Compositions Comprising Multispecific Binding Agents for Enhanced Immune Responses - Patent application

Abstract

Disclosed herein, in certain aspects, are materials and methods for improving single chain variable fragment containing molecules.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Patent Application No. 63 / 281,954, filed November 22, 2021, U.S. Patent Application No. 63 / 322,158, filed March 21, 2022, and U.S. Patent Application No. 63 / 393,750, filed July 29, 2022, the disclosures of each of which are incorporated by reference herein in their entireties.

[0002] (Sequence Listing) This application contains a computer readable sequence listing submitted herewith in XML file format, the entire contents of which are incorporated herein by reference in their entirety. The sequence listing XML file submitted herewith is named "14620-710-228_SEQ_LISTING.xml", was created on November 18, 2022, and is 94,758 bytes in size.

[0003] FIELD OF THE INVENTION Disclosed herein, in various aspects, are materials and methods for making and using multispecific molecules comprising improved single-chain variable fragments and their equivalents. Summary of the Invention

[0004] In one aspect, the disclosure provides materials and methods for molecules (e.g., binding molecules) capable of binding to a target. In one aspect, the molecules include an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), and a fragment crystallizable region (Fc region), wherein the scFv includes a heavy chain variable region (VH), a linker (L), and a light chain variable region (VL), wherein the scFv includes: a) A disulfide bond between a structurally conserved surface-exposed VH position mutated to a cysteine ​​(Cys) and L Cys; b) a disulfide bond between a structurally conserved, surface-exposed VL position mutated to Cys and an L Cys; c) 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. In some embodiments, the molecule has improved stability, expression yield, and / or quality compared to a comparable molecule lacking a disulfide bond or two disulfide bonds, e.g., lacking the first and second disulfide bonds.

[0005] In some embodiments, a) the VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position, and the L comprises an L Cys; b) the VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position, and the L comprises an L Cys; or c) the VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position, the VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position, and the L comprises a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys are capable of forming a disulfide bond, and the VL Cys and the second L Cys are capable of forming a disulfide bond.

[0006] In some embodiments, the distance between VH Cys and VL Cys is about 5 Å to about 10 Å or about 7 Å to about 9 Å.

[0007] In some embodiments, the VH Cys is at H3, H5, H40, H43, H46, or H105, where residue numbering is according to Chothia.

[0008] In some embodiments, the VL Cys is at L3, L5, L39, L42, L43, L45, L100, or L102, where residue numbering is according to Chothia.

[0009] In some embodiments, the VH Cys is at H105 and the VL Cys is at L42; or VH Cys is at H43 and VL Cys is at L100, or VH Cys is in H3 and VL Cys is in L3, or VH Cys is in H3 and VL Cys is in L5, or VH Cys is in H3 and VL Cys is in L39, or VH Cys is in H3 and VL Cys is in L42, or VH Cys is in H3 and VL Cys is in L45, or VH Cys is in H3 and VL Cys is in L100, or VH Cys is at H3 and VL Cys is at L102, or VH Cys is in H5 and VL Cys is in L3, or VH Cys is in H5 and VL Cys is in L5, or VH Cys is at H5 and VL Cys is at L39, or VH Cys is at H5 and VL Cys is at L42, or VH Cys is at H5 and VL Cys is at L45, or VH Cys is in H5 and VL Cys is in L100, or VH Cys is at H5 and VL Cys is at L102, or VH Cys is in H40 and VL Cys is in L3, or VH Cys is in H40 and VL Cys is in L5, or VH Cys is at H40 and VL Cys is at L39, or VH Cys is at H40 and VL Cys is at L42, or VH Cys is at H40 and VL Cys is at L45, or VH Cys is at H40 and VL Cys is at L100, or VH Cys is at H40 and VL Cys is at L102, or VH Cys is in H43 and VL Cys is in L3, or VH Cys is at H43 and VL Cys is at L5, or VH Cys is at H43 and VL Cys is at L39, or VH Cys is at H43 and VL Cys is at L42, or VH Cys is at H43 and VL Cys is at L45, or VH Cys is at H43 and VL Cys is at L102, or VH Cys is at H46 and VL Cys is at L3, or VH Cys is at H46 and VL Cys is at L5, or VH Cys is at H46 and VL Cys is at L39, or VH Cys is at H46 and VL Cys is at L42, or VH Cys is at H46 and VL Cys is at L45, or VH Cys is at H46 and VL Cys is at L100, or VH Cys is at H46 and VL Cys is at L102, or VH Cys is at H105 and VL Cys is at L3, or VH Cys is at H105 and VL Cys is at L5, or VH Cys is at H105 and VL Cys is at L39, or VH Cys is at H105 and VL Cys is at L45, or VH Cys is at H105 and VL Cys is at L100, or VH Cys is at H105 and VL Cys is at L102, or VH Cys is at H105 and VL Cys is at L43; Residue numbering is according to Chothia.

[0010] In some embodiments, L comprises a contiguous amino acid sequence derived from an immunoglobulin (Ig) hinge region. In some embodiments, the Ig hinge region is derived from a human Ig hinge region or a non-human Ig hinge region. In some embodiments, the Ig hinge region is derived from a human Ig hinge region.

[0011] In some embodiments, the human Ig hinge region is an IgG1, IgG2, IgG3, or IgG4 isotype.

[0012] In some embodiments, L is the amino acid sequence C(X) y C (SEQ ID NO: 23), wherein X is glycine (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), threonine (Thr), tryptophan (Trp), or tyrosine (Tyr), and y is an integer from 1 to 3.

[0013] In some embodiments, L is the amino acid sequence C(X) y C (SEQ ID NO: 24), wherein X is Gly, Ser, or Pro, and y is an integer from 1 to 3.

[0014] In some embodiments, L comprises the amino acid sequence CPC, CGC, CSC, CPPC (SEQ ID NO: 1), CGPC (SEQ ID NO: 28), CPGC (SEQ ID NO: 29), CGGC (SEQ ID NO: 30), CSPG (SEQ ID NO: 31), CPSC (SEQ ID NO: 32), CSSC (SEQ ID NO: 33), CGSC (SEQ ID NO: 34), CSGC (SEQ ID NO: 35), CPPPC (SEQ ID NO: 36), CGPPC (SEQ ID NO: 37), CPGPC (SEQ ID NO: 38), CPPGC (SEQ ID NO: 39), CGGPC (SEQ ID NO: 40), CPGGC (SEQ ID NO: 41), CGGGC (SEQ ID NO: 42), CSPPC (SEQ ID NO: 43), CPSPC (SEQ ID NO: 44), CPPSC (SEQ ID NO: 45), CSSPC (SEQ ID NO: 46), CPSSC (SEQ ID NO: 47), CSSSC (SEQ ID NO: 48), CGSPC (SEQ ID NO: 49), CPGSC (SEQ ID NO: 50), CSGPC (SEQ ID NO: 51), or CPSGC (SEQ ID NO: 52).

[0015] In some embodiments, L comprises about 14 to about 19 amino acids. In some embodiments, L comprises about 14, about 15, about 16, about 17, about 18, or about 19 amino acids. In some embodiments, L comprises about 14 to about 19 amino acids. In some embodiments, L is about 14, about 15, about 16, about 17, about 18, or about 19 amino acids in length.

[0016] 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.

[0017] 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.

[0018] In some embodiments, L is the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 27), wherein X is Gly or Pro, m is an integer of 6 to 9, y is an integer of 1 to 3, and n is an integer of 4 to 6.

[0019] In some embodiments, L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.

[0020] In some embodiments, the scFv is in a VL-L-VH orientation. In some embodiments, the scFv is in a VH-L-VL orientation.

[0021] In some embodiments, the VH comprises a Cys at H105, the VL comprises a Cys at L42, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VL-L-VH orientation.

[0022] In some embodiments, the VH comprises a Cys at H105, the VL comprises a Cys at L45, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VL-L-VH orientation.

[0023] In some embodiments, the VH comprises a Cys at H105, the VL comprises a Cys at L39, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VL-L-VH orientation.

[0024] In some embodiments, the VH comprises a Cys at H5, the VL comprises a Cys at L42, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VL-L-VH orientation.

[0025] In some embodiments, the VH comprises a Cys at H5, the VL comprises a Cys at L45, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VL-L-VH orientation.

[0026] In some embodiments, the VH comprises a Cys at H5, the VL comprises a Cys at L39, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VL-L-VH orientation.

[0027] In some embodiments, the VH comprises a Cys at H3, the VL comprises a Cys at L42, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VL-L-VH orientation.

[0028] In some embodiments, the VH comprises a Cys at H3, the VL comprises a Cys at L45, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VL-L-VH orientation.

[0029] In some embodiments, the VH comprises a Cys at H3, the VL comprises a Cys at L39, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VL-L-VH orientation.

[0030] In some embodiments, the VH comprises a Cys at H43, the VL comprises a Cys at L100, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0031] In some embodiments, the VH comprises a Cys at H43, the VL comprises a Cys at L102, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0032] In some embodiments, the VH comprises a Cys at H43, the VL comprises a Cys at L5, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0033] In some embodiments, the VH comprises a Cys at H43, the VL comprises a Cys at L3, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0034] In some embodiments, the VH comprises a Cys at H40, the VL comprises a Cys at L100, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0035] In some embodiments, the VH comprises a Cys at H40, the VL comprises a Cys at L102, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0036] In some embodiments, the VH comprises a Cys at H40, the VL comprises a Cys at L5, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0037] In some embodiments, the VH comprises a Cys at H40, the VL comprises a Cys at L3, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0038] In some embodiments, the VH comprises a Cys at H46, the VL comprises a Cys at L100, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0039] In some embodiments, the VH comprises a Cys at H46, the VL comprises a Cys at L102, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0040] In some embodiments, the VH comprises a Cys at H46, the VL comprises a Cys at L5, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0041] In some embodiments, the VH comprises a Cys at H46, the VL comprises a Cys at L3, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0042] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 7.

[0043] In some embodiments, the binding molecule comprises a heavy chain, a light chain, and a polypeptide, wherein the N-terminus of the heavy chain and the light chain form a Fab, the polypeptide comprises an scFv at its N-terminus, and the C-terminus of the polypeptide and the C-terminus of the heavy chain form an Fc region.

[0044] In some embodiments, the Fab binds to a tumor antigen and the scFv binds to a T cell antigen, hi some embodiments, the tumor antigen is BCMA and the T cell antigen is CD3.

[0045] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO:126 or SEQ ID NO:128.

[0046] In some embodiments, the Fab comprises (i) a VH comprising the amino acid sequence of SEQ ID NO: 132 and a VL comprising the amino acid sequence of SEQ ID NO: 129, or (ii) a VH comprising the amino acid sequence of SEQ ID NO: 137 and a VL comprising the amino acid sequence of SEQ ID NO: 135.

[0047] In some embodiments, the VH comprises a Cys at H105, the VL comprises a Cys at L43, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VL-L-VH orientation.

[0048] In one aspect, the present disclosure provides a molecule comprising an antigen-binding fragment (Fab) that binds to a first antigen, a single-chain variable fragment (scFv) that binds to a second antigen, and a fragment crystallizable region (Fc region), wherein the scFv comprises a means for stabilizing the scFv.

[0049] In some embodiments, the scFv comprises a heavy chain variable region (VH), a linker (L), and a light chain variable region (VL), and the means for stabilizing the scFv comprises a) a disulfide bond between a structurally conserved, surface-exposed VH cysteine ​​(Cys) and an L Cys, b) a disulfide bond between a structurally conserved, surface-exposed VL Cys and an L Cys, or c) 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.

[0050] In some embodiments, a) the VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position, and the L comprises an L Cys; b) the VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position, and the L comprises an L Cys; or c) the VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position, the VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position, and the L comprises a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys are capable of forming a disulfide bond, and the VL Cys and the second L Cys are capable of forming a disulfide bond.

[0051] In some embodiments, the distance between VH Cys and VL Cys is about 5 Å to about 10 Å or about 7 Å to about 9 Å.

[0052] In some embodiments, the VH Cys is at H3, H5, H40, H43, H46, or H105, and / or the VL Cys is at L3, L5, L39, L42, L43, L45, L100, or L102, where residue numbering is according to Chothia.

[0053] In some embodiments, the VH Cys is at H105 and the VL Cys is at L42; or VH Cys is at H43 and VL Cys is at L100, or VH Cys is in H3 and VL Cys is in L3, or VH Cys is in H3 and VL Cys is in L5, or VH Cys is in H3 and VL Cys is in L39, or VH Cys is in H3 and VL Cys is in L42, or VH Cys is in H3 and VL Cys is in L45, or VH Cys is in H3 and VL Cys is in L100, or VH Cys is at H3 and VL Cys is at L102, or VH Cys is in H5 and VL Cys is in L3, or VH Cys is in H5 and VL Cys is in L5, or VH Cys is at H5 and VL Cys is at L39, or VH Cys is at H5 and VL Cys is at L42, or VH Cys is at H5 and VL Cys is at L45, or VH Cys is in H5 and VL Cys is in L100, or VH Cys is at H5 and VL Cys is at L102, or VH Cys is in H40 and VL Cys is in L3, or VH Cys is in H40 and VL Cys is in L5, or VH Cys is at H40 and VL Cys is at L39, or VH Cys is at H40 and VL Cys is at L42, or VH Cys is at H40 and VL Cys is at L45, or VH Cys is at H40 and VL Cys is at L100, or VH Cys is at H40 and VL Cys is at L102, or VH Cys is in H43 and VL Cys is in L3, or VH Cys is at H43 and VL Cys is at L5, or VH Cys is at H43 and VL Cys is at L39, or VH Cys is at H43 and VL Cys is at L42, or VH Cys is at H43 and VL Cys is at L45, or VH Cys is at H43 and VL Cys is at L102, or VH Cys is at H46 and VL Cys is at L3, or VH Cys is at H46 and VL Cys is at L5, or VH Cys is at H46 and VL Cys is at L39, or VH Cys is at H46 and VL Cys is at L42, or VH Cys is at H46 and VL Cys is at L45, or VH Cys is at H46 and VL Cys is at L100, or VH Cys is at H46 and VL Cys is at L102, or VH Cys is at H105 and VL Cys is at L3, or VH Cys is at H105 and VL Cys is at L5, or VH Cys is at H105 and VL Cys is at L39, or VH Cys is at H105 and VL Cys is at L45, or VH Cys is at H105 and VL Cys is at L100, or VH Cys is at H105 and VL Cys is at L102, or VH Cys is at H105 and VL Cys is at L43; Residue numbering is according to Chothia.

[0054] In some embodiments, L comprises a contiguous amino acid sequence derived from an immunoglobulin (Ig) hinge region. In some embodiments, the Ig hinge region is derived from a human Ig hinge region or a non-human Ig hinge region. In some embodiments, the Ig hinge region is derived from a human Ig hinge region.

[0055] In some embodiments, the human Ig hinge region is an IgG1, IgG2, IgG3, or IgG4 isotype.

[0056] In some embodiments, L is the amino acid sequence C(X) y C (SEQ ID NO: 23), wherein X is glycine (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), threonine (Thr), tryptophan (Trp), or tyrosine (Tyr), and y is an integer from 1 to 3.

[0057] In some embodiments, L is the amino acid sequence C(X) y C (SEQ ID NO: 24), wherein X is Gly, Ser, or Pro, and y is an integer from 1 to 3.

[0058] In some embodiments, L comprises the amino acid sequence CPC, CGC, CSC, CPPC (SEQ ID NO: 1), CGPC (SEQ ID NO: 28), CPGC (SEQ ID NO: 29), CGGC (SEQ ID NO: 30), CSPG (SEQ ID NO: 31), CPSC (SEQ ID NO: 32), CSSC (SEQ ID NO: 33), CGSC (SEQ ID NO: 34), CSGC (SEQ ID NO: 35), CPPPC (SEQ ID NO: 36), CGPPC (SEQ ID NO: 37), CPGPC (SEQ ID NO: 38), CPPGC (SEQ ID NO: 39), CGGPC (SEQ ID NO: 40), CPGGC (SEQ ID NO: 41), CGGGC (SEQ ID NO: 42), CSPPC (SEQ ID NO: 43), CPSPC (SEQ ID NO: 44), CPPSC (SEQ ID NO: 45), CSSPC (SEQ ID NO: 46), CPSSC (SEQ ID NO: 47), CSSSC (SEQ ID NO: 48), CGSPC (SEQ ID NO: 49), CPGSC (SEQ ID NO: 50), CSGPC (SEQ ID NO: 51), or CPSGC (SEQ ID NO: 52).

[0059] In some embodiments, L comprises about 14 to about 19 amino acids. In some embodiments, L comprises about 14, about 15, about 16, about 17, about 18, or about 19 amino acids. In some embodiments, L comprises about 14 to about 19 amino acids. In some embodiments, L is about 14, about 15, about 16, about 17, about 18, or about 19 amino acids in length.

[0060] 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.

[0061] 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.

[0062] In some embodiments, L is the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 27), wherein X is Gly or Pro, m is an integer of 6 to 9, y is an integer of 1 to 3, and n is an integer of 4 to 6.

[0063] In some embodiments, L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.

[0064] In some embodiments, the scFv is in a VL-L-VH orientation.

[0065] In some embodiments, the scFv is in a VH-L-VL orientation.

[0066] In some embodiments, the VH comprises a Cys at H105, the VL comprises a Cys at L42, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VL-L-VH orientation.

[0067] In some embodiments, the VH comprises a Cys at H105, the VL comprises a Cys at L45, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VL-L-VH orientation.

[0068] In some embodiments, the VH comprises a Cys at H105, the VL comprises a Cys at L39, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VL-L-VH orientation.

[0069] In some embodiments, the VH comprises a Cys at H5, the VL comprises a Cys at L42, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VL-L-VH orientation.

[0070] In some embodiments, the VH comprises a Cys at H5, the VL comprises a Cys at L45, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VL-L-VH orientation.

[0071] In some embodiments, the VH comprises a Cys at H5, the VL comprises a Cys at L39, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VL-L-VH orientation.

[0072] In some embodiments, the VH comprises a Cys at H3, the VL comprises a Cys at L42, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VL-L-VH orientation.

[0073] In some embodiments, the VH comprises a Cys at H3, the VL comprises a Cys at L45, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VL-L-VH orientation.

[0074] In some embodiments, the VH comprises a Cys at H3, the VL comprises a Cys at L39, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VL-L-VH orientation.

[0075] In some embodiments, the VH comprises a Cys at H43, the VL comprises a Cys at L100, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0076] In some embodiments, the VH comprises a Cys at H43, the VL comprises a Cys at L102, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0077] In some embodiments, the VH comprises a Cys at H43, the VL comprises a Cys at L5, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0078] In some embodiments, the VH comprises a Cys at H43, the VL comprises a Cys at L3, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0079] In some embodiments, the VH comprises a Cys at H40, the VL comprises a Cys at L100, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0080] In some embodiments, the VH comprises a Cys at H40, the VL comprises a Cys at L102, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0081] In some embodiments, the VH comprises a Cys at H40, the VL comprises a Cys at L5, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0082] In some embodiments, the VH comprises a Cys at H40, the VL comprises a Cys at L3, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0083] In some embodiments, the VH comprises a Cys at H46, the VL comprises a Cys at L100, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0084] In some embodiments, the VH comprises a Cys at H46, the VL comprises a Cys at L102, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0085] In some embodiments, the VH comprises a Cys at H46, the VL comprises a Cys at L5, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0086] In some embodiments, the VH comprises a Cys at H46, the VL comprises a Cys at L3, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0087] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 7.

[0088] In some embodiments, the molecule comprises a heavy chain, a light chain, and a polypeptide, wherein the N-terminus of the heavy chain and the light chain form a Fab, the polypeptide comprises an scFv at its N-terminus, and the C-terminus of the polypeptide and the C-terminus of the heavy chain form an Fc region.

[0089] In some embodiments, the Fab binds to a tumor antigen and the scFv binds to a T cell antigen, hi some embodiments, the tumor antigen is BCMA and the T cell antigen is CD3.

[0090] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO:126 or SEQ ID NO:128.

[0091] In some embodiments, the Fab comprises (i) a VH comprising the amino acid sequence of SEQ ID NO: 132 and a VL comprising the amino acid sequence of SEQ ID NO: 129, or (ii) a VH comprising the amino acid sequence of SEQ ID NO: 137 and a VL comprising the amino acid sequence of SEQ ID NO: 135.

[0092] In some embodiments, the VH comprises a Cys at H105, the VL comprises a Cys at L43, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VL-L-VH orientation.

[0093] In one aspect, the present disclosure provides a polynucleotide encoding a molecule disclosed herein or a fragment thereof, or a polypeptide thereof.

[0094] In one aspect, the present disclosure provides a vector comprising a polynucleotide disclosed herein.

[0095] In one aspect, the present disclosure provides a host cell comprising a vector disclosed herein. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is a eukaryotic cell.

[0096] In one aspect, the present disclosure provides methods of producing a molecule of the present disclosure. In some embodiments, the method comprises culturing a host cell of the present disclosure under conditions such that the molecule is produced, and purifying the produced molecule. In some embodiments, the method comprises introducing a polynucleotide of the present disclosure into a host cell, culturing the host cell under conditions such that the molecule is produced, and purifying the produced molecule.

[0097] In one aspect, the disclosure provides a composition comprising a molecule of the disclosure. In some embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0098] In one aspect, the present disclosure provides means for producing the molecules of the present disclosure.

[0099] In one aspect, the disclosure provides a method for directing or engaging a cell to a target cell. In some embodiments, the method comprises contacting the target cell with a molecule of the disclosure. In some embodiments, the Fab binds to a first antigen on the target cell and the scFv binds to a second antigen on the cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the target cell is a tumor cell. In some embodiments, the method is for treating a disease or disorder in a subject. In some embodiments, the disease or disorder is a tumor. In some embodiments, the disease or disorder is cancer. In some embodiments, the subject is a human subject.

[0100] In one aspect, the present disclosure provides methods for eliminating or inhibiting target cells. In some embodiments, the methods comprise contacting the target cells with a molecule of the present disclosure.

[0101] In one aspect, the disclosure provides a method for treating a disease or disorder in a subject. In some embodiments, the method comprises administering to the subject a molecule of the disclosure.

[0102] In one aspect, the present disclosure provides a molecule of the present disclosure for use as a medicament. In one aspect, the present disclosure provides a molecule of the present disclosure for use in treating a disease or disorder. [Brief explanation of the drawings]

[0103] [Figure 1] 1 shows an exemplary design of a stabilized bispecific BCMA / CD3 antibody. The CD3 scFvs of the bispecific antibody are connected by a flexible linker, which is stabilized to an scFv (spFv) by a disulfide bond between the staple sequence and the anchor point in the linker. [Figure 2A]Figure 2A shows the improved thermal stability of the Cris7a / b domains due to stapling. Figure 2A: Overlay of DSC thermograms for Cris7a scFv, Cris7a spFv, Cris7b scFv, and Cris7b spFv. Parameters related to protein design and enthalpy characteristics from the analysis are listed in Tables 12 and 14. [Figure 2B] Improved thermal stability of the Cris7a / b domains due to stapling is shown in Figure 2B. SDS-PAGE of scFv and spFv proteins of Cris7a / Cris7b in the LH orientation. [Figure 3A] 1 shows size exclusion chromatography of Cris7b containing bispecific molecules. [Figure 3B] 1 shows size exclusion chromatography of Cris7b containing bispecific molecules. [Figure 3C] 1 shows size exclusion chromatography of Cris7b containing bispecific molecules. [Figure 3D] 1 shows size exclusion chromatography of Cris7b containing bispecific molecules. [Figure 4A] 1 shows size exclusion chromatography of CD3B219 containing bispecific molecules. [Figure 4B] 1 shows size exclusion chromatography of CD3B219 containing bispecific molecules. [Figure 4C] 1 shows size exclusion chromatography of CD3B219 containing bispecific molecules. [Figure 4D] 1 shows size exclusion chromatography of CD3B219 containing bispecific molecules. [Figure 5] 1 shows the thermal stability of CD3 / BCMA bispecific molecules. [Figure 6] Figure 1 shows the cytotoxicity of CD3 / BCMA bispecific molecules. CD3 / BCMA bispecific molecules killed BCMA+H929-GFP+ cells. [Figure 7] Activation of CD4+ / CD25+ effector cells by CD3 / BCMA bispecific molecules. [Figure 8]Activation of CD8+ / CD25+ effector cells by CD3 / BCMA bispecific molecules. [Figure 9] 1 shows the protein yields of spFv bispecific molecules. [Figure 10] 1 shows the aggregation resistance of spFv bispecific molecules. [Figure 11] 1 shows similar CD3 binding affinities of scFv and spFv bispecific molecules. [Figure 12] 1 shows similar CD3-mediated killing properties of scFv and spFv bispecific molecules. [Figure 13]ScFv stapling is shown. Figure 13A: scFv stapling to improve low stability and minimize respiratory-mediated aggregation. Figure 13B: Schematic of the "stapling" scheme, using the HL configuration as an example. A similar scheme is valid for LH constructs. The dashed line indicates the flexible linker connecting the C-terminus of the leading variable region to the stapling "CPPC" motif, and a second dashed line connects to the N-terminus of the trailing variable region. The segment in the middle of the linker, labeled "CPPC," showed one possible design of the "staple" naturally occurring in the IgG1 hinge. The anchor points (labeled "C(APL" and "C(APH")) mutated to Cys residues in VH and VL are shown as sticks. The short line between the staple Cys residue and the anchor point indicates staple disulfide bond formation. Figure 13C: Germline human antibody (PDB ID Schematic illustration of the geometric considerations (HL configuration) of anchor point selection mapped onto the Fv of human IgG (PDB 5DK3, GLk1). Cter: C-terminus of the preceding domain; Nter: N-terminus of the following domain; APH: anchor position on the preceding domain; APL: anchor position on the following domain; dAP: distance between APH and APH; d1-d4: various distances defined in the diagram. A similar diagram can be drawn for the LH orientation (not shown). The anchor points for the HL orientation were Chothia position 43 for the VH (H43C in the diagram) and position 100 for the VL (L100C in the diagram). For the LH, Chothia positions 42 in the VL (L42C) and 105 in the VH (H105C). Figures 13D and 13E show the hinge CPPC of human IgG (PDB 5DK3) and mouse IgG2a (PDB 5DK3). 1IGT) Cβ(Cys1)-Cβ(Cys2) distances between two Cys residues in the hinge CPPC (Figure 13E). These hinge Cβ(Cys1)-Cβ(Cys2) distances range from approximately 7 Å to approximately 9 Å. [Figure 14A] Structures and comparison of various scFv / spFv domains are shown in Figure 14A: GLk1 spFv LH. [Figure 14B] Structures and comparison of various scFv / spFv domains are shown in Figure 14B: GLk1 spFv HL. [Figure 14C]Structures and comparison of various scFv / spFv domains are shown in Figure 14C: GLk2 spFv HL. [Figure 14D] Structures and comparison of various scFv / spFv domains are shown. Figure 14D: 2mFo-dFc electron density (1.5σ contour) of the staple motifs CPPC and SS to the anchor point of Glk2 spFv HL. Circles indicate stapling disulfide density. [Figure 14E] Structures and comparison of different scFv / spFv domains are shown in Figure 14E: CAT2200b spFv HL. [Figure 14F] The structures and comparison of various scFv / spFv domains are shown in Figure 14F: Non-binding CAT2200b spFv HLL compared to CAT2200a scFv LH bound to IL-17. [Figure 14G] Structures and comparison of various scFv / spFv domains are shown. Figure 14G: Front and back views of unbound CAT2200b spFv HL compared to CAT2200a spFv LH bound to IL-17. [Figure 15] The conformations of the staples and linkers in five spFv structures are shown. For clarity, the CPPC motif has been relabeled as Cys1, Pro1, Pro2, and Cys2. The structures are superimposed on the backbone of the CPPC motif. Dashed lines indicate the Cα-Cα and Cβ-Cβ distances between the Cys1 and Cys2 residues. The range of Cβ-Cβ distances observed across all copies of the linker-staple Cys residues is shown. The N-terminus is indicated by "Nter," and the C-terminus is indicated by "Cter." [Figure 16A] Figure 16A shows improved yield, product quality, and predicted disulfide formation in the stapled linker of spFv bispecific molecules. Figure 16B shows the BCMA (Fab) x CD3 (scFv / spFv) bispecific molecule structure. HK in the Fc region indicates knob-in-hole (K, knob; H, hole) Fc heterodimerization mutations. RF (H435R and Y436F) mutations in the Fab-containing chain for purification to prevent Protein A binding to RF-containing chain monomers or homodimers. [Figure 16B] Improved yield, product quality, and predicted disulfide formation in stapled linkers of spFv bispecific molecules are shown in Figure 16B. Post-CH1 SEC profile of scFv / spFv Cris7b-containing molecules with BCMB749 shows the presence of oligomeric species (labeled O) in the scFv protein but lack of them in the spFv protein (monomer, M). [Figure 16C] Figure 16C shows improved yield, product quality, and predicted disulfide formation in stapled linkers of spFv bispecific molecules. Figure 16C: Schematic representation of predicted disulfides in stapled bispecific molecules. All Cys residues are indicated by their sequence position / number in each polypeptide chain. Predicted disulfide bonds are indicated by the lines connecting them. Dotted lines represent additional disulfide bonds in the stapled region of single-chain Fvs. Interchain disulfide bonds are indicated by solid double lines. [Figure 16D] The improved yield, product quality, and predicted disulfide formation in the stapled linker of spFv bispecific molecules are shown. Figure 16D: Total ion current (TIC) of the non-reduced LysC-ProAlanase digested bispecific molecule. The labeled chromatographic peak is the fully cleaved disulfide peptide. Other peaks in the TIC represent nonspecifically digested proteins of the predicted disulfide peptide. Disulfide bonds marked with an asterisk are representative species with XIC / MS1 / MS2 data shown in Figures 25 and 26. [Figure 17A] Figure 17A shows stable and retained binding affinity to CD3 of Cris7b-containing spFv bispecific molecules. Figure 17A: NanoDSF trace of Cris7b-containing scFv / spFv bispecific molecules with BCMB749 showed a ~10°C shift to a higher Tm with incorporation of the stapling mutation. [Figure 17B]Figures 17B and 17C show stable and retained binding affinity to CD3 of spFv bispecific molecules containing Cris7b. Cris7b spFv bispecific molecules were resistant to heat-induced aggregation. SEC traces (Figure 17B) and quantification of aggregation levels (Figure 17C) showed that spFv bispecific molecules containing Cris7b had a dramatic reduction in heat-induced aggregation over a 6-week time frame at either 4°C or 40°C. [Figure 17C] Figures 17B and 17C show stable and retained binding affinity to CD3 of spFv bispecific molecules containing Cris7b. Cris7b spFv bispecific molecules were resistant to heat-induced aggregation. SEC traces (Figure 17B) and quantification of aggregation levels (Figure 17C) showed that spFv bispecific molecules containing Cris7b had a dramatic reduction in heat-induced aggregation over a 6-week time frame at either 4°C or 40°C. [Figure 17D] Figure 17D shows stable and retained binding affinity to CD3 of spFv bispecific molecules containing Cris7b. BLI binding traces showed comparable binding characteristics (e.g., association and dissociation) for their binding to recombinant CD3. Light gray: Cris7b spFv; dark gray: Cris7b scFv Bird; dashed line: Cris7b G4S. [Figure 18A] Showing similar function between spFv bispecific molecules and their non-stapled counterparts, Figure 18A: spFv bispecific molecules exhibited potent killing activity of BCMA+ cancer cells. [Figure 18B] These results demonstrate similar function between spFv bispecific molecules and their non-stapled counterparts. Figures 18B and 18C: scFv / spFv bispecific molecules activated CD4 / CD25 (Figure 18B) and CD8 / CD25 (Figure 18C) T cells with similar potency. Null controls showed no killing or T cell activation activity. [Figure 18C]These results demonstrate similar function between spFv bispecific molecules and their non-stapled counterparts. Figures 18B and 18C: scFv / spFv bispecific molecules activated CD4 / CD25 (Figure 18B) and CD8 / CD25 (Figure 18C) T cells with similar potency. Null controls showed no killing or T cell activation activity. [Figure 19A] Anchor point selection in the VL and VH sequences is shown. Figure 19A: Proposed linker between VL (SEQ ID NO: 144) and VH (SEQ ID NO: 144). The variable number of amino acid residues (aa) provides flexibility and allows for proper linker-anchor disulfide formation, but is not long enough to allow disulfide scrambling. [Figure 19B] Anchor point selection in VL and VH sequences is shown. Figure 19B: VL and VH sequences are numbered according to the Chothia numbering scheme (Chothia and Lesk 1987) and show the sequences above. Anchor points are highlighted in pairs with a number (1 or 2) below the selected position. Two positions in VL and VH with the same highlight and below number represent pairs of positions used as anchor points for a particular spFv construct. Pairs 1 and 2 are for the LH and HL constructs, respectively. The anchor points for the HL orientation were Chothia position 43 for VH (H43C in the figure) and position 100 for VL (L100C in the figure), and for LH, Chothia positions 42 in VL (L42C) and 105 in VH (H105C). Glk1 VL (SEQ ID NO: 56); Glk1 VH (SEQ ID NO: 60); Glk2 VL (SEQ ID NO: 145); Glk2 VH (SEQ ID NO: 146); CAT2200 VL (SEQ ID NO: 147); CAT2200a VH (SEQ ID NO: 148). [Figure 20A]Disulfide bond geometries (Figure 20A). DS1: disulfide bond between Chothia positions L43 and H105; DS2: L100 and H44. (Figure 20A) Schematic showing the location of the Cα-Cα and Cβ-Cβ distances in the formed disulfide bond. The relative distance between Cα and Cβ residues strongly influenced the efficiency of disulfide bond formation. When evaluating the distance distributions in Figures 20B-20E, the two Cys residues at anchor positions are unlikely to form direct disulfide bonds because the distance, especially Cβ-Cβ, is much longer than typical for positions that form disulfide bonds. For VL / VH inter-disulfide bonds (DS1 and DS2, Figures 20D and 20E), the majority of VL / VH pairs had Cβ-Cβ distances much wider than the typical disulfide bond geometry, which is a likely structural reason why direct disulfide bonds often did not form or improve stability. All antibody Fab and scFv crystal structures in the human PDB (rcsb.org), including those of humanized and murine origin, with a resolution of 2.5 Å or higher were included in the distance calculations. All VL / VH pairs in these structures were included. Multiple copies in the asymmetric unit were treated as independent. For positions with Gly in the structure, the Gly residue was mutated to Ala without energy minimization to obtain the coordinates of the predicted Cβ position. A total of 2501 Fv structures were included in the distance calculations. All calculations were performed in MOE (CCG, Montreal) using custom scripts from CCG technical support, whose assistance is hereby acknowledged. (Figures 20A and 20B) Cα-Cα and Cβ-Cβ distances for the two anchor positions selected for LH and HL stapling, respectively. (Figures 20C and 20D) α-Cα and Cβ-Cβ distances for DS1 and DS2 in known antibody structures. Cα and Cβ protein disulfide bond distances are from Dani et al. (2003) Protein Eng. 16(3) 187-193. [Figure 20B]α-Cα and Cβ-Cβ distance distributions between anchor points for stapling (Figures 20B and 20C). DS1: disulfide bond between Chothia positions L43 and H105; DS2: L100 and H44. (Figure 20A) Schematic showing the location of Cα-Cα and Cβ-Cβ distances in the formed disulfide bond. The relative distances between Cα and Cβ residues strongly influenced the efficiency of disulfide bond formation. When evaluating the distance distributions in Figures 20B-20E, the two Cys residues at the anchor positions are unlikely to form a direct disulfide bond because the distance, especially Cβ-Cβ, is much longer than typical for positions that form S—S bonds. For VL / VH interdisulfide bonds (DS1 and DS2, Figures 20D and 20E), the majority of VL / VH pairs had Cβ-Cβ distances much wider than the typical disulfide bond geometry, likely explaining why direct disulfide bonds often failed to form or improve stability. All antibody Fab and scFv crystal structures in the human PDB (rcsb.org), including humanized and murine origins, with a resolution of 2.5 Å or higher were included in the distance calculations. All VL / VH pairs in these structures were included. Multiple copies in the asymmetric unit were treated as independent. For positions with Gly in the structure, the Gly residue was mutated to Ala without energy minimization to obtain the coordinates of the predicted Cβ position. A total of 2501 Fv structures were included in the distance calculations. All calculations were performed in MOE (CCG, Montreal) using custom scripts from CCG technical support, whose assistance is acknowledged herein. (Figures 20A and 20B) Cα-Cα and Cβ-Cβ distances for the two anchor positions selected for LH and HL stapling. (Figures 20C and 20D) α-Cα and Cβ-Cβ distances for DS1 and DS2 in known antibody structures. Distances of Cα and Cβ protein disulfide bonds are from Dani et al. (2003) Protein Eng. 16(3) 187-193. [Figure 20C]α-Cα and Cβ-Cβ distance distributions between anchor points for stapling (Figures 20B and 20C). DS1: disulfide bond between Chothia positions L43 and H105; DS2: L100 and H44. (Figure 20A) Schematic showing the location of Cα-Cα and Cβ-Cβ distances in the formed disulfide bond. The relative distances between Cα and Cβ residues strongly influenced the efficiency of disulfide bond formation. When evaluating the distance distributions in Figures 20B-20E, the two Cys residues at the anchor positions are unlikely to form a direct disulfide bond because the distance, especially Cβ-Cβ, is much longer than typical for positions that form S—S bonds. For VL / VH interdisulfide bonds (DS1 and DS2, Figures 20D and 20E), the majority of VL / VH pairs had Cβ-Cβ distances much wider than the typical disulfide bond geometry, likely explaining why direct disulfide bonds often failed to form or improve stability. All antibody Fab and scFv crystal structures in the human PDB (rcsb.org), including humanized and murine origins, with a resolution of 2.5 Å or higher were included in the distance calculations. All VL / VH pairs in these structures were included. Multiple copies in the asymmetric unit were treated as independent. For positions with Gly in the structure, the Gly residue was mutated to Ala without energy minimization to obtain the coordinates of the predicted Cβ position. A total of 2501 Fv structures were included in the distance calculations. All calculations were performed in MOE (CCG, Montreal) using custom scripts from CCG technical support, whose assistance is acknowledged herein. (Figures 20A and 20B) Cα-Cα and Cβ-Cβ distances for the two anchor positions selected for LH and HL stapling. (Figures 20C and 20D) α-Cα and Cβ-Cβ distances for DS1 and DS2 in known antibody structures. Distances of Cα and Cβ protein disulfide bonds are from Dani et al. (2003) Protein Eng. 16(3) 187-193. [Figure 20D]Distance distribution between the positions of direct interchain disulfide bonds (Figures 20D and 20E). DS1: disulfide bond between Chothia positions L43 and H105; DS2: L100 and H44. (Figure 20A) Schematic showing the positions of Cα-Cα and Cβ-Cβ distances in the formed disulfide bond. The relative distances between Cα and Cβ residues strongly influenced the efficiency of disulfide bond formation. When evaluating the distance distributions in Figures 20B-20E, the two Cys residues at the anchor positions are unlikely to form a direct disulfide bond because the distance, especially Cβ-Cβ, is much longer than typical for positions that form S—S bonds. For VL / VH interdisulfide bonds (DS1 and DS2, Figures 20D and 20E), the majority of VL / VH pairs had Cβ-Cβ distances much wider than the typical disulfide bond geometry, likely explaining why direct disulfide bonds often failed to form or improve stability. All antibody Fab and scFv crystal structures in the human PDB (rcsb.org), including humanized and murine origins, with a resolution of 2.5 Å or higher were included in the distance calculations. All VL / VH pairs in these structures were included. Multiple copies in the asymmetric unit were treated as independent. For positions with Gly in the structure, the Gly residue was mutated to Ala without energy minimization to obtain the coordinates of the predicted Cβ position. A total of 2501 Fv structures were included in the distance calculations. All calculations were performed in MOE (CCG, Montreal) using custom scripts from CCG technical support, whose assistance is acknowledged herein. (Figures 20A and 20B) Cα-Cα and Cβ-Cβ distances for the two anchor positions selected for LH and HL stapling. (Figures 20C and 20D) α-Cα and Cβ-Cβ distances for DS1 and DS2 in known antibody structures. Distances of Cα and Cβ protein disulfide bonds are from Dani et al. (2003) Protein Eng. 16(3) 187-193. [Figure 20E]Distance distribution between the positions of direct interchain disulfide bonds (Figures 20D and 20E). DS1: disulfide bond between Chothia positions L43 and H105; DS2: L100 and H44. (Figure 20A) Schematic showing the positions of Cα-Cα and Cβ-Cβ distances in the formed disulfide bond. The relative distances between Cα and Cβ residues strongly influenced the efficiency of disulfide bond formation. When evaluating the distance distributions in Figures 20B-20E, the two Cys residues at the anchor positions are unlikely to form a direct disulfide bond because the distance, especially Cβ-Cβ, is much longer than typical for positions that form S—S bonds. For VL / VH interdisulfide bonds (DS1 and DS2, Figures 20D and 20E), the majority of VL / VH pairs had Cβ-Cβ distances much wider than the typical disulfide bond geometry, likely explaining why direct disulfide bonds often failed to form or improve stability. All antibody Fab and scFv crystal structures in the human PDB (rcsb.org), including humanized and murine origins, with a resolution of 2.5 Å or higher were included in the distance calculations. All VL / VH pairs in these structures were included. Multiple copies in the asymmetric unit were treated as independent. For positions with Gly in the structure, the Gly residue was mutated to Ala without energy minimization to obtain the coordinates of the predicted Cβ position. A total of 2501 Fv structures were included in the distance calculations. All calculations were performed in MOE (CCG, Montreal) using custom scripts from CCG technical support, whose assistance is acknowledged herein. (Figures 20A and 20B) Cα-Cα and Cβ-Cβ distances for the two anchor positions selected for LH and HL stapling. (Figures 20C and 20D) α-Cα and Cβ-Cβ distances for DS1 and DS2 in known antibody structures. Distances of Cα and Cβ protein disulfide bonds are from Dani et al. (2003) Protein Eng. 16(3) 187-193. [Figure 21A]Figure 21A shows the stapling anchors to the terminal geometry. Figure 21A is a schematic diagram of distances. Nter: N-terminus; Cter: C-terminus; d: distance between the VL anchor point and the VH anchor point; d1-d4: d1: the preceding segment from domain 1, the distance from the C-terminus of domain 1 (VH, in the figure) to the Cys anchor residue of domain 1 (VH, in the figure); d2: the distance from the C-terminus of domain 1 (VH, in the figure) to the Cys anchor residue of domain 2 (VL, in the figure); d3: the corresponding subsequent segment of domain 2; the distance from the N-terminus of domain 2 (VL, in the figure) to the Cys anchor residue of domain 2 (VL, in the figure); d4: the distance from the N-terminus of domain 2 (VL, in the figure) to the Cys anchor residue of domain 1 (VH, in the figure). [Figure 21B] Stapling anchors to terminal geometries are shown. Figures 21B and 21C: Distance distributions for the same set of Fv fragments as in Figure S2 for LH and HL configurations. Methods were the same as provided in Figure 20. [Figure 21C] Stapling anchors to terminal geometries are shown. Figures 21B and 21C: Distance distributions for the same set of Fv fragments as in Figure S2 for LH and HL configurations. Methods were the same as provided in Figure 20. [Figure 22] The H-bonds between E1 of the subsequent VL domain and the backbone of the subsequent linker segment of the Glk2 spFv HL structure are shown. [Figure 23] Figure 1 shows the humanization and sequence alignment of BCMB749. Each sequence alignment includes the parent (top), selected human acceptor germline sequence (middle), and the CDRs with backmutations grafted in italics (bottom). CDRs are underlined. Bold: CDR-supported positions in the framework regions. Boxed: VL / VH interface residues. BCMB749_VL (SEQ ID NO: 129); BCMB749_VH (SEQ ID NO: 132); huKV1-12*01 (SEQ ID NO: 149); huHV1-3*01 (SEQ ID NO: 150); BCMB749h_VL SEQ ID NO: 135); BCMB749h_VH (SEQ ID NO: 137). [Figure 24]Analytical SEC traces comparing the product quality of small-scale produced CD3-containing bispecific samples with either scFv (left) or spFv (right) arms after purification are shown. The top plot shows bispecific molecules containing Cris7b variants. The bottom plot shows bispecific molecules containing alternative anti-CD3 binding variants. The left plot contains a murine anti-BCMA Fab arm, and the right plot contains a humanized anti-BCMA Fab arm. [Figure 25A] Mass spectrometry mapping of disulfides in Byos. Figure 25A: Calculated and observed mass results for all disulfide-linked dipeptide species after LysC and ProAlanase digestion. Figure 25B: Fc disulfide 262-322. Figure 25C: spFv disulfide 119-237. Figures 25B and 25C: (Top left panel) MS1 of the predicted mass is within 2 ppm of the calculated disulfide species. (Top right panel) Extracted ion chromatogram (XIC) showing the retention time of the predicted disulfide. The recovered peptide signal was in the mid-range. (Bottom panel) MS / MS coverage for both peptides of the disulfide. [Figure 25B] Mass spectrometry mapping of disulfides in Byos. Figure 25A: Calculated and observed mass results for all disulfide-linked dipeptide species after LysC and ProAlanase digestion. Figure 25B: Fc disulfide 262-322. Figure 25C: spFv disulfide 119-237. Figures 25B and 25C: (Top left panel) MS1 of the predicted mass is within 2 ppm of the calculated disulfide species. (Top right panel) Extracted ion chromatogram (XIC) showing the retention time of the predicted disulfide. The recovered peptide signal was in the mid-range. (Bottom panel) MS / MS coverage for both peptides of the disulfide. [Figure 25C]Mass spectrometry mapping of disulfides in Byos. Figure 25A: Calculated and observed mass results for all disulfide-linked dipeptide species after LysC and ProAlanase digestion. Figure 25B: Fc disulfide 262-322. Figure 25C: spFv disulfide 119-237. Figures 25B and 25C: (Top left panel) MS1 of the predicted mass is within 2 ppm of the calculated disulfide species. (Top right panel) Extracted ion chromatogram (XIC) showing the retention time of the predicted disulfide. The recovered peptide signal was in the mid-range. (Bottom panel) MS / MS coverage for both peptides of the disulfide. [Figure 26A] Stapling does not affect antibody binding. Figure 26A: ELISA titrations against recombinant CD3 show equivalent antigen binding regardless of the presence of scFv or spFv arms, indicating that the stapling mutations do not interfere with antigen binding. [Figure 26B] Stapling does not affect antibody binding. Figure 26B: ELISA titrations against recombinant BCMA show equivalent antigen binding regardless of the presence of scFv or spFv arms, supporting that the stapling mutations do not affect binding of the partner arms. [Figure 26C] Stapling does not affect antibody binding. Figure 26C: BLI binding traces for a pair of bispecific molecules (Cris7b scFv trilinker x BCMA, left; Cris7b spFv x BCMA, right) show equivalent binding to BCMA regardless of the presence of the scFv or spFv arm. [Figure 27] Figure 1 shows a comparison of the biophysical properties of scFv / spFv bispecifics and trispecifics. BsAb: bispecific antibody; TsAb: trispecific antibody, where 1 and 2 indicate targets 1 and 2. sc / sp indicates the format of the single chain portion (scFv / spFv). All affinity values ​​are by SPR. *Cell binding, EC50. Values ​​shown are for the scFv / spFv portion only. Detailed Description of the Invention

[0104] The methods and molecules of the present disclosure may be understood more readily by reference to the following detailed description taken in conjunction with the accompanying drawings, which form a part of this disclosure: It is to be understood that the methods and molecules of the present disclosure are not limited to the specific methods and molecules described and / or illustrated herein, and further, that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting.

[0105] All patents, published patent applications, and publications cited herein are incorporated by reference as if fully set forth herein.

[0106] Where lists are presented, unless otherwise stated, it is to be understood that each individual element of that list and every combination of that list is a separate embodiment. For example, a list of embodiments presented as "A, B, or C" should be interpreted to include the embodiments "A," "B," "C," "A or B," "A or C," "B or C," or "A, B, or C."

[0107] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

[0108] The transitional phrases "comprising," "consisting essentially of," and "consisting of" are intended to connote their generally accepted meanings in patent language, i.e., (i) "comprising" is synonymous with "comprising," "containing," or "characterized by" and is inclusive or open-ended and does not exclude other unrecited elements or method steps; (ii) "consisting of" excludes any element, step, or ingredient not specified in the claim; and (iii) "consisting essentially of" limits the claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics" of the claimed invention. Embodiments described with the phrase "comprising" (or its equivalents) also provide as embodiments embodiments described independently with "consisting of" and "consisting essentially of."

[0109] "About" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on the limitations of how the value is measured or determined, i.e., the measurement system. In the context of a particular assay, result, or embodiment, unless expressly stated otherwise in the Examples or elsewhere herein, "about" means within one standard deviation, or a range of up to 5%, whichever is greater, per practice in the art.

[0110] "Alternative scaffold" refers to a single-chain protein framework containing a structured core associated with conformationally tolerant variable domains that can be engineered and selected to bind specific antigens, allowing for polymorphism to be introduced without compromising the integrity of the scaffold.

[0111] "Antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refers to a mechanism of cell death induction that depends on the interaction of antibody-coated target cells with lytic effector cells, such as natural killer cells (NK), monocytes, macrophages, and neutrophils, via Fc gamma receptors (FcγR) expressed on the effector cells.

[0112] "Antibody-dependent cellular phagocytosis" or "ADCP" refers to the elimination of antibody-coated target cells by uptake by phagocytic cells such as macrophages or dendritic cells.

[0113] The term "antigen" refers to any molecule (e.g., a protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, portion thereof, or combination thereof) that can mediate an immune response. Non-limiting exemplary immune responses include antibody production and activation of immune cells such as T cells, B cells, or NK cells.

[0114] The term "antigen-binding fragment" or "antigen-binding domain" refers to a portion of a protein that binds to an antigen. Antigen-binding fragments may be synthetic, enzymatically obtainable, or recombinant polypeptides, and include heavy chain variable regions (VH), light chain variable regions (VL), Fab, Fab', F(ab'), Fd, and Fv fragments, domain antibodies (dAbs) consisting of VH or VL domains, camelized VH domains, VHH domains, minimal recognition units consisting of amino acid residues mimicking antibody CDRs such as FR3-CDR3-FR4 portions, portions of immunoglobulins that bind to antigens such as HCDR1, HCDR2, and / or HCDR3, and LCDR1, LCDR2, and / or LCDR3, surrogate scaffolds that bind to antigens, and multispecific molecules containing antigen-binding fragments. Antigen-binding fragments (such as VH and VL) can be linked together via linkers to form various types of single-chain antibody designs in which, when the VH and VL domains are expressed as separate single chains, the VH / VL domains can pair intramolecularly or intermolecularly to form monovalent antigen-binding domains, e.g., single-chain variable fragments (scFvs) or diabodies. Antigen-binding fragments can also be conjugated to other antibodies, proteins, antigen-binding fragments, or alternative scaffolds, which can be monospecific or multispecific, to genetically engineer bispecific and multispecific molecules.

[0115] The term "antibody" has a broad meaning and includes immunoglobulin molecules, including monoclonal antibodies (including murine, human, humanized, and chimeric monoclonal antibodies), antigen-binding fragments, multispecific antibodies (e.g., bispecific, trispecific, and tetraspecific), dimeric, tetrameric, or multimeric antibodies, single-chain antibodies, domain antibodies, and any other modified form of immunoglobulin molecule containing an antigen-binding site of the required specificity. A "full-length antibody" is composed of two heavy chains (HC) and two light chains (LC), and multimers thereof (e.g., IgM), interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (consisting of domains CH1, hinge, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with framework regions (FRs). Each VH and VL is composed of three CDR and four FR segments, arranged from amino- to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Immunoglobulins can be assigned to five major classes, namely, IgA, IgD, IgE, IgG, and IgM, depending on the amino acid sequence of the heavy-chain constant domain. IgA and IgG are further subdivided into isotypes, IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0116] The term "bispecific" refers to a molecule (e.g., an antibody) that specifically binds to two different antigens, or two different epitopes, within the same antigen. Bispecific molecules may be cross-reactive to other related antigens, e.g., the same antigen (homologues), from other species such as humans or monkeys, e.g., cynomolgus monkeys (Macaca cynomolgus, cyno) or chimpanzees (Pan troglodytes), or may bind to an epitope shared between two or more different antigens.

[0117] The term "BCMA" refers to B-cell maturation antigen. BCMA, also known as CD269 and TNFRSF17 (UniProt Q02223), is a member of the tumor necrosis receptor superfamily expressed in differentiated plasma cells. In some embodiments, the BCMA is human BCMA. An exemplary human BCMA nucleotide sequence is provided by GenBank Accession No. BC058291. There are four major haplotypes of the BCMA gene in the human genome (Kawasaki et al., Genes Immun. 2:276-9, 2001). According to the present disclosure, the term "BCMA" encompasses all four haplotypes. In some embodiments, the extracellular domain of human BCMA consists of amino acids 1-54 of the amino acid sequence having Uniprot Ref. No. Q02223-1. As used herein, the term "antibody against BCMA, i.e., anti-BCMA antibody" refers to an antibody that specifically binds to BCMA. In some embodiments, the anti-BCMA antibody is human BCMA. In some embodiments, the anti-BCMA antibody binds to a portion of human BCMA, hi some embodiments, the anti-BCMA antibody binds to the extracellular domain of human BCMA.

[0118] The term "chimeric antigen receptor" or "CAR" refers to an engineered T cell receptor that transfers ligand or antigen specificity onto immune cells, such as T cells (including, but not limited to, 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 immune receptors. In some embodiments, a CAR comprises an extracellular domain capable of binding to an antigen, a transmembrane domain, and at least one intracellular domain. In some embodiments, the intracellular domain comprises a polypeptide known to function as a domain that transduces a signal that causes activation or inhibition of a biological process within the cell. In some embodiments, the transmembrane domain comprises a peptide or polypeptide known to span the cell membrane and capable of functioning to connect the extracellular domain and the cytosolic domain. In some embodiments, a CAR further comprises a hinge domain that serves as a linker between the extracellular domain and the transmembrane domain.

[0119] "CD3" refers to an antigen expressed on T cells as part of the multimeric T cell receptor (TCR) complex. CD3 consists of a homodimer or heterodimer formed from the association of two or four receptor chains: CD3ε, CD3δ, CD3ζ, and CD3γ. In some embodiments, the CD3 antibodies provided herein bind to the CD3ε polypeptide, which, together with CD3γ, CD3δ, and CD3ζ, and T cell receptor α / β and γ / δ heterodimers, form the T cell receptor-CD3 complex. This complex plays a key role in antigen binding, recognizing several intracellular signaling pathways. The CD3 complex mediates signal transduction, leading to T cell activation and proliferation. CD3 is required for the immune response. The term "CD3" encompasses any CD3 variant, isoform, and species homolog that can be naturally expressed by cells (including T cells) or expressed on cells transfected with a gene or cDNA encoding a protein of interest. In certain embodiments, the CD3 is human CD3.

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

[0121] The term "complementarity-determining region" (CDR) refers to the region of an antibody that binds to an antigen. There are three CDRs in VH (HCDR1, HCDR2, and HCDR3), and three CDRs in VL (LCDR1, LCDR2, and LCDR3). CDRs can be defined using various descriptions, such as Kabat (Wu et al. (1970) J Exp Med 123:211-250; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al., (1987) J Mol Biol 196:901-17), IMGT (Lefranc et al., (2003) Dev Comp Immunol 27:55-77), and AbM (Martin and Thornton (1996) J Bmol Biol 263:800-815). The correspondence between various descriptions and the numbering of variable regions has been described (see, for example, Lefranc et al. (2003) Dev Comp Immunol 27:55-77; Honegger and Pluckthun, J Mol Biol (2001) 309:657-670; the International ImMunoGeneTics (IMGT) database; web resource http: / / www_imgt_org). CDRs can be described using available programs such as abYsis by UCL Business PLC. As used herein, the terms "CDR," "HCDR1," "HCDR2," "HCDR3," "LCDR1," "LCDR2," and "LCDR3" include CDRs defined by any of the Kabat, Chothia, IMGT, or AbM methods described above, unless otherwise expressly stated in the specification.

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

[0123] The terms "enhance," "promote," or "increase" generally refer to the ability of a test molecule to mediate a greater response (i.e., a downstream effect) when compared to a control or vehicle-mediated response. Non-limiting exemplary responses include binding of a protein to its antigen or receptor, enhanced binding to FcγRs or enhanced Fc effector function such as enhanced ADCC, CDC, and / or ADCP. A decrease can be a statistically significant difference in the measured response between the test molecule and the control (or vehicle), or about a 1.1-, 1.2-, 1.5-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, or 30-fold or greater decrease, e.g., a 500-, 600-, 700-, 800-, 900-, or 1000-fold or greater decrease.

[0124] The term "expression vector" means a vector that can be utilized in a biological system or reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0125] The term "heterologous" refers to a polypeptide or polynucleotide that comprises two or more polypeptides or two or more polynucleotides that are not found in the same relationship to each other in nature.

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

[0127] The term "heterologous polypeptide" refers to a polypeptide that comprises two or more polypeptides that are not found in the same relationship to each other in nature.

[0128] The term "human antibody" refers to an antibody optimized to elicit a minimal immune response when administered to a human subject. The variable regions of a human antibody are derived from human immunoglobulin sequences. If a human antibody contains a constant region or a portion of a constant region, the constant region is also derived from a human immunoglobulin sequence. A human antibody contains heavy and light chain variable regions "derived" from sequences of human origin when the variable regions of the human antibody are obtained from a system using human germline immunoglobulins or rearranged immunoglobulin genes. Exemplary such systems are phage-displayed human immunoglobulin gene libraries and transgenic nonhuman animals, such as mice or rats, carrying human immunoglobulin loci. "Human antibodies" typically contain amino acid differences compared to immunoglobulins expressed in humans due to differences in the systems used to obtain human antibodies and human immunoglobulin loci, the introduction of somatic mutations or intentional substitutions into frameworks or CDRs, or both. Typically, a "human antibody" is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by human germline immunoglobulin or rearranged immunoglobulin genes. Optionally, a "human antibody" can comprise a consensus framework sequence obtained from human framework sequence analysis as described, for example, in Knappik et al., (2000) J Mol Biol 296:57-86, or a synthetic HCDR3 incorporated into a phage-displayed human immunoglobulin gene library as described, for example, in Shi et al., (2010) J Mol Biol 397:385-396 and WO 2009 / 085462. Antibodies in which at least one CDR is derived from a non-human species are not included in the definition of "human antibody."

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

[0130] The term "isolated" refers to a homogenous population of molecules (e.g., an scFv, or an spFv of the disclosure, or a heterologous protein comprising an scFv or an spFv of the disclosure) that have been substantially separated and / or purified from other components of the system in which the molecule is produced, such as a recombinant cell, and to proteins that have been subjected to at least one purification or isolation step. The term "isolated" refers to molecules that are substantially free of other cellular material and / or chemicals and includes molecules isolated to greater degrees of purity, e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% purity.

[0131] The term "modulate" refers to either an enhanced or decreased ability of a test molecule to mediate a greater or lesser response (i.e., a downstream effect) when compared to a control or vehicle-mediated response.

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

[0133] The term "multispecific" refers to a molecule that binds to two or more different antigens, or to two or more different epitopes within the same antigen. Multispecific molecules may be cross-reactive to other related antigens, e.g., the same antigen (homologues) from other species such as humans or monkeys, e.g., cynomolgus monkeys (Macaca fascicularis) (cynomolgus, cyno) or chimpanzees (Pan troglodytes), or may bind to an epitope shared between two or more different antigens.

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

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

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

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

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

[0139] The term "staple" refers to an scFv linker that contains one or two Cys residues that can form a disulfide bond with the anchor point Cys.

[0140] The term "stapled single-chain Fv" or "spFv" refers to an scFv that contains one or more disulfide bonds between the VH and the linker or between the VL and the linker. In some embodiments, the spFv contains one disulfide bond between the VH and the linker, one disulfide bond between the VL and the linker, or two disulfide bonds, one between the VH and the linker and one between the linker and the VL and the linker. In some embodiments, scFv molecules that contain a disulfide bond between the VH and the VL are excluded from the term "spFv."

[0141] The term "subject" includes any human or non-human animal. "Non-human animals" include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. The terms "subject" and "patient" may be used interchangeably herein. In some embodiments, the subject is a human subject.

[0142] The term "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount may vary depending on factors such as the individual's medical condition, age, sex, and weight, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual.

[0143] The terms "treat," "treating," or "treatment" of a disease or disorder hereinafter refer to achieving one or more of: reducing the severity and / or duration of the disorder, inhibiting the worsening of symptoms characteristic of the disorder being treated, limiting or preventing the recurrence of the disorder in a subject who previously had the disorder, or limiting or preventing the recurrence of symptoms in a subject who was previously symptomatic for the disorder.

[0144] The term "trispecific" refers to a molecule (such as an antibody) that specifically binds to three different antigens, or three different epitopes, within the same antigen. Bispecific molecules may also be cross-reactive to other related antigens, e.g., the same antigen (homologues), from other species such as humans or monkeys, e.g., cynomolgus monkeys (Macaca cynomolgus, cyno) or chimpanzees (Pan troglodytes), or may bind to epitopes shared among three or more different antigens.

[0145] The terms "variant," "mutant," or "altered" refer to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide by one or more modifications, e.g., one or more substitutions, insertions, and / or deletions.

[0146] Throughout this disclosure, the numbering of amino acid residues in antibody constant regions follows the EU index as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), unless otherwise expressly stated in this disclosure.

[0147] Ig constant region mutations are designated as follows: L351Y_F405A_Y407V refers to the L351Y, F405A, and Y407V mutations in one immunoglobulin constant region, and L351Y_F405A_Y407V / T394W refers to the L351Y, F405A, and Y407V mutations in the first Ig constant region and the T394W mutation in the second Ig constant region present in the molecule.

[0148] Numbering of variable regions is according to Chothia unless explicitly stated otherwise.

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

[0150] The term "VL cysteine" or "VL Cys" refers to a Cys residue present in the VL framework.

[0151] The term "stabilized" refers to the scFv retaining comparable binding to antigen when compared to an unheated scFv sample, which is referred to as thermostability.

[0152] The term "improved stability" refers to an spFv of the present disclosure having an increased melting temperature (Tm) when compared to a parent scFv lacking the disulfide bond and Cys residues introduced into the spFv. The increased Tm can be an increase of about 2°C or more, for example, about 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.

[0153] The term "anchor point" refers to an scFv VH or VL framework Cys residue that can be mutated to a Cys without adversely affecting the overall scFv structure and that can form a disulfide bond with a Cys present in the scFv linker.

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

[0155] As used herein, the term "LTBR" refers to a polypeptide that is a cell surface receptor for lymphotoxin, a member of the tumor necrosis factor receptor superfamily that is involved in apoptosis and cytokine release. LTBR may also be referred to as "tumor necrosis factor receptor superfamily member 3 (TNFRSF3)." LTBR is expressed on the surface of many cell types, including cells of epithelial and myeloid lineages. LTBR can bind to the membrane form of lymphotoxin (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. In some embodiments, the LTBR is human LTBR. An exemplary human LTBR comprises the amino acid sequence having UniProt number P36941.

[0156] 4.1. Composition of the substance Antigen-binding single-chain variable fragments (scFvs) are molecules that can be used as therapeutic agents, imaging agents, diagnostic agents, or as part of heterologous molecules such as multispecific molecules, taking into account the broad teachings in the art and herein. The challenge with scFvs is their low stability and tendency to aggregate (see, for example, Worn and Pluckthun (2001) J Mol Biol 305:989-1010; Rothlisberger et al., (2005) J Mol Biol 347:773-789; Gross et al., (1989) Transplant Proc 21(1 Pt 1):127-130, Porter et al., (2011) J Cancer 2:331-332; Porter et al., (2011) N Engl J Med 365:725-733).

[0157] In this context, the inventors recognized the need for improved materials and methods for designing scFvs that can optionally be incorporated into a variety of molecules, including, but not limited to, multispecific and heterologous molecules. The present disclosure provides stabilized scFv molecules (referred to herein as spFvs (stapled Fvs)), heterologous and multispecific molecules comprising spFvs, polynucleotides encoding them, vectors, host cells, and methods of making and using them. The present disclosure is based, at least in part, on the identification of suitable residue positions in the VH and / or VL (referred to herein as VH anchor points or VL anchor points) and in the flexible linker (referred to herein as staples), which can be engineered into cysteine ​​residues that result in disulfide bond formation between the linker and variable domains in scFvs. The "stapling" strategy described herein is broadly applicable to a variety of molecules, including, but not limited to, all VH / VL domains and existing scFv molecules, particularly those that provide structural identity with scFvs with improved stability. The spFvs described herein can be conjugated to any heterologous protein, bispecific, or multispecific format, including chimeric antigen receptors (CARs), T cell-redirecting molecules, bispecific, and multispecific molecules, and can be used as therapeutic, diagnostic, and detection molecules.

[0158] 4.1.1 spFvs of the present disclosure The present disclosure provides various spFvs. 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) a disulfide bond between a structurally conserved, surface-exposed VH cysteine ​​(Cys) and an L Cys; b) a disulfide bond between a structurally conserved, surface-exposed VL Cys and an L Cys; or c) 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.

[0159] The present disclosure also provides an isolated scFv comprising a VH, L, and VL, wherein a) the VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position and the L comprises an L Cys, b) the VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position and the L comprises an L Cys, or c) the VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position and the VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position and the L comprises a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys are capable of forming a disulfide bond and the VL Cys and the second L Cys are capable of forming a disulfide bond. In some embodiments, the disulfide bond is typically formed during expression of the scFv.

[0160] In some embodiments, the spFv consists of one disulfide bond formed between the L Cys and the VH Cys or between the L Cys and the VL Cys. In some embodiments, such spFvs are referred to as "semi-rigid spFvs." The anchor position is the same for spFvs with one or two disulfide bonds. The linker Cys position can vary in semi-rigid molecules as long as it satisfies the distance and geometric requirements for disulfide bond formation by the anchor point. In some embodiments, the semi-rigid spFv constrains the relative movement of the VL / VH, similar to a VL / VH pair stabilized by two disulfide bonds. Thus, the semi-rigid spFv is also stabilized.

[0161] The VH and VL in spFvs can be fixed in any orientation. For example, in some embodiments, the N-terminus of the VH is fixed to the C-terminus of the VL. In some embodiments, the C-terminus of the VH is fixed to the N-terminus of the VL. The anchor positions also depend on the orientation of the VL and VH, and not all VL and VH anchor points can be paired.

[0162] In some embodiments, spFvs of the present disclosure have increased stability compared to a parent scFv lacking disulfide bond(s). Stability includes thermal stability and mechanical stability. Thermal stability can be assessed using differential thermal calorimetry (DSC), in which DSC scans are performed using a heated protein sample (such as a sample heated to 100°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, respectively, are recorded. In some embodiments, spFvs exhibit increased thermal stability when compared to a parent scFv lacking disulfide bonds. In some embodiments, the Tm of the spFv is about 10°C higher when compared to a parent scFv lacking disulfide bonds, regardless of the Tm of the parent scFv.

[0163] In some embodiments, spFvs of the present disclosure exhibit significantly improved yield and quality of bispecific monomers compared to parent scFvs lacking disulfide bond(s). In some embodiments, spFvs of the present disclosure exhibit reduced aggregation upon heat stress at high concentrations compared to parent scFvs lacking disulfide bond(s). In some embodiments, spFv molecules of the present disclosure are multispecific molecules. In some embodiments, spFv molecules are bispecific molecules. In some embodiments, spFv molecules are trispecific molecules. In certain embodiments, spFv molecules have improved developability compared to parent scFvs lacking disulfide bond(s). In some embodiments, stapling can increase the success of scFv conversion, thereby allowing more scFv molecules to become available as molecular building blocks for therapeutic constructs.

[0164] In some embodiments, the distance between VH Cys and VL Cys is about 5 Å to about 10 Å. In some embodiments, the distance between VH Cys and VL Cys is about 7 Å to about 9 Å. In some embodiments, the distance between VH Cys and VL Cys is about 7 Å. In some embodiments, the distance between VH Cys and VL Cys is about 8 Å. In some embodiments, the distance between VH Cys and VL Cys is about 9 Å.

[0165] In some embodiments, the VH Cys is at H3, H5, H40, H43, H46, or H105, where residue numbering is according to Chothia.

[0166] In some embodiments, the VH Cys is in H3.

[0167] In some embodiments, the VH Cys is at H5.

[0168] In some embodiments, the VH Cys is at H40.

[0169] In some embodiments, the VH Cys is at H43.

[0170] In some embodiments, the VH Cys is at H46.

[0171] In some embodiments, the VH Cys is at H105.

[0172] In some embodiments, the VL Cys is at L3, L5, L39, L42, L43, L45, L100, or L102, where residue numbering is according to Chothia.

[0173] In some embodiments, the VL Cys is in L3.

[0174] In some embodiments, the VL Cys is at L5.

[0175] In some embodiments, the VL Cys is at L39.

[0176] In some embodiments, the VL Cys is at L42.

[0177] In some embodiments, the VL Cys is at L43.

[0178] In some embodiments, the VL Cys is at L45.

[0179] In some embodiments, the VL Cys is at L100.

[0180] In some embodiments, the VL Cys is at L102.

[0181] In some embodiments, the VH Cys is at H105 and the VL Cys is at L42.

[0182] In some embodiments, the VH Cys is at H105 and the VL Cys is at L43.

[0183] In some embodiments, the VH Cys is at H43 and the VL Cys is at L100.

[0184] In some embodiments, the VH Cys is in H3 and the VL Cys is in L3.

[0185] In some embodiments, the VH Cys is in H3 and the VL Cys is in L5.

[0186] In some embodiments, the VH Cys is at H3 and the VL Cys is at L39.

[0187] In some embodiments, the VH Cys is at H3 and the VL Cys is at L42.

[0188] In some embodiments, the VH Cys is at H3 and the VL Cys is at L45.

[0189] In some embodiments, the VH Cys is at H3 and the VL Cys is at L100.

[0190] In some embodiments, the VH Cys is at H3 and the VL Cys is at L102.

[0191] In some embodiments, the VH Cys is in H5 and the VL Cys is in L3.

[0192] In some embodiments, the VH Cys is in H5 and the VL Cys is in L5.

[0193] In some embodiments, the VH Cys is at H5 and the VL Cys is at L39.

[0194] In some embodiments, the VH Cys is at H5 and the VL Cys is at L42.

[0195] In some embodiments, the VH Cys is at H5 and the VL Cys is at L45.

[0196] In some embodiments, the VH Cys is at H5 and the VL Cys is at L100.

[0197] In some embodiments, the VH Cys is at H5 and the VL Cys is at L102.

[0198] In some embodiments, the VH Cys is in H40 and the VL Cys is in L3.

[0199] In some embodiments, the VH Cys is at H40 and the VL Cys is at L5.

[0200] In some embodiments, the VH Cys is at H40 and the VL Cys is at L39.

[0201] In some embodiments, the VH Cys is at H40 and the VL Cys is at L42.

[0202] In some embodiments, the VH Cys is at H40 and the VL Cys is at L45.

[0203] In some embodiments, the VH Cys is at H40 and the VL Cys is at L100.

[0204] In some embodiments, the VH Cys is at H40 and the VL Cys is at L102.

[0205] In some embodiments, the VH Cys is in H43 and the VL Cys is in L3.

[0206] In some embodiments, the VH Cys is at H43 and the VL Cys is at L5.

[0207] In some embodiments, the VH Cys is at H43 and the VL Cys is at L39.

[0208] In some embodiments, the VH Cys is at H43 and the VL Cys is at L42.

[0209] In some embodiments, the VH Cys is at H43 and the VL Cys is at L45.

[0210] In some embodiments, the VH Cys is at H43 and the VL Cys is at L102.

[0211] In some embodiments, the VH Cys is at H46 and the VL Cys is at L3.

[0212] In some embodiments, the VH Cys is at H46 and the VL Cys is at L5.

[0213] In some embodiments, the VH Cys is at H46 and the VL Cys is at L39.

[0214] In some embodiments, the VH Cys is at H46 and the VL Cys is at L42.

[0215] In some embodiments, the VH Cys is at H46 and the VL Cys is at L45.

[0216] In some embodiments, the VH Cys is at H46 and the VL Cys is at L100.

[0217] In some embodiments, the VH Cys is at H46 and the VL Cys is at L102.

[0218] In some embodiments, the VH Cys is at H105 and the VL Cys is at L3.

[0219] In some embodiments, the VH Cys is at H105 and the VL Cys is at L5.

[0220] In some embodiments, the VH Cys is at H105 and the VL Cys is at L39.

[0221] In some embodiments, the VH Cys is at H105 and the VL Cys is at L45.

[0222] In some embodiments, the VH Cys is at H105 and the VL Cys is at L100.

[0223] In some embodiments, the VH Cys is at H105 and the VL Cys is at L102.

[0224] Residue numbering for the VH and VL regions is according to Chothia. Chothia numbering is well known. Other numbering systems, such as Kabat or IMGT numbering, or consecutive numbering, can also be used to number the VH and VL residue positions. Table 1 shows the correspondence between Chothia, Kabat, and consecutive numbering for an exemplary VH, GLk1 VH (SEQ ID NO: 60). Table 2 shows the correspondence between Chothia, Kabat, and consecutive numbering for an exemplary VL, GLk1 VL (SEQ ID NO: 56).

[0225] [Table 1-1]

[0226] [Table 1-2]

[0227] [Table 2]

[0228] In some embodiments, L comprises a contiguous amino acid sequence derived from an immunoglobulin (Ig) hinge region. 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 from mouse, rat, dog, chicken, and non-human primates such as monkey. In some embodiments, the Ig hinge region is derived from a human Ig hinge region. In some embodiments, the human Ig hinge region is an IgG1, IgG2, IgG3, IgG4, IgM, IgA, or IgE isotype.

[0229] In some embodiments, the Ig hinge region includes residue 216 and ends at residue 230 of human IgG, where residue numbering is according to the EU index. In some cases, the lower hinge region from about residue 231 to about residue 237 can also be included in the IgG hinge region. In some embodiments, the IgG1 hinge region includes the amino acid sequence of SEQ ID NO: 63, as provided below. In some embodiments, the IgG1 hinge region includes the amino acid sequence of SEQ ID NO: 64, as provided below. Hinge regions of other Ig isotypes are well known, and their amino acid sequences can be obtained, for example, from the ImMunoGeneTics website. In some embodiments, the Ig hinge region is an IgG2 hinge region. In some embodiments, the IgG2 includes the amino acid sequence of SEQ ID NO: 65, as provided below. EPKSCDKTHTCPPCP (SEQ ID NO: 63) EPKSCDKTHTCPPCPAPELLGG (SEQ ID NO: 64) ERKCCVECPPCP (SEQ ID NO: 65)

[0230] In some embodiments, L comprises a contiguous amino acid sequence derived from an Ig hinge region. Thus, in some embodiments, L comprises at least a portion of an Ig hinge region or at least a portion of an engineered Ig hinge region. An engineered Ig hinge region comprises one or more mutations when compared to a wild-type Ig hinge region. Non-limiting examples of mutations that may be introduced include substitution of a Cys residue (e.g., reducing the number of Cys in L to one or two), substitution of a Pro residue, or any conservative modification (e.g., conservative substitution).

[0231] "Conservative modifications" refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid modification. Conservative modifications include amino acid substitutions, additions, and deletions. A conservative amino acid substitution is one in which an amino acid is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are well-defined and include amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amides (e.g., asparagine, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine), and sulfur-containing side chains (cysteine, methionine). Additionally, any naturally occurring residue in the polypeptide can also be substituted with alanine, as previously described for alanine scanning mutagenesis (MacLennan et al., (1988) Acta Physiol Scand Suppl 643:55-67; ​​Sasaki et al., (1988) Adv Biophys 35:1-24). Amino acid substitutions can be made by known methods, such as PCR mutagenesis (U.S. Pat. No. 4,683,195). The resulting mutant hinges can be incorporated into spFv constructs of the present disclosure and tested for their properties, such as stability and binding to antigen, using known assays and those described herein.

[0232] In some embodiments, L is the amino acid sequence C(X) yC (SEQ ID NO: 23), where X is glycine (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), threonine (Thr), tryptophan (Trp), or tyrosine (Tyr), and y is an integer from 1 to 3. Pro can be included in L to provide rigidity. Gly can be included in L to allow for maximum flexibility. Any other amino acid, except Cys and Met, can also be used in L.

[0233] In some embodiments, L is the amino acid sequence C(X) y C (SEQ ID NO: 24), wherein X is Gly, Ser or Pro, and y is an integer from 1 to 3.

[0234] In some embodiments, L comprises the amino acid sequence CPC, CGC, CSC, CPPC (SEQ ID NO: 1), CGPC (SEQ ID NO: 28), CPGC (SEQ ID NO: 29), CGGC (SEQ ID NO: 30), CSPG (SEQ ID NO: 31), CPSC (SEQ ID NO: 32), CSSC (SEQ ID NO: 33), CGSC (SEQ ID NO: 34), CSGC (SEQ ID NO: 35), CPPPC (SEQ ID NO: 36), CGPPC (SEQ ID NO: 37), CPGPC (SEQ ID NO: 38), CPPGC (SEQ ID NO: 39), CGGPC (SEQ ID NO: 40), CPGGC (SEQ ID NO: 41), CGGGC (SEQ ID NO: 42), CSPPC (SEQ ID NO: 43), CPSPC (SEQ ID NO: 44), CPPSC (SEQ ID NO: 45), CSSPC (SEQ ID NO: 46), CPSSC (SEQ ID NO: 47), CSSSC (SEQ ID NO: 48), CGSPC (SEQ ID NO: 49), CPGSC (SEQ ID NO: 50), CSGPC (SEQ ID NO: 51), or CPSGC (SEQ ID NO: 52).

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

[0236] In some embodiments, L comprises the amino acid sequence CGC.

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

[0238] In some embodiments, L comprises the amino acid sequence CPPC (SEQ ID NO: 1).

[0239] In some embodiments, L comprises the amino acid sequence CGPC (SEQ ID NO: 28).

[0240] In some embodiments, L comprises the amino acid sequence CPGC (SEQ ID NO: 29).

[0241] In some embodiments, L comprises the amino acid sequence CGGC (SEQ ID NO: 30).

[0242] In some embodiments, L comprises the amino acid sequence CSPG (SEQ ID NO: 31).

[0243] In some embodiments, L comprises the amino acid sequence CPSC (SEQ ID NO: 32).

[0244] In some embodiments, L comprises the amino acid sequence CSSC (SEQ ID NO: 33).

[0245] In some embodiments, L comprises the amino acid sequence CGSC (SEQ ID NO: 34).

[0246] In some embodiments, L comprises the amino acid sequence CSGC (SEQ ID NO: 35).

[0247] In some embodiments, L comprises the amino acid sequence CPPPC (SEQ ID NO: 36).

[0248] In some embodiments, L comprises the amino acid sequence CGPPC (SEQ ID NO: 37).

[0249] In some embodiments, L comprises the amino acid sequence CPGPC (SEQ ID NO: 38).

[0250] In some embodiments, L comprises the amino acid sequence CPPGC (SEQ ID NO: 39).

[0251] In some embodiments, L comprises the amino acid sequence CGGPC (SEQ ID NO: 40).

[0252] In some embodiments, L comprises the amino acid sequence CPGGC (SEQ ID NO: 41).

[0253] In some embodiments, L comprises the amino acid sequence CGGGC (SEQ ID NO: 42).

[0254] In some embodiments, L comprises the amino acid sequence CSPPC (SEQ ID NO: 43).

[0255] In some embodiments, L comprises the amino acid sequence CPSPC (SEQ ID NO: 44).

[0256] In some embodiments, L comprises the amino acid sequence CPPSC (SEQ ID NO: 45).

[0257] In some embodiments, L comprises the amino acid sequence CSSPC (SEQ ID NO: 46).

[0258] In some embodiments, L comprises the amino acid sequence CPSSC (SEQ ID NO: 47).

[0259] In some embodiments, L comprises the amino acid sequence CSSSC (SEQ ID NO: 48).

[0260] In some embodiments, L comprises the amino acid sequence CGSPC (SEQ ID NO: 49).

[0261] In some embodiments, L comprises the amino acid sequence CPGSC (SEQ ID NO: 50).

[0262] In some embodiments, L comprises the amino acid sequence CSGPC (SEQ ID NO: 51).

[0263] In some embodiments, L comprises the amino acid sequence CPSGC (SEQ ID NO: 52).

[0264] In some embodiments, L comprises about 15 to about 20 amino acids. In some embodiments, L comprises about 15 to about 20 amino acids in length.

[0265] In some embodiments, L comprises about 14 to about 19 amino acids. In some embodiments, L comprises about 14 to about 19 amino acids in length. In some embodiments, L comprises about 14 amino acids. In some embodiments, L comprises about 14 amino acids in length. In some embodiments, L comprises about 15 amino acids. In some embodiments, L comprises about 15 amino acids in length. In some embodiments, L comprises about 16 amino acids in length. In some embodiments, L comprises about 16 amino acids in length. In some embodiments, L comprises about 17 amino acids in length. In some embodiments, L comprises about 17 amino acids in length. In some embodiments, L comprises about 18 amino acids in length. In some embodiments, L comprises about 18 amino acids in length. In some embodiments, L comprises about 19 amino acids in length. In some embodiments, L comprises about 19 amino acids in length. In some embodiments, L comprises about 20 amino acids in length. In some embodiments, L comprises about 20 amino acids in length.

[0266] In some embodiments, L is the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 25), wherein X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gln, His, Ile, leu, Lys, Phe Thr, Trp, or Tyr, m is an integer of 6 to 9, y is an integer of 1 to 3, and n is an integer of 4 to 6.

[0267] In some embodiments, L is the amino acid sequence (X)(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.

[0268] In some embodiments, L is the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 27), wherein X is Gly or Pro, m is an integer of 6 to 9, y is an integer of 1 to 3, and n is an integer of 4 to 6.

[0269] In some embodiments, L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.

[0270] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 7.

[0271] In some embodiments, L comprises a length of about 5 to about 10 amino acids. In some embodiments, L comprises about 5 amino acids. In some embodiments, L consists of about 5 amino acids. In some embodiments, L comprises 7 amino acids. In some embodiments, L consists of 7 amino acids. In some embodiments, L consists of 8 amino acids. In some embodiments, L consists of 8 amino acids. In some embodiments, L consists of 9 amino acids. In some embodiments, L consists of 9 amino acids. In some embodiments, L comprises about 10 amino acids. In some embodiments, L consists of about 10 amino acids.

[0272] In some embodiments, L further comprises a following segment. In some embodiments, the following segment is 4 amino acids in length. In some embodiments, the following segment is 5 amino acids in length.

[0273] In some embodiments, L comprises a 9+4+5 configuration.

[0274] In some embodiments, the spFv is in a VL-L-VH orientation. In some embodiments, the spFv is in a VH-L-VL orientation.

[0275] The present disclosure provides an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H105, the VL comprises a Cys at L42, and the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and the scFv is in a VL-L-VH orientation.

[0276] The present disclosure provides an scFv comprising a VH, L, and VL, wherein VH comprises a Cys at H105, VL comprises a Cys at L45, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and wherein the scFv is in a VL-L-VH orientation.

[0277] The present disclosure provides an scFv comprising a VH, L, and VL, wherein VH comprises a Cys at H105, VL comprises a Cys at L39, and L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and wherein the scFv is in a VL-L-VH orientation.

[0278] The present disclosure provides an scFv comprising a VH, L, and VL, wherein VH comprises a Cys at H5, VL comprises a Cys at L42, and L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and wherein the scFv is in a VL-L-VH orientation.

[0279] The present disclosure provides an scFv comprising a VH, L, and VL, wherein VH comprises a Cys at H5, VL comprises a Cys at L45, and L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and wherein the scFv is in a VL-L-VH orientation.

[0280] The present disclosure provides an scFv comprising a VH, L, and VL, wherein VH comprises a Cys at H5, VL comprises a Cys at L39, and L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and wherein the scFv is in a VL-L-VH orientation.

[0281] The present disclosure provides an scFv comprising a VH, L, and VL, wherein VH comprises a Cys at H3, VL comprises a Cys at L42, and L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and wherein the scFv is in a VL-L-VH orientation.

[0282] The present disclosure provides an scFv comprising a VH, L, and VL, wherein VH comprises a Cys at H3, VL comprises a Cys at L45, and L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and wherein the scFv is in a VL-L-VH orientation.

[0283] The present disclosure provides an scFv comprising a VH, L, and VL, wherein VH comprises a Cys at H3, VL comprises a Cys at L39, and L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and wherein the scFv is in a VL-L-VH orientation.

[0284] The present disclosure provides an scFv comprising a VH, L, and VL, wherein VH comprises a Cys at H43, VL comprises a Cys at L100, and L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and wherein the scFv is in a VH-L-VL orientation.

[0285] The present disclosure provides an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H43, the VL comprises a Cys at L102, and the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and the scFv is in a VH-L-VL orientation.

[0286] The present disclosure provides an scFv comprising a VH, L, and VL, wherein VH comprises a Cys at H43, VL comprises a Cys at L5, and L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and wherein the scFv is in a VH-L-VL orientation.

[0287] The present disclosure also provides an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H43, the VL comprises a Cys at L3, and L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and the scFv is in a VH-L-VL orientation.

[0288] The present disclosure also provides an scFv comprising a VH, L, and VL, wherein VH comprises a Cys at H40, VL comprises a Cys at L100, and L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and wherein the scFv is in a VH-L-VL orientation.

[0289] The present disclosure also provides an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H40, the VL comprises a Cys at L102, and the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and the scFv is in a VH-L-VL orientation.

[0290] The present disclosure also provides an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H40, the VL comprises a Cys at L5, and L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and the scFv is in a VH-L-VL orientation.

[0291] The present disclosure provides an scFv comprising a VH, L, and VL, wherein VH comprises a Cys at H40, VL comprises a Cys at L3, and L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and wherein the scFv is in a VH-L-VL orientation.

[0292] The present disclosure provides an scFv comprising a VH, L, and VL, wherein VH comprises a Cys at H46, VL comprises a Cys at L100, and L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and wherein the scFv is in a VH-L-VL orientation.

[0293] The present disclosure provides an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H46, the VL comprises a Cys at L102, and the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and the scFv is in a VH-L-VL orientation.

[0294] The present disclosure provides an scFv comprising a VH, L, and VL, wherein VH comprises a Cys at H46, VL comprises a Cys at L5, and L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and wherein the scFv is in a VH-L-VL orientation.

[0295] The present disclosure provides an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H46, the VL comprises a Cys at L3, and L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and the scFv is in a VH-L-VL orientation.

[0296] The present disclosure provides an scFv comprising a VH, L, and VL, wherein VH comprises a Cys at H105, VL comprises a Cys at L43, and L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and wherein the scFv is in a VL-L-VH orientation.

[0297] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 7.

[0298] 4.1.2. Molecules Comprising spFvs of the Disclosure The present disclosure provides molecules comprising spFvs of the present disclosure (e.g., those disclosed in Section 4.1.1). In some embodiments, the molecules are multispecific molecules. In some embodiments, the molecules are heterologous molecules.

[0299] Similar to unstabilized scFvs lacking disulfide bond(s), spFvs of the present disclosure can be conjugated to a second molecule, non-limiting examples of which are disclosed herein and include half-life extending moieties, imaging agents, therapeutic agents, various antibody formats and fragments thereof, antigen-binding domains, Fc regions, immunoglobulin heavy / light chains or fragments thereof.

[0300] In some embodiments, the molecule comprises 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 disulfide bond between a structurally conserved surface-exposed VH cysteine ​​(Cys) and an L Cys, a disulfide bond between a structurally conserved surface-exposed VL Cys and an 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.

[0301] In some embodiments, the molecule comprises an scFv comprising a VH, L, and VL, wherein the VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position and the L comprises an L Cys; the VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position and the L comprises an L Cys; or the VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position and the VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position and the L comprises a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys are capable of forming a disulfide bond and the VL Cys and the second L Cys are capable of forming a disulfide bond.

[0302] In some embodiments, the distance between VH Cys and VL Cys is about 5 Å to about 10 Å. In some embodiments, the distance between VH Cys and VL Cys is about 7 Å to about 9 Å.

[0303] In some embodiments, the VH Cys is at H3, H5, H40, H43, H46, or H105, where residue numbering is according to Chothia.

[0304] In some embodiments, the VL Cys is at L3, L5, L39, L42, L43, L45, L100, or L102, where residue numbering is according to Chothia.

[0305] In some embodiments, the VH Cys is at H105 and the VL Cys is at L42.

[0306] In some embodiments, the VH Cys is at H105 and the VL Cys is at L43.

[0307] In some embodiments, the VH Cys is at H43 and the VL Cys is at L100.

[0308] In some embodiments, the VH Cys is in H3 and the VL Cys is in L3.

[0309] In some embodiments, the VH Cys is in H3 and the VL Cys is in L5.

[0310] In some embodiments, the VH Cys is at H3 and the VL Cys is at L39.

[0311] In some embodiments, the VH Cys is at H3 and the VL Cys is at L42.

[0312] In some embodiments, the VH Cys is at H3 and the VL Cys is at L45.

[0313] In some embodiments, the VH Cys is at H3 and the VL Cys is at L100.

[0314] In some embodiments, the VH Cys is at H3 and the VL Cys is at L102.

[0315] In some embodiments, the VH Cys is in H5 and the VL Cys is in L3.

[0316] In some embodiments, the VH Cys is in H5 and the VL Cys is in L5.

[0317] In some embodiments, the VH Cys is at H5 and the VL Cys is at L39.

[0318] In some embodiments, the VH Cys is at H5 and the VL Cys is at L42.

[0319] In some embodiments, the VH Cys is at H5 and the VL Cys is at L45.

[0320] In some embodiments, the VH Cys is at H5 and the VL Cys is at L100.

[0321] In some embodiments, the VH Cys is at H5 and the VL Cys is at L102.

[0322] In some embodiments, the VH Cys is in H40 and the VL Cys is in L3.

[0323] In some embodiments, the VH Cys is at H40 and the VL Cys is at L5.

[0324] In some embodiments, the VH Cys is at H40 and the VL Cys is at L39.

[0325] In some embodiments, the VH Cys is at H40 and the VL Cys is at L42.

[0326] In some embodiments, the VH Cys is at H40 and the VL Cys is at L45.

[0327] In some embodiments, the VH Cys is at H40 and the VL Cys is at L100.

[0328] In some embodiments, the VH Cys is at H40 and the VL Cys is at L102.

[0329] In some embodiments, the VH Cys is in H43 and the VL Cys is in L3.

[0330] In some embodiments, the VH Cys is at H43 and the VL Cys is at L5.

[0331] In some embodiments, the VH Cys is at H43 and the VL Cys is at L39.

[0332] In some embodiments, the VH Cys is at H43 and the VL Cys is at L42.

[0333] In some embodiments, the VH Cys is at H43 and the VL Cys is at L45.

[0334] In some embodiments, the VH Cys is at H43 and the VL Cys is at L100.

[0335] In some embodiments, the VH Cys is at H43 and the VL Cys is at L102.

[0336] In some embodiments, the VH Cys is at H46 and the VL Cys is at L3.

[0337] In some embodiments, the VH Cys is at H46 and the VL Cys is at L5.

[0338] In some embodiments, the VH Cys is at H46 and the VL Cys is at L39.

[0339] In some embodiments, the VH Cys is at H46 and the VL Cys is at L42.

[0340] In some embodiments, the VH Cys is at H46 and the VL Cys is at L45.

[0341] In some embodiments, the VH Cys is at H46 and the VL Cys is at L100.

[0342] In some embodiments, the VH Cys is at H46 and the VL Cys is at L102.

[0343] In some embodiments, the VH Cys is at H105 and the VL Cys is at L3.

[0344] In some embodiments, the VH Cys is at H105 and the VL Cys is at L5.

[0345] In some embodiments, the VH Cys is at H105 and the VL Cys is at L39.

[0346] In some embodiments, the VH Cys is at H105 and the VL Cys is at L42.

[0347] In some embodiments, the VH Cys is at H105 and the VL Cys is at L45.

[0348] In some embodiments, the VH Cys is at H105 and the VL Cys is at L100.

[0349] In some embodiments, the VH Cys is at H105 and the VL Cys is at L102.

[0350] Residue numbering for the VH and VL regions is according to Chothia.

[0351] In some embodiments, L comprises a contiguous amino acid sequence derived from an immunoglobulin (Ig) hinge region. In some embodiments, the Ig hinge region is derived from a human Ig hinge region or a non-human Ig hinge region. Non-limiting examples of non-human Ig hinge regions include those from mouse, rat, dog, chicken, and non-human primates such as monkey. In some embodiments, the Ig hinge region is derived from a human Ig hinge region. In some embodiments, the human Ig hinge region is an IgG1, IgG2, IgG3, IgG4, IgM, IgA, or IgE isotype.

[0352] In some embodiments, L is the amino acid sequence C(X) yC (SEQ ID NO: 23), where X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gln, His, Ile, Leu, Lys, Phe, Thr, Trp, or Tyr, and y is an integer from 1 to 3. Pro can be included in the linker to provide rigidity. Gly can be included in the linker to allow for maximum flexibility. Any other amino acid, except Cys and Met, can also be used in L.

[0353] In some embodiments, L is the amino acid sequence C(X) y C (SEQ ID NO: 24), wherein X is Gly, Ser, or Pro, and y is an integer from 1 to 3.

[0354] In some embodiments, L comprises the amino acid sequence CPC, CGC, CSC, CPPC (SEQ ID NO: 1), CGPC (SEQ ID NO: 28), CPGC (SEQ ID NO: 29), CGGC (SEQ ID NO: 30), CSPG (SEQ ID NO: 31), CPSC (SEQ ID NO: 32), CSSC (SEQ ID NO: 33), CGSC (SEQ ID NO: 34), CSGC (SEQ ID NO: 35), CPPPC (SEQ ID NO: 36), CGPPC (SEQ ID NO: 37), CPGPC (SEQ ID NO: 38), CPPGC (SEQ ID NO: 39), CGGPC (SEQ ID NO: 40), CPGGC (SEQ ID NO: 41), CGGGC (SEQ ID NO: 42), CSPPC (SEQ ID NO: 43), CPSPC (SEQ ID NO: 44), CPPSC (SEQ ID NO: 45), CSSPC (SEQ ID NO: 46), CPSSC (SEQ ID NO: 47), CSSSC (SEQ ID NO: 48), CGSPC (SEQ ID NO: 49), CPGSC (SEQ ID NO: 50), CSGPC (SEQ ID NO: 51), or CPSGC (SEQ ID NO: 52).

[0355] In some embodiments, L comprises about 15 to about 20 amino acids. In some embodiments, L comprises about 15 to about 20 amino acids in length. In some embodiments, L comprises about 14 to about 19 amino acids in length. In some embodiments, L comprises about 14 to about 19 amino acids in length. In some embodiments, L comprises about 14 amino acids in length. In some embodiments, L comprises about 14 amino acids in length. In some embodiments, L comprises about 15 amino acids in length. In some embodiments, L comprises about 15 amino acids in length. In some embodiments, L comprises about 16 amino acids in length. In some embodiments, L comprises about 16 amino acids in length. In some embodiments, L comprises about 17 amino acids in length. In some embodiments, L comprises about 17 amino acids in length. In some embodiments, L comprises about 18 amino acids in length. In some embodiments, L comprises about 18 amino acids in length. In some embodiments, L comprises about 19 amino acids in length. In some embodiments, L comprises about 19 amino acids in length. In some embodiments, L comprises about 20 amino acids. In some embodiments, L comprises a length of about 20 amino acids.

[0356] In some embodiments, L is the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 25), wherein X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gln, His, Ile, leu, Lys, Phe, Thr, Trp, or Tyr; m is an integer of 6 to 9; y is an integer of 1 to 3; and n is an integer of 4 to 6.

[0357] 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.

[0358] In some embodiments, L is the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 27), wherein X is Gly or Pro, m is an integer of 6 to 9, y is an integer of 1 to 3, and n is an integer of 4 to 6.

[0359] In some embodiments, L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.

[0360] In some embodiments, the spFv is in a VL-L-VH orientation. In some embodiments, the spFv is in a VH-L-VL orientation.

[0361] In one embodiment, a molecule is provided comprising an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H105, the VL comprises a Cys at L42, and L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and the scFv is in a VL-L-VH orientation.

[0362] In one embodiment, a molecule is provided comprising an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H105, the VL comprises a Cys at L45, and L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and the scFv is in a VL-L-VH orientation.

[0363] In one embodiment, a molecule is provided comprising an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H105, the VL comprises a Cys at L39, and the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and the scFv is in a VL-L-VH orientation.

[0364] In one embodiment, a molecule is provided comprising an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H5, the VL comprises a Cys at L42, and L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and the scFv is in a VL-L-VH orientation.

[0365] In one embodiment, a molecule is provided comprising an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H5, the VL comprises a Cys at L45, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VL-L-VH orientation.

[0366] In one embodiment, a molecule is provided comprising an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H5, the VL comprises a Cys at L39, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VL-L-VH orientation.

[0367] In one embodiment, a molecule is provided comprising an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H3, the VL comprises a Cys at L42, and L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and the scFv is in a VL-L-VH orientation.

[0368] In one embodiment, a molecule is provided comprising an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H3, the VL comprises a Cys at L45, and L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and the scFv is in a VL-L-VH orientation.

[0369] In one embodiment, a molecule is provided comprising an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H3, the VL comprises a Cys at L39, and L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and the scFv is in a VL-L-VH orientation.

[0370] In one embodiment, a molecule is provided comprising an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H43, the VL comprises a Cys at L100, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0371] In one embodiment, a molecule is provided comprising an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H43, the VL comprises a Cys at L102, and the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and the scFv is in a VH-L-VL orientation.

[0372] In one embodiment, a molecule is provided comprising an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H43, the VL comprises a Cys at L5, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0373] In one embodiment, a molecule is provided comprising an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H43, the VL comprises a Cys at L3, and L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and the scFv is in a VH-L-VL orientation.

[0374] In one embodiment, a molecule is provided comprising an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H40, the VL comprises a Cys at L100, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0375] In one embodiment, a molecule is provided comprising an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H40, the VL comprises a Cys at L102, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0376] In one embodiment, a molecule is provided comprising an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H40, the VL comprises a Cys at L5, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0377] In one embodiment, a molecule is provided comprising an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H40, the VL comprises a Cys at L3, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0378] In one embodiment, a molecule is provided comprising an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H46, the VL comprises a Cys at L100, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0379] In one embodiment, a molecule is provided comprising an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H46, the VL comprises a Cys at L102, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0380] In one embodiment, a molecule is provided comprising an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H46, the VL comprises a Cys at L5, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0381] In one embodiment, a molecule is provided comprising an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H46, the VL comprises a Cys at L3, and L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the scFv is in a VH-L-VL orientation.

[0382] In one embodiment, a molecule is provided comprising an scFv comprising a VH, L, and VL, wherein the VH comprises a Cys at H105, the VL comprises a Cys at L43, and L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and the scFv is in a VL-L-VH orientation.

[0383] In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 7.

[0384] In some embodiments, an scFv of the disclosure is conjugated to a second protein, polynucleotide, therapeutic agent, cytotoxic agent, or detectable label.

[0385] In some embodiments, the second protein is a half-life extending moiety.

[0386] In some embodiments, the second protein is an antibody or a fragment thereof.

[0387] In some embodiments, the second protein is an antigen-binding fragment.

[0388] In some embodiments, the second protein is a therapeutic molecule.

[0389] 4.1.2.1. Molecules Comprising spFvs of the Disclosure and Half-Life Extending Moieties The present disclosure, in some embodiments, provides molecules comprising an spFv of the present disclosure (e.g., those disclosed in Section 4.1.1) and a half-life extending moiety. In some embodiments, the spFv of the present disclosure is conjugated to a half-life extending moiety.

[0390] Non-limiting examples of half-life extending moieties include immunoglobulins (Ig), fragments of Ig, Ig constant regions, fragments of Ig constant regions, Fc regions, transferrin, albumin, albumin variants, albumin binding domains, or polyethylene glycol (PEG). The amino acid sequences of human Igs are well known. Human Igs include IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE.

[0391] In some embodiments, an spFv of the present disclosure is conjugated to an Ig or a fragment thereof. In some embodiments, an spFv of the present disclosure is conjugated to an Fc region. In some embodiments, an spFv of the present disclosure is conjugated to transferrin. In some embodiments, an spFv of the present disclosure is conjugated to albumin. In some embodiments, an spFv of the present disclosure is conjugated to an albumin-binding protein. In some embodiments, an spFv of the present disclosure is conjugated to polyethylene glycol (PEG). Non-limiting examples of PEG include PEG5000 and PEG20000. In some embodiments, an spFv of the present disclosure is conjugated to a fatty acid or fatty acid ester, e.g., for desired properties. Non-limiting examples of illustrative fatty acids and fatty acid esters include laurate, myristate, stearate, arachidate, behenate, oleate, arachidonate, octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, polylysine, octane, carbohydrates (dextran, cellulose, oligo- or polysaccharides).

[0392] The half-life extending moiety can be a direct fusion to the spFv of the present disclosure and can be produced by standard cloning and expression techniques. Alternatively, well-known chemical coupling methods can be used to attach the moiety to a recombinantly produced spFv of the present disclosure.

[0393] 4.1.2.2. Molecules Comprising spFvs of the Disclosure and a Therapeutic Agent, Cytotoxic Agent, or Detectable Label The present disclosure provides molecules comprising spFvs of the present disclosure (e.g., those disclosed in Section 4.1.1), which are conjugated to a therapeutic agent, a cytotoxic agent, or a detectable label.

[0394] Such molecules can be used to direct therapeutic agents, mediate killing, visualize, identify, and / or purify cells expressing the antigen to which the spFv binds in vitro or in vivo.

[0395] Detectable labels include compositions that, when conjugated to an spFv of the disclosure, can be made detectable by, for example, spectroscopic, photochemical, biochemical, immunochemical, or chemical means.

[0396] Non-limiting examples of detectable labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., commonly used in ELISAs), biotin, digoxigenin, haptens, luminescent molecules, chemiluminescent molecules, fluorescent dyes, fluorophores, fluorescence quenchers, colored molecules, radioisotopes, scintillates, avidin, streptavidin, protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni 2+ , Flag tags, myc tags, heavy metals, enzymes, alkaline phosphatase, peroxidase, luciferase, electron donors / acceptors, acridinium esters, and colorimetric substrates.

[0397] A detectable label may emit a signal spontaneously, such as when the detectable label is a radioisotope. In other cases, the detectable label emits a signal as a result of being stimulated by an external field.

[0398] Non-limiting examples of radioisotopes include gamma-emitting, Auger-emitting, beta-emitting, alpha-emitting, and positron-emitting radioisotopes. 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 1 211 At 212 Bi 213 Bi 223 Ra 226 Ra 225 Ac, and 227 Ac are included.

[0399] In some embodiments, the metal atom is calcium, scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, bromine, krypton, rubidium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, indium, tin, antimony, tellurium, iodine, xenon, cesium, barium, lanthanum, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, mercury, thallium and metals having an atomic number greater than 20, including, but not limited to, uranium, lead, bismuth, francium, radium, actinium, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, thorium, protactinium, uranium, neptunium, plutonium, americium, curium, berkelium, californium, einsteinium, fermium, mendelevium, nobelium, or lawrencium atoms.

[0400] In some embodiments, the metal atom can be an alkaline earth metal having an atomic number greater than 20.

[0401] In some embodiments, the metal atom is a lanthanide. In some embodiments, the metal atom is an actinide. In some embodiments, the metal atom is a transition metal. In some embodiments, the metal atom is a base metal. In some embodiments, the metal atom is a gold atom, a bismuth atom, a tantalum atom, and a gadolinium atom.

[0402] In some embodiments, the metal atom is a metal having an atomic number between 53 (ie, iodine) and 83 (ie, bismuth).

[0403] In some embodiments, the metal atom is an atom suitable for magnetic resonance imaging.

[0404] In some embodiments, the metal atom is Ba 2+ , Bi 3+ , Cs + , Ca 2+ , Cr 2+ , Cr 3+ , Cr 6+ , Co 2+ , Co 3+ , Cu + , Cu 2+ , Cu 3+ , Ga 3+ , Gd 3+ , Au + , Au 3+ , Fe 2+ , Fe 3+ , F 3+ , Pb 2+ , Mn 2+ , Mn 3+ , Mn 4+ , Mn 7+ , Hg 2+ , Ni 2+ , Ni 3+ , Ag + , Sr 2+ , Sn 2+ , Sn 4+ , and Zn 2+ The metal ion may be in the form of a +1, +2, or +3 oxidation state, such as: The metal atom may comprise a metal oxide, including, but not limited to, iron oxide, manganese oxide, or gadolinium oxide.

[0405] Suitable dyes include any commercially available dye, including, but not limited to, for example, 5(6)-carboxyfluorescein, IRDye 680RD maleimide, IRDye 800CW, ruthenium polypyridyl dyes, and the like.

[0406] Suitable fluorophores include fluorescein isothiocyanate (FITC), fluorescein thiosemicarbazide, rhodamine, Texas Red, CyDye (e.g., Cy3, Cy5, Cy5.5), Alexa Fluors (e.g., Alexa488, Alexa555, Alexa594, Alexa647), near infrared (NIR) (700-900 nm) fluorescent dyes, and carbocyanine and aminostyryl dyes.

[0407] Heterologous molecules comprising an scFv of the present disclosure conjugated to a detectable label can be used as imaging agents.

[0408] In some embodiments, the detectable label is also a cytotoxic agent, hi some embodiments, the cytotoxic agent is a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioactive isotope (i.e., a radioconjugate).

[0409] In some embodiments, the cytotoxic agent is daunomycin, doxorubicin, methotrexate, vindesine, a bacterial toxin such as diphtheria toxin, ricin, geldanamycin, a maytansinoid, or calicheamicin. A cytotoxic agent may elicit its cytotoxic or cytostatic effect by mechanisms including, but not limited to, tubulin binding, DNA binding, or topoisomerase inhibition.

[0410] In some embodiments, the cytotoxic agent is an enzymatically active toxin such as diphtheria A chain, an unbound active fragment of diphtheria toxin, exotoxin A chain (Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes.

[0411] In some embodiments, the cytotoxic agent is 212 Bi, 131 I, 131 In, 90 Y, and 186 Radioactive nuclides such as Re.

[0412] In some embodiments, the cytotoxic agent is a dolastatin or peptide analogs and derivatives of dolostatin, auristatin, or monomethylauristatin phenylalanine. Exemplary molecules are disclosed in U.S. Patent Nos. 5,635,483 and 5,780,588. Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division, and to have anticancer and antifungal activity. The dolastatin or auristatin drug moiety can be attached to the spFv of the present disclosure via the N-terminus or C-terminus of the peptide drug moiety (see WO 02 / 088172), or via any cysteine ​​engineered into the antibody.

[0413] Conjugation to a detectable label can be carried out using known methods.

[0414] In some embodiments, the detectable label is complexed to a chelator.

[0415] In some embodiments, the detectable label is conjugated to the spFv of the disclosure via a linker.

[0416] The detectable label or cytotoxic agent can be linked directly or indirectly to the spFv of the disclosure using known methods. Suitable linkers are known in the art and include, but are not limited to, prosthetic groups, non-phenolic linkers (derivatives of N-succimidyl-benzoate, dodecaborate), chelating moieties of both macrocyclic and acyclic chelators, such as derivatives of 1,4,7,10-tetraazacyclododecane-1,4,7,10,tetraacetic acid (DOTA), derivatives of diethylenetriaminepentaacetic acid (DTPA), S-2-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (S-2-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid), and the like. derivatives of 1,4,8,11-tetraazacyclodocedan-1,4,8,11-tetraacetic acid (NOTA) and Examples of suitable peptide linkers include derivatives of N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), activated esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene), as well as other chelating moieties. Suitable peptide linkers are well known.

[0417] 4.1.2.3. Heterologous Molecules Comprising an spFv of the Disclosure and an Immunoglobulin (Ig) Constant Region or Fragment Thereof The spFvs of the present disclosure can be conjugated to an Ig constant region or a fragment thereof. The present disclosure provides molecules comprising an spFv of the present disclosure (e.g., those disclosed in Section 4.1.1) and an Ig constant region or a fragment thereof. In some embodiments, the Ig constant region or a fragment thereof can confer antibody-like properties, including Fc effector functions C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, or downregulation of cell surface receptors (e.g., B cell receptor (BCR)). The Ig constant region or a fragment thereof can also function as a half-life extending moiety as described herein. The spFvs of the present disclosure can also be engineered into full-length antibodies using standard methods. Full-length antibodies, including spFvs, can be further engineered as described herein.

[0418] The immunoglobulin heavy chain constant region consists of subdomains CH1, CH2, and CH3. The CH1 domain spans residues 118-215 on the heavy chain, the CH2 domain residues 231-340, and the CH3 domain residues 341-447, with residue numbering according to the EU index. In some cases, residue 341 is referred to as the CH2 domain residue. In some embodiments, the hinge includes residue 216 of human IgG1 and terminates at residue 230 of human IgG1. In some embodiments, the hinge includes the lower hinge region from about residue 231 to about residue 237 as described herein. The Ig Fc region includes at least the CH2 and CH3 domains of the Ig constant region and thus includes at least the region from about 231 to 447 of the Ig heavy chain constant region.

[0419] In some embodiments, the Ig constant region is a heavy chain constant region.

[0420] In some embodiments, the Ig constant region is a light chain constant region.

[0421] In some embodiments, the fragment of an Ig constant region comprises an Fc region. In some embodiments, the fragment of an Ig constant region comprises a CH2 domain. In some embodiments, the fragment of an Ig constant region comprises a CH3 domain. In other embodiments, the fragment of an Ig constant region comprises the CH2 domain and the CH3 domain. In other embodiments, the fragment of an Ig constant region comprises at least a portion of a hinge, the CH2 domain, and the CH3 domain. A portion of a hinge refers to one or more amino acid residues of an Ig hinge. In other embodiments, the fragment of an Ig constant region comprises the hinge, the CH2 domain, and the CH3 domain.

[0422] In some embodiments, the spFv is conjugated to the N-terminus of an Ig constant region or a fragment thereof. In some embodiments, the spFv of the disclosure is conjugated to the C-terminus of an Ig constant region or a fragment thereof.

[0423] Molecules comprising the spFv and Ig constant regions or fragments thereof of the present disclosure can be evaluated for their function using several known assays. Binding to a target antigen can be evaluated using the methods described herein. Altered properties conferred by an Ig constant domain or fragment thereof (e.g., an Fc region) can be assayed in Fc receptor binding assays using soluble forms of receptors such as FcγRI, FcγRII, FcγRIII, or FcRn, or using cell-based assays measuring, for example, ADCC, CDC, or ADCP.

[0424] ADCC activity can be assessed using an in vitro assay using cells expressing the antigen bound by the spFv of the present disclosure as target cells and NK cells as effector cells. Cytolysis can be detected by the release of a label (e.g., a radioactive substrate, a fluorescent dye, or a natural intracellular protein) from lysed cells. In an exemplary assay, target cells are used at a ratio of one target cell to four effector cells. Target cells are pre-labeled with BATDA and combined with effector cells and a test antibody. Samples are incubated for 2 hours, and cytolysis is measured by measuring BATDA released into the supernatant. Data are normalized to maximum cytotoxicity with 0.67% Triton X-100 (Sigma Aldrich), and a minimum control is determined by the spontaneous release of BATDA from target cells in the absence of any antibody.

[0425] ADCP can be assessed by using monocyte-derived macrophages as effector cells and any cells expressing the antigen to which the spFv of the present disclosure binds as target cells, and monocyte-derived macrophages as target cells engineered to express GFP or another marker molecule. In an exemplary assay, the effector:target cell ratio can be, for example, 4:1. Effector cells can be incubated with target cells for 4 hours, with or without the addition of an antibody of the present invention. After incubation, cells can be detached using activase. Macrophages can be identified using anti-CD11b and anti-CD14 antibodies conjugated to fluorescent labels, and the rate of phagocytosis can be measured using standard methods to measure CD11b and CD14 phagocytosis. + CD14 + It can be calculated based on the % GFP fluorescence in macrophages.

[0426] For example, Daudi cells were cultured at 1 × 10 in RPMI-B (RPMI supplemented with 1% BSA) for CDC. 5Lysate can be measured by plating 100 cells / well (50 μL / well), adding 50 μL of test protein to the well at a final concentration of 0-100 μg / mL, incubating the reaction at room temperature for 15 minutes, adding 11 μL of pooled human serum to the well, and incubating the reaction at 37°C for 45 minutes. The percentage of lysed cells can be detected as the % of propidium iodide-stained cells in a FACS assay using standard methods.

[0427] In some embodiments, the molecule comprises an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), and a fragment crystallizable region (Fc region), wherein the scFv comprises a heavy chain variable region (VH), a linker (L), and a light chain variable region (VL), wherein the scFv comprises a structurally conserved disulfide bond between a surface-exposed VH cysteine ​​(Cys) and an L Cys, a disulfide bond between a surface-exposed VL Cys and an L Cys, or a first disulfide bond between a surface-exposed VH Cys and a first L Cys, and a second disulfide bond between a surface-exposed VL Cys and a second L Cys, and the molecule has improved stability, expression yield, and / or quality compared to a molecule lacking disulfide bonds, e.g., a molecule lacking the first and second disulfide bonds.

[0428] In some embodiments, a) the VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position, and the L comprises an L Cys; b) the VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position, and the L comprises an L Cys; or c) the VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position, the VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position, and the L comprises a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys are capable of forming a disulfide bond, and the VL Cys and the second L Cys are capable of forming a disulfide bond.

[0429] In some embodiments, the distance between VH Cys and VL Cys is about 5 Å to about 10 Å. In some embodiments, the distance between VH Cys and VL Cys is about 7 Å to about 9 Å.

[0430] In some embodiments, the VH Cys is at H3, H5, H40, H43, H46, or H105, where residue numbering is according to Chothia. In some embodiments, the VL Cys is at L3, L5, L39, L42, L43, L45, L100, or L102, where residue numbering is according to Chothia.

[0431] In some embodiments, the VH Cys is at H105 and the VL Cys is at L42; or VH Cys is at H43 and VL Cys is at L100, or VH Cys is in H3 and VL Cys is in L3, or VH Cys is in H3 and VL Cys is in L5, or VH Cys is in H3 and VL Cys is in L39, or VH Cys is in H3 and VL Cys is in L42, or VH Cys is in H3 and VL Cys is in L45, or VH Cys is in H3 and VL Cys is in L100, or VH Cys is at H3 and VL Cys is at L102, or VH Cys is in H5 and VL Cys is in L3, or VH Cys is in H5 and VL Cys is in L5, or VH Cys is at H5 and VL Cys is at L39, or VH Cys is at H5 and VL Cys is at L42, or VH Cys is at H5 and VL Cys is at L45, or VH Cys is in H5 and VL Cys is in L100, or VH Cys is at H5 and VL Cys is at L102, or VH Cys is in H40 and VL Cys is in L3, or VH Cys is in H40 and VL Cys is in L5, or VH Cys is at H40 and VL Cys is at L39, or VH Cys is at H40 and VL Cys is at L42, or VH Cys is at H40 and VL Cys is at L45, or VH Cys is at H40 and VL Cys is at L100, or VH Cys is at H40 and VL Cys is at L102, or VH Cys is in H43 and VL Cys is in L3, or VH Cys is at H43 and VL Cys is at L5, or VH Cys is at H43 and VL Cys is at L39, or VH Cys is at H43 and VL Cys is at L42, or VH Cys is at H43 and VL Cys is at L45, or VH Cys is at H43 and VL Cys is at L102, or VH Cys is at H46 and VL Cys is at L3, or VH Cys is at H46 and VL Cys is at L5, or VH Cys is at H46 and VL Cys is at L39, or VH Cys is at H46 and VL Cys is at L42, or VH Cys is at H46 and VL Cys is at L45, or VH Cys is at H46 and VL Cys is at L100, or VH Cys is at H46 and VL Cys is at L102, or VH Cys is at H105 and VL Cys is at L3, or VH Cys is at H105 and VL Cys is at L5, or VH Cys is at H105 and VL Cys is at L39, or VH Cys is at H105 and VL Cys is at L45, or VH Cys is at H105 and VL Cys is at L100, or VH Cys is at H105 and VL Cys is at L102, or The VH Cys is at H105 and the VL Cys is at L43, residue numbering is according to Chothia.

[0432] In some embodiments, the molecule comprises an scFv (or spFv) that binds CD3 and a Fab that binds BCMA. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 125. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 126. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 127. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 128. In some embodiments, the scFv comprises a VH, L, and VL. L links the VH and VL. In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the linker comprises SEQ ID NO: 3. In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the VH comprises a Cys at H105 and the VL comprises a Cys at L43. In some embodiments, the VH comprises a Cys at H105 and the VL comprises a Cys at L43. In some embodiments, the scFv is in a VL-L-VH orientation.

[0433] In some embodiments, the scFv (or spFv) that binds CD3 is conjugated to an Ig constant region. In some embodiments, the Ig constant region comprises the amino acid sequence of SEQ ID NO: 133. In some embodiments, the Ig constant region comprises the amino acid sequence of SEQ ID NO: 139. In some embodiments, the molecule comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 140. In some embodiments, the molecule comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 141. In some embodiments, the molecule comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 142. In some embodiments, the molecule comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 143.

[0434] In some embodiments, a Fab that binds BCMA comprises a VH and a VL. In some embodiments, the VH of the Fab comprises the amino acid sequence of SEQ ID NO: 132. In some embodiments, the VH of the Fab comprises the amino acid sequence of SEQ ID NO: 137. In some embodiments, the VL of the Fab comprises the amino acid sequence of SEQ ID NO: 129. In some embodiments, the VL of the Fab comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the BCMA VH / VL comprises SEQ ID NO: 132 and SEQ ID NO: 129. In some embodiments, the VH of the Fab comprises the amino acid sequence of SEQ ID NO: 132 and the VL of the Fab comprises the amino acid sequence of SEQ ID NO: 135. In other embodiments, the VH of the Fab comprises SEQ ID NO: 137 and SEQ ID NO: 129. In some embodiments, the BCMA VH / VL comprises the amino acid sequence of SEQ ID NO: 137 and the VL of the Fab comprises the amino acid sequence of SEQ ID NO: 135. In further embodiments, the BCMA VH is conjugated to an Ig constant region. In some embodiments, the Ig constant region comprises the amino acid sequence of SEQ ID NO: 133. In some embodiments, the molecule comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 134. In some embodiments, the molecule comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 138.

[0435] In some embodiments, the molecule is a bispecific molecule. In some embodiments, the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 131 or SEQ ID NO: 136, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134 or SEQ ID NO: 138, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, or SEQ ID NO: 143.

[0436] In some embodiments, a bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 131, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 140. In some embodiments, a bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 131, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 141. In some embodiments, a bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 131, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 142. In some embodiments, a bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 131, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 143.

[0437] In some embodiments, a bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 131, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 138, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 140. In some embodiments, a bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 131, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 138, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 141. In some embodiments, a bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 131, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 138, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 142. In some embodiments, a bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 131, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 138, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 143.

[0438] In some embodiments, the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 136, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 140. In some embodiments, the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 136, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 141.

[0439] In some embodiments, the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 136, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 142. In some embodiments, the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 136, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 143.

[0440] In some embodiments, the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 136, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 138, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 140. In some embodiments, the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 136, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 138, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 141.

[0441] In some embodiments, the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 136, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 138, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 142. In some embodiments, the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 136, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 138, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 143.

[0442] 4.1.2.4. CARs Comprising spFvs of the Disclosure The present disclosure provides chimeric antigen receptors (CARs) comprising spFvs of the present disclosure (e.g., those disclosed in Section 4.1.1). CARs comprising spFvs of the present disclosure can be monospecific or multispecific, comprising one or more scFvs of the present disclosure as their extracellular domains.

[0443] Chimeric antigen receptors (CARs) are genetically engineered receptors. These engineered receptors can be easily inserted into and expressed by immune cells, including T cells, according to techniques known in the art. CARs allow a single receptor to recognize a specific antigen, and upon binding to that antigen, activate immune cells to attack and destroy cells bearing that antigen. If these antigens are present on target cells, immune cells expressing the CAR can target and kill the target cells.

[0444] In some embodiments, a CAR comprises an extracellular domain that binds to an antigen, an optional linker, a transmembrane domain, and an intracellular domain that includes a signaling domain.

[0445] The extracellular domain of a CAR can contain any polypeptide that binds to a desired antigen. In some embodiments, the extracellular domain of a CAR comprises an scFv (or spFv) disclosed herein. CARs can also be engineered to bind to two or more desired antigens, which can be arranged in tandem and separated by a linker sequence. For example, one or more scFvs (or spFvs), domain antibodies, llama VHH antibodies, or other VH-only antibody fragments of the present disclosure can be configured in tandem via a linker to generate a bispecific or multispecific CAR.

[0446] The transmembrane domain of CAR is the transmembrane domain of CD8, the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp 80(KLRFI), CD19, IL2Rβ, IL2Rγ, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, IT GAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT It can be derived from AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0447] In some embodiments, the intracellular domain of the CAR further comprises a costimulatory domain. The costimulatory domain can be derived from the intracellular domain of one or more costimulatory molecules. Costimulatory molecules are well-known cell surface molecules other than antigen receptors or Fc receptors that provide a second signal necessary for efficient activation and function of T lymphocytes upon binding to an antigen. Non-limiting examples of costimulatory domain molecules include 4-1BB, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70.

[0448] The intracellular signaling domain of a CAR can be derived from, for example, the signaling domain of CD3ζ, CD3ε, CD22, CD79a, CD66d, or CD39. The intracellular domain of a CAR refers to the portion of the CAR polypeptide that is involved in transducing the message of effective CAR binding to a target antigen inside an immune effector cell to elicit effector cell function, such as activation, cytokine production, proliferation, and cytotoxic activity (including release of cytotoxic factors into the CAR-bound target cell or other cellular response elicited after antigen binding to the extracellular CAR domain).

[0449] In some embodiments, the linker is located between the extracellular domain and the transmembrane domain. In some embodiments, the linker is a polypeptide between about 2 and 100 amino acids in length. The linker may contain or be composed of flexible residues such as glycine and serine, allowing adjacent protein domains to move freely relative to one another. Longer linkers can be used if it is desirable to ensure that two adjacent domains do not sterically interfere with one another. The linker may be cleavable or non-cleavable. An exemplary cleavable linker includes 2A.

[0450] An exemplary CAR comprises an extracellular domain comprising an scFv (or spFv) of the present disclosure, a transmembrane domain comprising the transmembrane domain of CD8, and an intracellular domain comprising the signaling domain of CD3ζ. An exemplary CAR comprises an extracellular domain comprising an scFv (or spFv) of the present disclosure, a transmembrane domain comprising the transmembrane domain of CD8 or the transmembrane domain of CD28, an intracellular domain comprising the signaling domain of CD3ζ, and a costimulatory domain comprising the intracellular domain of CD28, the intracellular domain of 4-1BB, or the intracellular domain of OX40.

[0451] CARs are produced by standard molecular biology techniques.

[0452] In some embodiments, the molecule is monospecific.

[0453] In some embodiments, the molecule is multispecific.

[0454] In some embodiments, the molecule is bispecific.

[0455] In some embodiments, the molecule is trispecific.

[0456] In some embodiments, the molecule is tetraspecific.

[0457] In some embodiments, provided herein are scFv (e.g., spFv) structures defined by the atomic coordinates provided in Table 18. In other embodiments, provided herein are scFv (e.g., spFv) structures defined by one or more subsets of the atomic coordinates provided in Table 18. In some embodiments, provided herein are scFv (e.g., spFv) structures defined by the atomic coordinates provided in Table 19. In other embodiments, provided herein are scFv (e.g., spFv) structures defined by one or more subsets of the atomic coordinates provided in Table 19. In some embodiments, provided herein are scFv (e.g., spFv) structures defined by the atomic coordinates provided in Table 20. In other embodiments, provided herein are scFv (e.g., spFv) structures defined by one or more subsets of the atomic coordinates provided in Table 20. In some embodiments, provided herein are scFv (e.g., spFv) structures defined by the atomic coordinates provided in Table 21. In other embodiments, provided herein are scFv (e.g., spFv) structures defined by one or more subsets of the atomic coordinates provided in Table 21. In some embodiments, provided herein are scFv (e.g., spFv) structures defined by the atomic coordinates provided in Table 22. In other embodiments, provided herein are scFv (e.g., spFv) structures defined by one or more subsets of the atomic coordinates provided in Table 22. In some embodiments, provided herein are scFv (e.g., spFv) structures defined by the atomic coordinates provided in Table 23. In other embodiments, provided herein are scFv (e.g., spFv) structures defined by one or more subsets of the atomic coordinates provided in Table 23.

[0458] 4.2. Generation of Cultured spFv-Containing Molecules of the Disclosure The spFvs of the present disclosure can be engineered into multispecific molecules of any known format using known recombinant techniques, expression and purification protocols.

[0459] The spFvs of the present disclosure can be engineered into full-length multispecific antibodies with one or more mutations in the CH3 domain that promote stability of the two halves of the molecule. These multispecific antibodies can be generated in vitro using Fab arm exchange or by coexpression of the various chains. For in vitro Fab arm exchange, two monospecific bivalent antibodies are engineered with one or more substitutions in the CH3 domain, and the antibodies are incubated together under reducing conditions sufficient to cause disulfide bond isomerization at the cysteines in the hinge region, thereby generating the multispecific antibody through Fab arm exchange. Incubation conditions can optimally be returned to non-reducing conditions. Non-limiting examples of reducing agents that can be used include 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol. In some embodiments, the reducing agent is selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. For example, incubation at a temperature of at least 20° C., in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreitol, at a pH of 5-8, e.g., pH 7.0 or pH 7.4, for at least 90 minutes can be used.

[0460] CH3 mutations that can be used include techniques such as knob-in-hole mutagenesis (Genentech), electrostatic match mutagenesis (Chugai, Amgen, NovoNordisk, Oncomed), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), Duobody® mutagenesis (Genmab), and other asymmetric mutagenesis (e.g., Zymeworks).

[0461] Knob-in-hole mutations, such as those disclosed in WO 1996 / 027011, include mutations at the interface of the CH3 domains in which an amino acid with a small side chain (the hole) is introduced into the first CH3 domain and an amino acid with a large side chain (the knob) is introduced into the second CH3 domain, resulting in preferential interactions between the first and second CH3 domains. Non-limiting examples of knob-and-hole-forming CH3 domain mutations include T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V.

[0462] Heavy chain heterodimer formation can be promoted using electrostatic interactions by substituting positively charged residues on the first CH3 region with negatively charged residues on the second CH3 region, as described in U.S. Patent Application Publication No. 2010 / 0015133, U.S. Patent Application Publication No. 2009 / 0182127, U.S. Patent Application Publication No. 2010 / 028637, or U.S. Patent Application Publication No. 2011 / 0123532.

[0463] Other asymmetric mutations that can be used to promote heavy chain heterodimerization include, but are not limited to, L351Y_F405A_Y407V / T394W, T366I_K392M_T394W / F405A_Y4, as described in U.S. Patent Application Publication No. 2012 / 0149876 or U.S. Patent Application Publication No. 2013 / 0195849 (Zymeworks). 07V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W.

[0464] SEED body mutations involve replacing selected IgG residues with IgA residues to promote heavy chain heterodimerization, as described in US Patent Application Publication No. 20070287170.

[0465] Other exemplary mutations that may be used include R409D_K370E / D399K_E357K, S354C_T366W / Y349C_T366W, and R409D_K370E / D399K_E357K, S354C_T366W / Y349C_T366W, as described in WO 2007 / 147901, WO 2011 / 143545, WO 2013 / 157954, WO 2013 / 096291, and U.S. Patent Application Publication No. 2018 / 0118849. T366S_L368A_Y407V, Y349C_T366W / S354C_T366S_L368A_Y407V, T366K / L351D, L351K / Y349E, L351K / Y349D, L351K / L368E, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, K392D / D399K, K392D / E356K, K253E_D282K_K322D / D239K_E240K_K292D, K392D_K409D / D356K_D399K.

[0466] Duobody® mutations (Genmab) are disclosed, for example, in U.S. Patent Application Publication No. 2014 / 0303356 and include the following mutations: F405L / K409R, wild-type / F405L_R409K, T350I_K370T_F405L / K409R, K370W / K409R, D399AFGHILMNRSTVWY / K409R, T366ADEFGHILMQVY / K409R, L368ADEGHNRSTVQ / K409AGRH, D399FHKRQ / K409AGRH, F405IKLSTVW / K409AGRH, and Y407LWQ / K409AGRH.

[0467] Additional bispecific or multispecific structures into which the spFvs of the present disclosure may be incorporated include Dual Variable Domain immunoglobulins (DVDs) (WO 2009 / 134776, where a DVD is a full-length antibody comprising a heavy chain with a VH1-linker-VH2-CH structure and a light chain with a VL1-linker-VL2-CL structure, where the linker is optional), structures comprising various dimerization domains for linking two antibody arms with different specificities, for example, leucine zippers or collagen dimerization domains (WO 2012 / 022811, U.S. Pat. Nos. 5,932,448, 6,833,441), two or more domain antibodies (dAbs) conjugated together, diabodies, heavy chain-only antibodies such as camelid antibodies and engineered camelid antibodies, dual targeting (DT)-Ig (GSK / Domantis), two-in-one antibodies (Genentech), cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star) and CovX-body (CovX / Pfizer), IgG-like bispecific (InnClone / Eli Lilly), Ts2Ab (MedImmune / AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idec) and TvAb (Roche), ScFv / Fc fusion (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS)), Dual Affinity Retargeting Technology (Fc-DART) (MacroGenics) and Dual(ScFv)2-Fab (National Research Center for Antibody Medicine--China), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol), and Fab-Fv (UCB-Celltech).ScFv antibodies, diabody-based antibodies, and domain antibodies include, but are not limited to, Bispecific T Cell Engager (BiTE) (Micromet), Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART) (MacroGenics), single-chain diabodies (Academic), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack), and COMBODY (Epigen Biotech), dual-targeting nanobodies (Ablynx), dual-targeting heavy chain-only domain antibodies.

[0468] The scFvs (or spFvs) of the present disclosure can also be engineered into multispecific molecules comprising three antigen-binding domains. In such designs, at least one antigen-binding domain is in the form of an scFv of the present disclosure. Exemplary designs include the following (where "1" indicates the first antigen-binding domain, "2" indicates the second antigen-binding domain, and "3" indicates the third antigen-binding domain): Design 1: Chain A) scFv1-CH2-CH3, Chain B) VL2-CL, Chain C) VH2-CH1-hinge-CH2-CH3 Design 2: Chain A) scFv1-hinge-CH2-CH3, Chain B) VL2-CL, Chain C) VH2-CH1-hinge-CH2-CH3 Design 3: Chain A) scFv1-CH1-hinge-CH2-CH3, Chain B) VL2-CL, Chain C) VH2-CH1-hinge-CH2-CH3 Design 4: Chain A) CH2-CH3-scFv1, Chain B) VL2-CL, Chain C) VH2-CH1-hinge-CH2-CH3

[0469] CH3 manipulations can be performed using, but are not limited to, L351Y_F405A_Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W as described in U.S. Patent Application Publication No. 2012 / 0149876 or U.S. Patent Application Publication No. 2013 / 0195849 (Zymeworks). / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W mutations can be incorporated into designs 1-4.

[0470] 4.3. Isotype, Allotype, and Fc Engineering The Ig constant region, or fragment thereof, such as the Fc region, present in the molecules of the present disclosure may be of any allotype or isotype.

[0471] In some embodiments, the Ig constant region or fragment thereof is an IgG1 isotype. In some embodiments, the Ig constant region or fragment thereof is an IgG2 isotype. In some embodiments, the Ig constant region or fragment thereof is an IgG3 isotype. In some embodiments, the Ig constant region or fragment thereof is an IgG4 isotype.

[0472] The Ig constant region or a fragment thereof can be of any allotype. In some embodiments, the allotype is not expected to affect the properties of the Ig constant region, such as binding or Fc-mediated effector function. The immunogenicity of therapeutic proteins containing fragment Ig constant regions is associated with an increased risk of infusion reactions and a shorter duration of therapeutic response (Baert et al., (2003) N Engl J Med 348:602-608). The extent to which therapeutic proteins containing fragment Ig constant regions induce an immune response in the host can be determined, in part, by the allotype of the Ig constant region (Stickler et al., (2011) Genes and Immunity 12:213-221). The allotype of an Ig constant region is related to variations in the amino acid sequence at specific positions in the constant region sequence of an antibody. Table 3 shows selected IgG1, IgG2, and IgG4 allotypes.

[0473] [Table 3]

[0474] The C-terminal lysine (CTL) can be removed from the Ig constant region by endogenous circulating carboxypeptidases in the bloodstream (Cai et al., (2011) Biotechnol Bioeng 108:404-412). During production, extracellular Zn was removed as described in U.S. Patent Application Publication No. 2014 / 0273092. 2+ , EDTA, or EDTA-Fe 3 By controlling the concentration of the CTL, the removal of CTL can be controlled to less than the maximum level. The CTL content of the protein can be measured using known methods.

[0475] In some embodiments, an spFv of the present disclosure conjugated to an 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 50% to about 80%. In some embodiments, the C-terminal lysine content is about 60% to about 80%. In some embodiments, the C-terminal lysine content is about 50% to about 70%. In some embodiments, the C-terminal lysine content is about 60% to about 70%. In some embodiments, the C-terminal lysine content is about 55% to about 70%. In some embodiments, the C-terminal lysine content is about 60%.

[0476] Fc region mutations can be made to the molecules of the present disclosure or fragments thereof comprising 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 in the Fc that modulate binding of the mutated Fc to activating FcγRs (FcγRI, FcγRIIa, FcγRIII), FcγRIIb, and / or FcRn.

[0477] In some embodiments, the molecules of the present disclosure comprise at least one mutation in an Ig constant region or fragment thereof, hi some embodiments, the at least one mutation is in the Fc region.

[0478] In some embodiments, the molecules of the disclosure comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations in the Fc region.

[0479] In some embodiments, the molecules of the disclosure comprise at least one mutation in the Fc region that modulates binding of the molecule to FcRn.

[0480] Positions in Fc that can be mutated to modulate half-life (e.g., binding to FcRn) include, but are not limited to, positions 250, 252, 253, 254, 256, 257, 307, 376, 380, 428, 434, and 435. Non-limiting examples of mutations that can be made alone or in combination include the mutations T250Q, M252Y, I253A, S254T, T256E, P257I, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A, and H435R. Exemplary, non-limiting examples of mutations that may be made, alone or in combination, to increase the half-life of an antibody include the mutations M428L / N434S, M252Y / S254T / T256E, T250Q / M428L, N434A, and T307A / E380A / N434A. Exemplary, non-limiting examples of mutations that may be made, alone or in combination, to decrease the half-life include the mutations H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A, and H435R.

[0481] In some embodiments, the molecules of the disclosure comprise M252Y / S254T / T256E mutations in the Fc region.

[0482] The molecules of the disclosure may comprise at least one mutation in the Fc region that reduces binding of the molecule to activating Fcγ receptors (FcγRs) and / or reduces an Fc effector function such as C1q binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and / or phagocytosis (ADCP).

[0483] Fc positions that may be mutated to reduce binding of the molecules of the present disclosure to activating FcγRs and subsequently reduce effector function include, but are not limited to, positions 214, 233, 234, 235, 236, 237, 238, 265, 267, 268, 270, 295, 297, 309, 327, 328, 329, 330, 331, and 365. Non-limiting examples of exemplary mutations that may be made alone or in combination include the mutations K214T, E233P, L234V, L234A, deletion of G236, V234A, F234A, L235A, G237A, P238A, P238S, D265A, D265S, S267E, H268A, H268Q, Q268A, N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F, A330S, and P331S in IgG1, IgG2, IgG3, or IgG4. Non-limiting examples of exemplary combinations of mutations that result in reduced ADCC include L234A / L235A in IgG1, L234A / L235A / D265S in IgG1, V234A / G237A / P238S / H268A / V309L / A330S / P331S in IgG2, F234A / L235A in IgG4, S228P / F234A / L235A in IgG4, N297A in all Ig isotypes, V234A / G237A in IgG2, K214T / E233P / L234V / L235A / G in IgG1, and K214T / E233P / L234V / L235A / G in IgG1. Examples of mutations include IgG2 deletion 236 / A327G / P331A / D365E / L358M, H268Q / V309L / A330S / P331S in IgG2, S267E / L328F in IgG1, L234F / L235E / D265A in IgG1, L234A / L235A / G237A / P238S / H268A / A330S / P331S in IgG1, S228P / F234A / L235A / G237A / P238S in IgG4, and S228P / F234A / L235A / G236 deletion / G237A / P238S in IgG4. Alternatively, a hybrid IgG2 / 4 Fc domain may be used, such as an Fc having residues 117-260 from IgG2 and residues 261-447 from IgG4.

[0484] An exemplary mutation that results in reduced CDC is the K322A mutation. The S228P mutation can be added to an IgG4 antibody to enhance the stability of the IgG4.

[0485] In some embodiments, a molecule of the disclosure comprises at least one mutation in the Fc region, wherein the at least one mutation is selected from the group consisting of K214T, E233P, L234V, L234A, deletion of G236, V234A, F234A, L235A, G237A, P238A, P238S, D265A, S267E, H268A, H268Q, Q268A, N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F, A330S, and P331S. In some embodiments, the at least one mutation comprises L234A, L235A, D265S. In some embodiments, the at least one mutation comprises L234A or L235A.

[0486] In some embodiments, the molecules of the present disclosure may comprise at least one mutation in the Fc region that enhances binding of the molecule to FcγR and / or enhances an Fc effector function, such as C1q binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and / or phagocytosis (ADCP).

[0487] Fc positions that can be mutated to increase binding of the molecule to activating FcγRs and / or enhance Fc effector function include, but are not limited to, 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). Non-limiting examples of mutations that may be made singly or in combination include G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305L, K326A, A330K, I332E, E333A, K334A, A339T, and P396L. Non-limiting examples of combinations of mutations that result in proteins with increased ADCC or ADCP include S239D / I332E, S298A / E333A / K334A, F243L / R292P / Y300L, F243L / R292P / Y300L / P396L, F243L / R292P / Y300L / V305I / P396L, and G236A / S239D / I332E.

[0488] Fc positions that can be mutated to enhance CDC include positions 267, 268, 324, 326, 333, 345, and 430. Non-limiting examples of mutations that can be made singly or in combination are S267E, F1268F, S324T, K326A, K326W, E333A, E345K, E345Q, E345R, E345Y, E430S, E430F, and E430T. Non-limiting examples of combination mutations that result in increased CDC include K326A / E333A, K326W / E333A, H268F / S324T, S267E / H268F, S267E / S324T, and S267E / H268F / S324T.

[0489] In some embodiments, the mutation is present in wild-type IgG1, wild-type IgG2, or wild-type IgG4. In some embodiments, the wild-type IgG1 comprises the amino acid sequence of SEQ ID NO: 66, as provided below. In some embodiments, the wild-type IgG2 comprises the amino acid sequence of SEQ ID NO: 67, as provided below. In some embodiments, the wild-type IgG4 comprises the amino acid sequence of SEQ ID NO: 68, as provided below. ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 66) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 67) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 68)

[0490] Binding of the molecules of the present disclosure to the FcγRs or FcRn of the present disclosure can be assessed in cells genetically engineered to express the respective receptor using flow cytometry.

[0491] 4.4. Glycoengineering The ability of the molecules of the present disclosure comprising an Ig constant region or a fragment thereof to mediate ADCC can be enhanced by manipulating the oligosaccharide components of the Ig constant region or a fragment thereof. Human IgG1 or IgG3 is N-glycosylated at Asn297, where the majority of glycans are in the known biantennary G0, G0F, G1, G1F, G2, or G2F forms. Ig constant region-containing proteins that can be produced by non-genetically engineered CHO cells typically have a glycan fucose content of at least about 85%. Removal of core fucose from the biantennary complex-type oligosaccharides added to the Ig constant region or a fragment thereof enhances the ADCC of the molecule by improving FcγRIIIa binding, without altering antigen binding or CDC activity. Such molecules have been reported to successfully express relatively highly defucosylated immunoglobulins with biantennary and complex Fc oligosaccharide types using different methods, such as controlling the culture osmolarity (Konno et al. (2012), Cytotechnology 64:249-265), applying the mutant CHO line Lec13 as a host cell line (Shields et al. (2002) J Biol Chem 277:26733-26740), applying the mutant CHO line EB66 as a host cell line (Olivier et al. (2010) MAbs;2:405-415), applying the rat hybridoma cell line YB2 / 0 as a host cell line (Shinkawa et al. (2003) J Biol Chem 278:3466-3473), and introducing small interfering RNA specific for the 1,6-fucosyltransferase (FUT8) gene (Mori et al. al., (2004) Biotechnol Bioeng 88:901-908), or co-expression of β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II or the potent α-mannosidase I inhibitor kifunensin (Ferrara et al., (2006) J Biol Chem 281:5032-5036).

[0492] In some embodiments, molecules of the disclosure comprising an Ig constant region or a fragment thereof have biantennary glycan structures with a fucose content of about 1% to about 15%, e.g., about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, molecules of the disclosure comprising an Ig constant region or a fragment thereof have glycan structures with a fucose content of about 50%, 40%, 45%, 40%, 35%, 30%, 25%, or 20%.

[0493] "Fucose content" refers to the amount of fucose monosaccharide in the glycan at Asn 297. The relative amount of fucose is the ratio of fucose-containing structures to total glycan structures. These glycostructures can be identified by several methods, including, for example, 1) using MALDI-TOF of N-glycosidase F treated samples (e.g., complex, hybrid, and oligo- and high mannose structures) as described in WO 2008 / 077546; 2) enzymatic release of the Asn297 glycan followed by derivatization and detection / quantification by HPLC with fluorescence detection (UPLC) and / or HPLC-MS (UPLC-MS); 3) intact protein analysis of native or reduced mAbs, with or without treatment of the Asn297 glycan with Endo S or other enzymes that cleave between the first and second GlcNAc monosaccharides, leaving the fucose attached to the first GlcNAc; 4) enzymatic digestion (e.g., trypsin or endopeptidase Lys-C) of the mAb into its constituent peptides followed by separation, detection, and quantification by HPLC-MS (UPLC-MS); 5) dehydrogenase analysis of the Asn297 glycan by enzymatic digestion (e.g., trypsin or endopeptidase Lys-C) followed by separation, detection, and quantification by HPLC-MS (UPLC-MS); mAb oligosaccharides can be characterized and quantified by isolating them from the mAb protein by specific enzymatic deglycosylation with PNGase F at position 297. The oligosaccharides thus released can be labeled with fluorophores and separated and identified by a variety of complementary techniques, including detailed characterization of glycan structures by matrix-assisted laser desorption ionization (MALDI) mass spectrometry by comparison of observed masses with theoretical masses, determination of the degree of sialylation by ion-exchange HPLC (GlycoSep C), separation and quantification of oligosaccharide types according to hydrophilicity criteria by normal-phase HPLC (GlycoSep N), and separation and quantification of oligosaccharides by high-performance capillary electrophoresis-laser-induced fluorescence (HPCE-LIF).

[0494] "Low fucose" or "low fucose content" refers to molecules of the present disclosure that comprise an Ig constant region or fragment thereof that has a fucose content of about 1% to about 15%.

[0495] "Normal fucose" or "normal fucose content" refers to a molecule of the present disclosure comprising an Ig constant region or fragment thereof having a fucose content of greater than about 50%, e.g., greater than about 80% or greater than about 85%.

[0496] 4.5. Anti-idiotype antibodies An anti-idiotype antibody is an antibody that specifically binds to an spFv of the present disclosure. The present disclosure also provides an anti-idiotype antibody that specifically binds to an spFv of the present disclosure.

[0497] In some embodiments, the anti-idiotype antibody binds to a disulfide bond in an spFv of the present disclosure. In some embodiments, the anti-idiotype antibody binds to the antigen-binding domain of an spFv of the present disclosure.

[0498] 4.6. Polynucleotides, Vectors, and Host Cells The present disclosure also provides polynucleotides encoding the spFvs of the disclosure. Additionally, vectors containing such polynucleotides are provided.

[0499] In some embodiments, the vector is an expression vector. Expression vectors can be plasmid vectors, viral vectors, vectors for baculovirus expression, vectors for prokaryotic expression, vectors for eukaryotic expression, transposon-based vectors, or any other vector suitable for introducing a polynucleotide of the present disclosure into a given cell or organism. The polynucleotide encoding the spFv of the present disclosure can be operably linked to regulatory sequences in the expression vector that promote expression of the spFv. Such regulatory elements can include, but are not limited to, a transcriptional promoter, a sequence encoding a suitable mRNA ribosomal binding site, and sequences controlling transcription and translation termination. The expression vector can also contain one or more non-transcribed elements, such as an origin of replication, other 5' or 3' flanking non-transcribed sequences, 5' or 3' non-translated sequences (such as necessary ribosomal binding sites), splice donor and acceptor sites, or a selectable marker. The polynucleotide can also be a cDNA. The promoter driving spFv expression can be a strong, weak, tissue-specific, inducible, or developmentally specific promoter. Non-limiting examples of promoters include hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin, human myosin, human hemoglobin, human muscle creatine, etc. In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for use in the described embodiments. Such viral promoters include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, the SV40 early and late promoters, the mouse mammary tumor virus (MMTV) promoter, the long terminal repeats (LTRs) of Moloney leukemia virus, human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), Rous sarcoma virus (RSV), and other retroviruses, and the herpes simplex virus thymidine kinase promoter.Inducible promoters, such as those containing one or more interferon-stimulated response elements (ISREs), include metallothionein promoters, tetracycline-inducible promoters, doxycycline-inducible promoters, protein kinase R 2',5'-oligoadenylate synthetase, Mx genes, and ADAR1. The vectors of the present disclosure may also contain one or more internal ribosome entry sites (IRES). The inclusion of an IRES sequence in a fusion vector may be beneficial for enhancing the expression of some proteins. The vectors of the present disclosure may be circular or linear. They may be prepared to contain replication systems functional in prokaryotic or eukaryotic host cells. Replication systems can be derived from, for example, ColE1, SV40, 2μ plasmid, λ, bovine papilloma virus, etc. Expression vectors may be designed for transient expression, stable expression, or both. Expression vectors may be constructed for constitutive or inducible expression.

[0500] Exemplary vectors that can be used are bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia), pEE6.4 (Lonza), and pEE12.4 (Lonza). Additional vectors include the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors such as λGT10, λGT11, λEMBL4, and λNM1149, λZapII (Stratagene) can be used. Exemplary plant expression vectors include pBI01, pBI01.2, pBI121, pBI101.3, and pBIN19 (Clontech). Exemplary animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The expression vector can be a viral vector, eg, a retroviral vector, eg, a gamma retroviral vector.

[0501] The present disclosure also provides a host cell comprising the vector of the present disclosure. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is a eukaryotic cell.

[0502] A "host cell" refers to a cell into which a vector has been introduced. It is understood that the term host cell is intended to refer not only to the particular subject cell, but also to the progeny of such a cell, as well as to stable cell lines generated from the particular subject cell. Because certain modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Such host cells may be eukaryotic, prokaryotic, plant, or archaeal cells. Examples of prokaryotic host cells are bacilli such as Escherichia coli and Bacillus subtilis, and other Enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species. Other microorganisms, such as yeast, are also useful for expression. Examples of suitable yeast host cells are Saccharomyces (e.g., S. cerevisiae) and Pichia. Exemplary eukaryotic cells may be of mammalian, insect, avian, or other animal origin. Mammalian eukaryotic cells include immortalized cell lines such as hybridomas, or myeloma cell lines such as SP2 / 0 (American Type Culture Collection (ATCC), Manassas, VA, CRL-1581), NS0 (European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646), and Ag653 (ATCC CRL-1580) mouse cell lines. An exemplary human myeloma cell line is U266 (ATCC CRL-TIB-196). Other useful cell lines include those derived from Chinese Hamster Ovary (CHO) cells, such as CHO-K1SV (Lonza Biologics, Walkersville, MD), CHO-K1 (ATCC CRL-61), or DG44.

[0503] The present disclosure also provides methods for producing an spFv of the present disclosure. In some embodiments, the method includes culturing a host cell of the present disclosure under conditions such that an spFv is produced, and recovering the spFv produced by the host cell. Methods for making scFvs and purifying them are known. Once synthesized (chemically or recombinantly), the spFv can be purified according to standard procedures, including, but not limited to, ammonium sulfate precipitation, affinity columns, column chromatography, high-performance liquid chromatography (HPLC) purification, gel electrophoresis, and the like (see generally, Scopes, Protein Purification (Springer-Verlag, NY, (1982))). The scFv can be substantially pure, e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or at least about 98% to 99% pure, or more, and can be free from contaminants such as cellular debris, macromolecules other than the protein of interest, and the like.

[0504] Polynucleotides encoding the spFv of the present disclosure can be incorporated into vectors using standard molecular biology techniques, and host cell transformation, culture, antibody expression, and purification are carried out using well-known methods.

[0505] 4.7. Pharmaceutical Compositions and Administration The present disclosure also provides compositions comprising the spFvs or molecules disclosed herein. In some embodiments, the composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier.

[0506] "Carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the spFv or molecule is administered. Such vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, and sesame oil. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally free of particulate matter. They can be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The composition can contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, stabilizers, thickeners, lubricants, and coloring agents. The concentration of the spFv or molecule in the composition can vary from less than about 0.5% by weight, usually at least about 1% by weight, and up to 15 or 20% by weight, and can be selected primarily based on the required dose, fluid volumes, viscosity, and the like, according to the selected method of administration. Suitable vehicles and formulations containing other human proteins, such as human serum albumin, are described, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, DB ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, see in particular pp. 958-989.

[0507] The mode of administration of the spFv, molecules, or compositions disclosed herein can be by any suitable route, including, but not limited to, parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous, or subcutaneous, transmucosal (oral, intranasal, intravaginal, rectal), or other means recognized by one of skill in the art.

[0508] 4.8. Processes for Preparing spFv of the Disclosure The present disclosure further provides a process for preparing cultured spFvs of the present disclosure (e.g., those disclosed in Section 4.1.1). In some embodiments, the process includes providing a heavy chain variable region (VH) and a light chain variable region (VL) that form an antigen-binding site, providing a linker (L) that includes or is engineered to include a first L Cys, engineering 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 the first L Cys to prepare a stabilized scFv.

[0509] In some embodiments, the method includes providing a VH and a VL that form an antigen binding site; providing an L that includes or is engineered to include a second L Cys; 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 scFv.

[0510] In some embodiments, the process comprises providing a heavy chain variable region (VH) and a light chain variable region (VL) that form an antigen-binding site; providing a linker (L) that comprises, or is engineered to comprise, a first L Cys and a second L Cys; engineering the VH to comprise a VH Cys at a structurally conserved, surface-exposed VH framework residue position; engineering the VL to comprise a VL Cys at a structurally conserved, surface-exposed VL framework residue position; forming a disulfide bond between the VH Cys and the first L Cys, and forming a disulfide bond between the VL Cys and the second L Cys, to prepare a stabilized scFv.

[0511] In some embodiments, disulfide bonds are typically formed during expression of the scFv.

[0512] Any known VH / VL pair of scFvs that form antigen-binding domains can be engineered into stabilized scFvs. Alternatively, antigen-binding VH / VL pairs of interest can be identified de novo using known methods, and the resulting VH / VL pair engineered into an spFv format.

[0513] For example, the hybridoma method of Kohler and Milstein can be used to identify VH / VL pairs that bind to the antigen of interest, and the resulting VH / VL pairs can be engineered as spFvs. Alternatively, transgenic animals, such as mice, rats, or chickens, carrying human immunoglobulin (Ig) loci in their genomes can be used to produce antigen-binding fragments; these are described, for example, in U.S. Pat. No. 6,150,584, WO 1999 / 45962, WO 2002 / 066630, WO 2002 / 43478, WO 2002 / 043478, and WO 1990 / 04036. The endogenous immunoglobulin loci of such animals can be disrupted or deleted, and at least one complete or partial human immunoglobulin locus can be inserted into the animal's genome using homologous or non-homologous recombination, using transchromosomes, or using minigenes. Companies such as Regeneron (http: / / _www_regeneron_com), Harbour Antibodies (http: / / _www_harbourantibodies_com), Open Monoclonal Technology, Inc. (OMT) (http: / / _www_omtinc_net), KyMab (http: / / _www_kymab_com), Trianni (http: / / _www.trianni_com) and Ablexis (http: / / _www_ablexis_com) may be working to provide human antibodies targeting selected antigens using the above technology. Phage display may also be used to generate antigen-binding fragments that can be engineered as spFvs.

[0514] In some embodiments, the spFv is humanized. In some embodiments, the spFv is human. In some embodiments, the spFv is non-human.

[0515] In some embodiments, the process comprises expressing a polynucleotide of the disclosure (eg, those disclosed in Section 4.6) in a host cell to produce a stabilized scFv.

[0516] The following examples are provided to further describe some of the embodiments disclosed herein and are intended to illustrate, but not limit, embodiments of the present disclosure.

[0517] 4.9. Exemplary spFvs, Molecules, and Linkers In some embodiments, the spFvs or molecules of the disclosure comprise one or more amino acid sequences set forth in Table 4.

[0518] [Table 4-1]

[0519] [Table 4-2]

[0520] [Table 4-3]

[0521] In some embodiments, the "Cris7a VL-VH scFv" comprises the amino acid sequence of SEQ ID NO:125.

[0522] In some embodiments, the "Cris7a VL-VH spFv" comprises the amino acid sequence of SEQ ID NO:126.

[0523] In some embodiments, the "Cris7b VL-VH scFv" comprises the amino acid sequence of SEQ ID NO: 127. In some embodiments, the "Cris7b VL-VH spFv" comprises the amino acid sequence of SEQ ID NO: 128. In some embodiments, the "BCMB749_VL" comprises the amino acid sequence of SEQ ID NO: 129.

[0524] In some embodiments, "human CL" comprises the amino acid sequence of SEQ ID NO:130.

[0525] In some embodiments, "BCMB749 LC" comprises the amino acid sequence of SEQ ID NO:131.

[0526] In some embodiments, "BCMB749_VH" comprises the amino acid sequence of SEQ ID NO: 132.

[0527] In some embodiments, "Human_HC_ConstantDomains 1" comprises the amino acid sequence of SEQ ID NO:133.

[0528] In some embodiments, "BCMB749 HC1" comprises the amino acid sequence of SEQ ID NO:134.

[0529] In some embodiments, "BCMB749h_VL" comprises the amino acid sequence of SEQ ID NO: 135.

[0530] In some embodiments, "BCMB749h LC" comprises the amino acid sequence of SEQ ID NO:136.

[0531] In some embodiments, "BCMB749h_VH" comprises the amino acid sequence of SEQ ID NO: 137.

[0532] In some embodiments, "BCMB749h HC1" comprises the amino acid sequence of SEQ ID NO:138.

[0533] In some embodiments, "Human_HC_ConstantDomains 2" comprises the amino acid sequence of SEQ ID NO:139.

[0534] In some embodiments, "Cris7b VL-VH scFv HC2" comprises the amino acid sequence of SEQ ID NO:140.

[0535] In some embodiments, "Cris7b VL-VH spFv HC2" comprises the amino acid sequence of SEQ ID NO:141.

[0536] In some embodiments, "CD3B219a99v scFv HC2" comprises the amino acid sequence of SEQ ID NO:142.

[0537] In some embodiments, "CD3B219a99v spFv HC2" comprises the amino acid sequence of SEQ ID NO:143.

[0538] In further embodiments, the scFv linker of the disclosure comprises an amino acid sequence shown in Table 5.

[0539] [Table 5]

[0540] In some embodiments, the "GLk1 scFv VL-VH" linker comprises the amino acid sequence of SEQ ID NO:2.

[0541] In some embodiments, the "GLk1 spFv VL-VH" linker comprises the amino acid sequence of SEQ ID NO:3.

[0542] In some embodiments, the "GLk1 scFv VH-VL" linker comprises the amino acid sequence of SEQ ID NO:2.

[0543] In some embodiments, the "GLk1 spFv VH-VL" linker comprises the amino acid sequence of SEQ ID NO:3.

[0544] In some embodiments, the "GLk2 scFv VL-VH" linker comprises the amino acid sequence of SEQ ID NO:2.

[0545] In some embodiments, the "GLk2 spFv VL-VH" linker comprises the amino acid sequence of SEQ ID NO:3.

[0546] In some embodiments, the "GLk2 scFv VH-VL" linker comprises the amino acid sequence of SEQ ID NO:2.

[0547] In some embodiments, the "GLk2 spFv VH-VL" linker comprises the amino acid sequence of SEQ ID NO:4.

[0548] In some embodiments, the "CAT2200a scFv VL-VH" linker comprises the amino acid sequence of SEQ ID NO:2.

[0549] In some embodiments, the "CAT2200a spFv VL-VH" linker of the present disclosure comprises the amino acid sequence of SEQ ID NO:5.

[0550] In some embodiments, the "CAT2200b scFv VL-VH" linker of the present disclosure comprises the amino acid sequence of SEQ ID NO:2.

[0551] In some embodiments, the "CAT2200a spFv VL-VH" linker of the present disclosure comprises the amino acid sequence of SEQ ID NO:6.

[0552] In some embodiments, the "CAT2200a scFv VH-VL" linker of the present disclosure comprises the amino acid sequence of SEQ ID NO:2.

[0553] In some embodiments, the "CAT2200b spFv VH-VL" linker of the present disclosure comprises the amino acid sequence of SEQ ID NO:7.

[0554] 5. Implementation form The present invention provides the following non-limiting embodiments.

[0555] In one set of embodiments, the following is provided: A1. A molecule comprising an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), and a fragment crystallizable region (Fc region), wherein the scFv comprises a heavy chain variable region (VH), a linker (L), and a light chain variable region (VL), wherein the scFv comprises: a) A disulfide bond between a structurally conserved surface-exposed VH position mutated to a cysteine ​​(Cys) and L Cys; b) a disulfide bond between a structurally conserved, surface-exposed VL position mutated to Cys and an L Cys; c) a structurally conserved first disulfide bond between the surface-exposed VH Cys and the first L Cys, and a second disulfide bond between the surface-exposed VL Cys and the second L Cys, and optionally the molecule has improved stability, expression yield, and / or quality compared to a comparable molecule lacking the disulfide bonds, e.g., a comparable molecule lacking the first and second disulfide bonds. A2. a) VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position and L comprises an L Cys; b) VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position and L comprises an L Cys; or c) The molecule of embodiment A1, wherein VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position, VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position, and L comprises a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys are capable of forming a disulfide bond, and the VL Cys and the second L Cys are capable of forming a disulfide bond. A3. The molecule of embodiment A1 or A2, wherein the distance between the VH Cys and the VL Cys is from about 5 Å to about 10 Å or from about 7 Å to about 9 Å. A4. The molecule of any one of embodiments A1-A3, wherein the VH Cys is at H3, H5, H40, H43, H46, or H105, and residue numbering is according to Chothia. A5. The molecule of any one of embodiments A1 to A4, wherein VL Cys is at L3, L5, L39, L42, L43, L45, L100, or L102, and residue numbering is according to Chothia. A6. a) VH Cys is at H105 and VL Cys is at L42; or b) VH Cys is at H43 and VL Cys is at L100; or c) VH Cys is in H3 and VL Cys is in L3; d) VH Cys is in H3 and VL Cys is in L5; or e) VH Cys is at H3 and VL Cys is at L39; or f) VH Cys is at H3 and VL Cys is at L42; g) VH Cys is at H3 and VL Cys is at L45; or h) VH Cys is in H3 and VL Cys is in L100; i) VH Cys is at H3 and VL Cys is at L102; or j) VH Cys is in H5 and VL Cys is in L3; or k) VH Cys is in H5 and VL Cys is in L5; or l) VH Cys is at H5 and VL Cys is at L39; or m) VH Cys is at H5 and VL Cys is at L42; or n) VH Cys is at H5 and VL Cys is at L45; or o) VH Cys is at H5 and VL Cys is at L100; or p) VH Cys is at H5 and VL Cys is at L102; or q) VH Cys is in H40 and VL Cys is in L3; or r) VH Cys is in H40 and VL Cys is in L5; or s) VH Cys is at H40 and VL Cys is at L39; or t) VH Cys is at H40 and VL Cys is at L42; or u) VH Cys is at H40 and VL Cys is at L45; or v) VH Cys is at H40 and VL Cys is at L100; or w) VH Cys is at H40 and VL Cys is at L102; or x) VH Cys is at H43 and VL Cys is at L3; or y) VH Cys is at H43 and VL Cys is at L5; or z) VH Cys is at H43 and VL Cys is at L39; or aa) VH Cys is at H43 and VL Cys is at L42; or bb) VH Cys is at H43 and VL Cys is at L45; or cc) VH Cys is at H43 and VL Cys is at L102; or dd) VH Cys is at H46 and VL Cys is at L3; or ee) VH Cys is at H46 and VL Cys is at L5; or ff) VH Cys is at H46 and VL Cys is at L39; or gg) VH Cys is at H46 and VL Cys is at L42; or hh) VH Cys is at H46 and VL Cys is at L45; or ii) VH Cys is at H46 and VL Cys is at L100; or jj) VH Cys is at H46 and VL Cys is at L102; or kk) VH Cys is at H105 and VL Cys is at L3, or ll) VH Cys is at H105 and VL Cys is at L5; or mm) VH Cys is at H105 and VL Cys is at L39, or nn) VH Cys is at H105 and VL Cys is at L45; or oo) VH Cys is at H105 and VL Cys is at L100; or pp) VH Cys is at H105 and VL Cys is at L102, or qq) The molecule of any one of embodiments A1 to A5, wherein the VH Cys is at H105 and the VL Cys is at L43, residue numbering according to Chothia. A7. The molecule of any one of embodiments A1 to A6, wherein L comprises a contiguous amino acid sequence derived from an immunoglobulin (Ig) hinge region. A8. The molecule of any one of embodiments A1 to A7, wherein the Ig hinge region is derived from a human Ig hinge region or a non-human Ig hinge region. A9. The molecule of any one of embodiments A1 to A8, wherein the Ig hinge region is derived from a human Ig hinge region. A10. The molecule of any one of embodiments A1-A9, wherein the human Ig hinge region is of the IgG1, IgG2, IgG3, or IgG4 isotype. A11.L is the amino acid sequence C(X) y C (SEQ ID NO: 23), wherein X is glycine (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), threonine (Thr), tryptophan (Trp), or tyrosine (Tyr); and y is an integer from 1 to 3. A12.L is the amino acid sequence C(X) y The molecule of embodiment A11, comprising C (SEQ ID NO: 24), wherein X is Gly, Ser, or Pro and y is an integer from 1 to 3. A13.L contains 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), CGGPC (SEQ ID NO: 41), CGGPC (SEQ ID NO: 42), CGGPC (SEQ ID NO: 43), CGGPC (SEQ ID NO: 44), CGGPC (SEQ ID NO: 45), CGGPC (SEQ ID NO: 46), CGGPC (SEQ ID NO: 47), CGGPC (SEQ ID NO: 48), CGGPC (SEQ ID NO: 49), CGGPC (SEQ ID NO: 50), CGGPC (SEQ ID NO: 51), CGGPC (SEQ ID NO: 52), CGGPC (SEQ ID NO: 53), CGGPC (SEQ ID NO: 54), CGGPC (SEQ ID NO: 55), CGGPC (SEQ ID NO: 56), CGGPC (SEQ ID NO: 57), CGGPC (SEQ ID NO: 58), CGGPC (SEQ ID NO: 59), CGGPC (SEQ ID NO: 60), CGGPC (SEQ ID NO: 61), CGGPC (SEQ ID NO: 62), CGGPC (SEQ ID NO: 63), CGGPC (SEQ ID NO: 64 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). A14. The molecule of any one of embodiments A1 to A13, wherein L comprises about 14 to about 19 amino acids, e.g., about 14, about 15, about 16, about 17, about 18, or about 19 amino acids, and / or L has a length of about 14 to about 19 amino acids, e.g., about 14, about 15, about 16, about 17, about 18, or about 19 amino acids. A15.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. A16.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. A17.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. A18.L is a molecule of any one of embodiments A1 to A17, comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. A19. The molecule of any one of embodiments A1 to A18, wherein the scFv is in a VL-L-VH orientation. A20. The molecule of any one of embodiments A1 to A18, wherein the scFv is in a VH-L-VL orientation. A21. a) VH contains Cys at H105; b) VL contains a Cys at L42; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments A1 to A19, wherein the scFv is in a VL-L-VH orientation. A22. a) VH contains Cys at H105; b) VL contains a Cys at L45; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments A1 to A19, wherein the scFv is in a VL-L-VH orientation. A23. a) VH contains Cys at H105; b) VL contains a Cys at L39; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments A1 to A19, wherein the scFv is in a VL-L-VH orientation. A24. a) VH contains Cys at H5; b) VL contains a Cys at L42; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments A1 to A19, wherein the scFv is in a VL-L-VH orientation. A25. a) VH contains Cys at H5; b) VL contains a Cys at L45; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments A1 to A19, wherein the scFv is in a VL-L-VH orientation. A26. a) VH contains Cys at H5; b) VL contains a Cys at L39; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments A1 to A19, wherein the scFv is in a VL-L-VH orientation. A27. a) VH contains Cys at H3; b) VL contains a Cys at L42; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments A1 to A19, wherein the scFv is in a VL-L-VH orientation. A28. a) VH contains Cys at H3; b) VL contains a Cys at L45; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments A1 to A19, wherein the scFv is in a VL-L-VH orientation. A29. a) VH contains Cys at H3; b) VL contains a Cys at L39; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments A1 to A19, wherein the scFv is in a VL-L-VH orientation. A30. a) VH contains Cys at H43; b) VL contains a Cys at L100; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments A1 to A18 and A20, wherein the scFv is in a VH-L-VL orientation. A31. a) VH contains Cys at H43; b) VL contains a Cys at L102; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments A1 to A18 and A20, wherein the scFv is in a VH-L-VL orientation. A32. a) VH contains Cys at H43; b) VL contains a Cys at L5; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments A1 to A18 and A20, wherein the scFv is in a VH-L-VL orientation. A33. a) VH contains Cys at H43; b) VL contains a Cys at L3; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments A1 to A18 and A20, wherein the scFv is in a VH-L-VL orientation. A34. a) VH contains Cys at H40; b) VL contains a Cys at L100; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments A1 to A18 and A20, wherein the scFv is in a VH-L-VL orientation. A35. a) VH contains Cys at H40; b) VL contains a Cys at L102; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments A1 to A18 and A20, wherein the scFv is in a VH-L-VL orientation. A36. a) VH contains Cys at H40; b) VL contains a Cys at L5; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments A1 to A18 and A20, wherein the scFv is in a VH-L-VL orientation. A37. a) VH contains Cys at H40; b) VL contains a Cys at L3; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments A1 to A18 and A20, wherein the scFv is in a VH-L-VL orientation. A38. a) VH contains Cys at H46; b) VL contains a Cys at L100; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments A1 to A18 and A20, wherein the scFv is in a VH-L-VL orientation. A39. a) VH contains Cys at H46; b) VL contains a Cys at L102; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments A1 to A18 and A20, wherein the scFv is in a VH-L-VL orientation. A40. a) VH contains Cys at H46; b) VL contains a Cys at L5; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments A1 to A18 and A20, wherein the scFv is in a VH-L-VL orientation. A41. a) VH contains Cys at H46; b) VL contains a Cys at L3; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments A1 to A18 and A20, wherein the scFv is in a VH-L-VL orientation. A42.L is a molecule according to any one of embodiments A21 to A41, comprising the amino acid sequence of SEQ ID NO:3. A43.L is a molecule of any one of embodiments A21 to A41 comprising the amino acid sequence of SEQ ID NO:6. A44.L is a molecule of any one of embodiments A21 to A41 comprising the amino acid sequence of SEQ ID NO:7. A45. The binding molecule comprises a heavy chain, a light chain, and a polypeptide; The N-termini of the heavy and light chains form Fab, the polypeptide comprises an scFv at the N-terminus, The molecule of any one of embodiments A1 to A44, wherein the C-terminus of the polypeptide and the C-terminus of the heavy chain form an Fc region. A46. The molecule of any one of embodiments A1 to A45, wherein the Fab binds to a tumor antigen and the scFv binds to a T cell antigen, optionally wherein the tumor antigen is BCMA and the T cell antigen is CD3. The molecule described in embodiment A46, wherein A47.scFv comprises the amino acid sequence of SEQ ID NO: 126 or SEQ ID NO: 128. A48. A molecule described in embodiment A46 or embodiment A47, wherein (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 132 and a VL comprising the amino acid sequence of SEQ ID NO: 129, or (ii) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 137 and a VL comprising the amino acid sequence of SEQ ID NO: 135. A49. a) VH contains Cys at H105; b) VL contains a Cys at L43; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of embodiment A47 or embodiment A48, wherein the scFv is in a VL-L-VH orientation. A50. A polynucleotide encoding a molecule or fragment thereof, or a polypeptide according to any one of embodiments A1 to A49. A51. A vector comprising a polynucleotide according to embodiment A50. A52. A host cell comprising the vector of embodiment A51. A53. A method for producing a binding molecule, comprising culturing a host cell according to embodiment A52 under conditions such that the molecule is produced, and purifying the binding molecule. A54. The method of embodiment A53, wherein the host cell is a prokaryotic cell. A55. The method of embodiment A53, wherein the host cell is a eukaryotic cell.

[0556] In one set of embodiments, the following is provided: B1. Optionally, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient, and optionally, the molecule comprises an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), and a fragment crystallizable region (Fc region), wherein the scFv comprises a heavy chain variable region (VH), a linker (L), and a light chain variable region (VL), wherein the scFv comprises: a) A disulfide bond between a structurally conserved surface-exposed VH cysteine ​​(Cys) and L Cys; b) a structurally conserved disulfide bond between a surface-exposed VL Cys and L Cys; or c) A composition comprising a molecule according to any one of embodiments A1 to A49, wherein the molecule comprises a first disulfide bond between a structurally conserved, surface-exposed VH Cys and a first L Cys, and a second disulfide bond between a structurally conserved, surface-exposed VL Cys and a second L Cys. B2. a) VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position and L comprises an L Cys; b) VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position and L comprises an L Cys; or c) The composition of embodiment B1, wherein VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position, VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position, and L comprises a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys are capable of forming a disulfide bond, and the VL Cys and the second L Cys are capable of forming a disulfide bond. B3. The composition of embodiment B1 or B2, wherein the distance between VH Cys and VL Cys is from about 5 Å to about 10 Å, or from about 7 Å to about 9 Å. B4. The composition of any one of embodiments B1-B3, wherein the VH Cys is at H3, H5, H40, H43, H46, or H105, and residue numbering is according to Chothia. B5. The composition of any one of embodiments B1-B4, wherein VL Cys is at L3, L5, L39, L42, L43, L45, L100, or L102, and residue numbering is according to Chothia. B6. a) VH Cys is at H105 and VL Cys is at L42; or b) VH Cys is at H43 and VL Cys is at L100; or c) VH Cys is in H3 and VL Cys is in L3; d) VH Cys is in H3 and VL Cys is in L5; or e) VH Cys is at H3 and VL Cys is at L39; or f) VH Cys is at H3 and VL Cys is at L42; g) VH Cys is at H3 and VL Cys is at L45; or h) VH Cys is in H3 and VL Cys is in L100; i) VH Cys is at H3 and VL Cys is at L102; or j) VH Cys is in H5 and VL Cys is in L3; or k) VH Cys is in H5 and VL Cys is in L5; or l) VH Cys is at H5 and VL Cys is at L39; or m) VH Cys is at H5 and VL Cys is at L42; or n) VH Cys is at H5 and VL Cys is at L45; or o) VH Cys is at H5 and VL Cys is at L100; or p) VH Cys is at H5 and VL Cys is at L102; or q) VH Cys is in H40 and VL Cys is in L3; or r) VH Cys is in H40 and VL Cys is in L5; or s) VH Cys is at H40 and VL Cys is at L39; or t) VH Cys is at H40 and VL Cys is at L42; or u) VH Cys is at H40 and VL Cys is at L45; or v) VH Cys is at H40 and VL Cys is at L100; or w) VH Cys is at H40 and VL Cys is at L102; or x) VH Cys is at H43 and VL Cys is at L3; or y) VH Cys is at H43 and VL Cys is at L5; or z) VH Cys is at H43 and VL Cys is at L39; or aa) VH Cys is at H43 and VL Cys is at L42; or bb) VH Cys is at H43 and VL Cys is at L45; or cc) VH Cys is at H43 and VL Cys is at L102; or dd) VH Cys is at H46 and VL Cys is at L3; or ee) VH Cys is at H46 and VL Cys is at L5; or ff) VH Cys is at H46 and VL Cys is at L39; or gg) VH Cys is at H46 and VL Cys is at L42; or hh) VH Cys is at H46 and VL Cys is at L45; or ii) VH Cys is at H46 and VL Cys is at L100; or jj) VH Cys is at H46 and VL Cys is at L102; or kk) VH Cys is at H105 and VL Cys is at L3, or ll) VH Cys is at H105 and VL Cys is at L5; or mm) VH Cys is at H105 and VL Cys is at L39, or nn) VH Cys is at H105 and VL Cys is at L45; or oo) VH Cys is at H105 and VL Cys is at L100; or pp) VH Cys is at H105 and VL Cys is at L102, or qq) VH Cys is at H105 and VL Cys is at L43; The composition of any one of embodiments B1 to B5, wherein residue numbering is according to Chothia. The composition of any one of embodiments B1-B6, wherein B7.L comprises a contiguous amino acid sequence derived from an immunoglobulin (Ig) hinge region. B8. The composition of any one of embodiments B1-B7, wherein the Ig hinge region is derived from a human Ig hinge region or a non-human Ig hinge region. B9. The composition of any one of embodiments B1-B8, wherein the Ig hinge region is derived from a human Ig hinge region. B10. The composition of any one of embodiments B1-B9, wherein the human Ig hinge region is of the IgG1, IgG2, IgG3, or IgG4 isotype. B11.L has the 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 (Glu), glutamine (Gln), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), threonine (Thr), tryptophan (Trp), or tyrosine (Tyr); and y is an integer from 1 to 3. B12.L has the amino acid sequence C(X) y The composition of embodiment B11, comprising C (SEQ ID NO: 24), wherein X is Gly, Ser or Pro and y is an integer from 1 to 3. B13.L contains 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), CGGPC (SEQ ID NO: 41), CGGPC (SEQ ID NO: 42), CGGPC (SEQ ID NO: 43), CGGPC (SEQ ID NO: 44), CGGPC (SEQ ID NO: 45), CGGPC (SEQ ID NO: 46), CGGPC (SEQ ID NO: 47), CGGPC (SEQ ID NO: 48), CGGPC (SEQ ID NO: 49), CGGPC (SEQ ID NO: 50), CGGPC (SEQ ID NO: 51), CGGPC (SEQ ID NO: 52), CGGPC (SEQ ID NO: 53), CGGPC (SEQ ID NO: 54), CGGPC (SEQ ID NO: 55), CGGPC (SEQ ID NO: 56), CGGPC (SEQ ID NO: 57), CGGPC (SEQ ID NO: 58), CGGPC (SEQ ID NO: 59), CGGPC (SEQ ID NO: 60), CGGPC (SEQ ID NO: 61), CGGPC (SEQ ID NO: 62), CGGPC (SEQ ID NO: 63), CGGPC (SEQ ID NO: 64 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 composition of any one of embodiments B1 to B13, wherein B14.L comprises about 14 to about 19 amino acids, e.g., about 14, about 15, about 16, about 17, about 18, or about 19 amino acids. B15.L has the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 25), where X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gln, His, Ile, leu, Lys, Phe, Thr, Trp, or Tyr; m is an integer from 6 to 9; y is an integer from 1 to 3; and n is an integer from 4 to 6. B16.L has 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. B17.L has 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. The composition of any one of embodiments B1-B17, wherein B18.L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. B19. The composition of any one of embodiments B1 to B18, wherein the scFv is in a VL-L-VH orientation. The composition of any one of embodiments B1 to B18, wherein the B20.scFv is in a VH-L-VL orientation. B21. a) VH contains Cys at H105; b) VL contains a Cys at L42; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The composition of any one of embodiments B1 to B19, wherein the scFv is in a VL-L-VH orientation. B22. a) VH contains Cys at H105; b) VL contains a Cys at L45; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The composition of any one of embodiments B1 to B19, wherein the scFv is in a VL-L-VH orientation. B23. a) VH contains Cys at H105; b) VL contains a Cys at L39; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The composition of any one of embodiments B1 to B19, wherein the scFv is in a VL-L-VH orientation. B24. a) VH contains Cys at H5; b) VL contains a Cys at L42; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The composition of any one of embodiments B1 to B19, wherein the scFv is in a VL-L-VH orientation. B25. a) VH contains Cys at H5; b) VL contains a Cys at L45; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The composition of any one of embodiments B1 to B19, wherein the scFv is in a VL-L-VH orientation. B26. a) VH contains Cys at H5; b) VL contains a Cys at L39; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The composition of any one of embodiments B1 to B19, wherein the scFv is in a VL-L-VH orientation. B27. a) VH contains Cys at H3; b) VL contains a Cys at L42; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The composition of any one of embodiments B1 to B19, wherein the scFv is in a VL-L-VH orientation. B28. a) VH contains Cys at H3; b) VL contains a Cys at L45; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The composition of any one of embodiments B1 to B19, wherein the scFv is in a VL-L-VH orientation. B29. a) VH contains Cys at H3; b) VL contains a Cys at L39; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The composition of any one of embodiments B1 to B19, wherein the scFv is in a VL-L-VH orientation. B30. a) VH contains Cys at H43; b) VL contains a Cys at L100; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The composition of any one of embodiments B1 to B18 and B20, wherein the scFv is in a VH-L-VL orientation. B31. a) VH contains Cys at H43; b) VL contains a Cys at L102; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The composition of any one of embodiments B1 to B18 and B20, wherein the scFv is in a VH-L-VL orientation. B32. a) VH contains Cys at H43; b) VL contains a Cys at L5; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The composition of any one of embodiments B1 to B18 and B20, wherein the scFv is in a VH-L-VL orientation. B33. a) VH contains Cys at H43; b) VL contains a Cys at L3; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The composition of any one of embodiments B1 to B18 and B20, wherein the scFv is in a VH-L-VL orientation. B34. a) VH contains Cys at H40; b) VL contains a Cys at L100; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The composition of any one of embodiments B1 to B18 and B20, wherein the scFv is in a VH-L-VL orientation. B35. a) VH contains Cys at H40; b) VL contains a Cys at L102; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The composition of any one of embodiments B1 to B18 and B20, wherein the scFv is in a VH-L-VL orientation. B36. a) VH contains Cys at H40; b) VL contains a Cys at L5; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The composition of any one of embodiments B1 to B18 and B20, wherein the scFv is in a VH-L-VL orientation. B37. a) VH contains Cys at H40; b) VL contains a Cys at L3; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The composition of embodiment B1, wherein the scFv is in a VH-L-VL orientation. B38. a) VH contains Cys at H46; b) VL contains a Cys at L100; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The composition of any one of embodiments B1 to B18 and B20, wherein the scFv is in a VH-L-VL orientation. B39. a) VH contains Cys at H46; b) VL contains a Cys at L102; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The composition of any one of embodiments B1 to B18 and B20, wherein the scFv is in a VH-L-VL orientation. B40. a) VH contains Cys at H46; b) VL contains a Cys at L5; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The composition of any one of embodiments B1 to B18 and B20, wherein the scFv is in a VH-L-VL orientation. B41. a) VH contains Cys at H46; b) VL contains a Cys at L3; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The composition of any one of embodiments B1 to D18 and B20, wherein the scFv is in a VH-L-VL orientation. The composition of any one of embodiments B21 to B41, wherein B42.L comprises the amino acid sequence of SEQ ID NO:3. The composition of any one of embodiments B21 to B41, wherein B43.L comprises the amino acid sequence of SEQ ID NO:6. The composition of any one of embodiments B21 to B41, wherein B44.L comprises the amino acid sequence of SEQ ID NO:7. B45. The binding molecule comprises a heavy chain, a light chain, and a polypeptide; The N-termini of the heavy and light chains form Fab, the polypeptide comprises an scFv at the N-terminus, The composition of any one of embodiments B1 to B43, wherein the C-terminus of the polypeptide and the C-terminus of the heavy chain form an Fc region. B46. The composition of any one of embodiments B1 to B45, wherein the Fab binds to a tumor antigen and the scFv binds to a T cell antigen, optionally wherein the tumor antigen is BCMA and the T cell antigen is CD3. The composition of embodiment B46, wherein B47.scFv comprises the amino acid sequence of SEQ ID NO: 126 or SEQ ID NO: 128. B48. The composition of embodiment B46 or embodiment B47, wherein (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 132 and a VL comprising the amino acid sequence of SEQ ID NO: 129, or (ii) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 137 and a VL comprising the amino acid sequence of SEQ ID NO: 135. B49. a) VH contains Cys at H105; b) VL contains a Cys at L43; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The composition of embodiment B47 or embodiment B48, wherein the scFv is in a VL-L-VH orientation.

[0557] In one set of embodiments, the following is provided: C1. A method for producing a binding molecule, comprising introducing a polynucleotide encoding the molecule or a fragment thereof into a host cell, culturing the host cell under conditions such that the molecule is produced, and purifying the binding molecule, wherein the molecule comprises an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), and a fragment crystallizable region (Fc region), wherein the scFv comprises a heavy chain variable region (VH), a linker (L), and a light chain variable region (VL), wherein the scFv comprises: a) A disulfide bond between a structurally conserved surface-exposed VH cysteine ​​(Cys) and L Cys; b) a structurally conserved disulfide bond between a surface-exposed VL Cys and L Cys; or c) 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. C2. a) VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position and L comprises an L Cys; b) VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position and L comprises an L Cys; or c) The method of embodiment C1, wherein VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position, VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position, and L comprises a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys are capable of forming a disulfide bond, and the VL Cys and the second L Cys are capable of forming a disulfide bond. C3. The method of embodiment C1 or C2, wherein the distance between VH Cys and VL Cys is from about 5 Å to about 10 Å or from about 7 Å to about 9 Å. C4. The method of any one of embodiments C1 to C3, wherein the VH Cys is at H3, H5, H40, H43, H46, or H105, and residue numbering is according to Chothia. C5. The method of any one of embodiments C1 to C4, wherein VL Cys is at L3, L5, L39, L42, L43, L45, L100, or L102, and residue numbering is according to Chothia. C6. a) VH Cys is at H105 and VL Cys is at L42; or b) VH Cys is at H43 and VL Cys is at L100; or c) VH Cys is in H3 and VL Cys is in L3; d) VH Cys is in H3 and VL Cys is in L5; or e) VH Cys is at H3 and VL Cys is at L39; or f) VH Cys is at H3 and VL Cys is at L42; g) VH Cys is at H3 and VL Cys is at L45; or h) VH Cys is in H3 and VL Cys is in L100; i) VH Cys is at H3 and VL Cys is at L102; or j) VH Cys is in H5 and VL Cys is in L3; or k) VH Cys is in H5 and VL Cys is in L5; or l) VH Cys is at H5 and VL Cys is at L39; or m) VH Cys is at H5 and VL Cys is at L42; or n) VH Cys is at H5 and VL Cys is at L45; or o) VH Cys is at H5 and VL Cys is at L100; or p) VH Cys is at H5 and VL Cys is at L102; or q) VH Cys is in H40 and VL Cys is in L3; or r) VH Cys is in H40 and VL Cys is in L5; or s) VH Cys is at H40 and VL Cys is at L39; or t) VH Cys is at H40 and VL Cys is at L42; or u) VH Cys is at H40 and VL Cys is at L45; or v) VH Cys is at H40 and VL Cys is at L100; or w) VH Cys is at H40 and VL Cys is at L102; or x) VH Cys is at H43 and VL Cys is at L3; or y) VH Cys is at H43 and VL Cys is at L5; or z) VH Cys is at H43 and VL Cys is at L39; or aa) VH Cys is at H43 and VL Cys is at L42; or bb) VH Cys is at H43 and VL Cys is at L45; or cc) VH Cys is at H43 and VL Cys is at L102; or dd) VH Cys is at H46 and VL Cys is at L3; or ee) VH Cys is at H46 and VL Cys is at L5; or ff) VH Cys is at H46 and VL Cys is at L39; or gg) VH Cys is at H46 and VL Cys is at L42; or hh) VH Cys is at H46 and VL Cys is at L45; or ii) VH Cys is at H46 and VL Cys is at L100; or jj) VH Cys is at H46 and VL Cys is at L102; or kk) VH Cys is at H105 and VL Cys is at L3, or ll) VH Cys is at H105 and VL Cys is at L5; or mm) VH Cys is at H105 and VL Cys is at L39, or nn) VH Cys is at H105 and VL Cys is at L45; or oo) VH Cys is at H105 and VL Cys is at L100; or pp) VH Cys is at H105 and VL Cys is at L102, or qq) VH Cys is at H105 and VL Cys is at L43; The method of any one of embodiments C1 to C5, wherein residue numbering is according to Chothia. The method of any one of embodiments C1 to C6, wherein C7.L comprises a contiguous amino acid sequence derived from an immunoglobulin (Ig) hinge region. C8. The method of any one of embodiments C1-C7, wherein the Ig hinge region is derived from a human Ig hinge region or a non-human Ig hinge region. C9. The method of any one of embodiments C1-C8, wherein the Ig hinge region is derived from a human Ig hinge region. C10. The method of embodiment C9, wherein the human Ig hinge region is of the IgG1, IgG1, IgG2, IgG3, or IgG4 isotype. C11.L has the amino acid sequence C(X) y C (SEQ ID NO:23), wherein X is glycine (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), threonine (Thr), tryptophan (Trp), or tyrosine (Tyr); and y is an integer from 1 to 3. C12.L has the amino acid sequence C(X) y The method of embodiment C11, comprising C (SEQ ID NO: 24), wherein X is Gly, Ser, or Pro, and y is an integer from 1 to 3. C13.L has 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), CGGPC (SEQ ID NO: 41), CGGPC (SEQ ID NO: 42), CGGPC (SEQ ID NO: 43), CGGPC (SEQ ID NO: 44), CGGPC (SEQ ID NO: 45), CGGPC (SEQ ID NO: 46), CGGPC (SEQ ID NO: 47), CGGPC (SEQ ID NO: 48), CGGPC (SEQ ID NO: 49), CGGPC (SEQ ID NO: 50), CGGPC (SEQ ID NO: 51), CGGPC (SEQ ID NO: 52), CGGPC (SEQ ID NO: 53), CGGPC (SEQ ID NO: 54), CGGPC (SEQ ID NO: 55), CGGPC (SEQ ID NO: 56), CGGPC (SEQ ID NO: 57), CGGPC (SEQ ID NO: 58), CGGPC (SEQ ID NO: 59), CGGPC (SEQ ID NO: 60), CGGPC (SEQ ID NO: 61), CGGPC (SEQ ID NO: 62), CGGPC (SEQ ID NO: 63), CGGPC (SEQ ID NO: 64 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). C14. The method of any one of embodiments C1 to C13, wherein L comprises about 14 to about 19 amino acids, e.g., about 14, about 15, about 16, about 17, about 18, or about 19 amino acids, and / or L has a length of about 14 to about 19 amino acids, e.g., about 14, about 15, about 16, about 17, about 18, or about 19 amino acids. C15.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. C16.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. C17.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. The method of any one of embodiments C1 to C17, wherein C18.L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. The method of any one of embodiments C1 to C18, wherein the C19.scFv is in a VL-L-VH orientation. The method of any one of embodiments C1 to C18, wherein the C20.scFv is in a VH-L-VL orientation. C21. a) VH contains Cys at H105; b) VL contains a Cys at L42; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; The method of any one of embodiments C1 to C19, wherein d) the scFv is in a VL-L-VH orientation. C22. a) VH contains Cys at H105; b) VL contains a Cys at L45; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; The method of any one of embodiments C1 to C19, wherein d) the scFv is in a VL-L-VH orientation. C23. a) VH contains Cys at H105; b) VL contains a Cys at L39; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; The method of any one of embodiments C1 to C19, wherein d) the scFv is in a VL-L-VH orientation. C24. a) VH contains Cys at H5; b) VL contains a Cys at L42; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; The method of any one of embodiments C1 to C19, wherein d) the scFv is in a VL-L-VH orientation. C25. a) VH contains Cys at H5; b) VL contains a Cys at L45; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; The method of any one of embodiments C1 to C19, wherein d) the scFv is in a VL-L-VH orientation. C26. a) VH contains Cys at H5; b) VL contains a Cys at L39; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; The method of any one of embodiments C1 to C19, wherein d) the scFv is in a VL-L-VH orientation. C27. a) VH contains Cys at H3; b) VL contains a Cys at L42; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; The method of any one of embodiments C1 to C19, wherein d) the scFv is in a VL-L-VH orientation. C28. a) VH contains Cys at H3; b) VL contains a Cys at L45; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; The method of any one of embodiments C1 to C19, wherein d) the scFv is in a VL-L-VH orientation. C29. a) VH contains Cys at H3; b) VL contains a Cys at L39; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; The method of any one of embodiments C1 to C19, wherein d) the scFv is in a VL-L-VH orientation. C30. a) VH contains Cys at H43; b) VL contains a Cys at L100; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments C1 to C18 and C20, wherein the scFv is in a VH-L-VL orientation. C31. a) VH contains Cys at H43; b) VL contains a Cys at L102; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments C1 to C18 and C20, wherein the scFv is in a VH-L-VL orientation. C32. a) VH contains Cys at H43; b) VL contains a Cys at L5; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments C1 to C18 and C20, wherein the scFv is in a VH-L-VL orientation. C33. a) VH contains Cys at H43; b) VL contains a Cys at L3; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments C1 to C18 and C20, wherein the scFv is in a VH-L-VL orientation. C34. a) VH contains Cys at H40; b) VL contains a Cys at L100; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments C1 to C18 and C20, wherein the scFv is in a VH-L-VL orientation. C35. a) VH contains Cys at H40; b) VL contains a Cys at L102; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments C1 to C18 and C20, wherein the scFv is in a VH-L-VL orientation. C36. a) VH contains Cys at H40; b) VL contains a Cys at L5; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments C1 to C18 and C20, wherein the scFv is in a VH-L-VL orientation. C37. a) VH contains Cys at H40; b) VL contains a Cys at L3; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments C1 to C18 and C20, wherein the scFv is in a VH-L-VL orientation. C38. a) VH contains Cys at H46; b) VL contains a Cys at L100; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments C1 to C18 and C20, wherein the scFv is in a VH-L-VL orientation. C39. a) VH contains Cys at H46; b) VL contains a Cys at L102; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments C1 to C18 and C20, wherein the scFv is in a VH-L-VL orientation. C40. a) VH contains Cys at H46; b) VL contains a Cys at L5; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments C1 to C18 and C20, wherein the scFv is in a VH-L-VL orientation. C41. a) VH contains Cys at H46; b) VL contains a Cys at L3; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments C1 to C18 and C20, wherein the scFv is in a VH-L-VL orientation. The method of any one of embodiments C21 to C41, wherein C42.L comprises the amino acid sequence of SEQ ID NO:3. The method of any one of embodiments C21 to C41, wherein C43.L comprises the amino acid sequence of SEQ ID NO:6. The method of any one of embodiments C21 to C41, wherein C44.L comprises the amino acid sequence of SEQ ID NO:7. C45. The binding molecule comprises a heavy chain, a light chain, and a polypeptide; The N-termini of the heavy and light chains form Fab, the polypeptide comprises an scFv at the N-terminus, The method of embodiment C1, wherein the C-terminus of the polypeptide and the C-terminus of the heavy chain form an Fc region. C46. The method of any one of embodiments C1 to C45, wherein the Fab binds to a tumor antigen and the scFv binds to a T cell antigen, optionally wherein the tumor antigen is BCMA and the T cell antigen is CD3. The method of embodiment C46, ​​wherein C47.scFv comprises the amino acid sequence of SEQ ID NO: 126 or SEQ ID NO: 128. C48. The method of embodiment C46 or embodiment C47, wherein (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 132 and a VL comprising the amino acid sequence of SEQ ID NO: 129, or (ii) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 137 and a VL comprising the amino acid sequence of SEQ ID NO: 135. C49. a) VH contains Cys at H105; b) VL contains a Cys at L43; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; The method of embodiment C47 or embodiment C48, wherein the scFv is in a VL-L-VH orientation. C50. The method of any one of embodiments C1-C49, wherein the host cell is a prokaryotic cell. C51. The method of any one of embodiments C1-C49, wherein the host cell is a eukaryotic cell.

[0558] In one set of embodiments, the following is provided: D1. A method for directing or engaging a cell to a target cell, comprising contacting the target cell with a binding molecule; The molecules include an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), and a fragment crystallizable region (Fc region), wherein the scFv includes a heavy chain variable region (VH), a linker (L), and a light chain variable region (VL), and wherein the scFv includes: a) A disulfide bond between a structurally conserved surface-exposed VH cysteine ​​(Cys) and L Cys; b) a structurally conserved disulfide bond between a surface-exposed VL Cys and L Cys; or c) 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 method wherein the Fab binds to a first antigen on the target cell and the scFv binds to a second antigen on the cell. D2. a) VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position and L comprises an L Cys; b) VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position and L comprises an L Cys; or c) The method of embodiment D1, wherein VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position, VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position, and L comprises a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys are capable of forming a disulfide bond, and the VL Cys and the second L Cys are capable of forming a disulfide bond. D3. The method of embodiment D1 or D2, wherein the distance between VH Cys and VL Cys is from about 7 Å to about 9 Å, or from about 7 Å to about 9 Å. D4. The method of any one of embodiments D1-D3, wherein the VH Cys is at H3, H5, H40, H43, H46, or H105, and residue numbering is according to Chothia. D5. The method of any one of embodiments D1-D4, wherein VL Cys is at L3, L5, L39, L42, L43, L45, L100, or L102, and residue numbering is according to Chothia. D6. a) VH Cys is at H105 and VL Cys is at L42; or b) VH Cys is at H43 and VL Cys is at L100; or c) VH Cys is in H3 and VL Cys is in L3; d) VH Cys is in H3 and VL Cys is in L5; or e) VH Cys is at H3 and VL Cys is at L39; or f) VH Cys is at H3 and VL Cys is at L42; g) VH Cys is at H3 and VL Cys is at L45; or h) VH Cys is in H3 and VL Cys is in L100; i) VH Cys is at H3 and VL Cys is at L102; or j) VH Cys is in H5 and VL Cys is in L3; or k) VH Cys is in H5 and VL Cys is in L5; or l) VH Cys is at H5 and VL Cys is at L39; or m) VH Cys is at H5 and VL Cys is at L42; or n) VH Cys is at H5 and VL Cys is at L45; or o) VH Cys is at H5 and VL Cys is at L100; or p) VH Cys is at H5 and VL Cys is at L102; or q) VH Cys is in H40 and VL Cys is in L3; or r) VH Cys is in H40 and VL Cys is in L5; or s) VH Cys is at H40 and VL Cys is at L39; or t) VH Cys is at H40 and VL Cys is at L42; or u) VH Cys is at H40 and VL Cys is at L45; or v) VH Cys is at H40 and VL Cys is at L100; or w) VH Cys is at H40 and VL Cys is at L102; or x) VH Cys is at H43 and VL Cys is at L3; or y) VH Cys is at H43 and VL Cys is at L5; or z) VH Cys is at H43 and VL Cys is at L39; or aa) VH Cys is at H43 and VL Cys is at L42; or bb) VH Cys is at H43 and VL Cys is at L45; or cc) VH Cys is at H43 and VL Cys is at L102; or dd) VH Cys is at H46 and VL Cys is at L3; or ee) VH Cys is at H46 and VL Cys is at L5; or ff) VH Cys is at H46 and VL Cys is at L39; or gg) VH Cys is at H46 and VL Cys is at L42; or hh) VH Cys is at H46 and VL Cys is at L45; or ii) VH Cys is at H46 and VL Cys is at L100; or jj) VH Cys is at H46 and VL Cys is at L102; or kk) VH Cys is at H105 and VL Cys is at L3, or ll) VH Cys is at H105 and VL Cys is at L5; or mm) VH Cys is at H105 and VL Cys is at L39, or nn) VH Cys is at H105 and VL Cys is at L45; or oo) VH Cys is at H105 and VL Cys is at L100; or pp) VH Cys is at H105 and VL Cys is at L102, or qq) VH Cys is at H105 and VL Cys is at L43; The method of any one of embodiments D1 to D5, wherein residue numbering is according to Chothia. The method of any one of embodiments D1-D6, wherein D7.L comprises a contiguous amino acid sequence derived from an immunoglobulin (Ig) hinge region. D8. The method of any one of embodiments D1-D7, wherein the Ig hinge region is derived from a human Ig hinge region or a non-human Ig hinge region. D9. The method of any one of embodiments D1-D8, wherein the Ig hinge region is derived from a human Ig hinge region. D10. The method of any one of embodiments D1-D9, wherein the human Ig hinge region is of the IgG1, IgG2, IgG3, or IgG4 isotype. D11.L has the amino acid sequence C(X) y C (SEQ ID NO: 23), wherein X is glycine (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), threonine (Thr), tryptophan (Trp), or tyrosine (Tyr); and y is an integer from 1 to 3. D12.L has the amino acid sequence C(X) y The method of embodiment D11, comprising C (SEQ ID NO: 24), wherein X is Gly, Ser, or Pro, and y is an integer from 1 to 3. D13.L contains 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), CGGPC (SEQ ID NO: 41), CGGPC (SEQ ID NO: 42), CGGPC (SEQ ID NO: 43), CGGPC (SEQ ID NO: 44), CGGPC (SEQ ID NO: 45), CGGPC (SEQ ID NO: 46), CGGPC (SEQ ID NO: 47), CGGPC (SEQ ID NO: 48), CGGPC (SEQ ID NO: 49), CGGPC (SEQ ID NO: 50), CGGPC (SEQ ID NO: 51), CGGPC (SEQ ID NO: 52), CGGPC (SEQ ID NO: 53), CGGPC (SEQ ID NO: 54), CGGPC (SEQ ID NO: 55), CGGPC (SEQ ID NO: 56), CGGPC (SEQ ID NO: 57), CGGPC (SEQ ID NO: 58), CGGPC (SEQ ID NO: 59), CGGPC (SEQ ID NO: 60), CGGPC (SEQ ID NO: 61), CGGPC (SEQ ID NO: 62), CGGPC (SEQ ID NO: 63), CGGPC (SEQ ID NO: 64 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). D14. The method of any one of embodiments D1 to D13, wherein L comprises about 14 to about 19 amino acids, e.g., about 14, about 15, about 16, about 17, about 18, or about 19 amino acids, and / or L has a length of about 14 to about 19 amino acids, e.g., about 14, about 15, about 16, about 17, about 18, or about 19 amino acids. D15.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 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. D16.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. D17.L has 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. The method of any one of embodiments D1 to D17, wherein D18.L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. D19. The method of any one of embodiments D1 to D18, wherein the scFv is in a VL-L-VH orientation. D20. The method of any one of embodiments D1 to D18, wherein the scFv is in a VH-L-VL orientation. D21. a) VH contains Cys at H105; b) VL contains a Cys at L42; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments D1 to D19, wherein the scFv is in a VL-L-VH orientation. D22. a) VH contains Cys at H105; b) VL contains a Cys at L45; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments D1 to D19, wherein the scFv is in a VL-L-VH orientation. D23. a) VH contains Cys at H105; b) VL contains a Cys at L39; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments D1 to D19, wherein the scFv is in a VL-L-VH orientation. D24. a) VH contains Cys at H5; b) VL contains a Cys at L42; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments D1 to D19, wherein the scFv is in a VL-L-VH orientation. D25. a) VH contains Cys at H5; b) VL contains a Cys at L45; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments D1 to D19, wherein the scFv is in a VL-L-VH orientation. D26. a) VH contains Cys at H5; b) VL contains a Cys at L39; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of embodiment D1, wherein the scFv is in a VL-L-VH orientation. D27. a) VH contains Cys at H3; b) VL contains a Cys at L42; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments D1 to D19, wherein the scFv is in a VL-L-VH orientation. D28. a) VH contains Cys at H3; b) VL contains a Cys at L45; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments D1 to D19, wherein the scFv is in a VL-L-VH orientation. D29. a) VH contains Cys at H3; b) VL contains a Cys at L39; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments D1 to D19, wherein the scFv is in a VL-L-VH orientation. D30. a) VH contains Cys at H43; b) VL contains a Cys at L100; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments D1 to D18 and D20, wherein the scFv is in a VH-L-VL orientation. D31. a) VH contains Cys at H43; b) VL contains a Cys at L102; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments D1 to D18 and D20, wherein the scFv is in a VH-L-VL orientation. D32. a) VH contains Cys at H43; b) VL contains a Cys at L5; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments D1 to D18 and D20, wherein the scFv is in a VH-L-VL orientation. D33. a) VH contains Cys at H43; b) VL contains a Cys at L3; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments D1 to D18 and D20, wherein the scFv is in a VH-L-VL orientation. D34. a) VH contains Cys at H40; b) VL contains a Cys at L100; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments D1 to D18 and D20, wherein the scFv is in a VH-L-VL orientation. D35. a) VH contains Cys at H40; b) VL contains a Cys at L102; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments D1 to D18 and D20, wherein the scFv is in a VH-L-VL orientation. D36. a) VH contains Cys at H40; b) VL contains a Cys at L5; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments D1 to D18 and D20, wherein the scFv is in a VH-L-VL orientation. D37. a) VH contains Cys at H40; b) VL contains a Cys at L3; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments D1 to D18 and D20, wherein the scFv is in a VH-L-VL orientation. D38. a) VH contains Cys at H46; b) VL contains a Cys at L100; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments D1 to D18 and D20, wherein the scFv is in a VH-L-VL orientation. D39. a) VH contains Cys at H46; b) VL contains a Cys at L102; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments D1 to D18 and D20, wherein the scFv is in a VH-L-VL orientation. D40. a) VH contains Cys at H46; b) VL contains a Cys at L5; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments D1 to D18 and D20, wherein the scFv is in a VH-L-VL orientation. D41. a) VH contains Cys at H46; b) VL contains a Cys at L3; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of any one of embodiments D1 to D18 and D20, wherein the scFv is in a VH-L-VL orientation. The method of any one of embodiments D21 to D41, wherein D42.L comprises the amino acid sequence of SEQ ID NO:3. The method of any one of embodiments D21 to D41, wherein D43.L comprises the amino acid sequence of SEQ ID NO:6. The method of any one of embodiments D21 to D41, wherein D44.L comprises the amino acid sequence of SEQ ID NO:7. D45. The binding molecule comprises a heavy chain, a light chain, and a polypeptide; The N-termini of the heavy and light chains form Fab, the polypeptide comprises an scFv at the N-terminus, The method of any one of embodiments D1 to D44, wherein the C-terminus of the polypeptide and the C-terminus of the heavy chain form an Fc region. D46. The method of any one of embodiments D1 to D45, wherein the Fab binds to a tumor antigen and the scFv binds to a T cell antigen, optionally wherein the tumor antigen is BCMA and the T cell antigen is CD3. The method of embodiment D46, wherein D47.scFv comprises the amino acid sequence of SEQ ID NO: 126 or SEQ ID NO: 128. D48. The method of embodiment D46 or embodiment D47, wherein the Fab comprises (i) a VH comprising the amino acid sequence of SEQ ID NO: 132 and a VL comprising the amino acid sequence of SEQ ID NO: 129, or (ii) a VH comprising the amino acid sequence of SEQ ID NO: 137 and a VL comprising the amino acid sequence of SEQ ID NO: 135. D49. a) VH contains Cys at H105; b) VL contains a Cys at L43; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; The method of embodiment B47 or embodiment B48, wherein the scFv is in a VL-L-VH orientation. D50. The method of any one of embodiments D1-D49, wherein the target cells are tumor cells, thereby eliminating the tumor cells. D51. The method of any one of embodiments D1-D50 for treating a disease or disorder in a subject. D52. The method of embodiment D51, wherein the disease or disorder is a tumor, optionally, the disease or disorder is cancer. D53. The method of embodiment D51, wherein the subject is a human subject. D54. The method of any one of embodiments D1-D53, wherein the cell is an immune cell. D54. The method of any one of embodiments D1-D53, wherein the cell is a T cell.

[0559] In one set of embodiments, the following is provided: E1. The present disclosure provides a molecule comprising an antigen-binding fragment (Fab) that binds to a first antigen, a single-chain variable fragment (scFv) that binds to a second antigen, and a fragment crystallizable region (Fc region), wherein the scFv comprises a means for stabilizing the scFv. The E2.scFv comprises a heavy chain variable region (VH), a linker (L), and a light chain variable region (VL), and the means for stabilizing the scFv comprises: a) A disulfide bond between a structurally conserved surface-exposed VH cysteine ​​(Cys) and L Cys; b) a structurally conserved disulfide bond between a surface-exposed VL Cys and L Cys; or c) The molecule of embodiment E1, 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. E3. a) VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position and L comprises an L Cys; b) VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position and L comprises an L Cys; or c) The molecule of embodiment E2, wherein VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position, VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position, and L comprises a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys are capable of forming a disulfide bond, and the VL Cys and the second L Cys are capable of forming a disulfide bond. E4. The molecule of embodiment E2 or E3, wherein the distance between the VH Cys and the VL Cys is from about 5 Å to about 10 Å or from about 7 Å to about 9 Å. E5. The molecule of any one of embodiments E1-E3, wherein the VH Cys is at H3, H5, H40, H43, H46, or H105, and / or the VL Cys is at L3, L5, L39, L42, L43, L45, L100, or L102, and wherein residue numbering is according to Chothia. E6. a) VH Cys is at H105 and VL Cys is at L42; or b) VH Cys is at H43 and VL Cys is at L100; or c) VH Cys is in H3 and VL Cys is in L3; d) VH Cys is in H3 and VL Cys is in L5; or e) VH Cys is at H3 and VL Cys is at L39; or f) VH Cys is at H3 and VL Cys is at L42; g) VH Cys is at H3 and VL Cys is at L45; or h) VH Cys is in H3 and VL Cys is in L100; i) VH Cys is at H3 and VL Cys is at L102; or j) VH Cys is in H5 and VL Cys is in L3; or k) VH Cys is in H5 and VL Cys is in L5; or l) VH Cys is at H5 and VL Cys is at L39; or m) VH Cys is at H5 and VL Cys is at L42; or n) VH Cys is at H5 and VL Cys is at L45; or o) VH Cys is at H5 and VL Cys is at L100; or p) VH Cys is at H5 and VL Cys is at L102; or q) VH Cys is in H40 and VL Cys is in L3; or r) VH Cys is in H40 and VL Cys is in L5; or s) VH Cys is at H40 and VL Cys is at L39; or t) VH Cys is at H40 and VL Cys is at L42; or u) VH Cys is at H40 and VL Cys is at L45; or v) VH Cys is at H40 and VL Cys is at L100; or w) VH Cys is at H40 and VL Cys is at L102; or x) VH Cys is at H43 and VL Cys is at L3; or y) VH Cys is at H43 and VL Cys is at L5; or z) VH Cys is at H43 and VL Cys is at L39; or aa) VH Cys is at H43 and VL Cys is at L42; or bb) VH Cys is at H43 and VL Cys is at L45; or cc) VH Cys is at H43 and VL Cys is at L102; or dd) VH Cys is at H46 and VL Cys is at L3; or ee) VH Cys is at H46 and VL Cys is at L5; or ff) VH Cys is at H46 and VL Cys is at L39; or gg) VH Cys is at H46 and VL Cys is at L42; or hh) VH Cys is at H46 and VL Cys is at L45; or ii) VH Cys is at H46 and VL Cys is at L100; or jj) VH Cys is at H46 and VL Cys is at L102; or kk) VH Cys is at H105 and VL Cys is at L3, or ll) VH Cys is at H105 and VL Cys is at L5; or mm) VH Cys is at H105 and VL Cys is at L39, or nn) VH Cys is at H105 and VL Cys is at L45; or oo) VH Cys is at H105 and VL Cys is at L100; or pp) VH Cys is at H105 and VL Cys is at L102, or qq) VH Cys is at H105 and VL Cys is at L43; The molecule of any one of embodiments E1 to E5, wherein residue numbering is according to Chothia. The molecule of any one of embodiments E1 to E6, wherein E7.L comprises a contiguous amino acid sequence derived from an immunoglobulin (Ig) hinge region. E8. The molecule of any one of embodiments E1-E7, wherein the Ig hinge region is derived from a human Ig hinge region or a non-human Ig hinge region. E9. The molecule of any one of embodiments E1-E8, wherein the Ig hinge region is derived from a human Ig hinge region. E10. The molecule of any one of embodiments E1-E9, wherein the human Ig hinge region is of the IgG1, IgG2, IgG3, or IgG4 isotype. E11.L has the amino acid sequence C(X) y C (SEQ ID NO:23), wherein X is glycine (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), threonine (Thr), tryptophan (Trp), or tyrosine (Tyr); and y is an integer from 1 to 3. E12.L has the amino acid sequence C(X) y The molecule of embodiment E11, comprising C (SEQ ID NO: 24), wherein X is Gly, Ser or Pro and y is an integer from 1 to 3. E13.L contains 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 ( The molecule of any one of embodiments E1 to E12, comprising 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). E14.L is a molecule of any one of embodiments E1 to E13 comprising about 14 to about 19 amino acids, e.g., about 14, about 15, about 16, about 17, about 18, or about 19 amino acids. E15.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 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. E16.L has 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. E17.L has 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. The molecule of any one of embodiments E1 to E17, wherein E18.L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. E19. The molecule of any one of embodiments E1 to E18, wherein the scFv is in a VL-L-VH orientation. E20. The molecule of any one of embodiments E1 to E18, wherein the scFv is in a VH-L-VL orientation. E21. a) VH contains Cys at H105; b) VL contains a Cys at L42; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments E1 to E19, wherein the scFv is in a VL-L-VH orientation. E22. a) VH contains Cys at H105; b) VL contains a Cys at L45; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments E1 to E18, wherein the scFv is in a VL-L-VH orientation. E23. a) VH contains Cys at H105; b) VL contains a Cys at L39; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments E1 to E18, wherein the scFv is in a VL-L-VH orientation. E24. a) VH contains Cys at H5; b) VL contains a Cys at L42; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments E1 to E18, wherein the scFv is in a VL-L-VH orientation. E25. a) VH contains Cys at H5; b) VL contains a Cys at L45; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments E1 to E18, wherein the scFv is in a VL-L-VH orientation. E26. a) VH contains Cys at H5; b) VL contains a Cys at L39; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments E1 to E18, wherein the scFv is in a VL-L-VH orientation. E27. a) VH contains Cys at H3; b) VL contains a Cys at L42; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments E1 to E18, wherein the scFv is in a VL-L-VH orientation. E28. a) VH contains Cys at H3; b) VL contains a Cys at L45; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments E1 to E18, wherein the scFv is in a VL-L-VH orientation. E29. a) VH contains Cys at H3; b) VL contains a Cys at L39; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments E1 to E18, wherein the scFv is in a VL-L-VH orientation. E30. a) VH contains Cys at H43; b) VL contains a Cys at L100; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments E1 to E18 and E20, wherein the scFv is in a VH-L-VL orientation. E31. a) VH contains Cys at H43; b) VL contains a Cys at L102; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments E1 to E18 and E20, wherein the scFv is in a VH-L-VL orientation. E32. a) VH contains Cys at H43; b) VL contains a Cys at L5; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments E1 to E18 and E20, wherein the scFv is in a VH-L-VL orientation. E33. a) VH contains Cys at H43; b) VL contains a Cys at L3; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments E1 to E18 and E20, wherein the scFv is in a VH-L-VL orientation. E34. a) VH contains Cys at H40; b) VL contains a Cys at L100; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments E1 to E18 and E20, wherein the scFv is in a VH-L-VL orientation. E35. a) VH contains Cys at H40; b) VL contains a Cys at L102; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments E1 to E18 and E20, wherein the scFv is in a VH-L-VL orientation. E36. a) VH contains Cys at H40; b) VL contains a Cys at L5; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments E1 to E18 and E20, wherein the scFv is in a VH-L-VL orientation. E37. a) VH contains Cys at H40; b) VL contains a Cys at L3; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments E1 to E18 and E20, wherein the scFv is in a VH-L-VL orientation. E38. a) VH contains Cys at H46; b) VL contains a Cys at L100; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments E1 to E18 and E20, wherein the scFv is in a VH-L-VL orientation. E39. a) VH contains Cys at H46; b) VL contains a Cys at L102; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments E1 to E18 and E20, wherein the scFv is in a VH-L-VL orientation. E40. a) VH contains Cys at H46; b) VL contains a Cys at L5; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments E1 to E18 and E20, wherein the scFv is in a VH-L-VL orientation. E41. a) VH contains Cys at H46; b) VL contains a Cys at L3; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The molecule of any one of embodiments E1 to E18 and E20, wherein the scFv is in a VH-L-VL orientation. The molecule of any one of embodiments E21 to E41, wherein E42.L comprises the amino acid sequence of SEQ ID NO:3. The molecule of any one of embodiments E21 to E41, wherein E43.L comprises the amino acid sequence of SEQ ID NO:6. The molecule of any one of embodiments E21 to E41, wherein E44.L comprises the amino acid sequence of SEQ ID NO:7. E45. The binding molecule comprises a heavy chain, a light chain, and a polypeptide; The N-termini of the heavy and light chains form Fab, the polypeptide comprises an scFv at the N-terminus, The molecule of any one of embodiments E1 to E44, wherein the C-terminus of the polypeptide and the C-terminus of the heavy chain form an Fc region. E46. The molecule of any one of embodiments E1 to E45, wherein the Fab binds to a tumor antigen and the scFv binds to a T cell antigen, optionally wherein the tumor antigen is BCMA and the T cell antigen is CD3. The molecule of embodiment E46, wherein E47.scFv comprises the amino acid sequence of SEQ ID NO: 126 or SEQ ID NO: 128. E48. The molecule of embodiment E46 or embodiment E47, wherein (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 132 and a VL comprising the amino acid sequence of SEQ ID NO: 129, or (ii) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 137 and a VL comprising the amino acid sequence of SEQ ID NO: 135. E49. a) VH contains Cys at H105; b) VL contains a Cys at L43; c) L comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; d) The method of embodiment E47 or embodiment E48, wherein the scFv is in a VL-L-VH orientation. E50. A means for producing a molecule according to any one of embodiments E1-E49. E51. A method for directing or engaging a cell to a target cell, comprising contacting the target cell with a molecule of any one of embodiments E1-E49. E52. A method for eliminating or inhibiting target cells, comprising contacting the target cells with a molecule according to any one of embodiments E1-E49. E53. A method for treating a disease or disorder in a subject, comprising administering to the subject a molecule according to any one of embodiments E1-E49.

[0560] In one set of embodiments, the following is provided: F1. A molecule of any one of embodiments A1 to A55 for use in medicine. F2. A molecule of any one of embodiments A1-A55 for use in treating a disease or disorder.

[0561] Specific embodiments of the present invention are described herein. Upon reading the foregoing description, it is expected that variations of the disclosed embodiments may become apparent to those skilled in the art, who may employ such variations as necessary. Accordingly, it is intended that the present invention be practiced otherwise than as specifically described herein, and that the present invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, any combination of the above-described elements in all possible variations thereof is encompassed herein unless otherwise indicated herein or otherwise clearly contradicted by context. Many embodiments of the present invention have been described. However, it will be understood that various modifications can be made without departing from the spirit and scope of the present invention. Accordingly, the description in the Examples section is intended to illustrate, but not limit, the scope of the invention as defined by the claims. [Example]

[0562] 6.1 Example 1: CD3 / CRIS7A and CD3 / CRIS7B scFv and SPFV stabilization 6.1.1 Expression and Purification of Anti-CD3 Cris7a and Cris7b scFv / spFv All scFv and spFv molecules were cloned into a CMV promoter-driven mammalian expression vector. These constructs were transfected into Expi293 cells using the manufacturer's protocol, and the cells were cultured for 5 days. Each protein was purified from the clarified supernatant on a 1 mL His-TRAPHP column (GE Healthcare) using the AktaXpress system (GE Healthcare). The column was prepared with a 0-100% gradient of elution buffer (wash buffer: 50 mM Tris, pH 7.5, 500 mM NaCl, 20 mM imidazole; elution buffer: 50 mM Tris, pH 7.5, 500 mM NaCl, 500 mM imidazole) to remove loosely bound nickel, and then re-equilibrated with DPBS. The clarified supernatant was first adjusted to 50 mM Tris, pH 7.5, and 20 mM imidazole and then loaded onto a 1 mL HisTRAP HP column at 0.8 mL / min at 4°C. The column was then washed with PBS until a stable baseline was obtained. The column was then further washed with 20 CV of wash buffer and eluted with elution buffer into a single injection loop. The column was then desalted on a 26 / 10 HiPrep desalting column in 1x DPBS, and fractions were collected. Fractions containing purified protein were then pooled and concentrated. The scFv and spFv proteins were dialyzed into DPBS for thermal stability measurements.

[0563] scFv / spFv stabilization by differential scanning calorimetry (DSC) The conformational stability of Cris7a or Cris7b scFv and their stapled spFvs was measured by differential scanning calorimetry (DSC) using a Microcal Capillary DSC instrument (Malvern Instruments) equipped with an autosampler. Samples containing matching buffer were scanned at a rate of 60 °C / h over the range of 25–100 °C without the feedback option. Six buffer-buffer-only scans were performed before the protein sample to establish a thermal history and a stable baseline. Raw DSC data were subjected to buffer blank subtraction and normalized by their protein concentration and baseline subtraction. The processed data were fitted using a non-two-state transition model using Origin 7 software (version 7.0552). Iterative curve fitting was performed to derive thermodynamic parameters related to melting, e.g., thermal stability, enthalpy.

[0564] 6.1.2 Surface plasmon resonance Binding of Cris7b scFv and stapled spFv to the CD3 antigen was measured by surface plasmon resonance using a Biacore 8K instrument (Cytiva, formerly GE Healthcare) at 25°C. Goat anti-human Fcγ protein (Jackson ImmunoResearch 109-005-098) was directly immobilized on a CM4 chip (Series S CM4 Sensor chip, Cat. No. BR100534) using standard amine coupling. Approximately 4000 Rus was ultimately immobilized on each channel. Samples containing Cris7b scFv or spFv-containing bispecific antibodies were captured by the anti-human Fcγ surface at levels ranging from 100 to 250 Rus, followed by binding of a series of five antigen concentrations, starting at 300 nM, of human CD3E-CD3D heterodimer protein (Acro Cat. No. CDDH52W1) in 3-fold dilutions (300 nM to 3.7 nM) using a single-cycle kinetic method. Association and dissociation times were 150 and 600 seconds, respectively. To remove captured / bound antibody / antigen complexes before the next interaction round, the surface was regenerated using 0.85% phosphoric acid with three short pulses of 20 seconds each at a flow rate of 50 μl / min. The running buffer was 0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.05% v / v surfactant P20. Raw binding data were processed by double referencing by subtracting 1) the signal from antigen binding to an empty chip surface (FC1 on each channel) and 2) the signal from an ongoing buffer blank injection. The processed data were then subjected to a 1:1 simple Langmuir binding model analysis to derive kinetic (k, k) and affinity (K) parameters using Biacore Insight Evaluation software version 2 (Cytiva).

[0565] 6.1.3 Comparison of Stability of Anti-CD3 Cris7a and Cris7b scFv / spFv Cris7a and Cris7b were derived from anti-CD3 variants of CRIS7 with T cell redirecting potential. The T of their scFv moieties was less than ideally thermostable (Figure 2A), with T of 59.7°C a...

Claims

1. A molecule comprising an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), and a fragment crystallizable region (Fc region), wherein the scFv comprises a heavy chain variable region (VH), a linker (L), and a light chain variable region (VL), and the scFv comprises: a) a disulfide bond between a structurally conserved surface-exposed VH position mutated to a cysteine ​​(Cys) and L Cys; b) a disulfide bond between a structurally conserved, surface-exposed VL position mutated to Cys and L Cys; or c) A 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.

2. 10. The molecule of claim 1, having improved stability, expression yield, and / or quality compared to a comparable molecule lacking disulfide bonds.

3. A molecule comprising an Fab that binds to a first antigen, an scFv that binds to a second antigen, and an Fc region, wherein the scFv comprises a means for stabilizing the scFv.

4. the scFv comprises a VH, L, and VL, and the means for stabilizing the scFv comprises: a) a structurally conserved disulfide bond between the surface-exposed VH Cys and L Cys; b) a structurally conserved disulfide bond between the surface-exposed VL Cys and L Cys; or c) 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.

5. a) the VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position, and the L comprises an L Cys; b) the VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position and the L comprises an L Cys; or c) The molecule of claim 1, wherein the VH comprises a VH Cys at a structurally conserved, surface-exposed VH framework residue position, the VL comprises a VL Cys at a structurally conserved, surface-exposed VL framework residue position, the L comprises a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys are capable of forming a disulfide bond, and the VL Cys and the second L Cys are capable of forming a disulfide bond.

6. 2. The molecule of claim 1, wherein the distance between the VH Cys and the VL Cys is from about 5 Å to about 10 Å or from about 7 Å to about 9 Å.

7. a) the VH Cys is at H3, H5, H40, H43, H46, or H105; and / or b) the VL Cys is at L3, L5, L39, L42, L43, L45, L100, or L102; The molecule of claim 1 , wherein residue numbering is according to Chothia.

8. 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; pp) the VH Cys is at H105 and the VL Cys is at L102; or qq) the VH Cys is at H105 and the VL Cys is at L43; The molecule of claim 1 , wherein residue numbering is according to Chothia.

9. L comprises a consecutive amino acid sequence derived from an immunoglobulin (Ig) hinge region; optionally, the Ig hinge region is derived from a human Ig hinge region or a non-human Ig hinge region, optionally, the Ig hinge region is derived from a human Ig hinge region; Optionally, the human Ig hinge region is of the IgG1, IgG2, IgG3, or IgG4 isotype.

10. The L is a) Amino acid sequence C(X) ｙ C (SEQ ID NO:23), wherein X is glycine (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), threonine (Thr), tryptophan (Trp), or tyrosine (Tyr), and y is an integer from 1 to 3. b) 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; Optionally, L is selected from the group consisting of 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), CPPGC (SEQ ID NO: 40), CPPPG (SEQ ID NO: 41), CPPPG (SEQ ID NO: 42), CPPPG (SEQ ID NO: 43), CPPPG (SEQ ID NO: 44), CPPPG (SEQ ID NO: 45), CPPPG (SEQ ID NO: 46), CPPPG (SEQ ID NO: 47), CPPPG (SEQ ID NO: 48), CPPPG (SEQ ID NO: 49), CPPPG (SEQ ID NO: 50), CPPPG (SEQ ID NO: 51), CPPPG (SEQ ID NO: 52), CPPPG (SEQ ID NO: 53), CPPPG (SEQ ID NO: 54), CPPPG (SEQ ID NO: 55), CPPPG (SEQ ID NO: 56), CPPPG (SEQ ID NO: 57), CPPPG (SEQ ID NO: 58), CPPPG (SEQ ID NO: 59), CPPPG (SEQ ID NO: 60), CPPPG (SEQ ID NO: 61), CPPPG (SEQ ID NO: 62), CPPPG (SEQ ID NO: 63), CPPPG (SEQ No. 39), CGGPC (SEQ ID NO: 40), CPGGC (SEQ ID NO: 41), CGGGC (SEQ ID NO: 42), CSPPC (SEQ ID NO: 43), CPSPC (SEQ ID NO: 44), CPPSC (SEQ ID NO: 45), CSSPC (SEQ ID NO: 46), CPSSC (SEQ ID NO: 47), CSSSC (SEQ ID NO: 48), CGSPC (SEQ ID NO: 49), CPGSC (SEQ ID NO: 50), CSGPC (SEQ ID NO: 51), or CPSGC (SEQ ID NO: 52), c) Amino acid sequence (X) ｍ C(X) ｙ C(X) ｎ (SEQ ID NO:25), wherein X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gln, His, Ile, leu, Lys, Phe, Thr, Trp, or Tyr; m is an integer from 6 to 9; y is an integer from 1 to 3; and n is an integer from 4 to 6. d) Amino acid sequence (X) ｍ C(X) ｙ C(X) ｎ (SEQ ID NO:26), wherein X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gln, His, He, Leu, Lys, Thr, or Tyr; m is an integer from 6 to 9; y is an integer from 1 to 3; and n is an integer from 4 to 6; or e) 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.

11. 2. The molecule of claim 1, wherein L has a length of about 14 to about 19 amino acids, optionally L has a length of about 14, about 15, about 16, about 17, about 18, or about 19 amino acids.

12. The molecule of claim 1 , wherein L comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:

7.

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

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

15. (i) (a) the VH comprises a Cys at H105, (b) the VL comprises a Cys at L42, (c) the L comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and (d) the scFv is in a VL-L-VH orientation. (ii) (a) the VH comprises a Cys at H105, (b) the VL comprises a Cys at L45, (c) the L comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and (d) the scFv is in a VL-L-VH orientation. (iii) (a) the VH comprises a Cys at H105, (b) the VL comprises a Cys at L39, (c) the L comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and (d) the scFv is in a VL-L-VH orientation. (iv) (a) the VH comprises a Cys at H5, (b) the VL comprises a Cys at L42, (c) the L comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or the amino acid sequence of SEQ ID NO:7, and (d) the scFv is in a VL-L-VH orientation. (v) (a) the VH comprises a Cys at H5, (b) the VL comprises a Cys at L45, (c) the L comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and (d) the scFv is in a VL-L-VH orientation. (vi) (a) the VH comprises a Cys at H5, (b) the VL comprises a Cys at L39, (c) the L comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and (d) the scFv is in a VL-L-VH orientation. (vii) (a) the VH comprises a Cys at H3, (b) the VL comprises a Cys at L42, (c) the L comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and (d) the scFv is in a VL-L-VH orientation. (viii) (a) the VH comprises a Cys at H3; (b) the VL comprises a Cys at L45; (c) the L comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; and (d) the scFv is in a VL-L-VH orientation; or (ix) The molecule of any one of claims 1-13, wherein (a) the VH comprises a Cys at H3, (b) the VL comprises a Cys at L39, (c) the L comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and (d) the scFv is in a VL-L-VH orientation.

16. (i) (a) the VH comprises a Cys at H43, (b) the VL comprises a Cys at L100, (c) the L comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and (d) the scFv is in a VH-L-VL orientation. (ii) (a) the VH comprises a Cys at H43, (b) the VL comprises a Cys at L102, (c) the L comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and (d) the scFv is in a VH-L-VL orientation. (iii) (a) the VH comprises a Cys at H43, (b) the VL comprises a Cys at L5, (c) the L comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and (d) the scFv is in a VH-L-VL orientation. (iv) (a) the VH comprises a Cys at H43, (b) the VL comprises a Cys at L3, (c) the L comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and (d) the scFv is in a VH-L-VL orientation. (v) (a) the VH comprises a Cys at H40, (b) the VL comprises a Cys at L100, (c) the L comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and (d) the scFv is in a VH-L-VL orientation. (vi) (a) the VH comprises a Cys at H40, (b) the VL comprises a Cys at L102, (c) the L comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and (d) the scFv is in a VH-L-VL orientation. (vii) (a) the VH comprises a Cys at H40, (b) the VL comprises a Cys at L5, (c) the L comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and (d) the scFv is in a VH-L-VL orientation. (viii) (a) the VH comprises a Cys at H40; (b) the VL comprises a Cys at L3; 9c) the L comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; and (d) the scFv is in a VH-L-VL orientation. (ix) (a) the VH comprises a Cys at H46, (b) the VL comprises a Cys at L100, (c) the L comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and (d) the scFv is in a VH-L-VL orientation. (x) (a) the VH comprises a Cys at H46, (b) the VL comprises a Cys at L102, (c) the L comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and (d) the scFv is in a VH-L-VL orientation. (xi) (a) the VH comprises a Cys at H46, (b) the VL comprises a Cys at L5, (c) the L comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and (d) the scFv is in a VH-L-VL orientation; or (xii) The molecule of claim 1, wherein (a) the VH comprises a Cys at H46, (b) the VL comprises a Cys at L3, (c) the L comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and (d) the scFv is in a VH-L-VL orientation.

17. 16. The molecule of claim 15, wherein L comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:6, or SEQ ID NO:

7.

18. the binding molecule comprises a heavy chain, a light chain, and a polypeptide; the N-termini of the heavy and light chains form a Fab; the polypeptide comprises the scFv at the N-terminus, and The molecule of claim 1 , wherein the C-terminus of the polypeptide and the C-terminus of the heavy chain form an Fc region.

19. 2. The molecule of claim 1, wherein the Fab binds to a tumor antigen and the scFv binds to a T cell antigen, optionally wherein the tumor antigen is BCMA and the T cell antigen is CD3.

20. 20. The molecule of claim 19, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 126 or SEQ ID NO:

128.

21. 20. The molecule of claim 19, wherein the Fab comprises (i) a VH comprising the amino acid sequence of SEQ ID NO: 132 and a VL comprising the amino acid sequence of SEQ ID NO: 129, or (ii) a VH comprising the amino acid sequence of SEQ ID NO: 137 and a VL comprising the amino acid sequence of SEQ ID NO:

135.

22. 22. The molecule of claim 21, wherein (a) the VH comprises a Cys at H105, (b) the VL comprises a Cys at L43, (c) the L comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and (d) the scFv is in a VL-L-VH orientation.

23. A polynucleotide encoding the molecule or fragment thereof according to any one of claims 1 to 22.

24. A vector comprising the polynucleotide of claim 23.

25. 25. A host cell comprising the vector of claim 24, optionally wherein the host cell is a prokaryotic or eukaryotic cell.

26. A method for producing a molecule in vitro, comprising: a) introducing the polynucleotide of claim 23 into a host cell; b) culturing said host cells under conditions such that said molecule is produced; and c) purifying said produced molecule.

27. A method for producing a molecule in vitro, comprising: a) culturing the host cell of claim 25 under conditions such that the molecule is produced; and b) purifying said produced molecule.

28. A pharmaceutical composition comprising a molecule according to any one of claims 1 to 22, and optionally a pharmaceutically acceptable excipient.

29. 23. A pharmaceutical composition for use in a method for directing or engaging a cell to a target cell, said method comprising contacting said target cell with a molecule according to any one of claims 1 to 22, wherein optionally said Fab binds to a first antigen on said target cell and said scFv binds to a second antigen on said cell.

30. 30. The pharmaceutical composition of claim 29, wherein the cell is an immune cell, optionally wherein the immune cell is a T cell.

31. 31. The pharmaceutical composition of claim 29 or 30, wherein the target cell is a tumor cell and / or the method is for treating a disease or disorder in a subject, optionally wherein a) the disease or disorder is a tumor, optionally wherein the tumor is cancer, and / or b) the subject is a human subject.

32. 23. A pharmaceutical composition for use in a method for eliminating or inhibiting target cells, said method comprising contacting said target cells with a molecule according to any one of claims 1 to 22.