Multispecific antibodies for use in avidity receptor cross-linking and immunomodulation

Abstract

Multispecific, optionally multivalent, antibodies comprising at least one antigen-binding portion in Fv format. Methods for producing such multispecific antibodies and their use for modulating immune responses and treating diseases such as cancer are also provided herein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of International Patent Application No. PCT / CN2022 / 073220, filed January 21, 2022, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been filed electronically in XML format and is incorporated by reference in its entirety. The XML copy created on January 17, 2023 is named 112238-0101-70011WO01_SEQ.xml and is 715,745 bytes in size. [Background technology]

[0003] Immune cell receptors, such as those in the tumor necrosis factor receptor (TNFR) superfamily members or CD3, play an important role in controlling immune responses against pathogens or diseased cells, including cancer cells and pathogen-infected cells. Antibodies targeting such immune cell receptors have been used to regulate immune responses and disease treatment. However, such therapeutic approaches may fail to achieve the desired clinical efficacy and / or present safety concerns. Therefore, it is of great interest to develop new immunotherapies that are both effective and safe. Summary of the Invention

[0004] The present disclosure is based, at least in part, on the design of a multispecific, optionally multivalent, antibody format that utilizes at least one antigen-binding moiety in Fv format. A multispecific antibody having a format as provided herein may bind to multiple immune cell receptors or may bind to both an immune cell receptor and a tumor-associated antigen (TAA). For example, a binding moiety in the Fv format may bind to an immune cell receptor. Since monovalent Fv fragments typically have relatively low binding affinity to target antigens, especially when the Fv fragment is connected to other antibody components via a peptide linker, it is expected that the multispecific antibodies disclosed herein will provide avidity-driven cross-linking of target antigens (e.g., immune cell receptors) through simultaneous binding of multiple antigen-binding moieties to their multiple target antigens so as to conditionally modulate the immune response. Such avidity-driven cross-linking of desired target receptors provides therapeutic activity (e.g., anti-tumor cell immune response), for example, in the tumor microenvironment, while avoiding the stimulation of a systemic immune response that may cause undesirable side effects.

[0005] Furthermore, any desired peptide linker, including flexible and rigid peptide linkers, may in some cases be used in the multispecific antibodies disclosed herein. Such peptide linkers may have at least the following advantageous features: (a) contribute to the functionality of the Fv fragment, (b) contribute to proper dimerization (e.g., heterodimerization) of the two polypeptides of the multispecific antibody, thereby stabilizing the entire molecule, and (c) reduce the binding affinity to the Fc gamma receptor, either alone or in combination with additional mutations in the heavy chain constant regions (e.g., CH2 and CH3 regions) used in the multispecific antibody, in order to reduce or eliminate Fc-mediated effector functions.

[0006] It is noteworthy that the advantageous features associated with the specific Fv-containing multispecific antibody format disclosed herein are not target antigen or antigen-binding moiety specific. This multispecific antibody format, comprising one or more Fv fragments and optionally specific peptide linkers disclosed herein, can be used to construct multispecific antibodies capable of binding to any desired target antigen and is expected to have the advantageous features disclosed herein, such as avidity-driven cross-linking of the target antigen.

[0007] Thus, provided herein are multispecific antibodies comprising at least one antigen-binding portion in Fv format (monovalent), optionally in multivalent form, methods for producing such multispecific antibodies, and methods of using such multispecific antibodies for modulating immune responses and for treating diseases.

[0008] In some embodiments, the disclosure features a multispecific antibody comprising a first binding moiety specific for a first target antigen and a second binding moiety specific for a second target antigen. The first target antigen is a first immune cell receptor. In some cases, the first immune cell receptor can be a first T cell receptor, e.g., a T cell activation receptor or a T cell checkpoint receptor. In some cases, the second target antigen is a second immune cell receptor, optionally a second T cell receptor (e.g., a T cell activation receptor or a T cell checkpoint receptor). In some examples, the second immune cell receptor is different from the first immune cell receptor. Alternatively, the second target antigen is a tumor-associated antigen (TAA) (first TAA). The first binding moiety is a first heavy chain variable region (V) that forms a heterodimer. H ) and the first light chain variable region (V L The first Fv fragment comprises H is linked to a first flexible peptide linker and a first rigid peptide linker. Lis linked to a second flexible peptide linker and a second rigid peptide linker. The first rigid peptide linker and the second rigid peptide linker form one or more disulfide bonds. In some cases, the second binding moiety is linked to the first V via the first flexible peptide linker. H or a first V via a second flexible peptide linker L is connected to one of the

[0009] In some embodiments, the first flexible peptide linker and the second flexible peptide linker are the same. Alternatively, the first flexible peptide linker and the second flexible peptide linker are different. In some examples, the first flexible peptide linker, the second flexible peptide linker, or both are G / S-rich peptide linkers. For example, a G / S-rich peptide linker is a G X S) n where X is an integer from 1 to 6, inclusive, and n is an integer from 1 to 10, inclusive. Specific examples are provided in Table 1, any of which may be used in the multispecific antibodies disclosed herein.

[0010] In some embodiments, the first rigid peptide linker, the second rigid peptide linker, or both, comprise the amino acid sequence of DKTHTCPPCPAPEAAGP (SEQ ID NO:21), DKTHTCPPCPAPELLGP (SEQ ID NO:9), or DKTHTCPPCPAPELLGGP (SEQ ID NO:27). In some cases, the rigid peptide linker may comprise the previously mentioned sequences flanked at the N-terminus and / or C-terminus by G / S-rich peptide linkers. In some examples, the first rigid peptide linker, the second rigid peptide linker, or both, comprise the amino acid sequence of DKTHTCPPCPAPEAAGP (SEQ ID NO:21), DKTHTCPPCPAPELLGP (SEQ ID NO:9), or DKTHTCPPCPAPELLGGP (SEQ ID NO:27). X S) nIn another example, the first rigid peptide linker, the second rigid peptide linker, or both, are connected to the N-terminus of DKTHTCPPCPAPELLGP (SEQ ID NO: 9) or connected to the N-terminus of DKTHTCPPCPAPELLGGP (SEQ ID NO: 27). X S) n motif, where X is an integer from 1 to 6, inclusive, and n is an integer from 1 to 10, inclusive. Examples of rigid peptide linkers are provided in Table 1, any of which may be used in the multispecific antibodies disclosed herein.

[0011] Any of the multispecific antibodies disclosed herein may further comprise a third binding moiety specific for a third target antigen.In some cases, the third target antigen is identical to the second target antigen, for example, the third binding moiety is identical to the second binding moiety.Alternatively, the third binding moiety is different from the second binding moiety.For example, the third target antigen is a second TAA that is different from the first TAA.

[0012] In some embodiments, the second binding moiety is connected to the first V via a first flexible peptide linker. H and a third binding moiety is linked to the first V via a second flexible peptide linker. L is linked to.

[0013] In some cases, the first V H is further linked to a first Fc fragment via a first rigid peptide linker. Lcan be further linked to a second Fc fragment via a second rigid peptide linker. Each Fc fragment can include a CH2 domain and / or a CH3 domain. In some cases, the Fc fragment can be derived from an IgG1 molecule. In some cases, the first Fc fragment and the second Fc fragment can include a mutation in the CH3 domain that enhances heterodimerization over homodimerization of the first and second Fc fragments and / or reduces Protein A binding compared to the wild-type counterpart. For example, the mutation can be a knob-in-hole mutation, a charge mutation, or a ZW1 mutation.

[0014] In some examples, the second binding moiety and / or the third binding moiety are in single chain variable fragment (scFv) format. Alternatively, the second binding moiety and / or the third binding moiety are in single domain antibody format, optionally in heavy chain (VHH) format, Fab format, or cross-Fab format. In certain examples, the second binding moiety is a Fab fragment comprising a first VH-CH1 fragment and a first VL-CL fragment. In other examples, the second binding moiety is a cross-Fab fragment comprising a first VH-CL fragment and a first VL-CH1 fragment. Alternatively or additionally, the third binding moiety is a Fab fragment comprising a second VH-CH1 fragment and a second VL-CL fragment. In other examples, the third binding moiety is a cross-Fab fragment comprising a second VH-CL fragment and a second VL-CH1 fragment.

[0015] In certain examples, the multispecific antibodies disclosed herein comprise (a) from N-terminus to C-terminus a first VH-CH1 or VH-CL fragment of a second binding moiety, a first flexible peptide linker, a first VH-CH2 or VH-CL fragment of a second binding moiety, a second VH-CH3 or VH-CL fragment of a second binding moiety, a third VH-CH4 or VH-CL fragment of a second binding moiety, a fourth VH-CH1 or VH-CL fragment of a second binding moiety, a fifth VH-CH2 or VH-CL fragment of a second binding moiety, a fifth VH-CH1 ... H (b) a first polypeptide comprising, from N-terminus to C-terminus, a second VH-CH1 or VH-CL fragment of a third binding moiety, a second flexible peptide linker, a first VH-CH1 or VH-CL fragment of a third binding moiety ... L(c) a second polypeptide comprising a first VL-CL or VL-CH1 fragment of the second binding moiety, and (d) a fourth polypeptide comprising a second VL-CL or VL-CH1 fragment of the third binding moiety. In one example, the third and fourth polypeptides are identical.

[0016] Any of the multispecific antibodies disclosed herein may further comprise a fourth binding moiety specific for a fourth antigen. In some embodiments, the fourth target antigen is a third immune receptor, optionally a third T cell activation receptor. In some examples, the third immune receptor is different from the first immune receptor and / or the second immune receptor. For example, the fourth target antigen is a third TAA, optionally different from either the first TAA or the second TAA.

[0017] In some embodiments, the fourth binding moiety is a second V H and the second V L A second Fv fragment comprising the second V H is linked to a first Fc fragment via a first peptide linker, and a second V L is linked to the second Fc fragment via a second peptide linker. In some cases, the first peptide linker is identical to the second peptide linker. In some cases, the first peptide linker, the second peptide linker, or both are G / S-rich peptide linkers. In one example, the G / S-rich peptide linker has the formula (GxS)n, where X is an integer from 1 to 6, inclusive, and n is an integer from 1 to 10, inclusive.

[0018] In some embodiments, the multispecific antibody comprises a second V H and the second V LIn some cases, the third peptide linker and the fourth peptide linker are a pair of rigid peptide linkers, optionally identical, that form one or more disulfide bonds. In some examples, the third peptide linker, the fourth peptide linker, or both, comprise the amino acid sequence of DKTHTCPPCPAPEAAGP (SEQ ID NO:21), DKTHTCPPCPAPELLGP (SEQ ID NO:9), or DKTHTCPPCPAPELLGGP (SEQ ID NO:27). Each of the sequences may be linked to a G / S-rich peptide linker. In certain examples, the third peptide linker, the fourth peptide linker, or both, may be connected to the N-terminus of DKTHTCPPCPAPEAAGP (SEQ ID NO:21), DKTHTCPPCPAPELLGP (SEQ ID NO:9), or DKTHTCPPCPAPELLGGP (SEQ ID NO:27) (G X S) n motif, wherein X is an integer from 1 to 6, inclusive, and n is an integer from 1 to 10, inclusive.

[0019] In certain examples, the multispecific antibodies disclosed herein comprise (a) from N-terminus to C-terminus a first VH-CH1 or VH-CL fragment of a second binding moiety, a first flexible peptide linker, a first VH-CH2 or VH-CL fragment of a second binding moiety, a second VH-CH3 or VH-CL fragment of a second binding moiety, a third VH-CH4 or VH-CL fragment of a second binding moiety, a fourth VH-CH1 or VH-CL fragment of a second binding moiety, a fifth VH-CH2 or VH-CL fragment of a second binding moiety, a fifth VH-CH1 ... H , a first rigid peptide linker, a first Fc fragment, a first peptide linker, a second V H and optionally a second peptide linker; and (b) a first polypeptide comprising, from N-terminus to C-terminus, a second VH-CH1 or VH-CL fragment of a third binding moiety, a second flexible peptide linker, a first V L , a second rigid peptide linker, a second Fc fragment, a third peptide linker, and a second V Land optionally a fourth peptide linker, (c) a third polypeptide comprising a first VL-CL or VL-CH1 fragment of the second binding moiety, and (d) a fourth polypeptide comprising a second VL-CL or VL-CH1 fragment of the third binding moiety. In one example, the third polypeptide is identical to the fourth polypeptide.

[0020] In some cases, the multispecific antibodies disclosed herein may further comprise a first heavy chain constant region fragment linked to a first flexible peptide linker and a second heavy chain constant region fragment linked to a second flexible peptide linker. In some cases, the first and / or second heavy chain constant region fragment are derived from an IgG1 molecule. Each of the first and second heavy chain constant region fragments comprises a hinge domain, a CH2 domain, and a CH3 domain. The second binding moiety is linked to either the first heavy chain constant region fragment or the second heavy chain constant region fragment. In some cases, the first heavy chain constant region fragment and the second heavy chain constant region fragment comprise a mutation in the CH3 domain that enhances heterodimerization over homodimerization of the first and second Fc fragments and / or reduces Protein A binding compared to the wild-type counterpart. In some cases, the mutation may be a knob-in-hole mutation, a charge mutation, or a ZW1 mutation.

[0021] In some examples, the multispecific antibody may further comprise a third binding moiety specific for a third target antigen. The second binding moiety may be linked to the first heavy chain constant region fragment, and the third binding moiety may be linked to the second heavy chain constant region fragment. In some examples, the third binding moiety is different from the second binding moiety. For example, the third target antigen is a second TAA that is different from the first TAA. In other examples, the third target antigen may be the same as the second target antigen. For example, the third binding moiety is the same as the second binding moiety.

[0022] In some examples, the second binding moiety and / or the third binding moiety are in single chain variable fragment (scFv) format. Alternatively, the second binding moiety and / or the third binding moiety are in single domain antibody format, optionally in heavy chain (VHH) format, Fab format, or cross-Fab format. In one example, the second binding moiety is a Fab fragment comprising a first VH-CH1 fragment and a first VL-CL fragment. In another example, the second binding moiety is a cross-Fab fragment comprising a first VH-CL fragment and a first VL-CH1 fragment. Alternatively or additionally, the third binding moiety is a Fab fragment comprising a second VH-CH1 fragment and a second VL-CL fragment. In another example, the third binding moiety is a cross-Fab fragment comprising a second VH-CL fragment and a second VL-CH1 fragment.

[0023] In certain examples, the multispecific antibodies disclosed herein comprise (a) from N-terminus to C-terminus a first VH-CH1 or VH-CL fragment of a second binding moiety, a first heavy chain constant region fragment, a first flexible peptide linker, a first VH-CH2 or VH-CL fragment of a second binding moiety, a first VH-CH3 or VH-CL fragment of a second binding moiety, a first VH-CH4 or VH-CL fragment of a second binding moiety, a first VH-CH1 ... H and a first rigid peptide linker; and (b) a first polypeptide comprising, from N-terminus to C-terminus, a second VH-CH1 or VH-CL fragment of a third binding moiety, a second heavy chain constant region fragment, a second flexible peptide linker, and a first VH-CH1 or VH-CL fragment of a third binding moiety. L (c) a second polypeptide comprising a first VL-CL or VL-CH1 fragment of the second binding moiety, and (d) a fourth polypeptide comprising a second VL-CL or VL-CH1 fragment of the third binding moiety. In one example, the third and fourth polypeptides are identical.

[0024] In another aspect, the disclosure features a multispecific antibody including a first binding moiety specific for a first target antigen and a second binding moiety specific for a second target antigen. The first target antigen is a first immune cell receptor, optionally a first T cell activation receptor, and the second target antigen is (i) a second immune cell receptor different from the first immune cell receptor, optionally a second T cell activation receptor, or (ii) a first tumor-associated antigen (TAA). The first binding moiety is a first heavy chain variable region (V H ) and the first light chain variable region (V L The first Fv fragment comprises H is linked to a first peptide linker and a first heavy chain constant region fragment, L is linked to a second peptide linker and a second heavy chain constant region fragment. The second binding moiety is connected to the first binding moiety via a first heavy chain constant region fragment or a second heavy chain constant region fragment, each of the first heavy chain constant region and the second heavy chain constant region comprising a hinge domain, a CH2 domain, and a CH3 domain. In some examples, the first and / or second heavy chain constant region fragment is derived from an IgG1 fragment.

[0025] In some embodiments, the first peptide linker, the second peptide linker, or both are G / S-rich peptide linkers. For example, a G / S-rich peptide linker is X S) n (SEQ ID NOs: 29 and 512 to 516), wherein X is an integer from 1 to 6 (inclusive) and n is an integer from 1 to 10 (inclusive).

[0026] In some embodiments, the multispecific antibody may further comprise a third binding moiety specific for a third target antigen. In some cases, the third target antigen is identical to the second target antigen. For example, the third binding moiety is identical to the second binding moiety. In some cases, the third binding moiety is different from the second binding moiety. For example, the third target antigen is a second TAA, optionally different from the first TAA. In some examples, the second binding moiety and / or the third binding moiety are in a single domain antibody format, Fab format, or cross-Fab format, optionally in a single chain variable fragment (scFv) format, or a heavy chain (VHH) format. In certain examples, the binding moieties are in scFv format and / or Fab format.

[0027] In some embodiments, the first heavy chain constant region fragment and the second heavy chain constant region fragment comprise a mutation in the CH3 domain that enhances heterodimerization over homodimerization of the first and second Fc fragments and / or reduces Protein A binding compared to the wild-type counterparts. Exemplary mutations include knobs-in-holes mutations, charge mutations, and / or ZW1 mutations.

[0028] In any of the multispecific antibodies disclosed herein that contain one or more heavy chain constant region fragments, such as an Fc fragment, such heavy chain constant region fragments may contain one or more mutations that alter the binding activity to an Fc receptor compared to the wild-type counterpart. In a specific example, the heavy chain constant region fragment may contain (i) a deletion at one or more of positions 236 to 238, (ii) an amino acid substitution at one or more of positions 239, 265, 297, 329, 330, and 332, or a combination thereof. In some examples, the heavy chain constant region fragment can include one or more of the following: (i) a deletion at position 237, (ii) at least two amino acid substitutions selected from L234A, L235A, and P329G, (iii) a deletion at position 237 and amino acid substitutions D265A and N297A, (iv) amino acid substitutions S239D, A330L, and I332E, and (v) a deletion at position 237 and the amino acid substitution P329G.

[0029] Any of the multispecific antibodies disclosed herein bind to at least one immune receptor (first immune receptor), and optionally additional immune receptors (second and / or third immune receptors), which may be CD3, CD28, PD-1, PD-L1, CTLA4, CD47, or members of the tumor necrosis factor receptor superfamily (TNFRSF). Examples of TNFRSF members include FAS, TNFRSF12A, 4-1BB / CD137, TNFRSF13B, TNFRSF13C, CD27 / TNFRSF7, CD30 / TNFRSF8, CD40 / TNFRSF5, DR3 / TNFRSF25, DR4 / TNFRSF10A, DR5 / TNFRSF10B, DR6 / TNFRSF21, GITR / TNFRSF18, HVEM / TNFRSF14. , LTβR, OX40 / TNFRSF4, TROY / TNFRSF19, RELT / TNFRSF19L, TL1A / TNFSF15, TNFRSF17, TNFRSF1A, TNFRSF11B, RANK / TNFRSF11A, TNFRSF11B, NGFR, EDA2R, and TNFRSF1B, TNFRSF6B, TNFRSF10C, TNFRSF10D, or TNFRSF13A.

[0030] In some embodiments, the multispecific antibodies disclosed herein may further bind to at least one TAA (a first TAA), and optionally additional TAAs (second and third TAAs), which may be one or more of B7H3, CD19, CD20, PSMA, HER2, CEA, BCMA, P53mut, DLL3, MET, EGFR, MAGE-A4, and PRAME.

[0031] In some embodiments, a multispecific antibody disclosed herein may bind to at least (a) CD3 and CD28, (b) CD3 and CD137, (c) CD137 and PD-1, (d) CD40 and PD-1, (e) CD40 and PD-L1, (f) CD137 and GITR, (g) CD137 and PD-L1, (h) CD137 and CD40, (i) CD137 and OX40, (j) CD3 and PD-1, (k) CD3 and PD-L1, or (l) CD3 and CTLA4.

[0032] In some examples, the multispecific antibodies disclosed herein are selected from the group consisting of: (1) B7H3, CD3, and CD137; (2) CD19, CD3, and CD137; (3) B7H3, CD3, and CD28; (4) CD19, CD3, and CD28; (5) B7H3, CD137, and PD-1; (6) B7H3, CD40, and PD-1; (7) PMSA, CD3, and CD137; (8) B7H3 and CD3; (9) B7H3 and CD137; (10) CD19 and CD3; (11) CD19 and CD137; (12) B7H4 and CD40; (13) HE R2, CD3, and CD137, (14) CEA, CD3, and CD137, (15) BCMA, CD3, and CD137, (16) P53 mutant, CD3, and CD137, (17) PD-1 and CD137, (18) PD-1 and CD40, (19) B7H3 and CD40, (20) PD-L1 and CD40, (21) PD-L1 and CD3, (22) PD-L1, CD3, and CD137, (23) PD-L1, CD3, and CD28, (24) PD-L1, CD137, and B7H3, (25) PD-L1, CD40, and B7H3, (26) PD-1, CD40, and CD137, (27) PD-1, CD137, and GITR, (28) CD19, CD20, CD3, and CD137, (29) CEA and CD3, (30) CEA and CD137, (31) P53 mutant and CD3, (32) P53 mutant and CD137, (33) PD-L1, CD40, and CD137, (34) PD-L1 and CD137, (35) MET, EGFR, and CD47, (36) BCMA and CD3, (37) BCMA and CD137, (38) PSMA and CD3, (39) HER2 and CD3, (4 0) HER2 and CD40, (41) HER2 and CD137, (42) PSMA and CD137, (43) HER2, MET, CD3, and CD137, (44) MAGE-A4, CD3, and CD137, (45) PRAME, CD3, and CD137, (46) HER2, MET, and CD3, (47) MAGE-A4 and CD3, (48) PRAME and CD3, (49) HER2, MET, and CD47, (50) B7H3, CD3, and PD-1, (51) B7H3, CD3, and PD-L1, (52) B7H3, CD3, and CTLA4,(53) PSMA, CD3, and CD137, (54) PSMA, CD3, and CD28, (55) HER2, CD3, and CD137, (56) HER2, CD3, and CD28, (57) CEA, CD3, and CD137, (58) CEA, CD3, and CD28, (59) BCMA, CD3, and CD137, (60) BCMA, CD3, and CD28, (61) CD19, CD19, CD3, and CD28.

[0033] Any of the multispecific antibodies may contain the same heavy chain complementarity determining regions (CDRs) and the same light chain CDRs as in one or more of the parent antibodies listed in Table 1. In some cases, the multispecific antibodies may contain the same V and V CDRs as in one or more of the parent antibodies. H and V L Includes.

[0034] Any of the multispecific antibodies disclosed herein may be multivalent. In some instances, the multispecific antibodies may be trivalent. In other instances, the multispecific antibodies may be bivalent or tetravalent.

[0035] Exemplary multispecific antibodies disclosed herein are provided in Table 2.

[0036] In another aspect, provided herein is a nucleic acid or set of nucleic acids that collectively encode the multispecific antibodies disclosed herein. In some embodiments, the nucleic acid or set of nucleic acids may be an expression vector or an expression vector set. Also provided herein is a host cell comprising a nucleic acid or set of nucleic acids encoding the multispecific antibodies disclosed herein. In some embodiments, the host cell is a mammalian host cell.

[0037] In yet another aspect, the disclosure features a method for producing a multispecific antibody, the method including: (i) culturing a host cell disclosed herein under conditions that allow for expression of the antibody; and (ii) recovering the antibody thus produced.

[0038] In addition, the disclosure features a pharmaceutical composition comprising any of the multispecific antibodies disclosed herein, or a nucleic acid or set of nucleic acids encoding such a multispecific antibody, and a pharma- ceutically acceptable carrier.

[0039] In another aspect, the disclosure features a method for modulating an immune response, the method comprising administering an effective amount of a multispecific antibody disclosed herein, a nucleic acid encoding such a multispecific antibody, or a pharmaceutical composition comprising the antibody or encoding nucleic acid to a subject in need thereof. In some embodiments, the subject can be a human patient having or suspected of having cancer.

[0040] Also within the disclosure are any of the multispecific antibodies disclosed herein for use in immunomodulation and therapy (e.g., cancer therapy), as well as the use of such multispecific antibodies for the manufacture of a medicament for the intended medical use.

[0041] The details of one or more embodiments of the invention are set forth in the specification below. Other features or advantages of the invention will be apparent from the following drawings and detailed description of certain embodiments, as well as from the appended claims. [Brief description of the drawings]

[0042] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawings in combination with the detailed description of specific embodiments presented herein.

[0043] [Figure 1A-1D]The figures provided herein include diagrams showing the schematic designs of exemplary multispecific antibodies. Figure 1A: Exemplary antigen-binding moieties in Fv format containing a heavy chain variable domain (VH) and a light chain variable domain (VL), each of which is connected to a flexible peptide linker and a rigid peptide linker. Figure 1B: Exemplary designs of multispecific antibodies with a central (hidden) Fv fragment. Figure 1C: Exemplary designs of multispecific antibodies with terminal Fv fragments. Figure 1D: Exemplary designs of multispecific antibodies with one central and one terminal Fv fragment. [Figure 2A-2B]Graphs showing the characterization of multispecific antibodies with different flexible peptide linkers are included. Figure 2A: B7H3 binding activity of Ly2364 [with a (G4S)5 motif (SEQ ID NO: 7) in the flexible peptide linker], Ly2365 [with a (G4S)3 motif (SEQ ID NO: 5) in the flexible peptide linker], Ly2366 [with a (G4S)2 motif (SEQ ID NO: 4) in the flexible peptide linker], Ly2367 (with a G4S motif in the flexible peptide linker), Ly2368 (without a G4S motif in the flexible peptide linker). B7H3 mAb Ly1612 was used as a positive control. Figure 2B: B7H3 binding activity of Ly2384 (with a G3S motif in the flexible peptide linker), Ly2385 (with a G2S motif in the flexible peptide linker), and control Ly1612. Figure 2C: CD3 binding activity of Ly2364, Ly2365, Ly2366, Ly2367, and Ly2368. CD3 mAb Ly305 was used as a positive control. Figure 2D: CD3 binding activity of Ly2384, Ly2385, and control Ly305. Figure 2E: B7H3 binding activity of Ly2136 [with a (G4S)5 motif (SEQ ID NO:7) in a flexible peptide linker], Ly2137 [with a (G4S)3 motif (SEQ ID NO:5) in a flexible peptide linker], Ly2138 [with a (G4S)2 motif (SEQ ID NO:4) in a flexible peptide linker], Ly2139 (with a G4S motif in a flexible peptide linker), and control Ly1612. Figure 2F: CD3 binding activity of Ly2136, Ly2137, Ly2138, Ly2139, and control Ly305. [Figure 3A-3B]Graphs showing the characterization of multispecific antibodies with different rigid peptide linkers are included. Figure 3A: Images showing SDS-PAGE analysis of multispecific antibodies with different rigid peptide linkers under both non-reducing and reducing conditions. Lanes 1 and 2: Clone Ly1891 under reducing and non-reducing conditions, respectively. Lanes 3 and 4: Clone Ly1899 under reducing and non-reducing conditions, respectively. Lanes 5 and 6: Clone Ly1963 under reducing and non-reducing conditions, respectively. Lanes 7 and 8: Clone Ly1800 under reducing and non-reducing conditions, respectively. Lanes 9 and 10: Clone Ly1803 under reducing and non-reducing conditions, respectively. Lanes 11 and 12: Clone Ly1961 under reducing and non-reducing conditions, respectively. M: molecular weight marker. Figure 3B: Chart showing the CD3 binding activity of the exemplary multispecific antibodies shown. The parent CD3 mAb Ly305 was used as a positive control. [Figure 4A-4C] Includes charts showing the CD3 binding activity of the exemplary multispecific antibodies shown. Figure 4A: CD3 binding activity of Ly2125 (no G4S motif in the rigid peptide linker) and Ly2128 (with G4S motif in the rigid peptide linker). Clone Ly305 was used as a positive control CD3 mAb. Figure 4B: CD3 binding activity of Ly2157 (no G4S motif in the rigid peptide linker) and Ly1963 (with G4S motif in the rigid peptide linker). Figure 4C: CD3 binding activity of Ly2167 (no G4S motif in the rigid peptide linker) and Ly1967 (with G4S motif in the rigid peptide linker). [Figure 5A-5I]Figures 5A-5D include figures showing CD3 agonist activity of the indicated exemplary multispecific antibodies comprising a central Fv fragment that binds to CD3. Agonist activity was measured at various concentrations when incubated alone or in co-culture with CHO cells expressing the target antigen. Figure 5A: CD3 agonist activity for clones Ly2128 and Ly305 (reference CD3 mAb) assessed alone. Figure 5B: CD3 agonist activity for clones Ly2128 and Ly305 when co-cultured with B7H3-overexpressing CHO cells. Figure 5C: CD3 agonist activity for clones Ly1966, Ly1967, and Ly305 assessed alone. Figure 5D: CD3 agonist activity for clones Ly1966, Ly1967, and Ly305 when co-cultured with CD137-overexpressing CHO cells. Figure 5E: CD3 agonist activity for clones Ly1963, Ly1965, and Ly305 assessed alone. Figure 5F: CD3 agonist activity for clones Ly1963, Ly1965, and Ly305 when co-cultured with CD137-overexpressing CHO cells. Figure 5G: CD3 agonist activity for clones Ly1963, Ly1965, and Ly305 when co-cultured with B7H3-overexpressing CHO cells. Figure 5H: CD3 agonist activity for clones Ly2278 and Ly305 assessed alone. Figure 5I: CD3 agonist activity for clones Ly2278 and Ly305 when co-cultured with CD19-overexpressing CHO cells. [Figure 6A-6B] Figures 6A-6C include diagrams showing cytotoxicity assays using target tumor cells, effector PBMCs, and exemplary multispecific antibodies as indicated. Lactate dehydrogenase (LDH) release was used as an indicator of cytotoxicity. Figure 6A: Cytotoxicity of clones Ly1963, Ly1965, and Ly2128. Figure 6B: Cytotoxicity of Ly1967 and Ly2278. [Figure 7A-7B] Figures showing the characterization of an exemplary multispecific antibody with a central Fv fragment that binds to CD137 are included. Figure 7A: CD137 binding activity of clone Ly2118 relative to the parent clone Ly1630. Figure 7B: CD137 binding activity of clone Ly2281 relative to the parent clone Ly1630. [Figure 8A-8E]Figures 8A-8D include figures showing activation of human CD137 signaling as indicated by IL-8 secretion in a reporter assay by exemplary multispecific antibodies co-cultured with CHO or Jurkat cells overexpressing the target antigen. Parental clone Ly1630 (anti-CD137) and control anti-CD137 clone TM173 were used as controls. Figure 8A: Agonistic activity of clones Ly2118 and Ly1630 alone. Figure 8B: Agonistic activity of clones Ly2118 and Ly1630 co-cultured with Jurkat cells overexpressing PD-1. Figure 8C: Agonistic activity of clones Ly2118 and Ly1630 co-cultured with CHO cells overexpressing B7H3. Figure 8D: Agonistic activity of clones Ly2281 and TM173 alone. FIG. 8E: Agonistic activity of clones Ly2281 and TM173 co-cultured with CHO cells overexpressing PD-1. [Figure 9A-9B] Figures showing CD40 binding activity of exemplary multispecific antibodies are included: Figure 9A: CD40 binding activity of Ly2121 compared to the parental CD40 mAb clone TM383. Figure 9B: CD40 binding activity of Ly2279 and Ly2280 compared to the parental CD40 mAb clone TM383. [Figure 10A-10F]Figures 10A and 10B show activation of human CD40 signaling by exemplary multispecific antibodies as indicated by IL8 secretion in a reporter assay. The agonist activity of these multispecific antibodies was evaluated either alone or in co-culture with CHO cells overexpressing other target antigens. Clone TM383 is the parental CD40 mAb used as a control. Figure 10A: Activation of human CD40 signaling by clone Ly2121 compared to TM383 alone. Figure 10B: Activation of human CD40 signaling by clone Ly2121 compared to TM383 during co-culture with PD-1 expressing CHO cells. Figure 10C: Activation of human CD40 signaling by clone Ly2121 compared to TM383 during co-culture with B7H3 expressing CHO cells. Figure 10D: Activation of human CD40 signaling by clones Ly2279 and Ly2280 compared to TM383 alone. Figure 10E: Activation of human CD40 signaling by clone Ly2279 compared to TM383 during co-culture with B7H3-expressing CHO cells. Figure 10F: Activation of human CD40 signaling by clone Ly2280 compared to TM383 during co-culture with PD-1-expressing CHO cells. [Figure 11] FIG. 1 shows the CD28 binding activity of exemplary multispecific antibodies Ly2132, Ly2133, Ly2134, Ly2135, and Ly2128, which contain different peptide linkers in relation to the CD28 binding moiety. [Figure 12A-12B] Figures 12A and 12B include diagrams showing activation of human CD3 / CD28 signaling. The agonist activity of exemplary multispecific antibodies was evaluated either alone or in co-culture with CHO cells overexpressing other target antigens. Clone Ly305 is the parent anti-CD3 antibody and is used as a control. Figure 12A: Agonist activity of Ly2132, Ly2133, Ly2134, Ly2135, and Ly2128 alone. Figure 12B: Agonist activity of Ly2132, Ly2133, Ly2134, Ly2135, and Ly2128 during co-culture with CHO cells expressing B7H3. [Figures 13A-13N]Figures 13A-13N include figures showing the characterization of exemplary multispecific antibodies with terminal Fv fragments that bind to CD28. Figure 13A: CD28 binding activity of clone Ly2956 and parental clone Ly224. Figure 13B: CD28 binding activity of clone Ly3103 and parental clone Ly224. Figure 13C: CD28 binding activity of clone Ly3169 and parental clone Ly224. Figure 13D: CD28 binding activity of clones Ly3190, Ly3196, and parental clone Ly224. Figures 13E-13N include figures showing Jurkat T cell reporter activation. Agonist activity of exemplary multispecific antibodies was assessed either alone or in co-culture with additional target expressing cells. Parental anti-CD28 mAb Ly224 was used as a control. The corresponding multispecific antibodies Ly1965, Ly2951, Ly3098, Ly2915, and Ly3152, which do not contain the CD28 binding terminal Fv fragment, were also included. Figure 13E: Agonistic activity of clones Ly2128, Ly1965, and Ly224 alone. Figure 13F: Agonistic activity of clones Ly2128, Ly1965, and Ly224 co-cultured with CHO cells overexpressing B7H3. Figure 13G: Agonistic activity of clones Ly2956, Ly2951, and Ly224 alone. Figure 13H: Agonistic activity of clones Ly2956, Ly2951, and Ly224 co-cultured with H929 cells expressing BCMA. Figure 13I: Agonistic activity of clones Ly3103, Ly3098, and Ly224 alone. Figure 13J: Agonistic activity of clones Ly3103, Ly3098, and Ly224 co-cultured with H929 cells expressing BCMA. Figure 13K: Agonistic activity of clones Ly2915, Ly3169, and Ly224 alone. Figure 13L: Agonistic activity of clones Ly2915, Ly3169, and Ly224 co-cultured with HEK293 cells overexpressing CEA. Figure 13M: Agonistic activity of clones Ly3152, Ly3190, Ly3196, and Ly224 alone. FIG. 13N: Agonist activity of clones Ly3152, Ly3190, Ly3196, and Ly224 co-cultured with HER2-overexpressing CHO cells. [Figures 14A-14C]Figures 14A-14C include figures showing the characterization of exemplary multispecific antibodies with terminal Fv fragments that bind PD-L1. Figure 14A: PD-L1 binding activity of clones Ly2846, Ly2847, and parental clones Ly076 and Ly2530. Figures 14B-14C include figures showing the blocking activity of human PD-1 / PD-L1 signaling. The blocking activity of exemplary multispecific antibodies was assessed either alone or in co-culture with CHO cells overexpressing B7H3. Figure 14B: Blocking activity of clones Ly2846, Ly2847, Ly076, and Ly2530 alone. Figure 14C: Blocking activity of clones Ly2846, Ly2847, Ly076, and Ly2530 co-cultured with CHO cells overexpressing B7H3. [Figure 15A-15D] Figures showing tumor antigen binding activity of exemplary multispecific anti-CD19 / CD20 antibodies. Parental anti-CD19 mAb Ly238 and parental anti-CD20 mAb Ly238 were used as controls. Figure 15A: Binding of clones Ly1966, Ly1967, Ly2326, Ly2278, and Ly238 to CHO cells overexpressing CD19. Figure 15B: Binding of clones Ly2800, Ly2802, Ly2943, Ly2944, Ly238, and Ly239 to CHO cells overexpressing CD19. Figure 15C: Binding of clones Ly2800, Ly2802, Ly2943, Ly2944, Ly238, and Ly239 to CHO cells overexpressing CD20. FIG. 15D: Binding of clones Ly2800, Ly2802, Ly2943, Ly2944, Ly238, and Ly239 to Raji cells. [Figure 16A-16B] Figures 16A and 16B show the median Fv target binding activity of exemplary multispecific anti-CD19 / CD20 antibodies. The parental anti-CD3 mAb Ly305 was used as a control. Figure 16A: CD3 binding activity for clones Ly1967, Ly2326, Ly2278, and Ly305. Figure 16B: CD3 binding activity for clones Ly2800, Ly2802, Ly2943, Ly2944, and Ly305. [Figure 17A-17B]Figures 17A-C include figures showing terminal Fv target binding activity of exemplary multispecific anti-CD19 / CD20 antibodies. The parental anti-CD137 mAb Ly1630 was used as a control. Figure 17A: CD137 binding activity for clones Ly1966, Ly1967, Ly2326, Ly2278, and Ly1630. Figure 17B: CD137 binding activity for clones Ly2800, Ly2802, Ly2943, Ly2944, and Ly1630. [Fig. 18A-18H] Figures 18A and 18B include diagrams showing central Fv target activation activity of exemplary multispecific anti-CD19 / CD20 antibodies. Parental anti-CD3 mAb Ly305 serves as a control. Figure 18A: Activation of human CD3 signaling for clones Ly1966, Ly1967, Ly2278, Ly2326, and Ly305 alone. Figure 18B: Activation of human CD3 signaling for clones Ly1966, Ly1967, Ly2278, Ly2326, and Ly305 co-cultured with CD19 expressing cells. Figure 18C: Activation of human CD3 signaling for clones Ly1966, Ly1967, Ly2278, Ly2326, and Ly305 co-cultured with CD137 expressing cells. Figure 18D: Activation of human CD3 signaling for clones Ly2800, Ly2802, Ly2943, Ly2944, and Ly305 alone. Figure 18E: Activation of human CD3 signaling for clones Ly2800, Ly2802, Ly2943, Ly2944, and Ly305 cocultured with CD19 expressing cells. Figure 18F: Activation of human CD3 signaling for clones Ly2800, Ly2802, Ly2943, Ly2944, and Ly305 cocultured with CD20 expressing cells. Figure 18G: Activation of human CD3 signaling for clones Ly2800, Ly2802, Ly2943, Ly2944, and Ly305 cocultured with Raji cells. FIG. 18H: Activation of human CD3 signaling for clones Ly2800, Ly2802, Ly2943, Ly2944, and Ly305 co-cultured with CD137-expressing cells. [Figures 19A-19G]Figures 19A and 19B include diagrams showing terminal Fv target activation activity of exemplary multispecific anti-CD19 / CD20 antibodies. Reference anti-CD137 mAb TM173 serves as a control. Figure 19A: Activation of human CD137 signaling for clones Ly1967, Ly2278, Ly2326, and TM173 alone. Figure 19B: Activation of human CD137 signaling for clones Ly1967, Ly2278, Ly2326, and TM173 co-cultured with CD19-expressing cells. Figure 19C: Activation of human CD137 signaling for clones Ly2800, Ly2802, Ly2943, Ly2944, and TM173 alone. Figure 19D: Activation of human CD137 signaling for clones Ly2800, Ly2802, Ly2943, Ly2944, and TM173 co-cultured with CD19-expressing cells. Figure 19E: Activation of human CD137 signaling for clones Ly2800, Ly2802, Ly2943, Ly2944, and TM173 co-cultured with CD20 expressing cells. Figure 19F: Activation of human CD137 signaling for clones Ly2800, Ly2802, Ly2943, Ly2944, and TM173 co-cultured with Raji cells. Figure 19G: Activation of human CD137 signaling for clones Ly2800, Ly2802, Ly2943, Ly2944, and TM173 co-cultured with Jurkat cells. [Figure 20A-20B] Figures showing the cytotoxic activity of exemplary multispecific antibodies are included. Figure 20A: Lactate dehydrogenase (LDH) release cytotoxicity assay for clones Ly1963, Ly1966, and Ly1967. Figure 20B: Luciferase transduced tumor cell cytotoxicity assay for clones Ly2800, Ly2802, Ly1967, Ly2943, Ly2944, Ly2948, and Ly2309. [Figure 21] FIG. 1 shows serum concentrations of CD19 / CD3 / CD137 multispecific antibody clone Ly1967 in mice administered a single dose of 5 mg / kg intraperitoneally. [Figures 22A-22C]Figures showing the anti-tumor activity of exemplary multispecific antibody clones are included. Figure 22A: Anti-tumor activity of clones Ly1967 and Ly2278 in a human PBMC-engrafted mouse model bearing Raji tumor cells. Figure 22B: Anti-tumor activity of Ly1967 and Ly531 in a MC38-huCD19-bearing mouse model of bone marrow transplanted from CD3 and CD137 knock-in mice. Figure 22C: Anti-tumor activity of Ly1967 and Ly2278 in a B16F10-huCD19-bearing mouse model of bone marrow transplanted from CD3 and CD137 knock-in mice. Figure 22D: Anti-tumor activity of clones Ly2800, Ly2802, Ly2307, and Ly531 in a human PBMC-engrafted mouse model bearing Raji tumor cells. FIG. 22E: Antitumor activity of clones Ly2943, Ly2944, Ly2307, and Ly531 in a human PBMC-engrafted mouse model bearing Raji tumor cells. [Fig. 23A-23H]

[0036] Figure 23A shows B7H3 binding activity of exemplary multispecific antibodies. Figure 23A: B7H3 binding activity for clones Ly1963, Ly1965, Ly2122, and parental anti-B7H3 clone Ly1612. Figure 23B: B7H3 binding activity for clones Ly2600 and Ly1612. Figure 23C: B7H3 binding activity for clones Ly2936, Ly2937, Ly2939, Ly2940, and Ly1612. Figure 23D: B7H3 binding activity for clones Ly2823 and Ly1612. Figure 23E: B7H3 binding activity for clones Ly2846, Ly2847, Ly2936, and Ly1612. Figure 23F: B7H3 binding activity for clones Ly2904, Ly2936, and Ly1612. Figure 23G: B7H3 binding activity for clones Ly2938 and Ly1612. Figure 23H: B7H3 binding activity for clones Ly2901, Ly2902, Ly2903, Ly2936, and Ly1612. [Fig. 24A-24H]24A-24C are charts showing CD3 binding activity of multispecific antibody clones containing anti-B7H3 binding moieties. The parental anti-CD3 mAb Ly305 serves as a control. Figure 24A: CD3 binding activity for clones Ly1963 and Ly1965. Figure 24B: CD3 binding activity for clones Ly2600 and Ly2936. Figure 24C: CD3 binding activity for clones Ly2936, Ly2937, and Ly2939. Figure 24D: CD3 binding activity for clone Ly2823. Figure 24E: CD3 binding activity for clones Ly2846, Ly2847, and Ly2936. Figure 24F: CD3 binding activity for clones Ly2904 and Ly2936. Figure 24G: CD3 binding activity for clones Ly2938, Ly2939, and Ly2940. FIG. 24H: CD3 binding activity for Ly2901, Ly2902, Ly2903, and Ly2936. [Figures 25A-25C] 25A-C are charts showing CD137 binding activity of exemplary B7H3-binding multispecific antibody clones. The parental anti-CD137 clone Ly1630 serves as a control. Figure 25A: CD137 binding activity for clones Ly1963 and Ly2122. Figure 25B: CD137 binding activity for clones Ly2936, Ly2937, Ly2940, and Ly2600. Figure 25C: CD137 binding activity for clone Ly2823. [Figure 26] 1 is a chart showing the CD28 binding activity of an exemplary anti-B7H3 / CD3 / CD28 clone, Ly2938. [Figure 27] 1 is a chart showing the PD-1 binding activity of an exemplary anti-B7H3 / CD3 / PD-1 clone Ly2904. [Figure 28] 1 is a chart showing the PD-L1 binding activity of exemplary anti-B7H3 / CD3 / PD-L1 clones Ly2846, Ly2847, and the parental anti-PD-L1 clone Ly2530. [Figure 29] 1 is a chart showing the CTLA-4 binding activity of exemplary anti-B7H3 / CD3 / CTLA4 clones Ly2901, Ly2902, Ly2903, and the reference anti-CTLA4 mAb Ly2896. [Fig. 30A-30Q]Includes charts showing activation of human CD3 signaling by the exemplary multispecific antibodies shown. The agonist activity of these multispecific antibodies was assessed either alone or in co-culture with additional target expressing cells. Figures 30A-30C: Agonist activity of clones Ly1963, Ly1965, and parental clone Ly305 alone (A), co-cultured with CD137 expressing CHO cells (B) or B7H3 expressing CHO cells (C). Figures 30D-30F: Agonist activity of clones Ly2600, Ly2936, Ly2937, Ly2939, Ly2940, Ly305, and Ly1761 alone (D), co-cultured with B7H3 expressing CHO cells (E) or CD137 expressing CHO cells (F). Figures 30G-30I: Agonistic activity of clones Ly2600, Ly2823, and Ly305 alone (G), co-cultured with B7H3-expressing CHO cells (H), or CD137-expressing CHO cells (I). Figures 30J-30L: Agonistic activity of clones Ly2846, Ly2847, and Ly305 alone (J), co-cultured with B7H3-expressing CHO cells (K), or PD-L1-expressing CHO cells (L). Figures 30M-30O: Agonistic activity of clones Ly2904 and Ly305 alone (M), co-cultured with B7H3-expressing CHO cells (N), or PD-1-expressing CHO cells (O). Figures 30P-30Q: Agonist activity of clones Ly2901, Ly2902, Ly2903, and Ly305 alone (P) and co-cultured with B7H3-expressing CHO cells (Q). [Fig. 31A-31Q]Includes charts showing activation of human CD137 signaling by the exemplary multispecific antibodies shown. Agonistic activity of these multispecific antibodies was assessed either alone or in co-culture with additional target expressing cells. Levels of IL-8 or luminescence produced by reporter cells indicate CD137 activation. Figures 31A-31C: Agonistic activity of clone Ly1963 and reference anti-CD137 mAb TM173 alone (A), co-cultured with B7H3 expressing CHO cells (B) or CD3 expressing cells (C). Figures 31D-31F: Agonistic activity of clones Ly2600, Ly2936, Ly2937, Ly2823, parental anti-CD137 clone Ly1630, and reference anti-CD137 mAb TM173 alone (D), co-cultured with B7H3 expressing CHO cells (E) or CD3 expressing cells (F). [Fig. 32A-32L]Figures showing cytotoxic activity and IFN-γ secretion of exemplary multispecific antibodies are included. Figure 32A: Killing of B7H3-expressing A375 cells by human PBMCs when treated with clones Ly1963, Ly1965, and Ly2128. Figure 32B: IFN-γ secretion by PBMCs when co-cultured with B7H3-expressing A375 cells and treated with clones Ly1963, Ly1965, and Ly2128. Figure 32C: Killing of A375-Luc cells by human PBMCs when treated with clones Ly2600, Ly2936, Ly2937, Ly2939, and Ly1963. Figure 32D: IFN-γ secretion by PBMCs when A375-Luc cells were co-cultured and treated with clones Ly2600, Ly2936, Ly2937, Ly2939, and Ly1963. Figure 32E: Killing of A375-Luc cells by human PBMCs when treated with clones Ly2938 and Ly1963. Figure 32F: IFN-γ secretion by PBMCs when A375-Luc cells were co-cultured and treated with clones Ly2938 and Ly1963. Figure 32G: Killing of A375-Luc cells by human PBMCs when treated with clones Ly2846, Ly2847, and Ly1963. Figure 32H: IFN-γ secretion by PBMCs when A375-Luc cells were co-cultured and treated with clones Ly2846, Ly2847, and Ly1963. Figure 32I: Killing of A375-Luc cells by human PBMCs when treated with clones Ly2904 and Ly1963. Figure 32J: IFN-γ secretion by PBMCs when A375-Luc cells were co-cultured and treated with clones Ly2904 and Ly1963. Figure 32K: Killing of A375-Luc cells by human PBMCs when treated with clones Ly2901, Ly2902, Ly2903, and Ly1963. Figure 32L: IFN-γ secretion by PBMCs when A375-Luc cells were co-cultured and treated with clones Ly2901, Ly2902, Ly2903, and Ly1963. [Diagram 33] 1 is a chart showing serum concentrations of multispecific antibody Ly1963 comprising an anti-B7H3 binding moiety in mice administered a single dose of 5 mg / kg intraperitoneally. [Fig. 34A-34L]Figures showing the anti-tumor activity of exemplary multispecific antibodies are included. Figure 34A: Anti-tumor activity of Ly1963, Ly1965, and Ly2122 in a LL2-huB7H3-bearing mouse model of bone marrow transplanted from CD3 and CD137 knock-in mice. Figures 34B-34L: Anti-tumor activity, group mean and individual tumor volumes for Ly2823, Ly2936, Ly2937, Ly2600, Ly2846, Ly2847, Ly2938, Ly2939, and Ly2940 in a human PBMC-engrafted mouse model bearing A375 tumor cells are shown in Figure 34B and Figures 34C-34L, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] The present disclosure provides multispecific antibodies, optionally in multivalent form, utilizing a format that includes one or more monovalent Fv fragments. Such monovalent Fv fragments will have a relatively low binding affinity to a target antigen, e.g., an immune cell receptor. In some cases, the multispecific antibodies disclosed herein may further include one or more high affinity binding moieties specific for a tumor-associated antigen and / or an immune cell receptor, such as a T cell activation receptor or an inhibitory receptor (e.g., a checkpoint receptor). Thus, the multispecific antibodies disclosed herein can bind to a target immune receptor at a minimal level so as not to stimulate the target immune receptor in the absence of the target antigen to which the high affinity binding moieties bind, thereby minimizing undesirable immune responses and potential side effects.

[0045] It has been reported herein that multispecific antibodies comprising Fv binding moieties activate signaling pathways mediated by the immune cell receptor to which the Fv fragment binds only in the presence of additional target antigens, e.g., in the tumor microenvironment. In addition, two or more Fv fragments contained in a multispecific antibody allow for simultaneous binding to two or more immune cell receptors, providing potential synergistic effects specific to those targets. Alternatively or additionally, the use of peptide linkers disclosed herein (including flexible and / or rigid peptide linkers) alone or in combination with engineered Fc fragments allows for the reduction or elimination of undesired Fc effector functions, reduction of undesired aggregation or homodimerization of polypeptides, and / or improved stability of multispecific antibodies comprising such.

[0046] Without being bound by theory, such multispecific antibodies may modulate desired immune responses and therapeutic activity by cross-linking multiple target antigens. The advantageous features of multispecific antibodies with specific Fv-containing formats disclosed herein have been demonstrated in multiple exemplary antibodies targeting different antigens. See, for example, the following examples. Thus, such advantageous features result from the specific design of the multispecific antibody formats disclosed herein, i.e., utilizing one or more antigen-binding moieties in the form of Fv fragments and optionally a desired peptide linker, as well as an engineered Fc fragment. In other words, such advantageous features are not antigen-specific and / or antibody sequence-specific.

[0047] As used herein, a multispecific antibody refers to an antibody that can bind to two or more target antigens. In some instances, the multispecific antibody disclosed herein can be a bispecific antibody, i.e., can bind to two different target antigens or can bind to two different epitopes of a target antigen. In some instances, the multispecific antibody disclosed herein can be, for example, a trispecific antibody that binds to three different target antigens or epitopes. Alternatively, the multispecific antibody disclosed herein can be, for example, a tetraspecific antibody that binds to four different target antigens or epitopes.

[0048] As used herein, a multivalent antibody refers to an antibody having two or more antigen-binding sites. In some examples, the multispecific antibody disclosed herein may have two antigen-binding sites. In some examples, the multispecific antibody disclosed herein may have three antigen-binding sites. In some examples, the multispecific antibody disclosed herein may have four antigen-binding sites.

[0049] Accordingly, provided herein are multispecific antibodies having the specific designs disclosed herein, nucleic acids encoding the multispecific antibodies, host cells harboring the encoding nucleic acids, methods of producing the multispecific antibodies, and methods of using the multispecific antibodies in immune response modulation and treatment of targeted diseases such as cancer.

[0050] I. Multispecific antibodies The multispecific antibodies disclosed herein comprise two or more antigen-binding moieties, which may be linked via a peptide linker (e.g., a flexible or rigid peptide linker as provided herein) or a heavy chain constant region fragment, such as an Fc fragment, also disclosed herein. At least one antigen-binding moiety in the disclosed multispecific antibodies is in the Fv format.

[0051] A. Antigen-binding portion Each antigen-binding portion in any of the multispecific antibodies disclosed herein may be an antigen-binding portion of any form, including, but not limited to, an intact (i.e., full-length) antibody, an antigen-binding fragment thereof (such as Fab, Fab', F(ab').sub.2, Fv, tribodies, triFabs, tandem linked Fabs, Fab-Fv, tandem linked V domains, tandem linked scFv, and other formats), single chain antibodies (scFv antibodies), single domain antibodies such as VHHs, crossed Fabs, and tetravalent antibodies.

[0052] At least one antigen-binding moiety in the multispecific antibody disclosed herein is in Fv format. Other antigen-binding moieties in the multispecific antibody may be in scFv format, single domain antibody format (e.g., VHH), Fab format, cross-Fab format, or combinations thereof. In some examples, at least one antigen-binding moiety in the multispecific antibody disclosed herein is in Fab format. In other examples, at least one antigen-binding moiety in the multispecific antibody disclosed herein is in cross-Fab format. Alternatively or additionally, at least one antigen-binding moiety in the multispecific antibody disclosed herein is in scFv format.

[0053] Any scFv fragment in a bispecific or multispecific antibody may be H →V L Alternatively, this may be in the V L →V H The antigen-binding portion of the Fab format contains two separate chains, one containing the VH-CH1 fragment and the other containing the VL-CL fragment. The CL domain may be a Ck domain or a Cl domain.

[0054] Single domain antibodies (sdAbs), also known as nanobodies, are antibody fragments consisting of a single monomeric variable antibody domain. In some embodiments, single domain antibodies may be heavy chain only (VHH) fragments that may be derived from camelid antibodies.

[0055] A Fab fragment typically contains two separate chains: one chain contains a heavy chain variable region (VH) in association with a heavy chain constant region fragment such as CH1, and the other chain contains a light chain variable region (VL) in association with a light chain constant region (e.g., Cκ or Cλ).

[0056] A crossed Fab fragment has a similar two-chain structure as a Fab fragment, but with swapped VH / VL and heavy / light chain constant region fragment connections. A crossed Fab fragment contains a first chain containing a VH connected to a light chain constant region (e.g., Cκ or Cλ) and a second chain containing a VL connected to a heavy chain constant region fragment such as CH1.

[0057] The antigen-binding moieties in the multispecific antibodies disclosed herein may specifically bind to multiple immune cell receptors (e.g., immune cell activation receptors such as T cell activation receptors), or may specifically bind to at least one immune cell receptor and at least one TAA. Exemplary immune cell receptor antigens include, but are not limited to, CD3, CD28, PD-1, PD-L1, CTLA4, CD47, and members of the tumor necrosis factor receptor superfamily (TNFRSF). Examples of TNFRSF family members include, but are not limited to, FAS, TNFRSF12A, 4-1BB / CD137, TNFRSF13B, TNFRSF13C, CD27 / TNFRSF7, CD30 / TNFRSF8, CD40 / TNFRSF5, DR3 / TNFRSF25, DR4 / TNFRSF10A, DR5 / TNFRSF10B, DR6 / TNFRSF21, GITR / TNFRSF18, HVEM / TNFRSF14, LTBR, ​​OX40 / TNFRSF4, TROY / TNFRSF19, RELT / TNFRSF19L, TNFRSF12A, TNFRSF13B, TL1A / TNFSF15, TNFRSF17, TNFRSF1A, TNFRSF11B, RANK / TNFRSF11A, TNFRSF11B, NGFR, EDA2R, and TNFRSF1B.

[0058] In some examples, the antigen-binding moieties in the multispecific antibodies disclosed herein may specifically bind to multiple immune cell receptors, such as immune cell checkpoint receptors, including, but not limited to, PD-1, PD-L1, and CD47.

[0059] Tumor-associated antigens (TAA) refer to antigens produced by tumor cells. TAAs are tumor markers for identifying tumor cells and therapeutic targets for use in cancer therapy. Exemplary TAAs include, but are not limited to, B7H3, CD19, CD20, PSMA, HER2, CEA, BCMA, p53, p53mut, DLL3, MET, EGFR, B7H4, CD20, FGF, HER2, HER3, BCMA, P53mut, MSLN, EPCAM, ROR1, MAGE (e.g., MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A10, MAGE-A12, MAGE-B, or MAGE-C), SSX2, CAGE, GAGE, NY-ESO-1, SPANX-A, SPANX-C, SPANX-D, PRAME, PECAM, ICAM-3, and HLA-DR, PI3K, RAS, RAF, MEK, and ERK.

[0060] The Fv fragments (e.g., the middle Fv fragment or the hidden Fv fragments) in the multispecific antibody can specifically bind to an immune cell receptor, such as a T cell receptor (e.g., a T cell activation receptor or a T cell checkpoint receptor). For example, the middle (hidden) Fv fragment in the multispecific antibody disclosed herein can be specific for CD3, CD137, CD40, CD28. In some cases, the multispecific antibody can contain a middle Fv fragment and a terminal Fv fragment. In some examples, the two Fv fragments can be specific for a pair of immune cell receptors, such as CD3 and CD28, CD3 and CD137, CD3 and PD-1, CD3 and PD-L1, CD3 and CTLA4, CD137 and PD-1, CD40 and PD-1, CD40 and PD-L1, CD137 and GITR, CD137 and PD-L1, CD137 and CD40, CD137 and OX40. For example, the central Fv fragment can be specific for CD3 and the terminal Fv fragments can be specific for different immune cell receptors, e.g., CD40, CD137, CD28, PD-1, PD-L1, CTLA4, FAS, TNFRSF12A, 4-1BB / CD137, TNFRSF13B, TNFRSF13C, CD27 / TNFRSF7, CD30 / TNFRSF8, CD40 / TNFRSF5, DR3 / TNFRSF25, DR4 / TNFRSF10A, DR5 / TNFRSF The gene may be specific for F10B, DR6 / TNFRSF21, GITR / TNFRSF18, HVEM / TNFRSF14, LTBR, ​​OX40 / TNFRSF4, TROY / TNFRSF19, RELT / TNFRSF19L, TNFRSF12A, TNFRSF13B, TL1A / TNFSF15, TNFRSF17, TNFRSF1A, TNFRSF11B, RANK / TNFRSF11A, TNFRSF11B, NGFR, EDA2R, or TNFRSF1B.In other examples, the central Fv fragment can be specific for an immune cell receptor such as CD3, CD137, CD40, CD47, or CD28, and the terminal Fv fragment can be specific for a TAA, e.g., any one of B7H3, CD19, CD20, MAGE-A4, PRAME, PSMA, HER2, CEA, BCMA, P53mut, DLL3, MET, and EGFR.

[0061] In addition to Fv fragments, the multispecific antibodies disclosed herein may further comprise additional antigen-binding moieties, such as, for example, scFv format, single domain antibodies such as heavy chain only (VHH) format, Fab format, or cross-Fab format, which may be specific for different immune cell receptors or different TAAs. Collectively, multispecific antibodies may bind to more than one immune cell receptor, or may bind to at least one immune cell receptor and at least one TAA.

[0062] In some embodiments, the multispecific antibodies disclosed herein may be bispecific antibodies that bind to two different immune cell receptors or that bind to one immune cell receptor (e.g., CD3, CD40, CD137, PD-1, CD47, or PD-L1) and one TAA (e.g., CD19, CD20, MAGE-A4, PRAME, B7H3, CEA, P53mut, PSMA, or HER2). Examples include, but are not limited to, CD3 and B7H3, CD137 and B7H3, CD3 and CD19, CD137 and CD19, CD137 and PD-1, CD40 and PD-1, CD40 and B7H3, CD40 and PD-L1, CD3 and PD-1, CD3 and CEA, CD3 and P53mut, CD137 and P53mut, CD137 and PD-L1, CD3 and BCMA, CD137 and BCMA, CD3 and PSMA, CD137 and PSMA, CD3 and HER2, CD137 and HER2, CD40 and HER2, B7H3 and CD137, B7H3 and CD3, B7H3 and CD28, CD19 and CD3, CEA and CD137, PD-1 and CD137, PD-1 and CD40, or PMSA and CD3.

[0063] In some embodiments, a multispecific antibody disclosed herein may be a trispecific antibody that binds to a mixture of three different antigens, which may be immune cell receptors and TAAs, e.g., those disclosed herein. In some cases, the trispecific antibody binds to two different immune cell receptors and one TAA. In other cases, the trispecific antibody binds to two different TAAs and one immune cell receptor. Exemplary antigen combinations include B7H3, CD3, and CD137; CD19, CD3, and CD137; B7H3, CD3, and CD28; CD19, CD3, and CD28; B7H3, CD137, and PD-1; B7H3, CD40, and PD-1; PMSA, CD3, and CD137; CD20, CD3, and CD137; HER2, CD3, and CD137; CEA, CD3, and CD137; BCMA, CD3, and CD137; P53 mutant, CD3, and CD137; PD-L1, CD3, and CD1 37; PD-L1, CD3, and CD28; PD-L1, CD137, and B7H3; PD-L1, CD40, and B7H3; PD-1, CD40, and CD137; PD-1, CD137, and GITR; CD19, CD20, CD3, and CD137; PD-L1, CD40, and CD137; MET, EGFR, and CD47; CD19, CD3, and CD137; B7H3, CD3, and PD-1; B7H3, CD3, and PD-L; B7H3, CD3, and CTLA4.

[0064] An antibody that "specifically binds" to an antigen or epitope is a term well understood in the art. A molecule is said to exhibit "specific binding" if it reacts more frequently, more rapidly, with a longer duration, and / or with a higher affinity to a particular target antigen than to alternative targets. An antibody "specifically binds" to a target antigen or epitope if it binds with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to an antigen (e.g., those described above) or an antigenic epitope therein is an antibody that binds to this target antigen with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other antigens or other epitopes within the same antigen. By this definition, it is also understood that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second or third target antigen. Thus, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. In some instances, an antibody that "specifically binds" to a target antigen or epitope thereof may not bind to other antigens or other epitopes within the same antigen (i.e., only baseline binding activity may be detected by conventional methods). Alternatively or additionally, the antibodies described herein specifically bind to a human antigen or fragment thereof compared to a monkey counterpart, or vice versa (e.g., have at least a 10-fold higher binding affinity for one antigen than the other, as determined in the same assay under the same assay conditions). In other instances, the antibodies described herein may cross-react with human and non-human antigens (e.g., monkeys), e.g., the difference in binding affinity for human and non-human antigens is less than 5-fold, e.g., less than 2-fold, or substantially similar.

[0065] In some embodiments, the antigen-binding portion of any of the bispecific or multispecific antibodies described herein has suitable binding affinity for a target antigen (e.g., an immune cell receptor or TAA disclosed herein) or an antigenic epitope thereof. As used herein, "binding affinity" refers to the apparent association constant or KA Refers to. A is the dissociation constant (K D The antibodies described herein have a binding affinity of at least 10 to a target antigen or antigen epitope. -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 M or lower binding affinity (K D The increase in binding affinity can be expressed as K D The higher affinity binding of an antibody to a first antigen compared to a second antigen corresponds to a decrease in the K A (or number K D ) for binding the first antigen. A (or a smaller number K D In such cases, the antibody has specificity for a first antigen (e.g., a first protein in a first conformation or a mimetic thereof) compared to a second antigen (e.g., the same first protein or a second protein in a second conformation or a mimetic thereof). The difference in binding affinity (e.g., for specificity or other comparison) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000, or 10 5 In some embodiments, any of the antibodies may be further affinity matured to increase the binding affinity of the antibody to the target antigen or antigenic epitope thereof.

[0066] Binding affinity (or binding specificity) can be determined by a variety of methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for evaluating binding affinity are HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v / v) Surfactant P20). These techniques can be used to measure the concentration of bound binding protein as a function of target protein concentration. The concentration of bound binding protein ([Bound]) is generally related to the concentration of free target protein ([Free]) by the following formula: [Bound]=[Free] / (Kd+[Free])

[0067] However, obtaining a quantitative measure of affinity, as determined using methods such as ELISA or FACS analysis, is not sufficient. A and can therefore be used for comparison, such as to determine whether a higher affinity, e.g., 2-fold higher, is to obtain a qualitative measure of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay, e.g., an in vitro or in vivo assay, so that K A It is not necessary to make a precise determination of the

[0068] Exemplary Parent Antibodies The antigen-binding portions of the multispecific antibodies disclosed herein may be derived from parent antibodies specific for any of the immune cell receptors or TAA target antigens disclosed herein. Exemplary parent antibodies from which any of the antigen-binding portions are derived are provided in Table 1 below (heavy and light chain CDRs based on the Kabat scheme are identified in bold).

[0069] As used herein, an antigen-binding portion of a multispecific antibody "derived from" a parent antibody means that the parent antibody is used as the starting material for creating one antigen-binding site in the multispecific antibody. The antigen-binding portion may contain the same heavy and / or light chain CDRs as those of the parent antibody. The same V Hand / or V L Two antibodies having CDRs mean that the CDRs are identical when determined by the same method (e.g., the Kabat definition, the Chothia definition, the AbM definition, and / or the contact definition known in the art).

[0070] In some cases, the antigen-binding portion derived from the parent antibody may be a functional variant of the parent antibody. Such functional variants are substantially similar to the reference antibody, both structurally and functionally. Functional variants have substantially the same V H and V L CDRs. For example, a functional variant may only contain up to five (e.g., four, three, two, or one) amino acid residue mutations in the entire heavy chain CDR region of the reference antibody and / or up to five (e.g., four, three, two, or one) amino acid residue mutations in the entire light chain CDR region of the reference antibody. In some examples, a functional variant may contain up to eight (e.g., seven, six, five, four, three, two, or one) amino acid residue mutations in the entire heavy and light chain CDRs compared to those of the reference antibody. Such functional variants may bind to the same epitope of B7H3 with substantially similar affinity (e.g., the same order of K D Alternatively or additionally, the amino acid residue variations are conservative amino acid residue substitutions as disclosed herein.

[0071] In some embodiments, the antigen-binding portion of the multispecific antibodies disclosed herein is the V H Alternatively or additionally, the antigen-binding portion may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the V CDRs as the parent antibody. L It may comprise light chain CDRs that, individually or collectively, are at least 80% (eg, 85%, 90%, 95%, or 98%) sequence identical to the CDRs.

[0072] In other embodiments, the antigen-binding portion is the VH Alternatively or additionally, the antigen-binding portion may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the V CDRs as the parent antibody. L It may comprise light chain CDRs that, individually or collectively, are at least 80% (eg, 85%, 90%, 95%, or 98%) sequence identical to the CDRs.

[0073] The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the present invention. When gaps exist between the two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0074] Alternatively or additionally, the amino acid residue mutation may be a conservative amino acid residue substitution. As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants may be prepared according to methods for altering polypeptide sequences known to those skilled in the art, which methods are found in references that collect such methods, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FMAusubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include substitutions made between amino acids within the following groups: (a) M, I, L, V, (b) F, Y, W, (c) K, R, H, (d) A, G, (e) S, T, (f) Q, N, and (g) E, D.

[0075] B. Multispecific Antibody Formats The multispecific antibodies disclosed herein are multi-chain molecules comprising two or more antigen-binding moieties, at least one of which is in the Fv format. The other antigen-binding moieties in the multispecific antibodies, if present, may be in any suitable format, such as scFv format, single domain antibody format (e.g., heavy chain antibody or VHH), Fab, or cross-Fab), or a combination thereof. In other examples, one or more of the antigen-binding moieties used to construct the multispecific antibodies disclosed herein may be in the BCR or TCR format.

[0076] In some embodiments, a multispecific antibody may comprise one Fv antigen-binding portion and one or more additional antigen-binding portions in the form of scFv and / or Fab. For example, a multispecific antibody may comprise one Fv fragment and two Fab fragments. The two Fab fragments may have different structures for binding to different antigens or different epitopes of the target antigen. Alternatively, the two Fab fragments may be identical. In another example, a multispecific antibody may comprise one Fv fragment and one Fab fragment.

[0077] In other embodiments, a multispecific antibody may comprise two Fv antigen-binding moieties specific for different target antigens, and optionally one or more additional antigen-binding moieties in the form of scFv and / or Fab. In some cases, a multispecific antibody may comprise two Fv fragments and two Fab fragments. The two Fab fragments may have different structures for binding to different antigens or different epitopes of the target antigen. Alternatively, the two Fab fragments may be identical. In other examples, a multispecific antibody may comprise two Fv fragments and one Fab fragment.

[0078] The multiple antigen-binding moieties in the multispecific antibodies disclosed herein can be linked via peptide linkers, such as the flexible and rigid peptide linkers disclosed herein.

[0079] In some cases, the two antigen-binding moieties are connected via a heavy chain constant region fragment, e.g., a fragment comprising the hinge domain, CH2, and CH3, and the disulfide bond formation characteristics of such a fragment (e.g., in the hinge domain) can be exploited to form a dimer between the two polypeptides of the multispecific antibody. In some cases, the multispecific antibody disclosed herein can further comprise a heavy chain constant region fragment, such as an Fc fragment (typically comprising CH2 and CH3 of the heavy chain constant chain), to facilitate the formation of the whole antibody molecule via disulfide bond formation and / or to further modulate the immune response via its Fc receptor binding activity.

[0080] (a) Fv binding part As used herein, an Fv fragment comprises heavy chain variable domains (V H ) and the light chain variable domain (V L ) (see, for example, FIG. 1A). In some embodiments, the Fv fragment in the multispecific antibodies disclosed herein may be located in the center of the antibody molecule, i.e., V H and V L Each of the fragments is located in the middle of the polypeptide with its N-terminus and C-terminus linked to other components of the antibody (e.g., other antigen-binding moieties). Such Fv fragments are also referred to as middle Fvs or hidden Fvs as disclosed herein. See, e.g., FIG. 1B. In other embodiments, the Fv fragments in the multispecific antibodies disclosed herein can be located at either end of the antibody molecule. H and V L Each of the fragments is located in the middle of a polypeptide with one of its N-terminus and C-terminus linked to another component of the antibody, such as another antigen-binding moiety. The other terminus may be the terminus of the entire polypeptide, or may be linked to a peptide linker rather than any other antigen-binding moiety or heavy chain constant region fragment, such as an Fc fragment. Such Fv fragments are also referred to as terminal Fvs, as disclosed herein. See, for example, FIG. 1C.

[0081] In some cases, the multispecific antibodies disclosed herein contain one central Fv fragment that may be specific for an immune cell receptor of interest as disclosed herein (e.g., CD3, CD137, CD40, GITR, OX40, CD47, PD-1, or CD28). In some cases, the multispecific antibodies disclosed herein may contain one terminal Fv fragment that may be specific for an immune cell receptor of interest as disclosed herein (e.g., CD3, CD137, CD40, GITR, OX40, CD47, CD28, PD-1, PD-L1, CTLA4). In some cases, the multispecific antibodies disclosed herein may comprise at least two Fv fragments (e.g., two Fv fragments), one being a central Fv and the other being a terminal Fv. See, e.g., FIG. 1D. The two Fv fragments may be specific for two different immune cell receptors, e.g., two immune cell receptors that provide complementary signaling for optimal immune regulation. Alternatively, one of the two Fv fragments (e.g., the middle Fv) can be specific for an immune cell receptor of interest such as those disclosed herein (e.g., CD3, CD137, CD40, GITR, OX40, CD47, or CD28), and the other (e.g., the terminal Fv) can be specific for a TAA such as those provided herein. Alternatively, the other (e.g., the terminal Fv) can be specific for a second immune cell receptor such as those disclosed herein.

[0082] Fv fragments in a multispecific antibody, specifically the central Fv, are expected to have a relatively low binding affinity to the target antigen (e.g., immune cell receptors such as those disclosed herein, e.g., CD3, CD137, CD40, GITR, OX40, CD47, PD-1, PD-L1, CTLA4, or CD28) in the absence of other antigens to which other antigen-binding moieties in the same multispecific antibody bind. Without being bound by theory, the presence of other antigens may restore or enhance the Fv fragment binding affinity and activation capacity, in some cases, to a similar or even higher level compared to that of their parental mAb clones. As shown in the examples below, the terminal Fvs show at least a 2- to 10-fold reduced binding affinity or activation capacity alone compared to their parental mAb clones. As for the central Fv, it shows an even lower 10- to 100-fold reduced or zero binding affinity or activation capacity alone compared to that of their parental mAb clones. However, when co-cultured with other target antigen-expressing cells, the binding affinity and activation potency of these middle or terminal Fvs are enhanced, in some cases, to levels similar to or even higher than those of their parental mAb clones.

[0083] Taking this approach to the design of the multispecific antibodies disclosed herein provides one or more binding arms to one or more target immune cell receptors or TAs of interest, but avoids eliciting an immune response in the absence of high affinity target antigens that may be accompanied by undesirable clinical side effects. Multispecific antibodies create synthetic biology that activates signaling pathways mediated by immune cell receptors that Fv fragments bind only in the presence of additional target antigens in the tumor microenvironment, via avidity-driven cross-linking of multiple target antigens, to achieve therapeutic efficacy.

[0084] (b) Peptide linker A peptide linker may be provided between two fragments in a polypeptide of a multispecific antibody disclosed herein, such as a V in an scFv fragment. H Part and V L Between the V and V portions of the Fv fragment H Part or VL It may be located between the moiety and the chain of another antigen-binding moiety, or between the VH or VL of an Fv fragment and a heavy chain constant region fragment, such as an Fc fragment.

[0085] Any of the peptide linkers described herein may include naturally occurring and / or non-naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamic acid (Glu), glutamine (Gin), glycine (Gly), histidine (His), isoleucine (He), leucine (Leu), lysine (Lys), methionine (Met), ornithine (Orn), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). Non-naturally occurring amino acids may include protected amino acids, such as naturally occurring amino acids protected with groups such as acetyl, formyl, tosyl, nitro, etc.Non-limiting examples of non-naturally occurring amino acids include azidohomoalanine, homopropargylglycine, homoallylglycine, p-bromophenylalanine, p-iodophenylalanine, azidophenylalanine, acetylphenylalanine or ethynylphenylalanine, amino acids containing an internal alkene such as trans-crotylalkene, serine allyl ether, allylglycine, propargylglycine, vinylglycine, pyrrolidine, N-sigma-o-azidobenzyloxycarbonyl-L-lysine (AzZLys), N-sigma -Propargyloxycarbonyl-L-lysine, N-sigma-2-azidoethoxycarbonyl-L-lysine, N-sigma-tert-butyloxycarbonyl-L-lysine (BocLys), N-sigma-allyloxycarbonyl-L-lysine (AlocLys), N-sigma-acetyl-L-lysine (AcLys), N-sigma-benzyloxycarbonyl-L-lysine (ZLys), N-sigma-cyclopentyloxycarbonyl-L-lysine (CycLys), N-sigma-D-prolyl-L-lysine, N-sigma-nicotinoyl -L-lysine (NicLys), N-Sigma-N-Me-anthraniloyl-L-lysine (NmaLys), N-Sigma-biotinyl-L-lysine, N-Sigma-9-fluorenylmethoxycarbonyl-L-lysine, N-Sigma-methyl-L-lysine, N-Sigma-dimethyl-L-lysine, N-Sigma-multimethyl-L-lysine, N-Sigma-isopropyl-L-lysine, N-Sigma-dansyl-L-lysine, N-Sigma-o,p-dinitrophenyl-L-lysine, N-Sigma-p-toluenesulfonyl-L-lysine, N-Sigma-D Included are L-2-amino-2 carboxyethyl-L-lysine, N-sigma-phenylpyruvamido-L-lysine, N-sigma-pyruvamido-L-lysine, azidohomoalanine, homopropargylglycine, homoallylglycine, p-bromophenylalanine, p-iodophenylalanine, azidophenylalanine, acetylphenylalanine or ethynylphenylalanine, amino acids containing an internal alkene such as trans-crotylalkene, serine allyl ether, allyl glycine, propargylglycine, and vinyl glycine.

[0086] The peptide linkers provided herein may contain about 5 to 160 amino acid residues, for example, about 10 to 120 amino acid residues, about 10 to 100 amino acid residues, about 10 to 80 amino acid residues, about 10 to 60 amino acid residues, about 10 to 50 amino acid residues, about 10 to 40 amino acid residues, about 10 to 30 amino acid residues, or about 10 to 20 amino acid residues.

[0087] In some embodiments, the peptide linker may be a flexible peptide linker, typically containing small flexible amino acid residues to connect various domains in a multispecific antibody without affecting their binding activity. In some examples, the flexible peptide linker is, for example, a Gly-rich linker containing a (GxS)n motif, where X is an integer of 1, 2, 3, 4, 5, or 6, and n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Exemplary flexible peptide linkers are provided in Table 1 below, any of which may be used to construct the multispecific antibodies disclosed herein.

[0088] In some embodiments, the peptide linker may be a so-called rigid peptide linker that contains at least one cysteine ​​residue (e.g., one or two cysteine ​​residues) so that it can form a disulfide bond with another rigid peptide linker. The use of one or more pairs of rigid peptide linkers in the multispecific antibodies disclosed herein can promote dimerization across multiple polypeptides of the antibody via disulfide formation, thereby forming an intact multi-chain antibody molecule.

[0089] In some cases, the rigid peptide linker may be derived from the hinge domain of an IgG molecule (positions 216-230 according to the EU numbering system or fragments thereof), such as an IgG1 molecule, taking advantage of the disulfide bond formation ability of such fragments. In some examples, such a peptide linker is a fragment of a wild-type IgG molecule (e.g., a human IgG1 molecule). Alternatively, the rigid peptide linker may contain one or more mutations compared to the wild-type counterpart.

[0090] In some instances, the rigid peptide linker may contain only the hinge domain of an IgG molecule or a fragment thereof. In other instances, the rigid peptide linker may contain the hinge domain or a fragment thereof, as well as a Gly-rich fragment (e.g., as disclosed herein) that may be linked to either the N-terminus and / or C-terminus of the hinge domain or a fragment thereof. Examples of rigid peptide linkers for use in any of the multispecific antibodies disclosed herein are provided in Table 1 below.

[0091] (c) Heavy chain constant region fragment and Fc fragment In some embodiments, the multispecific antibodies disclosed herein may contain a pair of heavy chain constant region fragments, such as an Fc fragment, each located on a separate polypeptide.

[0092] In some instances, the heavy chain constant region fragment contains the hinge domain, CH2 domain, and CH3 domain of a suitable immunoglobin (Ig) molecule, such as an IgG molecule. In some cases, the heavy chain constant region fragment is derived from an IgG1 molecule. In some instances, the heavy chain constant region fragment can be an Fc fragment containing the CH2 domain and CH3 domain of a suitable Ig molecule, such as an IgG, such as IgG1. In some instances, the heavy chain constant region fragment is a fragment of a wild-type Ig molecule (e.g., an IgG, such as IgG1). Alternatively, the heavy chain constant region fragment can contain an increasing number of mutations compared to the wild-type counterpart.

[0093] In some cases, a heavy chain constant region fragment, such as an Fc fragment, may contain one or more mutations that enhance heterodimer formation. Examples include "knobs-into-holes" (Ridgway et al., Protein Engineering, 9(7), pp. 617-21 (1996); Merchant et al., Nature Biotechnology, 16(7), pp. 677-681 (1998)), electrostatic (Gunasekaran et al., Journal of Biological Chemistry, 285(25), pp. 19637-19646 (2010)), or negative state designs (Kreudenstein et al., mAbs, 5(5), pp. 646-654 (2013); Leaver-Fay et al., Structure, 24(4), pp. 641-651 (2016)) (charge mutations). Other examples can be found, for example, in Brinkmann et al., MABS (2017), 9(2):182-212, the relevant disclosure of which is incorporated by reference for the subject matter and purposes mentioned herein.

[0094] In some examples, the mutations can be at position 366 (e.g., T366W or T366S), 368 (e.g., L368A), and / or 407 (e.g., G407V). In a particular example, one heavy chain constant region fragment in a multispecific antibody can contain mutations at positions 366 (e.g., T366W) and 407 (e.g., G407V) and a second heavy chain constant region fragment in the same multispecific antibody can contain mutations at positions 366 (e.g., T366S), 368 (e.g., L368A), and 407 (G407V). Unless explicitly called out, all numbers referring to positions in an Ig molecule follow the EU numbering system.

[0095] In some cases, mutations that reduce binding affinity to Protein A may be introduced into one or both of the heavy chain Fc regions in a multispecific antibody to facilitate purification of the multispecific antibody. Such mutations are known in the art. See, e.g., Tustian et al., mAbs 8:828-838 (2016), the relevant disclosure of which is incorporated by reference for purposes and subject matter noted herein.

[0096] Alternatively or additionally, mutations at positions involved in Fc receptor binding may be introduced into the heavy chain constant region fragment. Such mutations may modulate binding affinity and selectivity to the Fc receptor. Unless otherwise expressly indicated, the numbering of positions in the heavy or light chain of an antibody is according to the EU index numbering. In some cases, the heavy chain constant region fragment, such as the Fc fragment, may contain an amino acid substitution at one or more of positions 267, 273, 328, and 329. In some cases, the one or more mutations may be an amino acid substitution at one or more of positions 239, 265, 297, 329, 330, and 332. In one example, the heavy chain constant region fragment, such as the Fc fragment, may contain (i) a deletion at position 237 and (ii) two amino acid substitutions at positions 234 (e.g., L234A) and 235 (e.g., L235A). In another example, the heavy chain constant region fragment can include one or more of the following: (i) a deletion at position 237, (ii) two amino acid substitutions selected from L234A, L235A, and P329G, (iii) a deletion at position 237 and amino acid substitutions D265A and N297A, (iv) amino acid substitutions S239D, A330L, and I332E, and (v) a deletion at position 237 and the amino acid substitution P329G. In some examples, the Fc fragment disclosed herein may include (i) an amino acid substitution at position 329, optionally P329G; (ii) an amino acid substitution at positions 265 and 297, optionally D265A and N297A; (iii) an amino acid substitution at positions 239, 330, and 332, optionally S239D, A330L, and I332E; or a combination of any one of (i)-(iii).

[0097] Additional mutations of Fc fragments to modulate Fc receptor binding activity can be found, for example, in US-2020-0392227, the relevant disclosure of which is incorporated by reference for the subject matter and purposes mentioned herein.

[0098] C. Exemplary Multispecific Antibodies Exemplary designs of the multispecific antibodies disclosed herein are provided below.

[0099] (a) Multispecific antibody with a central Fv In some embodiments, the multispecific antibody disclosed herein comprises a first V H and the first V L The first Fv fragment contains the following: H Chain and V L Each of the chains is connected at one end to a flexible peptide linker (as disclosed herein, see Table 1 for examples) and at the other end to a rigid peptide linker (as disclosed herein, see Table 1 for examples). See FIG. 1A. Such multispecific antibodies include a first flexible peptide linker, V H and a first polypeptide comprising a first rigid peptide linker, V L and a second polypeptide comprising a second rigid peptide linker. In some cases, the first flexible peptide and the second flexible peptide linker are the same. Alternatively, the first flexible peptide and the second flexible peptide linker are different. The first rigid peptide linker and the second rigid peptide linker can form one or more disulfide bonds, e.g., the first polypeptide and the second polypeptide can form a heterodimer.

[0100] The multispecific antibody is connected via a first flexible peptide linker and / or a second flexible peptide linker to a V H and / or V L The second antigen-binding moiety, and optionally a third antigen-binding moiety, may be attached to the second antigen-binding moiety. The second antigen-binding moiety, and optionally the third antigen-binding moiety, may be in scFv or Fab format.

[0101] In some examples, the multispecific antibody contains a second antigen-binding portion in a Fab format that contains a VH-CH1 fragment and a VL-CL fragment. Either the VH-CH1 fragment or the VL-CL fragment is linked to a first flexible peptide linker to form the N-terminal fragment of the first polypeptide, and the other fragment is present as a separate polypeptide (the third polypeptide of the multispecific antibody). Alternatively, either the VH-CH1 fragment or the VL-CL fragment is linked to a second flexible peptide linker to form the N-terminal fragment of the second polypeptide, and the other fragment is present as a separate polypeptide (the third polypeptide of the multispecific antibody).

[0102] In some examples, the multispecific antibody contains a second antigen-binding portion that is in a Fab format (first Fab) and a third antigen-binding portion that can also be in a Fab format (second Fab). The first Fab contains a first VH-CH1 fragment and a first VL-CL fragment, and the second Fab contains a second VH-CH1 fragment and a second VL-CL fragment. Either the first VH-CH1 fragment or the first VL-CL fragment is linked to a first flexible peptide linker to form the N-terminal fragment of the first polypeptide, and the other fragment is present as a separate polypeptide (third polypeptide of the multispecific antibody). Furthermore, either the second VH-CH1 fragment or the second VL-CL fragment is linked to a second flexible peptide linker to form the N-terminal fragment of the second polypeptide, and the other fragment is present as a separate polypeptide (fourth polypeptide of the multispecific antibody). In some cases, the second antigen-binding portion and the third antigen-binding portion are the same (the third polypeptide and the fourth polypeptide are the same).

[0103] In some instances, the multispecific antibody contains a second antigen-binding portion in a cross-Fab format and a third antigen-binding portion that may be in a Fab format. The first cross-Fab contains a VH-CL (Cλ or Cκ) fragment and a first VL-CH1 fragment, and the Fab contains a VH-CH1 fragment and a VL-CL (Cλ or Cκ) fragment. Either the VH-CL fragment or the VL-CH1 fragment is linked to a first flexible peptide linker to form an N-terminal fragment of a first polypeptide, and the other fragment is present as a separate polypeptide (the third polypeptide of the multispecific antibody). Furthermore, either the VH-CH1 fragment or the VL-CL fragment is linked to a second flexible peptide linker to form an N-terminal fragment of a second polypeptide, and the other fragment is present as a separate polypeptide (the fourth polypeptide of the multispecific antibody). In some instances, the second antigen-binding portion and the third antigen-binding portion are identical (the third polypeptide and the fourth polypeptide are identical).

[0104] In some examples, the multispecific antibody contains a second antigen-binding portion that is in a cross-Fab format (first cross-Fab) and a third antigen-binding portion that can also be in a cross-Fab format (second cross-Fab). The first cross-Fab contains a first VH-CL (Cλ or Cκ) fragment and a first VL-CH1 fragment, and the second cross-Fab contains a second VH-CL (Cλ or Cκ) fragment and a second VL-CH1 fragment. Either the first VH-CL fragment or the first VL-CH1 fragment is linked to a first flexible peptide linker to form the N-terminal fragment of the first polypeptide, and the other fragment is present as a separate polypeptide (third polypeptide of the multispecific antibody). Furthermore, either the second VH-CL fragment or the second VL-CH1 fragment is linked to a second flexible peptide linker to form the N-terminal fragment of the second polypeptide, and the other fragment is present as a separate polypeptide (fourth polypeptide of the multispecific antibody). In some cases, the second antigen-binding portion and the third antigen-binding portion are the same (the third polypeptide and the fourth polypeptide are the same).

[0105] The multispecific antibodies disclosed herein comprise a first V H a first Fc fragment linked to a first V via a second rigid peptide linker L and a second Fc fragment linked to the first polypeptide. The Fc fragment may contain a mutation disclosed herein, e.g., a knobs-in-holes mutation to promote the formation of a heterodimer between the first and second polypeptides. See, e.g., FIG. 1B.

[0106] (b) Multispecific antibodies with terminal Fvs In some embodiments, the multispecific antibody disclosed herein comprises a first V H and the first V L The first Fv fragment contains the following: H Chain and VL Each of the chains is connected at one end to a first peptide linker, such as a first flexible peptide linker (disclosed herein, see Table 1 for examples), and optionally at the other end to another peptide linker, such as a rigid peptide linker (disclosed herein, see Table 1 for examples). See, e.g., FIG. 1A. Such multispecific antibodies include a first peptide linker (e.g., a first flexible peptide linker), V H and a first polypeptide comprising a second peptide linker (e.g., a first rigid peptide linker), and a third peptide linker (e.g., a second flexible peptide linker), V L and a second polypeptide comprising a fourth peptide linker (e.g., a second rigid peptide linker). In some cases, the first peptide and the third peptide linker are the same. Alternatively, the first peptide and the third peptide linker are different. The second peptide linker (e.g., the first rigid peptide linker) and the fourth peptide linker (e.g., the second rigid peptide linker) can form one or more disulfide bonds, e.g., the first polypeptide and the second polypeptide can form a heterodimer.

[0107] The multispecific antibodies disclosed herein comprise a first V H a first heavy chain constant region fragment linked to a first V via a second peptide linker; L and a second heavy chain constant region fragment linked to the first polypeptide. The heavy chain constant region fragment may contain a mutation disclosed herein, e.g., a knobs-in-holes mutation to promote heterodimer formation between the first and second polypeptides.

[0108] The multispecific antibody comprises a first heavy chain constant region fragment and / or a second heavy chain constant region fragment, and the V H and / or V LThe antibody may further comprise a second antigen-binding moiety, optionally a third antigen-binding moiety, which may be linked to the second antigen-binding moiety. The second antigen-binding moiety, and optionally the third antigen-binding moiety, may be in an scFv format, or a Fab or cross-Fab format. Alternatively, the second antigen-binding moiety and / or the third antigen-binding moiety may be in a BCR or TCR format.

[0109] In some examples, the multispecific antibody contains a second antigen-binding portion in a Fab format that contains a VH-CH1 fragment and a VL-CL fragment. Either the VH-CH1 fragment or the VL-CL fragment is linked to a first heavy chain constant region fragment to form the N-terminal fragment of a first polypeptide, and the other fragment is present as a separate polypeptide (the third polypeptide of the multispecific antibody). Alternatively, either the VH-CH1 fragment or the VL-CL fragment is linked to a second heavy chain constant region fragment to form the N-terminal fragment of a second polypeptide, and the other fragment is present as a separate polypeptide (the third polypeptide of the multispecific antibody).

[0110] In another example, the multispecific antibody contains a second antigen-binding portion in a cross-Fab format that contains a VH-Cκ or VH-Cλ fragment and a VL-CH1 fragment. Either the VH-Cκ or VH-Cλ fragment or the VL-CH1 fragment is linked to a first heavy chain constant region fragment to form the N-terminal fragment of a first polypeptide, and the other fragment is present as a separate polypeptide (the third polypeptide of the multispecific antibody). Alternatively, either the VH-Cκ or VH-Cλ fragment or the VL-CH1 fragment is linked to a second heavy chain constant region fragment to form the N-terminal fragment of a second polypeptide, and the other fragment is present as a separate polypeptide (the third polypeptide of the multispecific antibody).

[0111] In some examples, the multispecific antibody contains a second antigen-binding portion that is in a Fab format (first Fab) and a third antigen-binding portion that can also be in a Fab format (second Fab). The first Fab contains a first VH-CH1 fragment and a first VL-CL fragment, and the second Fab contains a second VH-CH1 fragment and a second VL-CL fragment. Either the first VH-CH1 fragment or the first VL-CL fragment is linked to a first heavy chain constant region fragment to form the N-terminal fragment of the first polypeptide, and the other fragment is present as a separate polypeptide (third polypeptide of the multispecific antibody). Furthermore, either the second VH-CH1 fragment or the second VL-CL fragment is linked to a second heavy chain constant region fragment to form the N-terminal fragment of the second polypeptide, and the other fragment is present as a separate polypeptide (fourth polypeptide of the multispecific antibody). In some cases, the second antigen-binding portion and the third antigen-binding portion are the same (the third polypeptide and the fourth polypeptide are the same). See, e.g., FIG. 1C.

[0112] In other examples, the multispecific antibody contains a second antigen-binding portion in a cross-Fab format and a third antigen-binding portion that may be in a Fab format. The cross-Fab contains a VH-Cκ or VH-Cλ fragment and a VL-CH1 fragment, and the Fab contains a VH-CH1 fragment and a VL-CL fragment. Either the VH-Cκ or VH-Cλ fragment or the VL-CH1 fragment is linked to a first heavy chain constant region fragment to form an N-terminal fragment of a first polypeptide, and the other fragment is present as a separate polypeptide (the third polypeptide of the multispecific antibody). Furthermore, either the VH-CH1 fragment or the VL-CL fragment is linked to a second heavy chain constant region fragment to form an N-terminal fragment of a second polypeptide, and the other fragment is present as a separate polypeptide (the fourth polypeptide of the multispecific antibody). In some cases, the second antigen-binding portion and the third antigen-binding portion are identical (the third polypeptide and the fourth polypeptide are identical).

[0113] In another example, the multispecific antibody contains a second antigen-binding portion that is in a cross-Fab format (first cross-Fab) and a third antigen-binding portion that may also be in a cross-Fab format (second cross-Fab). The first cross-Fab contains a first VH-Cκ or a first VH-Cλ fragment and a first VL-CH1 fragment, and the second cross-Fab contains a second VH-Cκ or a second VH-Cλ fragment and a second VL-CH1 fragment. Either the first VH-Cκ or the first VH-Cλ fragment or the first VL-CH1 fragment is linked to a first heavy chain constant region fragment to form the N-terminal fragment of the first polypeptide, and the other fragment is present as a separate polypeptide (third polypeptide of the multispecific antibody). Additionally, either the second VH-Cκ or the second VH-Cλ fragment and the second VL-CH1 fragment are linked to a second heavy chain constant region fragment to form the N-terminal fragment of the second polypeptide, and the other fragment exists as a separate polypeptide (the fourth polypeptide of the multispecific antibody). In some cases, the second antigen-binding moiety and the third antigen-binding moiety are the same (the third polypeptide and the fourth polypeptide are the same).

[0114] Alternatively, the first Fv fragment comprises a first V H and the first V L Contains V H Chain and V L Each of the chains is connected at one end to a flexible peptide linker (as disclosed herein, see Table 1 for examples) and does not contain a peptide linker at the other end. See Figure 1C.

[0115] (c) Multispecific antibodies with both central and terminal Fvs In some embodiments, the multispecific antibody disclosed herein comprises a first V H and the first V L The first Fv fragment (middle Fv) contains H Chain and V LEach of the chains is connected at one end to a flexible peptide linker (as disclosed herein, see Table 1 for examples) and at the other end to a rigid peptide linker (as disclosed herein, see Table 1 for examples). See FIG. 1A. Such multispecific antibodies include a first flexible peptide linker, V H and a first polypeptide comprising a first rigid peptide linker, V L and a second polypeptide comprising a second rigid peptide linker. In some cases, the first flexible peptide and the second flexible peptide linker are the same. Alternatively, the first flexible peptide and the second flexible peptide linker are different. The first rigid peptide linker and the second rigid peptide linker can form one or more disulfide bonds, e.g., the first polypeptide and the second polypeptide can form a heterodimer.

[0116] Multispecific antibodies have a second V H and the second V L The second V may further comprise a second Fv fragment (terminal Fv) comprising: H and the second V L Each of V is linked at its N-terminus to a peptide linker. The peptide linker can be a flexible peptide linker disclosed herein (see, e.g., Table 1 for examples). In some cases, the second V H The peptide linker connected to the second VL and the peptide linker connected to the second VL can be the same. H and the second V L may be associated with a different peptide linker. In some cases, the second V H and the second V L can be linked to a peptide linker at their C-terminus. Such a C-terminal peptide linker can be a rigid peptide linker for forming a disulfide bond. Examples are provided in Table 1 below. Alternatively, the second V H and the second V Lare not linked to a peptide linker at their C-terminus.

[0117] The first Fv fragment and the second Fv fragment in the multispecific antibody can be connected via the Fc fragment, e.g., the first V H - first rigid peptide linker-first Fc fragment-peptide linker-second V H and the first V L - second rigid peptide linker-second Fc fragment-peptide linker-second V L In some cases, the first V H and the second VL can be located on one polypeptide, L and the second V H In one polypeptide, for example, the first V H - first rigid peptide linker-first Fc fragment-peptide linker-second V L and the first V L - second rigid peptide linker-second Fc fragment-peptide linker-second V H The position may be in the form:

[0118] The multispecific antibody comprises a first VF of a first Fv (middle Fv) linked via a first flexible peptide linker and / or a second flexible peptide linker. H and / or the first V L The antibody may further comprise a second antigen-binding moiety, optionally a third antigen-binding moiety, which may be linked to the second antigen-binding moiety. The second antigen-binding moiety, and optionally the third antigen-binding moiety, may be in a single domain antibody, such as an scFv format, a VHH format, a Fab format, or a cross-Fab format. Alternatively, the second antigen-binding moiety, and optionally the third antigen-binding moiety, may be in a BCR or TCR format.

[0119] In some examples, the multispecific antibody contains a second antigen-binding portion in a Fab format that contains a VH-CH1 fragment and a VL-CL fragment. Either the VH-CH1 fragment or the VL-CL fragment is linked to a first flexible peptide linker to form the N-terminal fragment of the first polypeptide, and the other fragment is present as a separate polypeptide (the third polypeptide of the multispecific antibody). Alternatively, either the VH-CH1 fragment or the VL-CL fragment is linked to a second flexible peptide linker to form the N-terminal fragment of the second polypeptide, and the other fragment is present as a separate polypeptide (the third polypeptide of the multispecific antibody).

[0120] In another example, the multispecific antibody contains a second antigen-binding portion in a cross-Fab format containing a VH-Cκ or a second VH-Cλ fragment and a VL-CH1 fragment. Either the VH-Cκ or the second VH-Cλ fragment or the VL-CH1 fragment is linked to a first flexible peptide linker to form the N-terminal fragment of the first polypeptide, and the other fragment is present as a separate polypeptide (the third polypeptide of the multispecific antibody). Alternatively, either the VH-Cκ or the second VH-Cλ fragment or the VL-CH1 fragment is linked to a second flexible peptide linker to form the N-terminal fragment of the second polypeptide, and the other fragment is present as a separate polypeptide (the third polypeptide of the multispecific antibody).

[0121] In some examples, the multispecific antibody contains a second antigen-binding portion that is in a Fab format (first Fab) and a third antigen-binding portion that can also be in a Fab format (second Fab). The first Fab contains a first VH-CH1 fragment and a first VL-CL fragment, and the second Fab contains a second VH-CH1 fragment and a second VL-CL fragment. Either the first VH-CH1 fragment or the first VL-CL fragment is linked to a first flexible peptide linker to form the N-terminal fragment of the first polypeptide, and the other fragment is present as a separate polypeptide (third polypeptide of the multispecific antibody). Furthermore, either the second VH-CH1 fragment or the second VL-CL fragment is linked to a second flexible peptide linker to form the N-terminal fragment of the second polypeptide, and the other fragment is present as a separate polypeptide (fourth polypeptide of the multispecific antibody). In some cases, the second antigen-binding portion and the third antigen-binding portion are the same (the third polypeptide and the fourth polypeptide are the same). See, e.g., FIG. 1D.

[0122] In some instances, the multispecific antibody contains a second antigen-binding portion in a cross-Fab format and a third antigen-binding portion that may be in a Fab format. The cross-Fab contains a VH-Cκ or VH-Cλ fragment and a VL-CH1 fragment, and the Fab contains a VH-CH1 fragment and a VL-CL fragment. Either the VH-Cκ or VH-Cλ fragment or the VL-CH1 fragment is linked to a first flexible peptide linker to form an N-terminal fragment of a first polypeptide, and the other fragment is present as a separate polypeptide (the third polypeptide of the multispecific antibody). Additionally, either the VH-CH1 fragment or the VL-CL fragment is linked to a second flexible peptide linker to form an N-terminal fragment of a second polypeptide, and the other fragment is present as a separate polypeptide (the fourth polypeptide of the multispecific antibody). In some instances, the second antigen-binding portion and the third antigen-binding portion are identical (the third polypeptide and the fourth polypeptide are identical).

[0123] In yet another example, the multispecific antibody contains a second antigen-binding portion that is in a cross-Fab format (first cross-Fab) and a third antigen-binding portion that may also be in a cross-Fab format (second cross-Fab). The first cross-Fab contains a first VH-Cκ or VH-Cλ fragment and a first VL-CH1 fragment, and the second cross-Fab contains a second VH-Cκ or VH-Cλ fragment and a second VL-CH1 fragment. Either the first VH-Cκ / VH-Cλ fragment or the first VL-CH1 fragment is linked to a first flexible peptide linker to form the N-terminal fragment of the first polypeptide, and the other fragment is present as a separate polypeptide (the third polypeptide of the multispecific antibody). Additionally, either the second VH-Cκ / VH-Cλ fragment or the second VL-CH1 fragment is linked to a second flexible peptide linker to form the N-terminal fragment of the second polypeptide, and the other fragment exists as a separate polypeptide (the fourth polypeptide of the multispecific antibody). In some cases, the second antigen-binding moiety and the third antigen-binding moiety are the same (the third polypeptide and the fourth polypeptide are the same).

[0124] Exemplary multispecific antibodies disclosed herein are provided in Table 2 below.

[0125] III. Methods for Antibody Preparation Any of the multispecific antibodies, including bispecific and trispecific antibodies, described herein may be made by any method known in the art, see, e.g., Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

[0126] In some embodiments, multispecific antibodies can be produced through conventional recombinant techniques, for example as exemplified below.

[0127] Nucleic acids encoding multiple chains of the multispecific antibody described herein can be cloned into one expression vector, with each nucleotide sequence operably linked to a suitable promoter. In one example, each of the nucleotide sequences encoding the heavy and light chains is operably linked to a separate promoter. Alternatively, the nucleotide sequences encoding multiple chains can be operably linked to a single promoter, such that both the heavy and light chains are expressed from the same promoter. If necessary, an internal ribosome entry site (IRES) can be inserted between the coding sequences of the heavy and light chains.

[0128] In some instances, the nucleotide sequences encoding multiple chains of an antibody are cloned into two or more vectors, which can be introduced into the same or different cells. When multiple chains are expressed in different cells, each of them can be isolated from the host cell in which it is expressed, and the isolated multiple chains can be mixed and incubated under suitable conditions to allow the formation of a multi-chain antibody.

[0129] In general, a nucleic acid sequence encoding one or all chains of an antibody can be cloned into a suitable expression vector in operable linkage with a suitable promoter using methods known in the art. For example, the nucleotide sequence and the vector can be contacted with a restriction enzyme under suitable conditions to create complementary ends on each molecule that can pair with each other and join together with a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the ends of the gene. These synthetic linkers contain nucleic acid sequences that correspond to specific restriction sites in the vector. The choice of expression vector / promoter depends on the type of host cell used to produce the antibody.

[0130] A variety of promoters can be used for expression of the antibodies described herein, including, but not limited to, the cytomegalovirus (CMV) intermediate early promoter, viral LTRs such as rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, simian virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and herpes simplex tk virus promoter.

[0131] Regulatable promoters can also be used, including those that use the lac repressor from E. coli (Brown, M. et al., Cell, 49:603-612 (1987)) and those that use the tetracycline repressor (tetR) (Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)) as a transcriptional modulator to regulate transcription from mammalian cell promoters carrying the lac operator. Other systems include FK506 dimers, VP16 or p65 using astradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech, and Ariad.

[0132] Regulatable promoters containing repressors with operons can be used. In one embodiment, the lac repressor from E. coli can function as a transcriptional modulator to regulate transcription from a lac operator-bearing mammalian cell promoter (M. Brown et al., Cell, 49:603-612 (1987); Gossen and Bujard (1992); M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)) in combination with a tetracycline repressor (tetR) and a transcriptional activator (VP16) to create a tetR-mammalian cell transcriptional activator fusion protein, tTa (tetR-VP16), and a tetO-bearing minimal promoter from the human cytomegalovirus (hCMV) major immediate early promoter to create a tetR-tet operator system for controlling gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. When a tetracycline operator is appropriately placed downstream of the TATA element of the CMVIE promoter, the tetracycline repressor (tetR) alone, rather than a tetR-mammalian cell transcription factor fusion derivative, can function as a potent trans-modulator to control gene expression in mammalian cells (Yao et al., Human Gene Therapy, 10(16):1392-1399 (2003)). One particular advantage of this tetracycline-inducible switch is that it does not require the use of tetracycline repressor-mammalian cell transactivator or repressor fusion proteins, which may be toxic to cells in some cases, to achieve its regulatable effect (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)).

[0133] In addition, the vectors can include, for example, some or all of the following: a selectable marker gene such as a neomycin gene for selecting stable or transient transfectants in mammalian cells, an enhancer / promoter sequence from the immediate early gene of human CMV for high level transcription, transcription termination and RNA processing signals from SV40 for mRNA stability, the SV40 polyoma origin of replication and ColE1 for proper episomal replication, an internal ribosome binding site (IRES), a versatile multiple cloning site, and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art.

[0134] Examples of polyadenylation signals useful in carrying out the methods described herein include, but are not limited to, the human collagen I polyadenylation signal, the human collagen II polyadenylation signal, and the SV40 polyadenylation signal.

[0135] One or more vectors (e.g., expression vectors) containing nucleic acids encoding any of the antibodies can be introduced into a suitable host cell for producing the antibody. The host cells can be cultured under conditions suitable for expression of the antibody or any of its polypeptide chains. Such antibodies or their polypeptide chains can be recovered by the cultured cells (e.g., from the cells or culture supernatant) via conventional methods, such as affinity purification. If desired, the antibody polypeptide chains can be incubated under suitable conditions for a suitable period of time to allow for production of the antibody.

[0136] In some embodiments, the methods for preparing the antibodies described herein involve a recombinant expression vector encoding all of the multiple chains of the multispecific antibody also described herein. The recombinant expression vector can be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection. Positive transformant host cells can be selected and cultured under suitable conditions that allow the expression of multiple polypeptide chains (e.g., three or four) that form the antibody, which can be recovered from the cells or from the culture medium. If necessary, the multiple chains recovered from the host cells can be incubated under suitable conditions that allow the formation of a multi-chain antibody.

[0137] In one example, two or more recombinant expression vectors are provided, each encoding one or more of the antibody chains. The two or more recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection. Alternatively, each of the expression vectors can be introduced into a suitable host cell. Positive transformants can be selected and cultured under suitable conditions that allow the expression of the antibody polypeptide chains. If two or more expression vectors are introduced into the same host cell, the antibody produced therein can be recovered from the host cell or from the culture medium. If necessary, the polypeptide chains can be recovered from the host cell or from the culture medium and then incubated under suitable conditions that allow the formation of the antibody. If two or more expression vectors are introduced into different host cells, each of them can be recovered from the corresponding host cell or from the corresponding culture medium. The multiple polypeptide chains can then be incubated under suitable conditions for the formation of the antibody.

[0138] Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography using a Protein A or Protein G-bound matrix.

[0139] Any of the nucleic acids encoding multiple chains of the multispecific antibodies disclosed herein, vectors containing such (e.g., expression vectors), and host cells comprising the vectors are within the scope of this disclosure.

[0140] IV. Pharmaceutical Compositions Any of the multispecific antibodies, including bispecific or trispecific antibodies, disclosed herein, as well as the encoding nucleic acids or nucleic acid sets described herein, vectors containing such, or host cells containing vectors, can be mixed with a pharma- ceutically acceptable carrier (excipient) to form a pharmaceutical composition for use in treating a target disease. By "acceptable," it is meant that the carrier must be compatible with the active ingredient of the composition (and preferably be able to stabilize the active ingredient) and not deleterious to the subject being treated. Pharmaceutically acceptable excipients (carriers) include buffers, which are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K.E. Hoover.

[0141] The pharmaceutical compositions used in the methods of the present invention may contain pharma- ceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations used, and include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, serum albumins, and the like. The surfactants may include proteins such as albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextran, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants such as TWEEN, PLURONICS, or polyethylene glycol (PEG).

[0142] In some examples, the pharmaceutical compositions described herein include liposomes containing antibodies (or encoding nucleic acids) that can be prepared by methods known in the art, such as those described in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985), Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980), and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be generated by reverse phase evaporation using a lipid composition that includes phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to obtain liposomes with the desired diameter.

[0143] The antibodies or encoding nucleic acids may also be encapsulated in microcapsules prepared, for example, by droplet formation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacylate) microcapsules, respectively, colloid drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions. Such techniques are known in the art, see, for example, Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[0144] In other examples, the pharmaceutical compositions described herein can be formulated in sustained release form.Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, the matrices being in the form of shaped articles, such as films or microcapsules.Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactic acid (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), isobutyl sucrose acetate, and poly-D-(-)-3-hydroxybutyric acid.

[0145] Pharmaceutical compositions to be used for in vivo administration must be sterile. This is readily accomplished, for example, by filtration through sterile membranes. Therapeutic antibody compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0146] The pharmaceutical compositions described herein may be in unit dosage form such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories for oral, parenteral or rectal administration, or for administration by inhalation or insufflation.

[0147] To prepare solid compositions such as tablets, the primary active ingredient is mixed with pharmaceutical carriers, such as conventional tablet ingredients, such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gums, as well as other pharmaceutical diluents, such as water, to form a solid preformulation composition containing a homogenous mixture of the compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof. When these preformulation compositions are referred to as homogenous, it is meant that the active ingredient is evenly dispersed throughout the composition, such that the composition may be readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. Tablets or pills of the novel compositions may be coated or otherwise compounded to provide a dosage form that provides the advantage of prolonged action. For example, the tablet or pill may include an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0148] Suitable surfactants include non-ionic agents such as polyoxyethylene sorbitan (e.g., TWEEN 20, 40, 60, 80, or 85) and other sorbitans (e.g., SPAN 20, 40, 60, 80, or 85), among others. Compositions having surfactants conveniently contain 0.05-5% surfactant, and may be 0.1-2.5%. It will be appreciated that other ingredients, such as mannitol or other pharma-ceutically acceptable vehicles, may be added as required.

[0149] Suitable emulsions can be prepared using commercially available fat emulsions such as INTRALIPIDS, LIPOSYN, INFONUTROl, LIPOFUNDIN, and LIPIPHYSAN. The active ingredient can be dissolved in a premixed emulsion composition or, alternatively, in an emulsion formed upon mixing with oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and phospholipids (egg phospholipids, soybean phospholipids, or soybean lecithin) and water. It will be appreciated that other ingredients, such as glycerol or glucose, can be added to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20%, for example 5-20%, of oil. Fat emulsions contain fat droplets of 0.1-1.0 μm, especially 0.1-0.5 μm, and have a pH in the range of 5.5-8.0.

[0150] The emulsion composition may be prepared by mixing the antibody with INTRALIPID or its components (soybean oil, egg phospholipids, glycerol, and water).

[0151] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharma- ceutically acceptable aqueous or organic solvents, or mixtures thereof, as well as powders. Liquid or solid compositions may contain suitable pharma- ceutically acceptable excipients as described above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.

[0152] Compositions, preferably in sterile pharma- ceutically acceptable solvents, may be nebulized by the use of a gas. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device may be attached to a face mask, tent, or intermittent positive pressure respirator. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from devices that deliver the formulation in an appropriate manner.

[0153] V. Therapeutic uses Any of the multispecific antibodies disclosed herein can be used in clinical settings (e.g., therapeutic) or non-clinical settings (e.g., for research purposes).

[0154] In some aspects, provided herein are methods of using any of the antibodies disclosed herein to modulate immune responses or treat a target disease in a subject in need of treatment. To practice the methods disclosed herein, an effective amount of the pharmaceutical composition described herein can be administered to a subject (e.g., a human) in need of treatment via a suitable route, such as intravenous administration, for example, as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, inhalation or topical routes. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations can be directly nebulized, and lyophilized powders can be nebulized after reconstitution. Alternatively, the antibodies described herein can be aerosolized using fluorocarbon formulations and metered dose inhalers, or inhaled as lyophilized and milled powders.

[0155] The subject treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, livestock, sport animals, pets, primates, horses, dogs, cats, mice, and rats. The human subject in need of treatment can be a human patient who has, is at risk of, or is suspected of having a target disease / disorder, such as cancer, or an immune disorder, such as an autoimmune disease.

[0156] Examples of cancer include breast cancer; biliary tract cancer; bladder cancer; brain cancer, including glioblastoma and medulloblastoma; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; haematological malignancies, including acute lymphoblastic and myeloid leukemia, e.g. B-cell CLL; T-cell acute lymphoblastic leukemia / lymphoma; hairy cell leukemia; chronic myelogenous leukemia, multiple myeloma; AIDS-related leukemia and adult T-cell leukemia / lymphoma; intraepithelial neoplasia, including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas, including Hodgkin's disease and lymphocytic lymphoma; neuroblastoma; oral cancer, including squamous cell carcinoma; and cancers of the epithelial, stromal, germ cell and mesenchymal cells. These include, but are not limited to, ovarian cancer, including those arising from the thyroid gland; pancreatic cancer; prostate cancer; rectal cancer; sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer, including melanoma, Merkel cell carcinoma, Kaposi's sarcoma, basal cell carcinoma, and squamous cell carcinoma; testicular cancer, including seminoma, non-seminoma (teratoma, choriocarcinoma), stromal tumors, and embryonal tumors such as germ cell tumors; thyroid cancer, including thyroid carcinoma and medullary carcinoma; and renal cancer, including adenocarcinoma and Wilms' tumor.

[0157] In some cases, a multispecific antibody for use in the therapeutic methods disclosed herein contains one binding arm specific for a TAA, and a patient for treatment has cancer cells expressing the target TAA. For example, the multispecific antibody is specific for CD19, and the patient has a CD19+ cancer. In other examples, the multispecific antibody is specific for CD20, and the patient has a CD20+ cancer. In other examples, the multispecific antibody is specific for BCMA, and the patient has a BCMA+ cancer. In other examples, the multispecific antibody is specific for B7H3, and the patient has a B7H3+ cancer. In some examples, the multispecific antibody is specific for HER2, and the patient has a HER2+ cancer. In some examples, the multispecific antibody is specific for p53mut, and the patient has a P53mut+ cancer. In some examples, the multispecific antibody is specific for MET, and the patient has a MET+ cancer. In some examples, the multispecific antibody is specific for PSMA and the patient has a PSMA+ cancer. In some examples, the multispecific antibody is specific for CEA and the patient has a CEA+ cancer. In some examples, the multispecific antibody is specific for EGFR and the patient has an EGFR+ cancer. In some examples, the multispecific antibody is specific for DLL3 and the patient has a DLL3+ cancer. In some examples, the multispecific antibody is specific for MAGE-A4 and the patient has a MAGE-A4+ cancer. In some examples, the multispecific antibody is specific for PRAME and the patient has a PRAME+ cancer.

[0158] In some cases, a multispecific antibody for use in the therapeutic methods disclosed herein contains one binding arm specific for an immune cell receptor, and a patient for treatment possesses immune cells expressing such immune cell receptor. For example, the multispecific antibody is specific for PD-(L)1, and the patient possesses PD-(L)1+ immune cells. In some examples, the multispecific antibody is specific for CD137, and the patient possesses CD137+ immune cells. In some examples, the multispecific antibody is specific for CD3, and the patient possesses CD3+ immune cells. In some examples, the multispecific antibody is specific for CTLA4, and the patient possesses CTLA4+ immune cells. In some examples, the multispecific antibody is specific for CD40, and the patient possesses CD40+ immune cells. In some examples, the multispecific antibody is specific for GITR, and the patient possesses GITR+ immune cells. In some instances, the multispecific antibody is specific for at least two of immune cell receptors, such as PD-1, CD137, CD3, CTLA4, CD40, OX40, GITR, and the patient has immune cell expressing two such immune cell receptors.

[0159] Subjects with target cancers can be identified by routine medical tests, such as laboratory tests, organ function tests, CT scans, ultrasound, and / or genetic tests. In some embodiments, the subjects treated by the methods described herein can be human cancer patients who have undergone or are undergoing anti-cancer therapy, such as chemotherapy, radiation therapy, immunotherapy, or surgery.

[0160] Immune disorder refers to a malfunction of the immune system. Examples include autoimmune disease, immunodeficiency, or allergy. In some embodiments, the target disease for treatment is an autoimmune disease. Examples include rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), myasthenia gravis (MG), Graves' disease, idiopathic thrombocytopenic purpura (ITP), Guillain-Barre syndrome, autoimmune myocarditis, membranous glomerulonephritis, hyper-IgM syndrome, diabetes mellitus, type I or II diabetes, multiple sclerosis, Raynaud's syndrome, autoimmune thyroiditis, gastritis, celiac disease, vitiligo, hepatitis, primary biliary cirrhosis, inflammatory bowel disease, spondyloarthropathy, experimental autoimmune myocarditis, and rheumatoid arthritis. These include, but are not limited to, autoimmune encephalomyelitis, immune neutropenia, juvenile onset diabetes, and immune responses associated with delayed type hypersensitivity mediated by cytokines, T lymphocytes typically seen in tuberculosis, sarcoidosis, and polymyositis, polyarteritis nodosa, cutaneous vasculitis, pemphigus, pemphigoid, Goodpasture's syndrome, Kawasaki disease, systemic sclerosis, antiphospholipid syndrome, Sjogren's syndrome, graft versus host disease (GVH), and immune thrombocytopenia.

[0161] Subjects with a target autoimmune disease may be identified by routine medical tests, such as the presence of antinuclear antibodies, antimitochondrial autoantibodies, antineutrophil cytoplasmic antibodies, antiphospholipid antibodies, anticitrullinated peptide (anti-CCP), antirheumatoid factor, immunoglobulin A, C-reactive protein testing, complement testing, erythrocyte sedimentation rate (ESR) testing, blood clotting profile, and protein electrophoresis / immunofixation electrophoresis, and / or genetic testing, etc. In some embodiments, the subject treated by the methods described herein may be a human subject with an autoimmune disease who has undergone or is undergoing treatment for the autoimmune disease, such as immunosuppressive mediation, hormone replacement therapy, blood transfusions, anti-inflammatory drug therapy, and / or analgesic drug therapy.

[0162] A subject suspected of having any of these target diseases / disorders may exhibit one or more symptoms of the disease / disorder. A subject at risk of a disease / disorder may have one or more risk factors for the disease / disorder.

[0163] As used herein, "effective amount" refers to the amount of each active agent required to provide a therapeutic effect to a subject, either alone or in combination with one or more other active agents. Determining whether the amount of the composition described herein achieves a therapeutic effect will be apparent to one of skill in the art. As recognized by those skilled in the art, the effective amount will vary depending on individual patient parameters, including the specific condition being treated, the severity of the condition, age, physical condition, size, sex and weight, duration of treatment, the nature of concomitant therapy (if any), the specific route of administration, and similar factors within the knowledge and expertise of the medical practitioner. These factors are well known to those skilled in the art and can be addressed with only routine experimentation. In general, it is preferred to use the maximum dose of the individual components or combinations thereof, i.e., the highest safe dose according to sound medical judgment.

[0164] Empirical considerations such as half-life generally contribute to the determination of dosage. For example, an antibody compatible with the human immune system, such as a humanized antibody or a fully human antibody, may be used to extend the half-life of the antibody and prevent the antibody from being attacked by the host's immune system. The frequency of administration can be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on the treatment and / or suppression and / or improvement and / or delay of the target disease / disorder. Alternatively, sustained release formulations of antibodies may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0165] In one example, the dosage of the antibody described herein can be empirically determined in an individual who is given one or more doses of the antibody. The individual is given increasing doses of the agonist. To evaluate the effectiveness of the agonist, indicators of disease / disorder can be tracked.

[0166] In general, for administration of any of the antibodies described herein, the initial candidate dosage may be about 2 mg / kg. For purposes of this disclosure, a typical daily dosage may range anywhere from about 0.1 μg / kg to 3 μg / kg to 30 μg / kg to 300 μg / kg to 3 mg / kg, to 30 mg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or more, depending on the condition, treatment is sustained until desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved to alleviate the target disease or disorder, or its symptoms. An exemplary dosing regimen includes administering an initial dose of about 2 mg / kg, followed by a weekly maintenance dose of about 1 mg / kg of the antibody, or a maintenance dose of about 1 mg / kg every other week thereafter. However, other dosage regimens may be useful depending on the pattern of pharmacokinetic decay the physician wishes to achieve. For example, dosing from 1 to 4 times per week is contemplated. In some embodiments, a dosage range of about 3 μg / mg to about 2 mg / kg (e.g., about 3 μg / mg, about 10 μg / mg, about 30 μg / mg, about 100 μg / mg, about 300 μg / mg, about 1 mg / kg, and about 2 mg / kg) may be used. In some embodiments, the dosage frequency is once every week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks, or once every month, every 2 months, or every 3 months, or more. The progress of this therapy is easily monitored by conventional techniques and assays. The dosage regimen (including the antibody used) may be varied over time.

[0167] In some embodiments, a dose ranging from about 0.003 to 5.00 mg / kg can be administered to a normal weight adult patient. In some examples, the dosage of the antibodies described herein can be 10 mg / kg. The particular dosing regimen, i.e., dose, timing, and repetition, will depend on the particular individual and that individual's medical history, as well as the characteristics of the individual drug (such as the drug's half-life, and other considerations known in the art).

[0168] For purposes of this disclosure, appropriate dosages of antibodies described herein will depend on the particular antibody(ies), antibodies, and / or non-antibody peptides (or compositions thereof) used, the type and severity of the disease / disorder, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's medical history and response to the agonist, and the discretion of the attending physician. Typically, the clinician will administer the antibody until a dosage is reached that achieves the desired result. In some embodiments, the desired result is an increase in an anti-tumor immune response in the tumor microenvironment. Methods for determining whether a dosage has produced a desired result will be apparent to one of skill in the art. Administration of one or more antibodies may be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to the skilled physician. Administration of the antibody may be essentially continuous over a preselected period of time or may be a series of spaced doses, for example, either before, during, or after the onset of the target disease or disorder.

[0169] As used herein, the term "treating" refers to the application or administration of a composition comprising one or more active agents to a subject having a target disease or disorder, a symptom of a disease / disorder, or a predisposition to a disease / disorder, for the purpose of curing, healing, mitigating, alleviating, altering, relieving, improving, ameliorating, or affecting the disorder, a symptom of the disease, or a predisposition to a disease or disorder.

[0170] Alleviating the target disease / disorder includes delaying the onset or progression of the disease, or reducing the severity of the disease, or prolonging survival. Alleviating the disease or prolonging survival does not necessarily require a curative outcome. As used herein, "delaying" the onset of the target disease or disorder means suspending, hindering, slowing, delaying, stabilizing, and / or postponing the progression of the disease. This delay can be for a variety of lengths of time, depending on the history of the disease and / or the individual being treated. A method of "delaying" or alleviating the onset of a disease, or delaying the onset of a disease, is a method that reduces the likelihood of developing one or more symptoms of the disease within a given time frame and / or reduces the severity of symptoms within a particular time frame, when compared to not using the method. Such comparisons are typically based on clinical studies using a sufficient number of subjects to produce statistically significant results.

[0171] "Onset" or "progression" of a disease refers to the initial symptoms and / or subsequent progression of a disease. Onset of a disease can be detected and assessed using standard clinical techniques well known in the art. However, onset also refers to progression that may be undetectable. For purposes of this disclosure, onset or progression refers to the biological course of a symptom. "Onset" includes onset, recurrence, and onset. As used herein, "onset" or "onset" of a target disease or disorder includes initial onset and / or recurrence.

[0172] Depending on the type of disease or site of disease to be treated, the pharmaceutical composition can be administered to the subject using conventional methods known to those skilled in the medical field. The composition can also be administered via other conventional routes, for example, orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, intravaginally, or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, the subject can be administered via an injectable depot administration route, such as using 1-month, 3-month, or 6-month depot injectable or biodegradable materials and methods. In some examples, the pharmaceutical composition is administered intraocularly or intravitreally.

[0173] Injectable compositions may contain a variety of carriers, such as vegetable oils, dimethylactamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies may be administered by drip infusion, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. For intramuscular preparations, for example, a sterile formulation of a suitable soluble salt form of the antibody may be dissolved and administered in a pharmaceutical excipient, such as water for injection, 0.9% saline, or 5% glucose solution.

[0174] In one embodiment, the antibody is administered via site-specific or targeted local delivery techniques. Examples of site-specific or targeted local delivery techniques include various implantable depot sources of the antibody or local delivery catheters such as infusion catheters, indwelling catheters, or needle catheters, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site-specific carriers, direct injection, or direct application. See, e.g., PCT Publication No. WO00 / 53211 and U.S. Patent No. 5,981,568.

[0175] Targeted delivery of therapeutic compositions containing antisense polynucleotides, expression vectors, or subgenomic polynucleotides can also be used.Receptor-mediated DNA delivery techniques are described, for example, in Findeis et al., Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics: Methods and Applications Of Direct Gene Transfer (JA Wolff, ed.) (1994); Wu et al., J.Biol.Chem. (1988) 263:621; Wu et al., J.Biol.Chem. (1994) 269:542; Zenke et al., Proc.Natl.Acad.Sci.USA (1990) 87:3655; Wu et al., J.Biol.Chem. (1991) 266:338.

[0176] Therapeutic compositions containing polynucleotides (e.g., those encoding an antibody described herein) are administered in the range of about 100 ng to about 200 mg of DNA for local administration in gene therapy protocols. In some embodiments, concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg or more of DNA can also be used during gene therapy protocols.

[0177] The therapeutic polynucleotides and polypeptides described herein can be delivered using gene delivery vehicles. Gene delivery vehicles can be of viral or non-viral origin (see generally Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters and / or enhancers. Expression of the coding sequences can be constitutive or regulated.

[0178] Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90 / 07936, WO 94 / 03622, WO 93 / 25698, WO 93 / 25234, WO 93 / 11230, WO 93 / 10218, WO 91 / 02805, U.S. Pat. Nos. 5,219,740 and 4,777,127, British Patent No. 2,200,651, and European Patent No. 0345242), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246), and Venezuelan equine encephalitis virus (ATCC VR-1247). VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532), and adeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO94 / 12649, WO93 / 03769, WO93 / 19191, WO94 / 28938, WO95 / 11984, and WO95 / 00655). Administration of DNA linked to killed adenovirus, as described in Curiel, Hum. Gene Ther. (1992) 3:147, may also be used.

[0179] Non-viral delivery vehicles and methods may also be used, including, but not limited to, polycation-condensed DNA alone, with or without linkage to killed adenovirus (see, e.g., Curiel, Hum. Gene Ther. (1992) 3:147), ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989) 264:16985), eukaryotic cell delivery vehicle cells (see, e.g., U.S. Patent No. 5,814,482, PCT Application Nos. WO95 / 07994, WO96 / 17072, WO95 / 30763, and WO97 / 42338), and nuclear charge neutralization or fusion with cell membranes. Naked DNA may also be used. Exemplary naked DNA transfer methods are described in PCT Publication No. WO 90 / 11092 and U.S. Patent No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Patent No. 5,422,120, PCT Publication Nos. WO 95 / 13796, WO 94 / 23697, WO 91 / 14445, and European Patent No. 0524968. Additional techniques are described in Philip, Mol. Cell. Biol. (1994) 14:2411, and Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

[0180] The particular dosage regimen employed in the methods described herein, i.e., dosage, timing, and repetition, will depend on the particular subject and the subject's medical history.

[0181] In some embodiments, two or more antibodies, or a combination of an antibody and another suitable therapeutic agent, can be administered to a subject in need of treatment.Antibodies can also be used in combination with other agents that function to enhance and / or complement the effectiveness of the agent.The effectiveness of the treatment of the target disease / disorder can be evaluated by methods well known in the art.

[0182] When any of the antibodies described herein are used to treat cancer, they can be combined with anti-cancer therapies, such as those known in the art, including chemotherapy, surgery, radiation, immunotherapy, gene therapy, and the like.

[0183] Alternatively, the treatment of the present disclosure may be combined with chemotherapeutic agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine), purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); natural products such as the vinca alkaloids (vinblastine, vincristine, and vinorelbine); microtubule disruptors such as the taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones, and navelbine. antiproliferative / mitotic inhibitors including epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylneramine oxaliplatin, ifosfamide, melphalan, mechlorethamine, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, taxol, taxotere, teniposide, multiethylenethiophosphoramide, and etoposide (VP16)); dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), antibiotics such as bronchodilators, idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin; enzymes (L-asparaginase, which metabolizes L-asparagine systemically and deprives cells that do not have the ability to synthesize their own asparagine); antiplatelet agents, nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), and trazenes-dacarbazinine (DTIC) and other antiproliferative / mitotic inhibitor alkylating agents;Antiproliferative / mitotic antimetabolites such as folic acid analogues (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogues (estrogens, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other thrombin inhibitors); fibrinolytics (tissue plasminogen activator, streptokinase, urokinase, etc.), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents agents; secretory inhibitors (breveldin); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); antiangiogenic compounds (e.g., TNP-470, genistein, bevacizumab) and growth factor inhibitors (e.g., fibroblast growth factor (FGF) inhibitors); angiotensin receptor blockers; nitric oxide donors donors); antisense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, prenisolone); growth factor signaling kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; chromatin disruptors;

[0184] Any of the antibodies described herein, when used to treat immune disorders, can be used in combination with other immunomodulatory therapies, such as, for example, therapeutic vaccines (including but not limited to, GVAX, DC-based vaccines, etc.), or checkpoint inhibitors (including but not limited to, agents that block CTLA4, PD1, LAG3, TIM3, etc.). In some cases, the antibody can be combined with another therapy for autoimmune disease. Examples include, but are not limited to, intravenous Ig therapy; nonsteroidal anti-inflammatory drugs (NSAIDs); corticosteroids; cyclosporine, rapamycin, ascomycin; cyclophosphamide; azathioprine; methotrexate; brequinar; FTY720; leflunomide; mizoribine; mycophenolic acid; mycophenolate mofetil; 15-deoxyspergualin; immunosuppressants, or adhesion molecule inhibitors.

[0185] For examples of additional useful agents, see also Physician's Desk Reference, 59th edition, (2005), Thomson PDR, Montvale NJ; Gennaro et al., Eds. Remington's The Science and Practice of Pharmacy 20th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., Eds. Harrison's Principles of Internal Medicine, 15th edition, (2001), McGraw Hill, NY; Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway NJ.

[0186] When a second therapeutic agent is used, such agent can be administered simultaneously or sequentially (in any order) with the therapeutic agents described herein. When co-administered with additional therapeutic agents, the suitable therapeutically effective dosage of each agent may be reduced due to additive or synergistic effects.

[0187] VI. Kits Containing the Multispecific Antibodies Disclosed Herein The present disclosure also provides kits for use in treating or alleviating a target disease, such as cancer or an immune disorder, as described herein. Such kits may include one or more containers containing any of the multispecific antibodies disclosed herein, and, optionally, a second therapeutic agent for use in combination with the antibody, also as described herein.

[0188] In some embodiments, the kit can include instructions for use according to any of the methods described herein. The included instructions can include instructions for administering the antibody and, optionally, a second therapeutic agent to treat, delay the onset of, or alleviate a target disease, such as those described herein. The kit can further include instructions for selecting an individual suitable for treatment, for example, based on applying the diagnostic methods described herein to identify whether the individual has the target disease. In yet other embodiments, the instructions include instructions for administering the antibody to an individual at risk for the target disease.

[0189] The instructions for use of the antibody will generally include information regarding dosages, dosing schedules, and routes of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. The instructions provided with the kits of the invention are typically written instructions on a label or insert (e.g., a paper sheet included with the kit), although machine-readable instructions (e.g., instructions transmitted on a magnetic or optical storage disk) are also acceptable.

[0190] The label or package insert indicates that the composition is used for treating, delaying the onset of, and / or alleviating a disease, such as cancer or an immune disorder (e.g., an autoimmune disease). Instructions for performing any of the methods described herein can be provided.

[0191] The kit of the present invention is in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Packages for use in combination with specific devices, such as inhalers, nasal administration devices (e.g., nebulizers), or infusion devices, such as mini-pumps, are also contemplated. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is an antibody, such as those described herein.

[0192] The kit may optionally provide additional components such as buffers and instructional information. Typically, the kit comprises a container and a label or package insert on or associated with the container. In some embodiments, the invention provides an article of manufacture comprising the contents of the kit described above.

[0193] general technique The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are described in Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press, Oligonucleotide Synthesis (MJ Gait, ed. 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (JECellis, ed., 1989) Academic Press, Animal Cell Culture (RIFreshney, ed. 1987), Introuction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and DG Newell, eds. 1993-8) J. Wiley and Sons, Methods in Enzymology (Academic Press, Inc.), Handbook of Experimental Immunology (DMWeir and CCBlackwell, eds.), Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, eds., 1987), Current Protocols in Molecular Biology (FMAusubel, et al. eds. 1987), PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994), Current Protocols in Immunology (JEColigan et al., eds.,1991)、Short Protocols in Molecular Biology(Wiley and Sons,1999)、Immunobiology(CAJaneway and P.Travers,1997)、Antibodies(P.Finch,1997)、Antibodies: A Practice Approach(D.Catty.,ed.,IRL Press,1988-1989)、Monoclonal antibodies: a practical approach(P.Shepherd and C.Dean,eds.,Oxford University Press,2000)、Using antibodies:a laboratory manual(E.Harlow and D.Lane(Cold Spring Harbor Laboratory Press,1999). JDCapra,eds.Harwood Academic Publishers,1995)、DNA Cloning:A Practical Approach,Volumes I and II(DNGlover ed.1985)、Nucleic Acid Hybridization(BDHames&S.J.Higgins eds.(1985>>、Transcription and Translation(BDHames&S.J.Higgins,eds.(1984>>、Animal Cell Culture(RIFreshney,ed.(1986>>、Immobilized Cells and Enzymes(lRL Press,(1986>>、B.Perbal,A Practical Guide To Molecular Cloning(1984)) FMAusubel et al.(eds.) In this context.

[0194] Without further elaboration, it is believed that one skilled in the art can utilize the present invention to its fullest extent based on the above description. Accordingly, the specific embodiments provided herein are to be construed as merely illustrative, and not limiting in any way to the remainder of the disclosure. All publications cited herein are incorporated by reference for the purpose or subject matter referenced herein. EXAMPLES

[0195] Example 1: Construction and production of multispecific and multivalent antibodies Exemplary multispecific antibodies shown in Table 2 were produced by recombinant techniques and characterized for their antigen binding activity and biological activity as disclosed herein.

[0196] Briefly, the heavy chain variable regions (V H ) and the light chain variable region (V L cDNA encoding the multispecific antibodies was used as starting material for generating the multispecific antibodies. The coding sequences for the multiple chains of each exemplary multispecific antibody were cloned into an expression vector, which was transfected into CHO cells for transient expression. The multispecific antibodies produced in CHO cells were purified from culture supernatants by Protein A affinity chromatography. Antibody properties were examined using standard protocols or as described herein.

[0197] Example 2: Effect of flexible peptide linkers on multispecific antibody properties. This example examines the effect of flexible peptide linkers, which are G / S-rich peptide linkers, on the characteristics of multispecific antibodies that contain such flexible peptide linkers. In this example, multispecific antibodies that lack flexible peptide linkers of various lengths were tested.

[0198] Exemplary multispecific antibodies were produced in CHO cells as described above in Example 1. Binding of the multispecific antibodies to the corresponding target antigens was assessed by ELISA or FCM according to standard procedures.

[0199] As shown in Figures 2A-2F and Table 3, the length of the flexible peptide linker does not significantly affect the binding of the binding moieties connected by the flexible peptide linker, nor does it impair the overall quality of these multispecific antibodies (data not shown). Two sets of multispecific antibodies were tested with similar results. [Table 1]

[0200] Example 3: Effect of rigid peptide linkers on multispecific antibody properties. This example examines the effect of rigid peptide linkers capable of forming disulfide bonds between each other on the characteristics of multispecific antibodies that contain such.

[0201] (i) Disulfide-forming properties of rigid peptide linkers on antibody stability and binding Multispecific antibodies that compromise an exemplary rigid peptide linker, GGGGSDKTHTCPPCPAPEAAGP (SEQ ID NO: 13), which contains a Cys residue for disulfide bond formation, or the control peptide linker PAPEAAGP (SEQ ID NO: 28), which does not contain a Cys residue and therefore cannot form disulfide bonds, were tested in this example.

[0202] Exemplary multispecific antibodies containing the peptide linkers described above were produced in CHO cells as described in Example 1 above. Proteins were prepared in reducing or non-reducing LDS sample buffer and loaded onto Bis-Tris precast gels. Gels were run at 140 volts for 40 minutes with MOPS SDS buffer. After electrophoresis, gels were stained with Coomassie Blue. Figure 3A shows the mobility of antibodies Ly1891, Ly1899, and Ly1963 with a rigid peptide linker (SEQ ID NO: 13) and antibodies Ly1800, Ly1803, and Ly1961 with a control peptide linker (SEQ ID NO: 28). Antibodies Ly1891, Ly1899, and Ly1963 showed the expected mobility on SDS-PAGE under both reducing and non-reducing conditions. In contrast, antibodies Ly1800, Ly1803, and Ly1961 showed the expected mobility under reducing conditions, but under non-reducing conditions only half-mers of the intact molecule were visible, suggesting insufficient stability of the subsequent antibodies due to the absence of disulfide-forming segments.

[0203] Size exclusion chromatography (SEC) was performed using a high performance liquid chromatography system. Samples were injected into a Zenix-SEC-300 column or equivalent using DPBS, pH 7.4 as the mobile phase. SEC-HPLC analysis revealed that disulfide-free antibody constructs (Ly1800, Ly1803, and Ly1961) formed more aggregates compared to their disulfide-containing counterparts (Ly1891, Ly1899, and Ly1963), as demonstrated by the percentage of high molecular weight (HMW) peaks summarized in Table 4 below. [Table 2]

[0204] The binding of these antibody constructs to CD3-expressing Jurkat cells was assessed by flow cytometry (FCM) as in Example 2. FCM analysis revealed that the presence of the disulfide-forming segments Ly1899 and Ly1891 increased target-dependent binding to the cells compared to antibodies lacking the disulfide-forming segments (Ly1803 and Ly1800), consistent with the above observations of lower stability and more aggregates of constructs without linker disulfides (Figure 3B).

[0205] (ii) Contribution of G4S segments in rigid peptide linkers to antibody stability and aggregation G 4 The S motif is G 4 To investigate how having a rigid peptide linker containing an S motif affects the properties of a multispecific antibody, clones Ly2128, Ly1963, and Ly1967, which contain a linker of GGGGSDKTHTCPPCPAPEAAGP (SEQ ID NO: 13), and clones Ly2125, Ly2157, and Ly2167, which have the rigid peptide linker DKTHTCPPCPAPEAAGP (SEQ ID NO: 21), were tested.

[0206] SEC-HPLC analysis, performed as described in the previous section, revealed that the G 4 Antibodies without the S motif were shown to have relatively shorter retention times (RT), suggesting their ability to exist as oligomers (Table 5). Native mass spectrometry for Ly2125 revealed a mass of 398 kDa, indicating the desired dimeric structure of the intact molecule for this antibody. [Table 3]

[0207] The binding of these antibody constructs to the CD3 target antigen was evaluated by ELISA as in Example 2. As shown in Figures 4A-4C, the G in the rigid peptide linker (Ly2125, Ly2157, and Ly2167) 4 The absence of the S motif is G4 Compared with rigid peptide linkers with S motifs (Ly2128, Ly1963, and Ly1967), they increased CD3 binding. The higher binding capacity of Ly2125, Ly2157, and Ly2167 is likely the result of oligomerization.

[0208] Example 4: Activity of the central Fv binding moiety in a multispecific antibody This example evaluates the binding activity of the central Fv fragment in a multispecific antibody.

[0209] CD3 Modulators Several bispecific or trispecific antibodies were constructed as CD3 modulators targeting B7H3 and / or CD19. The trispecific antibodies have either CD137 or CD28 binding domains and a central (hidden) CD3 binding domain. Binding of these antibodies to CD3 was determined as described in Example 2, and they showed 10-50 fold weaker binding to CD3 compared to the parental anti-CD3 mAb Ly305 (Table 6 below and Figures 4A-4C).

[0210] A CD3 reporter assay was performed to determine the potency of these antibodies. A CD3 reporter assay system was used, including Jurkat / NFAT-Luc2P cells (Jurkat cells expressing a luciferase reporter driven by an NFAT response element). Briefly, Jurkat / NFAT-Luc2P cells were harvested, aliquoted at 50000 cells / well in 96-well plates, and co-cultured with or without additional target-expressing cells. Test antibodies were added and the plates were incubated at 37°C for an additional 6 hours, after which a Bright-Glo™ Luciferase Assay (Promega Cat. No. E2620) was performed. NFAT-mediated luminescence in this assay corresponds to the activation of CD3 by multispecific antibodies in the absence or presence of high affinity target antigen.

[0211] CD3 reporter assays showed greatly reduced potency for these antibodies in the absence of additional targets (no activity at 10 μg / mL) compared to the parental anti-CD3 mAb (Table 6). However, CD3 activity was greatly enhanced in the presence of additional targets for cross-linking, such as tumor antigens or immune receptors (Table 6 below and Figures 5A-5I). [Table 4]

[0212] Cell killing by exemplary multispecific antibodies was determined by measuring lactate dehydrogenase (LDH) release. Target cells and PBMC effector cells were seeded in 96-well plates and incubated with the test antibodies. Plates were incubated at 37° C. for 48 hours. LDH released from damaged cells was measured using an LDH assay kit (Promega) according to the manufacturer's instructions. In addition, immune cell activation was assessed by measuring IFN-γ levels using an HTRF assay kit from Cisbio according to the manufacturer's instructions. Figures 6A and 6B show, respectively, that CD3 activation induced T cell killing activity against tumor cells expressing specific TA for the multispecific antibodies.

[0213] These examples demonstrate that the central CD3 Fv binding module has significantly reduced CD3 binding and minimal ability to induce CD3 activation by itself. However, in a multispecific format, the CD3 Fv binding module has a higher clustering and activation potential due to the presence of secondary and / or tertiary binding partners, a much higher CD3 activation capacity compared to the parental mAb Ly305.

[0214] CD137 Modulators Several bispecific or trispecific antibodies, such as Ly2118 and Ly2281, were constructed as CD137 modulators and targeting B7H3 and / or PD-1. These bispecific or trispecific antibodies showed 20-30 fold weaker CD137 binding compared to the parent anti-CD137 mAb (Table 7 below or Figures 7A-7B).

[0215] CD137 reporter assays to determine the potency of these antibodies using GS-H2-huCD137 reporter cells expressing human CD137, and downstream signaling assays for IL-8 expression were performed. GS-H2-huCD137 reporter cells with or without additional target expressing cells were seeded in assay plates at 3000 cells / well and 25000 cells / well, respectively. Exemplary multispecific antibodies were added to the assay plates. Plates were incubated for 18-20 hours in a 37°C, CO2 (5%) incubator. 8 μL of supernatant from each well of the assay plate was subjected to a homogenous time-resolved fluorescence (HTRF) assay (Cisbio). Human interleukin-8 (reporter of CD137 activation) detection assay was performed in a 16 μL assay volume using a human IL-8 assay kit (Cisbio, catalog no. 62IL8PEB). Results were read using a Tecan F200pro.

[0216] Results from the CD137 reporter assay showed minimal activity for the antibodies tested, however activity was greatly enhanced when additional targets for cross-linking, such as tumor antigens or immune receptors, were available (Table 7 below and Figures 8A-8E). [Table 5]

[0217] As shown in Table 7 and Figures 7-8, the central Fv CD137 binding portion exhibited much lower binding affinity to CD137 compared to the parental anti-CD137 antibody (Ly1630), and neither the parental clone nor the multispecific antibody exhibited CD137 activation activity in the presence of GS-H2-huCD137 reporter cells. However, in the presence of cells expressing other target antigens (B7H3 or PD-1), the multispecific antibody exhibited high potency in activating CD137, whereas the parental anti-CD137 did not show such an effect.

[0218] These examples demonstrate that the central CD137 Fv binding module has significantly reduced CD137 binding and minimal ability to induce CD137 activation, however in a multispecific format the CD137 Fv binding module has high clustering and activation potential due to the presence of secondary and / or tertiary binding partners.

[0219] CD40 Modulators Several bispecific or trispecific antibodies were constructed that include Ly2121, Ly2279, and Ly2280 as CD40 modulators and target B7H3 and / or PD-1. These bispecific or trispecific antibodies showed 3-6 fold weaker CD40 binding compared to the parent anti-CD40 mAb (Table 8 below and Figures 9A-9B).

[0220] CD40 reporter assays to determine agonist activity of exemplary multispecific antibodies using reporter cells expressing human CD40, and downstream signaling assays for IL-8 expression were performed. GS-H2-huCD40 reporter cells with or without additional target expressing cells were seeded in assay plates at 1000 cells / well and 25000 cells / well, respectively. Exemplary multispecific antibodies were added to the assay plates. The assay plates were incubated in a 37°C, CO2 (5%) incubator for 18-20 hours. 8 μL of supernatant from each well of the assay plate was subjected to HTRF detection assay (Cisbio). Human interleukin 8 (reporter of CD40 activation) detection assay was performed in a 16 μL assay volume (Cisbio, catalogue no. 62IL8PEB). Results were read using a Tecan F200pro.

[0221] The CD40 reporter assay results showed reduced activity in the CD40 activation reporter assay, however, activity was greatly enhanced in the presence of additional targets for cross-linking, such as tumor antigens or immune receptors (Table 8 below and Figures 10A-10F). [Table 6]

[0222] These examples demonstrated that the central CD40 Fv binding module has significantly reduced CD40 binding and low ability to induce CD40 activation. However, in a multispecific format, the CD40 Fv binding module has high clustering and activation potential in the presence of secondary and / or tertiary binding partners, including the TAA B7H3 or the immune checkpoint inhibitor PD-1, suggesting synthetic biology of avidity-driven xLink (crosslinking) activity via high affinity target antigens and optimally modulating immune responses, where multivalent binding may readily occur, for example, in the tumor microenvironment.

[0223] Example 5: Contribution of peptide linker sequences to binding in trispecific antibodies This example examines the effect of peptide linkers in the context of Fvs on the characteristics of multispecific antibodies that contain such.

[0224] The trispecific B7H3 / CD3 / CD28 antibodies Ly2132, Ly2133, Ly2134, Ly2135, and Ly2128, all of which contain a terminal Fv CD28 binding module, were examined in this study. H and V L Each fragment was connected to the Fc fragment via a peptide linker (flexible or rigid) as shown below: Ly2134: GGGGS (SEQ ID NO: 3), Ly2133: GGGGSGGGGS (SEQ ID NO: 4), Ly2132: GGGGSGGGGSGGGGS (SEQ ID NO:5), Ly2128: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 6), Ly2135:DKTHTCPPCPGGGGSGGGGSGGGGS (sequence number 17).

[0225] FIG. 11 shows the effect of these peptide linkers on binding to the target antigen. 4 The S)n sequence correlates with higher binding activity (Ly2128>Ly2132>Ly2133>Ly2134) and shorter G 4 S was at Ly2134 with minimal binding. The presence of the DKTHTCPPCP (SEQ ID NO:8) peptide sequence in the linker adversely affected binding activity, as Ly2135 showed lower binding than Ly2132. The binding affinity of the Fv fragments in the multispecific antibody was found to be approximately 100-fold lower than that of the parent mAb (in IgG format).

[0226] 12A-12B show the agonistic properties of the trispecific antibody. 4S)n sequence correlated with agonist activity (Ly2128>Ly2132>Ly2133>Ly2134). The presence of the DKTHTCPPCP (SEQ ID NO:8) peptide sequence in the peptide linker adversely affected agonist activity, as Ly2135 showed lower agonist activity than Ly2132. A similar trend was observed when reporter cells were treated with the antibodies listed above along with B7H3-expressing CHO cells.

[0227] Taken together, the data demonstrated that the length and structure of the linker connecting the Fv moieties can finely tune the Fv binding capacity and thus further obtain desired functionality.

[0228] Example 6: Characterization of multispecific antibodies containing terminal Fv binding moieties Using the Fv CD137, CD40, CD28 or CD3 binding modules according to the invention at the C-terminus, several multispecific antibodies were constructed, including Ly1963, Ly1966, Ly1967, Ly1800, Ly1891, Ly2122, Ly2128, Ly2326, Ly2396, Ly2405, Ly2415, Ly2424, Ly2434, Ly2444, Ly2454, Ly2580, Ly2579, Ly2638, Ly2639, Ly2640, Ly2641, Ly2642, Ly2643 and Ly2644. Binding of these multispecific antibodies to the corresponding targets was assessed by FCM or ELISA performed as described above. Activation of CD137, CD40, CD28, or CD3 signaling by these multispecific antibodies was also assessed using the appropriate reporter systems described above.

[0229] CD137 Modulators Several B7H3, CD19 or PD-1 targeted bispecific or trispecific antibodies were constructed with CD137 modulators via their terminal Fv fragments, and these bispecific or trispecific antibodies showed weaker CD137 binding compared to the parent anti-CD137 mAb (Table 9 below, and Figures 17A-17B and 25A-25C).

[0230] As described in the above examples, CD137 reporter assay was performed to determine the potency of these antibodies. The results from CD137 reporter assay showed minimal activity for the antibodies tested in the absence of additional target. However, activity was greatly enhanced when additional target for crosslinking, such as tumor antigen or immune receptor, was available (Table 9 below and Figures 31A-31C). [Table 7]

[0231] As shown in Figures 17A-17B and Figures 25A-25C, the terminal Fv CD137 binding moieties exhibited 20-100 fold lower binding affinity to CD137 compared to the reference and parental anti-CD137 antibodies (TM173 and Ly1630), and none of the multispecific antibodies exhibited CD137 activating activity in the absence of additional targets. However, in the presence of cells expressing other target antigens (B7H3, CD19, PD-1, or CD3), the multispecific antibodies exhibited high potency in activating CD137, whereas the parental anti-CD137 mAb did not exhibit such a cross-linking effect. See also Figures 31A-31C.

[0232] These examples demonstrate that the CD137 Fv binding module at the C-terminus has significantly reduced CD137 binding and minimal ability to induce CD137 activation. However, in a multispecific format, the CD137 Fv binding module has high clustering and activation potential due to the presence of secondary and / or tertiary binding partners.

[0233] CD28 Modulators Several B7H3, BCMA, CEA, or HER2 targeted multispecific antibodies were constructed with CD28 modulators via their terminal Fv fragments.

[0234] As shown in Figures 13A-13D, the terminal Fv CD28 binding portion exhibited 20-100 fold lower binding affinity for CD28 compared to the parental anti-CD28 antibody Ly224.

[0235] Jurkat reporter assays were performed to determine the potency of these antibodies, as described in the above examples. Results from the reporter assays showed weak activity for the antibodies tested in the absence of additional targets. However, activity was greatly enhanced when additional targets for cross-linking, such as tumor antigens, were available (Figures 13E-13N).

[0236] These examples demonstrate that the C-terminal CD28 Fv binding module has significantly reduced CD28 binding, however in a multispecific format the CD28 Fv binding module has high clustering and activation potential due to the presence of secondary and / or tertiary binding partners, as summarized in Table 10 below. [Table 8]

[0237] PD-L1 Modulators Several B7H3-targeting multispecific antibodies were constructed with PD-L1 modulators via their terminal Fv fragments.

[0238] As shown in Figure 14A, the terminal Fv PD-L1 binding portions exhibited 20-100 fold lower binding affinity to PD-L1 compared to the parental anti-PD-L1 antibodies Ly076 and Ly2530.

[0239] Jurkat reporter assays were performed to determine the blocking ability of the terminal Fv PD-L1 binding moieties. Results from the reporter assays showed weak blocking activity for the antibodies tested in the absence of additional targets. However, activity was slightly enhanced when additional targets for cross-linking, such as tumor antigens, were available (Figures 14B and 14C).

[0240] These examples demonstrate that the C-terminal PD-L1 Fv binding module has significantly reduced PD-L1 binding and weak blocking activity. However, in a multispecific format, the PD-L1 Fv binding module has the potential for clustering and activation by the presence of secondary and / or tertiary binding partners, as summarized in Table 11 below. [Table 9]

[0241] Example 7: Evaluation of multispecific antibodies with hidden Fv modules for cancer immunotherapy This example tests the anti-tumor efficacy of an exemplary multispecific antibody with a cryptic Fv module and additional binding domains that target the tumor microenvironment.

[0242] A. Multispecific antibodies targeting CD3 and CD19 and / or CD20 (i) Binding to target antigen Bispecific antibodies targeting CD3 and CD19 or CD3 and CD20 have been shown to be effective against B-cell lymphoma. However, such bispecific antibodies have some drawbacks, including short half-life and high side effects. To address these issues, in this example, several multispecific antibodies targeting CD19 and / or CD20 with either or both anti-CD3 and anti-CD137 Fvs were evaluated, such as Ly1966, Ly1967, Ly2278, Ly2326, Ly2579, Ly2580, Ly2800, Ly2802, Ly2943, Ly2944, Ly2945, Ly2946, Ly2947, and Ly2948. The binding of these multispecific antibodies to their corresponding targets is assessed by FCM or ELISA as described above, and their binding to tumor antigen-expressing tumor cells, e.g., Raji cells (CD20+CD19+), is also examined. A summary of these data is presented in Table 12 and in Figures 4C and 15-17. These data demonstrate that the binding activity of the Fv binding modules is significantly reduced. [Table 10]

[0243] (ii) Reporter assay Reporter assays as described above were performed to determine activation of CD3 signaling by multispecific antibodies containing anti-CD19 / CD20 and / or CD137 binding moieties. These data are summarized in Table 13 and in Figures 5C-5I and 18A-18L. The presence of CD19, CD20, including tumor cells expressing these antigens (e.g., Raji cells), dramatically increased CD3 activation of multispecific antibodies containing Ly1966, Ly1967, Ly2278, Ly2800, Ly2802, Ly2943, and Ly2944. Binding to CD137 enhanced CD3 activation of multispecific antibodies Ly1966, Ly1967, Ly2800, Ly2802, Ly2943, and Ly2944, albeit at higher concentrations compared to CD19 binding, reflecting the lower affinity of the CD137-binding Fv module. [Table 11]

[0244] Activation of CD137 signaling by multispecific antibodies targeting CD19 or CD20 was also determined using a reporter assay, and the results are summarized in Table 14 and Figures 19A-19G. [Table 12]

[0245] Activation of immune receptors CD3 or CD137 by the multispecific antibodies was minimal but significantly increased under crosslinking conditions, supporting the avidity-mediated effect of the design.

[0246] (iii) Tumor cell killing, pharmacokinetics, and antitumor efficacy Tumor cell killing by immune cells induced by anti-CD19 / 20 polyspecific antibodies was examined in vitro.

[0247] Raji cells and human PBMCs were mixed and incubated with various test substances for 48 hours, and the release of LDH from damaged cells was measured using a kit from Promega according to the instructions. The cytotoxicity of the test substances was calculated based on the LDH levels in the culture supernatant. Ly1966 and Ly1967 showed very strong activity with EC50 values ​​of 10.8 pM and 8.9 pM, respectively (Figure 20A). The B7H3-targeted control Ly1963 showed no significant activity in this assay, supporting the CD19 target-specific cytotoxicity activated by Ly1966 and Ly1967 (Figure 20A).

[0248] Nalm-6-luc cells and human PBMCs were mixed and incubated with various test substances for 48 hours, and luminescence from tumor cells was measured using a kit from Promega according to the instructions. The cytotoxicity of the test substances was inversely correlated with the luminescence intensity. Anti-CD19( / CD20) antibodies incorporated with anti-CD3 and / or anti-CD137 Fv showed significant tumor cell killing activity in this assay, comparable to or superior to the anti-CD20 / CD3 bsAb reference Ly2309 (Figure 20B).

[0249] (iv) Pharmacokinetics Ly1967 was administered to mice for pharmacokinetic analysis. Briefly, C57BL / 6 mice (6-7 weeks old, 19-20 g, female, purchased from Vital River) were used for this study. Antibody was diluted in DPBS and administered via ip injection at 5 mg / kg to groups of 4 mice. Blood was collected by serial bleeding before administration, on days 1, 4, 7, 10, 14, 17, and 21. 10 μL of blood per time point was added to 40 μL of PBS-BSA solution. Samples were then mixed thoroughly and centrifuged at 2000xg for 5 min at 4°C. Supernatants were frozen on dry ice and stored at -70°C until analysis. Blood antibody concentrations were determined from target recombinant human protein binding by ELISA. After a single intraperitoneal injection of 5 mg / kg, serum concentrations of the active molecule were determined by double antigen-binding ELISA. As shown in FIG. 21, Ly1967 remained in the circulation for up to 3 weeks after administration, with a PK profile similar to that of a conventional antibody.

[0250] (v) Antitumor Efficacy Antitumor efficacy was examined in a bone marrow transplant syngeneic mouse model using the human CD19-overexpressing murine colon cancer cell line MC38, the murine melanoma cell line B16F10, or in a human PBMC-engrafted NCG mouse model using the human Burkitt's lymphoma cell line Raji. Test articles were injected i.p. Mice were weighed and tumor growth was measured twice weekly using calipers. Tumor growth was calculated using the formula 0.5 x (length x width). 2) was used to estimate tumor volume. Strong anti-tumor activity was achieved by both Ly1967 and Ly2278, as shown in Figure 22A. In addition, the fact that Ly1967 exhibits stronger anti-tumor activity than Ly531 (anti-CD19 / CD3 bispecific antibody) (Figure 22B), Ly1967 exhibits stronger anti-tumor activity than Ly2278 (Figure 22C), and Ly2800 / 2802 exhibits stronger anti-tumor activity than Ly2307 or Ly531 (Figure 22D), and Ly2943 / 2944 exhibits stronger anti-tumor activity than Ly2307 or Ly531 (Figure 22E), collectively suggesting an advantage of anti-CD19 / 20 multispecific antibodies over reference anti-CD3 bsAbs (Ly2278, Ly2307, and Ly531) in terms of anti-tumor activity in vivo.

[0251] B. Multispecific antibodies targeting CD3 and B7H3 B7H3-targeted CD3 bispecific T cell engagers have the potential to mediate immune killing of various solid tumors where high levels of B7H3 expression are common. Using the Fv CD3 and CD137, CD28, PD-1, PD-L1, or CTLA4 binding modules described in the present invention, several anti-B7H3 multispecific antibodies were generated, including Ly1965, Ly1963, Ly2128, Ly2122, Ly2936, Ly2937, Ly2939, and Ly2940.

[0252] (i) Binding to target antigen The binding of these multispecific antibodies to their corresponding targets was assessed by FCM or ELISA and is summarized in Table 15 below, as well as in Figures 4A-4C and Figures 23-29. The multispecific antibodies showed affinity binding to the TA target B7H3 and significantly weakened affinity to the immune targets CD3, CD137, CD28, PD-1, PD-L1, or CTLA4. [Table 13]

[0253] (ii) Reporter assay Activation of CD3 signaling by anti-B7H3 multispecific antibodies was assessed using a reporter system and summarized in Table 16 below, and Figures 5A-5I and 30A-30Q. The presence of B7H3 target antigen significantly increased CD3 activation of multispecific antibodies including Ly1963 and Ly1965. Binding to terminal Fv targets such as CD137, CD28, PD-1, or PD-L1 enhanced the CD3 activity of the corresponding multispecific antibodies. [Table 14]

[0254] Activation of CD137 signaling by anti-B7H3 multispecific antibodies was assessed using a reporter system and summarized in Table 17 below and Figures 31A-31F. [Table 15]

[0255] Blockade of PD-(L)1 signaling by anti-B7H3 multispecific antibodies was assessed using a reporter system and summarized in Table 18 below and Figures 14B-14C. [Table 16]

[0256] Activation of immune receptors CD3 or CD137 by the multispecific antibodies was minimal but significantly increased under crosslinking conditions, supporting the avidity-mediated effect of the design. The impact of the multispecific antibody crosslinking effect on PD-L1 signaling was also observed with the exemplary anti-B7H3 / CD3 / PD-L1 multispecific antibody.

[0257] (iii) Tumor cell killing, pharmacokinetics, and antitumor efficacy Tumor cell killing by immune cells induced by anti-B7H3 polyspecific antibodies was examined in vitro. A375 or A375-Luc cells and human PBMCs were mixed and incubated with various test antibodies for 48 hours, and LDH release or luminescence intensity was measured as previously described.

[0258] Figures 32A-32B provide a summary of these results in an in vitro assay for killing A375 cancer cells. Ly2128 and Ly1963 showed a trend toward stronger cytotoxicity than Ly1965, suggesting the contribution of additional CD28 or CD137 signaling induced by the corresponding terminal Fv. Ly2128 induced higher cytokine production than Ly1963, but there was no obvious difference in tumor cell killing.

[0259] Figures 32C-32D provide a summary of these results in an in vitro assay for killing of A375-luc cancer cells. Ly2600, Ly2936, and Ly1963 showed a trend toward stronger cytotoxicity than Ly2939, suggesting the contribution of additional CD137-terminal Fv.

[0260] Figures 32E-32F provide the results of an in vitro assay for killing of A375-luc cancer cells by Ly2938 carrying a CD28-terminal Fv.

[0261] Figures 32G-32H provide the results of an exemplary multispecific antibody of B7H3 / CD3 / PD-L1 in vitro assay for killing of A375-luc cancer cells. Ly2846 and Ly2847 showed similar or stronger cytotoxicity than Ly1963, and higher IFN-γ secretion than Ly1963, suggesting the contribution of an additional PD-L1 terminal Fv.

[0262] Figures 32I-32J provide the results of an exemplary B7H3 / CD3 / PD-1 multispecific antibody in vitro assay for killing of A375-luc cancer cells. Ly2904 exhibited similar strong cytotoxicity in terms of tumor cell killing and IFN-γ secretion as Ly1963, suggesting the contribution of the additional PD-1 terminal Fv.

[0263] Figures 32K-32L provide the results of an exemplary multispecific antibody of B7H3 / CD3 / CTLA4 in vitro assay for killing of A375-luc cancer cells. Ly2901, Ly2902, and Ly2903 showed variable and slightly lower cytotoxicity and IFN-γ secretion levels than Ly1963, suggesting the contribution of additional CTLA4-terminal Fv.

[0264] Pharmacokinetic analysis of these multispecific antibodies was performed using a mouse model as described above. Figure 33 shows the presence of circulating Ly1963 antibodies over a 21 day period, with a PK profile similar to that of a conventional antibody.

[0265] Antitumor efficacy was examined in a bone marrow transplant mouse model. Bone marrow from homozygous human CD3 and CD137 knock in mice was transplanted into wild-type C57 mice that had been subjected to total body irradiation 12-24 hours prior. Three weeks after bone marrow transplantation, human B7H3-expressing LL2 tumors were inoculated sc. Five days after tumor inoculation, mice were divided into groups and injected ip with test articles every week. Mice were weighed and tumor growth was measured twice weekly using calipers. Tumor volume was calculated using the formula 1 / 2(length × width) / (length × width). 2 ) Strong antitumor activity was achieved by Ly1963, as shown in Figure 34A. The trispecific antibody Ly1963 showed stronger antitumor efficacy than either the bispecific antibody Ly1965 or Ly2122, suggesting potential advantages in designing synthetic CD3 and CD137 antibodies for stronger antitumor immunity.

[0266] Antitumor efficacy was examined in a human PBMC engrafted mouse model. A375 tumors expressing human B7H3 were inoculated sc and human PBMCs were inoculated iv on day 0. Four days after tumor inoculation, mice were divided into groups and injected ip with test articles every week. Mice were weighed and tumor growth was measured twice weekly using calipers. Tumor volume was calculated using the formula 1 / 2(length × width) = 1 / 2 (length × width). 2 Antitumor activity was achieved by exemplary multispecific antibodies Ly2823, Ly2936, Ly2937, Ly2600, Ly2846, Ly2847, Ly2938, Ly2939, and Ly2940, as shown in Figures 34B-34L. It is noteworthy that bispecific antibody Ly2940 exhibits robust antitumor efficacy, suggesting a potential advantage of an anti-CD137 Fv on the C-terminus.

[0267] C. Other immune modulators and multispecific antibodies targeting B7H3 Bispecific antibodies targeting B7H3 and other immune modulators have been evaluated for their anti-tumor efficacy in clinical trials. Using the Fv CD137 and / or CD40 binding modules described in the present invention, several anti-B7H3 multispecific antibodies were generated, including Ly2279, Ly2424, Ly2428, Ly2434, Ly2641 Ly2642 Ly2512, Ly2513, Ly2514, and Ly2515.

[0268] (i) Binding to target antigen The binding of these multispecific antibodies to their corresponding targets was assessed by FCM or ELISA and is summarized below in Table 19. The multispecific antibodies showed affinity binding to the TAA target B7H3 and significantly reduced affinity to the immune targets CD40 or CD137. [Table 17]

[0269] The activation of CD137 or CD40 signaling by these multispecific antibodies is evaluated using a reporter system. The activation of immune cells and tumor cell killing induced by these multispecific antibodies are examined in vitro. These multispecific antibodies are administered to mice for pharmacokinetic analysis. The antitumor efficacy of these multispecific antibodies is examined in mouse models.

[0270] D. Trispecific and Bispecific Anti-PD-1 Antibodies Using the Fv CD137, CD40, or GITR binding modules described in the invention, a number of anti-PD-1 multispecific antibodies have been generated, including Ly2281, Ly2396, Ly2638, Ly2280, Ly2405, Ly2409, Ly2415, Ly2639, Ly2640, Ly2505, Ly2506, Ly2509, Ly2510, Ly2511, Ly2578, Ly2507, Ly2508, Ly2576, and Ly2577.

[0271] The binding of these multispecific antibodies to their corresponding targets was assessed by FCM or ELISA and is summarized below in Table 20. The multispecific antibodies showed affinity binding to the target PD-1 and significantly reduced affinity to the immune targets CD40, CD137, or GITR. [Table 18]

[0272] Activation of CD137 or CD40 or GITR, or inhibition of PD-1 signaling by these multispecific antibodies was assessed using the reporter systems described above or below.

[0273] A PD-1 reporter assay was performed to determine the potency of these antibodies. The assay consisted of two engineered cell lines, Jurkat T cells expressing human PD-1 and a luciferase reporter driven by NFκB response element, and Raji cells expressing human PD-L1. Jurkat and Raji cells with or without additional target expressing cells were seeded into an assay plate. Exemplary multispecific antibodies were added to the assay plate. The plate was incubated in a 37°C, CO2 (5%) incubator for 6 hours. Luminescence signals were examined with the Bright-glo kit from Promega.

[0274] The tumor cell killing by immune cells induced by these multispecific antibodies is examined in vitro.The pharmacokinetics is evaluated in mice by injecting multispecific antibodies and measuring the circulating levels of these antibodies for at least 21 days.The anti-tumor efficacy of these multispecific antibodies is examined using mouse tumor models.

[0275] E. Trispecific and Bispecific Anti-PD-L1 Antibodies The Fv CD137, CD40, CD28, or CD3 binding modules described in the invention are used to generate a number of anti-PD-L1 multispecific antibodies, including Ly2438, Ly2444, Ly2453, Ly2454, Ly2643, Ly2644, Ly2517, Ly2518, Ly2519, Ly2520, Ly2521, Ly2522, Ly2626, Ly2627, Ly2628, Ly2629, Ly2630, Ly2631, Ly2632, Ly2633, Ly2634, Ly2635, Ly2636, and Ly2637.

[0276] The binding of these multispecific antibodies to their corresponding targets was assessed by FCM or ELISA and is summarized below in Table 21. The multispecific antibodies showed affinity binding to the target PD-L1 and significantly weakened affinity to the immune targets CD40, CD137, CD3, or CD28. [Table 19]

[0277] Activation of CD137, CD40, CD28, or CD3 signaling and blockade of PD-1 / PD-L1 signaling by these multispecific antibodies are assessed using appropriate reporter systems as described above. Tumor cell killing by immune cells induced by these multispecific antibodies is examined in vitro. Pharmacokinetics is evaluated in mice by injecting the multispecific antibodies and measuring the circulating levels of these antibodies for at least 21 days. Antitumor efficacy of these multispecific antibodies is examined using mouse tumor models.

[0278] Example 8: Further characterization of multispecific antibodies Multispecific antibodies can be further characterized as exemplified below: (i) Trispecific and bispecific anti-PSMA antibodies Using the Fv CD3 binding module described in the present invention, several anti-PSMA multispecific antibodies are constructed, including Ly2083, Ly2084, Ly2086, Ly2606, Ly2607, Ly2608, and Ly2609. The binding of these multispecific antibodies to the corresponding targets is evaluated by FCM or ELISA. The activation of CD3 or CD137 signaling by these multispecific antibodies is evaluated using the appropriate reporter system described above. The tumor cell killing by immune cells induced by these multispecific antibodies is examined in vitro. The pharmacokinetics is evaluated in mice by injecting the multispecific antibodies and measuring the circulating levels of these antibodies for at least 21 days. The anti-tumor efficacy of these multispecific antibodies is examined using a mouse tumor model.

[0279] (ii) Trispecific and bispecific anti-BCMA antibodies Using the Fv CD3 binding module described in the present invention, several anti-BCMA multispecific antibodies are generated, including Ly2312, Ly2315, Ly2602, Ly2603, Ly2604, and Ly2605. The binding of these multispecific antibodies to the corresponding targets is evaluated by FCM or ELISA. The activation of CD3 or CD137 signaling by these multispecific antibodies is evaluated using the appropriate reporter system described above. The tumor cell killing by immune cells induced by these multispecific antibodies is examined in vitro. The pharmacokinetics is evaluated in mice by injecting the multispecific antibodies and measuring the circulating levels of these antibodies for at least 21 days. The antitumor efficacy of these multispecific antibodies is examined using mouse tumor models.

[0280] (iii) Trispecific and bispecific anti-HER2 antibodies Using the Fv CD3 binding module described in the present invention, several anti-HER2 multispecific antibodies are generated, including Ly2316, Ly2317, Ly2318, Ly2319, Ly2610, Ly2611, Ly2612, Ly2613, Ly2614, Ly2615, Ly2616, and Ly2617. The binding of these multispecific antibodies to the corresponding targets is evaluated by FCM or ELISA. The activation of CD3 or CD137 signaling by these multispecific antibodies is evaluated using the appropriate reporter system described above. The tumor cell killing by immune cells induced by these multispecific antibodies is examined in vitro. The pharmacokinetics is evaluated in mice by injecting the multispecific antibodies and measuring the circulating levels of these antibodies for at least 21 days. The anti-tumor efficacy of these multispecific antibodies is examined using a mouse tumor model.

[0281] (iv) Trispecific and bispecific anti-CEA antibodies Using the Fv CD3 binding module described in the present invention, several anti-CEA multispecific antibodies are generated, including Ly2320, Ly2321, Ly2322, Ly2618, Ly2619, Ly2620, Ly2621, Ly2622, and Ly2623. The binding of these multispecific antibodies to the corresponding targets is evaluated by FCM or ELISA. The activation of CD3 or CD137 signaling by these multispecific antibodies is evaluated using the appropriate reporter system described above. The tumor cell killing by immune cells induced by these multispecific antibodies is examined in vitro. The pharmacokinetics is evaluated in mice by injecting the multispecific antibodies and measuring the circulating levels of these antibodies for at least 21 days. The anti-tumor efficacy of these multispecific antibodies is examined using a mouse tumor model.

[0282] (v) Trispecific and bispecific anti-P53 R175H antibodies Using the Fv CD3 binding module described in the present invention, several anti-p53 R175H multispecific antibodies are generated, including Ly2288, Ly2624, and Ly2625. The binding of these multispecific antibodies to the corresponding targets is evaluated by FCM or ELISA. The binding of these multispecific antibodies to the corresponding targets is evaluated by FCM or ELISA. The activation of CD3 or CD137 signaling by these multispecific antibodies is evaluated using the appropriate reporter system described above. The tumor cell killing by immune cells induced by these multispecific antibodies is examined in vitro. The pharmacokinetics is evaluated in mice by injecting the multispecific antibodies and measuring the circulating levels of these antibodies for at least 21 days. The antitumor efficacy of these multispecific antibodies is examined using a mouse tumor model.

[0283] (vi) Trispecific and bispecific anti-MAGE-A4 antibodies Using the Fv CD3 binding module described in the present invention, several anti-MAGE-A4 multispecific antibodies are generated. The binding of these multispecific antibodies to the corresponding targets is evaluated by FCM or ELISA. The activation of CD3 or CD137 signaling by these multispecific antibodies is evaluated using the appropriate reporter system described above. The tumor cell killing by immune cells induced by these multispecific antibodies is examined in vitro. The pharmacokinetics is evaluated in mice by injecting the multispecific antibodies and measuring the circulating levels of these antibodies for at least 21 days. The antitumor efficacy of these multispecific antibodies is examined using a mouse tumor model.

[0284] (vii) Trispecific and bispecific anti-PRAME antibodies Using the Fv CD3 binding module described in the present invention, several anti-CEA multispecific antibodies are generated. The binding of these multispecific antibodies to the corresponding targets is evaluated by FCM or ELISA. The activation of CD3 or CD137 signaling by these multispecific antibodies is evaluated using the appropriate reporter system described above. The tumor cell killing by immune cells induced by these multispecific antibodies is examined in vitro. The pharmacokinetics is evaluated in mice by injecting the multispecific antibodies and measuring the circulating levels of these antibodies for at least 21 days. The antitumor efficacy of these multispecific antibodies is examined using mouse tumor models.

[0285] (viii) Trispecific and bispecific anti-CD47 antibodies Using the Fv CD47 binding module described in the present invention, several anti-CD47 multispecific antibodies, including Ly2147 and Ly2148, are generated. The binding of these multispecific antibodies to the corresponding targets is evaluated by FCM or ELISA. The phagocytosis of tumor cells induced by these multispecific antibodies is examined in vitro. The pharmacokinetics is evaluated in mice by injecting the multispecific antibodies and measuring the circulating levels of these antibodies for at least 21 days. The antitumor efficacy of these multispecific antibodies is examined using mouse tumor models.

[0286] Sequence Listing [Table 20-1] [Table 20-2] [Table 20-3] [Table 20-4] [Table 20-5] [Table 20-6] [Table 20-7] [Table 20-8]

Table 20-9

Table 21-1

Table 21-2

Table 21-3

Table 21-4

Table 21-5

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Table 21-7

Table 21-8

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Table 21-13

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Table 21-16

Table 21-17

Table 21-18

Table 21-19

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Table 21-21

Table 21-22

Table 21-23

Table 21-24

Table 21-25

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Table 21-27

Table 21-28

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Table 21-30

Table 21-31

Table 21-32

Table 21-33

Table 21-34

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Table 21-36

Table 21-37

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Table 21-62

Table 21-63

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Table 21-71

Table 21-72

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Table 21-79

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Table 21-83

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Table 21-89

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Table 21-96

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[0287] Other embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced with an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is merely an example of a generic series of equivalent or similar features.

[0288] From the above description, those skilled in the art can easily ascertain the essential features of the present disclosure, and can make various changes and modifications to adapt the disclosure to various usages and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are within the scope of the claims.

[0289] Equivalent While several embodiments of the invention have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein, and each such variation and / or modification is deemed to be within the scope of the embodiments of the invention described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application or applications for which the teachings of the invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Thus, the foregoing embodiments are presented by way of example only, and it will be understood that, within the scope of the appended claims and equivalents thereto, embodiments of the invention may be practiced otherwise than as specifically described and claimed. The inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more of such features, systems, articles, materials, kits, and / or methods is within the inventive scope of the present disclosure, provided that such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0290] All definitions and those used herein should be understood to control for any dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0291] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, in some cases including the entire document.

[0292] The indefinite articles "a" and "an," as used herein in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one." The phrase "and / or" as used in the specification and claims herein should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so conjoined. Other elements other than the elements specifically identified by the "and / or" clause may optionally be present, whether related to the elements specifically identified or not. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with open-ended language such as "comprising", may refer in one embodiment to only A (optionally including elements other than B), in another embodiment to only B (optionally including elements other than A), in yet another embodiment to both A and B (optionally including other elements), etc.

[0293] When used in the specification and claims herein, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., including at least one, but not more than one, of a number or list of elements, and optionally additional list items. Only terms clearly indicated to the contrary, such as "only one" or "exactly one," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, when used herein, the term "or" should only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by a term of exclusivity, such as "either," "one of," "only one of," or "exactly one of." When used in the claims, "consisting essentially of" has the ordinary meaning of the term as used in the field of patent law.

[0294] As used herein in the specification and claims, the phrase "at least one" in connection with a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but need not include at least one of each and every element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether or not related to the specifically identified elements, may optionally be present. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer in one embodiment to at least one A (optionally including more than one A) and no B (and optionally including elements other than B), in another embodiment to at least one B (optionally including more than one B) and no A (and optionally including elements other than A), in yet another embodiment to at least one A (optionally including more than one A) and at least one B (optionally including more than one B) (and optionally including other elements), etc.

[0295] It is also to be understood that, unless expressly stated to the contrary, in any method claimed herein that includes two or more steps or actions, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are recited.

Claims

1. 1. A multispecific antibody comprising a first binding moiety specific for a first target antigen and a second binding moiety specific for a second target antigen, the first target antigen is a first immune cell receptor and the second target antigen is (i) a second immune cell receptor that is different from the first immune cell receptor, or (ii) a first tumor-associated antigen (TAA); The first binding moiety comprises a first heavy chain variable region (V Ｈ ) and a first light chain variable region (V Ｌ a first Fv fragment comprising The first V Ｈ is linked to a first flexible peptide linker and a first rigid peptide linker, Ｌ is linked to a second flexible peptide linker and a second rigid peptide linker; A multispecific antibody, wherein the first rigid peptide linker and the second rigid peptide linker form one or more disulfide bonds.

2. 2. The multispecific antibody of claim 1, further comprising a third binding moiety specific for a third target antigen.

3. The first V Ｈ is further linked to a first Fc fragment via the first rigid peptide linker, and the first V Ｌ is further linked to a second Fc fragment via said second rigid peptide linker, and each Fc fragment comprises a CH2 domain and a CH3 domain.

4. 4. The multispecific antibody of claim 3, wherein the second binding moiety and / or the third binding moiety are in single-chain variable fragment (scFv) format, single-domain antibody format, Fab format, or cross-Fab format.

5. (a) from N-terminus to C-terminus, the first VH-CH1 or VH-CL fragment of the second binding moiety, the first flexible peptide linker, the first V Ｈ a first polypeptide comprising the first rigid peptide linker, and the first Fc fragment; (b) from N-terminus to C-terminus, a second VH-CH1 or VH-CL fragment of the third binding moiety, the second flexible peptide linker, the first V Ｌ a second polypeptide comprising the second rigid peptide linker, and the second Fc fragment; (c) a third polypeptide comprising the first VL-CL or VL-CH1 fragment of the second binding moiety; and and (d) a fourth polypeptide comprising the second VL-CL or VL-CH1 fragment of the third binding moiety.

6. and a fourth binding moiety specific for a fourth target antigen, said fourth binding moiety being a second V Ｈ and the second V Ｌ a second Fv fragment comprising said second V Ｈ is linked to the first Fc fragment via a first peptide linker, and the second V Ｌ is linked to the second Fc fragment via a second peptide linker.

7. The first flexible peptide linker, the second flexible peptide linker, the first peptide linker, and / or the second peptide linker are G / S-rich peptide linkers; (G Ｘ S) n, wherein X is an integer from 1 to 6, inclusive, and n is an integer from 1 to 10, inclusive; and / or 7. The multispecific antibody of claim 1 , wherein the first rigid peptide linker, the second rigid peptide linker, the third peptide linker, and / or the fourth peptide linker comprise the amino acid sequence DKTHTCPPCPAPEAAGP (SEQ ID NO: 21), DKTHTCPPCPAPELLGP (SEQ ID NO: 9), or DKTHTCPPCPAPELLGGP (SEQ ID NO: 27).

8. The multispecific antibody of claim 1, wherein the one or more rigid linkers, flexible linkers, and peptide linkers are listed in Table 1.

9. (a) from N-terminus to C-terminus, the first VH-CH1 or VH-CL fragment of the second binding moiety, the first flexible peptide linker, the first V Ｈ a first polypeptide comprising the first rigid peptide linker, the first Fc fragment, the first peptide linker, and the second VH; (b) from N-terminus to C-terminus, the second VH-CH1 or VH-CL fragment of the third binding moiety, the second flexible peptide linker, the first V Ｌ a second polypeptide comprising the second rigid peptide linker, the second Fc fragment, the third peptide linker, and a second VL; (c) a third polypeptide comprising the first VL-CL fragment or the first VL-CH1 of the second binding moiety; and and (d) a fourth polypeptide comprising the second VL-CL or VL-CH1 fragment of the third binding moiety.

10. 2. The multispecific antibody of claim 1, which is one of the multispecific antibodies listed in Table 2.

11. 7. The multispecific antibody of any one of claims 1 to 6, wherein the first, second and / or third immune receptor is selected from the group consisting of CD3, CD28, PD-1, PD-L1, CD47, and members of the tumor necrosis factor receptor superfamily (TNFRSF).

12. at least (a) CD3 and CD28; (b) CD3 and CD137; (c) CD137 and PD-1; (d) CD40 and PD-1; (e) CD40 and PD-L1; (f) CD137 and GITR; (g) CD137 and PD-L1; (h) CD137 and CD40; (i) CD137 and OX40; (j) CD3 and PD-1; (k) CD3 and PD-L1, or (l) The multispecific antibody according to any one of claims 1 to 6, which binds to CD3 and CTLA4.

13. 7. The multispecific antibody of any one of claims 1 to 6, wherein the first TAA, the second TAA and / or the third TAA is selected from the group consisting of B7H3, CD19, CD20, PSMA, HER2, CEA, BCMA, P53mut, DLL3, MET, and EGFR.

14. (1) B7H3, CD3, and CD137; (2) CD19, CD3, and CD137; (3) B7H3, CD3, and CD28; (4) CD19, CD3, and CD28; (5) B7H3, CD137, and PD-1; (6) B7H3, CD40, and PD-1; (7) PMSA, CD3, and CD137; (8) B7H3 and CD3, (9) B7H3 and CD137, (10) CD19 and CD3, (11) CD19 and CD137, (12) B7H4 and CD40, (13) HER2, CD3, and CD137, (14) CEA, CD3, and CD137; (15) BCMA, CD3, and CD137; (16) p53 mutant, CD3, and CD137; (17) PD-1 and CD137, (18) PD-1 and CD40, (19) B7H3 and CD40, (20) PD-L1 and CD40, (21) PD-L1 and CD3, (22) PD-L1, CD3, and CD137, (23) PD-L1, CD3, and CD28, (24) PD-L1, CD137, and B7H3, (25) PD-L1, CD40, and B7H3, (26) PD-1, CD40, and CD137, (27) PD-1, CD137, and GITR, (28) CD19, CD20, CD3, and CD137, (29) CEA and CD3, (30) CEA and CD137, (31) p53 mutant and CD3, (32) p53 mutant and CD137, (33) PD-L1, CD40, and CD137, (34) PD-L1 and CD137, (35) MET, EGFR, and CD47; (36) BCMA and CD3, (37) BCMA and CD137, (38) PSMA and CD3, (39) HER2 and CD3, (40) HER2 and CD40, (41) HER2 and CD137, (42) PSMA and CD137, (43) HER2, MET, CD3, and CD137, (44) MAGE-A4, CD3, and CD137, (45) PRAME, CD3, and CD137; (46) HER2, MET, and CD3, (47) MAGE-A4 and CD3, (48) PRAME and CD3, (49) HER2, MET, and CD47, (50) B7H3, CD3, and PD-1; (51) B7H3, CD3, and PD-L1, (52) B7H3, CD3, and CTLA4; (53) PSMA, CD3, and CD137, (54) PSMA, CD3, and CD28, (55) HER2, CD3, and CD137, (56) HER2, CD3, and CD28, (57) CEA, CD3, and CD137, (58) CEA, CD3, and CD28, (59) BCMA, CD3, and CD137; (60) BCMA, CD3, and CD28, or (61) The multispecific antibody according to any one of claims 1 to 6, which binds to CD19, CD19, CD3, and CD28.

15. 7. The multispecific antibody of any one of claims 1 to 6, wherein the multispecific antibody comprises the same heavy chain complementarity determining regions (CDRs) and the same light chain CDRs, and / or the same VH and VL as in one or more of the parent antibodies listed in Table 1.

16. A nucleic acid or set of nucleic acids collectively encoding the multispecific antibody of any one of claims 1 to 6.

17. A host cell comprising the nucleic acid or set of nucleic acids of claim 16.

18. 1. A method for producing a multispecific antibody, comprising: (i) culturing the host cell of claim 17 under conditions that allow expression of the antibody; (ii) recovering the antibody thus produced.

19. A pharmaceutical composition comprising the multispecific antibody of any one of claims 1 to 6, or a nucleic acid or a set of nucleic acids encoding said multispecific antibody, and a pharmaceutically acceptable carrier.

20. 10. A pharmaceutical composition for use in modulating an immune response in a subject, the pharmaceutical composition comprising the multispecific antibody of any one of claims 1 to 6, or a nucleic acid or a set of nucleic acids encoding said multispecific antibody, the pharmaceutical composition being administered to a subject in need thereof.