Trivalent and trispecific antibody constructs and methods of use thereof

Abstract

The present disclosure provides trivalent and trispecific antibody constructs capable of binding to two different antigens on one or more cytotoxic effector cells and a tumor-associated antigen (TAA) on a tumor cell. Pharmaceutical compositions comprising such antibody constructs, as well as methods of preparing and using such constructs and compositions (e.g., for the treatment of cancer), are also disclosed.

Description

[Technical Field]

[0001] cross reference This application claims priority to and benefit of U.S. Provisional Application No. 63 / 591,311, filed October 18, 2023, U.S. Provisional Application No. 63 / 465,137, filed May 9, 2023, U.S. Provisional Application No. 63 / 458,852, filed April 12, 2023, and U.S. Provisional Application No. 63 / 417,542, filed October 19, 2022, the entire contents of each of which are incorporated herein by reference for all purposes.

[0002] Technical Field The present disclosure generally relates to trivalent and trispecific trispecific T cell engaging antibody constructs that may include a first binding domain capable of binding to a first antigen on a first cytotoxic effector cell, a second binding domain capable of binding to a second antigen on a second cytotoxic effector cell, and a third binding domain capable of binding to a tumor-associated antigen (TAA) on a tumor cell. [Background technology]

[0003] Despite significant advances in cancer treatment over the past few decades, cancer continues to pose significant unmet medical needs. While current standard of care and more recently developed anticancer therapies have demonstrated some clinical progress, various indications, such as those with low T-cell tumor infiltration, still present significant clinical challenges. Summary of the Invention

[0004] In various embodiments, the present disclosure describes trivalent and trivalent antibody constructs capable of engaging two different antigens on one or more immune cells (e.g., T cells) and an antigen (e.g., a TAA) on a tumor cell. In certain embodiments of the present disclosure, the present specification describes trivalent and trispecific antibody constructs comprising three binding domains, the first binding domain being capable of binding to a first antigen on a first cytotoxic effector cell, a second binding domain being capable of binding to a second antigen on a second cytotoxic effector cell, and a third binding domain being capable of binding to a tumor-associated antigen (TAA) on a tumor cell.

[0005] In one embodiment, described herein is an antibody construct comprising: (i) a Fab domain capable of binding to a first antigen on a first cytotoxic effector cell; (ii) a first scFv domain and a second scFv domain, wherein one of the scFv domains is capable of binding to a second antigen on a second cytotoxic effector cell and the other scFv domain is capable of binding to a tumor-associated antigen (TAA) on a tumor cell; and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the first and second antigens on the one or more cytotoxic effector cells are different; (b) the Fab domain is linked to the N-terminus of the first Fc polypeptide; and (c) each of the first and second scFv domains is independently linked to either the N-terminus of the Fab domain, the C-terminus of the Fab domain, or the N-terminus of the second Fc polypeptide.

[0006] In one embodiment, described herein is an antibody construct comprising: (i) a Fab domain capable of binding to CD3 on a first cytotoxic effector cell; (ii) a first scFv domain and a second scFv domain, wherein one of the scFv domains is capable of binding to CD28 on a second cytotoxic effector cell and the other scFv domain is capable of binding to a tumor-associated antigen (TAA) on a tumor cell; and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of the first Fc polypeptide; and (b) each of the first and second scFv domains is independently linked to either the N-terminus of the Fab domain, the C-terminus of the Fab domain, the C-terminus of one of the Fc polypeptides, or the N-terminus of the second Fc polypeptide.

[0007] In one embodiment, described herein is an antibody construct comprising: (i) a Fab domain capable of binding to CD28 on a first cytotoxic effector cell; (ii) a first scFv domain and a second scFv domain, wherein one of the scFv domains is capable of binding to CD3 on a second cytotoxic effector cell and the other of the scFv domains is capable of binding to a tumor-associated antigen (TAA) on a tumor cell; and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of the first Fc polypeptide; and (b) each of the first and second scFv domains is independently linked to either the N-terminus of the Fab domain, the C-terminus of the Fab domain, the C-terminus of one of the Fc polypeptides, or the N-terminus of the second Fc polypeptide.

[0008] In one embodiment, provided herein is an antibody construct comprising: (i) a Fab domain capable of binding to CD3 on a first cytotoxic effector cell; (ii) a first scFv domain and a second scFv domain, wherein the first scFv domain is capable of binding to CD28 on a second cytotoxic effector cell and the second scFv domain is capable of binding to a tumor-associated antigen (TAA) on a tumor cell; and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of the first Fc polypeptide; and (b) the first scFv domain is linked to the V-terminus of the Fab domain. H and (c) a second scFv domain is linked to the N-terminus of a second Fc polypeptide. An antibody construct according to such an embodiment is shown, for example, in Figure 1A.

[0009] In one embodiment, provided herein is an antibody construct comprising: (i) a Fab domain capable of binding to CD28 on a first cytotoxic effector cell; (ii) a first scFv domain and a second scFv domain, wherein the first scFv domain is capable of binding to CD3 on a second cytotoxic effector cell and the second scFv domain is capable of binding to a tumor-associated antigen (TAA) on a tumor cell; and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of the first Fc polypeptide; and (b) the first scFv domain is linked to the V-terminus of the Fab domain. H and (c) a second scFv domain is linked to the N-terminus of a second Fc polypeptide. An antibody construct according to such an embodiment is shown, for example, in Figure 1A.

[0010] In one embodiment, provided herein is an antibody construct comprising: (i) a Fab domain capable of binding to CD3 on a first cytotoxic effector cell; (ii) a first scFv domain and a second scFv domain, wherein the first scFv domain is capable of binding to CD28 on a second cytotoxic effector cell and the second scFv domain is capable of binding to a tumor-associated antigen (TAA) on a tumor cell; and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of the first Fc polypeptide; and (b) the first scFv domain is linked to the C-terminus of the light chain of the Fab domain. L and (c) a second scFv domain is linked to the N-terminus of a second Fc polypeptide. An antibody construct according to such an embodiment is shown, for example, in Figure IB.

[0011] In one embodiment, provided herein is an antibody construct comprising: (i) a Fab domain capable of binding to CD28 on a first cytotoxic effector cell; (ii) a first scFv domain and a second scFv domain, wherein the first scFv domain is capable of binding to CD3 on a second cytotoxic effector cell and the second scFv domain is capable of binding to a tumor-associated antigen (TAA) on a tumor cell; and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of the first Fc polypeptide; and (b) the first scFv domain is linked to the C-terminus of the light chain of the Fab domain. L and (c) a second scFv domain is linked to the N-terminus of a second Fc polypeptide. An antibody construct according to such an embodiment is shown, for example, in Figure IB.

[0012] In one embodiment, the present invention provides an antibody construct comprising: (i) a Fab domain capable of binding to CD3 on a first cytotoxic effector cell; (ii) a first scFv domain and a second scFv domain, wherein the first scFv domain is capable of binding to CD28 on a second cytotoxic effector cell and the second scFv domain is capable of binding to a tumor-associated antigen (TAA) on a tumor cell; and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of the first Fc polypeptide, (b) the first scFv domain is linked to the C-terminus of the first Fc polypeptide, and (c) the second scFv domain is linked to the N-terminus of the second Fc polypeptide. An antibody construct according to such an embodiment is shown, for example, in FIG. 1F.

[0013] In one embodiment, the present invention provides an antibody construct comprising: (i) a Fab domain capable of binding to CD28 on a first cytotoxic effector cell; (ii) a first scFv domain and a second scFv domain, wherein the first scFv domain is capable of binding to CD3 on a second cytotoxic effector cell and the second scFv domain is capable of binding to a tumor-associated antigen (TAA) on a tumor cell; and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of the first Fc polypeptide, (b) the first scFv domain is linked to the C-terminus of the first Fc polypeptide, and (c) the second scFv domain is linked to the N-terminus of the second Fc polypeptide. An antibody construct according to such an embodiment is shown, for example, in FIG. 1F.

[0014] In one embodiment, provided herein is an antibody construct comprising: (i) a Fab domain capable of binding to CD3 on a first cytotoxic effector cell; (ii) a first scFv domain and a second scFv domain, wherein the first scFv domain is capable of binding to CD28 on a second cytotoxic effector cell and the second scFv domain is capable of binding to a tumor-associated antigen (TAA) on a tumor cell; and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of the first Fc polypeptide, (b) the first scFv domain is linked to the C-terminus of the first Fc polypeptide, and (c) the second scFv domain is linked to the V of the Fab domain. H

[0023] Antibody constructs are described in which the N-terminus of a domain is linked to the N-terminus of the domain. An antibody construct according to such an embodiment is shown, for example, in Figure 1C.

[0015] In one embodiment, provided herein is an antibody construct comprising: (i) a Fab domain capable of binding to CD28 on a first cytotoxic effector cell; (ii) a first scFv domain and a second scFv domain, wherein the first scFv domain is capable of binding to CD3 on a second cytotoxic effector cell and the second scFv domain is capable of binding to a tumor-associated antigen (TAA) on a tumor cell; and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of the first Fc polypeptide, (b) the first scFv domain is linked to the C-terminus of the first Fc polypeptide, and (c) the second scFv domain is linked to the V of the Fab domain. H

[0023] Antibody constructs are described in which the N-terminus of a domain is linked to the N-terminus of the domain. An antibody construct according to such an embodiment is shown, for example, in Figure 1C.

[0016] In some embodiments, described herein are pharmaceutical compositions comprising a trivalent and trispecific antibody construct of the present disclosure and a pharmaceutically acceptable carrier, excipient, diluent, or combination thereof.

[0017] In some embodiments, described herein are nucleic acid molecules or sets of nucleic acid molecules that encode one or more, two or more, or three or more polypeptide chains that form the trivalent and trispecific antibody constructs of the present disclosure.

[0018] In some embodiments, described herein are vectors or sets of vectors comprising a nucleic acid molecule or set of nucleic acid molecules encoding one or more, two or more, or three or more polypeptide chains of the trivalent and trispecific antibody constructs of the present disclosure.

[0019] In certain embodiments, the present disclosure relates to a method of producing a trivalent and trispecific antibody construct, the method comprising: (a) obtaining a host cell culture comprising at least one host cell that comprises one or more nucleic acid molecules encoding one or more, two or more, or three or more polypeptide chains that form the antibody construct; and (b) recovering the antibody construct from the host cell culture.

[0020] In certain embodiments, the present disclosure relates to a method of eliciting an anti-tumor immune response in a cell population comprising immune cells and tumor cells expressing a TAA, the method comprising contacting the cell population with an effective amount of a trivalent and trispecific antibody construct of the present disclosure, wherein the immune cells express a first and a second antigen and the tumor cells express the TAA.

[0021] In certain embodiments, the present disclosure relates to a method of inhibiting the growth of tumor cells that express a TAA, the method comprising contacting a cell population comprising tumor cells and immune cells with an effective amount of a trivalent and trispecific antibody construct of the present disclosure, wherein the immune cells express a first and a second antigen, and the tumor cells express the TAA.

[0022] In certain embodiments, the present disclosure relates to a method of killing tumor cells that express a TAA, the method comprising contacting a cell population comprising tumor cells and immune cells with an effective amount of a trivalent and trispecific antibody construct of the present disclosure, wherein the immune cells express a first and a second antigen, and the tumor cells express the TAA.

[0023] In some of these embodiments, the first antigen can be CD3 or CD28, the second antigen can be CD3 or CD28, and the first and second antigens are different antigens. Further, in some embodiments, the TAA can be MSLN or Cldn18.2.

[0024] In certain embodiments, the present disclosure relates to methods of treating cancer in a subject in need thereof, the method comprising administering to the subject a trivalent and trispecific antibody construct of the present disclosure, or a medicament comprising a trivalent and trispecific antibody construct of the present disclosure. In some embodiments, such methods may further comprise eliciting a cytotoxic immune response against the cancer in the subject with the trivalent and trispecific antibody construct, thereby treating the cancer in the subject.

[0025] In some embodiments, the present disclosure relates to trivalent and trispecific antibody constructs for use in the treatment of cancer.

[0026] In some embodiments, the present disclosure relates to a trivalent and trispecific antibody construct in the manufacture of a medicament for the treatment of cancer.

[0027] In some embodiments, provided herein is an antibody construct comprising a binding domain capable of binding to CD28, wherein the binding domain comprises a VHDR1 having the sequence SX1GVH (SEQ ID NO: 302), an HCDR2 having the sequence VIWX2GGX3TNFNSALMS (SEQ ID NO: 306), and an HCDR3 having the sequence DRAX4GX5YX6X7AMDY (SEQ ID NO: 312). HLCDR1 having the sequence RASESVEYYX8TSLMQ (SEQ ID NO: 315), and LCDR2 having the sequence AASX9VX 10 V comprising an LCDR2 having the sequence QQSRKVPFT (SEQ ID NO: 320), and an LCDR3 having the sequence QQSRKVPFT (SEQ ID NO: 321). L Antibody constructs are described that comprise the sequence, and have one or more of the following amino acid substitutions at the positions specified in the CDR sequences: X1: Y to A (i.e., residue Y is replaced by residue A), X2: P to A, X3: G to S, X4: S to Y, X5: N to A, X6: L to N, X7: S to Y, X8: G to V, X9: N to A, or X 10 :E to D.

[0028] In some embodiments, provided herein is an antibody construct comprising a binding domain capable of binding to Cldn18.2, wherein the binding domain comprises a VHCDR1 having the sequence SNPMI (SEQ ID NO: 333), an HCDR2 having the sequence IIDTDGSTYYADWAKG (SEQ ID NO: 334), and an HCDR3 having the sequence RLHGSSNGYYDDL (SEQ ID NO: 335). H and a V comprising an LCDR1 having the sequence QASQSIYSYLS (SEQ ID NO: 336), an LCDR2 having the sequence KASTLAS (SEQ ID NO: 337), and an LCDR3 having the sequence QQGYTVTNVDKNT (SEQ ID NO: 338). L Antibody constructs are described, including sequences. [Brief explanation of the drawings]

[0029] Implementations disclosed herein are illustrated by way of example, and not by way of limitation, in the accompanying drawings, which are merely illustrative and aid in understanding and are not intended as a definition of the limits of the antibody constructs, pharmaceutical compositions, and methods of the present disclosure.

[0030] [Figure 1A]1 shows a schematic diagram of the format and geometry of a trivalent and trispecific antibody construct comprising binding domains capable of engaging CD3, CD28, and a TAA according to an embodiment of the present disclosure. [Figure 1B] 1 shows a schematic diagram of the format and geometry of a trivalent and trispecific antibody construct comprising binding domains capable of engaging CD3, CD28, and a TAA according to an embodiment of the present disclosure. [Figure 1C] 1 shows a schematic diagram of the format and geometry of a trivalent and trispecific antibody construct comprising binding domains capable of engaging CD3, CD28, and a TAA according to an embodiment of the present disclosure. [Figure 1D] 1 shows a schematic diagram of the format and geometry of a trivalent and trispecific antibody construct comprising binding domains capable of engaging CD3, CD28, and a TAA according to an embodiment of the present disclosure. [Figure 1E] 1 shows a schematic diagram of the format and geometry of a trivalent and trispecific antibody construct comprising binding domains capable of engaging CD3, CD28, and a TAA according to an embodiment of the present disclosure. [Figure 1F] 1 shows a schematic diagram of the format and geometry of a trivalent and trispecific antibody construct comprising binding domains capable of engaging CD3, CD28, and a TAA according to an embodiment of the present disclosure. [Figure 1G] 1 shows a schematic diagram of the format and geometry of a trivalent and trispecific antibody construct comprising binding domains capable of engaging CD3, CD28, and a TAA according to an embodiment of the present disclosure. [Figure 1H] FIG. 1 shows a schematic diagram of the bispecific control construct used and described in this disclosure. [Figure 1I] FIG. 1 shows a schematic diagram of the bispecific control construct used and described in this disclosure. [Figure 1J] FIG. 1 shows a schematic diagram of the bispecific control construct used and described in this disclosure. [Figure 1K]FIG. 1 shows a schematic diagram of the bispecific control construct used and described in this disclosure. [Figure 1L] FIG. 1 shows a schematic diagram of the bispecific control construct used and described in this disclosure. [Figure 2A] Shown is binding of selected anti-CD3(Fab) / anti-CD28(scFv) / anti-MSLN(scFv) trivalent and trispecific constructs (v34914, v34915, v34916 and v34917) to CD4+ (left) and CD8+ (right) T cells as measured by flow cytometry. [Figure 2B] Shown is binding of selected anti-CD3 (scFv) / anti-CD28 (Fab) / anti-MSLN (scFv) trivalent and trispecific constructs (v34913 and v34918) to CD4+ (left) and CD8+ (right) T cells as measured by flow cytometry. [Figure 3A] We demonstrate that selected anti-CD3 (Fab) / anti-CD28 (scFv) / anti-MSLN (scFv) trivalent and trispecific constructs (v34914, v34915, v34916, and v34917) direct T cells from healthy donors to MSLN+H292 target cells and induce T cell-mediated killing of tumor cells. [Figure 3B] We demonstrate that defined anti-CD3 (scFv) / anti-CD28 (Fab) / anti-MSLN (scFv) trivalent and trispecific constructs (v34913 and v34918) direct T cells from healthy donors to MSLN+H292 target cells and induce T cell-mediated killing of tumor cells. [Figure 4A] Figure 1 shows that selected anti-CD3 (Fab) / anti-CD28 (scFv) / anti-MSLN (scFv) trivalent and trispecific constructs (v34914, v34915, v34916, and v34917) induced interleukin-2 (IL-2, left) and / or tumor necrosis factor alpha (TNFα, right) production from T cells when co-incubated with MSLN+H292 cells (2:1 E:T) for 72 hours (hr). [Figure 4B]Figure 1 shows that the given anti-CD3 (scFv) / anti-CD28 (Fab) / anti-MSLN (scFv) trivalent and trispecific constructs (v34913 and v34918) induced interleukin-2 (IL-2, left) and / or tumor necrosis factor alpha (TNFα, right) production from T cells when co-incubated with MSLN+H292 cells (2:1 E:T) for 72 hours (h). [Figure 5A] Figure 1 shows in vitro T cell-dependent cytotoxicity induced by the trivalent and trispecific antibody construct v34914 compared to the bispecific anti-(MSLN x CD3) benchmark control construct v34919 against MSLN-high H292 cells (152,986 MSLN / cell) and MSLN-low OVTOKO cells (9,752 MSLN / cell). [Figure 5B] Figure 1 shows in vitro T cell-dependent cytotoxicity induced by the trivalent and trispecific antibody construct v34916 compared to the bispecific anti-(MSLN x CD3) benchmark control construct v34919 against MSLN-high H292 cells (152,986 MSLN / cell) and MSLN-low OVTOKO cells (9,752 MSLN / cell). [Figure 5C] Figure 1 shows in vitro T cell-dependent cytotoxicity induced by the trivalent and trispecific antibody construct v34913 compared to the bispecific anti-(MSLN x CD3) benchmark control construct v34919 against MSLN-high H292 cells (152,986 MSLN / cell) and MSLN-low OVTOKO cells (9,752 MSLN / cell). [Figure 5D] Figure 1 shows in vitro T cell-dependent cytotoxicity induced by the trivalent and trispecific antibody construct v34918 compared to the bispecific anti-(MSLN x CD3) benchmark control construct v34919 against MSLN-high H292 cells (152,986 MSLN / cell) and MSLN-low OVTOKO cells (9,752 MSLN / cell). [Figure 6A]We demonstrate that trivalent and trispecific antibody constructs v34913, v34916, and v34918 directed T cells from healthy donors to kill MSLN+H292 target cells expressing moderate levels of MSLN. This long-term T cell-dependent cytotoxicity (TDCC) study utilized a low E:T ratio of 1:5 and a 3-day incubation period, e.g., long-term co-culture at a low E:T ratio to more accurately reflect conditions in a given (e.g., solid) tumor type. [Figure 6B] We demonstrate that trivalent and trispecific antibody constructs v34913, v34916, and v34918 directed T cells from healthy donors to kill MSLN+H292 target cells expressing moderate levels of MSLN. This long-term T cell-dependent cytotoxicity (TDCC) study utilized a low E:T ratio of 1:5 and a 7-day incubation period, e.g., long-term co-culture at a low E:T ratio to more accurately reflect conditions in a given (e.g., solid) tumor type. [Figure 7A] Figure 1 shows that trivalent and trispecific antibody constructs v34914, v34916, and v34917 containing anti-CD3 Fab domains induced proliferation of T cells (10:1 E:T) co-incubated with MSLN+OVCAR3 cells for 5 days when compared to control constructs v34919, v34927, and v31926. [Figure 7B] Figure 1 shows that trivalent and trispecific antibody constructs v34913 and v34918 containing anti-CD28 Fab domains induced proliferation of T cells (10:1 E:T) co-incubated with MSLN+OVCAR3 cells for 5 days when compared to control constructs v34919, v34927, and v31926. [Figure 7C] Shown are T cell proliferation data measured after 3, 5, and 7 days of co-culture with H292 cells (E:T ratio = 2:1) for the potent trivalent and trispecific construct v34913, the corresponding bispecific anti-(MSLN x CD3) control construct v34919, and the negative control v22277. [Figure 8A]Figure 1 shows the binding of selected trivalent and trispecific anti-CD3 / anti-CD28 / anti-Cldn18.2 antibody constructs to CLDN18.2+SNU601 cells as measured by flow cytometry. Both constructs containing anti-CD3 Fab and anti-CD28 scFv domains (v37633, v37634, and v37635) and those containing anti-CD28 Fab and anti-CD3 scFv domains (v37638, v37640, and v37642) were compared to an anti-(Cldn18.2 x CD3) bispecific construct (v37663), a one-arm anti-Cldn18.2 antibody (v37675), a benchmark control (v35923, also known as AMG910), an anti-Cldn18.2 monoclonal antibody (mAb), and the anti-RSV protein F mAb palivizumab. [Figure 8B] Figure 1 shows the binding of selected trivalent and trispecific anti-CD3 / anti-CD28 / anti-Cldn18.2 antibody constructs to CLDN18.2+SNU601 cells as measured by flow cytometry. Both constructs containing anti-CD3 Fab and anti-CD28 scFv domains (v37633, v37634, and v37635) and those containing anti-CD28 Fab and anti-CD3 scFv domains (v37638, v37640, and v37642) were compared to an anti-(Cldn18.2 x CD3) bispecific construct (v37663), a one-arm anti-Cldn18.2 antibody (v37675), a benchmark control (v35923, also known as AMG910), an anti-Cldn18.2 monoclonal antibody (mAb), and the anti-RSV protein F mAb palivizumab. [Figure 9A] Shown is binding of selected anti-CD3 (Fab) / anti-CD28 (scFv) / anti-Cldn18.2 (scFv) trivalent and trispecific constructs (v37633-v37637, Figure 9A) to CD4+ (left) and CD8+ (right) T cells as measured by flow cytometry. [Figure 9B]Shown is binding of selected anti-CD3 (scFv) / anti-CD28 (Fab) / anti-Cldn18.2 (scFv) trivalent and trispecific constructs (v37638-v37642, Figure 9B) to CD4+ (left) and CD8+ (right) T cells as measured by flow cytometry. [Figure 10A] We demonstrate that selected anti-CD3 (Fab) / anti-CD28 (scFv) / anti-Cldn18.2 (scFv) trivalent and trispecific constructs (v37633-v37637) directed T cells from healthy donors to kill CLDN18.2+SNU601 target cells. These trispecific constructs were compared against a bispecific anti-(Cldn18.2 x CD3) control (v37663), a bispecific anti-(Cldn18.2 x CD28) control (v37665), and a negative control (a Het-Fc version of palivizumab, v22277). [Figure 10B] We demonstrate that selected anti-CD3 (scFv) / anti-CD28 (Fab) / anti-Cldn18.2 (scFv) trivalent and trispecific constructs (v37638-v37642) directed T cells from healthy donors to kill CLDN18.2+SNU601 target cells. These trispecific constructs were compared against a bispecific anti-(Cldn18.2 x CD3) control (v37663), a bispecific anti-(Cldn18.2 x CD28) control (v37665), and a negative control (a Het-Fc version of palivizumab, v22277). [Figure 11A] Figure 1 shows that the selected anti-CD3 (Fab) / anti-CD28 (scFv) / anti-Cldn18.2 (scFv) trivalent and trispecific constructs (v37633-v37637) induced IL-2 (top) and / or TNFα (bottom) production from T cells co-incubated with CLDN18.2+SNU601 cells (2:1 E:T) for 72 hours. [Figure 11B]Figure 1 shows that the selected anti-CD3 (scFv) / anti-CD28 (Fab) / anti-Cldn18.2 (scFv) trivalent and trispecific constructs (v37638-v37642) induced IL-2 (top) and / or TNFα (bottom) production from T cells co-incubated with CLDN18.2+SNU601 cells (2:1 E:T) for 72 hours. [Figure 12A] Figure 1 shows in vitro T cell-dependent cytotoxicity induced by the trivalent and trispecific antibody construct v37633 against CLDN18.2-high SNU601 and CLDN18.2-low SKOV-3 target cells and compared to the bispecific anti-(MSLN x CD3) benchmark control construct v35923. [Figure 12B] Figure 1 shows in vitro T cell-dependent cytotoxicity induced by the trivalent and trispecific antibody construct v37634 against CLDN18.2-high SNU601 and CLDN18.2-low SKOV-3 target cells and compared to the bispecific anti-(MSLN x CD3) benchmark control construct v35923. [Figure 12C] Figure 1 shows in vitro T cell-dependent cytotoxicity induced by the trivalent and trispecific antibody construct v37638 against CLDN18.2-high SNU601 and CLDN18.2-low SKOV-3 target cells and compared to the bispecific anti-(MSLN x CD3) benchmark control construct v35923. [Figure 12D] Figure 1 shows in vitro T cell-dependent cytotoxicity induced by the trivalent and trispecific antibody construct v37642 against CLDN18.2-high SNU601 and CLDN18.2-low SKOV-3 target cells and compared to the bispecific anti-(MSLN x CD3) benchmark control construct v35923. [Figure 13A]Concentration-response curves for long-term (7-day) TDCC studies of tested anti-CLDN18.2 trivalent and trispecific constructs (v37633-v36735) containing anti-CD3 (Fab) / anti-CD28 (scFv) / anti-CLDN18.2 (scFv) are shown, demonstrating that the constructs directed T cells from healthy donors to kill CLDN18.2+ target cells expressing various levels of CLDN18.2 using a 1:1 E:T ratio. Top left: SNU-601 (276,125 CLDN18.2 / cell); top right: KATO-III (63,566 CLDN18.2 / cell); bottom: DAN-G (33,164 CLDN18.2 / cell). [Figure 13B] Concentration-response curves for long-term (7-day) TDCC studies for tested anti-CLDN18.2 trivalent and trispecific constructs (v37638, v37640, v37642) containing anti-CD3 (scFv) / anti-CD28 (Fab) / anti-Cldn18.2 (scFv) are shown, demonstrating that the constructs directed T cells from healthy donors to kill CLDN18.2+ target cells expressing various levels of CLDN18.2 using a 1:1 E:T ratio. Top left: SNU-601 (276,125 CLDN18.2 / cell); top right: KATO-III (63,566 CLDN18.2 / cell); bottom: DAN-G (33,164 CLDN18.2 / cell). [Figure 14A] Figure 1 shows that selected anti-CLDN18.2 trivalent and trispecific constructs (v37633-v36735) that contained anti-CD3 (Fab) / anti-CD28 (scFv) / anti-CLDN18.2 (scFv) were able to induce the production of several cytokines (i.e., IFNγ (top left), IL-2 (top right), TNFα (bottom)) in T cells in the presence of CLDN18.2 + target SNU601, compared to selected bispecific and benchmark control constructs, using a 72-hour incubation period and an E:T ratio of 2:1. [Figure 14B]Figure 1 shows that selected anti-CLDN18.2 trivalent and trispecific constructs (v37638, v37640, v37642) that comprised anti-CD3 (scFv) / anti-CD28 (Fab) / anti-CLDN18.2 (scFv) were able to induce the production of several cytokines (i.e., IFNγ (top left), IL-2 (top right), TNFα (bottom)) in T cells in the presence of CLDN18.2 + target SNU601, compared to selected bispecific and benchmark control constructs, using a 72 hour incubation period and an E:T ratio of 2:1. [Figure 15] 1 shows binding curves to CD3+CD28+ Jurkat cells for several anti-CLDN18.2 trivalent and trispecific constructs v37638, v37683, v37689, v37692, and v37694 containing anti-CD28 binding domains with mutation(s) in their VH or VL domains and therefore varying affinities for CD28 as measured by flow cytometry. [Figure 16] We show that several tested anti-CLDN18.2 trivalent and trispecific constructs, v37638, v37683, v37689, v37692, and v37694, with varying affinities for CD28, directed T cells from healthy donors to kill CLDN18.2+SNU601 cells, compared to bispecific (v37663) and negative (v22277) control constructs. [Figure 17A] We show that several tested anti-CLDN18.2 trivalent and trispecific constructs, v37638, v37683, v37689, v37692, and v37694, with varying affinities for CD28, induced IL-2 production from T cells (2:1 E:T) co-incubated with CLDN18.2+SNU601 cells for 72 hours, compared to bispecific (v37663) and negative (v22277) control constructs. [Figure 17B]Several tested anti-CLDN18.2 trivalent and trispecific constructs, v37638, v37683, v37689, v37692, and v37694, with varying affinities for CD28, induced TNFα production from T cells co-incubated with CLDN18.2+SNU601 cells (2:1 E:T) for 72 hours compared to bispecific (v37663) and negative (v22277) control constructs. [Figure 18] 1 shows upregulation of Bcl-xL expression in activated T cells using either the trivalent and trispecific antibody construct v37634 of the present disclosure or the selected bispecific control constructs. [Figure 19] 1 shows upregulation of T cell proliferation in activated T cells using either the trivalent and trispecific antibody construct v37634 of the present disclosure or the designated bispecific control constructs. [Figure 20] Figure 1 shows in vivo anti-tumor activity in donor X-engrafted mice treated with either the bispecific control constructs v35923 or v38417, or the trispecific and trivalent antibody construct v37634. [Figure 21] 20 shows the body weights of treated mice over the course of the in vivo efficacy study referenced in FIG. [Figure 22A] 1 shows a library of conventional agonist paratope variants with varying CD28 binding affinities as determined by SPR. [Figure 22B] 1 shows a library of agonist paratope variants with different CD3 binding affinities as determined by SPR. [Figure 22C] Shown is a diagram of the effect of paratope format (scFv vs. Fab) and geometry of an antibody construct on binding affinity to CD3 and CD28 for a subset of formats with the same CD3 and CD28 paratopes (left), and a diagram of affinity after CD3 and CD28 paratope engineering for one antibody construct format that can be transferred between formats to generate a large panel of antibody constructs (right). [Figure 23A] 1 shows that a representative trivalent and trispecific antibody construct, v37634, showed no significant change in purity after five cycles of freezing at −80° C. followed by thawing at 4° C. [Figure 23B] This shows that v37634 showed minimal change in purity after 14 days of incubation at 40° C. in an accelerated stress test. The construct concentration was 1 mg / mL. [Figure 23C] 1 shows that the trivalent and trispecific antibody construct v37634 showed no significant change in purity after 10 weeks of storage at −80° C. [Figure 23D] 1 shows that the trivalent and trispecific antibody construct v37634 showed no significant change in purity after 14 days of storage at 4° C. [Figure 23E] 1 shows that the trivalent and trispecific antibody construct v37634 did not show significant purity changes after 3 hours under low (pH 3.5) pH conditions. [Figure 23F] 1 shows that the trivalent and trispecific antibody construct v37634 did not show significant purity changes after 3 hours under high (pH 9) pH conditions. DETAILED DESCRIPTION OF THE INVENTION

[0031] Detailed Description In various embodiments, the present disclosure describes trivalent and trispecific antibody constructs capable of binding two different antigens located either on a cytotoxic effector cell (e.g., a T cell) or on two different cytotoxic effector cells (i.e., each antigen is located on a different cell), and a TAA on a tumor cell. Such antibody constructs may include a first binding domain capable of binding to a first antigen on a first cytotoxic effector cell, a second binding domain capable of binding to a second antigen on a second cytotoxic effector cell, and a third binding domain capable of binding to a TAA on a tumor cell. Thus, in certain embodiments, the present disclosure describes trivalent and trispecific T cell-engaging antibody constructs capable of costimulating one or more effector cells (e.g., T cells) by engaging two effector cell antigens (e.g., CD3 and CD28).

[0032] In some embodiments, and as further described herein, the trivalent and trispecific antibody constructs of the present disclosure may comprise (i) a fragment antigen binding (Fab) domain (i.e., first binding domain) capable of binding to a first antigen on a first cytotoxic effector cell, (ii) a first single-chain variable fragment (scFv) domain (i.e., second binding domain) capable of binding to a second antigen on a second cytotoxic effector cell, and (iii) a second scFv domain (i.e., third binding domain) capable of binding to a TAA on a tumor cell. Such trivalent and trispecific antibody constructs comprise a first Fc polypeptide and a second Fc polypeptide, and may further comprise Fc domains (e.g., heterodimeric Fc domains) in which the various binding domains are linked either directly (e.g., without a linker) or indirectly, e.g., via a linker and / or via another binding domain (e.g., in an example in which a first binding domain is indirectly linked to an Fc polypeptide via a second binding domain, e.g., the C-terminus of the first binding domain is linked to the N-terminus of the second binding domain, and such second binding domain is in turn linked to the N-terminus of the Fc polypeptide, thereby indirectly linking the first binding domain to the Fc polypeptide).

[0033] As further described herein, Figures 1A-1G illustrate certain antibody construct geometries having different relative orientations of the three binding domains according to certain embodiments of the present disclosure.

[0034] Further described herein are pharmaceutical compositions comprising one or more of the trivalent and trispecific antibody constructs disclosed herein.

[0035] Other embodiments of the present disclosure relate to a nucleic acid molecule or set of nucleic acid molecules that encode one or more (e.g., two, three, or more) polypeptide chains of the antibody constructs described herein. Some embodiments relate to a vector or set of vectors that comprise a nucleic acid molecule or set of nucleic acid molecules that encode an antibody construct of the present disclosure.

[0036] Other embodiments of the present disclosure describe methods of making and using the trivalent and trispecific antibody constructs described herein, e.g., for the treatment of cancer in a subject (e.g., a rodent or a human).

[0037] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0038] The term "about," when used herein in connection with a numerical value or range, typically refers to ±10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or ±1% of the stated or claimed numerical value or range, unless otherwise specified. In various embodiments, the term "about" refers to approximately a ±10% variation from a given value or range. In other embodiments, the term "about" refers to approximately a ±5% variation from a given value or range. In still other embodiments, the term "about" refers to approximately a ±1% variation from a given value or range. It should be understood that such a variation is always included in any given value provided herein, whether or not it is specifically mentioned.

[0039] The use of the terms "a" or "an," when used herein in conjunction with the term "comprising," can mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0040] As used herein, the terms "comprising," "having," "including," and "containing," and grammatical variants thereof, are inclusive or open-ended and do not exclude additional, unrecited elements and / or method steps. The term "consisting essentially of," when used herein in conjunction with a construct, composition, use, or method, indicates that additional features, elements, and / or method steps may be present, but that these additions do not materially affect the manner in which the described construct, composition, method, or use functions. The term "consisting of," when used herein in conjunction with a construct, composition, use, or method, excludes the presence of additional elements and / or method steps. An antibody construct, composition, use, or method described herein as comprising certain elements and / or steps may also, in certain embodiments, consist essentially of those elements and / or steps, and in other embodiments, consist of those elements and / or steps (whether or not these embodiments are specifically mentioned).

[0041] The terms "subject" and "patient" are used interchangeably herein and generally refer to an animal in need of treatment. The animal in need of treatment can be a human or a non-human animal, e.g., a mammal, a bird, or a fish. In certain embodiments, the subject or patient is a mammal. In some embodiments, the subject is a human. In other embodiments, the subject is a rodent or a non-human primate.

[0042] In terms of a particular result to be achieved, an "effective amount" of a trivalent and trispecific antibody construct described herein, or a pharmaceutical composition comprising such an antibody construct, is an amount sufficient to achieve the desired result. For example, an "effective amount" of an antibody construct or pharmaceutical composition, when referred to in terms of killing cancer cells, refers to the amount of the antibody construct or composition comprising the antibody construct sufficient to produce the killing effect.

[0043] Unless otherwise specified, the terms "Fc region," "Fc," and "Fc domain" are used interchangeably herein to refer to the C-terminal region of an immunoglobulin (Ig) heavy chain containing at least a portion of the constant region. In various embodiments, the Fc domain herein may be a dimer. Such a dimeric Fc domain may comprise a first Fc polypeptide and a second Fc polypeptide, and each Fc polypeptide may comprise a CH2 domain and a CH3 domain. Such a dimeric Fc may be either a homodimer, i.e., a first and second Fc polypeptide having identical amino acid sequences, or a heterodimer, i.e., a first and second Fc polypeptide having different amino acid sequences, e.g., sequences sharing about 95%, 96%, 97%, 98%, or about 99% sequence identity. In some embodiments, the antibody constructs of the present disclosure comprise a homodimeric Fc domain. In yet other embodiments, and as further described herein, the antibody construct comprises a heterodimeric Fc domain wherein at least one of the CH2 and / or CH3 domains of the first and second Fc polypeptides have amino acid sequences that share less than about 99%, less than about 98%, or less than about 97% sequence identity.

[0044] The term "multispecific," as used herein in the context of an antibody construct, refers to a biologically functional protein (e.g., an antibody construct described herein) that is "at least bispecific." That is, it comprises at least a first binding domain and a second binding domain, and such first and second binding domains can specifically bind to two distinct epitopes, e.g., a first epitope and a second epitope. Such first and second epitopes can be located on the same antigen or on different antigens (e.g., a first epitope on Cluster of Differentiation 3 (CD3) or Cluster of Differentiation 28 (CD28) and a second epitope on a TAA). Thus, in some embodiments, an antibody construct according to the present disclosure can comprise specificity for at least two different antigens or for at least three different antigens or targets. Thus, the term "multispecific", in the context of antibody constructs herein, encompasses antibody constructs that are at least bispecific (i.e., containing two binding domains with specificity for two different antigens or targets), or at least trispecific (i.e., containing three binding domains with specificity for three different antigens or targets, e.g., CD3, CD28, and a TAA).

[0045] The term "trispecific," as used herein in the context of an antibody construct, refers to a biologically functional protein (e.g., an antibody construct described herein) that is "at least trispecific." That is, it comprises at least a first binding domain, a second binding domain, and a third binding domain, and such first, second, and third binding domains can specifically bind to three distinct epitopes, e.g., a first epitope, a second epitope, and a third epitope. Such first, second, and third epitopes can be located on the same antigen or different antigens (e.g., a first epitope on CD3, a second epitope on CD28, and a third epitope on a TAA (e.g., a claudin (Cldn), e.g., Cldn6 or Cldn18.2, or mesothelin (MSLN)). Thus, in some embodiments, a trispecific antibody construct according to the present disclosure can comprise specificity for at least three different antigens or targets. Thus, specificity in the context of an antibody construct herein refers to the total number of different epitopes and / or antigens to which the antibody construct can specifically bind; for example, a monospecific antibody construct contains one or more binding domains with specificity for one epitope or antigen, a trispecific antibody construct contains three or more binding domains with specificity for three different epitopes and / or antigens, etc.

[0046] The term "trivalent," as used herein, in the context of an antibody construct, refers to a biologically functional protein (e.g., an antibody construct described herein) that is "at least trivalent," i.e., it comprises three binding domains, e.g., at least a first binding domain, a second binding domain, and a third binding domain, each of which is capable of specifically binding to an epitope and / or antigen, e.g., CD3, CD28, or a TAA. The three binding domains may have specificity for three different epitopes or antigens, or two or more of the three binding domains may have specificity for the same epitope or antigen. Thus, in the context of an antibody construct herein, valency, e.g., monovalent, bivalent, or trivalent, refers to the total number of antigen-binding domains of the antibody construct. Thus, the valency of an antibody construct must be at least equal to its specificity, i.e., a trispecific antibody construct must be at least trivalent. In embodiments in which the antibody construct is trivalent and trispecific, each of the three binding domains of the construct is capable of binding to a different epitope or antigen, and the construct thus monovalently engages each of the three epitopes and / or antigens.

[0047] As used herein, the term "format," in the context of the antibody constructs described herein, generally describes the attributes of the antibody construct, including its antigen valency (e.g., the construct is monovalent or bivalent for a given antigen), the type of binding domain present in the antibody construct (e.g., possessing one or more scFv domains, Fab domains, etc.), and the presence, absence, and / or type of Fc domain (e.g., homodimer, heterodimer, containing one or more constant heavy domains, CH2, CH3, etc.). By way of example, in some embodiments, the antibody constructs of the present disclosure may be trivalent and trispecific in a 1+1+1 format, indicating that the construct contains three binding domains, each binding domain having affinity for a different antigen (e.g., CD3, CD28, and TAA), i.e., the trivalent construct is monovalent (denoted by "1") for each of the three antigens.

[0048] As used herein, the term "geometry" in the context of the antibody constructs described herein generally describes the overall structure of the antibody construct, including the relative spatial localization and arrangement and / or connectivity of the various domains of the antibody construct, e.g., the relative arrangement and connectivity of the binding and Fc domains, as further described herein and as shown in Figures 1A-1G according to certain embodiments of the present disclosure.

[0049] Generally, and unless otherwise specified, the amino acid sequences of polypeptides described herein are described and defined in the N-to-C-terminal direction. By way of example, a polypeptide described as comprising an scFv domain linked to an Fc polypeptide is defined herein as a polypeptide in which the C-terminus of the scFv domain is linked, with or without a linker, to the N-terminus of the Fc polypeptide, and the domain structure is referred to as scFv-Fc or, with the inclusion of a linker, as scFv-linker. scFv-Fc -Fc.

[0050] As used herein, abbreviations such as "H1" and "H2," or "A" and "B," are generally used as generic heavy chain identifiers and refer broadly to the first and second heavy chains, respectively, of an antibody construct, and thus are not intended to be limited to any particular heavy chain amino acid (or polynucleotide) sequence.

[0051] The term "amino acid modification," when used herein in the context of an amino acid sequence of a polypeptide, generally refers to an amino acid sequence of a polypeptide into which one or more amino acid substitutions, one or more amino acid insertions, and / or one or more amino acid deletions have been introduced relative to the corresponding unmodified (e.g., WT or reference) amino acid sequence of the polypeptide.

[0052] References to antibody constructs such as "anti-(Cldn18.2xCD28xCD3)" and "anti-CD3 / anti-CD28 / anti-Cldn18.2" are used interchangeably herein and may generally refer to antibody constructs that are at least trispecific and thus contain at least three binding domains capable of binding to epitopes on Cldn18.2, CD28, and CD3, respectively. In various embodiments, such references refer to trivalent and trispecific antibody constructs having three binding domains capable of binding to epitopes on Cldn18.2, CD28, and CD3, respectively.

[0053] It should be understood that the positive recitation of a feature in one embodiment generally serves as a basis for excluding that feature in an alternative embodiment. In particular, where a list of alternatives is presented for a given embodiment or claim, it should be understood that one or more alternatives may be deleted from the list, and that the shortened list may form an alternative embodiment, whether or not such alternative embodiment is specifically referenced.

[0054] It is further contemplated that any embodiment discussed herein can be implemented with respect to any antibody construct, method, use, or composition disclosed herein, and vice versa. Moreover, modifications of the specific embodiments described herein that will be apparent to those skilled in the art are intended to be included within the scope of the claims set forth herein.

[0055] II. Antibody Constructs In various embodiments, the present disclosure relates to trivalent and trispecific T cell-engaging antibody constructs comprising three binding domains capable of engaging two different antigens on one or more cytotoxic effector cells and a TAA on a tumor cell.

[0056] In some embodiments, a trivalent and trispecific antibody construct of the present disclosure comprises (i) a first binding domain capable of binding to a first antigen on a first cytotoxic effector cell, (ii) a second binding domain capable of binding to a second antigen on a second cytotoxic effector cell, (iii) a third binding domain capable of binding to a TAA on a tumor cell, and (iv) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide. The antibody constructs described herein can have a variety of formats and geometries, e.g., binding domains, and the Fc domains can have a variety of relative spatial localizations and configurations, as further described herein.

[0057] A. Antibody Construct Format and Geometry In various embodiments, the antibody constructs described herein are trivalent and trispecific, comprising three antigen-binding domains, each capable of binding to a different antigen. Thus, in various embodiments, the trivalent and trispecific antibody constructs of the present disclosure may have a 1+1+1 format, meaning that each of the three binding domains binds to a different antigen, and thus such constructs bind each antigen in a monovalent manner.

[0058] In some embodiments, such trivalent and trispecific antibody constructs may comprise one or more different types of binding domains. Types of binding domains that may be used in the antibody constructs described herein include scFv domains, Fab domains, single-domain antibodies (sdAbs), etc. Thus, in certain embodiments, the antibody constructs described herein may comprise one or more scFv domains and / or one or more Fab domains. In some embodiments, the antibody constructs may comprise one or more scFv domains and one or more Fab domains.

[0059] In some embodiments, a trivalent and trispecific antibody construct of the present disclosure comprises two scFv domains, e.g., a first scFv domain and a second scFv domain, and one Fab domain. Such an antibody construct may further comprise an Fc domain. Thus, the two scFv domains, the Fab domain, and the Fc domain may be linked to each other in various relative spatial arrangements to generate various construct geometries, e.g., as shown in Figures 1A-1G. In some embodiments, the Fc domain is a heterodimeric Fc domain.

[0060] In certain embodiments herein, the trivalent and trispecific antibody constructs of the present disclosure comprise, for example, either N to C-terminal or C to N-terminal, (V H / L -V L / H ) scFv1 -(V H / L -V L / H ) scFv2The antibody constructs described herein do not contain polypeptide chains comprising two or more scFv domains linked in tandem to one another (such chains may optionally contain one or more linkers linking the VH and VL domains together, both within the scFv domain or between the two scFv domains), according to the domain structure of ( ). Expression of an antibody construct comprising a polypeptide chain containing two or more scFv domains linked in tandem may reduce the productivity and stability (e.g., thermostability) of the antibody construct. Thus, in various embodiments, the antibody constructs described herein may have higher productivity and / or thermostability compared to conventional constructs comprising polypeptide chains with two or more scFv domains linked in tandem.

[0061] In some embodiments, described herein is an antibody construct comprising: (i) a Fab domain capable of binding to a first antigen on a first cytotoxic effector cell; (ii) a first scFv domain and a second scFv domain, wherein one of the scFv domains is capable of binding to a second antigen on a second cytotoxic effector cell and the other scFv domain is capable of binding to a tumor-associated antigen (TAA) on a tumor cell; and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the first and second antigens are different; (b) the Fab domain is linked to the N-terminus of the first Fc polypeptide; and (c) the first and second scFv domains are independently linked to either the N-terminus of the Fab domain, the C-terminus of the Fab domain, the C-terminus of one of the Fc polypeptides, or the N-terminus of the second Fc polypeptide.

[0062] In some embodiments, described herein is an antibody construct comprising: (i) a Fab domain capable of binding to a first antigen on a first cytotoxic effector cell; (ii) a first scFv domain and a second scFv domain, wherein one of the scFv domains is capable of binding to a second antigen on a second cytotoxic effector cell and the other scFv domain is capable of binding to a tumor-associated antigen (TAA) on a tumor cell; and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the first and second antigens are different; (b) the Fab domain is linked to the N-terminus of the first Fc polypeptide; and (c) the first and second scFv domains are independently linked to either the N-terminus of the Fab domain, the C-terminus of the Fab domain, or the N-terminus of the second Fc polypeptide.

[0063] In one embodiment, the present disclosure provides a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to a first antigen on a first cytotoxic effector cell; (ii) a first scFv domain capable of binding to a second antigen on a second cytotoxic effector cell; (iii) a second scFv domain capable of binding to a tumor-associated antigen (TAA); and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain (a) a first scFv domain is linked to the N-terminus of a first Fc polypeptide or the N-terminus of a second Fc polypeptide; (b) a first scFv domain is linked to either the N-terminus of a Fab domain, the C-terminus of a Fab domain, the C-terminus of the first Fc polypeptide, or the N-terminus of a second Fc polypeptide; and (c) a second scFv domain is linked to either the N-terminus of the first Fc polypeptide, the N-terminus of the second Fc polypeptide, or the N-terminus of a Fab domain.

[0064] In one embodiment, the present disclosure describes a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to a first antigen on a first cytotoxic effector cell; (ii) a first scFv domain capable of binding to a second antigen on a second cytotoxic effector cell; (iii) a second scFv domain capable of binding to a tumor-associated antigen (TAA); and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of the first Fc polypeptide; (b) the first scFv domain is linked to either the N-terminus of the Fab domain, the C-terminus of the Fab domain, or the C-terminus of the first Fc polypeptide; and (c) the second scFv domain is linked to the N-terminus of the second Fc polypeptide.

[0065] In some embodiments, the first antigen is CD3 or CD28, and the first antigen and the second antigen are different antigens.

[0066] In some embodiments, the second antigen is CD3 or CD28, and the first antigen and the second antigen are different antigens.

[0067] In certain embodiments of the present disclosure, the first antigen on the first cytotoxic effector cell is CD28 and the second antigen on the second cytotoxic effector cell is CD3, hi other embodiments, the first antigen is CD3 and the second antigen is CD28.

[0068] In one embodiment, the present disclosure describes a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to CD28 on a first cytotoxic effector cell; (ii) a first scFv domain capable of binding to CD3 on a second cytotoxic effector cell; (iii) a second scFv domain capable of binding to a TAA on a tumor cell; and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of the first Fc polypeptide; (b) the first scFv domain is linked to either the N-terminus of the Fab domain, the C-terminus of the Fab domain, or the C-terminus of the first Fc polypeptide; and (c) the second scFv domain is linked to the N-terminus of the second Fc polypeptide.

[0069] In one embodiment, the present disclosure describes a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to CD3 on a first cytotoxic effector cell; (ii) a first scFv domain capable of binding to CD28 on a second cytotoxic effector cell; (iii) a second scFv domain capable of binding to a TAA on a tumor cell; and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of the first Fc polypeptide; (b) the first scFv domain is linked to either the N-terminus of the Fab domain, the C-terminus of the Fab domain, or the C-terminus of the first Fc polypeptide; and (c) the second scFv domain is linked to the N-terminus of the second Fc polypeptide.

[0070] In some embodiments, provided herein is an antibody construct comprising: (i) a Fab domain capable of binding to a first antigen on a first cytotoxic effector cell; (ii) a first scFv domain and a second scFv domain, wherein one of the scFv domains is capable of binding to a second antigen on a second cytotoxic effector cell and the other scFv domain is capable of binding to a tumor-associated antigen (TAA) on a tumor cell; and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain binds to a first and (b) the first and second scFv domains are independently linked to either (i) the N-terminus of the Fab domain, (ii) the C-terminus of the Fab domain, (iii) the C-terminus of one of the Fc polypeptides, or (iv) the N-terminus of the second Fc polypeptide, wherein one of the scFv domains is linked to the C-terminus of one of the Fc polypeptides, and the first antigen is CD3 and the second antigen is CD28, or the first antigen is CD28 and the second antigen is CD3, and the first scFv domain and the second scFv domain are not linked in tandem to each other.

[0071] In some embodiments of the trivalent and trispecific antibody constructs described herein, the first scFv domain is linked to the N-terminus of the Fab domain. In such embodiments, the first scFv domain is linked to the V of the heavy chain of the Fab domain. H In other embodiments, the first scFv domain may be linked to the V of the light chain of the Fab domain. L It may be linked to the N-terminus of the sequence.

[0072] In certain embodiments, the trivalent and trispecific antibody construct of the present disclosure comprises a) from N-terminus to C-terminus: (i) a first scFv V L First scFv V linked to sequence H Array(V H -V L ) or the first scFv VH First scFv V linked to sequence L Array(V L -V H (ii) a first scFv domain comprising either Fab C H1 Fab V linked to the sequence H and (iii) a first heavy chain polypeptide comprising a first Fc polypeptide; b) from N-terminus to C-terminus: (i) a second scFv V L A second scFv V linked to the sequence H Array(V H -V L ) or a second scFv V H A second scFv V linked to the sequence L Array(V L -V H ), and (ii) a second heavy chain polypeptide comprising a second Fc polypeptide; and c) from the N-terminus to the C-terminus: Fab C L Fab V linked to the sequence L the heavy chain Fab sequence and the light chain polypeptide form a Fab domain, and the first Fc polypeptide and the second Fc polypeptide form a heterodimeric Fc domain.

[0073] In other embodiments of the trivalent and trispecific antibody construct, the first scFv domain is linked to the C-terminus of the first Fc polypeptide. In such embodiments, the trivalent and trispecific antibody construct comprises: a) from N-terminus to C-terminus: (i) Fab C H1 Fab V linked to the sequence H (ii) a heavy chain Fab sequence comprising the sequence, (iii) a first Fc polypeptide, and (iv) a first scFv V L First scFv V linked to sequence H Array(V H -V L ) or the first scFv V H First scFv V linked to sequence L Array(V L -V H(i) a first heavy chain polypeptide comprising a first scFv domain comprising, from N-terminus to C-terminus: (i) a second scFv V L A second scFv V linked to the sequence H Array(V H -V L ) or a second scFv V H A second scFv V linked to the sequence L Array(V L -V H (ii) a second heavy chain polypeptide comprising a second Fc polypeptide; and c) a Fab C L Fab V linked to the sequence L The antibody may comprise a light chain polypeptide comprising a heavy chain Fab sequence, wherein the heavy chain Fab sequence and the light chain polypeptide form a Fab domain, and the first Fc polypeptide and the second Fc polypeptide form a heterodimeric Fc domain. One embodiment of such a trivalent and trispecific antibody construct is shown in Figure IF.

[0074] In yet other embodiments of the trivalent and trispecific antibody constructs described herein, the first scFv domain comprises the C of the light chain of the Fab domain. L In such embodiments, the antibody construct comprises a) from N-terminus to C-terminus: (i) Fab C H1 Fab V linked to the sequence H and (ii) a first heavy chain polypeptide comprising a first Fc polypeptide; and b) from N-terminus to C-terminus: (i) a second scFv V L A second scFv V linked to the sequence H Array(V H -V L ) or a second scFv V H A second scFv V linked to the sequence L Array(V L -V H and (ii) a second heavy chain polypeptide comprising, from N-terminus to C-terminus: (i) Fab C LFab V linked to the sequence L and (ii) a light chain Fab sequence comprising the sequence L First scFv V linked to sequence H Array(V H -V L ) or the first scFv V H First scFv V linked to sequence L Array(V L -V H ), wherein the heavy chain Fab sequence and the Fab light chain polypeptide sequence form a Fab domain, and the first Fc polypeptide and the second Fc polypeptide form a heterodimeric Fc domain. One embodiment of such a trivalent and trispecific antibody construct is shown in Figure 1B.

[0075] In one embodiment, the present disclosure describes a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to CD28 on a first cytotoxic effector cell; (ii) a first scFv domain capable of binding to CD3 on a second cytotoxic effector cell; (iii) a second scFv domain capable of binding to a TAA on a tumor cell; and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of either the first Fc polypeptide or the second Fc polypeptide; (b) the first scFv domain is linked to either the N-terminus of the first Fc polypeptide, the C-terminus of the first Fc polypeptide, or the N-terminus of the second Fc polypeptide; and (c) the second scFv domain is linked to the N-terminus of the Fab domain. Certain embodiments of such trivalent and trispecific antibody constructs are shown in Figures 1D and 1F.

[0076] In another embodiment, the present disclosure describes a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to CD3 on a first cytotoxic effector cell; (ii) a first scFv domain capable of binding to CD28 on a second cytotoxic effector cell; (iii) a second scFv domain capable of binding to a TAA on a tumor cell; and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of either the first Fc polypeptide or the second Fc polypeptide; (b) the first scFv domain is linked to either the N-terminus of the first Fc polypeptide, the C-terminus of the first Fc polypeptide, or the N-terminus of the second Fc polypeptide; and (c) the second scFv domain is linked to the N-terminus of the Fab domain. Certain embodiments of such trivalent and trispecific antibody constructs are shown in Figures 1D and 1F.

[0077] In certain embodiments, the Fab domain is linked to the N-terminus of the first Fc polypeptide and the first scFv domain is linked to the C-terminus of the first Fc polypeptide. Exemplary embodiments are shown in Figures 1C and 1F. In one such embodiment, for example, as shown in Figure 1C, the antibody construct comprises: a) from N-terminus to C-terminus: (i) the second scFv V L A second scFv V linked to the sequence H Array(V H -V L ) or a second scFv V H A second scFv V linked to the sequence L Array(V L -V H (ii) a second scFv domain comprising either Fab C H1 Fab V linked to the sequence H (iii) a heavy chain Fab sequence comprising the sequence, (iv) a first Fc polypeptide, and (v) a first scFv V L First scFv V linked to sequence H Array(VH -V L ) or the first scFv V H First scFv V linked to sequence L Array(V L -V H a) a first heavy chain polypeptide comprising a first scFv domain comprising either a Fab C or a Fab C; b) a second heavy chain polypeptide comprising or consisting of a second Fc polypeptide; and c) from N-terminus to C-terminus: Fab C or a ... L Fab V linked to the sequence L The first Fc polypeptide and the second Fc polypeptide may comprise a light chain polypeptide comprising a light chain Fab sequence comprising the sequence, wherein the heavy chain Fab sequence and the light chain polypeptide form a Fab domain, and the first Fc polypeptide and the second Fc polypeptide form a heterodimeric Fc domain.

[0078] In another embodiment of the trivalent and trispecific antibody constructs described herein, the Fab domain is linked to the N-terminus of a first Fc polypeptide and the first scFv domain is linked to the N-terminus of a second Fc polypeptide, for example as shown in Figure IE. In such an embodiment, the antibody construct comprises: a) from N-terminus to C-terminus: (i) the second scFv V L A second scFv V linked to the sequence H Array(V H -V L ) or a second scFv V H A second scFv V linked to the sequence L Array(V L -V H (ii) a second scFv domain comprising either Fab C H1 Fab V linked to the sequence H and (iii) a first heavy chain polypeptide comprising a first Fc polypeptide; b) from N-terminus to C-terminus: (i) a first scFv V L First scFv V linked to sequence H Array(V H -V L ) or the first scFv V H First scFv V linked to sequence L Array(VL -V H ), and (ii) a second heavy chain polypeptide comprising a second Fc polypeptide; and c) from the N-terminus to the C-terminus: Fab C L Fab V linked to the sequence L The first Fc polypeptide and the second Fc polypeptide may comprise a light chain polypeptide comprising a light chain Fab sequence comprising the sequence, wherein the heavy chain Fab sequence and the light chain polypeptide form a Fab domain, and the first Fc polypeptide and the second Fc polypeptide form a heterodimeric Fc domain.

[0079] In another embodiment, the Fab domain is linked to the N-terminus of the second Fc polypeptide and the first scFv domain is linked to the N-terminus of the first Fc polypeptide, for example as shown in Figure ID. In such an embodiment, the antibody construct comprises: a) from N-terminus to C-terminus: (i) the first scFv V L First scFv V linked to sequence H Array(V H -V L ) or the first scFv V H First scFv V linked to sequence L Array(V L -V H (i) a first scFv domain comprising, from N-terminus to C-terminus: (i) a second scFv V; and (ii) a first heavy chain polypeptide comprising, from N-terminus to C-terminus: (i) a second scFv V; L A second scFv V linked to the sequence H Array(V H -V L ) or a second scFv V H A second scFv V linked to the sequence L Array(V L -V H (ii) a second scFv domain comprising either Fab C H1 Fab V linked to the sequence H and (iii) a second heavy chain polypeptide comprising a second Fc polypeptide; and c) from the N-terminus to the C-terminus: Fab C L Fab V linked to the sequence LThe first Fc polypeptide and the second Fc polypeptide may comprise a light chain polypeptide comprising a light chain Fab sequence comprising the sequence, wherein the heavy chain Fab sequence and the light chain polypeptide form a Fab domain, and the first Fc polypeptide and the second Fc polypeptide form a heterodimeric Fc domain.

[0080] In some embodiments, described herein are antibody constructs in which each of the first and second scFv domains may be independently linked to the N-terminus of the Fab domain, the C-terminus of the Fab domain, the C-terminus of one of the Fc polypeptides, or the N-terminus of the second Fc polypeptide.

[0081] In such embodiments, the antibody construct may comprise: (i) a Fab domain capable of binding to CD3 on a first cytotoxic effector cell; (ii) a first scFv domain and a second scFv domain, wherein one of the scFv domains is capable of binding to CD28 on a second cytotoxic effector cell and the other scFv domain is capable of binding to a tumor-associated antigen (TAA) on a tumor cell; and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of the first Fc polypeptide; and (b) each of the first and second scFv domains is independently linked to the N-terminus of the Fab domain, the C-terminus of the Fab domain, the C-terminus of one of the Fc polypeptides, or the N-terminus of the second Fc polypeptide.

[0082] In other embodiments, the antibody construct may comprise: (i) a Fab domain capable of binding to CD28 on a first cytotoxic effector cell; (ii) a first scFv domain and a second scFv domain, wherein one of the scFv domains is capable of binding to CD3 on a second cytotoxic effector cell and the other scFv domain is capable of binding to a tumor-associated antigen (TAA) on a tumor cell; and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of the first Fc polypeptide; and (b) the first and second scFv domains are each independently linked to either the N-terminus of the Fab domain, the C-terminus of the Fab domain, the C-terminus of one of the Fc polypeptides, or the N-terminus of the second Fc polypeptide.

[0083] In some of these embodiments, the first scFv domain is linked to the N-terminus of the Fab domain and the second scFv is linked to the N-terminus of the second Fc polypeptide. In certain embodiments, the first scFv domain is linked to the V of the heavy chain of the Fab domain. H In another embodiment, the first scFv domain is linked to the N-terminus of the V of the light chain of the Fab domain. L It is linked to the N-terminus of the sequence.

[0084] In some embodiments, a trivalent and trispecific antibody construct may comprise or consist of three polypeptide chains that can associate to form an antibody construct. In some embodiments, such a trivalent and trispecific antibody construct comprises a) from N-terminus to C-terminus: (i) a first scFv V L First scFv V linked to sequence H Array(V H -V L ) or the first scFv V H First scFv V linked to sequence L Array(V L -V H(ii) a first scFv domain comprising either Fab C H1 Fab V linked to the sequence H and (iii) a first heavy chain polypeptide comprising a first Fc polypeptide; b) from N-terminus to C-terminus: (i) a second scFv V L A second scFv V linked to the sequence H Array(V H -V L ) or a second scFv V H A second scFv V linked to the sequence L Array(V L -V H ), and (ii) a second heavy chain polypeptide comprising a second Fc polypeptide; and c) from the N-terminus to the C-terminus: Fab C L Fab V linked to the sequence L the heavy chain Fab sequence and the light chain polypeptide form a Fab domain, and the first Fc polypeptide and the second Fc polypeptide form an Fc domain.

[0085] In some embodiments, a first scFv domain is linked to the C-terminus of a Fab domain and a second scFv domain is linked to the N-terminus of a second Fc polypeptide. In such embodiments, the trivalent and trispecific antibody construct comprises the following polypeptide chains: a) from N-terminus to C-terminus: (i) Fab C H1 Fab V linked to the sequence H and (ii) a first heavy chain polypeptide comprising a first Fc polypeptide; and b) from N-terminus to C-terminus: (i) a second scFv V L A second scFv V linked to the sequence H Array(V H -V L ) or a second scFv V H A second scFv V linked to the sequence L Array(V L -V Hand (ii) a second heavy chain polypeptide comprising, from N-terminus to C-terminus: (i) Fab C L Fab V linked to the sequence L and (ii) the first scFv V L First scFv V linked to sequence H Array(V H -V L ) or the first scFv V H First scFv V linked to sequence L Array(V L -V H ), wherein the heavy chain Fab sequence and the Fab sequence of the light chain polypeptide form the Fab domain, and the first Fc polypeptide and the second Fc polypeptide form the Fc domain.

[0086] In some embodiments of the trivalent and trispecific antibody constructs described herein, a first scFv domain is linked to the N-terminus of a Fab domain and a second scFv domain is linked to the C-terminus of one of the Fc polypeptides. In such embodiments, the first scFv domain is linked to the V of the Fab domain. H In other embodiments, the first scFv domain may be linked to the N-terminus of the V domain of the Fab domain. L In some of these embodiments, the second scFv domain is linked to the N-terminus of the first Fc polypeptide. In yet other such embodiments, the second scFv domain is linked to the C-terminus of the first Fc polypeptide.

[0087] In some of these embodiments, the trivalent and trispecific antibody construct comprises the following polypeptide chains: a) from N-terminus to C-terminus: (i) a first scFv V L First scFv V linked to sequence H Array(V H -V L ) or the first scFv V HFirst scFv V linked to sequence L Array(V L -V H (ii) a first scFv domain comprising either Fab C H1 Fab V linked to the sequence H (iii) a heavy chain Fab sequence comprising the sequence, (iv) a first Fc polypeptide, and (v) a second scFv V L A second scFv V linked to the sequence H Array(V H -V L ) or a second scFv V H A second scFv V linked to the sequence L Array(V L -V H a) a first heavy chain polypeptide comprising a second scFv domain comprising either a) a first heavy chain polypeptide comprising a second Fc polypeptide; and c) a Fab C L Fab V linked to the sequence L The heavy chain Fab sequence and the light chain polypeptide form a Fab domain, and the first Fc polypeptide and the second Fc polypeptide form an Fc domain.

[0088] In other embodiments, the trivalent and trispecific antibody construct comprises the following polypeptide chains: a) from N-terminus to C-terminus: (i) a first scFv V L First scFv V linked to sequence H Array(V H -V L ) or the first scFv V H First scFv V linked to sequence L Array(V L -V H (ii) a first scFv domain comprising either Fab C H1 Fab V linked to the sequence H and (iii) a first heavy chain polypeptide comprising a first Fc polypeptide; b) from N-terminus to C-terminus: (i) a second scFv V L A second scFv V linked to the sequence H Array(V H-V L ) or a second scFv V H A second scFv V linked to the sequence L Array(V L -V H (ii) a second heavy chain polypeptide comprising a second Fc polypeptide; and c) a Fab C L Fab V linked to the sequence L The heavy chain Fab sequence and the light chain polypeptide form a Fab domain, and the first Fc polypeptide and the second Fc polypeptide form an Fc domain.

[0089] In some embodiments of the trivalent and trispecific antibody constructs described herein comprising a first scFv domain and a second scFv domain, the first scFv domain is capable of binding to a TAA and the second scFv domain is capable of binding to CD28 or CD3. In still other embodiments, the second scFv domain is capable of binding to a TAA and the first scFv domain is capable of binding to CD28 or CD3.

[0090] In one embodiment, provided herein is an antibody construct comprising: (i) a Fab domain capable of binding to CD3 on a first cytotoxic effector cell; (ii) a first scFv domain and a second scFv domain, wherein the first scFv domain is capable of binding to CD28 on a second cytotoxic effector cell and the second scFv domain is capable of binding to a tumor-associated antigen (TAA) on a tumor cell; and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of the first Fc polypeptide; and (b) the first scFv domain is linked to the V-terminus of the Fab domain. H and (c) a second scFv domain is linked to the N-terminus of a second Fc polypeptide.

[0091] In one embodiment, provided herein is an antibody construct comprising: (i) a Fab domain capable of binding to CD28 on a first cytotoxic effector cell; (ii) a first scFv domain and a second scFv domain, wherein the first scFv domain is capable of binding to CD3 on a second cytotoxic effector cell and the second scFv domain is capable of binding to a tumor-associated antigen (TAA) on a tumor cell; and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of the first Fc polypeptide; and (b) the first scFv domain is linked to the V-terminus of the Fab domain. H and (c) a second scFv domain is linked to the N-terminus of a second Fc polypeptide.

[0092] In one embodiment, provided herein is an antibody construct comprising: (i) a Fab domain capable of binding to CD3 on a first cytotoxic effector cell; (ii) a first scFv domain and a second scFv domain, wherein the first scFv domain is capable of binding to CD28 on a second cytotoxic effector cell and the second scFv domain is capable of binding to a tumor-associated antigen (TAA) on a tumor cell; and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of the first Fc polypeptide; and (b) the first scFv domain is linked to the C-terminus of the Fab domain. L and (c) a second scFv domain is linked to the N-terminus of a second Fc polypeptide.

[0093] In one embodiment, provided herein is an antibody construct comprising: (i) a Fab domain capable of binding to CD28 on a first cytotoxic effector cell; (ii) a first scFv domain and a second scFv domain, wherein the first scFv domain is capable of binding to CD3 on a second cytotoxic effector cell and the second scFv domain is capable of binding to a tumor-associated antigen (TAA) on a tumor cell; and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of the first Fc polypeptide; and (b) the first scFv domain is linked to the C-terminus of the Fab domain. L and (c) a second scFv domain is linked to the N-terminus of a second Fc polypeptide.

[0094] In one embodiment, described herein is an antibody construct comprising: (i) a Fab domain capable of binding to CD3 on a first cytotoxic effector cell; (ii) a first scFv domain and a second scFv domain, wherein the first scFv domain is capable of binding to CD28 on a second cytotoxic effector cell and the second scFv domain is capable of binding to a tumor-associated antigen (TAA) on a tumor cell; and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of the first Fc polypeptide, (b) the first scFv domain is linked to the C-terminus of the first Fc polypeptide, and (c) the second scFv domain is linked to the N-terminus of the second Fc polypeptide.

[0095] In one embodiment, described herein is an antibody construct comprising: (i) a Fab domain capable of binding to CD28 on a first cytotoxic effector cell; (ii) a first scFv domain and a second scFv domain, wherein the first scFv domain is capable of binding to CD3 on a second cytotoxic effector cell and the second scFv domain is capable of binding to a tumor-associated antigen (TAA) on a tumor cell; and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of the first Fc polypeptide, (b) the first scFv domain is linked to the C-terminus of the first Fc polypeptide, and (c) the second scFv domain is linked to the N-terminus of the second Fc polypeptide.

[0096] In one embodiment, provided herein is an antibody construct comprising: (i) a Fab domain capable of binding to CD3 on a first cytotoxic effector cell; (ii) a first scFv domain and a second scFv domain, wherein the first scFv domain is capable of binding to CD28 on a second cytotoxic effector cell and the second scFv domain is capable of binding to a tumor-associated antigen (TAA) on a tumor cell; and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of the first Fc polypeptide, (b) the first scFv domain is linked to the C-terminus of the first Fc polypeptide, and (c) the second scFv domain is linked to the V of the Fab domain. H Antibody constructs are described in which the N-terminus of a domain is linked to the N-terminus of the domain.

[0097] In one embodiment, provided herein is an antibody construct comprising: (i) a Fab domain capable of binding to CD28 on a first cytotoxic effector cell; (ii) a first scFv domain and a second scFv domain, wherein the first scFv domain is capable of binding to CD3 on a second cytotoxic effector cell and the second scFv domain is capable of binding to a tumor-associated antigen (TAA) on a tumor cell; and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of the first Fc polypeptide, (b) the first scFv domain is linked to the C-terminus of the first Fc polypeptide, and (c) the second scFv domain is linked to the V of the Fab domain. H Antibody constructs are described in which the N-terminus of a domain is linked to the N-terminus of the domain.

[0098] In various embodiments, the dimeric Fc domain of the trivalent and trispecific antibody construct is a heterodimeric Fc domain.

[0099] In some embodiments, the first scFv domain comprises, from N to C terminus, V H -V L As further described herein, the first scFv domain has a domain structure of the first scFv V H The sequence is given as the first scFv V L Linker to connect to the sequence scFv1 It may further include:

[0100] In certain other embodiments, the first scFv domain comprises, from N to C terminus, V L -V H As further described herein, the first scFv domain has a domain structure of the first scFv V L The sequence is H Linker to connect to the sequence scFv1 It may further include:

[0101] In some embodiments, the second scFv domain comprises, from N to C terminus, V H -V L As further described herein, the second scFv domain has a domain structure of the second scFv V H The sequence was converted to the second scFv V L Linker to connect to the sequence scFv2 It may further include:

[0102] In certain embodiments, the second scFv domain comprises, from N to C terminus, V L -V H As further described herein, the second scFv domain has a domain structure of the second scFv V L The sequence was converted to the second scFv V H Linker to connect to the sequence scFv2 It may further include:

[0103] In some embodiments, the first scFv domain comprises, from N to C terminus, V L -V H The second scFv domain has a domain structure of, from the N- to C-terminus, V L -V H It has a domain structure of

[0104] In some embodiments, the first scFv domain comprises, from N to C terminus, V H -V L The second scFv domain has a domain structure of, from the N- to C-terminus, V H -V L It has a domain structure of

[0105] In some embodiments, the first scFv domain comprises, from N to C terminus, V L -V H The second scFv domain has a domain structure of, from the N- to C-terminus, V H -V L It has a domain structure of

[0106] In some embodiments, the first scFv domain comprises, from N to C terminus, V H -V L The second scFv domain has a domain structure of, from the N- to C-terminus, V L -V H It has a domain structure of

[0107] In various embodiments of the trivalent and trispecific antibody constructs of the present disclosure, the TAA is MSLN or Cldn18.2, as further described herein.

[0108] As further described herein, the antibody constructs of the present disclosure may further comprise one or more linkers. Such one or more linkers may be one or more peptide linkers. The one or more linkers may link one, two, or more domains and / or sequences of the antibody construct to one another. For example, the linkers scFv scFv V H The sequence is scFv V L It can be linked to the sequence.

[0109] B. Domains of the Antibody Construct The trivalent and trispecific antibody constructs of the present disclosure may comprise one or more antibody domains. In various embodiments, the trivalent and trispecific antibody constructs of the present disclosure comprise multiple (i.e., two or more) antibody domains. Such multiple antibody domains include: (i) one or more Fc domains, which may comprise a first Fc polypeptide and a second Fc polypeptide, and may be either homodimers or heterodimers; (ii) one or more Fab domains, which may comprise a heavy variable domain (V) and a heavy variable domain (F) that are fused to one or more Fc domains; H ) sequence and heavy constant domain (C H1 ) sequence and a heavy chain polypeptide comprising a light variable domain (V L ) sequence and the light constant domain (C L) one or more Fab domains, which may comprise a light chain polypeptide comprising the sequence: scFv V L scFv V linked to sequence H The antibody construct may comprise one or more scFv domains, which may comprise the sequence: The various domains that the antibody construct may comprise are further described herein.

[0110] The Ig structural unit consists of two pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kilodaltons (kD)) and one "heavy" chain (approximately 50-70 kD). Light chains can be classified as either kappa or lambda. The "class" of an Ig refers to the type of constant domain carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the different classes of immunoglobulins (Ig) are called alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), respectively.

[0111] In various embodiments, the trivalent and trispecific antibody constructs described herein are based on IgG class immunoglobulins, e.g., IgG1, IgG2, IgG3, or IgG4 immunoglobulins. In some embodiments, the antibody constructs described herein are based on IgG1, IgG2, or IgG4 immunoglobulins. In certain embodiments, the antibody constructs described herein are based on IgG1 immunoglobulins. In the context of the present disclosure, when an antibody construct is based on a specified Ig isotype, it refers to an antibody construct that includes either all or a portion of the constant region (i.e., Fc domain) of the specified Ig isotype. It should be understood that the antibody constructs may also include hybrids of isotypes and / or subclasses, in accordance with certain embodiments of the present disclosure.

[0112] Typically, in antibodies, the N-terminal domain of each polypeptide chain is typically a variable region (e.g., V) of about 100-110 amino acids or more in length that is primarily responsible for antigen recognition. H or V L ) defines the variable light chain (V L ) and variable heavy chain (V H The terms V and V ) refer to these domains in the light and heavy chains, respectively. As described herein, in various embodiments, the trivalent and trispecific antibody constructs of the present disclosure can comprise two or more variable domain sequences. In various embodiments, the trivalent and trispecific antibody constructs of the present disclosure comprise two variable domain sequences per binding domain, and are thus trivalent (i.e., contain three binding domains) and trispecific (i.e., each of the three binding domains targets a different antigen), resulting in a total of six variable domain sequences, e.g., three V H Domain sequence and three V L It may comprise a domain sequence.

[0113] In some embodiments, two or more of such variable domains are, for example, V (from either the N- or C-terminus) L V linked to domain H As described for scFv-type binding domains containing variable domains, or where the scFv domain (containing two variable domain sequences linked in tandem to each other) is a Fab variable domain sequence (e.g., Fab V H or Fab V L In some embodiments, the heavy chain domains are linked to each other in tandem and in a single polypeptide chain format, as described for constructs linked to a Fab domain via any of the following sequences: H -V L ) or (V L -V H )] scFv -V H -C H1 -Fc may be included.

[0114] Thus, an antibody construct of the present disclosure derived from an Ig molecule may comprise different Ig domains within its heavy and light chain(s). The heavy chain domain may include, for example, an Fc domain (or Fc region) comprising a CH2 domain and a CH3 domain, a hinge domain (or hinge region), a variable heavy domain (V H ) and constant heavy domain (C H1 ), and the light chain domain may include a heavy chain Fab domain containing a variable light domain (V L ) and light constant domain (C L In some embodiments, according to the given nomenclature, an "Fc domain" may include CH2 and CH3 domains and a hinge domain (or hinge region).

[0115] B.1 Complementarity-Determining Regions (CDRs) and Binding Domains V of the antibody constructs herein H Domain and V L Each of the domains has three loops that are hypervariable in sequence and form the antigen-binding site. Each of these loops is called a "hypervariable region" or "HVR," or a "complementarity-determining region" or "CDR." The terms hypervariable region (HVR) and complementarity-determining region (CDR) refer to the variable domains (e.g., V H or V L ) are used interchangeably herein with respect to the moiety. H With the exception of CDR1 in V, CDRs typically comprise amino acid residues that form hypervariable loops. H Domain and V L A domain consists of multiple relatively invariant stretches called framework regions (FRs), each about 15-30 amino acids long, separated by shorter CDRs, each of which is typically about 5-15 amino acids long, but can be longer or shorter in some cases. H and V L The three CDRs and four FRs constituting a domain are arranged from the N- to C-terminus as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0116] Several different definitions and numbering conventions for CDR regions in Ig molecules are commonly used, including those described by Kabat et al. (1983, Sequences of Proteins of Immunological Interest, NIH Publication No. 369-847, Bethesda, MD), Chothia et al. (1987, J Mol Biol, 196:901-917), and the IMGT, AbM, and Contact definitions. These different definitions include overlapping or subsets of amino acid residues when compared against each other. By way of example, CDR definitions according to Kabat, Chothia, IMGT, AbM, and Contact are provided in Table 1 below.

[0117] Therefore, as will be readily apparent to one skilled in the art, the exact numbering and arrangement of the CDRs may vary based on the numbering system used. H It should be understood that the disclosure herein of a variable light domain (V) includes the disclosure of the associated (unique) heavy chain CDRs (HCDRs) as defined by any known numbering system. L It should be understood that the disclosure herein of a heavy chain CDR (HCDR) of a known antibody includes disclosure of the relevant (unique) heavy chain CDRs (HCDRs) as defined by any known numbering system. One of skill in the art will appreciate that a limited number of amino acid substitutions can be made to the CDR sequences or VDRs of known antibodies without the antibody losing its ability to bind its target, e.g., without a reduction in binding affinity of at least about 1000-fold or more. H or L It will be understood that candidate amino acid substitutions can be introduced into the sequence. Candidate amino acid substitutions can be identified by techniques such as computer modeling or alanine scanning, and the resulting variants can be analyzed by standard techniques to determine binding activity (e.g., expressed as binding affinity, e.g., measured EC 50The antibody constructs described herein are tested for CDRs (i.e., heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3) that have 90% or more, 95% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 321-326, respectively, and the binding domains retain or substantially retain the ability to bind to CD3. In this context, the term "substantially" refers to a change in binding affinity of less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%. [Table 1]

[0118] In some embodiments, the antibody constructs described herein comprise at least one Ig domain from a mammalian Ig, such as bovine Ig, human Ig, camelid Ig, rat Ig, or mouse Ig. In some embodiments, the antibody constructs herein may be chimeric constructs comprising two or more Ig domains, with at least one domain from a first mammalian Ig, such as human Ig, and at least a second domain from a second mammalian Ig, such as mouse or rat Ig. In other embodiments, the antibody constructs may be derived from Igs from different species; for example, the antibody constructs may be chimeric or humanized. A "chimeric antibody construct" typically refers to an antibody comprising at least one variable domain from a rodent antibody (usually a murine antibody) and at least one constant domain from a human antibody. A "humanized antibody construct" is a type of chimeric antibody that contains minimal sequence derived from a non-human antibody. In some embodiments, the antibody constructs herein may comprise at least one Ig constant domain from a human Ig. In various embodiments, all domains of the antibody constructs described herein may be from (or may be derived from) human Ig.

[0119] In some embodiments, and as further described herein, modifications (e.g., to the amino acid sequence) to one or more domains of the antibody construct may be made to further refine the properties and performance (e.g., antigen affinity, stability, and / or pharmacokinetics, etc.) of the antibody construct. For example, framework region (FR) residues of a human Ig may be replaced by corresponding non-human residues, or a humanized antibody may comprise residues that are not found in either the recipient antibody or the donor antibody. Typically, the variable domain in a humanized antibody or humanized antibody domain will contain all or substantially all of the hypervariable regions from a non-human Ig and all or substantially all of the FRs from a human Ig sequence. As further described herein, modifications in the Fc domain may enable preferential pairing of Fc polypeptides to form heterodimeric Fc domains rather than homodimeric Fc domains.

[0120] In some embodiments, the present disclosure relates to antibody constructs that may have different valencies, e.g., bivalent or trivalent. Thus, in various embodiments, the antibody constructs herein comprise two or three antigen-binding domains, i.e., are at least bivalent or at least trivalent. In various embodiments of the present disclosure, the antibody constructs may be trispecific and trivalent, and thus, such antibody constructs may comprise three binding domains. Each of the three binding domains may have a unique binding specificity for an antigen (either the same epitope / antigen or different ones). In some of these embodiments, the trispecific and trivalent antibody constructs of the present disclosure comprise three binding domains, e.g., one or more Fab domains and / or one or more scFv domains, capable of binding to three different antigens (e.g., CD3, CD28, and TAA).

[0121] In some embodiments, antibody constructs of the present disclosure may comprise (i) one or more Fab domains, (ii) one or more scFv domains, and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide. In various embodiments, the trivalent and trispecific antibody constructs described herein comprise (i) a Fab domain capable of binding to a first antigen, (ii) a first scFv domain capable of binding to a second antigen, and (iii) a second scFv domain capable of binding to a third antigen, and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide.

[0122] Generally, a "Fab domain" as used herein refers to the constant domain of the light chain (C L ) and the first constant domain of the heavy chain (C H1 ), and a variable domain V on the light chain and heavy chain comprising the CDRs described herein. L and V H and a variable region comprising each of the Fab light and heavy chain constant chains. In some embodiments, the Fab domain can be a single-chain Fab. A single-chain Fab can be a Fab molecule in which a Fab light chain and a Fab heavy chain constant chain are connected by a peptide linker to form a single polypeptide chain. In such embodiments, typically, in a single-chain Fab molecule, the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain, although other formats are encompassed herein. However, in various embodiments herein, the Fab domain of an antibody construct is formed by two separately expressed polypeptide chains, i.e., a light chain and a heavy chain (or portions thereof). However, the heavy and light chain portions of a Fab domain can be interconnected by a covalent bond, e.g., a disulfide bond.

[0123] An "scFv domain," as used herein, refers to a heavy chain variable domain (V), typically in the form of a single polypeptide chain. H ) and the light chain variable domain (V L The scFv contains a V that can assist the scFv in forming a functional structure for antigen binding.H Domains and V L The scFv domains herein may optionally contain a peptide linker between the domains. Thus, in various embodiments, the scFv domains herein may contain a linker scFv By V H V linked via its C-terminus to the N-terminus of the domain L domain, i.e., the scFv domain may have the domain structure: V L -Linker scFv -V H or alternatively, the scFv may have a linker scFv By V L V connected by its C-terminus to the N-terminus of H i.e., the domain structure: V H -Linker scFv -V L It has.

[0124] In some embodiments, the antibody constructs described herein may further comprise another domain or moiety that may not be derived from an Ig molecule. Such a non-Ig domain may be referred to as a moiety. Such a moiety may be a detectable label (e.g., a radioactive or fluorescent label), a low molecular weight (e.g., <750 Da) drug molecule, another peptide (e.g., a signal peptide(s)) or polypeptide molecule, or a combination thereof.

[0125] B.2 Binding domains for antigens on cytotoxic effector cells As further described herein, the antibody constructs of the present disclosure may comprise at least two binding domains capable of binding to one or more molecules, e.g., polypeptides, on the surface of one or more cytotoxic effector cells. Such one or more cytotoxic effector cells may be one or more immune cells. Such one or more immune cells may include T cells, macrophages, dendritic cells, neutrophils, B cells, NK cells, or a combination thereof.

[0126] In various embodiments, the trivalent and trispecific antibody constructs of the present disclosure comprise at least one binding domain capable of binding to a first antigen on a first cytotoxic effector cell and at least one binding domain capable of binding to a second antigen on a second cytotoxic effector cell.

[0127] In various embodiments, the first and second cytotoxic effector cells are different cells, i.e., the first and second antigens are located on the surface of different cells. In other embodiments, the first and second cytotoxic effector cells are the same cell, e.g., the first and second antigens engaged by the antibody constructs are expressed by the same cell, i.e., are located on the same cell surface. In some embodiments, the first and second cytotoxic effector cell(s) comprise or consist of T cell(s).

[0128] In some embodiments, the first antigen is cluster of differentiation 3 (CD3) and the second antigen is CD28. In such embodiments, both CD3 and CD28 can be engaged by a trivalent and trispecific antibody construct when located on the same cytotoxic effector cell surface. In other embodiments, both antigens CD3 and CD28 can be engaged by a trivalent and trispecific antibody construct when located on the surface of different cytotoxic effector cells.

[0129] Thus, in some embodiments, the present disclosure relates to a trivalent and trispecific antibody construct comprising a first binding domain capable of binding to CD3, a second binding domain capable of binding to CD28, and a third binding domain capable of binding to a TAA. Thus, the antibody constructs described herein may also be referred to as "T cell engagers," "TCEs," or "T cell engager molecules," referring to the construct's ability to bind to both an antigen on one or more T cells and a TAA on tumor cells. In some embodiments, for example, in the tumor (micro)environment, engagement of the trivalent and trispecific antibody construct with two different antigens on one or more T cells and an antigen on tumor cells may be simultaneous (at least transiently), thereby establishing a TCR-independent immune synapse and directing T cell-mediated cytotoxic activity to the tumor environment containing tumor cells expressing the TAA. In various embodiments, and as further described herein, the trivalent and trispecific antibody constructs may result in significantly reduced immune cell (e.g., T cell) activation in the absence of a TAA, for example, if an immune synapse cannot adequately form due to the absence of a TAA.

[0130] In various embodiments, the first antigen-binding domain capable of binding to CD3 on a first cytotoxic effector cell can be a Fab domain or scFv domain described herein. In these embodiments, the second antigen-binding domain capable of binding to CD28 on a second cytotoxic effector cell can also be a Fab domain or scFv domain described herein.

[0131] Thus, in some embodiments, both binding domains capable of binding to CD3 and CD28 on the first and second cytotoxic effector cells, respectively, are Fab domains.

[0132] In other embodiments, in some embodiments, both binding domains capable of binding to CD3 and CD28 on the first and second cytotoxic effector cells, respectively, are scFv domains.

[0133] In yet other embodiments, the first binding domain capable of binding to CD28 on a first cytotoxic effector cell is a Fab domain, and the second binding domain capable of binding to CD3 on a second cytotoxic effector cell is an scFv domain.

[0134] In another embodiment, the first binding domain capable of binding to CD28 on a first cytotoxic effector cell is an scFv domain, and the second binding domain capable of binding to CD3 on a second cytotoxic effector cell is a Fab domain.

[0135] In various embodiments, the Fab domain capable of binding to either CD3 or CD28 comprises a heavy chain constant domain (C) comprising or consisting of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 107. H1 In some embodiments, such C H1 The domain sequence comprises or consists of an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 107. In some embodiments, such a C H1 The domain sequence comprises or consists of an amino acid sequence having at least about 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 107. In some embodiments, such a C H1 The domain sequence comprises or consists of an amino acid sequence having at least about 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 107. In some embodiments, such a C H1 The domain sequence comprises or consists of an amino acid sequence having at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 107. In some embodiments, such a C H1 The domain sequence comprises or consists of the amino acid sequence shown in SEQ ID NO:107.

[0136] B.2.1 Binding domain for CD3 As described herein, in some embodiments, the trivalent and trispecific antibody constructs of the present disclosure comprise a binding domain capable of binding to CD3.

[0137] In various embodiments, such anti-CD3 binding domains (e.g., scFv domains or Fab domains) of the antibody constructs herein have an affinity for CD3 (EC for binding to CD3) of about 1 nM, 5 nM, 10 nM, 20 nM or less, or about 30 nM or less. 50 Thus, in various embodiments, the anti-CD3 binding domains herein may have an EC value for binding to CD3 of about 20 nM to about 80 nM, about 30 nM to about 60 nM, or about 40 nM to about 50 nM or less. 50 In some embodiments, the anti-CD3 binding domains herein have an EC value for binding to CD3 that is about 30 nM, 40 nM, 50 nM, or about 60 nM or less. 50 In various embodiments, the antibody constructs herein have an EC value for binding to CD3 of about 20 nM to about 40 nM, e.g., about 30 nM. 50 The antibody comprises an anti-CD3 binding domain having a value.

[0138] In some embodiments, the anti-CD3 binding domain is capable of binding to CD3 with a dissociation constant of about 20 nM to about 200 nM, about 30 nM to about 150 nM, about 40 nM to about 100 nM, or 50 nM to about 80 nM.

[0139] In various embodiments, the antibody constructs herein comprise a binding domain capable of binding to CD3 on a T cell, wherein such binding domain has an EC for binding to CD3 of about 20 nM to about 40 nM, e.g., about 30 nM. 50 V having the value shown in SEQ ID NOs: 321 to 323 H CDR sequences of the sequences and V sequences shown in SEQ ID NOs: 324 to 326 L The sequence includes the CDR sequences of the sequence.

[0140] In various embodiments, the antibody constructs herein comprise a binding domain capable of binding to CD3 on a T cell, wherein such binding domain has an EC for binding to CD3 of about 20 nM to about 40 nM, e.g., about 30 nM. 50 V having a value of 0.001 and including an HCDR1 comprising the sequence GVTFNYYG (SEQ ID NO: 321), an HCDR2 comprising the sequence ITSSGGRI (SEQ ID NO: 322), and an HCDR3 comprising the sequence TLDGRDGWVAY (SEQ ID NO: 323). H domain, and a V domain comprising an LCDR1 comprising the sequence TGNIGSNY (SEQ ID NO: 324), an LCDR2 comprising the sequence RND (SEQ ID NO: 325), and an LCDR3 comprising the sequence QSYSSGFI (SEQ ID NO: 326). L Includes the domain.

[0141] In some embodiments, the anti-CD3 binding domain comprises or consists of an amino acid sequence having at least about 80%, 90%, 95%, 97%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 102. H domain, and V comprising or consisting of an amino acid sequence having at least about 80%, 90%, 95%, 97%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 103. L In certain embodiments, the anti-CD3 binding domain of the antibody constructs herein comprises a V domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 102. H domain, and V comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 103 L Includes the domain.

[0142] In some embodiments, the CDRs of the anti-CD3 paratopes used in the antibody constructs of the present disclosure comprise one or more amino acid modifications in one or more of the CDR sequences set forth in SEQ ID NOs: 321-326, and retain at least about 80%, 90%, or 95% of the binding affinity to CD3 compared to the paratope without such amino acid modifications.

[0143] In some embodiments, the anti-CD3 binding domain comprised by the antibody constructs described herein may comprise or consist of an scFv domain or a Fab domain.

[0144] V of the anti-CD3 scFv or Fab domain of the antibody construct herein H The V domain may comprise or consist of an amino acid sequence having at least about 80%, 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 102. In some embodiments, the V domain of such an scFv or Fab domain may comprise or consist of an amino acid sequence having at least about 80%, 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 102. H The V domain comprises or consists of an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 102. In some embodiments, the V domain of such an scFv or Fab domain comprises H The V domain comprises or consists of an amino acid sequence having at least about 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 102. In some embodiments, the V domain of such an scFv or Fab domain comprises H In some embodiments, the V domain of such an scFv or Fab domain comprises or consists of an amino acid sequence having at least about 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 102. H In yet other embodiments, the V domain of such an scFv or Fab domain comprises or consists of an amino acid sequence having at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 102. H The domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:102.

[0145] In the case of a Fab domain, the V of the anti-CD3 scFv or Fab domain of the antibody construct herein may be part of a light chain (e.g., L1) paired with the anti-CD3 Fab domain sequence of the heavy chain. LThe V domain may comprise or consist of an amino acid sequence having at least about 80%, 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 103. In some embodiments, the V domain of such an scFv or Fab domain may comprise or consist of an amino acid sequence having at least about 80%, 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 103. L The V domain comprises or consists of an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 103. In some embodiments, the V domain of such an scFv or Fab domain L The V domain comprises or consists of an amino acid sequence having at least about 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 103. In some embodiments, the V domain of such an scFv or Fab domain L In some embodiments, the V domain of such an scFv or Fab domain comprises or consists of an amino acid sequence having at least about 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 103. L The V domain comprises or consists of an amino acid sequence having at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 103. In various embodiments, the V domain of such an scFv or Fab domain L The domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:103.

[0146] The anti-CD3 domain is C in its heavy chain and C in its light chain, respectively. H1 Domain and C L In embodiments where the antibody construct herein further comprises a Fab domain, the C of the anti-CD3 Fab domain of the antibody construct herein further comprises a C H1 The C domain may comprise or consist of an amino acid sequence having at least about 80%, 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 107. In some embodiments, the C domain of such a Fab domain may comprise or consist of an amino acid sequence having at least about 80%, 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 107. H1 In some embodiments, the C of such a Fab domain comprises or consists of an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 107.H1 In some embodiments, the C of such a Fab domain comprises or consists of an amino acid sequence having at least about 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 107. H1 In some embodiments, the C of such a Fab domain comprises or consists of an amino acid sequence having at least about 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 107. H1 In some embodiments, the C of such a Fab domain comprises or consists of an amino acid sequence having at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 107. H1 The domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:107.

[0147] Moreover, the C of the anti-CD3 Fab domain of the antibody construct herein L The C domain may comprise or consist of an amino acid sequence having at least about 80%, 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 121. In some embodiments, the C domain of such a Fab domain may comprise or consist of an amino acid sequence having at least about 80%, 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 121. L In some embodiments, the C of such a Fab domain comprises or consists of an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 121. L In some embodiments, the C of such a Fab domain comprises or consists of an amino acid sequence having at least about 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 121. L In some embodiments, the C of such a Fab domain comprises or consists of an amino acid sequence having at least about 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 121. L In yet another embodiment, the C of such a Fab domain comprises or consists of an amino acid sequence having at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 121. LThe domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:121.

[0148] In some embodiments, the anti-CD3 binding domain of the antibody construct is an scFv domain. In some embodiments, such an anti-CD3 scFv domain comprises, from the N- to C-terminus, H -Linker scFv -V L In another embodiment, the anti-CD3 scFv domain has a domain structure of, from N to C-terminus, V L -Linker scFv -V H In any of these embodiments, and as further described herein, V H The domain may comprise or consist of an amino acid sequence having at least about 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 102, and may be a V L The domain may comprise or consist of an amino acid sequence having at least about 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 102, and the linker scFv may comprise or consist of the amino acid sequence shown in SEQ ID NO:104.

[0149] Thus, in some embodiments, the anti-CD3 scFv domain comprises, from N to C terminus: V H -Linker scFv -V L or V L -Linker scFv -V H and V H The domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 102, L The domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 103, and the linker scFv comprises or consists of the amino acid sequence set forth in SEQ ID NO: 104. In some of these embodiments, the anti-CD3 scFv domain comprises, from N to C-terminus, V H -Linker scFv -VL In another embodiment, the anti-CD3 scFv domain has a domain structure of, from N to C-terminus, V L -Linker scFv -V H It has a domain structure of

[0150] In some embodiments, the anti-CD3 binding domain of the antibody construct is a Fab domain. In some embodiments, such an anti-CD3 Fab domain comprises a heavy chain, or a portion thereof (e.g., where the heavy chain further comprises an Fc portion, etc.), and a light chain. The heavy chain of an anti-CD3 Fab domain comprises, from the N- to C-terminus, C H1 V linked to domain H In various embodiments, the anti-CD3 Fab domain may comprise or consist of (i) a V domain comprising or consisting of an amino acid sequence having at least about 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 102. H (ii) a V domain comprising or consisting of an amino acid sequence having at least about 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 103. L (iii) a C domain comprising or consisting of an amino acid sequence having at least about 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 107. H1 domain, and (iv) a C domain comprising or consisting of an amino acid sequence having at least about 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 121. L Includes the domain.

[0151] In certain embodiments, the anti-CD3 Fab domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:102. H domain, comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 107 H1 Domain, V comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 103 Ldomain, and a C comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 121. L Includes the domain.

[0152] In various embodiments, and as further described herein, the anti-CD3 Fab domain of the antibody construct comprises V H Domain and C H1 The portion of the heavy chain (e.g., H1) containing the domain is V L In some embodiments, a V H and C H1 The heavy chain, or portion thereof, comprising the domain and forming the anti-CD3 Fab domain may have the amino acid sequence set forth in SEQ ID NO: 154, and the corresponding light chain that pairs with the anti-CD3 portion of the heavy chain to form the anti-CD3 Fab domain may have the amino acid sequence set forth in SEQ ID NO: 120.

[0153] In some embodiments, the anti-CD3 binding domain of the antibody constructs herein comprises or consists of an scFv domain. Such scFv domains have the V H Domain and V L In some embodiments, the V H The domain is the linker scFv via V L In some embodiments, the V H The domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 102, L The domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 103. Thus, in certain embodiments, the anti-CD3 binding domain of the antibody constructs herein comprises a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 104. scFv V comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 103 via L V comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 102 linked to the domain HThe scFv domain comprises or consists of a domain.

[0154] As further described herein, in some embodiments, the binding affinity of a trivalent and trispecific antibody construct of the present disclosure for one or more particular targets (e.g., CD3) may depend, at least in part, on its format and / or geometry. By way of example, the relative orientation and proximity of anti-CD3 binding domains in an antibody construct may affect the ability of the binding domain to interact with the target epitope to which it binds, e.g., through steric hindrance, conformational changes that occur when the construct interacts with one or more of its targets (e.g., constrained degrees of freedom for the binding domain, such as its steric flexibility), etc.

[0155] In further embodiments, the CD3 binding affinity of the anti-CD3 binding domain of the antibody constructs herein may be engineered and altered (e.g., increased / decreased compared to the unmodified domain), for example, by using one or more amino acid modifications. In some embodiments, the trispecific and trivalent antibody constructs of the present disclosure may comprise a variant anti-CD3 binding domain that comprises one or more amino acid modifications in its VH and / or VL domain(s) compared to the anti-CD3 binding domain described herein, e.g., comprising the VH sequence set forth in SEQ ID NO: 102 and the VL sequence set forth in SEQ ID NO: 103. Such one or more amino acid modifications may reduce or increase the binding affinity of the variant anti-CD3 binding domain to CD3 when compared to the binding affinity of the corresponding anti-CD3 binding domain that does not comprise such one or more amino acid modifications.

[0156] In some embodiments, the one or more amino acid modifications used to alter the binding affinity of the anti-CD3 binding domain can include one or more amino acid substitutions, one or more amino acid additions, and / or one or more amino acid deletions. In certain embodiments, the one or more amino acid modifications used to alter the binding affinity of the anti-CD3 binding domain comprise or consist of one or more amino acid substitutions relative to the unmodified binding domain sequence (e.g., the anti-CD3 VH or VL sequence).

[0157] In some embodiments, the anti-CD3 affinity of the affinity-engineered CD3 binding domain may be about ±2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher or lower than the corresponding parent anti-CD3 binding domain.

[0158] In some embodiments, the trivalent and trispecific antibody constructs of the present disclosure may have a variety of different anti-CD3 and anti-CD28 (described further below) binding affinities, providing various constructs with different target engagement profiles, particularly when considering that construct format and geometry can further influence antigen binding (see, e.g., Figures 22A-22C).

[0159] B.2.2 Binding Domain for CD28 As described herein, in various embodiments, the trivalent and trispecific antibody constructs of the present disclosure comprise a binding domain capable of binding to CD28.

[0160] In various embodiments, such anti-CD28 binding domains (e.g., scFv domains or Fab domains) of the antibody constructs herein have an affinity (EC ) for CD28 of about 10 nM to about 500 nM or about 20 nM to about 250 nM. 50 , i.e., K for binding to CD28 D The value may be given as

[0161] In some embodiments, the antibody constructs herein comprise the V H The CDRs of the sequences and the V sequences shown in SEQ ID NOs: 313, 316, and 320 L The sequence includes the CDRs.

[0162] In some embodiments, the antibody constructs herein comprise a binding domain (e.g., an scFv domain or a Fab domain) capable of binding to CD28 on a T cell, wherein such binding domain has an affinity for CD28 of about 15 nM to about 35 nM, and comprises an anti-CD28 V domain comprising an HCDR1 having the sequence SYGVH (SEQ ID NO: 300), an HCDR2 having the sequence VIWPGGGTNFNSALMS (SEQ ID NO: 303), and an HCDR3 having the sequence DRAYGNYLYAMDY (SEQ ID NO: 307). H and an anti-CD28 V antibody comprising an LCDR1 having the sequence RASESVEYYVTSLMQ (SEQ ID NO: 313), an LCDR2 having the sequence AASNVDS (SEQ ID NO: 316), and an LCDR3 having the sequence QQSRKVPFT (SEQ ID NO: 320). L Contains arrays.

[0163] In such embodiments, the antibody constructs herein comprise a binding domain (e.g., an scFv domain or a Fab domain) capable of binding to CD28 on a T cell, wherein such binding domain comprises or consists of an amino acid sequence having at least about 80%, 90%, 95%, 97%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 106. H domain, and V comprising or consisting of an amino acid sequence having at least about 80%, 90%, 95%, 97%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:116. L In some of these embodiments, the anti-CD28 binding domain comprises a V domain comprising or consisting of the sequence set forth in SEQ ID NO: 106. H domain, and V comprising or consisting of the sequence set forth in SEQ ID NO: 116 L Includes the domain.

[0164] In other embodiments, the trivalent and trispecific antibody construct of the present disclosure comprises (i) a V comprising a sequence having one or more amino acid substitutions compared to the sequence set forth in SEQ ID NO: 106. H domain, and / or (ii) a V comprising a sequence having one or more amino acid substitutions compared to the sequence set forth in SEQ ID NO: 116. L The positions of such amino acid substitutions are provided according to the IMGT numbering system.

[0165] In some embodiments, anti-CD28 V H domain and / or anti-CD28 V L Such one or more amino acid substitutions in any of the domains may be used to identify a V having such amino acid substitutions. H and / or V L an anti-CD28 binding domain that does not contain the sequence (e.g., the V shown in SEQ ID NOs: 106 and 116, respectively); H and V L When compared to the binding affinity of a corresponding anti-CD28 binding domain (a binding domain comprising the sequence), the binding affinity of the corresponding anti-CD28 binding domain may be reduced by about 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 2.0-fold, 3.0-fold, 3.3-fold, 3.5-fold, 3.7-fold, 3.9-fold, 5.0-fold, 5.2-fold, 5.5-fold, 6.0-fold, 7.0-fold, 8.0-fold, 8.5-fold, 9.0-fold, 10-fold, 20-fold, or 25-fold, or by about 1.5-fold to about 25-fold, about 2.0-fold to about 20-fold, about 3.0-fold to about 20-fold, or about 5.0-fold to about 10-fold.

[0166] Thus, in some embodiments, the anti-CD28 binding domain (e.g., scFv domain or Fab domain) of the antibody constructs herein has an affinity (EC ) for CD28 of about 10 nM to about 500 nM, about 20 nM to about 600 nM, about 20 nM to about 250 nM, about 20 nM to about 150 nM, about 20 nM to about 100 nM, or about 20 nM to about 50 nM. 50 , i.e., K for binding to CD28 D The value may be given as

[0167] In some embodiments, the anti-CD28 binding domain herein comprises a V nucleotide sequence comprising the amino acid substitution P1058A relative to the amino acid sequence set forth in SEQ ID NO: 106. H In some of these embodiments, the anti-CD28 V domain H The domain comprises the amino acid sequence set forth in SEQ ID NO:204.

[0168] In some embodiments, the anti-CD28 binding domain herein comprises a V nucleotide sequence comprising the amino acid substitution G1064S relative to the amino acid sequence set forth in SEQ ID NO: 106. H In some of these embodiments, the anti-CD28 V domain H The domain comprises the amino acid sequence set forth in SEQ ID NO:207.

[0169] In some embodiments, the anti-CD28 binding domain herein comprises a V nucleotide sequence comprising the amino acid substitution V1035G relative to the amino acid sequence set forth in SEQ ID NO: 116. L In some of these embodiments, the anti-CD28 V domain L The domain comprises the amino acid sequence set forth in SEQ ID NO:200.

[0170] In some embodiments, the anti-CD28 binding domain herein comprises a V nucleotide sequence comprising the amino acid substitution D1068E relative to the amino acid sequence set forth in SEQ ID NO: 116. L In some of these embodiments, the anti-CD28 V domain L The domain comprises the amino acid sequence set forth in SEQ ID NO:209.

[0171] In some embodiments, the anti-CD28 binding domain herein comprises a V nucleotide sequence comprising the amino acid substitution E1080K relative to the amino acid sequence set forth in SEQ ID NO: 106. H In some of these embodiments, the anti-CD28 V domain H The domain comprises the amino acid sequence set forth in SEQ ID NO:203.

[0172] In some embodiments, the anti-CD28 binding domain herein comprises a V nucleotide sequence comprising the amino acid substitution Y1110S relative to the amino acid sequence set forth in SEQ ID NO: 106. H In some of these embodiments, the anti-CD28 V domain H The domain comprises the amino acid sequence set forth in SEQ ID NO:201.

[0173] In some embodiments, the anti-CD28 binding domain herein comprises a V sequence comprising the amino acid substitution N1111aA relative to the amino acid sequence set forth in SEQ ID NO: 106. H In some of these embodiments, the anti-CD28 V domain H The domain comprises the amino acid sequence set forth in SEQ ID NO:208.

[0174] In some embodiments, the anti-CD28 binding domain herein comprises a V nucleotide sequence comprising the amino acid substitution Y1112S relative to the amino acid sequence set forth in SEQ ID NO: 106. H In some of these embodiments, the anti-CD28 V domain H The domain comprises the amino acid sequence set forth in SEQ ID NO:113.

[0175] In some embodiments, the anti-CD28 binding domain herein comprises a V sequence comprising the amino acid substitution L1112aN relative to the amino acid sequence set forth in SEQ ID NO: 106. H In some of these embodiments, the anti-CD28 V domain H The domain comprises the amino acid sequence set forth in SEQ ID NO:206.

[0176] In some embodiments, the anti-CD28 binding domain herein comprises a V nucleotide sequence comprising the amino acid substitution Y1113S relative to the amino acid sequence set forth in SEQ ID NO: 106. H In some of these embodiments, the anti-CD28 V domain H The domain comprises the amino acid sequence set forth in SEQ ID NO:210.

[0177] In some embodiments, the anti-CD28 binding domain herein comprises a V nucleotide sequence comprising the amino acid substitution Y1037A relative to the amino acid sequence set forth in SEQ ID NO: 106. H In some of these embodiments, the anti-CD28 V domain H The domain comprises the amino acid sequence set forth in SEQ ID NO:205.

[0178] In some embodiments, the anti-CD28 binding domain herein comprises a V nucleotide sequence comprising the amino acid substitution Y1031A relative to the amino acid sequence set forth in SEQ ID NO: 116. L In some of these embodiments, the anti-CD28 V domain L The domain comprises the amino acid sequence set forth in SEQ ID NO:151.

[0179] In some embodiments, the anti-CD28 binding domain herein comprises a V nucleotide sequence comprising the amino acid substitution N1066A relative to the amino acid sequence set forth in SEQ ID NO: 116. L In some of these embodiments, the anti-CD28 V domain L The domain comprises the amino acid sequence set forth in SEQ ID NO:202.

[0180] In some embodiments, V H and / or V L Antibody constructs comprising an anti-CD28 binding domain containing one or more of the domain substitutions may exhibit reduced non-specific activity in vitro and / or in vivo. In one such embodiment, antibody constructs comprising such mutated anti-CD28 binding domains (compared to the huTN228 wild-type sequence) may induce lower non-specific immune cell activity, e.g., with reduced non-specific cytokine production by immune cells. In certain embodiments, the V is reduced compared to huTN228 wild-type. L An antibody construct comprising an anti-CD28 binding domain carrying an N1066A substitution in the V domain may induce reduced non-specific T cell activity, e.g., cytokine production, relative to huTN228 wild type. LAntibody constructs comprising an anti-CD28 binding domain bearing a Y1031A substitution in the domain can induce reduced non-specific T cell activity, e.g., cytokine production. In some embodiments, non-specific T cell activity, e.g., cytokine production, e.g., TNFα, IL-2, etc., can be reduced by about 10-fold, 20-fold, 30-fold, 50-fold, 60-fold, 70-fold, or about 100-fold.

[0181] In various embodiments, the antibody constructs herein comprise a binding domain capable of binding to CD28 on a T cell, wherein such binding domain has an EC for binding to CD28 of about 20 nM to about 600 nM. 50 and a V having an HCDR1 with the sequence SX1GVH (SEQ ID NO: 302), an HCDR2 with the sequence VIWX2GGX3TNFNSALMS (SEQ ID NO: 306), and an HCDR3 with the sequence DRAX4GX5YX6X7AMDY (SEQ ID NO: 312). H LCDR1, sequence AASX9VX, having the domain and sequence RASESVEYYX8TSLMQ (SEQ ID NO: 315) 10 V comprising an LCDR2 having the sequence QQSRKVPFT (SEQ ID NO: 320), and an LCDR3 having the sequence QQSRKVPFT (SEQ ID NO: 321). L X1 = Y, A; X2 = P, A; X3 = G, S; X4 = S, Y; X5 = N, A; X6 = L, N; X7 = S, Y; X8 = G, V; X9 = N, A; and X 10 =E, D.

[0182] In some embodiments, the anti-CD28 binding domain (e.g., scFv domain or Fab domain) of the antibody constructs herein comprises or consists of an amino acid sequence having at least about 80%, 90%, 95%, 97%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 106. H domain, and V comprising or consisting of an amino acid sequence having at least about 80%, 90%, 95%, 97%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:116. LIn some of these embodiments, the anti-CD28 binding domain of the antibody constructs herein comprises a V domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 106. H domain, and V comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 116 L Includes the domain.

[0183] In some embodiments, the anti-CD28 binding domain (e.g., scFv domain or Fab domain) of the antibody constructs herein comprises a V domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 204. H domain, and V comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 116 L Includes the domain.

[0184] In some embodiments, the anti-CD28 binding domain (e.g., scFv domain or Fab domain) of the antibody constructs herein comprises a V domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 207. H domain, and V comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 116 L Includes the domain.

[0185] In some embodiments, the anti-CD28 binding domain (e.g., scFv domain or Fab domain) of the antibody constructs herein comprises a V domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 106. H domain, and V comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 200 L Includes the domain.

[0186] In some embodiments, the anti-CD28 binding domain (e.g., scFv domain or Fab domain) of the antibody constructs herein comprises a V domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 106. H domain, and V comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 209 L Includes the domain.

[0187] In some embodiments, the anti-CD28 binding domain (e.g., scFv domain or Fab domain) of the antibody constructs herein comprises a V domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 203. H domain, and V comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 116 L Includes the domain.

[0188] In some embodiments, the anti-CD28 binding domain (e.g., scFv domain or Fab domain) of the antibody constructs herein comprises a V domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 201. H domain, and V comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 116 L Includes the domain.

[0189] In some embodiments, the anti-CD28 binding domain (e.g., scFv domain or Fab domain) of the antibody constructs herein comprises a V domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 208. H domain, and V comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 116 L Includes the domain.

[0190] In some embodiments, the anti-CD28 binding domain (e.g., scFv domain or Fab domain) of the antibody constructs herein comprises a V domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 113. H domain, and V comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 116 L Includes the domain.

[0191] In some embodiments, the anti-CD28 binding domain (e.g., scFv domain or Fab domain) of the antibody constructs herein comprises a V domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 206. Hdomain, and V comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 116 L Includes the domain.

[0192] In some embodiments, the anti-CD28 binding domain (e.g., scFv domain or Fab domain) of the antibody constructs herein comprises a V domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 210. H domain, and V comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 116 L Includes the domain.

[0193] In some embodiments, the anti-CD28 binding domain (e.g., scFv domain or Fab domain) of the antibody constructs herein comprises a V domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 205. H domain, and V comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 116 L Includes the domain.

[0194] In some embodiments, the anti-CD28 binding domain (e.g., scFv domain or Fab domain) of the antibody constructs herein comprises a V domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 106. H domain, and V comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 151 L Includes the domain.

[0195] In some embodiments, the anti-CD28 binding domain (e.g., scFv domain or Fab domain) of the antibody constructs herein comprises a V domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 106. H domain, and V comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 202 L Includes the domain.

[0196] In embodiments where the anti-CD28 binding domain of the antibody construct is an scFv domain, such anti-CD28 scFv domain may be linked at either the N- to C-terminus or the C- to N-terminus by a linker scFv (The sequence is (G n S) m wherein n and m can independently be 1, 2, 3, 4, or 5, and are as set forth in SEQ ID NO: 348), through SEQ ID NO: 116, 151, 200, 202, or 209. L V comprising the amino acid sequence set forth in any one of SEQ ID NOs: 106, 113, 201, 203, 204, 205, 206, 207, 208, or 210 linked to a domain H In some of these examples, the linker domain may be included. scFv comprises or consists of the amino acid sequence set forth in SEQ ID NO: 104. In some embodiments, the anti-CD28 scFv domain also comprises, from N to C-terminus, V H -Linker scFv -V L In another embodiment, the anti-CD28 scFv domain has a domain structure of, from N to C-terminus, V L -Linker scFv -V H It has a domain structure of

[0197] In some embodiments, the anti-CD28 scFv domain of the antibody construct herein comprises or consists of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 119. In some embodiments, the anti-CD28 scFv domain of the antibody construct herein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 119.

[0198] In embodiments where the anti-CD28 binding domain of the antibody construct is a Fab domain, such anti-CD28 Fab domain has at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 117.L V having at least about 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 116 linked to a sequence L C having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 107, paired with a light chain comprising the sequence H1 The heavy chain may comprise a VH sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:106 linked to a sequence.

[0199] In some embodiments, the anti-CD28 Fab domain of the antibody construct herein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 117. L V comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 116, 151, 200, 202, or 209 linked to a sequence L C comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 107, paired with a light chain comprising the sequence H1 V comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 106, 113, 201, 203, 204, 205, 206, 207, 208, or 210 linked to a sequence H The heavy chain may comprise a sequence.

[0200] In some embodiments, the anti-CD28 Fab domain of the antibody construct herein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 117. L V comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 116 linked to the sequence L C comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 107, paired with a light chain comprising the sequence H1 V comprising or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 106 linked to a sequence H The heavy chain may comprise a sequence.

[0201] In certain embodiments, provided herein is an antibody construct comprising a binding domain capable of binding to CD28, wherein the binding domain comprises a VHV comprising an HCDR1 having the sequence SX1GVH (SEQ ID NO: 302), an HCDR2 having the sequence VIWX2GGX3TNFNSALMS (SEQ ID NO: 306), and an HCDR3 having the sequence DRAX4GX5YX6X7AMDY (SEQ ID NO: 312). H LCDR1 having the sequence RASESVEYYX8TSLMQ (SEQ ID NO: 315), and LCDR2 having the sequence AASX9VX 10 V comprising an LCDR2 having the sequence QQSRKVPFT (SEQ ID NO: 320), and an LCDR3 having the sequence QQSRKVPFT (SEQ ID NO: 321). L and contains one or more of the following amino acid substitutions at the positions specified in the CDR sequences: X1: Y to A, X2: P to A, X3: G to S, X4: S to Y, X5: N to A, X6: L to N, X7: S to Y, X8: G to V, X9: N to A, or X 10 :E to D, antibody constructs are described.

[0202] In some embodiments, the antibody construct comprises an anti-CD28 binding domain comprising substitutions X1:Y to A. In some embodiments, the antibody construct comprises an anti-CD28 binding domain comprising substitutions X2:P to A. In some embodiments, the antibody construct comprises an anti-CD28 binding domain comprising substitutions X3:G to S. In some embodiments, the antibody construct comprises an anti-CD28 binding domain comprising substitutions X4:S to Y. In some embodiments, the antibody construct comprises an anti-CD28 binding domain comprising substitutions X5:N to A. In some embodiments, the antibody construct comprises an anti-CD28 binding domain comprising substitutions X6:L to N. In some embodiments, the antibody construct comprises an anti-CD28 binding domain comprising substitutions X7:S to Y. In some embodiments, the antibody construct comprises an anti-CD28 binding domain comprising substitutions X8:G to V. In some embodiments, the antibody construct comprises an anti-CD28 binding domain comprising substitutions X9:N to A. In some embodiments, the antibody construct comprises an anti-CD28 binding domain comprising substitutions X 10: Contains the anti-CD28 binding domain comprising E to D.

[0203] In some embodiments, such antibody constructs may have binding affinity for CD28 that is reduced by about 1.5-fold to about 25-fold, about 2.0-fold to about 20-fold, about 3.0-fold to about 20-fold, or about 5.0-fold to about 10-fold when compared to the binding affinity of an antibody construct comprising an anti-CD28 binding domain that does not contain one or more amino acid substitutions in one or more of the CDR sequences.

[0204] In some embodiments, such antibody constructs comprise the V H The CDRs of the domains and V shown in SEQ ID NOs: 313, 316, and 320 L The anti-CD28 binding domain may comprise the CDRs of the sequence:

[0205] In some embodiments, such an antibody construct comprises an anti-CD28 V domain comprising an HCDR1 having the sequence SYGVH (SEQ ID NO: 300), an HCDR2 having the sequence VIWPGGGTNFNSALMS (SEQ ID NO: 303), and an HCDR3 having the sequence DRAYGNYLYAMDY (SEQ ID NO: 307). H and an anti-CD28 V antibody comprising an LCDR1 having the sequence RASESVEYYVTSLMQ (SEQ ID NO: 313), an LCDR2 having the sequence AASNVDS (SEQ ID NO: 316), and an LCDR3 having the sequence QQSRKVPFT (SEQ ID NO: 320). L The antibody may comprise an anti-CD28 binding domain comprising the sequence

[0206] C. Binding domain for tumor-associated antigen (TAA) As described herein, in various embodiments, the trivalent and trispecific antibody constructs of the present disclosure comprise binding domains capable of binding to a TAA. In various embodiments, and as further described herein, the antibody constructs herein may be trivalent and trispecific, comprising a first binding domain capable of binding to a first antigen on a first cytotoxic effector cell, a second binding domain capable of binding to a second antigen on a second cytotoxic effector cell, and a third binding domain, where such third binding domain is capable of binding to a TAA. The TAA may be any antigenic substance expressed on the surface of a tumor cell.

[0207] As described herein, in various embodiments, the anti-TAA binding domain of the antibody construct may be capable of binding to mesothelin (MSLN).

[0208] In other embodiments, the anti-TAA binding domain of the antibody construct may be capable of binding to Claudin18.2 (Cldn18.2).

[0209] Typically, the anti-TAA binding domain of the antibody construct herein may be an scFv domain or a Fab domain.

[0210] In various embodiments, the anti-TAA binding domain of the trivalent and trispecific antibody constructs herein is an scFv domain.

[0211] In various embodiments, the anti-TAA binding domain (e.g., scFv domain) of the antibody constructs herein is capable of binding to Cldn18.2. In some embodiments, such an anti-Cldn18.2 binding domain comprises an HCDR1 having the sequence SNPMI (SEQ ID NO: 333), an HCDR2 having the sequence IIDTDGSTYYADWAKG (SEQ ID NO: 334), and an HCDR3 having the sequence RLHGSSNGYYDDL (SEQ ID NO: 335), and an LCDR1 having the sequence QASQSIYSYLS (SEQ ID NO: 336), an LCDR2 having the sequence KASTLAS (SEQ ID NO: 337), and an LCDR3 having the sequence QQGYTVTNVDKNT (SEQ ID NO: 338). L Contains arrays.

[0212] In some embodiments, the anti-Cldn18.2 binding domain (e.g., scFv domain) of the antibody constructs herein comprises or consists of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 127. H and V comprising or consisting of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 128. L Contains arrays.

[0213] In various embodiments, the anti-Cldn18.2 binding domain of the antibody constructs herein is an scFv domain and is connected, either N- to C-terminally or C- to N-terminally, to a linker having the amino acid sequence set forth in SEQ ID NO: 104. scFv V comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 128 via L V comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 127 linked to the sequence H Contains arrays.

[0214] In various embodiments, the anti-TAA binding domain (e.g., scFv domain) of the antibody constructs herein is capable of binding to MSLN. In some embodiments, such an anti-MSLN binding domain comprises an HCDR1 having the sequence GYTMN (SEQ ID NO: 327), an HCDR2 having the sequence LITPYSGASSYAQKFQG (SEQ ID NO: 328), and an HCDR3 having the sequence GGYDGRGFDY (SEQ ID NO: 329). H and a V comprising an LCDR1 having the sequence SASSSVSYMH (SEQ ID NO: 330), an LCDR2 having the sequence DTSKLAS (SEQ ID NO: 331), and an LCDR3 having the sequence QQWSGHPLT (SEQ ID NO: 332). L Contains arrays.

[0215] In some embodiments, the anti-MSLN binding domain of the antibody construct herein is an scFv domain and comprises or consists of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 112. In some embodiments, the anti-MSLN scFv domain of the antibody construct herein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 112.

[0216] In certain embodiments, the V H CDRs of the sequences and Vs shown in SEQ ID NOs: 336 to 338 L An antibody construct is described that comprises an anti-Cldn18.2 binding domain comprising the CDRs of the sequences:

[0217] In some embodiments, such an antibody construct comprises a V HCDR1 having the sequence SNPMI (SEQ ID NO: 333), an HCDR2 having the sequence IIDTDGSTYYADWAKG (SEQ ID NO: 334), and an HCDR3 having the sequence RLHGSSNGYYDDL (SEQ ID NO: 335). Hand a V comprising an LCDR1 having the sequence QASQSIYSYLS (SEQ ID NO: 336), an LCDR2 having the sequence KASTLAS (SEQ ID NO: 337), and an LCDR3 having the sequence QQGYTVTNVDKNT (SEQ ID NO: 338). L The antibody may comprise an anti-Cldn18.2 binding domain comprising the sequence

[0218] In some embodiments, such antibody constructs comprise an anti-Cldn18.2 binding domain comprising or consisting of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 127, and a V domain comprising or consisting of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 128. L In certain embodiments, such an anti-Cldn18.2 binding domain may comprise a V that comprises or consists of the amino acid sequence set forth in SEQ ID NO:127. H and V comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 128. L Contains arrays.

[0219] D. Fc domain As described herein, the trivalent and trispecific antibody constructs of the present disclosure can comprise an Fc domain (or Fc region) comprising a first Fc polypeptide and a second Fc polypeptide. In various embodiments, the Fc domain is a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein the first Fc polypeptide and the second Fc polypeptide share about 90%, 95%, 97%, or about 99% amino acid sequence identity and each comprise one or more asymmetric amino acid substitutions that can promote preferential pairing of the Fc polypeptides to form the heterodimeric Fc domain relative to the formation of their respective homodimeric Fc domains.

[0220] The term "Fc domain," as used herein, includes native (or wild-type) sequence Fc domains and variant Fc domains containing one or more amino acid modifications compared to the corresponding native or wild-type Fc domain. Unless otherwise specified herein, the numbering of amino acid residues in an Fc domain or constant region follows the EU numbering system, also known as the EU index, as described, for example, in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). The "Fc polypeptide" of a dimeric (e.g., heterodimeric) Fc domain refers to one of the two polypeptide chains (e.g., the first and second Fc polypeptides) that form the dimeric (e.g., heterodimeric) Fc domain. In some embodiments, the Fc polypeptide can include a C-terminal constant region of an Ig heavy chain capable of stable self-association. In various embodiments, and as further described herein, an Fc polypeptide (e.g., a first or second Fc polypeptide) comprises at least one of a CH2 domain and / or a CH3 domain. In certain embodiments, the Fc polypeptide of the antibody constructs described herein comprises a CH2 domain and a CH3 domain.

[0221] As disclosed herein, trivalent and trispecific antibody constructs can comprise an Fc domain, and such an Fc domain can be a heterodimeric Fc domain. An Fc domain of an antibody construct, e.g., a heterodimeric Fc domain, comprises a first Fc polypeptide and a second Fc polypeptide, unless otherwise specified. Typically, each Fc polypeptide of a (e.g., heterodimeric) Fc domain can comprise a CH2 domain, a CH3 domain, or, as described in various embodiments herein, both a CH2 domain and a CH3 domain.

[0222] In certain embodiments, the antibody construct comprises an Fc domain based on a human IgG Fc domain. In some embodiments, the antibody construct comprises an Fc domain based on a human IgG1 Fc domain. In various embodiments, the antibody construct comprises two different Fc polypeptides, e.g., a heterodimeric IgG Fc domain comprising a first Fc polypeptide and a second polypeptide, wherein the first and second Fc polypeptides have different amino acid sequences, e.g., amino acid sequences that have about 90%, 95%, 97%, or 99% sequence identity when compared and aligned with each other, as further described herein. In some embodiments, the difference in the amino acid sequences of the first and second Fc polypeptides can be due to asymmetric amino acid substitutions that can be introduced into each Fc polypeptide chain to promote preferential pairing of heavy chains to form the heterodimeric Fc domain, compared to the corresponding homodimeric Fc domain.

[0223] In various embodiments, the trivalent and trispecific antibody constructs herein comprise Fc domains that are modified IgG Fc domains, wherein at least the CH3 domain of at least one Fc polypeptide comprises one or more amino acid modifications compared to the respective wild-type CH3 domain. In some embodiments, the antibody constructs herein comprise Fc domains that are modified IgG Fc domains, wherein at least the CH2 domain of at least one Fc polypeptide comprises one or more amino acid modifications compared to the respective wild-type CH2 domain. In some embodiments, the antibody constructs herein comprise Fc domains that are modified IgG Fc domains, wherein both the CH3 domain and the CH2 domain of at least one Fc polypeptide comprise one or more amino acid modifications compared to the respective wild-type CH3 and CH2 domains. In various embodiments, both Fc polypeptides of a heterodimeric Fc domain may comprise one or more amino acid modifications in their CH3 domains. In some embodiments, both Fc polypeptides of a heterodimeric Fc domain may comprise one or more amino acid modifications in their CH2 domains. In yet other embodiments, both Fc polypeptides of a heterodimeric Fc domain may comprise one or more amino acid modifications in their CH2 and CH3 domains.

[0224] Modified Fc domain In some embodiments, the present disclosure relates to trivalent and trispecific antibody constructs that may include heterodimeric Ig Fc domains comprising modified heterodimeric CH3 domains, wherein the modified heterodimeric CH3 domains comprise one or more asymmetric amino acid modifications, i.e., one or both of the first and second Fc polypeptides each comprise one or more amino acid modifications in their CH3 domain sequences compared to their respective wild-type sequences. As used herein, the term "asymmetric amino acid modification" generally refers to a modification in which an amino acid at a particular position on a first Fc polypeptide is different from the amino acid at the corresponding position on a second Fc polypeptide. These asymmetric amino acid modifications may include modification of only one of the two amino acids at corresponding positions on each Fc polypeptide, or they may include modification of both amino acids at corresponding positions on each of the first and second Fc polypeptides. In various embodiments, an "asymmetric amino acid modification" is an asymmetric amino acid substitution.

[0225] In some embodiments, the antibody constructs herein comprise a heterodimeric Fc domain (i.e., a heterodimeric CH3 domain consisting of two CH3 domains of a first and a second Fc polypeptide) comprising a modified CH3 domain, wherein the modified CH3 domain comprises one or more asymmetric amino acid modifications that promote the formation of the heterodimeric Fc domain (e.g., pairing of a first Fc polypeptide with a second Fc polypeptide) over the formation of a corresponding homodimeric Fc domain (e.g., pairing of a first Fc polypeptide with another first Fc polypeptide). Amino acid modifications that can be made to the CH3 domain of an Fc domain to promote the formation of heterodimeric Fc domains are known in the art, and include, for example, the techniques described in International Publication No. WO 96 / 027011 ("knobs-into-holes"), Gunasekaran et al., 2010, J Biol Chem, 285, 19637-46 ("electrostatic steering"), Davis et al., 2010, Prot Eng Des Sel, 23(4):195-202 (strand-exchange engineered domain (SEED) technology), and Labrijn et al., 2013, Proc Natl Acad Sci USA, 110(13):5145-50 (Fab arm exchange). Other examples include approaches that combine positive and negative strategies to generate stable asymmetrically modified Fc regions, as described in International Publication Nos. WO2012 / 058768 and WO2013 / 063702.

[0226] In certain embodiments, the antibody constructs described herein comprise a heterodimeric Fc domain, wherein at least one, or both, of the Fc polypeptide chains comprises a modified heterodimeric CH3 domain comprising one or more amino acid modifications, as described in International Publication No. WO2012 / 058768 or International Patent Publication No. WO2013 / 063702.

[0227] In some embodiments, the antibody constructs described herein comprise a heterodimeric human IgG1 Fc domain with a modified CH3 domain. Table 2 herein lists the full-length human IgG1 heavy chain (e.g., V H , C H1 The present invention provides an amino acid sequence of a human IgG1 Fc domain sequence (e.g., a sequence from which a first and / or second Fc polypeptide may be derived) identified in SEQ ID NO: 1, corresponding to amino acids 231 to 447 of the full-length human IgG1 heavy chain (including the hinge, hinge, CH2 domain, and CH3 domain). The CH2 domain is typically defined as comprising amino acids 231 to 340 of the full-length human IgG1 heavy chain, and the CH3 domain is typically defined as comprising amino acids 341 to 447 of the full-length human IgG1 heavy chain.

[0228] As described herein, an antibody construct can comprise a heterodimeric Fc domain having a modified CH3 domain comprising one or more asymmetric amino acid modifications that promote the formation of heterodimeric Fc domains over homodimeric Fc domains, wherein the modified CH3 domain comprises a first Fc polypeptide comprising amino acid modifications at positions F405 and Y407 relative to SEQ ID NO: 1, and a second Fc polypeptide comprising amino acid modifications at positions T366 and T394 relative to SEQ ID NO: 1. In various embodiments, the one or more amino acid modifications comprise one or more amino acid substitutions. Thus, in some embodiments, the amino acid modification at position F405 of the first Fc polypeptide of the modified CH3 domain is F405A, F405I, F405M, F405S, F405T, or F405V. In some embodiments, the amino acid modification at position Y407 of the first Fc polypeptide of the modified CH3 domain is Y407I or Y407V. In some embodiments, the amino acid modification at position T366 in the second Fc polypeptide of the modified CH3 domain is T366I, T366L, or T366M. In some embodiments, the amino acid modification at position T394 in the second Fc polypeptide of the modified CH3 domain is T394W. In some embodiments, the modified CH3 domain of the first Fc polypeptide further comprises an amino acid modification at position L351 relative to SEQ ID NO: 1. In some embodiments, the amino acid modification at position L351 in the first Fc polypeptide of the modified CH3 domain is L351Y. In some embodiments, the second Fc polypeptide of the modified CH3 domain further comprises an amino acid modification at position K392 relative to SEQ ID NO: 1. In some embodiments, the amino acid modification at position K392 in the second Fc polypeptide of the modified CH3 domain is K392F, K392L, or K392M. In some embodiments, one or both of the first and second Fc polypeptides of the modified CH3 domain further comprises the amino acid modification T350V.

[0229] In certain embodiments, the antibody constructs herein comprise heterodimeric Fc domains having modified CH3 domains comprising one or more asymmetric amino acid modifications that promote heterodimeric Fc domain formation over homodimeric Fc domain formation, wherein the modified CH3 domains comprise a first Fc polypeptide comprising the amino acid modifications F405A, F405I, F405M, F405S, F405T, or F405V together with the amino acid modification Y407I or Y407V relative to SEQ ID NO: 1, and a second Fc polypeptide comprising the amino acid modifications T366I, T366L, or T366M together with the amino acid modification T394W relative to SEQ ID NO: 1. In some embodiments, the first Fc polypeptide of the modified CH3 domain further comprises the amino acid modification L351Y. In some embodiments, the second Fc polypeptide of the modified CH3 domain further comprises the amino acid modification K392F, K392L, or K392M. In some embodiments, one or both of the first and second Fc polypeptides having a modified CH3 domain further comprise the amino acid modification T350V. [Table 2]

[0230] In certain embodiments, the antibody constructs herein comprise a heterodimeric Fc domain comprising a modified CH3 domain with a first Fc polypeptide comprising amino acid modifications at positions F405 and Y407, and optionally further comprising an amino acid modification at position L351, relative to SEQ ID NO: 1, and a second Fc polypeptide comprising amino acid modifications at positions T366 and T394, and optionally further comprising an amino acid modification at position K392, wherein the first Fc polypeptide comprises amino acid modifications at positions S400 or Q347, or and / or the second Fc polypeptide further comprises an amino acid modification at one or both of positions K360 or N390, wherein the amino acid modification at position S400 is S400E, S400D, S400R, or S400K; the amino acid modification at position Q347 is Q347R, Q347E, or Q347K; the amino acid modification at position K360 is K360D or K360E; and the amino acid modification at position N390 is N390R, N390K, or N390D.

[0231] In some embodiments, the antibody construct comprises a heterodimeric Fc domain comprising a modified CH3 domain comprising any one of variant 1, variant 2, variant 3, variant 4, or variant 5 modifications as shown in Table 2.

[0232] In various embodiments, an antibody construct of the present disclosure may comprise a heterodimeric Fc domain comprising a first Fc polypeptide (A) and a second Fc polypeptide (B), wherein the first Fc polypeptide (A) and the second Fc polypeptide (B) comprise amino acid substitutions in their CH3 domains according to Variant #1 as shown in Table 2. In other embodiments, an antibody construct of the present disclosure may comprise a heterodimeric Fc domain comprising a first Fc polypeptide (A) and a second Fc polypeptide (B), wherein the first Fc polypeptide (A) and the second Fc polypeptide (B) comprise amino acid substitutions in their CH3 domains according to Variant #2 as shown in Table 2. In some embodiments, an antibody construct of the present disclosure may comprise a heterodimeric Fc domain comprising a first Fc polypeptide (A) and a second Fc polypeptide (B), wherein the first Fc polypeptide (A) and the second Fc polypeptide (B) comprise amino acid substitutions in their CH3 domains according to Variant #3 as shown in Table 2. In some embodiments, antibody constructs of the present disclosure may comprise a heterodimeric Fc domain comprising a first Fc polypeptide (A) and a second Fc polypeptide (B), wherein the first Fc polypeptide (A) and the second Fc polypeptide (B) comprise amino acid substitutions in their CH3 domains according to variant #4 as shown in Table 2. In yet other embodiments, antibody constructs of the present disclosure may comprise a heterodimeric Fc domain comprising a first Fc polypeptide (A) and a second Fc polypeptide (B), wherein the first Fc polypeptide (A) and the second Fc polypeptide (B) comprise amino acid substitutions in their CH3 domains according to variant #5 as shown in Table 2.

[0233] In certain embodiments, the CH3 domain of the first Fc polypeptide of the antibody construct herein has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 97%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 110. In certain embodiments, the CH3 domain of the second Fc polypeptide of the antibody construct herein has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 97%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 114. In some embodiments, the CH3 domain of the first Fc polypeptide of the antibody construct herein has the amino acid sequence set forth in SEQ ID NO: 110, and the second Fc polypeptide of the antibody construct herein has the amino acid sequence set forth in SEQ ID NO: 114.

[0234] In certain embodiments, the antibody constructs herein comprise a heterodimeric Fc domain based on an IgG Fc domain with a modified CH2 domain (i.e., a heterodimeric CH2 domain consisting of two CH2 domain sequences from respective first and second Fc polypeptides). In some embodiments, the antibody construct comprises a heterodimeric Fc domain based on an IgG Fc domain with a modified CH2 domain, wherein the modification(s) in the CH2 domain result in altered (e.g., reduced or attenuated) binding to one or more Fc receptors (FcR), e.g., receptors of the FcγRI, FcγRII, and FcγRIII subclasses.

[0235] Several amino acid modifications to the CH2 domain of the first and / or second Fc polypeptide(s) of an Fc domain that selectively alter the affinity of such Fc domains for different Fcγ receptors are known in the art. Both amino acid modifications that increase binding and amino acid modifications that decrease binding may be useful in certain indications. For example, increasing the binding affinity of an Fc to FcγRIIIa (an activating receptor) may result in increased antibody-dependent cell-mediated cytotoxicity (ADCC), which in turn may result in increased lysis of target cells. Decreased binding to FcγRIIb (an inhibitory receptor) may similarly be beneficial in some situations. In certain indications, reducing or eliminating ADCC and complement-mediated cytotoxicity (CDC) may be desirable. In such embodiments, modified CH2 domains containing amino acid modifications that result in increased binding to FcγRIIb or that may reduce or eliminate binding of the Fc region to all Fcγ receptors ("knockout" variants) may be useful.

[0236] Non-limiting examples of amino acid modifications to the CH2 domain that alter binding of the Fc domain by Fcγ receptors include, but are not limited to, the following: S298A / E333A / K334A and S298A / E333A / K334A / K326A (increased affinity for FcγRIIIa) (Lu, et al., 2011, J Immunol Methods, 365(1-2):132-41); F243L / R292P / Y300L / V305I / P396L (increased affinity for FcγRIIIa) (Stavenhagen, et al., 2007, Cancer Res, 67(18):8882-90); F243L / R292P / Y300L / L235V / P396L (increased affinity for FcγRIIIa) (Nordstrom JL, et al. al., 2011, Breast Cancer Res, 13(6):R123); F243L (increased affinity for FcγRIIIa) (Stewart, et al., 2011, Protein Eng Des Sel., 24(9):671-8); S298A / E333A / K334A (increased affinity for FcγRIIIa) (Shields, et al., 2001, J Biol Chem, 276(9):6591-604); S239D / I332E / A330L and S239D / I332E (increased affinity for FcγRIIIa) (Lazar, et al., 2006, Proc Natl Acad Sci USA, 103(11):4005-10), and S239D / S267E and S267E / L328F (increased affinity for FcγRIIb) (Chu, et al., 2008, Mol Immunol, 45(15):3926-33). Additional modifications that affect Fc domain binding to Fcγ receptors are described in Therapeutic Antibody Engineering (Strohl & Strohl, Woodhead Publishing series in Biomedicine No 11, ISBN 1 907568 37 9, October 2012, page 283).

[0237] In various embodiments, the antibody constructs of the present disclosure comprise heterodimeric Fc domains based on an IgG Fc domain with a modified CH2 domain, wherein one or both of the CH2 sequences (i.e., of the first / second Fc polypeptides) of the modified dimeric CH2 domains comprise one or more amino acid modifications (i.e., "knockout" or "KO" variants) that can result in reduced or eliminated binding of the Fc domain to one or more, or all, of the Fcγ receptors.

[0238] Various publications describe strategies that have been used to engineer antibodies to generate "knockout" Fc variants (see, e.g., Strohl, 2009, Curr Opin Biotech 20:685-691, and Strohl & Strohl, "Antibody Fc engineering for optimal antibody performance" In Therapeutic Antibody Engineering, Cambridge: Woodhead Publishing, 2012, pp 225-249). These strategies include modifying glycosylation, using an IgG2 / IgG4 scaffold, or reducing effector function by introducing mutations in the hinge or CH2 domain of the Fc (see also U.S. Patent Publication No. 2011 / 0212087, International Publication No. WO2006 / 105338, U.S. Patent Publication No. 2012 / 0225058, U.S. Patent Publication No. 2012 / 0251531, and Strop et al., 2012, J. Mol. Biol., 420:204-219).

[0239] In some embodiments, the Fc domain of the antibody construct may comprise one or more of the known amino acid modifications to reduce FcγR and / or complement binding of the Fc domain. In some embodiments, such modifications may include those identified in Table 3. [Table 3]

[0240] Additional examples herein include, for example, an engineered Fc domain to contain the amino acid modifications L235A / L236A / D265S, based on the sequence set forth in SEQ ID NO: 1. Asymmetric amino acid modifications in the CH2 domain that reduce Fc binding to all Fcγ receptors are also described in International Publication No. WO2014 / 190441.

[0241] In certain embodiments, the CH2 domain of the first and second Fc polypeptides herein comprises or consists of an amino acid sequence having at least about 80%, about 85%, about 90%, about 95%, about 97%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 109. In some embodiments, the CH2 domain of the first and / or second Fc polypeptides herein comprises or consists of the sequence set forth in SEQ ID NO: 109.

[0242] In certain embodiments, the antibody constructs herein comprise a heterodimeric Fc domain with modified native glycosylation. As is known in the art, glycosylation of Fc can be modified to increase or decrease effector function. For example, mutation of the conserved asparagine residue at position 297 to alanine, glutamine, lysine, or histidine (i.e., N297A, Q, K, or H) results in a deglycosylated Fc that lacks all effector function (Bolt et al., 1993, Eur. J. Immunol., 23:403-411; Tao & Morrison, 1989, J. Immunol., 143:2595-2601). Conversely, removal of fucose from the heavy chain N297-linked oligosaccharides has been shown to improve ADCC based on improved binding to FcγRIIIa (see, e.g., Shields et al., 2002, J. Biol. Chem., 277:26733-26740, and Niwa et al., 2005, J. Immunol. Methods, 306:151-160). Such low-fucose antibody constructs can be produced, for example, in knockout Chinese hamster ovary (CHO) cells lacking fucosyltransferase (FUT8) (Yamane-Ohnuki et al., 2004, Biotechnol. Bioeng., 87:614-622), in the variant CHO cell line Lec13 with reduced ability to attach fucose to N297-linked glycans (International Publication No. WO 03 / 035835), or in other cells that produce defucosylated antibodies (e.g., Li et al., 2006, Nat Biotechnol., 24:210-215; Shields et al., 2002, ibid., and Shinkawa et al., 2003, J. Biol. Chem., 278:3466-3473). Additionally, International Publication No. WO2009 / 135181 describes the addition of a fucose analogue to the culture medium during antibody production to inhibit the incorporation of fucose into the sugar chains on the antibody.

[0243] E. Linker In various embodiments of the present disclosure, the trivalent and trispecific antibody constructs described herein may comprise one or more linkers. In some embodiments, such one or more linkers are peptide (also referred to herein as "peptitics") linkers comprising or consisting of an amino acid sequence of about 1, 2, 3, 5, 10, 15, 20, 25, 30, 40, or about 50 consecutive amino acid residues in length. The one or more peptide linkers of the antibody constructs may comprise or consist of an amino acid sequence of 1 to about 50, 2 to about 40, 3 to about 30, or 5 to about 25 consecutive amino acid residues in length.

[0244] Such peptide linkers can connect or join together two or more polypeptide sequences and / or domains of an antibody construct. In various embodiments, the linker herein is a peptide linker that is linked to a first polypeptide chain, e.g., a heavy chain constant domain (C H1 ) to the Fc polypeptide. Thus, the linker can connect, from the N- to C-terminus, one domain of the antibody construct to another, e.g., a Fab domain to an Fc domain (e.g., a linker Fab-Fc ), scFv domain to Fab domain (e.g., linker scFv-Fab ), V H Domain V L to the domain (e.g., linker scFv ) or the like. A linker in which both scFv domains of an antibody construct have the same amino acid sequence can be used to link them. scFv In embodiments, such antibody constructs may comprise a linker scFv1 and linker scFv2 Instead of specifying that the linker scFv However, the linker between both scFv domains can be described as scFv In embodiments where the amino acid sequences of the linker scFv1 and linker scFv2 It can be described as including:

[0245] The linker may be, for example, a heavy chain variable domain (V H ) can be, for example, a light chain variable domain (V L ), the linker may be of sufficient length to allow both domains to elicit their biological functions. In addition to providing spacing functionality, linkers (e.g., peptide linkers) herein may provide suitable flexibility or rigidity for proper orientation of one or more domains of the antibody construct, both within the antibody construct itself and between the antibody construct and its target(s).

[0246] Furthermore, the linkers (e.g., peptide linkers) herein can (i) support expression of a full-length fusion protein, e.g., the full-length polypeptide chains H1, L1, H2, etc., of an antibody construct, and (ii) increase the stability of the purified protein both in vitro and in vivo after administration to a subject in need thereof, e.g., a human. One or more linkers used in the antibody constructs herein are typically non-immunogenic or poorly immunogenic in the mammalian subject to which the construct may be administered. In certain embodiments, one or more of the linkers used in the antibody constructs herein may comprise part or all of a human Ig hinge region, a stalk region of a C-type lectin, a family of type II membrane proteins, or a combination thereof. In certain embodiments, one or more of the linkers used in the antibody constructs herein comprise part or all of a human Ig hinge region, e.g., an IgG1 hinge region, e.g., a linker Fab-Fc or linker scFv-Fc may include:

[0247] In certain embodiments, each linker used in the antibody constructs herein can comprise or consist of an amino acid sequence having a length of 2 to about 50 amino acids. In some embodiments, each linker used in the antibody constructs herein can comprise or consist of an amino acid sequence having a length of about 3 to about 40 amino acids, about 10 to about 50 amino acids, about 2 to about 40 amino acids, about 5 to about 30 amino acids, about 5 to about 25 amino acids, about 4 to about 30 amino acids, about 10 to about 30 amino acids, or about 15 to about 25 amino acids. In some embodiments, one or more linkers of an antibody construct may each comprise an amino acid sequence comprising or consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive amino acids.

[0248] In certain embodiments, the linkers of the antibody constructs herein (e.g., linkers scFv , linker scFv-Fab , linker Fab-Fc etc.) is the amino acid sequence (EAAAK) n (wherein n is an integer from 1 to 5) (SEQ ID NO: 339). In some embodiments, the linker comprises or consists of the sequence EAAAK (SEQ ID NO: 340). In some embodiments, the linker comprises or consists of the sequence EAAAKEAAAK (SEQ ID NO: 341). In some embodiments, the linker comprises a polyproline linker, e.g., having the amino acid sequence of PPP (SEQ ID NO: 342) or PPPP (SEQ ID NO: 343). In certain embodiments, the linker is a glycine (G)-proline (P) polypeptide linker, e.g., comprising or consisting of one or more of GPPPG (SEQ ID NO: 344), GGPPPGG (SEQ ID NO: 345), GPPPPG (SEQ ID NO: 346), or GGPPPPGG (SEQ ID NO: 347). In some embodiments, the linker herein isn S) m linker (wherein n and m are independently integers from 1 to 5) (SEQ ID NO: 348). In certain embodiments, the linker is (G3S) n (G4S)1 (SEQ ID NO: 349), (G3S)1(G4S) n (SEQ ID NO: 350), (G3S) n (G4S) n (SEQ ID NO: 351), or (G4S) n (SEQ ID NO: 352), wherein each n is an integer from 1 to 5. In certain embodiments, the linker herein is suitable for connecting two different domains of an antibody construct, and includes a glycine-serine linker, such as, but not limited to, (G m S) n -GG (SEQ ID NO: 352), (SG n ) m (SEQ ID NO: 353), or (SEG n ) m (SEQ ID NO: 354) (wherein m and n are independently integers of 1 to 20).

[0249] In some embodiments, the antibody constructs described herein comprise any one or more of one or more of the linkers described herein. In some embodiments, the antibody constructs comprise, for example, one or more linkers Fab-Fc , one or more linkers scFv-Fab , and / or one or more linkers scFv The linker may comprise 1, 2, 3, 4, or 5 linkers.

[0250] In certain embodiments, the linker of the antibody constructs herein scFv is the amino acid sequence (G n S) m wherein n and m are independently integers from 1 to 5 (SEQ ID NO: 348). In such embodiments, n and m may both be 4, and thus one or more linkers of the antibody construct may comprise scFvmay comprise or consist of the sequence (G4S)4 (SEQ ID NO: 104). In some embodiments, one or more linkers of the antibody construct scFv (e.g., linkers having the same amino acid sequence) scFv1 and linker scFv2 ) may comprise or consist of an amino acid sequence having about 80%, 90%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:104.

[0251] In some embodiments, the antibody constructs of the present disclosure comprise a linker linking the C-terminus of the scFv domain to the N-terminus of the Fab domain. scFv-Fab Such a linker may comprise scFv-Fab may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 105, or a sequence having about 80%, 90%, or 100% sequence identity thereto.

[0252] In some embodiments, the antibody constructs of the present disclosure comprise a linker linking the C-terminus of the Fc polypeptide to the N-terminus of the scFv domain. Fc-scFv In some embodiments, such a linker Fc-scFv may also comprise or consist of the amino acid sequence set forth in SEQ ID NO: 105, or a sequence having about 80%, 90%, or 100% sequence identity thereto.

[0253] In certain embodiments herein, one or more of the linker-antibody constructs may be an amino acid sequence obtained, derived, or designed from an antibody hinge region sequence. In some embodiments, such linkers may have at least one cysteine ​​capable of participating in at least one disulfide bond under physiological conditions or other standard peptide conditions (e.g., peptide purification conditions, conditions for peptide storage, etc.). In certain embodiments, linkers corresponding to or similar to an Ig hinge peptide retain a cysteine ​​corresponding to the hinge cysteine ​​positioned toward the amino (or N) terminus of the hinge. In further embodiments, the linker may be derived from an IgG1 hinge and modified to remove any cysteine ​​residues, or the linker is an IgG1 hinge with one cysteine ​​or two cysteines corresponding to the hinge cysteines.

[0254] In certain embodiments, the linker of the antibody constructs described herein may comprise a "modified wild-type Ig hinge region" or "modified Ig hinge region." Such an altered hinge region may be a wild-type Ig hinge region having (a) up to 30 percent amino acid changes (e.g., up to 25 percent, 20 percent, 15 percent, 10 percent, or 5 percent amino acid substitutions, insertions, or deletions), (b) a wild-type Ig hinge region having up to 30 percent amino acid changes (e.g., up to 25 percent, 20 percent, 15 percent, 10 percent, or 5 percent amino acid substitutions, insertions, or deletions), or (c) a wild-type Ig hinge region having up to 30 percent amino acid changes (e.g., up to 25 percent, 20 percent, 15 percent, 10 percent, or 5 percent amino acid substitutions, insertions, or deletions). (c) a portion of a wild-type Ig hinge region that is at least 10 amino acids in length (e.g., at least 12, 13, 14, or 15 amino acids), (d) a portion of a wild-type Ig hinge region that includes the core hinge region (the portion can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length), or (d) a combination of any of (a)-(c). In certain embodiments, one or more cysteine ​​residues in a wild-type Ig hinge region, such as an IgG1 hinge, including the upper and core regions, can be substituted with one or more other amino acid residues (e.g., one or more serine residues). The modified Ig hinge region may alternatively or additionally have the proline residue of a wild-type Ig hinge region, such as an IgG1 hinge, including the upper and core regions, replaced by another amino acid residue (e.g., a serine residue).

[0255] Thus, in some embodiments, an antibody construct of the present disclosure comprises a linker comprising or consisting of an amino acid sequence having at least about 80%, 90%, 95%, 97%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 108. In some embodiments, an antibody construct comprises a linker comprising or consisting of an amino acid sequence having at least about 80%, 90%, 95%, 97%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 111. In some embodiments, an antibody construct comprises a linker comprising or consisting of an amino acid sequence having at least about 80%, 90%, 95%, 97%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 112.

[0256] In various embodiments, the antibody constructs of the present disclosure comprise a linker, which is a linker connecting the Fab domain to the first Fc polypeptide. Fab-Fc (Linker Fab-Fc comprises or consists of the amino acid sequence set forth in SEQ ID NO: 108), and a linker which is another linker connecting the first or second scFv domain to the first or second Fc polypeptide. scFv-Fc (Linker scFv-Fc comprises or consists of the amino acid sequence set forth in SEQ ID NO: 111 or SEQ ID NO: 112).

[0257] F. Certain Embodiments of Antibody Constructs In one embodiment, provided herein is a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to CD3 on a first immune cell, the Fab domain comprising, from N to C-terminus: C L V concatenated into an array L paired with a light chain comprising, from N to C terminus, C H1 V concatenated into an array H (ii) a first scFv domain capable of binding to CD28 on a second immune cell, the first scFv domain comprising, from N- to C-terminus, a first linker scFvvia the first scFv V L First scFv V linked to sequence H (iii) a second scFv domain capable of binding to MSLN on tumor cells, the second scFv domain comprising, from the N- to C-terminus, a second linker scFv via the second scFv V L A second scFv V linked to the sequence H and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein a) the Fab domain comprises Fab C H1 via the C-terminus of the sequence and the linker Fab-Fc and b) the C-terminus of the first scFv domain is linked to the N-terminus of the first Fc polypeptide via a linker scFv-Fab Via Fab V H and c) the C-terminus of the second scFv domain is linked to the N-terminus of the second scFv domain. scFv-Fc

[0010] A trivalent and trispecific antibody construct is described in which a first Fc polypeptide is linked to the N-terminus of a second Fc polypeptide via a

[0258] In one embodiment, provided herein is a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to CD3 on a first immune cell, the Fab domain comprising, from N to C-terminus: C L V concatenated into an array L paired with a light chain comprising, from N to C terminus, C H1 V concatenated into an array H (ii) a first scFv domain capable of binding to CD28 on a second immune cell, the first scFv domain comprising, from N- to C-terminus, a first linker scFv via the first scFv V H First scFv V linked to sequence L(iii) a second scFv domain capable of binding to MSLN on tumor cells, the second scFv domain comprising, from the N- to C-terminus, a second linker scFv via the second scFv V L A second scFv V linked to the sequence H and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein a) the Fab domain comprises Fab C H1 via the C-terminus of the sequence and the linker Fab-Fc and b) the N-terminus of the first scFv domain is linked to the N-terminus of the first Fc polypeptide via a linker Fab-scFv Via Fab C L and c) the C-terminus of the second scFv domain is linked to the C-terminus of the second scFv domain. scFv-Fc

[0010] A trivalent and trispecific antibody construct is described in which a first Fc polypeptide is linked to the N-terminus of a second Fc polypeptide via a nucleotide sequence.

[0259] In one embodiment, provided herein is a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to CD3 on a first immune cell, the Fab domain comprising, from N to C-terminus: C L V concatenated into an array L paired with a light chain comprising, from N to C terminus, C H1 V concatenated into an array H (ii) a first scFv domain capable of binding to CD28 on a second immune cell, the first scFv domain comprising, from N- to C-terminus, a first linker scFv First scFv V via H First scFv V linked to sequence L (iii) a second scFv domain capable of binding to MSLN on tumor cells, the second scFv domain comprising, from the N- to C-terminus, a second linker scFv via the second scFv V LA second scFv V linked to the sequence H and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein a) the Fab domain comprises Fab C H1 via the C-terminus of the sequence and the linker Fab-Fc and b) the N-terminus of the first scFv domain is linked to the N-terminus of the first Fc polypeptide via a linker Fc-scFv and c) the C-terminus of the second scFv domain is linked to the C-terminus of the first Fc polypeptide via a linker scFv-Fc

[0010] A trivalent and trispecific antibody construct is described in which a first Fc polypeptide is linked to the N-terminus of a second Fc polypeptide via a

[0260] In one embodiment, provided herein is a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to CD3 on a first immune cell, the Fab domain comprising, from N to C-terminus: C L V concatenated into an array L paired with a light chain comprising, from N to C terminus, C H1 V concatenated into an array H (ii) a first scFv domain capable of binding to CD28 on a second immune cell, the first scFv domain comprising, from N- to C-terminus, a first linker scFv via the first scFv V H First scFv V linked to sequence L (iii) a second scFv domain capable of binding to MSLN on tumor cells, the second scFv domain comprising, from the N- to C-terminus, a second linker scFv via the second scFv V L A second scFv V linked to the sequence H and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein a) the Fab domain comprises Fab C H1 via the C-terminus of the sequence and the linkerFab-Fc and b) the N-terminus of the first scFv domain is linked to the N-terminus of the first Fc polypeptide via a linker Fc-scFv and c) the C-terminus of the second scFv domain is linked to the C-terminus of the first Fc polypeptide via a linker scFv-Fab Via Fab V H A trivalent and trispecific antibody construct is described in which the N-terminally linked sequences of the antibody are linked to each other.

[0261] In one embodiment, provided herein is a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to CD3 on a first immune cell, the Fab domain comprising, from N to C-terminus: C L V concatenated into an array L paired with a light chain comprising, from N to C terminus, C H1 V concatenated into an array H (ii) a first scFv domain capable of binding to CD28 on a second immune cell, the first scFv domain comprising, from N- to C-terminus, a first linker scFv First scFv V via H First scFv V linked to sequence L (iii) a second scFv domain capable of binding to MSLN on tumor cells, the second scFv domain comprising, from the N- to C-terminus, a second linker scFv via the second scFv V L A second scFv V linked to the sequence H and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein a) the Fab domain comprises Fab C H1 via the C-terminus of the sequence and the linker Fab-Fc and b) the C-terminus of the first scFv domain is linked to the N-terminus of the second Fc polypeptide via a linker scFv-Fc and c) the C-terminus of the second scFv domain is linked to the N-terminus of the first Fc polypeptide via a linker scFv-FabVia Fab V H A trivalent and trispecific antibody construct is described in which the N-terminally linked sequences of the antibody are linked to each other.

[0262] In one embodiment, provided herein is a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to CD28 on a first immune cell, the Fab domain comprising, from N to C-terminus: C L V concatenated into an array L paired with a light chain comprising, from N to C terminus, C H1 V concatenated into an array H (ii) a first scFv domain capable of binding to CD3 on a second immune cell, the first scFv domain comprising, from N- to C-terminus, a first linker scFv via the first scFv V L First scFv V linked to sequence H (iii) a second scFv domain capable of binding to MSLN on tumor cells, the second scFv domain comprising, from the N- to C-terminus, a second linker scFv via the second scFv V L A second scFv V linked to the sequence H and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein a) the Fab domain comprises Fab C H1 via the C-terminus of the sequence and the linker Fab-Fc and b) the C-terminus of the first scFv domain is linked to the N-terminus of the first Fc polypeptide via a linker scFv-Fab Via Fab V H and c) the C-terminus of the second scFv domain is linked to the N-terminus of the second scFv domain. scFv-Fc

[0010] A trivalent and trispecific antibody construct is described in which a first Fc polypeptide is linked to the N-terminus of a second Fc polypeptide via a

[0263] In one embodiment, provided herein is a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to CD28 on a first immune cell, the Fab domain comprising, from N to C-terminus: C L V concatenated into an array L paired with a light chain comprising, from N to C terminus, C H1 V concatenated into an array H (ii) a first scFv domain capable of binding to CD3 on a second immune cell, the first scFv domain comprising, from N- to C-terminus, a first linker scFv First scFv V via H First scFv V linked to sequence L (iii) a second scFv domain capable of binding to MSLN on tumor cells, the second scFv domain comprising, from the N- to C-terminus, a second linker scFv via the second scFv V L A second scFv V linked to the sequence H and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein a) the Fab domain comprises Fab C H1 via the C-terminus of the sequence and the linker Fab-Fc and b) the N-terminus of the first scFv domain is linked to the N-terminus of the first Fc polypeptide via a linker Fab-scFv Via Fab C L and c) the C-terminus of the second scFv domain is linked to the C-terminus of the second scFv domain. scFv-Fc

[0010] A trivalent and trispecific antibody construct is described in which a first Fc polypeptide is linked to the N-terminus of a second Fc polypeptide via a nucleotide sequence.

[0264] In one embodiment, provided herein is a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to CD28 on a first immune cell, the Fab domain comprising, from N to C-terminus: C L V concatenated into an array Lpaired with a light chain comprising, from N to C terminus, C H1 V concatenated into an array H (ii) a first scFv domain capable of binding to CD3 on a second immune cell, the first scFv domain comprising, from N- to C-terminus, a first linker scFv via the first scFv V H First scFv V linked to sequence L (iii) a second scFv domain capable of binding to MSLN on tumor cells, the second scFv domain comprising, from the N- to C-terminus, a second linker scFv via the second scFv V L A second scFv V linked to the sequence H and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein a) the Fab domain comprises Fab C H1 via the C-terminus of the sequence and the linker Fab-Fc and b) the N-terminus of the first scFv domain is linked to the N-terminus of the first Fc polypeptide via a linker Fc-scFv and c) the C-terminus of the second scFv domain is linked to the C-terminus of the first Fc polypeptide via a linker scFv-Fc

[0010] A trivalent and trispecific antibody construct is described in which a first Fc polypeptide is linked to the N-terminus of a second Fc polypeptide via a

[0265] In one embodiment, provided herein is a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to CD28 on a first immune cell, the Fab domain comprising, from N to C-terminus: C L V concatenated into an array L paired with a light chain comprising, from N to C terminus, C H1 V concatenated into an array H(ii) a first scFv domain capable of binding to CD3 on a second immune cell, the first scFv domain comprising, from N- to C-terminus, a first linker scFv First scFv V via H First scFv V linked to sequence L (iii) a second scFv domain capable of binding to MSLN on tumor cells, the second scFv domain comprising, from the N- to C-terminus, a second linker scFv via the second scFv V L A second scFv V linked to the sequence H and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein a) the Fab domain comprises Fab C H1 via the C-terminus of the sequence and the linker Fab-Fc and b) the N-terminus of the first scFv domain is linked to the N-terminus of the first Fc polypeptide via a linker Fc-scFv and c) the C-terminus of the second scFv domain is linked to the C-terminus of the first Fc polypeptide via a linker scFv-Fab Via Fab V H A trivalent and trispecific antibody construct is described in which the N-terminally linked sequences of the antibody are linked to each other.

[0266] In one embodiment, provided herein is a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to CD28 on a first immune cell, the Fab domain comprising, from N to C-terminus: C L V concatenated into an array L paired with a light chain comprising, from N to C terminus, C H1 V concatenated into an array H (ii) a first scFv domain capable of binding to CD3 on a second immune cell, the first scFv domain comprising, from N- to C-terminus, a first linker scFv First scFv V via LFirst scFv V linked to sequence H (iii) a second scFv domain capable of binding to MSLN on tumor cells, the second scFv domain comprising, from the N- to C-terminus, a second linker scFv via the second scFv V L A second scFv V linked to the sequence H and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein a) the Fab domain comprises Fab C H1 via the C-terminus of the sequence and the linker Fab-Fc and b) the C-terminus of the first scFv domain is linked to the N-terminus of the first Fc polypeptide via a linker scFv-Fc and c) the C-terminus of the second scFv domain is linked to the N-terminus of the second Fc polypeptide via a linker scFv-Fab Via Fab V H A trivalent and trispecific antibody construct is described in which the N-terminally linked sequences of the antibody are linked to each other.

[0267] In one embodiment, provided herein is a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to CD3 on a first immune cell, the Fab domain comprising, from N to C-terminus: C L V concatenated into an array L paired with a light chain comprising, from N to C terminus, C H1 V concatenated into an array H (ii) a first scFv domain capable of binding to CD28 on a second immune cell, the first scFv domain comprising, from N- to C-terminus, a first linker scFv via the first scFv V L First scFv V linked to sequence H (iii) a second scFv domain capable of binding to Cldn18.2 on tumor cells, the second scFv domain comprising, from the N- to C-terminus, a second linker scFvvia the second scFv V L A second scFv V linked to the sequence H and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein a) the Fab domain comprises Fab C H1 via the C-terminus of the sequence and the linker Fab-Fc and b) the C-terminus of the first scFv domain is linked to the N-terminus of the first Fc polypeptide via a linker scFv-Fab Via Fab V H and c) the C-terminus of the second scFv domain is linked to the N-terminus of the second scFv domain. scFv-Fc

[0010] A trivalent and trispecific antibody construct is described in which a first Fc polypeptide is linked to the N-terminus of a second Fc polypeptide via a

[0268] In one embodiment, provided herein is a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to CD3 on a first immune cell, the Fab domain comprising, from N to C-terminus: C L V concatenated into an array L paired with a light chain comprising, from N to C terminus, C H1 V concatenated into an array H (ii) a first scFv domain capable of binding to CD28 on a second immune cell, the first scFv domain comprising, from N- to C-terminus, a first linker scFv via the first scFv V H First scFv V linked to sequence L (iii) a second scFv domain capable of binding to Cldn18.2 on tumor cells, the second scFv domain comprising, from the N- to C-terminus, a second linker scFv via the second scFv V L A second scFv V linked to the sequence Hand (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein a) the Fab domain comprises Fab C H1 via the C-terminus of the sequence and the linker Fab-Fc and b) the N-terminus of the first scFv domain is linked to the N-terminus of the first Fc polypeptide via a linker Fab-scFv Via Fab C L and c) the C-terminus of the second scFv domain is linked to the C-terminus of the second scFv domain. scFv-Fc

[0010] A trivalent and trispecific antibody construct is described in which a first Fc polypeptide is linked to the N-terminus of a second Fc polypeptide via a

[0269] In one embodiment, provided herein is a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to CD3 on a first immune cell, the Fab domain comprising, from N to C-terminus: C L V concatenated into an array L paired with a light chain comprising, from N to C terminus, C H1 V concatenated into an array H (ii) a first scFv domain capable of binding to CD28 on a second immune cell, the first scFv domain comprising, from N- to C-terminus, a first linker scFv via the first scFv V H First scFv V linked to sequence L (iii) a second scFv domain capable of binding to Cldn18.2 on tumor cells, the second scFv domain comprising, from the N- to C-terminus, a second linker scFv via the second scFv V L A second scFv V linked to the sequence H and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein a) the Fab domain comprises Fab C H1 via the C-terminus of the sequence and the linker Fab-Fcand b) the N-terminus of the first scFv domain is linked to the N-terminus of the first Fc polypeptide via a linker Fc-scFv and c) the C-terminus of the second scFv domain is linked to the C-terminus of the first Fc polypeptide via a linker scFv-Fc

[0010] A trivalent and trispecific antibody construct is described in which a first Fc polypeptide is linked to the N-terminus of a second Fc polypeptide via a

[0270] In one embodiment, provided herein is a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to CD3 on a first immune cell, the Fab domain comprising, from N to C-terminus: C L V concatenated into an array L paired with a light chain comprising, from N to C terminus, C H1 V concatenated into an array H (ii) a first scFv domain capable of binding to CD28 on a second immune cell, the first scFv domain comprising, from N- to C-terminus, a first linker scFv via the first scFv V H First scFv V linked to sequence L (iii) a second scFv domain capable of binding to Cldn18.2 on tumor cells, the second scFv domain comprising, from the N- to C-terminus, a second linker scFv via the second scFv V L A second scFv V linked to the sequence H and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein a) the Fab domain comprises Fab C H1 via the C-terminus of the sequence and the linker Fab-Fc and b) the N-terminus of the first scFv domain is linked to the N-terminus of the first Fc polypeptide via a linker Fc-scFv and c) the C-terminus of the second scFv domain is linked to the C-terminus of the first Fc polypeptide via a linker scFv-Fab Via Fab VH A trivalent and trispecific antibody construct is described in which the N-terminally linked sequences of the antibody are linked to each other.

[0271] In one embodiment, provided herein is a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to CD3 on a first immune cell, the Fab domain comprising, from N to C-terminus: C L V concatenated into an array L paired with a light chain comprising, from N to C terminus, C H1 V concatenated into an array H (ii) a first scFv domain capable of binding to CD28 on a second immune cell, the first scFv domain comprising, from N- to C-terminus, a first linker scFv First scFv V via H First scFv V linked to sequence L (iii) a second scFv domain capable of binding to Cldn18.2 on tumor cells, the second scFv domain comprising, from the N- to C-terminus, a second linker scFv via the second scFv V L A second scFv V linked to the sequence H and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein a) the Fab domain comprises Fab C H1 via the C-terminus of the sequence and the linker Fab-Fc and b) the C-terminus of the first scFv domain is linked to the N-terminus of the second Fc polypeptide via a linker scFv-Fc and c) the C-terminus of the second scFv domain is linked to the N-terminus of the first Fc polypeptide via a linker scFv-Fab Via Fab V H A trivalent and trispecific antibody construct is described in which the N-terminally linked sequences of the antibody are linked to each other.

[0272] In one embodiment, provided herein is a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to CD28 on a first immune cell, the Fab domain comprising, from N to C-terminus: C L V concatenated into an array L paired with a light chain comprising, from N to C terminus, C H1 V concatenated into an array H (ii) a first scFv domain capable of binding to CD3 on a second immune cell, the first scFv domain comprising, from N- to C-terminus, a first linker scFv via the first scFv V L First scFv V linked to sequence H (iii) a second scFv domain capable of binding to Cldn18.2 on tumor cells, the second scFv domain comprising, from the N- to C-terminus, a second linker scFv via the second scFv V L A second scFv V linked to the sequence H and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein a) the Fab domain comprises Fab C H1 via the C-terminus of the sequence and the linker Fab-Fc and b) the C-terminus of the first scFv domain is linked to the N-terminus of the first Fc polypeptide via a linker scFv-Fab Via Fab V H and c) the C-terminus of the second scFv domain is linked to the N-terminus of the second scFv domain. scFv-Fc

[0010] A trivalent and trispecific antibody construct is described in which a first Fc polypeptide is linked to the N-terminus of a second Fc polypeptide via a

[0273] In one embodiment, provided herein is a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to CD28 on a first immune cell, the Fab domain comprising, from N to C-terminus: C L V concatenated into an array Lpaired with a light chain comprising, from N to C terminus, C H1 V concatenated into an array H (ii) a first scFv domain capable of binding to CD3 on a second immune cell, the first scFv domain comprising, from N- to C-terminus, a first linker scFv via the first scFv V H First scFv V linked to sequence L (iii) a second scFv domain capable of binding to Cldn18.2 on tumor cells, the second scFv domain comprising, from the N- to C-terminus, a second linker scFv via the second scFv V L A second scFv V linked to the sequence H and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein a) the Fab domain comprises Fab C H1 via the C-terminus of the sequence and the linker Fab-Fc and b) the N-terminus of the first scFv domain is linked to the N-terminus of the first Fc polypeptide via a linker Fab-scFv Via Fab C L and c) the C-terminus of the second scFv domain is linked to the C-terminus of the second scFv domain. scFv-Fc

[0010] A trivalent and trispecific antibody construct is described in which a first Fc polypeptide is linked to the N-terminus of a second Fc polypeptide via a

[0274] In one embodiment, provided herein is a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to CD28 on a first immune cell, the Fab domain comprising, from N to C-terminus: C L V concatenated into an array L paired with a light chain comprising, from N to C terminus, C H1 V concatenated into an array H(ii) a first scFv domain capable of binding to CD3 on a second immune cell, the first scFv domain comprising, from N- to C-terminus, a first linker scFv via the first scFv V H First scFv V linked to sequence L (iii) a second scFv domain capable of binding to Cldn18.2 on tumor cells, the second scFv domain comprising, from the N- to C-terminus, a second linker scFv via the second scFv V L A second scFv V linked to the sequence H and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein a) the Fab domain comprises Fab C H1 via the C-terminus of the sequence and the linker Fab-Fc and b) the N-terminus of the first scFv domain is linked to the N-terminus of the first Fc polypeptide via a linker Fc-scFv and c) the C-terminus of the second scFv domain is linked to the C-terminus of the first Fc polypeptide via a linker scFv-Fc

[0010] A trivalent and trispecific antibody construct is described in which a first Fc polypeptide is linked to the N-terminus of a second Fc polypeptide via a

[0275] In one embodiment, provided herein is a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to CD28 on a first immune cell, the Fab domain comprising, from N to C-terminus: C L V concatenated into an array L paired with a light chain comprising, from N to C terminus, C H1 V concatenated into an array H (ii) a first scFv domain capable of binding to CD3 on a second immune cell, the first scFv domain comprising, from N- to C-terminus, a first linker scFv via the first scFv V HFirst scFv V linked to sequence L (iii) a second scFv domain capable of binding to Cldn18.2 on tumor cells, the second scFv domain comprising, from the N- to C-terminus, a second linker scFv via the second scFv V L A second scFv V linked to the sequence H and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein a) the Fab domain comprises Fab C H1 via the C-terminus of the sequence and the linker Fab-Fc and b) the N-terminus of the first scFv domain is linked to the N-terminus of the first Fc polypeptide via a linker Fc-scFv and c) the C-terminus of the second scFv domain is linked to the C-terminus of the first Fc polypeptide via a linker scFv-Fab Via Fab V H A trivalent and trispecific antibody construct is described in which the N-terminally linked sequences of the antibody are linked to each other.

[0276] In one embodiment, provided herein is a trivalent and trispecific antibody construct comprising: (i) a Fab domain capable of binding to CD28 on a first immune cell, the Fab domain comprising, from N to C-terminus: C L V concatenated into an array L paired with a light chain comprising, from N to C terminus, C H1 V concatenated into an array H (ii) a first scFv domain capable of binding to CD3 on a second immune cell, the first scFv domain comprising, from N- to C-terminus, a first linker scFv via the first scFv V L First scFv V linked to sequence H (iii) a second scFv domain capable of binding to Cldn18.2 on tumor cells, the second scFv domain comprising, from the N- to C-terminus, a second linker scFvvia the second scFv V L A second scFv V linked to the sequence H and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein a) the Fab domain comprises Fab C H1 via the C-terminus of the sequence and the linker Fab-Fc and b) the C-terminus of the first scFv domain is linked to the N-terminus of the first Fc polypeptide via a linker scFv-Fc and c) the C-terminus of the second scFv domain is linked to the N-terminus of the second Fc polypeptide via a linker scFv-Fab Via Fab V H A trivalent and trispecific antibody construct is described in which the N-terminally linked sequences of the antibody are linked to each other.

[0277] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 100 or 118; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 111 or 125; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 115 or 120.

[0278] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 100; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 111; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 115.

[0279] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 118; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 125; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 120.

[0280] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 122; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 111; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 123.

[0281] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 124; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 111; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 115.

[0282] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 124; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 125; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 115.

[0283] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 129; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 111; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 120.

[0284] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 122; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 125; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 123.

[0285] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 129; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 125; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 120.

[0286] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 100; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 125; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 115.

[0287] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 134; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 125; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 135.

[0288] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 130; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 131; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 120.

[0289] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 132; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 133; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 120.

[0290] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 136; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 131; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 115.

[0291] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 137; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 138; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 115.

[0292] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 139; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 125; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 115.

[0293] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 140; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 125; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 115.

[0294] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 100; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 125; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 141.

[0295] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 100; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 125; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 142.

[0296] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 143-149; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 125; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 115.

[0297] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 143; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 125; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 115.

[0298] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 144; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 125; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 115.

[0299] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 145; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 125; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 115.

[0300] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 146; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 125; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 115.

[0301] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 147; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 125; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 115.

[0302] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 148; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 125; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 115.

[0303] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 149; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 125; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 115.

[0304] In one embodiment, described herein is a trivalent and trispecific antibody construct comprising: (i) a first heavy chain polypeptide (H1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 100; (ii) a second heavy chain polypeptide (H2) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 125; and (iii) a light chain polypeptide (L1) comprising, or consisting of, an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 150 or SEQ ID NO: 152.

[0305] In certain embodiments, the trivalent and trispecific antibody constructs of the present disclosure are those in which the first scFv domain and the second scFv domain are not linked to each other in tandem, i.e., in the form of scFv1-scFv2 or scFv2-scFv (N to C termini and with or without a linker sequence between scFv1 and scFv2).

[0306] G. Prescribed Properties of Trivalent and Trispecific Antibody Constructs The trivalent and trispecific antibody constructs of the present disclosure may have several particular properties due to their format, geometry and antigen affinity.

[0307] Thus, in some embodiments, for example, in a tumor (micro)environment, engagement of a trivalent and trispecific antibody construct with two different antigens on one or more T cells and an antigen on a tumor cell can be simultaneous (at least transiently), thereby establishing a TCR-independent immune synapse and directing T cell-mediated cytotoxic activity to the tumor environment containing tumor cells expressing a TAA. In various embodiments, and as further described herein, trivalent and trispecific antibody constructs can cause significantly reduced immune cell (e.g., T cell) activation in the absence of a TAA, for example, if an immune synapse cannot adequately form due to the absence of a TAA. Such properties can be advantageous over those of conventional constructs, for example, because they enable activation of a subject's immune system in a more TAA-dependent manner, and thus can cause fewer off-target effects in a subject compared to conventional constructs of antibodies that act in a less TAA-dependent manner.

[0308] In some embodiments, the antibody constructs described herein may possess improved anti-tumor activity in tumors with relatively low T cell infiltration when compared to conventional constructs that target only one immune cell antigen (e.g., CD3 or CD28) due to their costimulatory activity by being able to engage both CD3 and CD28 either on the same immune cell (e.g., T cell) or on two different, e.g., adjacent, immune cells (e.g., T cells).

[0309] Typically, and as will be understood by those skilled in the art, natural T cell activation can require both TCR (e.g., involving CD3) and CD28 stimulation. The antibody constructs of the present disclosure are specifically designed, for example, via their format and geometry, to provide both CD3 and CD28 costimulation. Furthermore, and in accordance with various embodiments of the present disclosure, the anti-CD3 and anti-CD28 binding affinities of the anti-CD3 and anti-CD28 binding domains (e.g., scFv, Fab, etc.) of the antibody constructs described herein, in combination with their relative arrangement within the construct, are specifically selected and engineered to generate a signal for immune cell (e.g., T cell) activation with appropriate strength to reduce both T cell anergy on the one side and T cell hyperresponsiveness and dysfunction on the other side of the spectrum, and to provide an improved ratio of anti-tumor on-target to healthy tissue off-target activity. In various embodiments, such improved on-target versus off-target activity was achieved by optimizing the format and geometry of the antibody construct in such a way that engagement of all three antigens, i.e., CD3, CD28 and TAA, simultaneously enables the most potent anti-tumor activity, as compared to, for example, when only CD3 and CD28 are engaged and bound by the construct.

[0310] As further described herein, the antibody constructs of the present disclosure may be trivalent and trispecific, and may monovalently bind to each antigen, e.g., CD3, CD28, TAA, via one of its three antigen-binding domains.

[0311] In some embodiments, the trivalent and trispecific antibody construct has a binding affinity for the TAA of at least about 40 nM, 30 nM, 20 nM, 10 nM, or 5 nM, or about 40 nM to about 5 nM, or about 30 nM to about 10 nM, e.g., using SPR or other methods known in the art. In one embodiment, the TAA is MSLN. In another embodiment, the TAA is Cldn18.2.

[0312] In some embodiments, the trivalent and trispecific antibody construct has a melting temperature at Tm1, Tm2, ​​and / or Tm3 that is within 10°C, within 5°C, within 2°C, or within 1°C of that of a bivalent and monospecific IgG1 monoclonal antibody, as measured, for example, using differential scanning calorimetry (DSC) or differential scanning fluorimetry (DSF). In some embodiments, such a bivalent and monospecific IgG1 monoclonal antibody can be any conventional IgG1 antibody capable of binding to a specific antigen. In certain embodiments, such a monoclonal monospecific and bivalent IgG1 antibody is one that comprises two Fab domains as described herein in the context of a trispecific antibody construct. In some embodiments, the bivalent and monospecific IgG1 monoclonal antibody comprises two anti-CD3 Fab domains described herein, two anti-CD28 Fab domains described herein, two anti-MSLN Fab domains comprising the anti-MSLN VH and VL sequences described herein, or two anti-Cldn18.2 Fab domains comprising the anti-Cldn18.2 VH and VL sequences described herein.

[0313] In some embodiments, trivalent and trispecific antibody constructs of the present disclosure bind to cytotoxic effector cells (e.g., T cells) expressing CD3 and CD28 with an affinity of about 5 nM to about 100 pM, about 1 nM to about 100 pM, about 1 nM to about 250 pM, about 1 nM to about 500 pM, or about 1 nM to about 750 pM. In certain embodiments, such antibody constructs bind to cytotoxic effector cells expressing CD3 and CD28 with an affinity that is about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or about 200-fold higher and / or about 2-fold to about 200-fold, about 2-fold to about 150-fold, about 2-fold to about 100-fold, or about 20-fold to about 200-fold higher than that of the corresponding bispecific anti-CD3 x TAA and / or anti-CD28 x TAA antibody constructs.

[0314] In some embodiments, for example, using engineered anti-CD28 binding domains with reduced CD28 affinity compared to the parent huTN228 paratope can result in trivalent and trispecific antibody constructs that can induce lower CD28-mediated toxicity.

[0315] In some embodiments, the trivalent and trispecific antibody constructs of the present disclosure have an IC of about 50 pM to about 0.01 pM, about 25 pM to about 0.01 pM, about 10 pM to about 0.05 pM, about 10 pM to about 0.1 pM, about 10 pM to about 1 pM, or about 5 pM to about 1 pM for killing TAA-expressing tumor cells expressing at least about 100,000 TAAs / cell by TDCC in the presence of cytotoxic effector cells using a 2:1 E:T ratio and a 72 hour incubation period. 50 In some such embodiments, the antibody construct achieves maximal killing of at least about 60%, 65%, 70%, 75%, or 80%, 85%, or 90%, 100%, or between about 60% and about 100%, between about 70% and about 90%, or between about 75% and about 85% of TAA-expressing tumor cells.

[0316] In some embodiments, the trivalent and trispecific antibody constructs of the present disclosure are capable of inducing production of one or more cytokines by cytotoxic effector cells in the range of about 300 pg / mL to about 9000 pg / mL in the presence of TAA-expressing cells expressing at least about 100,000 TAAs / cell and using a 2:1 E:T ratio and a 72 hour incubation period.

[0317] Furthermore, in various embodiments, the trivalent and trispecific antibody constructs of the present disclosure can provide a strictly target cell (e.g., tumor cell) dependent cytotoxicity profile, as shown herein, for example, when cytokine release is significantly reduced in the presence of isolated T cells alone compared to conditions where the T cells are in co-culture with TAA-expressing tumor cells. See, e.g., Example 25.

[0318] In some embodiments, the trivalent and trispecific antibody constructs disclosed herein may have a thermal stability of at least about 90%, 95%, 97%, 98%, or 99% of the intact construct when measured at 40° C. and over a period of about 2, 3, 5, 7, 10, or 14 days, as measured, for example, using size exclusion chromatography or other methods known in the art. In certain embodiments, such constructs may comprise one Fab domain capable of binding either CD3 or CD28, and two scFv domains, where one such scFv domain is capable of binding either CD3 or CD28 (e.g., the one not bound by the Fab domain), and one scFv domain is capable of binding a TAA (e.g., as in construct v37634). In some embodiments, the trispecific and trivalent stability over a 14-day period at 40° C. is at least about 97% or 98% percent (i.e., at least about 97% or 98% of the construct is intact as measured, for example, using size exclusion chromatography). In some embodiments, the concentration of the construct in such stability experiments is about 1 mg / mL.

[0319] H. Trivalent and Trispecific Antibody Constructs Comprising Light Chains Comprising Fab and scFv Portions Certain embodiments of the present disclosure relate to trivalent and trispecific antibody constructs comprising (i) a light chain comprising a Fab portion comprising a first VL sequence and a CL sequence, linked (either N- to C-terminally or C- to N-terminally) to (ii) an scFv domain comprising a second VL sequence and a VH sequence.

[0320] In some embodiments, such trivalent and trispecific antibody constructs comprise a light chain with the following domain structure, from N to C-terminus, VL-CL-scFv, where the scFv domain may comprise, from N to C-terminus, a VH linked to a VL sequence, or a VL sequence linked to a VH sequence. Thus, in certain embodiments, the light chain comprises, from N to C-terminus, (VL-CL)Fab -(VL-VH) scFv In another embodiment, the light chain has a domain structure from N to C-terminus: (VL-CL) Fab -(VH-VL) scFv It has a domain structure of

[0321] In certain embodiments, the present disclosure provides a polypeptide having the following domain structure from N to C terminus: (VL-CL) Fab -(VL-VH) scFv

[0010] Trivalent and trispecific antibody constructs are described that comprise a light chain having the following structure:

[0011] In some embodiments, such a light chain may further comprise one or more linkers, as further described herein. In some embodiments, the light chain comprises a peptide linker between the Fab and scFv portions. Fab-scFv which links the Fab portion to the scFv portion to form the domain structure (VL-CL) Fab -Linker Fab-scFv -(VL-VH) scFv linker Fab-scFv may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 105. As further described herein, the scFv domain of the light chain may also comprise a linker that connects the VL sequence to the VH sequence. scFv Thus, in certain embodiments, the trivalent and trispecific antibody construct may comprise the domain structure (VL-CL) Fab -Linker Fab-scFv -(VL-linker scFv -VH) scFv The light chain comprises a linker scFv may comprise or consist of the amino acid sequence shown in SEQ ID NO:104.

[0322] In certain embodiments, described herein are trivalent and trispecific antibody constructs comprising a light chain comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 123.

[0323] In some embodiments, provided herein is a trivalent and trispecific antibody construct comprising a light chain comprising a Fab portion and an scFv portion, the antibody construct comprising: (i) a Fab domain comprising a heavy chain comprising a VH sequence and a CH1 sequence and a light chain comprising a VL sequence and a CL sequence, wherein the Fab domain is capable of binding to CD3; (ii) a first scFv domain comprising a first VH sequence and a first VL sequence, wherein the first scFv domain is capable of binding to CD28; and (iii) a second VH sequence and a second VL sequence. and (iv) a dimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein a) the Fab domain is linked to the N-terminus of the first Fc polypeptide via its CH1 sequence, b) the first scFv domain is linked to the C-terminus of the CL sequence of the Fab light chain, and c) the second scFv domain is linked to the N-terminus of the second Fc polypeptide.

[0324] In some embodiments, provided herein is a trivalent and trispecific antibody construct comprising a light chain comprising a Fab portion and an scFv portion, the antibody construct comprising: (i) a Fab domain capable of binding to CD3 on a first immune cell, the Fab domain comprising, from N to C-terminus: C L V concatenated into an array L paired with a light chain comprising, from N to C terminus, C H1 V concatenated into an array H (ii) a first scFv domain capable of binding to CD28 on a second immune cell, the first scFv domain comprising, from N- to C-terminus, a first linker scFv via the first scFv V H First scFv V linked to sequence L(iii) a second scFv domain capable of binding to Cldn18.2 on tumor cells, the second scFv domain comprising, from the N- to C-terminus, a second linker scFv via the second scFv V L A second scFv V linked to the sequence H and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein a) the Fab domain comprises Fab C H1 via the C-terminus of the sequence and the linker Fab-Fc and b) the N-terminus of the first scFv domain is linked to the N-terminus of the first Fc polypeptide via a linker Fab-scFv Via Fab C L and c) the C-terminus of the second scFv domain is linked to the C-terminus of the second scFv domain. scFv-Fc

[0010] A trivalent and trispecific antibody construct is described in which a first Fc polypeptide is linked to the N-terminus of a second Fc polypeptide via a nucleotide sequence.

[0325] In various embodiments, such trivalent and trispecific antibody constructs comprise the domain structure (VL-CL) Fab -Linker Fab-scFv -(VL-linker scFv -VH) scFv In certain embodiments, such a light chain comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 123. In some embodiments, such a construct comprises a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 123.

[0326] In various embodiments, trivalent and trispecific antibody constructs comprising light chains comprising Fab and scFv portions may possess certain properties that may be unique to constructs with a specified format and geometry.

[0327] In some embodiments, trivalent and trispecific antibody constructs herein comprising light chains comprising a Fab portion and an scFv portion do not reduce T cell viability by more than 5%, more than 4%, more than 3%, more than 2%, more than 1%, or more than 0% compared to T cells treated with a negative control construct that does not contain the binding domain for Cldn18.2, measured after 48 hours of incubation of T cells with the respective construct. In some embodiments, the trivalent and trispecific antibody constructs do not reduce T cell viability by more than 5%, more than 3%, more than 1%, or more than 0%. In some embodiments, the trivalent and trispecific antibody constructs do not reduce T cell viability by more than 5% compared to the control construct. In some embodiments, the trivalent and trispecific antibody constructs do not reduce T cell viability by more than 4% compared to the control construct. In some embodiments, the trivalent and trispecific antibody constructs do not reduce T cell viability by more than 3% compared to the control construct. In some embodiments, the trivalent and trispecific antibody constructs do not reduce T cell viability by more than 2% compared to a control construct. In some embodiments, the trivalent and trispecific antibody constructs do not reduce T cell viability by more than 1% compared to a control construct. In some embodiments, the trivalent and trispecific antibody constructs do not reduce T cell viability compared to a control construct.

[0328] In some embodiments, trivalent and trispecific antibody constructs herein comprising a light chain comprising a Fab portion and an scFv portion reduce T cell viability by about 1.5-fold to about 2-fold, about 1.5-fold to about 3-fold, or about 2-fold to about 3-fold less than antibody constructs in which the first scFv domain and the second scFv domain are linked to either the N-terminus of the Fab heavy chain or the N-terminus of the second Fc polypeptide, as measured when each antibody construct is incubated with T cells for 48 hours. In some embodiments, trivalent and trispecific antibody constructs herein comprising a light chain comprising a Fab portion and an scFv portion reduce T cell viability by about 1.5-fold to about 2-fold less than antibody constructs in which the first scFv domain and the second scFv domain are linked to either the N-terminus of the Fab heavy chain or the N-terminus of the second Fc polypeptide. In some embodiments, trivalent and trispecific antibody constructs comprising a light chain comprising a Fab portion and an scFv portion reduce T cell viability by about 1.5 to about 3 times less than antibody constructs in which the first scFv domain and the second scFv domain are linked to either the N-terminus of the Fab heavy chain or the N-terminus of the second Fc polypeptide. In some embodiments, trivalent and trispecific antibody constructs comprising a light chain comprising a Fab portion and an scFv portion reduce T cell viability by about 2 to about 3 times less than antibody constructs in which the first scFv domain and the second scFv domain are linked to either the N-terminus of the Fab heavy chain or the N-terminus of the second Fc polypeptide.

[0329] In some embodiments, the trivalent and trispecific antibody constructs herein comprising a light chain comprising a Fab portion and an scFv portion exhibit enhanced binding to human CD3 as compared to antibody constructs in which the first scFv domain and the second scFv domain are independently linked to either the N-terminus of the Fab heavy chain or the N-terminus of the second Fc polypeptide. + In assays containing only T cells, the antibody constructs induced approximately 80-2000-fold, approximately 100-1000-fold, or approximately 100-500-fold less cytokines, and each antibody construct expressed human CD3 +In some embodiments, trivalent and trispecific antibody constructs comprising a light chain comprising a Fab portion and an scFv portion exhibit increased affinity to human CD3 T cells compared to antibody constructs in which a first scFv domain and a second scFv domain are independently linked to either the N-terminus of the Fab heavy chain or the N-terminus of the second Fc polypeptide. + In some embodiments, trivalent and trispecific antibody constructs comprising a light chain comprising a Fab portion and an scFv portion induce about 80- to about 2000-fold less cytokines than human CD3 T cells compared to antibody constructs in which a first scFv domain and a second scFv domain are independently linked to either the N-terminus of the Fab heavy chain or the N-terminus of the second Fc polypeptide. + In some embodiments, trivalent and trispecific antibody constructs comprising a light chain comprising a Fab portion and an scFv portion induce about 100-fold to about 1000-fold less cytokines than human CD3 T cells compared to antibody constructs in which a first scFv domain and a second scFv domain are independently linked to either the N-terminus of the Fab heavy chain or the N-terminus of the second Fc polypeptide. + Induce about 100-fold to about 500-fold less cytokine than in assays involving only T cells. In some embodiments, trivalent and trispecific antibody constructs comprising a light chain comprising a Fab portion and an scFv portion induce less than about 10 pg / mL of cytokine, e.g., between about 10 pg / mL and 0.5 pg / mL of cytokine.

[0330] In some embodiments, trivalent and trispecific antibody constructs herein comprising a light chain comprising a Fab portion and an scFv portion induce about 5-fold to about 900-fold, about 5-fold to about 500-fold, or about 5-fold to about 300-fold less cytokines in an assay including only human PBMCs compared to an antibody construct in which a first scFv domain and a second scFv domain are independently linked to either the N-terminus of the Fab heavy chain or the N-terminus of the second Fc polypeptide, wherein each antibody construct is incubated with T cells for 48 hours. In some embodiments, trivalent and trispecific antibody constructs herein comprising a light chain comprising a Fab portion and an scFv portion induce about 5-fold to about 900-fold less cytokines in an assay including only human PBMCs compared to an antibody construct in which a first scFv domain and a second scFv domain are independently linked to either the N-terminus of the Fab heavy chain or the N-terminus of the second Fc polypeptide. In some embodiments, trivalent and trispecific antibody constructs comprising light chains comprising Fab and scFv portions induce about 5-fold to about 500-fold less cytokine in assays involving only human PBMCs compared to antibody constructs in which the first scFv domain and the second scFv domain are independently linked to either the N-terminus of the Fab heavy chain or the N-terminus of the second Fc polypeptide. In some embodiments, trivalent and trispecific antibody constructs comprising light chains comprising Fab and scFv portions induce about 5-fold to about 300-fold less cytokine in assays involving only human PBMCs compared to antibody constructs in which the first scFv domain and the second scFv domain are independently linked to either the N-terminus of the Fab heavy chain or the N-terminus of the second Fc polypeptide. In some embodiments, trivalent and trispecific antibody constructs comprising light chains comprising Fab and scFv portions induce less than about 20 pg / mL of cytokine, e.g., about 20 pg / mL to 0.5 pg / mL of cytokine.

[0331] In certain embodiments, the cytokine comprises one or more of IL-2, interleukin-6 (IL-6), IFNγ, and TNFα.

[0332] In some embodiments, trivalent and trispecific antibody constructs herein comprising light chains comprising Fab and scFv portions induce different amounts of memory T cell subsets upon stimulation of a given immune cell when compared to trivalent and trispecific antibody constructs having different formats and / or geometries. In some embodiments, trivalent and trispecific antibody constructs herein comprising light chains comprising Fab and scFv portions induce similar amounts of memory T cell subsets upon stimulation of a given immune cell when compared to a combination treatment of two bivalent and bispecific antibody constructs that together target the same antigen as a trivalent and trispecific antibody construct comprising light chains comprising Fab and scFv portions.

[0333] In some of these embodiments, the trivalent and trispecific antibody construct comprising a light chain comprising a Fab portion and an scFv portion comprises: (i) a Fab domain capable of binding to CD3; (ii) a first scFv domain capable of binding to CD28; (iii) a second scFv domain capable of binding to Claudin18.2 (Cldn18.2); and (iv) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein a) the Fab domain has a C H1 a) the first scFv domain is linked to the N-terminus of the first Fc polypeptide via a C sequence of the Fab light chain; L and c) a second scFv domain is linked to the N-terminus of the second Fc polypeptide.

[0334] In certain embodiments, the trivalent and trispecific antibody construct comprising a light chain comprising a Fab portion and an scFv portion is v37634.

[0335] III. Sequence Identity of Amino Acid and Nucleic Acid Sequences As described elsewhere in this disclosure, certain embodiments herein relate to an isolated polypeptide or set of isolated polypeptides (e.g., polypeptide chains H1, H2, L1, etc., or portions thereof, e.g., domains) of a trivalent and trispecific antibody construct, as well as a polynucleotide or set of polynucleotides encoding one or more polypeptide chains of the antibody constructs described herein. A polynucleotide in this context can encode all or a portion of an antibody construct, e.g., one or more polypeptide chains (e.g., H1, H2, L1, etc.) of an antibody construct.

[0336] In some embodiments, described herein are nucleic acid molecules or sets of nucleic acid molecules that encode one or more, two or more, or three or more polypeptide chains that form any of the trivalent and trispecific antibody constructs disclosed herein.

[0337] The terms "nucleic acid," "nucleic acid molecule," and "polynucleotide" are used interchangeably herein to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.

[0338] In some embodiments, the present specification describes a vector or a set of vectors comprising a nucleic acid molecule or a set of nucleic acid molecules encoding one or more polypeptide chains (e.g., one or more of H1, H2, L1, etc.) of the antibody constructs disclosed herein.

[0339] A polynucleotide "encoding" a given polypeptide is one that is transcribed (in the case of DNA) and translated into a polypeptide (in the case of mRNA) in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A transcription termination sequence may be located 3' to the coding sequence.

[0340] In certain embodiments, the present disclosure relates to polynucleotide and / or polypeptide sequences that are identical or substantially identical to another polynucleotide and / or polypeptide sequence. The term "identical," with respect to two or more polynucleotide or polypeptide sequences, refers to two or more sequences or subsequences that are identical, i.e., have the same sequence of nucleotide or amino acid monomers (i.e., 100% sequence identity), respectively. Polypeptide or polynucleotide sequences herein share "sequence identity" if they have a percentage or specified number of amino acid residues or nucleotides that are at least about 80%, about 85%, about 90%, about 95%, about 97%, or at least about 99% identical over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region, as determined using one of the commonly used sequence comparison algorithms known to those of skill in the art or by manual alignment and visual inspection. This definition also refers to the complement of a test polynucleotide sequence. Identity can exist over a region that is at least about 50 amino acids or nucleotides in length, or over a region that is about 75 to about 100 amino acids or nucleotides in length, or, if not specified, over the entire polypeptide or polynucleotide sequence. For sequence comparison, a test sequence is typically compared to a designated reference sequence. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence relative to the reference sequence based on the program parameters.

[0341] The term "comparison window," as used herein, refers to a segment of a sequence comprising about 20 to about 1,000 contiguous amino acid or nucleotide positions, e.g., about 50 to about 600, or about 100 to about 300, or about 150 to about 200 contiguous amino acid or nucleotide positions, over which a test sequence can be compared to a reference sequence over the same number of contiguous positions after optimally aligning the two sequences. Longer segments (up to the full-length sequence) may also be used as the comparison window in certain embodiments. Methods for aligning sequences for comparison purposes are known to those of skill in the art. Optimal sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, 1970, Adv. Appl. Math., 2:482c, by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol., 48:443, by the search for similarity method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA, 85:2444, or by computer implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, or TFASTA (Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI)), or by manual alignment and visual inspection (see, for example, Ausubel et al., Current Protocols in Molecular Biology, (1995 supplement), Cold Spring Harbor Laboratory Press). Examples of suitable available algorithms for determining percent sequence identity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1997, Acids Res., 25:3389-3402, and Altschul et al., 1990, J. Mol. Biol., 215:403-410, respectively.Software for performing BLAST analyses is publicly available from the website of the National Center for Biotechnology Information (NCBI).

[0342] Certain embodiments described herein include variant sequences (e.g., variant V) that contain one or more amino acid modifications, e.g., one or more amino acid insertions, one or more amino acid deletions, and / or one or more amino acid substitutions, when compared to a reference, e.g., a wild-type sequence. H In certain embodiments, the one or more amino acid modifications of the variant sequence include one or more amino acid substitutions when compared to a reference, such as a wild-type sequence. In such embodiments, the one or more amino acid substitutions are one or more non-conservative substitutions. In other embodiments, the one or more amino acid substitutions are one or more conservative substitutions. Generally, a "conservative substitution," as used herein, is considered to be the substitution of one amino acid for another amino acid with similar physical, chemical, and / or structural properties. Common conservative substitutions are listed in column 1 of Table 4. [Table 4]

[0343] Those skilled in the art will understand that, although the primary factors determining what constitutes a conservative substitution are usually the size of the amino acid side chain and its physical / chemical properties, in certain circumstances a wider range of amino acids than those listed in column 1 of Table 4 can be substituted for a given amino acid. These additional amino acids tend to either have similar properties to the amino acid being substituted but are significantly different in size, or similar in size but have significantly different physical / chemical properties. This broad range of conservative substitutions is listed in column 2 of Table 4. Those skilled in the art will be able to readily determine the most appropriate set of substituents to select, given the particular protein environment in which the amino acid substitution is to be made.

[0344] IV. Pharmaceutical Compositions In certain embodiments, the present disclosure relates to pharmaceutical compositions that may include one or more of the trivalent and trispecific antibody constructs described herein. In various embodiments, the pharmaceutical compositions herein may further include a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other substance familiar to those skilled in the art. Such substances are typically non-toxic and do not interfere with the efficacy of the active ingredient (i.e., the antibody construct). The precise nature of the carrier or other substance may depend on the route of administration. Thus, the pharmaceutical compositions herein may be formulated for a variety of uses and routes of administration, for example, oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular, or intraperitoneal routes of administration.

[0345] Pharmaceutical compositions for oral administration can be in tablet, capsule, powder, or liquid form. Tablets can contain solid carriers such as gelatin or adjuvants. Liquid pharmaceutical compositions usually contain liquid carriers such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol can be included.

[0346] For intravenous, cutaneous or subcutaneous injection, or injection into an affected area (e.g., a tumor site), the active ingredient (i.e., antibody construct) may be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art are well able to prepare suitable solutions using isotonic vehicles such as sodium chloride, Ringer's injection, lactated Ringer's injection, etc. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included, as necessary.

[0347] For antibody constructs according to the present disclosure to be administered to a subject, the administration is preferably in a "therapeutically effective amount" sufficient to show benefit to the individual, as further described herein. The actual amount administered, as well as the rate and time-course of administration, may depend on the nature and severity of the disease (e.g., cancer) being treated. Prescribing treatment, e.g., determining dosage, etc., is the responsibility of a general practitioner or other physician, and typically takes into account the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to physicians. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0348] In some embodiments, the pharmaceutical compositions may include a second active ingredient (e.g., another protein or a small molecule) in addition to the antibody constructs described herein.

[0349] Accordingly, also described herein are pharmaceutical compositions comprising any one or more of the trivalent and trispecific antibody constructs disclosed herein and a pharmaceutically acceptable carrier, excipient, diluent, or combination thereof.

[0350] V. Kit The present disclosure also describes kits comprising one or more of the trivalent and trispecific antibody constructs described herein, or pharmaceutical compositions comprising such antibody construct(s) described herein, and instructions for use. Thus, in certain embodiments, the present disclosure describes kits comprising vectors for expressing the antibody constructs described herein and instructions for use. In certain embodiments, the present disclosure describes kits comprising host cells comprising vectors for expressing the antibody constructs and instructions for use. In some embodiments, the present disclosure relates to kits comprising purified antibody constructs and instructions for use. The purified antibody constructs may be lyophilized or provided in a dried form, e.g., a powder or granules, and the kit may additionally contain a suitable solvent for reconstitution of the lyophilized or dried component(s).

[0351] The kit may further comprise a container and a label and / or package insert on or associated with the container. The label or package insert contains instructions customarily included in commercial packaging of therapeutic products providing information or instructions regarding the indications, uses, dosages, administration, contraindications, and / or warnings for the use of such therapeutic products (e.g., antibody constructs described herein). The label or package insert may further include a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceutical or biological products, the notice indicating approval by such agency for the manufacture, use, or sale for administration to humans or animals. The container may hold a composition comprising an antibody construct of the present disclosure. In some embodiments, the container may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper that can be punctured by a hypodermic injection needle.

[0352] In addition to the container containing the composition comprising the antibody construct, the kit may further comprise one or more additional containers containing other components of the kit. For example, a pharmaceutically acceptable buffer (e.g., bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, or dextrose solution), or other buffer or diluent may be included in such a kit.

[0353] Suitable containers may include, for example, bottles, vials, syringes, intravenous solution bags, etc. The containers may be formed from a variety of materials, such as glass or plastic. Where appropriate, one or more components of the kit (e.g., antibody construct) may be lyophilized or provided in a dried form, e.g., a powder or granules, and the kit may additionally contain a suitable solvent for the reconstitution of the lyophilized or dried component(s).

[0354] The kits herein may further include other materials desirable from a commercial or user standpoint, including filters, needles, and syringes.

[0355] VI. Method Further described herein are methods of making and using the trivalent and trispecific antibody constructs of the present disclosure.

[0356] A. Methods for generating antibody constructs In some embodiments, the present disclosure relates to methods for preparing the trivalent and trispecific antibody constructs described herein. In various embodiments, the antibody constructs of the present disclosure can be produced using standard recombinant methods known in the art (see, e.g., U.S. Patent No. 4,816,567 and "Antibodies: A Laboratory Manual," 2002). nd Edition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014).

[0357] For recombinant production of the antibody constructs described herein, a polynucleotide or set of polynucleotides encoding the antibody construct can be generated and inserted into one or more vectors for further cloning and / or expression in a host cell. The polynucleotide(s) encoding the antibody construct can be generated by standard methods known in the art (e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1994 & update, and "Antibodies: A Laboratory Manual," 2002). nd (See, for example, "Antibody Constructs: A Guide to Gene Expression," Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014). As will be appreciated by those skilled in the art, the number of polynucleotides required for expression of an antibody construct can depend on the format and / or geometry of the antibody construct, including, for example, the number of polypeptide chains comprising the antibody construct. For example, if an antibody construct includes three polypeptide chains (e.g., H1, H2, and L1), three polynucleotides, each encoding one polypeptide chain, can be used. In embodiments in which two or more polynucleotides are used, such two or more polynucleotides can be incorporated into one vector or multiple vectors (e.g., two or three separate vectors).

[0358] Typically, for expression, a polynucleotide or set of polynucleotides encoding an antibody construct herein may be incorporated into an expression vector along with one or more regulatory elements, such as transcription elements, that can be used for efficient transcription of the polynucleotide(s). Examples of such regulatory elements include, but are not limited to, promoters, enhancers, terminators, and polyadenylation signals. Those skilled in the art will appreciate that the selection of regulatory elements may depend on the host cell selected for expression of the antibody construct polypeptide and that such regulatory elements may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. Expression vectors may optionally further contain heterologous nucleic acid sequences that facilitate expression or purification of the expressed protein. Examples include, but are not limited to, signal peptides and affinity tags, such as metal affinity tags, histidine tags, avidin / streptavidin coding sequences, glutathione-S-transferase (GST) coding sequences, and biotin coding sequences. Expression vectors may be extrachromosomal or integrating vectors. Thus, in some embodiments, the amino acid sequences of the polypeptide chains of the expressed antibody constructs described herein, e.g., chains H1, H2, L1, etc., may comprise a signal peptide sequence. Such signal peptide sequences may vary depending on the expression system and conditions used to generate the antibody construct. Exemplary signal peptide sequences may include, e.g., for H1, H2, etc., the amino acid sequences METDTLLLWVLLLWVPGSTG (SEQ ID NO: 155) or MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO: 156), or may include, e.g., for L1, L2, etc., MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO: 156) or MGWSCIILFLVATATGVHS (SEQ ID NO: 157). In certain embodiments, one or more heavy chains (e.g., H1, H2, etc.) of the antibody constructs described herein may comprise a C-terminal lysine residue after expression of the polypeptide chain in a cell.In various embodiments, such C-terminal lysine residues may be enzymatically cleaved from the polypeptide chain prior to further processing (e.g., purification, formulation, etc.) and prior to use of the corresponding antibody construct, e.g., prior to administration of the construct to a subject in need thereof.

[0359] Certain embodiments for producing the antibody constructs of the present disclosure relate to vectors (such as expression vectors) containing one or more polynucleotides encoding at least a portion of the antibody constructs described herein. The polynucleotide(s) may be contained in a single vector or in multiple vectors. In some embodiments, the polynucleotides are contained in a multicistronic vector. Expression vectors that can be used to express polynucleotides include, but are not limited to, pTT5 and pUC15 cells containing vectors encoding the antibody constructs.

[0360] Suitable host cells for cloning or expressing antibody construct polypeptides include a variety of prokaryotic or eukaryotic cells known in the art. Eukaryotic host cells include, for example, mammalian cells, plant cells, insect cells, and yeast cells (e.g., Saccharomyces or Pichia cells). Prokaryotic host cells include, for example, E. coli, A. salmonicida, or B. subtilis cells. In certain embodiments, antibody constructs can be produced in bacteria, particularly when glycosylation and Fc effector functions are not necessary or desirable for the intended purpose of the antibody construct, as described, for example, in U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523, and Charlton, Methods in Molecular Biology, Vol. 248, pp. 245-254, BKC Lo, ed., Humana Press, Totowa, NJ, 2003. Eukaryotic microbes, such as filamentous fungi or yeast, particularly fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of antibodies with partial or fully human glycosylation patterns, are suitable expression host cells in certain embodiments (see, e.g., Gerngross, 2004, Nat. Biotech. 22:1409-1414, and Li et al., 2006, Nat. Biotech. 24:210-215).

[0361] Suitable host cells for expression of glycosylated antibody constructs are, in various embodiments, eukaryotic cells. For example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 describe the PLANTIBODIES™ technology for producing antibodies and portions thereof (e.g., scFv(s), Fab(s), etc.) in transgenic plants. Mammalian cell lines adapted to growth in suspension are particularly useful for expression of the antibody constructs described herein. Examples include SV40-transformed monkey kidney line CV1 (COS-7), human embryonic kidney (HEK) line 293 or 293 cells (see, e.g., Graham et al., 1977, J. Gen Virol., 36:59), baby hamster kidney cells (BHK), mouse Sertoli TM4 cells (see, e.g., Mather, 1980, Biol Reprod., 23:243-251); monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma (HeLa) cells, canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (HepG2), mouse mammary tumor (MMT060562), TRI cells (see, e.g., Mather et al., 1982, Annals of NY Acad. Sci, 383:44-68), MRC5 cells, FS4 cells, Chinese hamster ovary (CHO) cells (including DHFR-CHO cells; Urlaub et al., 1980, Proc Natl Acad Sci USA, 77:4216), and myeloma cell lines (such as Y0, NS0, and Sp2 / 0). Exemplary mammalian host cell lines suitable for antibody production are reviewed in Yazaki & Wu, Methods in Molecular Biology, Vol. 248, pp. 255-268 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003).

[0362] In certain embodiments, the host cells used to produce the trivalent and trispecific antibody constructs herein are transient or stable higher eukaryotic cell lines, e.g., mammalian cell lines. In some embodiments, the host cells are mammalian HEK293T, CHO, HeLa, NS0, or COS cells. In some embodiments, the host cells are stable cell lines that allow mature glycosylation of the antibody constructs.

[0363] Host cells containing expression vector(s) encoding the antibody construct may be cultured using conventional methods for producing antibody constructs. Alternatively, in some embodiments, host cells containing expression vector(s) encoding the antibody construct may be used therapeutically or prophylactically to deliver the antibody construct to a subject, or the polynucleotide or expression vector may be administered ex vivo to cells derived from a subject, which may then be returned to the subject's body.

[0364] In some embodiments, the host cell expresses the V of the binding domain of the antibody construct described herein. L and V H In some embodiments, the host cell comprises (e.g., has been transformed with) a vector comprising a polynucleotide encoding a full-length polypeptide chain of an antibody construct described herein, e.g., H1, H2, or L1 described herein. In another example ... L a first vector comprising a polynucleotide encoding a corresponding binding domain V Hand a second vector comprising a polynucleotide encoding the polypeptide. In various embodiments, the host cell is a eukaryotic organism, such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a lymphoid cell (e.g., a Y0, NS0, or Sp20 cell). In certain embodiments, the host cell is Expi293™ (Thermo Fisher, Waltham, MA). In certain embodiments, the host cell used herein is a CHO-S cell (National Research Council Canada) or a HEK293 cell.

[0365] Certain embodiments of the present disclosure relate to methods of producing an antibody construct, comprising culturing host cells into which one or more polynucleotides encoding the antibody construct, or one or more expression vectors encoding the antibody construct, have been introduced under conditions suitable for expression of the antibody construct. Such methods may further comprise recovering the antibody construct from the host cells (or from the culture medium of the host cells). In some embodiments, such methods may further comprise purifying the antibody construct.

[0366] Cell culture media that can be used include, but are not limited to, DMEM (Thermo Fisher, Waltham, MA), Opti-MEM™ (Thermo Fisher, Waltham, MA), Opti-MEM™ I Reduced Serum Medium (Thermo Fisher, Waltham, MA), RPMI-1640 medium, Expi293™ Expression Medium (Thermo Fisher, Waltham, MA), and FreeStyle CHO Expression Medium (Thermo Fisher Scientific, Waltham, MA). Cell culture media can be supplemented with serum, such as fetal bovine serum (FBS), amino acids, such as L-glutamine, antibiotics, such as penicillin and streptomycin, and / or antimycotics, such as amphotericin, or any other supplements routinely used to support cell culture.

[0367] In various embodiments, the antibody constructs of the present disclosure are purified after expression. Proteins, such as the antibody constructs of the present disclosure, can be isolated or purified by a variety of methods known to those of skill in the art (e.g., Protein Purification: Principles and Practice, 3). rd (See, Ed., Scopes, Springer-Verlag, NY, 1994). Standard purification methods that can be used for the antibody constructs disclosed herein include chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and reversed phase, performed at atmospheric or elevated pressure using systems such as FPLC and HPLC. Additional purification methods include electrophoretic, immunological, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques can also be used in conjunction with protein concentration. As is well known in the art, a variety of natural proteins bind to the Fc domain and other structural elements of antibody constructs, and in some embodiments, these proteins can be used for the purification of antibody constructs. For example, bacterial proteins A and G can bind to the Fc domain of some antibody constructs. Similarly, bacterial protein L can bind to the Fab domain of some antibody constructs. Purification can also be enabled by specific fusion partners. For example, antibody constructs can be bound to glutathione resins when GST fusions are used, or Ni when His tags are used. +2 Purification can be achieved using affinity chromatography or, if a flag tag is used, immobilized anti-flag antibodies. The degree of purification required can vary depending on the antibody construct used. Thus, in some embodiments, purification may not be necessary.

[0368] In certain embodiments, the antibody constructs of the present disclosure are substantially pure. The term "substantially pure" (or "substantially purified"), when used with reference to the antibody constructs described herein, refers to an antibody construct that is substantially or essentially free from components that normally accompany or interact with the protein as found in its naturally occurring environment, e.g., in natural cells or, in the case of recombinantly produced antibody constructs, in host cells. In certain embodiments, a substantially pure antibody construct is an antibody construct that has been purified to contain less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 2% (by dry weight) of other contaminating protein species.

[0369] Assessment of the purity and / or homogeneity of antibody constructs can be performed by any method known in the art, including, but not limited to, non-reducing / reducing CE-SDS, non-reducing / reducing SDS-PAGE, ultra-performance liquid chromatography-size exclusion chromatography (UPLC-SEC), high performance liquid chromatography (HPLC), mass spectrometry, multi-angle light scattering (MALS), and dynamic light scattering (DLS).

[0370] In certain embodiments, the antibody constructs described herein may comprise one or more post-translational modifications. Such post-translational modifications may occur in vivo or may be performed in vitro after isolation of the antibody construct from a host cell.

[0371] Post-translational modifications can include a variety of modifications known in the art (see, e.g., Proteins—Structure and Molecular Properties, 2nd Ed., TECreighton, W.H. Freeman and Company, New York, 1993; Post-Translational Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York, pp. 1-12, 1983; Seifter et al., 1990, Meth. Enzymol., 182:626-646, and Rattan et al., 1992, Ann. N.Y. Acad. Sci., 663:48-62). In those embodiments in which an antibody construct includes one or more post-translational modifications, the antibody construct can include the same type of modification at one or several sites (e.g., amino acid residues), or it can include different modifications at different sites.

[0372] Examples of post-translational modifications may include glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, formylation, oxidation, reduction, proteolytic cleavage or specific chemical cleavage (by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease or NaBH4).

[0373] Other examples of post-translational modifications may include, for example, the addition or removal of N- or O-linked glycans, chemical modification of N- or O-linked glycans, N- or C-terminal processing, conjugation of chemical moieties to the amino acid backbone, and the addition or deletion of N-terminal methionine residues resulting from expression in prokaryotic host cells. Post-translational modifications may also include modification with detectable labels, such as enzymes, fluorescent, isotopic, or affinity labels, to enable protein detection and isolation. Examples of suitable enzymatic labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase. Examples of suitable prosthetic group complexes include, but are not limited to, streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent substances include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin. An example of a luminescent material is luminol, examples of bioluminescent materials include luciferase, luciferin and aequorin, and examples of suitable radioactive materials include iodine, carbon, sulfur, tritium, indium, technetium, thallium, gallium, palladium, molybdenum, xenon, and fluorine.

[0374] Additional examples of post-translational modifications can include acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, pegylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0375] In some embodiments, described herein are methods for producing a trivalent and trispecific antibody construct of the present disclosure, the method comprising: (a) obtaining a host cell culture comprising at least one host cell comprising one or more nucleic acid molecules encoding one or more, two or more, or three or more polypeptide chains that form the antibody construct; and (b) recovering the antibody construct from the host cell culture. In some embodiments, such methods may further comprise purifying the antibody construct after step (b).

[0376] B. Methods of Using the Antibody Constructs of the Disclosure In certain embodiments, the present disclosure relates to methods of using the trivalent and trispecific antibody constructs of the present disclosure. In some embodiments, described herein are methods of using the antibody constructs described herein for the treatment of a disease or condition in a subject in need thereof.

[0377] Such methods may include administering a trivalent and trispecific antibody construct, or a pharmaceutical composition comprising such an antibody construct, to a subject in need thereof. In certain embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0378] In some embodiments, the present disclosure relates to a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a trivalent and trispecific antibody construct of the present disclosure, or a pharmaceutical composition comprising such an antibody construct. Cancers that can be treated using the methods and antibody constructs disclosed herein can include, but are not limited to, hematological neoplasms (including leukemia, myeloma, and lymphoma), carcinomas (including adenocarcinoma and squamous cell carcinoma), melanoma, and sarcoma. Carcinomas and sarcomas are also frequently referred to as "solid tumors." In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a leukemia. In some embodiments, the cancer is a lymphoma.

[0379] When used in the methods described herein, the antibody constructs of the present disclosure may exert either a cytotoxic or cytostatic effect, resulting in a reduction in tumor size in a tumor-bearing subject, a slowing or prevention of tumor growth in size, an increase in disease-free survival from the disappearance or removal of a tumor until its recurrence, prevention of initial or subsequent development of a tumor (e.g., metastasis), an increase in the time to progression, a reduction in one or more adverse symptoms associated with a tumor, an increase in overall survival, or one or more combinations of the above.

[0380] The methods described herein may include administering a trivalent and trispecific antibody construct to a subject in need thereof. The antibody construct may be administered to a subject by any suitable route of administration. As will be appreciated by one of skill in the art, the route and / or mode of administration may vary depending on the desired therapeutic outcome. In various embodiments, the antibody construct of the present disclosure may be administered by systemic or local administration. Local administration may be to the tumor site or tumor-draining lymph nodes. Typically, the antibody construct may be administered parenterally, e.g., intravenously, intramuscularly, intradermally, intraperitoneally, subcutaneously, or spinally, e.g., by injection or infusion.

[0381] Treatment (e.g., of cancer in a subject) can be achieved by administering a therapeutically effective amount of a trivalent and trispecific antibody construct to a subject in need thereof. A "therapeutically effective amount," as used herein, generally refers to an amount of an antibody construct described herein that is effective, at the dosage and for the duration necessary, to achieve a desired therapeutic result. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject. A therapeutically effective amount is also one in which the therapeutically beneficial effects outweigh any potential toxic or adverse effects of the antibody construct. A "sufficient amount" generally refers to an amount sufficient to produce a desired effect, e.g., an amount sufficient to generate an anti-tumor immune response against target (e.g., tumor) cells or tissues, for example, by engaging immune cells (e.g., T cells) using the trivalent and trispecific antibody constructs described herein.

[0382] Suitable dosages of the trivalent and trispecific antibody constructs described herein can be determined by one skilled in the art of medicine. The selected dose level may vary depending on various pharmacokinetic factors, including the activity (e.g., antigen affinity(ies)) of the particular antibody construct used, the route of administration, the time of administration, the rate of excretion of the construct, the duration of treatment, other drugs, compounds, and / or materials used in combination with the antibody construct, e.g., anti-cancer drugs, the age, sex, weight, condition, general health, and previous medical history of the subject being treated, and similar factors well known in the medical arts.

[0383] In some embodiments, methods of treating a disease (e.g., cancer) in a subject include administering a second active ingredient (e.g., another protein or small molecule) in addition to an antibody construct described herein. Such second active ingredient may be administered simultaneously or sequentially with the antibody construct, depending on the condition being treated.

[0384] In some embodiments, the present disclosure provides a method for eliciting an anti-tumor immune response in a cell population comprising immune cells expressing CD3 and CD28 and tumor cells expressing MSLN and / or Cldn18.2, the method comprising contacting the cell population with an effective amount of a trivalent and trispecific antibody construct of the present disclosure. In some embodiments, such a trivalent and trispecific antibody construct binds to CD3 and CD28 on one or more immune cells and MSLN or Cldn18.2 on tumor cells, thereby forming a TCR-independent immune synapse, and comprising: (i) a Fab domain capable of binding to either CD3 or CD28 on a first immune cell; (ii) a first scFv domain capable of binding to either CD3 or CD28, however, the Fab domain and the first scFv domain do not bind to the same antigen; and (iii) MSLN or Cldn18 on tumor cells. and (iv) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein (a) the Fab domain is linked to the N-terminus of either the first or second Fc polypeptide, (b) the first scFv domain is linked to either the N-terminus of the Fab domain, the N-terminus of the first or second Fc polypeptide, the C-terminus of the Fab light chain, or the C-terminus of the first Fc polypeptide, and (c) the second scFv domain is linked to the N-terminus of the Fab domain or the N-terminus of the first or second Fc polypeptide.

[0385] In some embodiments, the present disclosure provides a method for inhibiting the proliferation of tumor cells expressing MSLN and / or Cldn18.2 in a cell population comprising tumor cells and immune cells expressing CD3 and CD28...

Claims

1. (i) a Fab domain capable of binding to a first antigen on a first cytotoxic effector cell; (ii) a first scFv domain and a second scFv domain, wherein one of the scFv domains is capable of binding to a second antigen on a second cytotoxic effector cell and the other scFv domain is capable of binding to a tumor-associated antigen (TAA) on a tumor cell; and (iii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide; Including, (a) the Fab domain is linked to the N-terminus of the first Fc polypeptide; (b) the first and second scFv domains are independently linked to either (i) the N-terminus of the Fab domain, (ii) the C-terminus of the Fab domain, (iii) the C-terminus of one of the Fc polypeptides, or (iv) the N-terminus of the second Fc polypeptide; provided that when one of the scFv domains is linked to the C-terminus of one of the Fc polypeptides, the first antigen is CD3 and the second antigen is CD28, or the first antigen is CD28 and the second antigen is CD3. Antibody constructs.

2. 2. The antibody construct of claim 1, wherein the first scFv domain and the second scFv domain are not linked in tandem to each other.

3. 3. The antibody construct of claim 1, wherein the first antigen is CD28 and the second antigen is CD3.

4. 3. The antibody construct of claim 1, wherein the first antigen is CD3 and the second antigen is CD28.

5. 5. The antibody construct of claim 1, wherein the first scFv domain is linked to the N-terminus of the Fab domain and the second scFv is linked to the N-terminus of the second Fc polypeptide.

6. The first scFv domain comprises the V of the heavy chain of the Fab domain. Ｈ The antibody construct of claim 5 , wherein the sequence is linked to the N-terminus of the sequence.

7. The first scFv domain comprises the V of the light chain of the Fab domain. Ｌ The antibody construct of claim 5 , wherein the sequence is linked to the N-terminus of the sequence.

8. The antibody construct comprises: a) From N-terminus to C-terminus: (i) first scFv V Ｌ First scFv V linked to sequence Ｈ Sequence (V Ｈ -V Ｌ ) or the first scFv V Ｈ First scFv V linked to sequence Ｌ Sequence (V Ｌ -V Ｈ (ii) the first scFv domain comprising either Fab C or Ｈ１ Fab V linked to sequence Ｈ (iii) a first heavy chain polypeptide comprising said first Fc polypeptide; b) From the N-terminus to the C-terminus: (i) the second scFv V Ｌ A second scFv V linked to the sequence Ｈ Sequence (V Ｈ -V Ｌ ) or a second scFv V Ｈ A second scFv V linked to the sequence Ｌ Sequence (V Ｌ -V Ｈ (ii) a second heavy chain polypeptide comprising the second scFv domain comprising either (i) a first Fc polypeptide or (ii) a second scFv domain comprising either (i) a first Fc polypeptide or (ii) a second heavy chain polypeptide comprising the second scFv domain; and c) N-terminus to C-terminus: Fab C Ｌ Fab V linked to sequence Ｌ a light chain polypeptide comprising the sequence Including, the heavy chain Fab sequence and the light chain polypeptide associate to form the Fab domain; the first Fc polypeptide and the second Fc polypeptide associate to form the Fc domain. The antibody construct of claim 5 or 6.

9. The first scFv domain comprises a light chain C of the Fab domain. Ｌ The antibody construct of any one of claims 1 to 4, wherein the first scFv domain is linked to the C-terminus of the second Fc polypeptide, and the second scFv domain is linked to the N-terminus of the second Fc polypeptide.

10. The antibody construct comprises: a) From N-terminus to C-terminus: (i) Fab C Ｈ１ Fab V linked to sequence Ｈ (ii) a first heavy chain polypeptide comprising said first Fc polypeptide; b) From the N-terminus to the C-terminus: (i) the second scFv V Ｌ A second scFv V linked to the sequence Ｈ Sequence (V Ｈ -V Ｌ ) or a second scFv V Ｈ A second scFv V linked to the sequence Ｌ Sequence (V Ｌ -V Ｈ (ii) a second heavy chain polypeptide comprising the second scFv domain comprising either (i) a first Fc polypeptide or (ii) a second scFv domain comprising either (i) a first Fc polypeptide or (ii) a second heavy chain polypeptide comprising the second scFv domain; and c) From N-terminus to C-terminus: (i) Fab C Ｌ Fab V linked to sequence Ｌ and (ii) a light chain Fab sequence comprising the sequence: Ｌ First scFv V linked to sequence Ｈ Sequence (V Ｈ -V Ｌ ) or the first scFv V Ｈ First scFv V linked to sequence Ｌ Sequence (V Ｌ -V Ｈ a light chain polypeptide comprising the first scFv domain comprising any one of Including, the heavy chain Fab sequence and the light chain Fab sequence associate to form the Fab domain; the first Fc polypeptide and the second Fc polypeptide associate to form the Fc domain. The antibody construct of claim 9.

11. The antibody construct of any one of claims 1 to 4, wherein the first scFv domain is linked to the N-terminus of the Fab domain and the second scFv domain is linked to one C-terminus of the Fc polypeptide.

12. The first scFv domain is a V of the Fab domain. Ｈ The antibody construct of claim 11, wherein the domain is linked to the N-terminus of the domain.

13. The antibody construct of any one of claims 11 to 12, wherein the second scFv domain is linked to the C-terminus of the first Fc polypeptide.

14. The antibody construct of any one of claims 11 to 12, wherein the second scFv domain is linked to the C-terminus of the second Fc polypeptide.

15. The antibody construct comprises: a) From N-terminus to C-terminus: (i) first scFv V Ｌ First scFv V linked to sequence Ｈ Sequence (V Ｈ -V Ｌ ) or the first scFv V Ｈ First scFv V linked to sequence Ｌ Sequence (V Ｌ -V Ｈ (ii) the first scFv domain comprising either Fab C or Ｈ１ Fab V linked to sequence Ｈ (iii) a heavy chain Fab sequence comprising the sequence, (iii) the first Fc polypeptide, and (iv) a second scFv V Ｌ A second scFv V linked to the sequence Ｈ Sequence (V Ｈ -V Ｌ ) or a second scFv V Ｈ A second scFv V linked to the sequence Ｌ Sequence (V Ｌ -V Ｈ a first heavy chain polypeptide comprising the second scFv domain comprising either b) a second heavy chain polypeptide comprising said second Fc polypeptide; and c) Fab C Ｌ Fab V linked to sequence Ｌ A light chain polypeptide comprising the sequence Including, the heavy chain Fab sequence and the light chain polypeptide associate to form the Fab domain; the first Fc polypeptide and the second Fc polypeptide associate to form the Fc domain. The antibody construct according to any one of claims 11 to 13.

16. The antibody construct comprises: a) From N-terminus to C-terminus: (i) first scFv V Ｌ First scFv V linked to sequence Ｈ Sequence (V Ｈ -V Ｌ ) or the first scFv V Ｈ First scFv V linked to sequence Ｌ Sequence (V Ｌ -V Ｈ (ii) the first scFv domain comprising either Fab C or Ｈ１ Fab V linked to sequence Ｈ (iii) a first heavy chain polypeptide comprising said first Fc polypeptide; b) from N-terminus to C-terminus: (i) the second Fc polypeptide, and (ii) the second scFv V Ｌ A second scFv V linked to the sequence Ｈ Sequence (V Ｈ -V Ｌ ) or a second scFv V Ｈ A second scFv V linked to the sequence Ｌ Sequence (V Ｌ -V Ｈ a second heavy chain polypeptide comprising the second scFv domain comprising either c) Fab C Ｌ Fab V linked to sequence Ｌ A light chain polypeptide comprising the sequence Including, the heavy chain Fab sequence and the light chain polypeptide associate to form the Fab domain; the first Fc polypeptide and the second Fc polypeptide associate to form the Fc domain. An antibody construct according to any one of claims 11 to 12 or 14.

17. The antibody construct of any one of claims 1 to 16, wherein the first scFv domain is capable of binding to the TAA and the second scFv domain is capable of binding to the second antigen on the second cytotoxic effector cell.

18. The antibody construct of any one of claims 1 to 16, wherein the second scFv domain is capable of binding to the TAA and the first scFv domain is capable of binding to the second antigen on the second cytotoxic effector cell.

19. (i) the Fab domain capable of binding to CD3; (ii) the first scFv domain capable of binding to CD28; (iii) the second scFv domain capable of binding to Claudin18.2 (Cldn18.2); and (iv) the Fc domain comprising the first Fc polypeptide and the second Fc polypeptide. Including, a) the Fab domain has its C Ｈ１ linked to the N-terminus of the first Fc polypeptide via a sequence b) the first scFv domain is C of the Fab light chain Ｌ linked to the C-terminus of the sequence, c) the second scFv domain is linked to the N-terminus of the second Fc polypeptide; 11. The antibody construct of any one of claims 1, 9 or 10.

20. 20. The antibody construct of claim 19, wherein the antibody construct reduces T cell viability by more than 5%, more than 3%, not more than 1%, and not 0% compared to T cells treated with a negative control construct that does not contain a binding domain for Cldn18.2, and wherein the antibody construct is incubated with the T cells for 48 hours.

21. 21. The antibody construct of claim 19 or claim 20, wherein the antibody construct reduces T cell viability by about 1.5-fold to about 2-fold, about 1.5-fold to about 3-fold, or about 2-fold to about 3-fold less than an antibody construct in which the first scFv domain and the second scFv domain are independently linked to either the N-terminus of the Fab heavy chain or the N-terminus of the second Fc polypeptide, wherein each of the antibody constructs is incubated with the T cells for 48 hours.

22. The antibody construct has a higher affinity for human CD3 than an antibody construct in which the first scFv domain and the second scFv domain are independently linked to either the N-terminus of the Fab heavy chain or the N-terminus of the second Fc polypeptide. ＋ 22. The antibody construct of any one of claims 19 to 21, which induces about 80-fold to about 2000-fold, about 100-fold to about 1000-fold, or about 100-fold to about 500-fold less cytokine in an assay comprising T cells, wherein each said antibody construct is incubated with the T cells for 48 hours.

23. 23. The antibody construct of any one of claims 19 to 22, wherein the antibody construct induces about 5-fold to about 900-fold, about 5-fold to about 500-fold, or about 5-fold to about 300-fold less cytokines in an assay involving human PBMCs compared to an antibody construct in which the first scFv domain and the second scFv domain are independently linked to either the N-terminus of the Fab heavy chain or the N-terminus of the second Fc polypeptide, and wherein each of the antibody constructs is incubated with the T cells for 48 hours.

24. The antibody construct of any one of claims 22 to 23, wherein the cytokine comprises one or more of IL-2, TNFα, IFNγ, and IL-6.

25. The first scFv domain comprises, from N to C terminus: V Ｈ -V Ｌ The antibody construct according to any one of claims 1 to 24, having a domain structure of:

26. The first scFv domain comprises, from N to C terminus: V Ｌ -V Ｈ The antibody construct according to any one of claims 1 to 24, having a domain structure of:

27. The second scFv domain comprises, from N to C terminus: V Ｈ -V Ｌ The antibody construct according to any one of claims 1 to 26, having a domain structure of:

28. The second scFv domain comprises, from N to C terminus: V Ｌ -V Ｈ The antibody construct according to any one of claims 1 to 26, having a domain structure of:

29. The antibody construct of any one of claims 1 to 28, wherein the antibody construct comprises one or more linkers.

30. 30. The antibody construct of claim 29, wherein the one or more linkers are peptide linkers each comprising or consisting of an amino acid sequence of 1 to about 50, 2 to about 40, 3 to about 30, or 5 to about 25 contiguous amino acid residues in length.

31. The first scFv domain may comprise a linker ｓｃＦｖ１ The antibody construct of any one of claims 29 to 30, comprising:

32. The linker ｓｃＦｖ１ is the V Ｈ The N- or C-terminus of the domain is Ｌ 32. The antibody construct of claim 31 , comprising or consisting of an amino acid sequence linked to the C- or N-terminus of the domain, respectively, and having approximately 80%, 90%, or 100% sequence identity with the amino acid sequence set forth in SEQ ID NO:

104.

33. The second scFv domain may comprise a linker ｓｃＦｖ２ The antibody construct of any one of claims 29 to 32, comprising:

34. The linker ｓｃＦｖ２ is the V Ｈ The N- or C-terminus of the domain is Ｌ 34. The antibody construct of claim 33, comprising or consisting of an amino acid sequence linked to the C- or N-terminus of the domain, respectively, and having approximately 80%, 90%, or 100% sequence identity with the amino acid sequence set forth in SEQ ID NO:

104.

35. The Fab domain capable of binding to the first antigen on the first cytotoxic effector cell comprises a heavy chain constant domain (C) comprising or consisting of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

107. Ｈ１ 35. The antibody construct of claim 1, comprising:

36. The antibody construct has a dissociation constant (K) for CD28 of about 10 nM to about 500 nM, about 20 nM to about 600 nM, about 20 nM to about 250 nM, about 20 nM to about 150 nM, about 20 nM to about 100 nM, or about 20 nM to about 50 nM, as measured using SPR. Ｄ 36. The antibody construct of any one of claims 1 to 35, which is capable of binding to human CD28 at the C3 domain.

37. The antibody construct comprises the sequence SX １ HCDR1 with GVH (SEQ ID NO: 302), sequence VIWX ２ GGX ３ HCDR2 with TNFNSALMS (SEQ ID NO: 306), and sequence DRAX ４ GX ５ YX ６ X ７ Anti-CD28 V comprising HCDR3 with AMDY (SEQ ID NO: 312) Ｈ The sequence, as well as the sequence RASESVEYYX ８ LCDR1 with TSLMQ (SEQ ID NO: 315), sequence AASX ９ VX １０ anti-CD28 V comprising an LCDR2 having the sequence QQSRKVPFT (SEQ ID NO: 319), and an LCDR3 having the sequence QQSRKVPFT (SEQ ID NO: 320); Ｌ comprising the sequence X １ = Y or A; X ２ = P or A; X ３ = G or S; X ４ = S or Y; X ５ = N or A; X ６ = L or N; X ７ = S or Y; X ８ = G or V; X ９ = N or A; and X １０ 37. The antibody construct of any one of claims 1 to 36, wherein: =E or D.

38. The antibody construct may comprise an anti-CD28 V antibody having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

106. Ｈ and anti-CD28 V having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

116. Ｌ 38. The antibody construct of any one of claims 1 to 37, comprising the sequence:

39. The anti-CD28 V Ｈ The sequence comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 201, 203-208, and 210, Ｌ 39. The antibody construct of claim 38, wherein the sequence comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 200, 202, and 209.

40. The antibody construct comprises an anti-CD28 V antibody comprising an HCDR1 having the sequence SYGVH (SEQ ID NO: 300), an HCDR2 having the sequence VIWPGGGTNFNSALMS (SEQ ID NO: 303), and an HCDR3 having the sequence DRAYGNYLYAMDY (SEQ ID NO: 307). Ｈ and an LCDR1 having the sequence RASESVEYYVTSLMQ (SEQ ID NO: 313), an LCDR2 having the sequence AASNVDS (SEQ ID NO: 316), and an LCDR3 having the sequence QQSRKVPFT (SEQ ID NO: 320). Ｌ 40. The antibody construct of any one of claims 1 to 39, comprising the sequence:

41. The antibody constructs have a dissociation constant (K) for CD3 of about 20 nM to about 200 nM, about 30 nM to about 150 nM, about 40 nM to about 100 nM, or 50 nM to about 80 nM, as measured using SPR. Ｄ 41. The antibody construct of any one of claims 1 to 40, which is capable of binding to human CD3 at the C3 domain.

42. The antibody construct comprises an anti-CD3 V antibody comprising HCDR1-HCDR3 sequences set forth in SEQ ID NOs: 321-323, respectively. Ｈ and anti-CD3 V comprising LCDR1-3 sequences shown in SEQ ID NOs: 324-326, respectively. Ｌ 42. The antibody construct of any one of claims 1 to 41, comprising the sequence:

43. The antibody construct comprises an anti-CD3 V antibody comprising an HCDR1 having the sequence GVTFNYYG (SEQ ID NO: 321), an HCDR2 having the sequence ITSSGGRI (SEQ ID NO: 322), and an HCDR3 having the sequence TLDGRDGWVAY (SEQ ID NO: 323). Ｈ and an anti-CD3 V comprising an LCDR1 having the sequence TGNIGSNY (SEQ ID NO: 324), an LCDR2 having the sequence RND (SEQ ID NO: 325), and an LCDR3 having the sequence QSYSSGFI (SEQ ID NO: 326). Ｌ 43. The antibody construct of any one of claims 1 to 42, comprising the sequence:

44. The antibody construct may comprise an anti-CD3 V antibody having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

102. Ｈ and an anti-CD3 V having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

103. Ｌ 44. The antibody construct of any one of claims 1 to 43, comprising the sequence:

45. The antibody construct of any one of claims 1 to 44, wherein the first cytotoxic effector cell and the second cytotoxic effector cell are different cells.

46. The antibody construct of any one of claims 1 to 44, wherein the first cytotoxic effector cell and the second cytotoxic effector cell are the same cell.

47. The antibody construct of any one of claims 1 to 46, wherein the first and second antigens are present on a T cell.

48. The antibody construct of any one of claims 1 to 47, wherein the TAA is Cldn18.

2.

49. The antibody construct comprises an anti-Cldn18.2 V antibody comprising HCDR1-HCDR3 sequences set forth in SEQ ID NOs: 333-335, respectively. Ｈ and anti-Cldn18.2 V comprising LCDR1-3 sequences set forth in SEQ ID NOs: 336-338, respectively. Ｌ 49. The antibody construct of claim 48, comprising the sequence:

50. The antibody construct comprises an anti-Cldn18.2 V antibody construct comprising an HCDR1 having the sequence SNPMI (SEQ ID NO: 333), an HCDR2 having the sequence IIDTDGSTYYADWAKG (SEQ ID NO: 334), and an HCDR3 having the sequence RLHGSSNGYYDDL (SEQ ID NO: 335). Ｈ and an anti-Cldn18.2 V antibody comprising an LCDR1 having the sequence QASQSIYSYLS (SEQ ID NO: 336), an LCDR2 having the sequence KASTLAS (SEQ ID NO: 337), and an LCDR3 having the sequence QQGYTVTNVDKNT (SEQ ID NO: 338). Ｌ 50. The antibody construct of claim 49, comprising the sequence:

51. The anti-Cldn18.2 V Ｈ The sequence comprises or consists of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 127, and the anti-Cldn18.2 V Ｌ 51. The antibody construct of any one of claims 48-50, wherein the sequence comprises or consists of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 128, and optionally wherein the antibody construct binds to human Cldn18.2 with an affinity that is about 1 nM to about 80 nM, about 10 nM to about 60 nM, about 10 nM to about 50 nM, or about 20 nM to about 50 nM, as measured by flow cytometry.

52. 52. The antibody construct of any one of claims 1 to 51, wherein the first Fc polypeptide and the second Fc polypeptide of the Fc domain comprise or consist of a CH2 sequence and a CH3 sequence, respectively.

53. 53. The antibody construct of claim 52, wherein at least one of the CH2 sequences of the first and second Fc polypeptides is an IgG1 or IgG4 CH2 sequence and comprises one or more amino acid modifications when compared to an unmodified wild-type IgG1 or IgG4 CH2 sequence.

54. 54. The antibody construct of claim 53, wherein the CH2 sequences of both the first and second Fc polypeptides are IgG1 or IgG4 CH2 sequences and comprise one or more amino acid modifications when compared to an unmodified wild-type IgG1 or IgG4 CH2 sequence.

55. 55. The antibody construct of claims 53 to 54, wherein the one or more amino acid modifications to the CH2 sequence reduce or eliminate interaction of the Fc domain with one or more Fc receptors, optionally one or more Fcγ receptors.

56. 56. The antibody construct of any one of claims 52 to 55, wherein the CH2 sequences of both the first and second Fc polypeptides comprise or consist of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

109.

57. 57. The antibody construct of any one of claims 52-56, wherein at least one of the CH3 sequences of the first and second Fc polypeptides is an IgG1 or IgG4 CH3 sequence and comprises one or more amino acid modifications when compared to an unmodified wild-type IgG1 or IgG4 CH3 sequence, and optionally the first and second Fc polypeptides have different amino acid sequences and form a heterodimeric Fc domain.

58. 58. The antibody construct of claim 57, wherein the CH3 sequences of both the first and second Fc polypeptides are IgG1 or IgG4 CH3 sequences and comprise one or more amino acid modifications that promote preferential pairing of the first and second Fc polypeptides to form the heterodimeric Fc domain compared to formation of the corresponding homodimeric Fc domains.

59. the CH3 sequence of one of the Fc polypeptides comprises a set of amino acid substitutions selected from the group consisting of L351Y_F405A_Y407V, T350V_L351Y_F405A_Y407V and T350V_L351Y_S400E_F405A_Y407V, and the CH3 sequence of the other Fc polypeptide comprises a set of amino acid substitutions selected from the group consisting of T366L_K392M_T394W, T366L_K392L_T3 59. The antibody construct of any one of claims 57 to 58, comprising a set of amino acid substitutions selected from the group consisting of T350V_T366L_K392L_T394W, T350V_T366L_K392M_T394W, T350V_T366L_K392M_T394W and T350V_T366L_N390R_K392M_T394W, wherein the numbering of amino acid residues in the Fc polypeptide is according to the EU numbering system.

60. 60. The antibody construct of any one of claims 52 to 59, wherein the CH3 sequence of one Fc polypeptide comprises or consists of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

110.

61. 61. The antibody construct of any one of claims 52 to 60, wherein the CH3 sequence of the other Fc polypeptide comprises or consists of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

114.

62. The antibody construct of any one of claims 1 to 61, wherein the antibody construct is trivalent and trispecific, binding monovalently to each antigen.

63. 63. The antibody construct of any one of claims 1 to 62, wherein the antibody construct has a melting temperature at Tm1, Tm2, ​​and / or Tm3 that is within 10°C, within 5°C, within 2°C, or within 1°C of that of a corresponding bivalent and monospecific IgG1 monoclonal antibody.

64. 64. The antibody construct of any one of claims 1 to 63, wherein the antibody construct binds to the cytotoxic effector cells expressing CD3 and / or CD28 with about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or about 200-fold higher affinity than that of the corresponding bivalent and bispecific anti-CD3xTAA and / or anti-CD28xTAA antibody construct.

65. The antibody construct exhibits an IC of about 50 pM to about 0.01 pM, about 25 pM to about 0.01 pM, about 10 pM to about 0.05 pM, about 10 pM to about 0.1 pM, about 10 pM to about 1 pM, or about 5 pM to about 1 pM for killing TAA-expressing tumor cells expressing at least about 100,000 TAAs / cell by TDCC in the presence of the cytotoxic effector cells using an E:T ratio of 2:1 and an incubation period of 72 hours. ５０ 65. The antibody construct of any one of claims 1 to 64, wherein the antibody construct exhibits a value.

66. 66. The antibody construct of claim 65, wherein the antibody construct achieves maximal killing of at least about 60%, 65%, 70%, 75%, or 80%, 85%, or 90%, 100%, or about 60% to about 100%, about 70% to about 90%, or about 75% to about 85% of TAA-expressing tumor cells.

67. 67. The antibody construct of any one of claims 1 to 66, wherein the antibody construct is capable of inducing production of one or more cytokines by the cytotoxic effector cells in the range of about 300 pg / mL to about 9000 pg / mL in the presence of TAA-expressing cells expressing at least about 100,000 TAA / cell and using an E:T ratio of 2:1 and an incubation period of 72 hours.

68. 68. A pharmaceutical composition comprising the antibody construct of any one of claims 1 to 67 and a pharmaceutically acceptable carrier, excipient, diluent, or combination thereof.

69. A nucleic acid molecule or set of nucleic acid molecules encoding one or more, two or more, or three or more polypeptide chains forming the antibody construct of any one of claims 1 to 67.

70. 70. A vector or set of vectors comprising the nucleic acid molecule or set of nucleic acid molecules of claim 69.

71. 71. A cell comprising the nucleic acid molecule or set of nucleic acid molecules of claim 69 or the vector or set of vectors of claim 70.

72. 68. A method for producing an antibody construct according to any one of claims 1 to 67, comprising the steps of: (a) obtaining a host cell culture comprising at least one host cell containing one or more nucleic acid molecules encoding one or more, two or more, or three or more polypeptide chains forming said antibody construct; and (b) recovering the antibody construct from the host cell culture. The method comprising:

73. 73. The method of claim 72, further comprising purifying the antibody construct after step (b).

74. 100. A method for inducing an anti-tumor immune response in a cell population comprising immune cells and tumor cells, the method comprising contacting the cell population with an effective amount of an antibody construct of any one of claims 1 to 67, wherein the immune cells express the first and second antigens and the tumor cells express the TAA.

75. 68. A method for inhibiting tumor cell proliferation, comprising contacting a cell population comprising the tumor cells and immune cells with an effective amount of the antibody construct of any one of claims 1 to 67, wherein the immune cells express the first and second antigens and the tumor cells express the TAA.

76. 68. A method for killing tumor cells, comprising contacting a cell population comprising the tumor cells and immune cells with an effective amount of an antibody construct of any one of claims 1 to 67, wherein the immune cells express the first and second antigens and the tumor cells express the TAA.

77. 77. The method of any one of claims 74 to 76, wherein the immune cells comprise T cells.

78. 78. The method of any one of claims 74 to 77, wherein the TAA is Cldn18.

2.

79. 79. The method of any one of claims 74 to 78, wherein the antibody construct binds to CD3 and CD28 on one T cell or on two different T cells and to the TAA on a tumor cell.

80. 80. The method of claim 79, wherein the binding of the antibody construct to the first and second antigens and the TAA forms a TCR-independent artificial immune synapse between the one or more immune cells and the tumor cell, thereby eliciting a cytotoxic immune response of the immune cell against the tumor cell.

81. 81. The method of any one of claims 74 to 80, wherein the cell population is in a subject.

82. 68. A method for treating cancer in a subject in need thereof, comprising administering to the subject an antibody construct of any one of claims 1 to 67.

83. 83. The method of claim 82, wherein a cytotoxic immune response against the cancer is elicited in the subject, thereby treating the cancer in the subject.

84. 68. An antibody construct according to any one of claims 1 to 67 for use in the treatment of cancer.

85. 68. Use of an antibody construct according to any one of claims 1 to 67 in the manufacture of a medicament for the treatment of cancer.

86. 1. An antibody construct comprising a binding domain capable of binding to CD28, said binding domain having the sequence SX １ HCDR1 with GVH (SEQ ID NO: 302), sequence VIWX ２ GGX ３ HCDR2 with TNFNSALMS (SEQ ID NO: 306), and sequence DRAX ４ GX ５ YX ６ X ７ V comprising HCDR3 with AMDY (SEQ ID NO: 312) Ｈ The sequence, as well as the sequence RASESVEYYX ８ LCDR1 with TSLMQ (SEQ ID NO: 315), sequence AASX ９ VX １０ V comprising an LCDR2 having the sequence QQSRKVPFT (SEQ ID NO: 319), and an LCDR3 having the sequence QQSRKVPFT (SEQ ID NO: 320). Ｌ The antibody construct comprising the sequence and having one or more of the following amino acid substitutions at the positions specified in the CDR sequences: X １ : Y to A, X ２ : P to A, X ３ : G to S, X ４ : S to Y, X ５ : N to A, X ６ : L to N, X ７ : S to Y, X ８ : G to V, X ９ : N to A and / or X １０ :E to D.

87. The binding domain comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 201, 203-208, and 210. Ｈ and V comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 200, 202, and 209. Ｌ 87. The antibody construct of claim 86, comprising the sequence:

88. An antibody construct comprising a binding domain capable of binding to Cldn18.2, the binding domain comprising an HCDR1 having the sequence SNPMI (SEQ ID NO: 333), an HCDR2 having the sequence IIDTDGSTYYADWAKG (SEQ ID NO: 334), and an HCDR3 having the sequence RLHGSSNGYYDDL (SEQ ID NO: 335). Ｈ and a V comprising an LCDR1 having the sequence QASQSIYSYLS (SEQ ID NO: 336), an LCDR2 having the sequence KASTLAS (SEQ ID NO: 337), and an LCDR3 having the sequence QQGYTVTNVDKNT (SEQ ID NO: 338). Ｌ The antibody construct comprising the sequence:

89. The binding domain may comprise or consist of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

127. Ｈ and V comprising or consisting of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

128. Ｌ 89. The antibody construct of claim 88, comprising the sequence:

90. 90. The antibody construct of any one of claims 86 to 89, further comprising one or more additional binding domains capable of binding to one or more additional antigens.

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