VRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKST STAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSS

[0722] SEQ ID NO: 83 (HCB 21M N-terminal stapled BHA10 (VL-VH), I gG1s, with knob, pA mutations) DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQK PGCAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQ PEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPC GSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHW VRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKST STAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSS GGGGSGGGGSGGGGSGGGGSGGGGSQITLKESGPTLVKPT QTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYW DDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATY YCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLM ISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSI EKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0723] Sequence number 79 (HC B21M (RSV) IgG1s knob with pA mutation) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWI RQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQ VVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAG ASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCRE EMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRF TQKSLSLSPGK

[0724] Sequence number 80 (HC B21M(RSV)IgG1s hole) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWI RQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQ VVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAG ASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSRE EMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK

[0725] Sequence number 81 [LC B21M (RSV)] DIVMTQSPDSLAVSLGERATINCRASQSVDYNGISYMHW YQQKPGQPPKLLIYAASNPESGVPDRFSGSGSGTDFTLTI SSLQAEDVAVYYCQQIIEDPWTFGQGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

[0726] iii. COVA1486 is an anti - RSV carrying a C - terminal stapled scFv BHA10 (VH - V L - orientation sequence number 77) fusion (including sequence number 84, sequence number 79) Heavy chain (HC, sequence number 80) of the anti - RSV B21M antibody of the anti - RSV B21M antibody heavy chain and light chain (L C, sequence number 81) generated by co - expression (Figure 15C).

[0727] Sequence number 77 [Stapled scFv BHA10 (VH - VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQ APGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTA YMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGG SGGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKAS QNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSG SGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEI K

[0728] Sequence number 84 (HC B21M C-terminal stapling BHA (VH-VL), IgG 1s, with knob, pA mutation) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWI RQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQ VVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAG ASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCRE EMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRF TQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKP GSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPG NVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVY YCARSWEGFPYWGQGTTVTVSSGGGSGGGSGCPPCGGGGD IQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPG KAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPE DFATYFCQQYDTYPFTFGCGTKVEIK

[0729] Sequence number 79 (HC B21M (RSV) IgG1s knob with pA mutation) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWI RQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQ VVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAG ASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCRE EMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRF TQKSLSLSPGK

[0730] Accession number 80 (HC B21M(RSV)IgG1s hole) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWI RQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQ VVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAG ASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSRE EMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK

[0731] Accession number 81 [LC B21M(RSV)] DIVMTQSPDSLAVSLGERATINCRASQSVDYNGISYMHW YQQKPGQPPKLLIYAASNPESGVPDRFSGSGSGTDFTLTI SSLQAEDVAVYYCQQIIEDPWTFGQGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

[0732] iv. COVA1487 is an anti-RSV B 21M antibody heavy chain (HC, SEQ ID NO: 80) and light chain (LC, SEQ ID NO: 81) of the anti-RSV B 21M antibody carrying a C-terminal staple-processed scFv BHA10 (VL-VH orientation SEQ ID NO: 82) fusion (including SEQ ID NO: 85, SEQ ID NO: 79). generated by co-expression with the heavy chain of the anti-RSV B 21M antibody and the light chain (LC , SEQ ID NO: 81) (Figure 15D).

[0733] SEQ ID NO: 82 [staple-processed scFv BHA10 (VL-VH)] DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQK PGCAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQ PEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPC GSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHW VRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKST STAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSS

[0734] SEQ ID NO: 85 (HC B21M C staple-processed BHA (VL-VH), IgG1s , knob, with pA mutation) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWI RQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQ VVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAG ASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCRE EMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRF TQKSLSLSPGKGGGGSGGGGSGGGGSDIQMTQSPSSLSAS VGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYR YSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTY PFTFGQGTKVEIKGGSGGSGGCPPCGSGGQVQLVQSGAEV KKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWI YPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDT AVYYCARSWEGFPYWGCGTTVTVSS

[0735] Sequence number 79 (HC B21M (RSV) IgG1s knob with pA mutation) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWI RQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQ VVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAG ASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCRE EMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRF TQKSLSLSPGK

[0736] Sequence number 80 (HC B21M (RSV) IgG1s hole) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWI RQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQ VVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAG ASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSRE EMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK

[0737] Sequence number 81 [LC B21M (RSV)] DIVMTQSPDSLAVSLGERATINCRASQSVDYNGISYMHW YQQKPGQPPKLLIYAASNPESGVPDRFSGSGSGTDFTLTI SSLQAEDVAVYYCQQIIEDPWTFGQGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

[0738] v.COVA1480 is an anti-EDB antibody E that carries an N-terminal staple-processed scFv BHA10 (VH-VL arrangement sequence number 77) fusion (including sequence numbers 86 and 87). The heavy chain (HC, sequence number 88) and light chain (LC, sequence number 89) of the anti-EDB antibody EDBmAb1 of the anti-EDB antibody EDBmAb1 heavy chain were generated by co-expression (Figure 15E).

[0739] Sequence number 77 [Staple-processed scFv BHA10 (VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQ APGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTA YMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGG SGGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKAS QNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSG SGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEI K

[0740] Accession number 86 (HC L19 N staple treatment BHA10 (VH-VL), IgG1 s, with knob, pA mutation) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQ APGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTA YMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGG SGGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKAS QNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSG SGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEI KGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQP GGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGS SGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY YCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISR TPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0741] Sequence number 87 (HC L19 IgG1s knob with pA mutation) VQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQA PGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVF LFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKN QVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSL SLSPGK

[0742] Sequence number 88 (HC L19 IgG1s whole) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQ APGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTL YLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSV FLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTK NQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK

[0743] Sequence number 89 (LC L19) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQ KPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRL EPEDFAVYYCQQTGRIPPTFGQGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC

[0744] vi. COVA1481 is an anti - EDB antibody carrying an N - terminal stapled scFv BHA10 (VL - VH orientation sequence number 82) fusion (including sequence number 90, sequence number 87). The heavy chain (HC, sequence number 88) of the anti - EDB antibody EDBmAb1 and the light chain (LC, sequence number 89) of EDBmAb1 were co - expressed to generate it (Figure 15F).

[0745] Accession No. 82 [Staple-processed scFv BHA10 (VL-VH)] DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQK PGCAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQ PEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPC GSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHW VRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKST STAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSS

[0746] Accession No. 90 (HC L19 N Staple-processed BHA10 (VL-VH), IgG1 s, with knob, pA mutation) DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQK PGCAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQ PEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPC GSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHW VRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKST STAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSS GGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPG GSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSS GTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY CAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRT PEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTI SKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0747] Sequence number 87 (HC L19 IgG1s knob with pA mutation) VQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQA PGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVF LFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKN QVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSL SLSPGK

[0748] Sequence number 88 (HC L19 IgG1s hole) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQ APGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTL YLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSV FLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTK NQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK

[0749] Sequence number 89 (LC L19) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQ KPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRL EPEDFAVYYCQQTGRIPPTFGQGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC

[0750] vii. COVA1482 is an anti-EDB antibody carrying a C-terminal stapled scFv BHA10 (VH-V L orientation array number 77) fusion (including array numbers 91 and 87), and is generated by co-expression with the heavy chain (HC, array number 88) and light chain (LC, array number 89) of the anti-EDB antibody EDBmAb1 of the anti-EDB antibody EDBmAb1 heavy chain (Figure 15G).

[0751] Array number 77 [Stapled scFv BHA10 (VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQ APGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTA YMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGG SGGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKAS QNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSG SGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEI K

[0752] Array number 91 (HC L19 C stapled BHA10 (VH-VL), IgG1 s, with knob, pA mutations) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQ APGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTL YLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSV FLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTK NQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKS LSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSV KVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHA QYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAR SWEGFPYWGQGTTVTVSSGGGSGGGSGCPPCGGGGDIQMT QSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPK SLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFAT YFCQQYDTYPFTFGCGTKVEIK

[0753] Sequence number 87 (HC L19 IgG1s knob with pA mutation) VQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQA PGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVF LFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKN QVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSL SLSPGK

[0754] Sequence number 88 (HC L19 IgG1s hole) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQ APGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTL YLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSV FLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTK NQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK

[0755] SEQ ID NO: 89 (LC L19) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQ KPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRL EPEDFAVYYCQQTGRIPPTFGQGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC

[0756] viii. COVA1483 is an anti-EDB antibody carrying a C-terminal stapled scFv BHA10 (VL- VH orientation SEQ ID NO: 82) fusion (including SEQ ID NO: 92, SEQ ID NO: 87). The heavy chain (HC, SEQ ID NO: 88) of the anti-EDB antibody EDBmAb1 and the light chain (LC, SEQ ID NO: 89) of the anti-EDB antibody EDBmAb1 were co-expressed to generate it (Figure 15H).

[0757] SEQ ID NO: 82 [Stapled scFv BHA10 (VL-VH)] DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQK PGCAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQ PEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPC GSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHW VRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKST STAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSS

[0758] SEQ ID NO: 92 (HC L19 C staple treated BHA10 (VL-VH), IgG1 s, with knob, pA mutations) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQ APGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTL YLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSV FLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTK NQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKS LSLSPGKGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDR VTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGV PSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTF GQGTKVEIKGGSGGSGGCPPCGSGGQVQLVQSGAEVKKPG SSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGN VHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYY CARSWEGFPYWGCGTTVTVSS

[0759] Sequence number 87 (HC L19 IgG1s knob with pA mutation) VQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQA PGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVF LFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKN QVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSL SLSPGK

[0760] Sequence number 88 (HC L19 IgG1s whole) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQ APGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTL YLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSV FLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTK NQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK

[0761] Sequence number 89 (LC L19) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQ KPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRL EPEDFAVYYCQQTGRIPPTFGQGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC

[0762] ix. COVA14107 is the C-terminal stapled scFv BHA10 (VH-V L orientation, VL3 Y36F_S49Y_F87Y sequence number 93) fusion (sequence number 94, arrangement The anti-EDB antibody EDBmAb1 heavy chain carrying (including column number 87) anti-EDB antibody EDBm Generated by co-expression with the heavy chain (HC, SEQ ID NO: 88) and light chain (LC, SEQ ID NO: 89) of Ab1 (Figure 15I).

[0763] SEQ ID NO: 93 [Stapled scFv (VL3_Y36F_S49Y_F87Y)B HA10 (VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQ APGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTA YMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGG SGGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKAS QNVGINVAWFQQKPGKAPKSLIYSASYRYSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQYDTYPFTFGCGTKVEI K

[0764] SEQ ID NO: 94 (HC L19 C stapled (VL3_Y36F S49Y_F8 7Y)BHA (VH-VL), IgG1s, knob, with pA mutation) EVQLL...

Claims

1. An isolated monoclonal antibody comprising a heavy chain variable region (VH), a linker (L) and a light chain variable region (VL). A fragment chain variable (scFv), a) Between a structurally conserved surface-exposed VH cysteine ​​(Cys) and the first L Cys the first disulfide bond of b) A second disulfide residue between the structurally conserved surface-exposed VL Cys and the second L Cys. Sulfide bond, or c) a front end between the structurally conserved surface-exposed VH Cys and the first L Cys; the first disulfide bond, and the structurally conserved surface-exposed VL Cys and the second disulfide bond between two L Cys.

2. An isolated scFv comprising a VH, L and VL, a) the VH comprises a VH C ys, and said L comprises a first L Cys; b) the VL has a VL C at a structurally conserved, surface-exposed VL framework residue position; L comprises a second L Cys; or c) the VH is modified to include structurally conserved, surface-exposed VH framework residue positions; Cys, and said VL comprises a structurally conserved surface-exposed VL framework residue position wherein the VL Cys is selected from the group consisting of the first L Cys and the second L Cys. wherein the VH Cys and the first L Cys form a disulfide bond. and the VL Cys and the second L Cys form a disulfide bond. An isolated scFv capable of

3. The distance between the VH Cys and the VL Cys is about 7 Å to about 9 Å.

3. An scFv according to claim 1 or 2.

4. the VH Cys is at H3, H5, H40, H43, H46, or H105; The scFv according to any one of claims 1 to 3, wherein the numbering is according to Chothia.

5. The VL Cys is L3, L5, L39, L42, L45, L100 or L102. and the residue numbering is according to Chothia. cFv.

6. a) the VH Cys is at H105 and the VL Cys is at L42; b) the VH Cys is at H43 and the VL Cys is at L100; c) the VH Cys is in H3 and the VL Cys is in L3; d) the VH Cys is in H3 and the VL Cys is in L5; e) the VH Cys is in H3 and the VL Cys is in L39; f) the VH Cys is in H3 and the VL Cys is in L42; g) the VH Cys is in H3 and the VL Cys is in L45; h) the VH Cys is in H3 and the VL Cys is in L100; i) the VH Cys is at H3 and the VL Cys is at L102; j) the VH Cys is in H5 and the VL Cys is in L3; k) the VH Cys is in H5 and the VL Cys is in L5; l) the VH Cys is at H5 and the VL Cys is at L39; m) the VH Cys is at H5 and the VL Cys is at L42; n) the VH Cys is at H5 and the VL Cys is at L45; o) the VH Cys is at H5 and the VL Cys is at L100; p) the VH Cys is at H5 and the VL Cys is at L102; q) the VH Cys is in H40 and the VL Cys is in L3; r) the VH Cys is in H40 and the VL Cys is in L5; s) the VH Cys is at H40 and the VL Cys is at L39; t) the VH Cys is at H40 and the VL Cys is at L42; u) the VH Cys is at H40 and the VL Cys is at L45; v) the VH Cys is at H40 and the VL Cys is at L100; w) the VH Cys is at H40 and the VL Cys is at L102; x) the VH Cys is in H43 and the VL Cys is in L3; y) the VH Cys is in H43 and the VL Cys is in L5; z) the VH Cys is at H43 and the VL Cys is at L39; aa) the VH Cys is at H43 and the VL Cys is at L42; bb) the VH Cys is at H43 and the VL Cys is at L45; cc) the VH Cys is at H43 and the VL Cys is at L102; dd) the VH Cys is in H46 and the VL Cys is in L3; ee) the VH Cys is at H46 and the VL Cys is at L5; ff) the VH Cys is at H46 and the VL Cys is at L39; gg) the VH Cys is at H46 and the VL Cys is at L42; hh) the VH Cys is at H46 and the VL Cys is at L45; ii) the VH Cys is at H46 and the VL Cys is at L100; jj) the VH Cys is at H46 and the VL Cys is at L102; kk) the VH Cys is at H105 and the VL Cys is at L3; ll) the VH Cys is at H105 and the VL Cys is at L5; mm) the VH Cys is at H105 and the VL Cys is at L39; nn) the VH Cys is at H105 and the VL Cys is at L45; oo) the VH Cys is at H105 and the VL Cys is at L100; or teeth pp) the VH Cys is at H105 and the VL Cys is at L102; The scFv according to any one of claims 1 to 5, wherein the numbering is according to Chothia.

7. wherein L comprises a contiguous amino acid sequence derived from an immunoglobulin (Ig) hinge region. The scFv according to any one of claims 1 to 6.

8. The method of claim 7, wherein the Ig hinge region is derived from a human or non-human Ig hinge region. scFv.

9. The scF of claim 8, wherein the Ig hinge region is derived from a human Ig hinge region. v.

10. The human Ig hinge region is of the IgG1, IgG2, IgG3 or IgG4 isotype. The scFv of claim 9 .

11. The L is an amino acid sequence C(X) ｙ C (SEQ ID NO:23), where X is glycine. (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Gl u), 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. The scFv according to any one of claims 1 to 10.

12. The L is an amino acid sequence C(X) ｙ C (SEQ ID NO:24), wherein X is GIy, The scFv of claim 11, wherein y is Ser or Pro and y is an integer from 1 to 3.

13. The L is the amino acid sequence CPC, CGC, CSC, CPPC (SEQ ID NO: 1), CG PC (SEQ ID NO: 28), CPGC (SEQ ID NO: 29), CGGC (SEQ ID NO: 30), CSP G (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), CGPP C (SEQ ID NO: 37), CPGPC (SEQ ID NO: 38), CPPGC (SEQ ID NO: 39), CG GPC (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). The scFv shown above.

14. The L is from about 14 to about 19 amino acids, for example, about 14, about 15, about 16, about 17, about 1 14. An scFv according to any one of claims 1 to 13, comprising 8, or about 19 amino acids.

15. The L is an amino acid sequence (X): ｍ C(X) ｙ C(X) ｎ (SEQ ID NO:25), , X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gln, His, Ile, leu, Lys, Phe, Thr, Trp or Tyr, m is 6 y is an integer from 1 to 3, and n is an integer from 4 to 6.

14. An scFv according to any one of claims 14.

16. The L is an amino acid sequence (X): ｍ C(X) ｙ C(X) ｎ (SEQ ID NO:26), , X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gln, His, Ile, Leu, Lys, Thr, or Tyr, and m is an integer from 6 to 9.

16. The scFv according to claim 15, wherein y is an integer from 1 to 3 and n is an integer from 4 to 6. 。

17. The L is an amino acid sequence (X): ｍ C(X) ｙ C(X) ｎ (SEQ ID NO:27), X is Gly or Pro, m is an integer from 6 to 9, and y is an integer from 1 to 3. The scFv of claim 16, wherein n is an integer from 4 to 6.

18. Claims 1 to 17, wherein L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7. The scFv described in any one of claims 1 to 4.

19. 19. The method of claim 1, wherein the scFv is in a VL-L-VH orientation. scFv.

20. 19. The method of claim 1, wherein the scFv is in a VH-L-VL orientation. scFv.

21. A scFv comprising a VH, L, and VL, a) the VH comprises a Cys at H105; b) the VL comprises a Cys at L42; c) L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; d) An scFv, wherein said scFv is in a VL-L-VH orientation.

22. A scFv comprising a VH, L, and VL, a) the VH comprises a Cys at H105; b) the VL comprises a Cys at L45; c) L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; d) An scFv, wherein said scFv is in a VL-L-VH orientation.

23. A scFv comprising a VH, L, and VL, a) the VH comprises a Cys at H105; b) the VL comprises a Cys at L39; c) L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; d) An scFv, wherein said scFv is in a VL-L-VH orientation.

24. A scFv comprising a VH, L, and VL, a) the VH comprises a Cys at H5; b) the VL comprises a Cys at L42; c) L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; d) An scFv, wherein said scFv is in a VL-L-VH orientation.

25. A scFv comprising a VH, L, and VL, a) the VH comprises a Cys at H5; b) the VL comprises a Cys at L45; c) L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; d) An scFv, wherein said scFv is in a VL-L-VH orientation.

26. A scFv comprising a VH, L, and VL, a) the VH comprises a Cys at H5; b) the VL comprises a Cys at L39; c) L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; d) An scFv, wherein said scFv is in a VL-L-VH orientation.

27. A scFv comprising a VH, L, and VL, a) the VH comprises a Cys at H3; b) the VL comprises a Cys at L42; c) L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; d) An scFv, wherein said scFv is in a VL-L-VH orientation.

28. A scFv comprising a VH, L, and VL, a) the VH comprises a Cys at H3; b) the VL comprises a Cys at L45; c) L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; d) An scFv, wherein said scFv is in a VL-L-VH orientation.

29. A scFv comprising a VH, L, and VL, a) the VH comprises a Cys at H3; b) the VL comprises a Cys at L39; c) L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; d) An scFv, wherein said scFv is in a VL-L-VH orientation.

30. A scFv comprising a VH, L, and VL, a) the VH comprises a Cys at H43; b) the VL comprises a Cys at L100; c) L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; d) an scFv, wherein said scFv is in a VH-L-VL orientation.

31. A scFv comprising a VH, L, and VL, a) the VH comprises a Cys at H43; b) the VL comprises a Cys at L102; c) L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; d) an scFv, wherein said scFv is in a VH-L-VL orientation.

32. A scFv comprising a VH, L, and VL, a) the VH comprises a Cys at H43; b) the VL comprises a Cys at L5; c) L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; d) an scFv, wherein said scFv is in a VH-L-VL orientation.

33. A scFv comprising a VH, L, and VL, a) the VH comprises a Cys at H43; b) the VL comprises a Cys at L3; c) L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; d) an scFv, wherein said scFv is in a VH-L-VL orientation.

34. A scFv comprising a VH, L, and VL, a) the VH comprises a Cys at H40; b) the VL comprises a Cys at L100; c) L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; d) an scFv, wherein said scFv is in a VH-L-VL orientation.

35. A scFv comprising a VH, L, and VL, a) the VH comprises a Cys at H40; b) the VL comprises a Cys at L102; c) L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; d) an scFv, wherein said scFv is in a VH-L-VL orientation.

36. A scFv comprising a VH, L, and VL, a) the VH comprises a Cys at H40; b) the VL comprises a Cys at L5; c) L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; d) an scFv, wherein said scFv is in a VH-L-VL orientation.

37. A scFv comprising a VH, L, and VL, a) the VH comprises a Cys at H40; b) the VL comprises a Cys at L3; c) L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; d) an scFv, wherein said scFv is in a VH-L-VL orientation.

38. A scFv comprising a VH, L, and VL, a) the VH comprises a Cys at H46; b) the VL comprises a Cys at L100; c) L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; d) an scFv, wherein said scFv is in a VH-L-VL orientation.

39. A scFv comprising a VH, L, and VL, a) the VH comprises a Cys at H46; b) the VL comprises a Cys at L102; c) L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; d) an scFv, wherein said scFv is in a VH-L-VL orientation.

40. A scFv comprising a VH, L, and VL, a) the VH comprises a Cys at H46; b) the VL comprises a Cys at L5; c) L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; d) an scFv, wherein said scFv is in a VH-L-VL orientation.

41. A scFv comprising a VH, L, and VL, a) the VH comprises a Cys at H46; b) the VL comprises a Cys at L3; c) L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7; d) an scFv, wherein said scFv is in a VH-L-VL orientation.

42. 42. The method of claim 21, wherein L comprises the amino acid sequence of SEQ ID NO:

3. scFv.

43. 42. The method of claim 21, wherein L comprises the amino acid sequence of SEQ ID NO:

6. scFv.

44. 42. The method of claim 21, wherein L comprises the amino acid sequence of SEQ ID NO:

7. scFv.

45. The scFv of any one of claims 1 to 44, wherein the scFv is conjugated to a second molecule. cFv.

46. The scFv of claim 45, wherein the second molecule is a half-life extending moiety.

47. The half-life extending moiety may be an immunoglobulin (Ig), a fragment of an Ig, an Ig constant region, a A fragment of the Ig constant region, an Fc region, transferrin, albumin, an albumin binding domain The scFv of claim 46, wherein the scFv is polyvinyl chloride or polyethylene glycol.

48. 46. ​​The method of claim 45, wherein the second molecule is a cytotoxic agent or a detectable label. cFv.

49. The scFv of claim 48, wherein the second molecule is an antibody or a fragment thereof.

50. 5. The scFv and the antibody or fragment thereof bind to different antigens.

9. An scFv as described in

51. The scFv of claim 45, wherein the second molecule is a chimeric antigen receptor (CAR). 。

52. A composition comprising the scFv of any one of claims 1 to 51 and a pharma- ceutically acceptable carrier. M, a pharmaceutical composition.

53. A polynucleotide encoding the scFv of any one of claims 1 to 44.

54. A vector comprising the polynucleotide of claim 53.

55. A host cell comprising the vector of claim 54.

56. A method for producing an scFv according to any one of claims 1 to 44, comprising the steps of:

56. Culturing the host cell of claim 55 under conditions in which scFv is produced; and isolating the scFv. and manufacturing the same.

57. 57. The method of claim 56, wherein the host cell is a prokaryotic cell.

58. 57. The method of claim 56, wherein the host cell is a eukaryotic cell.

59. An anti-idiotype antibody that binds to an scFv according to any one of claims 1 to 44.

60. A kit comprising an scFv according to any one of claims 1 to 44.

61. A multispecific molecule comprising an scFv according to any one of claims 1 to 44.

62. The multispecific molecule of claim 61 , wherein the multispecific molecule comprises an antibody or an antibody fragment. 。

63. The multispecific protein comprises an Ig constant region or a fragment of an Ig constant region.

62. The multispecific molecule of claim 61.

64. 64. The multispecific polypeptide of claim 63, wherein said fragment of the Ig constant region comprises an Fc region. Stuffy.

65. 64. The multispecific antibody of claim 63, wherein the fragment of the Ig constant region comprises a CH2 domain. sex molecule.

66. 64. The multispecific antibody of claim 63, wherein the fragment of the Ig constant region comprises a CH3 domain. sex molecule.

67. The fragment of the Ig constant region comprises the CH2 domain and the CH3 domain. The multispecific molecule of claim 63.

68. The fragment of the Ig constant region comprises at least a portion of a hinge, a CH2 domain, and 64. The multispecific molecule of claim 63, comprising the CH3 domain.

69. The fragment of the Ig constant region comprises the hinge, the CH2 domain, and the CH3 domain.

64. The multispecific molecule of claim 63, comprising:

70. The scFv is N-terminal to the Ig constant region or the fragment of the Ig constant region.

70. The multispecific molecule of any one of claims 63 to 69, conjugated to:

71. The scFv is C-terminal to the Ig constant region or N-terminal to the fragment of the Ig constant region.

70. The multispecific molecule of any one of claims 63 to 69, conjugated to:

72. The Ig constant region or the fragment of the Ig constant region is selected from the group consisting of IgG1, IgG2, and Ig The multiple specific antibodies according to any one of claims 63 to 71, which are of the IgG3 or IgG4 isotype. Isomer molecules.

73. The Ig constant region or the fragment of the Ig constant region is an FcγR 73. The method according to claim 63, comprising at least one mutation that reduces binding to The multispecific molecules described herein.

74. The at least one mutation that reduces binding of the multispecific molecule to an FcγR is 34A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331 S, F234A / L235A, S228P / F234A / L235A, N297A, V2 34A / G237A, K214T / E233P / L234V / L235A / G236-missing Lost / A327G / P331A / D365E / L358M, H268Q / V309L / A3 30S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331 S, S228P / F234A / L235A / G237A / P238S and S228P / F 234A / L235A / G236-deletion / G237A / P238S 74. The multispecific molecule of claim 73, wherein residue numbering is according to the EU index.

75. The Ig constant region or the fragment of the Ig constant region is an FcγR 73. The method according to claim 63, comprising at least one mutation that enhances binding to The multispecific molecules described herein.

76. The at least one mutation that enhances binding of the multispecific molecule to an FcγR is S2 39D / I332E, S298A / E333A / K334A, F243L / R292P / Y300L, F243L / R292P / Y300L / P396L, F243L / R292 From P / Y300L / V305I / P396L and G236A / S239D / I332E The polynucleotide according to claim 75, wherein the residue numbering is according to the EU index. Heavy specificity molecules.

77. FcγR is FcγRI, FcγRIIA, FcγRIIB, or FcγRIII; or any combination thereof. Isomer molecules.

78. The Ig constant region or a fragment of the Ig constant region modulates the half-life of the multispecific molecule.

73. The multispecific antibody according to any one of claims 63 to 72, comprising at least one mutation that molecule.

79. The at least one mutation that modulates the half-life of the multispecific molecule is H435A , P257I / N434H, D376V / N434H, M252Y / S254T / T25 6E / H433K / N434F, T308P / N434A and H435R The multispecific molecule of claim 78, wherein the residue numbering is according to the EU index. child.

80. The Ig constant region or a fragment of the Ig constant region is at least one 73. The multispecific molecule of any one of claims 63 to 72, comprising one mutation.

81. The at least one mutation in the CH3 domain is T350V, L351Y, F405A, Y407V, T366Y, T366W, F405W, T394W, T394 S, Y407T, Y407A, T366S / L368A / Y407V, L351Y / F4 05A / Y407V, T366I / K392M / T394W, F405A / Y407V, T366L / K392M / T394W, L351Y / Y407A, T366A / K409 F, L351Y / Y407A, T366V / K409F, T366A / K409F, T3 50V / L351Y / F405A / Y407V and T350V / T366L / K392L / T394W, and the residue numbering is according to the EU index.

81. The multispecific molecule of claim 80.

82. 82. The multispecific molecule of claim 61, wherein the multispecific molecule is bispecific. Multispecific molecules.

83. 82. The multispecific molecule of claim 61, wherein the multispecific molecule is trispecific. Multispecific molecules.

84. 82. The multispecific molecule of any one of claims 61 to 81, wherein the multispecific molecule is tetraspecific. Multispecific molecules.

85. A multispecific molecule according to any one of claims 61 to 84 and a pharma- ceutically acceptable carrier.

13. A pharmaceutical composition comprising:

86. A heterologous molecule comprising an scFv according to any one of claims 1 to 44.

87. The scFv is linked to a second protein, polynucleotide, therapeutic agent, cytotoxic agent, or 87. The heterologous molecule of claim 86, which is conjugated to a detectable label.

88. 88. The heterologous molecule of claim 87, wherein the second protein is an antibody or a fragment thereof.

89. 88. The heterologous molecule of claim 87, wherein the second protein is an alternative scaffold.

90. 9. The method of claim 8, wherein the second protein is a chimeric antigen receptor (CAR) or a fragment thereof.

8. A heterologous molecule according to claim 7.

91. The heterologous molecule according to any one of claims 86 to 90, wherein the heterologous molecule is monospecific. child.

92. The heterologous molecule according to any one of claims 86 to 90, wherein the heterologous molecule is multispecific. child.

93. The heterologous molecule of claim 92, wherein the heterologous molecule is bispecific.

94. The heterologous molecule of claim 92, wherein the heterologous molecule is trispecific.

95. The heterologous molecule of claim 92, wherein the heterologous molecule is tetraspecific.

96. A heterologous molecule according to any one of claims 86 to 95 and a pharma- ceutically acceptable carrier. A pharmaceutical composition comprising:

97. 1. A process for preparing a stabilized scFv comprising: Provide a heavy chain variable region (VH) and a light chain variable region (VL) that form an antigen-binding domain. And, Providing a linker (L) that includes or is engineered to include a first L Cys And, to include VH Cys at structurally conserved, surface-exposed VH framework residue positions Manipulating the VH; A disulfide bond is formed between the VH Cys and the first L Cys, preparing a stabilized scFv.

98. 1. A process for preparing a stabilized scFv comprising: providing a VH and a VL which form an antigen-binding domain; providing an L that includes or is engineered to include a second L Cys; to include a VL Cys at a structurally conserved, surface-exposed VL framework residue position. Manipulating the VL; A disulfide bond is formed between the VL Cys and the second L Cys, preparing a stabilized scFv.

99. 1. A process for preparing a stabilized scFv comprising: providing a VH and a VL which form an antigen-binding domain; Contains or is engineered to contain a first L Cys and a second L Cys. Providing L; to include VH Cys at structurally conserved, surface-exposed VH framework residue positions Manipulating the VH; to include a VL Cys at a structurally conserved, surface-exposed VL framework residue position. Manipulating the VL; A disulfide bond is formed between the VH Cys and the first L Cys, and the V A disulfide bond is formed between the L Cys and the second L Cys to form the stabilized s and preparing a cFv.

100. The stabilized scFv is an scFv according to any one of claims 1 to 44.

100. The process of any one of paragraphs 97 to 99.

101. The stabilized scFv was compared to a control scFv lacking the disulfide bond. The process according to any one of claims 97 to 100, which binds to the antigen with equal affinity when vinegar.

102. 1. A process for preparing a stabilized scFv 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 a Cys at H3 and the VL comprises a Cys at L3; iv. the VH comprises a Cys at H3 and the VL comprises a Cys at 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; mosquito, ix. the VH comprises a Cys at H3 and the VL comprises a Cys at L102; x. the VH comprises a Cys at H5 and the VL comprises a Cys at L3; xi. the VH comprises a Cys at H5 and the VL comprises a 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 a Cys at H5 and the VL comprises a 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; mosquito, 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 iCys at H40 and the VL comprises Cys at L45; mosquito, xxii. the VH comprises Cys at H40 and the VL comprises Cys at L100; Ugh, xxiii. the VH comprises Cys at H40 and the VL comprises Cys at L102; Includes or xxiv. The VH comprises Cys at H43 and the VL comprises Cys at L3 、 xxv. the VH comprises a Cys at H43 and the VL comprises a Cys at L5; xxvi. the VH comprises Cys at H43 and the VL comprises Cys at L39; mosquito, xxvii. the VH comprises Cys at H43 and the VL comprises Cys at L42; Ugh, xxviii. the VH comprises Cys at H43 and the VL comprises Cys at L45; Includes or xxix. the VH comprises Cys at H43 and the VL comprises Cys at L102; Ugh, 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; Ugh, xxxiii. the VH comprises Cys at H46 and the VL comprises Cys at L42; Includes or xxxiv. the VH comprises Cys at H46 and the VL comprises Cys at L45; Ugh, xxxv. the VH comprises Cys at H46 and the VL comprises Cys at L100; Ugh, xxxvi. the VH comprises Cys at H46 and the VL comprises Cys at L102; Includes or xxxvii. the VH comprises a Cys at H105 and the VL comprises a Cys at L3 Includes or xxxviii. the VH comprises Cys at H105 and the VL comprises Cys at L5 Contains or xxxix. the VH comprises Cys at H105 and the VL comprises Cys at L39; Includes or xl. the VH comprises Cys at H105 and the VL comprises Cys at L45; 、 xli. the VH comprises Cys at H105 and the VL comprises Cys at L100; Muka, or xlii. the VH comprises Cys at H105 and the VL comprises Cys at L102; Including, providing that the residue numbering is according to Chothia; b) L comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, or 7; c) expressing the polynucleotide in a host cell to produce the stabilized scFv. A process comprising:

103. 103. The method of claim 102, wherein the host cell is a prokaryotic cell.

104. 103. The method of claim 102, wherein the host cell is a eukaryotic cell.

105. It comprises a heavy chain variable region (VH), a means for linking (L), and a light chain variable region (VL). An isolated single chain variable fragment (scFv) comprising: a) Between a structurally conserved surface-exposed VH cysteine ​​(Cys) and the first L Cys the first disulfide bond of b) A second disulfide residue between the structurally conserved surface-exposed VL Cys and the second L Cys. Sulfide bond, or c) a front end between the structurally conserved surface-exposed VH Cys and the first L Cys; the first disulfide bond and the structurally conserved surface-exposed VL Cys and the second disulfide bond the second disulfide bond between L Cys of said scFv.

106. An isolated antibody comprising a means for antigen binding, a linker (L), and a light chain variable region (VL). A single chain variable fragment (scFv) comprising: a) A structurally conserved surface-exposed antigen binding means cysteine ​​(Cys) and a first L Cy a first disulfide bond between b) A second disulfide residue between the structurally conserved surface-exposed VL Cys and the second L Cys. Sulfide bond, or c) the structurally conserved surface-exposed antigen binding means Cys and the first L Cys; and the structurally conserved surface-exposed VL Cys and the second L and the second Cys.

107. An isolated antibody comprising a heavy chain variable region (VH), a linker (L), and a means for antigen binding. A single chain variable fragment (scFv) comprising: a) Between a structurally conserved surface-exposed VH cysteine ​​(Cys) and the first L Cys the first disulfide bond of b) a second amino acid sequence between the structurally conserved surface-exposed antigen-binding means Cys and the second L Cys; 2 disulfide bonds, or c) a front end between the structurally conserved surface-exposed VH Cys and the first L Cys; the first disulfide bond and the structurally conserved surface-exposed antigen binding means Cys and the second L and the second Cys.

108. It comprises a heavy chain variable region (VH), a means for linking (L), and a light chain variable region (VL). A multispecific molecule comprising a single chain variable fragment (scFv) comprising: a) Between a structurally conserved surface-exposed VH cysteine ​​(Cys) and the first L Cys the first disulfide bond of b) A second disulfide residue between the structurally conserved surface-exposed VL Cys and the second L Cys. Sulfide bond, or c) a front end between the structurally conserved surface-exposed VH Cys and the first L Cys; the first disulfide bond and the structurally conserved surface-exposed VL Cys and the second disulfide bond and said second disulfide bond between the L Cys of.

109. A single-chain variable region comprising a linker (L) and a light chain variable region (VL) for antigen binding. A multispecific molecule comprising a fragment variable (scFv), said scFv comprising: a) A structurally conserved surface-exposed antigen binding means cysteine ​​(Cys) and a first L Cy a first disulfide bond between b) A second disulfide residue between the structurally conserved surface-exposed VL Cys and the second L Cys. Sulfide bond, or c) the structurally conserved surface-exposed antigen binding means cysteine ​​Cys and the first L The first disulfide bond between Cys and the structurally conserved surface-exposed VL and the second disulfide bond between the L Cys and the second L Cys. sex molecule.

110. It comprises a heavy chain variable region (VH), a linker (L) and a means for antigen binding (VL). A multispecific molecule comprising a single chain variable fragment (scFv) comprising: a) Between a structurally conserved surface-exposed VH cysteine ​​(Cys) and the first L Cys the first disulfide bond of b) a second L Cys between the structurally conserved surface-exposed antigen binding means Cys and the second L Cys or c) a front end between the structurally conserved surface-exposed VH Cys and the first L Cys; the first disulfide bond and the structurally conserved surface-exposed antigen binding means Cys and and said second disulfide bond between the second L Cys.

111. It comprises a heavy chain variable region (VH), a means for linking (L), and a light chain variable region (VL). A heterologous molecule comprising a single chain variable fragment (scFv) comprising: a) Between a structurally conserved surface-exposed VH cysteine ​​(Cys) and the first L Cys the first disulfide bond of b) A second disulfide residue between the structurally conserved surface-exposed VL Cys and the second L Cys. Sulfide bond, or c) a front end between the structurally conserved surface-exposed VH Cys and the first L Cys; the first disulfide bond and the structurally conserved surface-exposed VL Cys and the second disulfide bond and said second disulfide bond between the L Cys of.

112. A single-chain variable region comprising a linker (L) and a light chain variable region (VL) for antigen binding. A heterologous molecule comprising a fragment variable (scFv), said scFv comprising: a) A structurally conserved surface-exposed antigen binding means cysteine ​​(Cys) and a first L Cy a first disulfide bond between b) A second disulfide residue between the structurally conserved surface-exposed VL Cys and the second L Cys. Sulfide bond, or c) the structurally conserved surface-exposed antigen binding means Cys and the first L Cys; and the structurally conserved surface-exposed VL Cys and the second L and said second Cys.

113. A single chain variable region comprising a heavy chain variable region (VH), a linker (L), and a means for antigen binding. A heterologous molecule comprising a fragment variable (scFv), said scFv comprising: a) Between a structurally conserved surface-exposed VH cysteine ​​(Cys) and the first L Cys the first disulfide bond of b) a second L Cys between the structurally conserved surface-exposed antigen binding means Cys and the second L Cys or c) a front end between the structurally conserved surface-exposed VH Cys and the first L Cys; the first disulfide bond and the structurally conserved surface-exposed antigen binding means Cys and the second L and said second Cys.

114. A means for encoding the scFv according to any one of claims 105 to 113.

115. A means for replicating said vector 114.

116. A composition comprising a means for stabilizing an scFv.

117. A composition comprising a means for increasing the thermal stability of an scFv.

118. The means is between VH and L, between VL and the L, or between the VH and the L and 116 or 1, comprising forming a disulfide bond between the VL and the L.

18. The composition described in 17.

119. Multispecific molecules comprising means for stabilizing scFvs.

120. Multispecific molecules comprising means for increasing the thermal stability of scFvs.

121. The means is between VH and L, between VL and the L, or between the VH and the L and 119 or 1, comprising forming a disulfide bond between the VL and the L.

21. The multispecific molecule according to claim 20.

122. A heterologous molecule comprising a means for stabilizing the scFv.

123. A heterologous molecule comprising a means for increasing the thermal stability of the scFv.

124. The means is between VH and L, between VL and the L, or between the VH and the L and 122 or 1, comprising forming a disulfide bond between the VL and the L.

24. The heterologous molecule according to 23.

125. A means for producing a composition according to any one of claims 116 to 118.

126. A means for producing a multispecific molecule according to any one of claims 119 to 121.

127. A means for producing a heterologous molecule according to any one of claims 122 to 124.

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