Multispecific antibodies integrating dual immunomodulatory moieties and their use in immunotherapy

Abstract

This invention relates to multispecific antibodies comprising a first Fv fragment targeting CD3, a second Fv fragment targeting an immune receptor such as CD137 or PD-L1, and one or more binding moieties targeting tumor-associated antigens. The therapeutic use of these multispecific antibodies in cancer therapy is also provided herein.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of International Application No. PCT / CN2023 / 109322, filed on July 26, 2023, the entire contents of which are incorporated herein by reference.

[0003] sequence list

[0004] This application contains a sequence list that has been electronically submitted in XML format and is hereby incorporated in its entirety by reference. The XML copy created on July 22, 2024, is named “063588-512001WO_Seq-Listing_ST26.xml” and has a size of 137,925 bytes. Background Technology

[0005] The immune cell receptor CD3, along with co-stimulatory or co-inhibitory receptors such as CD137 and PD-(L)1, plays a crucial role in regulating immune cell function, thereby controlling the immune response against pathogens or diseased cells, such as cancer cells and pathogen-infected cells. Antibodies targeting these immune cell receptors have been used to modulate immune responses and treat diseases. However, such treatments may not achieve the desired clinical efficacy and / or raise safety concerns. Therefore, the development of effective and safe novel immunotherapies is of great significance. Summary of the Invention

[0006] This disclosure is based, at least in part, on the development of multispecific antibodies comprising two binding moieties (dual immunomodulatory constructs) that regulate immune responses. Dual immunomodulatory constructs may target CD3 and CD137 or CD3 and PD-L1. The two binding moieties targeting CD3 / CD137 or CD3 / PD-L1 may be linked by an Fc fragment, which may contain one or more mutations relative to its wild-type counterpart, such as a deletion at position 237 (237Δ) and an amino acid substitution at position P329 (e.g., P329G). Unless explicitly stated otherwise, all numbers relating to positions in the Ig molecule (including positions in the Fc fragment) follow the EU numbering system. The multispecific antibodies provided herein may further comprise one or more antigen-binding moieties specific to tumor-associated antigens (TAAs).

[0007] As reported in this paper, multispecific antibodies containing dual immunomodulatory constructs exhibited better tumor cell killing, CD8 T cell proliferation, and in vivo antitumor efficacy compared to multispecific antibodies containing only one immune target moiety. Furthermore, as reported in this paper, multispecific antibodies containing the 237Δ / P329G Fc variant eliminated Fc receptor binding activity, while Fc variants with 237Δ or P329G mutations still maintained FcR-mediated crosslinking effects at a certain level. This feature can help avoid inducing systemic immune responses mediated by Fc-FcR interactions or Fc effector function, thereby reducing the risk of side effects.

[0008] In summary, the multispecific antibodies presented in this paper can simultaneously target two immune receptors, thereby inducing affinity-driven cross-linking of immune target antigens (e.g., CD3 / CD137 or CD3 / PD-L1) to conditionally modulate the immune response. It is anticipated that such multispecific antibodies presented in this paper will exhibit excellent antitumor activity, for example, in the tumor microenvironment, while avoiding stimulation of systemic immune responses that may cause undesirable side effects.

[0009] Therefore, in some aspects, this disclosure provides a multispecific antibody comprising: (i) a first antigen-binding portion that binds to CD3, (ii) a second antigen-binding portion that binds to CD137 or PD-L1, (iii) a third antigen-binding portion that binds to a first tumor-associated antigen (TAA); and optionally (iv) a fourth antigen-binding portion that binds to a second TAA.

[0010] The first antigen-binding region (anti-CD3) contains the first heavy chain variable region (V... H ) and the first light chain variable region (V L The first Fv fragment of the antigen-binding region. Similarly, the second antigen-binding region (anti-CD137 or anti-PD-L1) contains the second V. H Second V L The second Fv fragment. The first antigen-binding portion and the second antigen-binding portion are linked via an Fc fragment. The third antigen-binding portion (anti-TAA) is linked to the first antigen-binding portion, for example, via a first peptide linker. An optional fourth binding portion (anti-TAA) may be linked to the first antigen-binding portion, for example, via a second peptide linker.

[0011] The Fc fragments of the multispecific antibodies provided herein may comprise a hinge domain and a CH2 domain. In some cases, the Fc fragment contains a deletion (237Δ) at position 237 and an amino acid substitution (e.g., P329G) at position P329. In some embodiments, the Fc fragment is an IgG1 Fc fragment that optionally comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, any of the Fc fragments provided herein may further comprise a CH3 domain. In some instances, the CH3 domain is derived from a wild-type immunoglobulin molecule (e.g., an IgG molecule), for example, comprising the amino acid sequence of SEQ ID NO: 92. In other instances, the CH3 domain may comprise one or more mutations relative to the wild-type counterpart that enhance heterodimerization of the Fc fragment containing the one or more mutations compared to homodimerization and / or reduce protein A binding. Exemplary CH3 domain variants may comprise the amino acid sequence of SEQ ID NO: 93, 94, or 95.

[0012] Of any of the multispecific antibodies presented in this article, the anti-CD3 portion V H (First V) H It may contain the same heavy chain CDR as the heavy chain complementarity determination region (CDR) in SEQ ID NO: 7. Alternatively or additionally, the V of the anti-CD3 portion L (First V) L () may contain the same light chain CDR as the light chain CDR in SEQ ID NO: 8. In some embodiments, the first V H It may contain the amino acid sequence of SEQ ID NO: 7, and / or the first V L It may contain the amino acid sequence of SEQ ID NO: 8.

[0013] In some embodiments, the second antigen-binding portion binds to CD137. In some cases, the V of the anti-CD137 portion... H (Second V) H It may contain the same heavy chain CDR as the heavy chain complementarity determination region (CDR) in SEQ ID NO: 9. Alternatively or additionally, the V of the anti-CD137 portion L (Second V) L It may contain the same light chain CDR as the light chain CDR in SEQ ID NO: 10. In some instances, the second V H Containing the amino acid sequence of SEQ ID NO: 9, and / or the second V LThe amino acid sequence includes SEQ ID NO: 10. An exemplary multispecific antibody comprising anti-CD3 and anti-CD137 portions may comprise a first polypeptide and a second polypeptide, the first polypeptide comprising the amino acid sequence of SEQ ID NO: 3 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 4.

[0014] In other embodiments, the second antigen-binding portion binds to PD-L1. In some cases, the V of the anti-PD-L1 portion... H (Second V) H It may contain the same heavy chain CDR as the heavy chain complementarity determination region (CDR) in SEQ ID NO: 11. Alternatively or additionally, the VL (second V) of the anti-PD-L1 portion L It may contain the same light chain CDR as the light chain CDR in SEQ ID NO: 12. In some instances, the second V H Containing the amino acid sequence of SEQ ID NO: 11, and / or the second V L The amino acid sequence includes SEQ ID NO:12. An exemplary multispecific antibody comprising anti-CD3 and anti-PD-L1 moieties may comprise a first polypeptide and a second polypeptide, the first polypeptide comprising the amino acid sequence of SEQ ID NO:5 and the second polypeptide comprising the amino acid sequence of SEQ ID NO:6.

[0015] The multispecific antibodies described herein contain one or more binding moieties against tumor-associated antigens (TAAs). In some cases, one or more of the anti-TAA moieties may be Fab fragments.

[0016] Exemplary TAAs include, but are not limited to, B7H3, CD19, CD20, PSMA, HER2, CEA, BCMA, P53mut, DLL3, MET, and EGFR. In some instances, multispecific antibodies comprise one or more antigen-binding moieties specific to HER2, CEA, BCMA, B7H3, or CD19. In one specific instance, the anti-TAA moieties of a multispecific antibody bind to HER2. In another specific instance, the anti-TAA moieties bind to CEA. In yet another specific instance, the anti-TAA moieties bind to BCMA. In some cases, the multispecific antibodies provided herein may comprise two anti-TAA moieties that can bind to two different TAAs. In other cases, the multispecific antibodies provided herein may comprise two anti-TAA moieties that can bind to the same TAA (e.g., to different epitopes of the TAA). In still other cases, the multispecific antibodies provided herein may comprise two identical anti-TAA moieties.

[0017] In some instances, the multispecific antibodies disclosed herein bind to HER2, CD3, and CD137. In some instances, the multispecific antibodies disclosed herein bind to HER2, CD3, and PD-L1. In some instances, the multispecific antibodies disclosed herein bind to CEA, CD3, and CD137. In some instances, the multispecific antibodies disclosed herein bind to CEA, CD3, and PD-L1. In some instances, the multispecific antibodies disclosed herein bind to BCMA, CD3, and CD137. In some instances, the multispecific antibodies disclosed herein bind to BCMA, CD3, and PD-L1.

[0018] The exemplary multispecific antibodies provided herein, having specific binding sites for CD3, CD137, and TAA, may include:

[0019] (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 23, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 24, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 25, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 26;

[0020] (ii) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 39, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 40, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 41;

[0021] (iii) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 55, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 56, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 57;

[0022] (iv) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 58, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 59, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 60;

[0023] (v) A first polypeptide containing the amino acid sequence of SEQ ID NO: 54, a second polypeptide containing the amino acid sequence of SEQ ID NO: 63, a third polypeptide containing the amino acid sequence of SEQ ID NO: 66, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 69;

[0024] (vi) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 74, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 77, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 80, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 83;

[0025] (vii) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 74, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 63, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 80, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 69;

[0026] (viii) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 54, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 102, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 103, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 104; or

[0027] (ix) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 54, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 63, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 103, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 69.

[0028] The exemplary multispecific antibodies provided herein, having specific binding moieties to CD3, PD-L1, and TAA, may include:

[0029] (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 27, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 25, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 26;

[0030] (ii) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 46, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 47, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 41;

[0031] (iii) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 67, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 68, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 60;

[0032] (iv) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 81, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 82, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 57;

[0033] (v) A first polypeptide containing the amino acid sequence of SEQ ID NO: 105, a second polypeptide containing the amino acid sequence of SEQ ID NO: 106, a third polypeptide containing the amino acid sequence of SEQ ID NO: 66, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 69;

[0034] (vi) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 107, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 108, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 80, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 83;

[0035] (vii) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 107, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 106, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 80, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 69;

[0036] (viii) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 105, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 109, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 103, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 104; or

[0037] (ix) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 105, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 106, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 103, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 69.

[0038] On the other hand, this document provides a nucleic acid or nucleic acid set that collectively encodes any of the multispecific antibodies disclosed herein. In some embodiments, a nucleic acid comprises the coding sequences for all polypeptides of the multispecific antibodies disclosed herein. In other embodiments, the coding sequences for polypeptides are located on two or more nucleic acids that together (“collectively”) encode all polypeptides of the multispecific antibodies disclosed herein. In some cases, the nucleic acid or nucleic acid set may be an expression vector or a set of expression vectors.

[0039] Furthermore, this disclosure features a host cell comprising the nucleic acid or nucleic acid group provided herein, which encodes or collectively encodes a polypeptide chain of a multispecific antibody as disclosed herein. In some embodiments, the host cell may be a mammalian host cell.

[0040] This document also provides a method for generating multispecific antibodies, the method comprising: (i) culturing a host cell containing a nucleic acid encoding the antibody under conditions that allow expression of the multispecific antibody disclosed herein; and (ii) collecting the antibody thereby generated.

[0041] In another aspect, this disclosure provides a pharmaceutical composition comprising any multispecific antibody or nucleic acid or group of nucleic acids encoding such multispecific antibody disclosed herein, and a pharmaceutically acceptable carrier.

[0042] Furthermore, this disclosure features a method for modulating the immune response of a subject, the method comprising administering to a subject in need an effective amount of the multispecific antibody disclosed herein, a nucleic acid encoding the multispecific antibody, or a pharmaceutical composition comprising the antibody or encoding the nucleic acid. In some embodiments, the subject is a human patient who has or is suspected of having cancer.

[0043] Details of one or more embodiments of the invention are set forth in the following description. Other features or advantages of the invention will become apparent from the following drawings and the following detailed description of several embodiments, and also from the appended claims. Attached Figure Description

[0044] The following drawings form part of this specification and are included to further illustrate certain aspects of this disclosure, which can be better understood by referring to the combination of the drawings and detailed descriptions of the specific embodiments presented herein.

[0045] Figure 1A-1B This includes a diagram illustrating a schematic design of an exemplary multispecific antibody provided herein. Figure 1A An exemplary dual immunomodulatory construct contains a central CD3-binding domain and a terminal CD137 or PD-L1-binding domain. Figure 1B Exemplary design of a multispecific antibody having a dual immunomodulatory construct and an additional antigen-binding fragment.

[0046] Figure 2A-2B This includes a diagram illustrating the cytotoxic activity and cytokine secretion of an exemplary multispecific antibody. Figure 2ACytotoxicity and secretion of cytokines (IL2, IL6, IFN-γ and TNF-α) by human PBMCs co-cultured with TA-expressing tumor cells in the presence of Ly2909. Figure 2B Cytotoxicity and secretion of cytokines (IL2, IL6, IFN-γ and TNF-α) by PBMCs co-cultured with TA-expressing tumor cells in the presence of clone Ly2323.

[0047] Figure 3 This is a graph showing the percentage of CD25+ in CD8+ T cells obtained by measuring T cell activation through co-culturing TA-expressing tumor cells with spleen cells from huCD3 / huCD137 KI mice in the absence (control) or presence of the indicated antibodies Ly2909, Ly2915, and Ly2323, as indicated.

[0048] Figure 4 This is a graph showing the average fluorescence intensity of CD25 in CD8+ T cells obtained by measuring T cell activation through co-culturing TA-expressing tumor cells with spleen cells from huCD3 / huCD137 KI mice in the absence (control) or presence of antibodies Ly2949, Ly2951, and Ly2313.

[0049] Figure 5 This figure shows the antitumor activity of antibodies Ly3096, Ly3098, and Ly2314 in a mouse model carrying EL4-huBCMA, which has bone marrow transplanted from human CD3 and CD137 knock-in mice.

[0050] Figure 6 This figure shows the antitumor activity of antibodies Ly2949, Ly2951, and Ly2313 in a mouse model carrying LL2-huBCMA that has bone marrow transplanted from human CD3 and CD137 knock-in mice.

[0051] Figure 7 This figure shows the antitumor activity of antibodies Ly2323, Ly2909, and Ly2915 in a mouse model of human PBMCs carrying LS 174T tumor cells.

[0052] Figure 8 This figure shows the antitumor activity of antibodies Ly2323, Ly2915, and Ly3060 in a mouse model of human PBMCs carrying LS 174T tumor cells.

[0053] Figure 9 This figure shows the antitumor activity of antibodies Ly2313 and Ly2959 in a mouse model of human PBMCs implanted with NCI-H929 tumor cells.

[0054] Figure 10 This figure shows the antitumor activity of antibodies Ly3098, 3106, and Ly2314 in a mouse model of human PBMCs carrying MM.1R tumor cells.

[0055] Figure 11A-11F Includes a graph showing the binding activity of exemplary multispecific antibodies against HER2, CD3, CD137, or PD-L1. Figure 11A : Binding activity of human HER2 protein against antibodies Ly3151 and TM737. Figure 11B : Binding activity of human HER2 protein against antibodies Ly3188 and TM737. Figure 11C : Binding activity of antibodies Ly3151 and Ly305 to human CD3D / E protein. Figure 11D : Binding activity of antibodies Ly3188 and Ly305 to human CD3D / E protein. Figure 11E : Binding activity of human CD137 protein against antibodies Ly3151 and Ly1630. Figure 11F The binding activity of antibodies Ly3188 and Ly076 to human PD-L1 protein was measured. Parental anti-HER2 mAb TM737, anti-CD3 mAb Ly305, anti-CD137 mAb Ly1630, and anti-PD-L1 mAb Ly076 were used as controls.

[0056] Figure 12A-12F Includes a graph illustrating the activation of human CD3 signaling by an exemplary multispecific antibody as indicated. Figures 12A-12C Activational activity of antibodies Ly3151, Ly3152, parental anti-CD137 mAb Ly1630, and parental anti-CD3 mAb Ly305 in Jurkat-NFAT-luminescent reporter assays (A), co-culture with TA-expressing CHO cells (B), or co-culture with CD137-expressing CHO cells (C). Figure 12D-12F The agonistic activity of antibody Ly3188, parental anti-CD3 mAb Ly305, and parental anti-PD-L1 mAb Ly076 in Jurkat-NFAT-luminescent reporter assays alone (D), co-cultured with CHO cells expressing TA (E), or co-cultured with CHO cells expressing PD-L1 (F). The luminescence produced by reporter cells indicates CD3 activation.

[0057] Figures 13A-13CIncludes graphs illustrating the activation of human CD137 signaling by exemplary multispecific antibodies as indicated. In CD137 reporter assays, the agonistic activity of these multispecific antibodies, either alone or co-cultured with additional target-expressing cells, is evaluated. Luminescence produced by reporter cells indicates CD137 activation. Figures 13A-13C Activational activity of the antibody Ly3151 alone, parental anti-CD137 mAb Ly1630, anti-CD3 mAb Ly305, anti-HER2 mAbTM737 and control anti-CD137 mAbTM173 (A), co-cultured with TA-expressing CHO cells (B) or co-cultured with CD3-expressing cells (C).

[0058] Figures 14A-14B Includes a graph illustrating the cytotoxic activity of an exemplary multispecific antibody as indicated. Figure 14A Human PBMCs kill HER2-expressing EBC-1-Luc cells in the presence of antibodies Ly3151 and Ly2935. Figure 14B Human PBMCs killed EBC-1-Luc cells in the presence of antibodies Ly3188 and Ly2935. Relative luminescent units (RLU) represent the number of viable tumor cells remaining after 48 hours of incubation.

[0059] Figure 15 This figure shows the antitumor activity of antibodies Ly3151 and Ly2935 in a mouse model of implanted human PBMCs carrying EBC-1 tumor cells.

[0060] Figure 16 This figure shows the antitumor activity of antibodies Ly3188 and Ly2935 in a mouse model of implanted human PBMCs carrying EBC-1 tumor cells.

[0061] Figure 17A-17G Includes a graph showing the binding activity of exemplary multispecific antibodies against CEA, CD3, or CD137 as indicated. Figure 17A : Binding activity of human CEA protein against antibodies Ly2909 and Ly312. Figure 17B : Binding activity of antibodies Ly2909 and Ly312 on human CEA-expressing LT174T cells. Figure 17C : Binding activity of antibodies Ly2909 and Ly312 on human CEA-expressing CHO cells. Figure 17D : Binding activity of antibodies Ly2909 and Ly305 to human CD3D / E protein. Figure 17E The binding activity of antibodies Ly2909 and Ly305 to Jurkat cells expressing human CD3. Figure 17F: Binding activity of human CD137 protein against antibodies Ly2909 and Ly1630. Figure 17G The binding activity of antibodies Ly2909 and Ly1630 on human CD137-expressing CHO cells was measured. Parental anti-CEA mAb Ly312, anti-CD3 mAb Ly305, and anti-CD137 mAb Ly1630 were used as controls.

[0062] Figures 18A-18D Includes a graph illustrating the activation of human CD3 signaling by an exemplary multispecific antibody as indicated in the Jurkat-NFAT-luminescent reporter assay. Figures 18A-18D The agonistic activity of the antibodies Ly2909, Ly2915, Ly2918, Ly3060 alone and the anti-CD3 mAb Ly305 (A), co-cultured with cells overexpressing CEA (B), co-cultured with cells overexpressing CD137 (C), and co-cultured with cells overexpressing PD-L1 (D). Luminescence produced by reporter cells indicates CD3 activation.

[0063] Figures 19A-19D Includes a graph illustrating the cytotoxic activity of an exemplary multispecific antibody. Figure 19A Killing occurs via PBMCs co-cultured with LS 174T-Luc cells in the presence of antibodies Ly2909 and Ly2323. Figure 19B Killing occurs via PBMCs co-cultured with HT29-Luc cells in the presence of antibodies Ly2909 and Ly2323. Figure 19C Killing occurs via PBMCs co-cultured with HT29-Luc cells in the presence of antibodies Ly3060 and Ly2323. Figure 19D Killing occurs via PBMCs co-cultured with LS 174T-Luc cells in the presence of antibodies Ly3060 and Ly2323.

[0064] Figure 20 This figure shows the antitumor activity of antibodies Ly2909 and Ly2323 in a mouse model of human PBMCs carrying LS 174T tumor cells.

[0065] Figure 21 This figure shows the antitumor activity of antibodies Ly2915, Ly3060, and Ly2323 in a mouse model implanted with human PBMCs carrying LS 174T tumor cells.

[0066] Figure 22A-22R Includes a graph showing the binding activity of exemplary multispecific antibodies against BCMA, CD3, CD137, or PD-L1 as indicated. Figure 22A: Binding activity of human BCMA protein against antibodies Ly2949 and Ly560. Figure 22B : Binding activity of human BCMA protein against antibodies Ly2959 and Ly560. Figure 22C : Binding activity of human BCMA protein against antibodies Ly3096 and Ly560. Figure 22D : Binding activity of human BCMA protein against antibodies Ly3106 and Ly560. Figure 22E : Binding activity of antibodies Ly2949 and Ly560 expressed in human BCMA-containing NCI-H929 cells. Figure 22F : Binding activity of antibodies Ly3096 and Ly3019 expressed in human BCMA-containing NCI-H929 cells. Figure 22G : Binding activity of antibodies Ly2949 and Ly560 on HEK293 cells overexpressing human BCMA. Figure 22H : Binding activity of antibodies Ly3096 and Ly3019 on HEK293 cells overexpressing human BCMA. Figure 22I : Binding activity of human CD3D / E protein against antibodies Ly2949 and Ly305. Figure 22J : Binding activity of antibodies Ly2959 and Ly305 to human CD3D / E protein. Figure 22K : Binding activity of antibodies Ly3096 and Ly305 to human CD3D / E protein. Figure 22L : Binding activity of antibodies Ly3106 and Ly305 to human CD3D / E protein. Figure 22M : Binding activity of antibodies Ly2949 and Ly305 on CD3-expressing Jurkat cells. Figure 22N : Binding activity of antibodies Ly3096 and Ly305 on CD3-expressing Jurkat cells. Figure 22O : Binding activity of human CD137 protein against antibodies Ly2949 and Ly1630. Figure 22P : Binding activity of human CD137 protein against antibodies Ly3096 and Ly1630. Figure 22Q : Binding activity of human PD-L1 protein against antibodies Ly2959 and Ly076. Figure 22R The binding activity of antibodies Ly3106 and Ly076 to human PD-L1 protein was measured. Parental anti-BCMA mAb Ly560, anti-CD3 mAb Ly305, anti-PD-L1 mAb Ly076, and anti-CD137 mAb Ly1630 were used as controls.

[0067] Figures 23A-23H Includes a graph illustrating the activation of human CD3 signaling by an exemplary multispecific antibody as indicated in the Jurkat-NFAT-luminescent reporter assay. Figures 23A-23D The agonistic activity of the individual antibodies Ly2949, Ly2951, Ly2952, Ly2959, anti-PD-L1 mAb Ly076, anti-BCMA mAb Ly560, anti-CD137 mAb Ly1630 and anti-CD3 mAb Ly305 (A), co-cultured with NCI-H929 cells expressing BCMA (B), co-cultured with CHO cells overexpressing CD137 (C), and co-cultured with CHO cells overexpressing PD-L1 (D). Figure 23E-23G The agonistic activity of the antibodies Ly3096, Ly3098, Ly3099, Ly3106, anti-CD137 mAb Ly1630, and anti-CD3 mAb Ly305 (E), co-cultured with NCI-H929 cells (F), co-cultured with CHO cells overexpressing CD137 (G), and co-cultured with CHO cells overexpressing PD-L1 (H) was evaluated. Luminescence generated by reporter cells indicated CD3 activation.

[0068] Figures 24A-24F Includes a graph illustrating the activation of human CD137 signaling by an exemplary multispecific antibody as indicated in a CD137 reporter gene assay. Figures 24A-24C Activational activity of the antibodies Ly2949, Ly2951, Ly2952, anti-CD137 mAb Ly1630, and anti-CD137 mAb TM173 (A), co-cultured with NCI-H929 cells (B), and co-cultured with Jurkat cells (C). Figure 24D-24F Activatory activity of the antibodies Ly3096, Ly3098, Ly3099, Ly1630, and TM173 alone (D), co-cultured with NCI-H929 cells (E), and co-cultured with Jurkat cells (F). Luminescent induction of CD137 activation by reporter cells.

[0069] Figure 25 This figure shows the antitumor activity of antibodies Ly2949 and Ly2323 in a mouse model of human PBMCs implanted with NCI-H929 tumor cells.

[0070] Figure 26 This figure shows the antitumor activity of antibodies Ly2949, Ly2951, and Ly2313 in a mouse model carrying LL2-huBCMA that has bone marrow transplanted from human CD3 and CD137 knock-in mice.

[0071] Figure 27This figure shows the antitumor activity of antibodies Ly3096, Ly3098, and Ly2314 in a mouse model carrying EL4-huBCMA, which has bone marrow transplanted from human CD3 and CD137 knock-in mice.

[0072] Figure 28 This figure shows the antitumor activity of antibodies Ly2949, Ly2959, and Ly2313 in a mouse model implanted with human PBMCs carrying NCI-H929 tumor cells.

[0073] Figure 29 This figure shows the antitumor activity of antibodies Ly3098, Ly3106, and Ly2314 in a mouse model of human PBMCs carrying MM.1R tumor cells.

[0074] Figures 30A-30F The diagram includes illustrations of CD3 activation in the Jurkat-NFAT-luminescent reporter assay for individual anti-CD3 mAb (A), parental CHO cells (B), CHO cells expressing FCGRI (C), CHO cells expressing FCGRIIA (D), CHO cells expressing FCGRIIB (E), and CHO cells expressing FCGRIIA (F). The antibodies tested were anti-CD3 mAbs carrying different Fc variants as described herein: Ly2863: 237 deletion; Ly2864: P329G; Ly2865: 237 deletion + P329G; Ly2873: L234A + L235A + 237 deletion; Ly1761: IgG1 wt.

[0075] Figure 31 This diagram illustrates CD3 activation in the Jurkat-NFAT luminescent reporter assay using an exemplary antibody alone (A), parental CHO cells (B), CHO cells expressing FCGRI (C), CHO cells expressing FCGRIIA (D), CHO cells expressing FCGRIIB (E), and CHO cells expressing FCGRIIA (F). The antibodies tested were multispecific antibodies carrying different Fc variants as described herein: Ly2600: 237 deletion + P329G; Ly1963: L234A + L235A + 237 deletion. Anti-CD3 mAb Ly305 was used as a control.

[0076] Figure 32This diagram illustrates CD3 activation in the Jurkat-NFAT luminescent reporter assay using an exemplary antibody alone (A), parental CHO cells (B), CHO cells expressing FCGRI (C), CHO cells expressing FCGRIIA (D), CHO cells expressing FCGRIIB (E), and CHO cells expressing FCGRIIA (F). The antibodies tested were multispecific antibodies carrying different Fc variants as described herein: Ly2601: 237 deletion + P329G; Ly1967: L234A + L235A + 237 deletion. Anti-CD3 mAb Ly305 was used as a control. Detailed Implementation

[0077] Bispecific antibodies (bsAbs) targeting tumor-associated antigens (TAAs) and CD3 can induce T-cell activation in the tumor microenvironment (TME), thereby generating immune protection against tumors. However, CD3 activation alone has limitations in cancer therapy. First, pan-T-cell activation via CD3 can stimulate high levels of cytokine release, which may lead to cytokine release syndrome, a serious side effect of T-cell conjugate therapy. Second, CD3 stimulation alone can promote T-cell unresponsiveness or activation-induced T-cell death, thus limiting the efficacy or durability of the anti-tumor response. Anti-TAA / CD3bsAbs have achieved some success in treating hematologic malignancies. However, a large proportion of patients do not respond to treatment, or even if they initially respond, eventually relapse.

[0078] This disclosure provides a multispecific antibody comprising a binding moiety targeting two immune receptors, CD3 / CD137 or CD3 / PD-L1, and a binding moiety targeting a TAA. CD137 activation is expected to induce secondary signaling to promote the survival of activated T cells, which may be triggered by CD3 activation. Compared to corresponding CD3 bsAbs, the exemplary multispecific antibody provided herein, containing a monovalent agonist anti-CD137 moiety, exhibits stronger in vivo antitumor efficacy. In vitro, dual CD3 / CD137 activation also increases CD8 T cell activation, which plays an important role in tumor prevention. Enhanced CD8 T cell activation is accompanied by reduced cytokine release, indicating a larger therapeutic window. Furthermore, as demonstrated by the experimental data provided herein, combining the CD3 binding moiety with the anti-PD-L1 moiety is expected to enhance antitumor efficacy.

[0079] Furthermore, the multispecific antibodies presented herein may contain an Fc fragment to link a binding portion therein, for example, linking the binding portion to CD3 and linking the binding portion to CD137 or PD-L1. The Fc fragment may contain one or more mutations relative to the wild-type parent, for example, a deletion at position 237 and an amino acid substitution at position 329 following the EU numbering system (e.g., Fc variant 237Δ / P329G). As shown herein, such Fc variants do not bind to the Fc receptor or induce Fc receptor-mediated crosslinking effects, thereby reducing the risk of inducing a systemic immune response or Fc effector function, which could potentially lead to undesirable side effects.

[0080] Therefore, this article provides multispecific antibodies that target tumor antigens and immune receptors (e.g., CD3 / CD137 or CD3 / PD-L1) and optionally involve Fc variants that bind little or no to the Fc receptor, nucleic acids encoding such multispecific antibodies, and host cells containing the encoding nucleic acids, which can be used to generate multispecific antibodies. This article also provides information on the use of multispecific antibodies in cancer therapy.

[0081] I. Multispecific antibodies

[0082] The multispecific antibodies disclosed herein comprise antigen-binding moieties specific to two immune receptors (e.g., CD3 / CD137 or CD3 / PD-L1) and one or more antigen-binding moieties targeting tumor-associated antigens. Each of the receptor-specific antigen-binding moieties is in the form of an Fv, which contains a heavy chain variable domain (V... H ) and light chain variable structural domain (V L Two antigen-binding moieties specific to the immune receptor can be linked via an Fc fragment, which may contain a hinge domain and a CH2 domain. In some cases, the Fc fragment contains a 237Δ deletion following the EU numbering system and an amino acid substitution at position P329 (e.g., P329G). The antigen-binding moieties specific to the TAA can be in any suitable form, such as single-chain variable fragments (scFv) or Fab. In some cases, the antigen-binding moieties are linked to, for example, a CD3-specific antigen-binding moieties (anti-CD3 moieties) via peptide linkers. In some cases, the multispecific antibodies presented herein are multivalent. An exemplary illustration of multispecific antibodies is provided in Figure 1.

[0083] As used herein, a multispecific antibody is an antibody capable of binding to two or more target antigens. In some instances, the multispecific antibodies disclosed herein may be bispecific antibodies, i.e., binding to two different target antigens or two different epitopes of a target antigen. In some instances, the multispecific antibodies disclosed herein may be trispecific antibodies, for example, binding to three different target antigens or epitopes. Alternatively, the multispecific antibodies disclosed herein may be tetraspecific antibodies, for example, binding to four different target antigens or epitopes.

[0084] As used herein, a multivalent antibody is an antibody having two or more antigen-binding sites. In some instances, the multispecific antibodies disclosed herein may have two antigen-binding sites. In some instances, the multispecific antibodies disclosed herein may have three antigen-binding sites. In some instances, the multispecific antibodies disclosed herein may have four antigen-binding sites.

[0085] A. antigen-binding portion

[0086] Each of the following fragments is in Fv form: an antigen-binding moiety specific to CD3 (anti-CD3 moiety), an antigen-binding moiety specific to CD137 (anti-CD137 moiety), or an antigen-binding moiety specific to PD-L1 (anti-PD-L1 moiety). The Fv fragment contains V... H Chain and V L These are monovalent antibody fragments, which are individual polypeptide chains. In some embodiments, the multispecific antibodies provided herein comprise an anti-CD3 moiety and an anti-CD137 moiety. In other embodiments, the multispecific antibodies provided herein comprise an anti-CD3 moiety and an anti-PD-L1 moiety.

[0087] Each of the antigen-binding portions (anti-TAA portions) that are specific to TAA can be in any suitable form, including but not limited to full-length antibodies, their antigen-binding fragments (such as Fab, Fab', F(ab')2, Fv, trifunctional antibodies, triFab, tandemly linked Fab, Fab-Fv, tandemly linked V domains, tandemly linked scFv and other forms), single-chain antibodies (scFv antibodies), single-domain antibodies (such as VHH), cross-Fab, and tetravalent antibodies. In some instances, one or more anti-TAA portions of the multispecific antibodies disclosed herein may be in Fab form. In other instances, one or more anti-TAA portions of the multispecific antibodies disclosed herein may be in cross-Fab form. Alternatively or additionally, one or more anti-TAA portions of the multispecific antibodies disclosed herein may be in scFv form.

[0088] Any scFv fragment in a multispecific antibody can be in V H V L Orientation. Alternatively, it can be in V. L V H Orientation. Single-domain antibodies (sdAbs), also known as nanobodies, are antibody fragments composed of a single monomeric variable antibody domain. In some embodiments, single-domain antibodies can be heavy-chain-only (VHH) fragments, which may be derived from camel antibodies.

[0089] Fab fragments typically contain two separate chains: one chain contains a heavy chain variable region (VH) connected to a heavy chain constant region segment (such as CH1), and the other chain contains a light chain variable region (VL) connected to a light chain constant region segment (such as Cκ or Cλ). Cross-Fab fragments have a similar bi-chain structure to Fab fragments, but the connections between the VH / VL and the heavy / light chain constant region segments are interchanged. A cross-Fab fragment comprises 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 segment (such as CH1).

[0090] Tumor-associated antigens (TAAs) are antigens produced by tumor cells. TAAs are tumor markers used to identify tumor cells and therapeutic targets for 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. In some cases, multispecific antibodies contain one or more anti-TAA moieties capable of binding to B7H3, CD19, CD20, PSMA, HER2, CEA, BCMA, P53mut, DLL3, MET, and EGFR. In specific instances, the TAA is HER2, CEA, or BCMA.

[0091] In some instances, the multispecific antibodies provided herein comprise one anti-TAA moiety, for example, binding to B7H3, CD19, CD20, PSMA, HER2, CEA, BCMA, P53mut, DLL3, MET, and EGFR (e.g., HER2, CEA, or BCMA). In other instances, the multispecific antibodies provided herein comprise two anti-TAA moiety, for example, binding to B7H3, CD19, CD20, PSMA, HER2, CEA, BCMA, P53mut, DLL3, MET, and EGFR (e.g., HER2, CEA, or BCMA). The two anti-TAA moiety may bind to two different TAAs. Alternatively, the two anti-TAA moiety may bind to one TAA, for example, to different epitopes of the TAA. In other instances, the multispecific antibodies provided herein comprise two identical anti-TAA moiety.

[0092] Antibodies that “specifically bind” to an antigen or epitope are a well-known term in the art. A molecule is said to exhibit “specific binding” if it reacts with a particular target antigen more frequently, more rapidly, for a longer duration, and / or with greater affinity than it does with alternative targets. An antibody is said to “specifically bind” to a target antigen or epitope if it binds with greater affinity, higher affinity, more readily, and / or for a longer duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to an antigen (e.g., those listed above) or an antigenic epitope therein is an antibody that binds to that target antigen with greater affinity, higher affinity, more readily, and / or for a longer duration than it binds to other antigens or other epitopes of the same antigen. Using this definition, it should also be understood that, for example, an antibody that specifically binds to a first target antigen may or may not specifically bind to or preferentially bind to a second or third target antigen. Thus, “specific binding” or “preferential binding” does not necessarily require (although it may include) exclusive binding. In some instances, antibodies that “specifically bind” to a target antigen or its epitope may not bind to other antigens or other epitopes of the same antigen (i.e., baseline binding activity can only be detected in conventional methods). Alternatively or additionally, the antibodies described herein may specifically bind to human antigens or fragments thereof, or vice versa, relative to their monkey counterparts (e.g., binding affinity for one antigen is at least 10 times greater than binding affinity for another antigen, 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., monkey antigens), for example, with a difference in binding affinity for human and non-human antigens of less than 5-fold, such as less than 2-fold, or substantially similar.

[0093] In some embodiments, the antigen-binding portion of any of the multispecific antibodies described herein has a suitable binding affinity for a target antigen (e.g., an immune cell receptor such as CD3 / CD137 or CD3 / PD-L1, or TAA, as disclosed herein) or its antigenic epitope. As used herein, “binding affinity” refers to the apparent association constant or K0. A K A The dissociation constant (K) D The reciprocal of ). The binding affinity (K) of the antibody described in this article to the target antigen or antigenic epitope. D ) can be at least 10 -5 10 -6 10 -7 10 -8 10 -9 10 -10 M or lower. Increased binding affinity corresponds to decreased K. D The higher affinity of an antibody for the first antigen compared to the second antigen can be achieved through a higher affinity for the first antigen compared to the binding of the second antigen to the K+ antigen. A (or numerical value K) D Higher K binding to the first antigen A (or a smaller value K) D This is indicated by [the specific antigen]. In such cases, the antibody is specific to the first antigen (e.g., the same first protein or its analogue in the first conformation) relative to the second antigen (e.g., the first protein in the second conformation; or the second protein). Differences in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, 37.5 times, 50 times, 70 times, 80 times, 91 times, 100 times, 500 times, 1000 times, 10,000 times, or 10 [folds]. 5 In some embodiments, either antibody may undergo further affinity maturation to increase its binding affinity to the target antigen or its epitope.

[0094] Binding affinity (or binding specificity) can be determined using various methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using fluorescence assays). An exemplary condition for assessing binding affinity is in 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 the bound protein as a function of the target protein concentration. The concentration of the bound protein ([bound]) is typically correlated with the concentration of the free target protein ([free]) by the following equation:

[0095] [Binded] = [Free] / (Kd + [Free])

[0096] However, it is not always necessary to target K. A Precise determination is necessary because sometimes, for example, activity in a functional assay (e.g., in vitro or in vivo assay) is sufficient to obtain a quantitative measurement of affinity, a qualitative measurement of affinity, or an inference of affinity, which is determined, for example, using methods such as ELISA or FACS analysis, and is related to K. A Proportional and therefore can be used for comparisons, such as determining whether a higher affinity is, for example, twice as high.

[0097] Exemplary parental antibodies

[0098] The antigen-binding portion of the multispecific antibodies disclosed herein may be derived from a parent antibody that is specific to any of the immune cell receptors CD3, CD137, and / or PD-L1 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 protocol are identified in bold).

[0099] For example, the anti-CD3 portion can be derived from the parental anti-CD3 clone Ly305. The anti-CD137 portion can be derived from the parental anti-CD137 clone Ly1630. Alternatively, the anti-CD137 portion can be derived from the parental anti-CD137 clone TM173. The anti-PD-L1 portion can be derived from the parental anti-PD-L1 clone Ly076. Alternatively, the anti-PD-L1 portion can be derived from the parental anti-PD-L1 clone Ly2530.

[0100] Alternatively or additionally, the multispecific antibodies provided herein may comprise an anti-HER2 moiety derived from the anti-HER2 parental clone Ly591 or TM737. In some cases, the multispecific antibody may comprise a first anti-HER2 moiety derived from Ly591 and a second anti-HER2 moiety derived from TM737.

[0101] In some cases, the multispecific antibodies presented herein may contain an anti-CEA portion derived from the anti-CEA parent clone Ly312.

[0102] In some cases, the multispecific antibodies described herein may contain an anti-BCMA moiety derived from the anti-BCMA parental clones Ly560 or Ly3019. In some cases, the multispecific antibodies may contain a primary anti-BCMA moiety derived from Ly560 and a secondary anti-BCMA moiety derived from Ly3019.

[0103] Exemplary antigen-binding portion

[0104] The antigen-binding portion of any of the multispecific antibodies disclosed herein may be derived from any of the corresponding parent antibodies (e.g., those provided in Table 1 below).

[0105] As used herein, "antigen-binding moiety in a multispecific antibody derived from" the parent antibody means using the parent antibody as the starting material for preparing one of the antigen-binding moieties in a multispecific antibody. The antigen-binding moiety may contain the same heavy and / or light chain CDRs as those in the parent antibody. Both antibodies have the same V0. H and / or V L A CDR means that when determined by the same method (e.g., the Kabat definition, Chothia definition, AbM definition, and / or contact definition known in the art), its CDR is the same.

[0106] In some cases, the antigen-binding moiety derived from the parent antibody can be a functional variant of the parent antibody. Such functional variants are substantially similar to the reference antibody in both structure and function. The functional variant contains a V that is substantially the same as the reference antibody. H and V L CDRs. For example, a CDR region of the reference antibody may contain only up to 5 (e.g., 4, 3, 2, or 1) amino acid residue variations in the total heavy chain CDR region, and / or only up to 5 (e.g., 4, 3, 2, or 1) amino acid residue variations in the total light chain CDR region. In some instances, relative to those of the reference antibody, functional variants may contain up to 8 (e.g., 7, 6, 5, 4, 3, 2, or 1) amino acid residue variations in both the total heavy chain and light chain CDRs. Such functional variants may have substantially similar affinities (e.g., K with the same order). D (Value) binds to the same epitope of the antigen targeted by the parent antibody. Alternatively or otherwise, amino acid residue variations are made to substitute conserved amino acid residues as disclosed herein.

[0107] In some embodiments, the V of the corresponding parental antibody H Compared to CDRs, the antigen-binding portion of multispecific antibodies disclosed herein may contain heavy chain CDRs, individually or collectively having at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity. Alternatively or additionally, with V as a parent antibody... L Compared to CDRs, the antigen-binding portion may contain light chain CDRs that individually or collectively have at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity.

[0108] In other embodiments, V with the corresponding parental antibody HCompared to a CDR, the antigen-binding portion may contain a heavy chain CDR, either alone or collectively, having at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity. Alternatively or additionally, it may be associated with V as a parent antibody. L Compared to CDRs, the antigen-binding portion may contain light chain CDRs that individually or collectively have at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity.

[0109] The "percentage of identity" between the two amino acid sequences was determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, as modified in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. This algorithm was 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 using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecule of this invention. In cases where there is a gap between two sequences, a gapped BLAST, as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997, can be used. When using the BLAST procedure and the gapped BLAST procedure, the default parameters of the respective procedures (e.g., XBLAST and NBLAST) can be used.

[0110] Alternatively or additionally, amino acid residue variations can be conserved amino acid residue substitutions. As used herein, “conserved amino acid substitution” means an amino acid substitution that does not alter the relative charge or size properties of the protein to which the substitution is made. Variants can be prepared according to methods for altering polypeptide sequences known to those skilled in the art, such as those found in references compiling such methods, for example, *Molecular Cloning: A Laboratory Manual*, J. Sambrook et al., eds., 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or *Current Protocols in Molecular Biology*, FM Ausubel et al., eds., John Wiley & Sons, Inc., New York. Conserved substitution of amino acids includes substitutions between amino acids within the following group: (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.

[0111] B. Forms of multispecific antibodies

[0112] The multispecific antibodies disclosed herein can comprise a dual immunomodulatory construct consisting of two antigen-binding moieties specific to two immune receptors, CD3 / CD137 or CD3 / PD-L1, and one or more anti-TAA moieties (such as Fab) that can be in a suitable form. See, for example Figure 1A and 1B .

[0113] (a) Dual immune modulation construct

[0114] In a dual immunomodulatory construct, both the anti-CD3 moiety and the anti-CD137 or anti-PD-L1 moiety are in Fv form, and the two antigen-binding moieties are linked via an Fc fragment (e.g., an Fc variant, such as those provided herein). Figure 1A An example demonstration is provided in the document.

[0115] Fv joint part

[0116] The anti-CD3 Fv fragment disclosed in this article contains V located on a separate chain. H and V L Similarly, the anti-CD137 or anti-PD-L1 Fv fragments disclosed herein contain V fragments on separate strands. H and V LIn some cases, V anti-CD3 Fv H It can be linked to the V-band of anti-CD137 or anti-PD-L1 Fv via the first Fc fragment. H And V that resists CD3 Fv L V is linked to anti-CD137 or anti-PD-L1 via the second Fc fragment. L In other cases, V resists CD3 Fv. H It can be linked to the V-band of anti-CD137 or anti-PD-L1 Fv via the first Fc fragment. L And V that resists CD3 Fv L V is linked to anti-CD137 or anti-PD-L1 via the second Fc fragment. H .

[0117] The anti-CD3 Fv fragment, anti-CD137 Fv fragment, and anti-PD-L1 Fv fragment can be derived from any of the corresponding parental antibodies provided in Table 1 below. See also the disclosure above.

[0118] In some instances, the first and second Fc fragments are identical. In other instances, the first and second Fc fragments are different (e.g., containing matching mutations to, for example, enhance heterodimerization compared to homodimerization and / or reduce protein A binding).

[0119] Fc fragment

[0120] The Fc fragment used to link the anti-CD3 and anti-CD137 or anti-PD-L1 portions may contain a hinge domain and a CH2 domain, and optionally a CH3 domain derived from a suitable immunoglobulin (Ig) molecule (e.g., an IgG molecule). In some cases, the Fc fragment is derived from an IgG1 molecule. In some instances, the Fc fragment is a fragment of a wild-type Ig molecule (e.g., IgG, such as IgG1). Alternatively, the Fc fragment may contain more or more mutations relative to its wild-type counterpart.

[0121] In some embodiments, the Fc fragment is an Fc variant containing one or more mutations relative to the wild-type counterpart. Such mutations can modulate binding affinity and selectivity to the Fc receptor. In some cases, the Fc fragment may contain amino acid substitutions at one or more of positions 267, 273, 328, and 329. In some cases, one or more mutations may be amino acid substitutions at one or more of positions 239, 265, 297, 329, 330, and 332. In one instance, a heavy chain constant region fragment (such as an Fc fragment) may contain (i) a deletion at position 237 and (ii) two amino acid substitutions, namely an amino acid substitution at position 234 (e.g., L234A) and an amino acid substitution at position 235 (e.g., L235A). In another instance, the heavy chain constant region segment may contain 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 of D265A and N297A, (iv) amino acid substitutions of S239D, A330L and I332E, and (v) a deletion at position 237 and amino acid substitution of P329G. In some instances, such as the Fc fragment disclosed herein, the Fc fragment may contain (i) an amino acid substitution at position 329, optionally P329G, (ii) amino acid substitutions at positions 265 and 297, optionally D265A and N297A, (iii) amino acid substitutions at positions 239, 330 and 332, optionally S239D, A330L and I332E, or a combination of any of (i)-(iii).

[0122] In one instance, the Fc variant may have a deletion at position 237 (237D) and an amino acid residue substitution at position P329 (e.g., P329G). Such Fc variants may comprise the amino acid sequence of SEQ ID NO: 91. Such Fc variants, their encoding nucleic acids, and antibodies containing them are also within the scope of this disclosure.

[0123] Additional mutations in the Fc fragment used to regulate Fc receptor binding activity can be found, for example, in US-2020-0392227, the relevant disclosure of which is incorporated herein by reference for the purposes and purposes of this document.

[0124] Alternatively or additionally, the Fc fragments used to construct the multispecific antibodies presented herein 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)), electrostatics (Gunasekaran et al., Journal of Biological Chemistry, 285 (25), pp. 19637–19646 (2010)), or negative design (Kreudenstein et al., mAbs, 5 (5), pp. 646–654 (2013); Leaver-Fay et al., Structure, 24 (4), pp. 641–651 (2016)) (charged mutations). Other examples can be found, for instance, in Brinkmann et al., MABS (2017), 9(2):182-212, and related publications are incorporated herein by reference for the purposes and purposes of this article.

[0125] In some instances, mutations may be located at positions 366 (e.g., T366W or T366S), 368 (e.g., L368A), and / or 407 (e.g., G407V). In specific instances, a heavy chain constant region fragment in a multispecific antibody may contain a mutation at position 366 (e.g., T366W) and a mutation at position 407 (e.g., G407V), and a second heavy chain constant region fragment in the same multispecific antibody may contain a mutation at position 366 (e.g., T366S), a mutation at position 368 (e.g., L368A), and a mutation at position 407 (G407V). Unless explicitly stated otherwise, all numbers relating to positions in Ig molecules follow the EU numbering system.

[0126] In some cases, mutations that reduce the binding affinity for protein A can be introduced into one or both of the heavy chain Fc regions of multispecific antibodies to facilitate the purification of the multispecific antibody. Such mutations are known in the art. See, for example, Tustian et al., mAbs 8:828-838 (2016), the relevant disclosure of which is incorporated herein by reference for the purposes and subject matter of this document.

[0127] Table 1 below provides exemplary CH3 domain sequences that include club-shaped mutations, mortar-shaped mutations, and mutations affecting protein A binding.

[0128] The dual immunomodulatory constructs used to prepare the multispecific antibodies provided herein comprise an anti-CD3 Fv fragment and an anti-CD137 or anti-PD-L1 Fv fragment linked via an Fc fragment (such as the Fc variants disclosed herein, e.g., an Fc variant with the 237Δ / P329G mutation). In some cases, the dual immunomodulatory constructs contain two peptides. The first peptide comprises an anti-CD3 Fv VH, a first Fc fragment as disclosed herein, and an anti-CD137 or anti-PD-L1 VH, and the second peptide comprises an anti-CD3 Fv VL, a second Fc fragment as disclosed herein, and an anti-CD137 or anti-PD-L1 VL. Alternatively, the first peptide comprises an anti-CD3 Fv VH, a first Fc fragment as disclosed herein, and an anti-CD137 or anti-PD-L1 VL, and the second peptide comprises an anti-CD3 Fv VL, a second Fc fragment as disclosed herein, and an anti-CD137 or anti-PD-L1 VH. The first and second Fc fragments can be identical. Alternatively, they can be different (e.g., containing matching club / mortise mutations as also disclosed herein). In some cases, peptide linkers (e.g., those listed in Table 1 below) can be inserted between the antibody fragment and the Fc fragment.

[0129] In some specific examples, the dual immunomodulatory construct targeting CD3 and CD137 used in the multispecific antibodies disclosed herein may comprise a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 3 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 4. In other specific examples, the dual immunomodulatory construct targeting CD3 and PD-L1 used in the multispecific antibodies disclosed herein may comprise a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 5 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 6.

[0130] (b) Anti-TAA component

[0131] One or more anti-TAA moieties in a multispecific antibody can be in any suitable form, see, for example, those disclosed herein. In some instances, the anti-TAA moieties are in Fab form.

[0132] The anti-TAA moiety can be linked to the anti-CD3 Fv moiety in a multispecific antibody via, for example, a peptide linker. In some instances, the multispecific antibody may contain an anti-TAA moiety that can be linked to either the VH chain or the VL chain of the anti-CD3 Fv antibody. In some cases, the anti-TAA moiety is in Fab form, comprising a VH-CH1 chain and a VL-CL chain. In some cases, the VH-CH1 chain can be linked to the VH chain of the anti-CD3 Fv antibody, and the VL-CL chain can be paired with the VH-CH1 chain via one or more disulfide bonds. Alternatively, the VL-CL chain can be linked to the VH chain of the anti-CD3 Fv antibody, and the VH-CL chain can be paired with the VL-CL chain via one or more disulfide bonds. In other cases, the VH-CH1 chain can be linked to the VL chain of the anti-CD3 Fv antibody, and the VL-CL chain can be paired with the VH-CH1 chain via one or more disulfide bonds. Alternatively, the VL-CL chain can be linked to the CD3 Fv-resistant VL chain, and the VH-CL chain can be paired with the VL-CL chain via one or more disulfide bonds.

[0133] In some embodiments, the multispecific antibody provided herein may comprise, for example, two anti-TAA moieties in the form of Fab, each of which may be linked to the VH and VL chains of the anti-CD3 Fv moieties in the multispecific antibody.

[0134] In some instances, the anti-TAA portion of a multispecific antibody may be derived from the parental antibodies provided in Table 1 below. See also the above disclosures.

[0135] (c) Peptide linkers

[0136] The antigen-binding portion or fragment thereof in the multispecific antibodies disclosed herein can be linked via a peptide linker. The peptide linker can be located between two fragments in the polypeptide of the multispecific antibodies disclosed herein, for example, the V fragment in the scFv segment. H With V L Between parts, V of Fv segment H or V L Between chains that bind to another antigen, or between the VH or VL of the Fv fragment and the Fc fragment.

[0137] Any of the peptide linkers described herein may comprise naturally occurring amino acids and / or non-naturally occurring amino acids. Non-naturally occurring amino acids may include protected amino acids, such as naturally occurring amino acids protected with groups such as acetyl, formyl, toluenesulfonyl, nitro, etc. Non-limiting examples of non-naturally occurring amino acids include azido-homalanine, homopropynylglycine, homoallylglycine, p-bromophenylalanine, p-iodophenylalanine, azidophenylalanine, acetylphenylalanine, or ethynylphenylalanine, amino acids containing an internal olefin (such as trans-crotonyl olefin), serine allyl ether, allylglycine, propynylglycine, vinylglycine, pyrrolidone, N-σ-o-azidobenzyloxycarbonyl-L-lysine (AzZLys), and N-σ-propynyloxycarbonyl. N-σ-2-azidoethoxycarbonyl-L-lysine, N-σ-tert-butoxycarbonyl-L-lysine (BocLys), N-σ-allyloxycarbonyl-L-lysine (AlocLys), N-σ-acetyl-L-lysine (AcLys), N-σ-benzyloxycarbonyl-L-lysine (ZLys), N-σ-cyclopentyloxycarbonyl-L-lysine (CycLys), N-σ-D-prolyl-L-lysine, N-σ-nicotinyl-L-lysine Lysine (NicLys), N-σ-N-Me-o-aminobenzoyl-L-lysine (NmaLys), N-σ-biotin-L-lysine, N-σ-9-fluorenylmethoxycarbonyl-L-lysine, N-σ-methyl-L-lysine, N-σ-dimethyl-L-lysine, N-σ-polymethyl-L-lysine, N-σ-isopropyl-L-lysine, N-σ-dansyl-L-lysine, N-σ-o,p-dinitrophenyl-L-lysine, N-σ-p-toluenesulfonyl- L-lysine, N-σ-DL-2-amino-2-carboxyethyl-L-lysine, N-σ-phenylacetoneamide-L-lysine, N-σ-acetoneamide-L-lysine, azido-homalanine, homopropylglycine, homoallylglycine, p-bromophenylalanine, p-iodophenylalanine, azidophenylalanine, acetylphenylalanine or ethynylphenylalanine, amino acids containing internal alkenes such as trans-crotyl alkenes, serine allyl ethers, allylglycine, propargylglycine and vinylglycine.

[0138] The peptide linkers provided in this article may contain approximately 5-160 amino acid residues, for example, approximately 10-120 amino acid residues, approximately 10-100 amino acid residues, approximately 10-80 amino acid residues, approximately 10-60 amino acid residues, approximately 10-50 amino acid residues, approximately 10-40 amino acid residues, approximately 10-30 amino acid residues, or approximately 10-20 amino acid residues.

[0139] In some embodiments, the peptide linker may be a flexible peptide linker, which typically contains small, flexible amino acid residues to connect various domains in a multispecific antibody without affecting its binding activity. In some instances, the flexible peptide linker is a Gly-rich linker, for example, comprising 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 peptide linkers are provided in Table 1 below, any of which can be used to construct the multispecific antibodies disclosed herein.

[0140] In some embodiments, the peptide linker may be a so-called rigid peptide linker containing at least one cysteine ​​residue (e.g., one or two cysteine ​​residues), such that it may 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 the dimerization of multiple polypeptides across the antibody via disulfide formation, thereby forming a complete multichain antibody molecule.

[0141] In some cases, utilizing the disulfide bond-forming ability of such fragments, rigid peptide linkers can originate from the hinge domains (positions 216-230 or fragments thereof, following the EU numbering system) of IgG molecules (e.g., IgG1 molecules). In some instances, such peptide linkers are fragments of wild-type IgG molecules (e.g., human IgG1 molecules). Alternatively, rigid peptide linkers can contain one or more mutations relative to their wild-type counterparts.

[0142] In some instances, rigid peptide linkers may contain only a hinge domain of an IgG molecule or a fragment thereof. In other instances, rigid peptide linkers may contain a hinge domain or a fragment thereof, as well as a Gly-rich fragment (e.g., the Gly-rich fragment disclosed herein), which may be attached to the N-terminus and / or C-terminus of the hinge domain or the fragment thereof. Examples of rigid peptide linkers for any of the multispecific antibodies disclosed herein are provided in Table 1 below. See also International Application No. PCT / US2023 / 061051, the disclosure of which is incorporated herein by reference for the purposes and purposes of this application.

[0143] C. Exemplary multispecific antibodies

[0144] In some instances, the multispecific antibodies disclosed herein comprise dual immunomodulatory constructs targeting CD3 and CD137, or CD3 and PD-L1. Such immunomodulatory constructs can have… Figure 1AThe structures shown are as follows. An exemplary anti-CD3 / CD137 dual immunomodulatory construct may comprise a first polypeptide and a second polypeptide, the first polypeptide comprising the amino acid sequence of SEQ ID NO: 3 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 4. An exemplary anti-CD3 / PD-L1 construct may comprise a first polypeptide and a second polypeptide, the first polypeptide comprising the amino acid sequence of SEQ ID NO: 5 and the second polypeptide comprising the amino acid sequence of SEQ ID NO: 6.

[0145] Dual immunomodulatory constructs can be linked to one or more anti-TAA moieties to generate the multispecific antibodies disclosed herein. See, for example... Figure 1B The structure is shown. In some cases, the anti-TAA moiety is a Fab fragment containing both a VH-CH1 chain and a VL-CL chain. The anti-TAA moiety can be linked to the anti-CD3 moiety. For example, the VH-CH1 chain of the anti-TAA Fab can be linked to the VH or VL of the anti-CD3 Fv fragment in a dual immunomodulatory construct, and the VL-CL chain can pair with the VH-CH1 fragment to form the TAA-binding moiety. A peptide linker can be inserted between the anti-TAA chain and the anti-CD3 chain.

[0146] In some instances, the multispecific antibodies provided herein may comprise (i) a first polypeptide comprising, from the N-terminus to the C-terminus, a VH-CH1 chain of anti-TAA Fab, a VH and Fc fragment of the anti-CD3 moiety, and a VH of the anti-CD137 or anti-PD-L1 moiety; (ii) a second polypeptide comprising, from the N-terminus to the C-terminus, a VH-CH1 chain of anti-TAA Fab, a VL and Fc fragment of the anti-CD3 moiety, and a VL of the anti-CD137 or anti-PD-L1 moiety; and (iii) a third polypeptide comprising a VL-CL of anti-TAA Fab. One or more peptide linkers (such as those provided in Table 1 below) may be used to link any two adjacent antibody fragments of any of the polypeptides of the multispecific antibody.

[0147] In other examples, the multispecific antibodies provided herein may comprise (i) a first polypeptide comprising, from the N-terminus to the C-terminus, a VH-CH1 chain of a first anti-TAA Fab, a VH and Fc fragment of an anti-CD3 moiety, and a VH fragment of an anti-CD137 or anti-PD-L1 moiety; (ii) a second polypeptide comprising, from the N-terminus to the C-terminus, a VH-CH1 chain of a second anti-TAA Fab, a VL and Fc fragment of an anti-CD3 moiety, and a VL fragment of an anti-CD137 or anti-PD-L1 moiety; (iii) a third polypeptide comprising a VL-CL of a first anti-TAA Fab; and (iv) a fourth polypeptide comprising a VL-CL chain of a second anti-TAA Fab. One or more peptide linkers (such as those provided in Table 1 below) may be used to link any two adjacent antibody fragments of any of the polypeptides of the multispecific antibody.

[0148] Table 2 below provides exemplary multispecific antibodies as disclosed herein. In specific examples, the exemplary multispecific antibodies provided herein include Ly3151 (anti-HER2 / CD3 / CD137), Ly3188 (anti-HER2 / CD3 / PD-L1), Ly2909 (anti-CEA / CD3 / CD137), Ly3060 (anti-CEA / CD3 / PD-L1), Ly2949 (anti-BCMA / CD3 / CD137), Ly3096 (anti-BCMA / CD3 / CD137), Ly3106 (anti-BCMA / CD3 / PD-L1), and Ly2959. (BCMA / CD3 / PD-L1), Ly3232 (anti-HER2 / CD3 / CD137), Ly3233 (anti-HER2 / CD3 / CD137), Ly3234 (anti-HER2 / CD3 / CD137), Ly3235 (anti-HER2 / CD3 / CD137), Ly3236 (anti-HER2 / CD3 / CD137), Ly3353 (anti-HER2 / CD3 / PD-L1), Ly3354 (anti-HER2 / CD3 / PD-L1), Ly3355 (anti-HER2 / CD3 / PD-L1), Ly3356 (anti-HER2 / CD3 / PD-L1), and Ly3357 (anti-HER2 / CD3 / PD-L1).

[0149] II. Methods for preparing multispecific antibodies

[0150] Any of the multispecific antibodies described herein (including bispecific and trispecific antibodies) can be prepared by any method known in the art. See, for example, Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

[0151] In some embodiments, multispecific antibodies may be generated via conventional recombinant techniques, such as those illustrated below.

[0152] Nucleic acids encoding multiple strands of a multispecific antibody, as described herein, can be cloned into an expression vector, with each nucleotide sequence operatively linked to a suitable promoter. In one example, each of the nucleotide sequences encoding the heavy and light chains is operatively linked to a different promoter. Alternatively, nucleotide sequences encoding multiple chains can be operatively 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 heavy and light chain coding sequences.

[0153] In some instances, the nucleotide sequences encoding multiple chains of antibodies 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 expressing such a chain, and the isolated chains can be mixed and incubated under suitable conditions that allow for the formation of multi-chain antibodies.

[0154] Typically, nucleic acid sequences encoding one or all strands of an antibody can be cloned into a suitable expression vector and operatively ligated to 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 generate complementary ends on each molecule that can pair with each other and be linked together with a ligase. Alternatively, synthetic nucleic acid adapters can be ligated to the ends of a gene. These synthetic adapters contain nucleic acid sequences corresponding to specific restriction sites in the vector. The choice of expression vector / promoter will depend on the type of host cell used to produce the antibody.

[0155] Various promoters can be used to express 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-1LTR), simian virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and herpes simplex virus TK promoter.

[0156] Adjustable promoters can also be used. Such adjustable promoters include those that use lac repressors from E. coli as transcription regulators to regulate the transcription of mammalian cell promoters carrying lac operators (Brown, M. et al., Cell, 49:603-612 (1987)) and those that use tetracycline repressors (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)). Other systems include FK506 dimer, VP16 or p65 using astradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech, and Ariad.

[0157] A tunable promoter comprising a repressor with an operon can be used. In one embodiment, a lac repressor from *E. coli* can be used as a transcription modulator to regulate transcription of a mammalian cell promoter carrying a lac operator (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)). This mammalian cell promoter combines a tetracycline repressor (tetR) with an activator of transcription (VP16) to produce the tetR-mammalian cell activator of transcription fusion protein tTa (tetR-VP16), which combines with a minimal promoter carrying tetO derived from the major immediate early promoter of human cytomegalovirus (hCMV) to produce the tetR-tet operator system, thereby controlling gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. Tetracycline repressor alone (tetR), rather than tetR-mammalian cell transcription factor fusion derivatives, can be used as potent trans-regulators to control gene expression in mammalian cells when the tetracycline operon is properly located downstream of the TATA element of the CMVIE promoter (Yao et al., Human Gene Therapy, 10(16):1392-1399 (2003)). A particular advantage of this tetracycline-inducible switch is that it does not require the use of a tetracycline repressor-mammalian cell trans-activator or repressor fusion protein to achieve its regulated effect, which in some cases may be toxic to cells (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)).

[0158] Additionally, the vector may contain some or all of the following: selectable marker genes, such as the neomycin gene for selecting stable or transient transfectants in mammalian cells; enhancer / promoter sequences from the immediate early gene of human CMV for high-level transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 multitumor origin of replication and ColE1 for appropriate appendage replication; internal ribosome binding site (IRES), universal 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.

[0159] Examples of polyadenylation signals that can be used to practice the methods described herein include, but are not limited to, human collagen I polyadenylation signals, human collagen II polyadenylation signals, and SV40 polyadenylation signals.

[0160] One or more vectors (e.g., expression vectors) containing nucleic acids encoding any of the antibodies can be introduced into host cells suitable for antibody production. Host cells can be cultured under suitable conditions for expression of the antibody or any of its polypeptide chains. Such antibodies or their polypeptide chains can be recovered from the cultured cells (e.g., from cells or culture supernatant) via conventional methods (e.g., affinity purification). If necessary, the antibody polypeptide chain can be incubated under suitable conditions for a suitable period of time allowing antibody production.

[0161] In some embodiments, the method for preparing the antibodies described herein involves a recombinant expression vector encoding all multiple chains of a multispecific antibody as described herein. The recombinant expression vector can be introduced into suitable host cells (e.g., dhfr-CHO cells) by conventional methods (e.g., calcium phosphate-mediated transfection). Positive transformant host cells can be selectively cultured under suitable conditions that allow expression of multiple polypeptide chains (e.g., three or four) that form antibodies, 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 for the formation of multi-chain antibodies.

[0162] In one example, two or more recombinant expression vectors are provided, each encoding one or more of a plurality of antibody chains. The two or more recombinant expression vectors can be introduced into suitable host cells (e.g., dhfr-CHO cells) using conventional methods (e.g., calcium phosphate-mediated transfection). Alternatively, each of the expression vectors can be introduced into a suitable host cell. Positive transformants can be selectively cultured under suitable conditions that allow the expression of the polypeptide chain containing the antibody. When the 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 chain can be recovered from the host cell or from the culture medium and then incubated under suitable conditions that allow antibody formation. When the two or more expression vectors are introduced into different host cells, each of the two or more expression vectors can be recovered from the corresponding host cell or from the corresponding culture medium. Multiple polypeptide chains can then be incubated under suitable conditions for antibody formation.

[0163] Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture host cells, and recover antibodies from the culture medium. For example, some antibodies can be separated using affinity chromatography with a protein A or protein G conjugate matrix.

[0164] The nucleic acid encoding multiple strands of a multispecific antibody as disclosed herein, a vector containing such a vector (e.g., an expression vector), or a host cell containing the vector are all within the scope of this disclosure.

[0165] III. Pharmaceutical Composition

[0166] As described herein, any of the multispecific antibodies disclosed herein, as well as those encoding nucleic acids or nucleic acid groups, vectors containing such nucleic acids or nucleic acid groups, or host cells containing such vectors, can be mixed with pharmaceutically acceptable excipients to form a pharmaceutical composition for treating a target disease. "Acceptable" means that the excipient must be compatible with (and preferably, able to stabilize) the active ingredient of the composition and harmless to the subject to be treated. Pharmaceutically acceptable excipients include buffers, which are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (2000), Lippincott Williams and Wilkins, editor KE Hoover.

[0167] Pharmaceutical compositions used in the methods of the present invention may comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. (Remington: The Science and Practice of Pharmacy, 20th Edition (2000), Lippincott Williams and Wilkins, edited by KE Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used and may include: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, etc. Examples of active ingredients include serum 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 nonionic surfactants such as TWEEN, PLURONICS, or polyethylene glycol (PEG).

[0168] In some instances, the pharmaceutical compositions described herein comprise liposomes containing antibodies (or encoding nucleic acids), which 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 cycle times are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be produced by a reverse-phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter with defined pore sizes to obtain liposomes with the desired diameter.

[0169] Antibodies or encoding nucleic acids can also be embedded in microcapsules prepared by, for example, coagulation techniques or interfacial polymerization (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or crude emulsions. These techniques are known in the art; see, for example, Remington, The Science and Practice of Pharmacy, 20th edition, Mack Publishing (2000).

[0170] In other instances, the pharmaceutical compositions described herein may be formulated into sustained-release forms. Suitable examples of sustained-release formulations include semi-permeable matrices of solid hydrophobic polymers containing antibodies, in the form of molded articles such as membranes or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactide (US Patent No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamic acid, 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)), sucrose isobutyrate acetate, and poly-D-(-)-3-hydroxybutyric acid.

[0171] Pharmaceutical compositions intended for internal administration must be sterile. This is readily achieved through filtration, for example, using a sterile filter membrane. Therapeutic antibody compositions are typically placed in containers with sterile access ports, such as intravenous solution bags or vials with stoppers that can be punctured by a hypodermic needle.

[0172] The pharmaceutical compositions described herein may be in unit dosage forms, such as tablets, pills, capsules, powders, granules, solutions or suspensions or suppositories, for oral, parenteral or rectal administration or administration by inhalation or blowing.

[0173] To prepare solid compositions (such as tablets), the main active ingredient can be mixed with a drug carrier (e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gum) and other drug diluents (e.g., water) to form a homogeneous mixture of solid preformed compositions containing the compounds of the present invention or a pharmaceutically acceptable non-toxic salt thereof. When referring to these preformed compositions as homogeneous, this means that the active ingredient is uniformly dispersed throughout the composition, allowing the composition to be readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. The solid preformed composition is then subdivided into unit dosage forms of the type described above, each containing 0.1 to about 500 mg of the active ingredient of the present invention. Tablets or pills of the new composition can be coated or otherwise formulated to provide a dosage form that offers the advantage of prolonged action. For example, tablets or pills may include an internal dose component and an external dose component, the latter being coated over the former. These two components can be separated by an enteric coating, which resists disintegration in the stomach and allows the internal components to be delivered intact into the duodenum or released with a delay. A variety of materials can be used for this enteric coating or coating, including various polymeric acids and mixtures of polymeric acids with such materials as shellac, hexadecyl alcohol, and cellulose acetate.

[0174] Suitable surfactants specifically include nonionic 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). Compositions containing surfactants will conveniently include surfactants at concentrations between 0.05% and 5%, and may be between 0.1% and 2.5%. It should be understood that other ingredients, such as mannitol or other pharmaceutically acceptable mediators, may be added if necessary.

[0175] Suitable emulsions can be prepared using commercially available fat emulsions such as INTRALIPID, LIPOSYN, INFONUTROL, LIPOFUNDIN, and LIPIPHYSAN. The active ingredient can be dissolved in the premixed emulsion composition, or alternatively, the active ingredient can be dissolved in oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and in an emulsion formed by mixing with phospholipids (e.g., egg phospholipid, soybean phospholipid, or soybean lecithin) and water. It should be understood that other ingredients (e.g., glycerol or glucose) can be added to adjust the emulsion's tension. A suitable emulsion will typically contain up to 20% oil, for example, between 5% and 20%. Fat emulsions can contain fat droplets between 0.1 and 1.0 μm, specifically between 0.1 and 0.5 μm, and the pH can be in the range of 5.5 to 8.0.

[0176] Emulsion compositions can be those prepared by mixing an antibody with INTRALIPID or its components (soybean oil, lecithin, glycerol, and water).

[0177] Pharmaceutical compositions for inhalation or inhalation comprise solutions and suspensions, as well as powders, in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as listed above. In some embodiments, these compositions are administered via the oral or nasal route for local or systemic effects.

[0178] Compositions in preferably sterile, pharmaceutically acceptable solvents can be nebulized using a gas. The nebulized solution can be inhaled directly from the nebulizer, or the nebulizer can be attached to a face mask, oxygen tent, or intermittent positive pressure ventilator. The solution, suspension, or powder composition can be administered orally or nasally from a device that delivers the formulation in a suitable manner.

[0179] IV. Therapeutic Applications

[0180] Any of the multispecific antibodies disclosed herein may be used in a clinical setting (e.g., for treatment) or a non-clinical setting (e.g., for research purposes).

[0181] In some respects, this document provides methods for modulating immune responses and / or treating cancer in subjects requiring treatment using any of the multispecific antibodies disclosed herein. To practice the methods disclosed herein, an effective amount of the pharmaceutical composition described herein may be administered to a subject requiring treatment (e.g., a human) via a suitable route, such as intravenous administration (e.g., bolus injection or continuous infusion over a period of time), intramuscular, intraperitoneal, intraspinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, inhalation, or local route. Commercially available nebulizers (including jet nebulizers and ultrasonic nebulizers) for use with the liquid formulation may be used for administration. The liquid formulation may be nebulized directly, and the lyophilized powder may be nebulized after reconstitution. Alternatively, the antibodies described herein may be nebulized using fluorocarbon formulations and metered-dose inhalers or inhaled as lyophilized powders and ground powders.

[0182] Subjects to be treated by the methods described herein may be mammals, more preferably humans. Mammals include, but are not limited to, livestock, sport animals, pets, primates, horses, dogs, cats, mice, and rats. Human subjects requiring treatment may be human patients who have, are at risk of having, or are suspected of having a target disease / condition (such as cancer or an immune condition, such as an autoimmune disease).

[0183] Examples of cancers include, but are not limited to, breast cancer; bile duct cancer; bladder cancer; brain cancer, including glioblastoma and medulloblastoma; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematologic malignancies, including acute lymphoblastic leukemia and myeloid leukemia, such as B-cell CLL; T-cell acute lymphoblastic leukemia / lymphoma; hairy cell leukemia; chronic myeloid 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; and lymphomas, including Hodgkin's disease. Diseases and lymphocytic lymphomas; neuroblastomas; oral cancers, including squamous cell carcinomas; ovarian cancers, including those caused by epithelial cells, stromal cells, germ cells, and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancers, including melanoma, Merkel cell carcinoma, Kaposi's sarcoma, basal cell carcinoma, and squamous cell carcinoma; testicular cancers, including germ cell tumors such as seminoma, non-seminomatous tumors (teratomas, choriocarcinomas), stromal tumors, and germ cell tumors; thyroid cancers, including adenocarcinoma and medullary carcinoma; and kidney cancers, including adenocarcinoma and Wilms' tumor.

[0184] In some cases, the multispecific antibodies used in the treatments disclosed herein contain a binding arm specific to a target TAA, and the patient being treated carries cancer cells expressing the target TAA. For example, the multispecific antibody is specific to CD19, and the patient carries CD19+ cancer. In other instances, the multispecific antibody is specific to CD20, and the patient carries CD20+ cancer. In other instances, the multispecific antibody is specific to BCMA, and the patient carries BCMA+ cancer. In other instances, the multispecific antibody is specific to B7H3, and the patient carries B7H3+ cancer. In some instances, the multispecific antibody is specific to HER2, and the patient carries HER2+ cancer. In some instances, the multispecific antibody is specific to p53mut, and the patient carries p53mut+ cancer. In some instances, the multispecific antibody is specific to MET, and the patient carries MET+ cancer. In some instances, the multispecific antibody is specific to PSMA, and the patient carries PSMA+ cancer. In some instances, the multispecific antibody is specific to CEA, and the patient carries CEA+ cancer. In some instances, the multispecific antibody is specific to EGFR, and the patient has EGFR+ cancer. In some instances, the multispecific antibody is specific to DLL3, and the patient has DLL3+ cancer. In some instances, the multispecific antibody is specific to MAGE-A4, and the patient has MAGE-A4+ cancer. In some instances, the multispecific antibody is specific to PRAME, and the patient has PRAME+ cancer.

[0185] Subjects with target cancer can be identified through routine medical examinations (e.g., laboratory tests, organ function tests, CT scans, ultrasound, and / or genetic testing). In some embodiments, subjects to be treated using the methods described herein can be human cancer patients who have undergone or are undergoing anticancer therapies (e.g., chemotherapy, radiation therapy, immunotherapy, or surgery).

[0186] Subjects suspected of having any of these target diseases / conditions (such as cancer) may exhibit one or more symptoms of that disease / condition. Subjects at risk of having a disease / condition may have one or more of the risk factors for that disease / condition.

[0187] As used herein, "effective amount" means the amount of each active agent required, alone or in combination with one or more other active agents, to impart a therapeutic effect to a subject. Determining whether a given amount of antibody achieves a therapeutic effect will be apparent to those skilled in the art. As will be recognized by those skilled in the art, the effective amount varies depending on the specific condition being treated, the severity of the condition, individual patient parameters (including age, physical condition, body type, sex, and weight), the duration of treatment, the nature of concurrent therapies (if present), the specific route of administration, and similar factors within the knowledge and expertise of a healthcare professional. These factors are well known to those skilled in the art and can be resolved through routine experimental procedures alone. Generally, it is preferred to use the maximum dose of the individual component or its combination, i.e., the highest safe dose based on reasonable medical judgment.

[0188] Empirical considerations, such as half-life, often aid in dosage determination. For example, antibodies compatible with the human immune system, such as humanized or fully human antibodies, can be used to prolong the antibody's half-life and prevent it from being attacked by the host's immune system. Dosage frequency can be determined and adjusted during therapy, and is generally, but not necessarily, based on the treatment and / or inhibition and / or mitigation and / or delay of the target disease / symptom. Alternatively, formulations for sustained, continuous release of antibodies may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0189] In one instance, the dosage of the antibody, as described herein, can be determined empirically in individuals who have received one or more doses. Individuals are given escalating doses of the agonist. To assess the efficacy of the agonist, indicators of the disease / symptom can be followed.

[0190] Typically, for the administration of any antibody described herein, the initial candidate dose may be about 2 mg / kg. For the purposes of this disclosure, a typical daily dose range may be any one of 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 described above. For repeated administration over several days or longer, treatment continues until desired symptom suppression is achieved or until a therapeutic level sufficient to alleviate the target disease or condition or its symptoms is reached, depending on the condition. Exemplary dosing regimens include an initial dose of about 2 mg / kg, followed by a weekly maintenance dose of about 1 mg / kg of antibody, or a maintenance dose of about 1 mg / kg every other week. However, other dosing regimens may be useful depending on the pharmacokinetic decay pattern desired by the practitioner. For example, dosing once to four times per week is considered. In some embodiments, the dosage range that can be used is from about 3 µg / mg to about 2 mg / kg (such as 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). In some embodiments, the dosing frequency is once weekly, once every 2 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, or once every 10 weeks; or once monthly, once every 2 months, once every 3 months, or longer. Progression of this therapy can be easily monitored using routine techniques and assays. The dosing regimen (including the antibodies used) may vary over time.

[0191] In some embodiments, the dosage range that can be administered to a normal-weight adult patient is about 0.003 to 5.00 mg / kg. In some instances, the dosage of the antibody described herein may be 10 mg / kg. The specific dosing regimen (i.e., dosage, time, and repetition) will depend on the particular individual and that individual's medical history, as well as the nature of the individual drug (such as the half-life of the drug and other considerations well known in the art).

[0192] For the purposes of this disclosure, the appropriate dose of the antibody as described herein will depend on the specific antibody, antibody and / or non-antibody peptide (or combination thereof) used, the type and severity of the disease / symptom, whether the antibody is administered for prophylactic or therapeutic purposes, prior therapy, the patient's clinical history and response to agonists, and the attending physician's judgment. Clinicians will typically administer antibodies up to a dose that achieves the desired outcome. In some embodiments, the desired outcome is an increase in the antitumor immune response in the tumor microenvironment. Methods for determining whether a dose produces the desired outcome will be apparent to those skilled in the art. The administration of one or more antibodies may be continuous or intermittent, depending on factors such as the recipient's physiological condition, whether the administration is therapeutic or prophylactic, and other factors known to a skilled practitioner. Antibody administration may be substantially continuous over a preselected time period or may be administered, for example, as a series of interval doses before, during, or after the development of the target disease or symptom.

[0193] As used herein, the term “treatment” refers to the application or administration of a composition comprising one or more active agents to a subject who suffers from a target disease or condition, symptoms of the disease / condition, or is susceptible to the disease / condition, with the aim of treating, curing, alleviating, relieving, altering, remedying, improving, modifying, or influencing the condition, symptoms of the disease, or susceptibility to the disease or condition.

[0194] Reducing the severity of a target disease / symptom includes delaying the development or progression of the disease, or reducing its severity or prolonging survival. Disease reduction or prolonged survival does not necessarily require a cure. As used herein, “delaying” the development of a target disease or symptom means postponing, hindering, slowing, stabilizing, and / or delaying the progression of the disease. This delay can vary in length, depending on the history of the disease and / or the individual being treated. Methods of “delaying” or reducing the development of the disease or the onset of the disease involve reducing the likelihood of developing one or more symptoms of the disease within a given time frame and / or reducing the degree of symptom reduction within a given time frame compared to not using this method. Such comparisons are typically based on clinical studies using a large number of subjects sufficient to provide statistically significant results.

[0195] The term "development" or "progression" of a disease refers to the initial presentation and / or subsequent progression of the disease. The development of a disease can be detectable and can be assessed using standard clinical techniques well known in the art. However, development also refers to progression that may not be detectable. For the purposes of this disclosure, development or progression refers to the biological process of symptoms. "Development" includes occurrence, relapse, and onset. As used herein, an "onset" or "occurrence" of a target disease or condition includes an initial onset and / or relapse.

[0196] Depending on the type or location of the disease to be treated, the pharmaceutical composition may be administered to the subject using conventional methods known to those skilled in the art. This composition may also be administered via other conventional routes, such as oral, parenteral, inhalation spray, topical, rectal, nasal, buccal, vaginal, or via an implantable receptacle. As used herein, the term “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. Additionally, it may be administered to the subject via injectable receptacle routes, such as using injectable or biodegradable materials and methods with 1-month, 3-month, or 6-month receptacles. In some instances, the pharmaceutical composition is administered intraocularly or intravitreal.

[0197] Injectable compositions may contain various carriers, such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies can be administered via infusion, thereby infusing a pharmaceutical formulation containing antibodies and physiologically acceptable excipients. Physiologically acceptable excipients may include, for example, 5% dextran, 0.9% saline, Ringer's solution, or other suitable excipients. Intramuscular preparations (e.g., sterile formulations in the form of suitable soluble salts of antibodies) can be dissolved and administered in pharmaceutical excipients such as water for injection, 0.9% saline, or 5% glucose solution.

[0198] In one embodiment, the antibody is administered via a site-specific or targeted local delivery technique. Examples of site-specific or targeted local delivery techniques include various implantable reservoir sources or local delivery catheters (such as infusion catheters, indwelling catheters or needle catheters, synthetic grafts, outer membrane wrappings, shunts and stents or other implantable devices), site-specific loads, direct injection, or direct application. See, for example, PCT Publication WO 00 / 53211 and U.S. Patent No. 5,981,568.

[0199] Targeted delivery can also be achieved using therapeutic compositions containing antisense polynucleotides, expression vectors, or subgenomic polynucleotides. 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 GeneTransfer (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.

[0200] For topical application in gene therapy regimens, a therapeutic composition containing a polynucleotide (e.g., a polynucleotide encoding an antibody described herein) is administered in the range of about 100 ng to about 200 mg of DNA. In some embodiments, during a gene therapy regimen, concentrations 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 of DNA or higher may also be used.

[0201] The therapeutic polynucleotides and peptides described herein can be delivered using gene delivery agents. These agents 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 these coding sequences can be induced using endogenous mammalian or heterologous promoters and / or enhancers. Expression of the coding sequences can be constitutive or regulated.

[0202] Virus-based vectors for delivering desired polynucleotides and expressing them in desired cells are well known in the art. Exemplary virus-based vectors include, but are not limited to, recombinant retroviruses (see, for example, PCT Publications WO 90 / 07936; WO 94 / 03622; WO 93 / 25698; WO 93 / 25234; WO 93 / 11230; WO 93 / 10218; WO 91 / 02805; U.S. Patents 5,219,740 and 4,777,127; GB Patent 2,200,651; and EP Patent 0 345242), alphavirus-based vectors (e.g., Sindbis virus vector, 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-923; ATCC VR-1250; ATCC VR-1246). 1249; ATCC VR-532) and adeno-associated virus (AAV) vectors (see, for example, PCT Publications WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655). Administration of DNA linked to a cytotoxic adenovirus (as described in Curiel, Hum. Gene Ther. (1992) 3:147) may also be used.

[0203] Non-viral delivery media and methods may also be used, including, but not limited to, polycationically concentrated DNA, either alone or unconjugated with a cytotoxic adenovirus (see, for example, Curiel, Hum. Gene Ther. (1992) 3:147); ligand-linked DNA (see, for example, Wu, J. Biol. Chem. (1989) 264:16985); eukaryotic cell delivery mediators (see, for example, U.S. Patent No. 5,814,482; PCT Publications WO 95 / 07994; WO 96 / 17072; WO 95 / 30763; and WO 97 / 42338); and nuclear charge neutralization or fusion with the cell membrane. Naked DNA may also be used. Exemplary methods for introducing naked DNA are described in PCT Publication WO 90 / 11092 and U.S. Patent No. 5,580,859. Liposomes that can be used as gene delivery mediators are described in U.S. Patent No. 5,422,120; PCT Publications Nos. WO 95 / 13796; WO 94 / 23697; WO 91 / 14445; and EP Patent No. 0524968. Additional methods are described in Philip, Mol. Cell. Biol. (1994) 14:2411 and Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

[0204] The specific dosing regimen used in the methods described herein, namely the dosage, timing, and repetition, will depend on the specific subject and the subject's medical history.

[0205] In some embodiments, more than one antibody or a combination of an antibody and another suitable therapeutic agent may be administered to a subject requiring treatment. Antibodies may also be used in combination with other agents to enhance and / or complement the effectiveness of the therapeutic agent. The therapeutic efficacy against the target disease / condition can be evaluated using methods well known in the art.

[0206] When any of the antibodies described herein are used to treat cancer, they can be combined with anticancer therapies (e.g., anticancer therapies known in the art). Other anticancer therapies include chemotherapy, surgery, radiation, immunotherapy, gene therapy, etc.

[0207] Alternatively, the treatment disclosed herein can be combined with: chemotherapeutic agents, such as pyrimidine analogs (5-fluorouracil, fluorouridine, capecitabine, gemcitabine, and cytarabine), purine analogs, folic acid antagonists, and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine, cladribine); antiproliferative / antimitotic agents, including natural products (such as vinca alkaloids (vincrine, vincristine, and vinorelbine), microtubule disruptors (such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, etc.). Nocodazole, epothilone, and navelbine; epipodophyllotoxin (etoposide, teniposide); DNA damaging agents (actinomycin, amsacrine, anthracycline, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytox an), dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethyhnelamine, oxaliplatin, ifosfamide, melphalan, mercchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine Taxol, taxotere, teniposide, multiethylenethiophosphoramide, and etoposide (VP16); antibiotics such as actinomycin D, daunorubicin, doxorubicin (adriamycin), idarubicin, anthracycline, mitoxantrone, bleomycin, purcamycin (mithramycin), and mitomycin; enzymes (L-asparaginases that systemically metabolize L-asparagine and deprive cells that do not have the ability to synthesize their own asparagine); antiplatelet agents;Antiproliferative / antimitotic alkylating agents, such as nitrogen mustard (dichloromethyldiethylamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethyleneimine and methylmelamine (hexamethylmelamine and thiotepa), alkyl sulfonates-bustano, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), and trazenes-dacarbazinine. (DTIC); antiproliferative / antimitotic antimetabolites, such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogens, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole) zole); anticoagulants (heparin, synthetic heparin salts and other thrombin inhibitors); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigration agents; antisecretory agents (breveldin); immunosuppressants (cyclosporine, tacrolimus). (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil; anti-angiogenic compounds (e.g., TNP-470, genistein, bevacizumab) and growth factor inhibitors (e.g., fibroblast growth factor (FGF) inhibitors); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin);mTOR inhibitors, topoisomerase inhibitors (doxorubicin, acridine, camptothecin, daunorubicin, geniposide, epirubicin, etoposide, idarubicin, mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, and prednisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and chromatin destroyers.

[0208] When any of the antibodies described herein is used to treat an autoimmune disease, it may be used in combination with other immunomodulatory therapies, such as 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 may be combined with another therapy for an 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; FTY 720; leflunomide; mizoribine; mycophenolic acid; mycophenolate mofetil; 15-deoxyguanidine; immunosuppressants; or adhesion molecule inhibitors.

[0209] For examples of other useful agents, see also Physician's Desk Reference, Supplement 59, (2005), Thomson PDR, Montvale NJ; Gennaro et al., eds., Remington's The Science and Practice of Pharmacy, Supplement 20, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., eds., Harrison's Principles of Internal Medicine, Supplement 15, (2001), McGraw Hill, NY; Berkow et al., eds., The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway NJ.

[0210] When using a second therapeutic agent, such an agent may be administered simultaneously or sequentially (in any order) with the therapeutic agents described herein. When administered co-administered with other therapeutic agents, the appropriate therapeutically effective dose of each agent may be reduced due to additive or synergistic effects.

[0211] V. Kits containing the multispecific antibodies disclosed herein

[0212] This disclosure also provides kits for treating or alleviating target diseases, such as cancers or immune disorders as described herein. Such kits may include one or more containers comprising any of the multispecific antibodies disclosed herein, and optionally a second therapeutic agent to be used in conjunction with the antibody, which is also described herein.

[0213] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The included instructions may include a description of administering an antibody for treating a target disease (such as those described herein), delaying the onset of a target disease, or alleviating a target disease, and optionally a second therapeutic agent. The kit may further include a description of selecting individuals suitable for treatment based on identifying whether the individual has a target disease (e.g., using diagnostic methods as described herein). In still other embodiments, the instructions include a description of administering an antibody to an individual at risk of a target disease.

[0214] Instructions for use related to antibodies typically include information about the dosage, dosing regimen, and route of administration for the intended treatment. Containers may be unit dose, bulk packaging (e.g., multi-dose packaging), or subunit dose. Instructions provided with the kits of this invention are typically written instructions on a label or packaging insert (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0215] The label or packaging insert indicates that the composition is intended to treat diseases (such as cancer or immune conditions, e.g., autoimmune diseases), delay their onset, and / or alleviate them. Instructions for use in practicing any of the methods described herein may be provided.

[0216] The kit of the present invention is packaged in a suitable manner. Suitable packaging includes, but is not limited to, vials, bottles, wide-mouth bottles, flexible packaging (e.g., sealed Mylar or plastic bags), etc. Packaging for use in conjunction with specific devices (such as inhalers, nasal application devices (e.g., nebulizers), or infusion devices (such as micropumps)) is also contemplated. The kit may have a sterile inlet port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be punctured by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be punctured by a hypodermic needle). At least one active agent in the composition is an antibody, such as those described herein.

[0217] The kit may optionally provide additional components such as buffer solutions, as well as explanatory information. Typically, the kit includes a container and a label or packaging insert on or associated with the container. In some embodiments, the invention provides an article of manufacture containing the contents of the kit described above.

[0218] General technology

[0219] Unless otherwise indicated, the practice of this disclosure will employ conventional molecular biology (including recombinant technologies), microbiology, cell biology, biochemistry, and immunology techniques within the scope of the art. These techniques are well explained in the following literature, such as *Molecular Cloning: A Laboratory Manual, 2nd 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* (JE Cellis, ed., 1989), Academic Press; *Animal Cell Culture* (RIFreshney, ed., 1987); *Introduction to Cell and Tissue Culture* (JP Mather and PE Roberts, 1998), Plenum Press; *Cell and Tissue Culture: Laboratory Procedures* (A. Doyle, JB Griffiths, and DG Newell, ed., 1993–8), J. Wiley and Sons; *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (DM Weir and CC Blackwell, ed.): Gene Transfer Vectors for Mammalian. Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); Current Protocols in Immunology (JE...Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: an apractical 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); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (DN Glover, ed., 1985); Nucleic Acid Hybridization (BD Hames and SJ Higgins, eds., 1985); Transcription and Translation (BD Hames and SJ Higgins, eds., 1984); Animal Cell Culture (RI Freshney, ed., 1986); Immobilized Cells and Enzymes (IRL Press, 1986); and B. Perbal, A practical guide to Molecular Cloning (1984); FM Ausubel et al. (eds.).

[0220] Without further detail, it is believed that those skilled in the art can utilize the invention to the fullest extent based on the above description. Therefore, the following specific embodiments should be interpreted as illustrative only and do not limit the remainder of this disclosure in any way. All disclosures referenced herein are incorporated by reference for the purposes or subject matter of this document.

[0221] Example

[0222] Example 1: Construction and generation of multispecific and multivalent antibodies

[0223] The exemplary multispecific antibodies shown in Table 2 were generated using recombinant technology and characterized for their antigen-binding activity and biological activity as disclosed herein.

[0224] In short, encoding Figure 1A The cDNA of the dual anti-CD3 / CD137 (SEQ ID NO: 3-4) or anti-CD3 / PD-L1 (SEQ ID NO: 5-6) immunomodulatory constructs shown in the figure is used for the preparation of Figure 1B The starting materials for the multispecific antibodies illustrated herein are shown in Table 1. The VH and VL sequences from the parental antibody clones shown in Table 1 were used to construct exemplary multispecific antibodies. The coding sequences of multiple strands of each exemplary multispecific antibody (see Table 2) were cloned into an expression vector, which was then transfected into CHO cells for transient expression. The multispecific antibodies produced from CHO cells were purified from the culture supernatant using protein A affinity chromatography. Antibody properties were investigated using standard protocols or as described herein.

[0225] Example 2: Characterization of a dual anti-CD3 / CD137 immunomodulatory construct

[0226] This example explores the activity of the dual immunomodulatory construct described in Example 1 above and its effect on multispecific antibodies containing it, which bind to CD3 and CD137.

[0227] As described in Example 1 above, exemplary multispecific antibodies were generated in CHO cells. Several multispecific antibodies were constructed using an anti-CD3 / CD137 dual modulator as a backbone and including portions for further binding to tumor antigens such as HER2, CEA, or BCMA. The forms of these multispecific antibodies are shown in... Figure 1B middle.

[0228] (i) Tumor cell killing

[0229] In vitro, tumor cell killing induced by immune cells was studied using the exemplary anti-CEA / CD3 / CD137 multispecific antibody Ly2909, which contains a dual CD3 / CD137 regulatory construct. Briefly, LS174T-Luc cells and human PBMCs were mixed and incubated for 48 hours in the presence of various test antibodies, and the luminescence of tumor cells was measured using a kit from Promega following the manufacturer's instructions. The cytotoxicity of the test antibodies was negatively correlated with the luminescence intensity. The CEA / CD3 bispecific antibody Ly2323 was used as a control.

[0230] Figure 2A-2B A summary of the results obtained from the in vitro assays described above is provided. Ly2909 exhibited stronger cytotoxicity than Ly2323, indicating that the additional CD137 signaling induced by the dual immunomodulatory construct played a role. Furthermore, at its relatively effective tumor-killing dose, Ly2909 induced less cytokine release than Ly2323, suggesting that Ly2909 has a larger therapeutic window than Ly2323, as the cytokine profile can predict adverse reactions.

[0231] (ii) In vitro CD8 T cell activation

[0232] To determine whether multispecific antibodies containing a dual CD3 / CD137 immunomodulatory construct could enhance cellular immune function, the in vivo morphology of T cells was evaluated. MC38 cells expressing human CEA and spleen cells from human CD3 / CD137 knock-in (KI) mice were mixed and incubated for 3 days in the presence of exemplary anti-CEA / CD3 / CD137 multispecific antibody Ly2909 or CEA / CD3 bispecific antibodies Ly2915 or Ly2323. Ly2909-induced activation levels of CD8 T cells were significantly higher than those induced by Ly2915 or Ly2323. Figure 3 As shown. Similarly, the anti-BCMA / CD3 / CD137 multispecific antibody Ly2949 stimulated CD8 cell activation at a much higher level than that induced by the BCMA / CD3 bispecific antibody controls Ly2951 or Ly2313, as... Figure 4 As shown in the figures, these data indicate that dual CD3 / CD137 activation is necessary to maximize the activation of CD8 T cells, which may enhance antitumor activity.

[0233] (iii) Anti-tumor effects

[0234] To determine whether a multispecific antibody containing a dual anti-CD3 / CD137 regulatory construct could provide better protection against tumor growth in vivo than a CD3 bispecific antibody (bsAb), antitumor efficacy was investigated in genotypical mouse models of bone marrow transplantation, inoculated with EL4 and LL2 cell lines expressing human BCMA, respectively. Mice were grouped thirteen or fifteen days after tumor cell inoculation and injected with the test product as instructed. Mice were weighed, and tumor growth was measured twice weekly using calipers. Tumor volume was estimated as 1 / 2 x length x width. 2 .

[0235] Compared with the bispecific anti-BCMA / CD3 bsAb controls Ly2314 and Ly3098, Ly3096 achieved stronger antitumor activity, such as Figure 5 As shown. Similarly, Ly2949 exhibited stronger antitumor activity than the anti-BCMA CD3 bsAb controls Ly2951 and Ly2313, such as Figure 6 As shown, the data further demonstrate the contribution of dual CD3 / CD137 activation to the in vivo antitumor efficacy.

[0236] Furthermore, the antitumor efficacy was investigated in a mouse model implanted with human PBMCs. On day 0, LS174T tumors expressing human CEA were subcutaneously inoculated and followed by intravenous inoculation with human PBMCs. Five days after tumor cell inoculation, mice were grouped and injected with the test product intraperitoneally weekly. Mice were weighed, and tumor growth was measured twice weekly using calipers. Tumor volume was estimated as 1 / 2 (length x width). 2 Compared to anti-CEA / CD3 bsAbs Ly2915 and Ly2323, the exemplary multispecific antibody Ly2909 achieved stronger antitumor activity, such as... Figure 7 As shown, this demonstrates the benefits of dual CD3 / CD137 activation in antitumor activity.

[0237] In summary, compared to bispecific antibodies lacking a portion that activates CD137 signaling, the multispecific antibodies tested in this paper containing the dual CD3 / CD137 regulatory construct and the binding portion targeting tumor antigens described in Example 1 above exhibited more desirable in vitro activity (e.g., enhanced T cell activation) and stronger in vivo antitumor efficacy.

[0238] Example 3: Characterization of a dual anti-CD3 / PD-L1 immunomodulatory construct

[0239] This example explores the activity of the dual immunomodulatory construct described in Example 1 above and its effect on multispecific antibodies containing it, which bind to CD3 and PD-L1.

[0240] The binding of exemplary multispecific antibodies to the tumor antigens and immune targets CD3 and PD-L1 involved was tested. Cytotoxicity assays were performed using co-culture assays of tumor cells and hPBMCs.

[0241] To evaluate the contribution of the additional PD-L1 binding effect in multispecific antibodies to their in vivo antitumor efficacy, exemplary multispecific antibodies containing a dual CD3 / PD-L1 immunomodulatory construct and a portion binding to tumor antigens such as CEA and BCMA were constructed. Their antitumor efficacy was investigated in a mouse model implanted with human PBMCs inoculated with cell lines expressing the corresponding tumor antigens.

[0242] The anti-CEA / CD3 / PD-L1 multispecific antibody Ly3060 showed stronger antitumor activity than its corresponding anti-CEA / CD3 bsAb Ly2915 and the control anti-CEA / CD3 bispecific Ly2323. Figure 8 As shown. For anti-BCMA antibodies, Ly2959 and Ly3106, containing a dual CD3 / PD-L1 regulatory construct and an anti-BCMA binding moiety, exhibited higher anti-tumor efficacy than their corresponding anti-BCMA / CD3 bsAbs Ly2313 and Ly3098 or Ly2314, such as Figure 9 and 10 As shown.

[0243] The promising antitumor efficacy data presented in this example strongly suggest that multispecific antibodies containing a dual CD3 / PD-L1 immunomodulatory construct have the potential to develop effective therapeutics, due to the addition of PD-L1 binding that enhances the cytotoxic effect of T cells on cancer cells.

[0244] Example 4: Evaluation of multispecific antibodies targeting HER2

[0245] This example evaluates the functionality of the multispecific antibody targeting HER2, which comprises a dual immunomodulatory construct disclosed herein and binds to CD3 and CD137 or CD3 and PD-L1.

[0246] (i) Binding to target antigen

[0247] As described in Example 1 above, exemplary multispecific antibodies were generated in CHO cells. Following standard procedures, the binding of these multispecific antibodies to their corresponding target antigens was assessed by ELISA or FCM. These antibodies bound to HER2 with high affinity; however, they showed much weaker binding to CD3 and CD137 compared to their corresponding parental anti-CD3 mAb or anti-CD137 mAb, as shown in Table 3 below. Figure 11A-11F The conclusions drawn.

[0248] Table 3. Binding of anti-HER2 multispecific antibodies to target antigens

[0249]

[0250] (ii) Reporter gene assay

[0251] Perform CD3 reporter gene assays as described in Example 3 to determine the potency of these antibodies.

[0252] Compared to the parental anti-CD3 mAb Ly305, CD3 reporter gene assays showed that the potency of these antibodies was significantly reduced in the absence of additional targets (no activity at 10 µg / mL) (Table 4). However, in the presence of additional targets for cross-linking (such as tumor antigens or immune receptors), the activity of multispecific antibodies was significantly enhanced (Table 4 below). Figure 12A-12F ).

[0253] Table 4. CD3 binding and activation

[0254]

[0255] As described in the examples above, CD137 reporter gene assays were performed to determine the potency of these antibodies. Results from the CD137 reporter gene assays show the minimum activity of the antibodies tested in the absence of additional targets. However, the activity is significantly enhanced when additional targets, such as tumor antigens or immune receptors, are available for cross-linking (Table 5 below). Figures 13A-13C ).

[0256] Table 5. CD137 binding and activation

[0257]

[0258] (iii) Tumor cell killing

[0259] In vitro studies were conducted on tumor cell killing induced by immune cells from anti-HER2 multispecific antibodies.

[0260] EBC-1-luc cells and human PBMCs were mixed and incubated with various test products for 48 hours, and tumor cell luminescence was measured using a kit from Promega following the manufacturer's instructions. Cytotoxicity of the test products was negatively correlated with luminescence intensity. In this assay, anti-HER2 antibodies integrated with dual CD3 / CD137 or CD3 / PD-L1 immunomodulatory fragments showed significant tumor cell killing activity comparable to or superior to the anti-HER2 / CD3 / CD28 trispecific antibody reference Ly2935. Figures 14A-14B ).

[0261] (iv) Anti-tumor effects

[0262] The antitumor efficacy was investigated in a mouse model implanted with human PBMCs from the EBC-1 lung squamous cell carcinoma cell line expressing human HER2. The test product was administered via intraperitoneal injection (ip). Mice were weighed, and tumor growth was measured twice weekly using calipers. The growth rate was calculated using the formula 0.5 x (length × width). 2(Estimation of tumor volume.) Compared to the reference HER2 / CD3 / CD28 trispecific antibody (Ly2935), the anti-HER2 / CD3 / CD137 multispecific antibody Ly3151 or the anti-HER2 / CD3 / PD-L1 multispecific antibody Ly3188, which contain the dual regulatory constructs described above, achieved stronger anti-tumor activity, such as... Figure 15 and Figure 16 As shown separately.

[0263] These examples demonstrate that, compared to their parental mAbs, the dual immunomodulatory constructs exhibit significantly reduced binding affinity to immune target antigens (CD3 and CD137 / PD-L1), resulting in minimal immunomodulatory activity. However, in the multispecific form, the regulatory potential is greatly enhanced in the presence of the added target in reporter gene assay systems. The two exemplary multispecific antibodies also achieved better in vitro and in vivo antitumor activity compared to the reference HER2 / CD3 / CD28 trispecific antibody (Ly2935).

[0264] Example 5: Evaluation of multispecific antibodies targeting CEA

[0265] CEA-targeting T-cell conjugates have the potential to mediate immune killing in various solid tumors, among which high levels of CEA expression are common. Exemplary anti-CEA multispecific antibodies Ly2909 and Ly3060, comprising the anti-CD3 / CD137 and anti-CD3 / PD-L1 dual-regulatory constructs provided herein, were generated following the methods described above.

[0266] (i) Binding to target antigen

[0267] The binding of exemplary anti-CEA multispecific antibodies to their corresponding target antigens was assessed by FCM or ELISA, and the results are summarized in Table 6 below. Figure 17A-17G In this study, multispecific antibodies exhibited high affinity for CEA, while their affinity for immune target antigens CD3, CD137, or PD-L1 was significantly weaker.

[0268] Table 6. Target binding of anti-CEA multispecific antibodies

[0269]

[0270] (ii) Reporter gene assay

[0271] The activation of CD3 signaling by exemplary anti-CEA multispecific antibodies was evaluated using a reporter gene system, and the results are summarized in Table 7 below. Figures 18A-18DThe presence of CEA target antigens significantly increases CD3 activation by multispecific antibodies, including Ly2909 (CEA / CD3 / CD137), Ly2915 (CEA / CD3), and Ly3060 (CEA / CD3 / PD-L1). Binding to another immune target antigen, such as CD137 or PD-L1, enhances CD3 activation by the corresponding multispecific antibody.

[0272] Table 7. Activation of CD3 signaling by anti-CEA multispecific antibodies

[0273]

[0274] Note: N / A = Not applicable

[0275] The reporter gene system was used to evaluate the blocking of CD137 signaling and anti-CEA multispecific antibody against PD-(L)1 signaling.

[0276] Multispecific antibodies exhibit minimal activation of the immune receptor CD3, but this activation significantly increases under cross-linking conditions, supporting the affinity-mediated effects of multispecific antibodies.

[0277] (iii) Tumor cell killing

[0278] Tumor cell killing induced by anti-CEA multispecific antibodies was studied in vitro. LS174T-Luc or HT29-Luc cells were mixed with human PBMCs and incubated with various test antibodies for 48 hours, and luminescence intensity was measured as described above. The CEA / CD3 bispecific antibody Ly2323 was used as a control.

[0279] The killing effect and cytokine release of LS174T-Luc cells were measured in vitro, and the results are summarized in [the table / document / etc.]. Figures 19A-19B Ly2909 (anti-CEA / CD3 / CD137) showed stronger cytotoxicity than Ly2323 (anti-CEA / CD3), indicating the contribution of additional CD137 signaling induced by the dual immunomodulatory construct in the multispecific antibody.

[0280] Figure 19C-19D This provides a summary of results obtained from in vitro assays regarding the killing of LS174T-Luc or HT29-Luc cancer cells. Ly3060 (anti-CEA / CD3 / PD-L1) showed stronger cytotoxicity than Ly2323 (anti-CEA / CD3), indicating the contribution of additional PD-L1 binding effect from the multispecific antibody.

[0281] (iv) Anti-tumor effects

[0282] Antitumor efficacy was investigated in a mouse model implanted with human PBMCs. On day 0, LS174T tumors expressing human CEA were subcutaneously injected (sc) followed by intravenous inoculation with human PBMCs. Five days post-tumor inoculation, mice were grouped and administered the test product via intraperitoneal injection (ip) weekly. Mice were weighed, and tumor growth was measured twice weekly using calipers. Tumor volume was estimated as 1 / 2 (length x width). 2 The antitumor activity of the exemplary multispecific antibody Ly2909 (anti-CEA / CD3 / CD137) is shown in... Figure 20 The robust antitumor efficacy achieved by the multispecific antibody Ly2909 suggests potential benefits arising from dual CD3 / CD137 activation, and potential advantages over the anti-CEA / CD3 bsAb Ly2323.

[0283] Compared to anti-CEA / CD3 bsAb Ly2915 and Ly2323, the dual CD3 / PD-L1 antibody Ly3060, containing a dual immunomodulatory construct for anti-CEA, also demonstrated stronger anti-tumor activity. Figure 21 As shown.

[0284] Example 6: Evaluation of multispecific antibodies targeting BCMA

[0285] The antitumor efficacy of bispecific antibodies targeting BCMA and other immunomodulators has been evaluated in clinical trials. The exemplary anti-BCMA multispecific antibodies described herein, comprising dual anti-CD3 / CD137 or anti-CD / PD-L1 immunomodulatory constructs, are derived in accordance with the disclosures herein and include Ly2949 (anti-BCMA / CD3 / CD137), Ly2959 (anti-BCMA / CD3 / PD-L1), Ly3096 (anti-BCMA / CD3 / CD137), and Ly3106 (anti-BCMA / CD3 / PD-L1).

[0286] (i) Binding to target antigen

[0287] The binding of exemplary anti-BCMA multispecific antibodies to their corresponding target antigens was assessed using FCM or ELISA. Results are summarized in Table 8 below. Figure 22A-22R The multispecific antibody exhibited high binding affinity to the TAA target BCMA, while its binding affinity to immune target antigens CD3 and CD137 or PD-L1 was significantly lower.

[0288] Table 8. Target binding of anti-BCMA multispecific antibodies

[0289]

[0290] Note: N / A = Not applicable

[0291] (ii) Reporter gene assay

[0292] The activation of CD3 signaling by anti-BCMA multispecific antibodies was evaluated using a reporter gene system, and the results are summarized in Table 9 below. Figures 23A-23H The presence of BCMA target antigens significantly increases CD3 activation by all anti-BCMA multispecific antibodies. Binding to secondary immune targets (such as CD137 or PD-L1) enhances CD3 activation by the corresponding multispecific antibodies.

[0293] Table 9. Activation of CD3 signaling by anti-BCMA multispecific antibodies

[0294]

[0295] The activation of CD137 signaling by anti-BCMA multispecific antibodies was evaluated using a reporter gene system, and the results are summarized in Table 10 below. Figures 24A-24F middle.

[0296] Table 10. Activation of CD137 signaling by anti-BCMA multispecific antibodies

[0297]

[0298] Note: N / A = Not applicable

[0299] Multispecific antibodies exhibit minimal activation of the immune receptors CD3 or CD137, but this activation is significantly increased under cross-linking conditions, supporting the designed affinity-mediated effects.

[0300] (iv) Tumor cell killing and anti-tumor effects

[0301] Tumor cell killing induced by exemplary anti-BCMA multispecific antibodies was studied in vitro. Antitumor efficacy was investigated in syngeneic mouse models of bone marrow transplantation using PBMCs implanted with BCMA-expressing NCI-H929 and MM1.R cells, or in syngeneic mouse models using LL2 and EL4 cells overexpressing human BCMA. Clones Ly2949, Ly3096, Ly2959, and Ly3106, containing dual anti-CD3 / CD137 or anti-CD3 / PD-L1 dual immunomodulatory constructs, demonstrated stronger antitumor activity compared to anti-BCMA / CD3 bsAbLy2951, Ly2314, Ly2313, or Ly3098. See also Figure 25-29 .

[0302] Example 7: Effects of Fc variants in antibodies

[0303] This example explores the effects of Fc variants (e.g., 237 deletion, L234A, L235A, P329G substitution, or combinations thereof) contained in antibodies as provided herein.

[0304] A CD3 reporter assay was performed to detect any cross-linking effect of the multispecific antibody tested in this study, via the binding of the Fc fragment contained in the multispecific antibody to the Fc receptor. The CD3 reporter assay used in this example involved Jurkat / NFAT-Luc2P cells (Jurkat cells expressing a luciferase reporter gene driven by the NFAT response element). Briefly, Jurkat / NFAT-Luc2P cells were harvested and aliquoted at 50,000 cells / well into 96-well plates and co-cultured with or without Fc receptor-expressing cells. The test antibody was added, and the plates were incubated at 37°C for an additional 6 hours, followed by a Bright-Glo™ luciferase assay (Promega catalog number E2620). In this assay, NFAT-mediated luminescence corresponds to antibody activation of CD3 in the absence or presence of the Fc receptor.

[0305] (i) The effect of Fc variants in anti-CD3 monoclonal antibodies

[0306] Exemplary anti-CD3 monoclonal antibodies derived from Ly1761 (IgG1 wt) with one or more mutations in their Fc fragment were tested in CD3 reporter gene assays, including Ly2863 (237 deletion), Ly2864 (P329G), Ly2865 (237 deletion + P329G), Ly2873 (L234A + L235A + 237 deletion), and Ly1761 (IgG1 wt). Figures 30A-30F As shown, Fc variants with a combination of 237 deletion and P329G substitution do not exhibit crosslinking effects due to Fc-FcR binding elimination, while Fc variants carrying individual mutations (individual 237 deletion and individual P329G substitution) or a combination of L234A+ L235A+ 237 deletion maintain a certain level of crosslinking effect when co-cultured with CHO cells expressing, for example, FCGRI or FCGRIIA.

[0307] (ii) Fc variant effect in multispecific antibodies

[0308] Exemplary multispecific antibodies Ly2600 (anti-B7H3 / CD3 / CD137) and Ly2601 (anti-CD19 / CD3 / CD137), both containing a 237 deletion and a P329G mutation, were tested in a CD3 reporter gene assay. Sequence information for these two antibody clones is provided in Table 2 below. Clones Ly1963 and Ly1967 were used as controls, each containing the same antigen-binding moiety as Ly2600 and Ly2601, along with substitutions for L234A, L235A, and the 237 deletion, respectively. Figure 31 and 32 As shown, the mutation of the combination of 237 deletion and P329G in the Fc variant fragment eliminates the crosslinking effect generated by Fc-FcR binding; such complete elimination of the crosslinking effect was not observed in Fc variants containing the combination of L234A+ L235A+ 237 deletion when co-cultured with FCGRIIA, as... Figure 31 D and Figure 32 As shown in D.

[0309] sequence list

[0310] Table 1: Components in Bispecific / Multispecific Antibodies

[0311]

[0312] Table 2: Sequences of exemplary bi- and multi-specific antibodies

[0313]

[0314] Other embodiments

[0315] All features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Therefore, unless otherwise expressly stated, each disclosed feature is merely an example of a general series of equivalent or similar features.

[0316] From the above description, those skilled in the art can readily determine the essential characteristics of the present invention, and various changes and modifications can be made to adapt it to various uses and conditions without departing from the spirit and scope of the invention. Therefore, other embodiments are also within the scope of the claims.

[0317] equivalent

[0318] Although several embodiments of the invention have been described and illustrated herein, those skilled in the art will readily conceive of various other means and / or structures for performing functions and / or obtaining results and / or one or more of the advantages described herein, and each of such variations and / or modifications is considered to be within the scope of the embodiments of the invention described herein. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are exemplary, and actual parameters, dimensions, materials, and / or configurations will depend on one or more specific applications using the teachings of this invention. Those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments of the invention described herein using only conventional experimentation. Therefore, it should be understood that the foregoing embodiments are presented by way of example only, and that embodiments of the invention may be practiced in ways other than those specifically described and claimed within the scope of the appended claims and their equivalents. The embodiments of the invention disclosed herein relate to each individual feature, system, article of manufacture, material, kit, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles of manufacture, materials, kits, and / or methods is included within the scope of this disclosure if such features, systems, articles of manufacture, materials, kits, and / or methods do not contradict each other.

[0319] It should be understood that all definitions, as defined and used herein, take precedence over dictionary definitions, definitions in referenced documents, and / or the general meaning of the qualified terms.

[0320] All references, patents and patent applications disclosed herein are incorporated by reference in relation to their respective subjects, and in some cases may cover the entire document.

[0321] Unless otherwise expressly stated otherwise, the indefinite articles “a” and “an” as used herein in the specification and claims shall be understood to mean “at least one (type)”.

[0322] As used herein, the phrase “and / or” in the specification and claims should be understood to mean “any one or both” of the elements so combined, i.e., elements that exist jointly in some cases and separately in others. Multiple elements listed with “and / or” should be interpreted in the same way, i.e., “one or more” of the elements so combined. Other elements may optionally be present, whether related to or unrelated to those specifically identified by the “and / or” clause. Thus, as a non-limiting example, in one embodiment, when used in conjunction with open-ended language such as “comprising,” a reference to “A and / or B” may refer only to A (optionally including elements other than B); in another embodiment, only to B (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); and so on.

[0323] As used herein in the specification and claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when items are listed separately in a list, “or” or “and / or” should be interpreted as inclusive, that is, including several elements or at least one of the elements in the list, but also including more than one, and optionally including additional unlisted items. Only terms that clearly indicate otherwise, such as “only one of…” or “exact one of…” or “consisting of…” when used in the claims, will refer to the inclusion of multiple elements or exactly one of the elements in the list. Generally, when preceded by an exclusive term (such as “any,” “one of,” “only one of,” or “exact one of”), the term “or” as used herein should be interpreted only to indicate an exclusive alternative (i.e., “one or the other, but not both”). When used in the claims, “consisting substantially of…” should have its ordinary meaning as used in the field of patent law.

[0324] As used herein in the specification and claims, the phrase "at least one" relating to a list having one or more elements should be understood to mean at least one element selected from any one or more elements in the element list, but not necessarily including at least one of every element specifically listed in the element list, and does not exclude any combination of elements in the element list. This definition also allows for the optional presence of elements other than those specifically identified in the element list referred to by the phrase "at least one," whether related to or unrelated to those specifically identified elements. Thus, as a non-limiting example, in one embodiment, "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") may refer to at least one, optionally including more than one A and without B (and optionally including elements other than B); in another embodiment, it refers to at least one, optionally including more than one B and without A (and optionally including elements other than A); in yet another embodiment, it refers to at least one, optionally including more than one A, and refers to at least one, optionally including more than one B (and optionally including other elements); and so on.

[0325] It should also be understood that, unless explicitly stated otherwise, in any method claimed herein that comprises more than one step or operation, the order of the steps or operations of the method is not necessarily limited to the order of the steps or operations of the method as described herein.

Claims

1. A multispecific antibody comprising: (i) Binding to the first antigen-binding portion of CD3, wherein the first antigen-binding portion comprises a first heavy chain variable region (V H ) and the first light chain variable region (V L The first Fv segment; (ii) A second antigen-binding portion that binds to CD137 or PD-L1, wherein the second antigen-binding portion comprises a second V H Second V L The second Fv segment; (iii) An Fc fragment connecting the first antigen-binding portion and the second antigen-binding portion, wherein the Fc fragment comprises a hinge domain and a CH2 domain, and wherein the Fc fragment comprises a deletion at position 237 and an amino acid substitution at position P329, optionally P329G, following the EU numbering system; and (iv) A third antigen-binding portion of a first tumor-associated antigen; wherein the third antigen-binding portion is optionally linked to the first antigen-binding portion via a first peptide linker.

2. The multispecific antibody of claim 1, further comprising (v) a fourth antigen-binding portion that binds to a second tumor-associated antigen; wherein the fourth antigen-binding portion is optionally linked to the first antigen-binding portion via a second peptide linker.

3. The multispecific antibody according to claim 1 or claim 2, wherein the Fc fragment of (iii) is an IgG1Fc fragment, which optionally contains the amino acid sequence of SEQ ID NO:

91.

4. The multispecific antibody according to any one of claims 1 to 3, wherein the Fc fragment further comprises a CH3 domain.

5. The multispecific antibody of claim 4, wherein the CH3 domain comprises one or more mutations relative to the wild-type counterpart, the one or more mutations causing heterodimerization of the Fc fragment comprising the one or more mutations to be enhanced compared to homodimerization and / or reducing protein A binding.

6. The multispecific antibody according to claim 5, wherein the CH3 domain comprises the amino acid sequence of SEQ ID NO: 93, 94 or 95.

7. The multispecific antibody according to any one of claims 1 to 6, wherein the first V H Contains the same heavy chain CDR as the heavy chain complementarity determination region (CDR) in SEQ ID NO: 7, and the first V L Contains the same light chain CDR as the light chain CDR in SEQ ID NO:

8.

8. The multispecific antibody according to claim 7, wherein the first V H Contains the amino acid sequence of SEQ ID NO: 7, and the first V L It contains the amino acid sequence of SEQ ID NO:

8.

9. The multispecific antibody according to any one of claims 1 to 8, wherein the second antigen-binding portion binds to CD137.

10. The multispecific antibody according to claim 9, wherein the second V H Contains the same heavy chain CDR as the heavy chain complementarity determination region (CDR) in SEQ ID NO: 9, and the second V L Contains the same light chain CDR as the light chain CDR in SEQ ID NO:

10.

11. The multispecific antibody according to claim 10, wherein the second V H Contains the amino acid sequence of SEQ ID NO: 9, and the second V L It contains the amino acid sequence of SEQ ID NO:

10.

12. The multispecific antibody according to claim 9, comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 3 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:

4.

13. The multispecific antibody according to any one of claims 1 to 8, wherein the second antigen-binding portion binds to PD-L1.

14. The multispecific antibody according to claim 13, wherein the second V H Contains the same heavy chain CDR as the heavy chain complementarity determination region (CDR) in SEQ ID NO: 11, and the second V L Contains the same light chain CDR as the light chain CDR in SEQ ID NO:

12.

15. The multispecific antibody according to claim 14, wherein the second V H Contains the amino acid sequence of SEQ ID NO: 11, and the second V L It contains the amino acid sequence of SEQ ID NO:

12.

16. The multispecific antibody according to claim 9, comprising a first polypeptide and a second polypeptide, the first polypeptide comprising the amino acid sequence of SEQ ID NO: 5, and the second polypeptide comprising the amino acid sequence of SEQ ID NO:

6.

17. The multispecific antibody according to any one of claims 1 to 16, wherein the first tumor-associated antigen is selected from the group consisting of: B7H3, CD19, CD20, PSMA, HER2, CEA, BCMA, P53mut, DLL3, MET, and EGFR.

18. The multispecific antibody according to any one of claims 2 to 17, wherein the second tumor-associated antigen is selected from the group consisting of: B7H3, CD19, CD20, PSMA, HER2, CEA, BCMA, P53mut, DLL3, MET, and EGFR; optionally wherein the second tumor-associated antigen is the same as the first tumor-associated antigen.

19. The multispecific antibody according to claim 17 or claim 18, wherein the first tumor-associated antigen and / or the second tumor-associated antigen is HER2, CEA, BCMA, B7H3 or CD19; optionally wherein the first tumor-associated antigen and / or the second tumor-associated antigen is HER2, CEA or BCMA.

20. The multispecific antibody according to any one of claims 2 to 19, wherein the third antigen-binding portion and the fourth antigen-binding portion bind to different epitopes of the tumor-associated antigen.

21. The multispecific antibody according to any one of claims 1 to 20, wherein the third antigen-binding portion and optionally the fourth antigen-binding portion are Fab fragments.

22. The multispecific antibody according to any one of claims 1 to 21, wherein the multispecific antibody binds to: (a) HER2, CD3, and CD137; (b) HER2, CD3, and PD-L1; (c) CEA, CD3 and CD137; (d) CEA, CD3 and PD-L1; (e) BCMA, CD3, and CD137; and (f) BCMA, CD3 and PD-L1.

23. The multispecific antibody according to claim 9, comprising: (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 23, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 24, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 25, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 26; (ii) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 39, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 40, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 41; (iii) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 55, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 56, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 57; (iv) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 58, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 59, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 60; (v) A first polypeptide containing the amino acid sequence of SEQ ID NO: 54, a second polypeptide containing the amino acid sequence of SEQ ID NO: 63, a third polypeptide containing the amino acid sequence of SEQ ID NO: 66, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 69; (vi) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 74, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 77, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 80, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 83; (vii) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 74, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 63, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 80, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 69; (viii) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 54, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 102, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 103, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 104; or (ix) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 54, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 63, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 103, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO:

69.

24. The multispecific antibody according to claim 13, comprising: (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 27, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 28, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 25, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 26; (ii) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 46, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 47, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 41; (iii) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 67, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 68, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 60; (iv) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 81, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 82, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 57; (v) A first polypeptide containing the amino acid sequence of SEQ ID NO: 105, a second polypeptide containing the amino acid sequence of SEQ ID NO: 106, a third polypeptide containing the amino acid sequence of SEQ ID NO: 66, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 69; (vi) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 107, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 108, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 80, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 83; (vii) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 107, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 106, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 80, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 69; (viii) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 105, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 109, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 103, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 104; or (ix) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 105, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 106, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 103, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO:

69.

25. A nucleic acid or nucleic acid group that collectively encodes a multispecific antibody according to any one of claims 1 to 24.

26. The nucleic acid or nucleic acid group according to claim 25, wherein it is an expression vector or expression vector group.

27. A host cell comprising the nucleic acid or nucleic acid group according to claim 25 or claim 26.

28. The host cell according to claim 27, wherein it is a mammalian host cell.

29. A method for generating multispecific antibodies, the method comprising: (i) Culture the host cells according to claim 27 or claim 28 under conditions that allow expression of the antibody; as well as (ii) Collect the antibodies generated therefrom.

30. A pharmaceutical composition comprising a multispecific antibody or a nucleic acid or group of nucleic acids encoding said multispecific antibody according to any one of claims 1 to 24, and a pharmaceutically acceptable carrier.

31. A method for modulating an immune response in a subject, the method comprising administering to a subject in need an effective amount of a multispecific antibody according to any one of claims 1 to 24, a nucleic acid encoding the multispecific antibody, or a pharmaceutical composition comprising the antibody or encoding a nucleic acid.

32. The method of claim 31, wherein the subject is a human patient who has or is suspected of having cancer.

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