Protein complexes and methods of use thereof

Abstract

The present disclosure relates to a novel platform for the design of multi-specific antibodies, incorporating innovative splits of antibodies, cytokines, or other proteins at various positions within a single antibody framework. This design aims to reduce toxicity and enhance therapeutic efficacy, particularly in applications such as immunotherapy and antibody-drug conjugates (ADCs). Multi-specific antibodies—including monospecific, bispecific, and multi-specific forms—comprise one or more antigen-binding domains that engage one or more epitopes of the same or different antigens. This disclosure encompasses methods for producing antibodies with one, two, or multi antigen-binding domains, which may consist of immunoglobulin heavy chain variable domains alone or in combination with other functional domains.

Description

[0001] PROTEIN COMPLEXES AND METHODS OF USE THEREOF

[0002] CLAIM OF PRIORITY

[0003] This application claims priority' to U. S. Provisional Application No. 63 / 714,514, filed on October 31. 2024 and U. S. Provisional Application No. 63 / 794,589. filed on April 25, 2025. The entire contents of the foregoing applications are incorporated herein by reference in their entireties.

[0004] TECHNICAL FIELD

[0005] The present disclosure relates to a novel platform for the design of multi-specific antibodies, incorporating innovative splits of antibodies, cytokines, or other proteins at various positions within a single antibody framework. This disclosure also encompasses methods for producing antibodies with one, two, or multiple antigen-binding domains, which may consist of immunoglobulin heavy chain variable domains alone or in combination with other functional domains.

[0006] BACKGROUND

[0007] Monoclonal antibody therapeutics harness the body’s immune system to target and eliminate diseases, particularly cancer. Traditional monoclonal antibodies typically exhibit monovalent affinity, binding exclusively to a single epitope. In contrast, multi-specific antibodies can be engineered to broaden the therapeutic scope of a single monoclonal antibody by incorporating multiple binding domains. For example, bispecific antibodies (BsAbs), which feature two binding sites directed against distinct target antigens, enable simultaneous binding and dual functionality.

[0008] The advantages of multi-specific antibodies extend beyond additive effects, including enhanced T cell engagement, checkpoint inhibition, and improved tumor penetration.

[0009] Simultaneous targeting of multiple antigens offers a therapeutic benefit that surpasses that of traditional monoclonal antibodies. Numerous bispecific and multi-specific antibody technology platforms have been developed, such as the tandem diabody (U. S. Patent Application No. 20030060354A1) and the hexavalent antibody formats (U. S. Patent No. 8828985B2). However, the preparation of multi-specific antibodies poses significant challenges due to potential mismatches between heavy and light chains, leading to difficulties in the purification and characterization of target products, ultimately resulting in low industrial efficiency and the presence of impurities. Monoclonal antibodies targeting cancer represent one of the fastest-growing fields in modem drug therapy. With hundreds of therapeutic antibodies and derivatives currently in preclinical and clinical studies, some approaches focus on retargeting cytotoxic T lymphocytes to malignant cells. Notable examples include chimeric antigen receptors (CARs) and bispecific T cell-engaging antibodies (BiTEs), both utilizing monospecific single-chain variable fragments (ScFvs) as targeting devices. These derivatives primarily target differentiation antigens present on malignant cells and their non-transformed counterparts, which can lead to serious adverse events due to cross-reactivity.

[0010] Despite the advancements in bispecific and multi-specific antibody platforms, designing formats that achieve a favorable therapeutic window characterized by strong efficacy and minimal toxicity remains a significant challenge. The rarity of true tumorspecific antigens suitable for antibody -based therapies complicates this landscape. Thus, there is a need to develop new formats of therapeutic antibodies with strong efficacy and minimal toxicity.

[0011] SUMMARY

[0012] Antibodies that target high-expression antigens may demonstrate significant efficacy but also incur substantial on-target, off-tumor toxicity. Conversely, those targeting low-expression antigens often exhibit improved tumor specificity and reduced toxicity but lack sufficient efficacy for effective treatment. The ideal therapeutic antibody should strike a balance between efficacy and safety, providing a wide therapeutic window. Innovative approaches are required to engineer antibodies with enhanced specificity and reduced off-target effects. Recent advancements in antibody engineering, such as those described in U. S. Patent No. 10100229B2, detail the use of modular antibody formats to improve specificity and affinity, highlighting the need for continued exploration of new multi-specific antibody platforms.

[0013] In light of these challenges, the present disclosure addresses the need for an innovative platform that facilitates the synthesis and engineering of multi-specific antibodies with optimized therapeutic profiles. By utilizing advanced protein engineering techniques and novel split designs, this platform aims to create antibodies that are not only effective in targeting tumor cells but also possess favorable safety profiles. This disclosure has the potential to significantly advance the field of antibody therapeutics, providing new options for patients with challenging malignancies. The present disclosure relates to novel multi-specific antibodies comprising split building blocks, such as antibodies, cytokines, or other proteins, designed at various positions within a single antibody to reduce toxicity and enhance efficacy. The disclosure also pertains to the use of these antibodies for therapeutic applications, including immunotherapy and antibody-drug conjugates (ADCs). In some embodiments, the split building blocks and the building blocks can include VH, VL, cytokine, VHH, ScFv, Fab, diabody, mini-protein, protein, cytokine splits, split of proteins, or combinations of these components. In some embodiments, the split building blocks are designed at various positions (as shown in FIGS.

[0014] 1-3) within a single antibody. A single antibody can encompass all building blocks and the Fc region within the same antibody molecule. The split building blocks can also be designed at different positions across multiple antibodies for combination treatment.

[0015] In one aspect, the disclosure is related to a protein complex comprising a first split building block, a second split building block, and an Fc region, wherein the first and second split building blocks are located at any two of the positions 1-4 in FIG. 1, any two of the positions 1-6 in FIG. 2, or any two of the positions 1-8 in FIG. 3. In some embodiments, the first split building block is a heavy chain variable region (VH) and the second split building block is a light chain variable region (VL). In some embodiments, the first split building block is a VL and the second split building block is a VH. In some embodiments, the first and second split building blocks are two portions of a cytokine or protein (e.g., any of the cytokines and protein described herein).

[0016] As shown in FIG. 1, the protein complex described herein may include an Fc region. In some embodiments, the first split building block can be located at position 1 and the second split building block can be located at position 2; the first split building block can be located at position 1 and the second split building block can be located at position 3; the first split building block can be located at position 1 and the second split building block can be located at position 4; the first split building block can be located at position 2 and the second split building block can be located at position 1; the first split building block can be located at position 2 and the second split building block can be located at position 3; the first split building block can be located at position 2 and the second split building block can be located at position 4; the first split building block can be located at position 3 and the second split building block can be located at position 1; the first split building block can be located at position 3 and the second split building block can be located at position 2; the first split building block can be located at position 3 and the second split building block can be located at position 4; the first split building block can be located at position 4 and the second split building block can be located at position 1; the first split building block can be located at position 4 and the second split building block can be located at position 2; or the first split building block can be located at position 4 and the second split building block can be located at position 3.

[0017] As shown in FIG. 2, the protein complex described herein may include a Fab region that is connected to an Fc region. In some embodiments, the first split building block can be located at position 1 and the second split building block can be located at position 2; the first split building block can be located at position 1 and the second split building block can be located at position 3; the first split building block can be located at position 1 and the second split building block can be located at position 4; the first split building block can be located at position 1 and the second split building block can be located at position 5; the first split building block can be located at position 1 and the second split building block can be located at position 6; the first split building block can be located at position 2 and the second split building block can be located at position 1; the first split building block can be located at position 2 and the second split building block can be located at position 3; the first split building block can be located at position 2 and the second split building block can be located at position 4; the first split building block can be located at position 2 and the second split building block can be located at position 5; the first split building block can be located at position 2 and the second split building block can be located at position 6; the first split building block can be located at position 3 and the second split building block can be located at position 1; the first split building block can be located at position 3 and the second split building block can be located at position 2; the first split building block can be located at position 3 and the second split building block can be located at position 4; the first split building block can be located at position 3 and the second split building block can be located at position 5; the first split building block can be located at position 3 and the second split building block can be located at position 6; the first split building block can be located at position 4 and the second split building block can be located at position 1; the first split building block can be located at position 4 and the second split building block can be located at position 2; the first split building block can be located at position 4 and the second split building block can be located at position 3; the first split building block can be located at position 4 and the second split building block can be located at position 5; the first split building block can be located at position 4 and the second split building block can be located at position 6 the first split building block can be located at position 5 and the second split building block can be located at position 1; the first split building block can be located at position 5 and the second split building block can be located at position 2; the first split building block can be located at position 5 and the second split building block can be located at position 3; the first split building block can be located at position 5 and the second split building block can be located at position 4; the first split building block can be located at position 5 and the second split building block can be located at position 6; the first split building block can be located at position 6 and the second split building block can be located at position 1; the first split building block can be located at position 6 and the second split building block can be located at position 2; the first split building block can be located at position 6 and the second split building block can be located at position 3; the first split building block can be located at position 6 and the second split building block can be located at position 4; or the first split building block can be located at position 6 and the second split building block can be located at position 5.

[0018] As shown in FIG. 3, the protein complex described herein may include two Fab regions that are connected to an Fc region. In some embodiments, the first split building block can be located at position 1 and the second split building block can be located at position 2; the first split building block can be located at position 1 and the second split building block can be located at position 3; the first split building block can be located at position 1 and the second split building block can be located at position 4; the first split building block can be located at position 1 and the second split building block can be located at position 5; the first split building block can be located at position 1 and the second split building block can be located at position 6; the first split building block can be located at position 1 and the second split building block can be located at position 7; the first split building block can be located at position 1 and the second split building block can be located at position 8; the first split building block can be located at position 2 and the second split building block can be located at position 1; the first split building block can be located at position 2 and the second split building block can be located at position 3; the first split building block can be located at position 2 and the second split building block can be located at position 4; the first split building block can be located at position 2 and the second split building block can be located at position 5; the first split building block can be located at position 2 and the second split building block can be located at position 6; the first split building block can be located at position 2 and the second split building block can be located at position 7; the first split building block can be located at position 2 and the second split building block can be located at position 8; the first split building block can be located at position 3 and the second split building block can be located at position 1; the first split building block can be located at position 3 and the second split building block can be located at position 2; the first split building block can be located at position 3 and the second split building block can be located at position 4; the first split building block can be located at position 3 and the second split building block can be located at position 5; the first split building block can be located at position 3 and the second split building block can be located at position 6; the first split building block can be located at position 3 and the second split building block can be located at position 7; the first split building block can be located at position 3 and the second split building block can be located at position 8; the first split building block can be located at position 4 and the second split building block can be located at position 1; the first split building block can be located at position 4 and the second split building block can be located at position 2; the first split building block can be located at position 4 and the second split building block can be located at position 3; the first split building block can be located at position 4 and the second split building block can be located at position 5; the first split building block can be located at position 4 and the second split building block can be located at position 6; the first split building block can be located at position 4 and the second split building block can be located at position 7; the first split building block can be located at position 4 and the second split building block can be located at position 8; the first split building block can be located at position 5 and the second split building block can be located at position 1; the first split building block can be located at position 5 and the second split building block can be located at position 2; the first split building block can be located at position 5 and the second split building block can be located at position 3; the first split building block can be located at position 5 and the second split building block can be located at position 4; the first split building block can be located at position 5 and the second split building block can be located at position 6; the first split building block can be located at position 5 and the second split building block can be located at position 7; the first split building block can be located at position 5 and the second split building block can be located at position 8; the first split building block can be located at position 6 and the second split building block can be located at position 1; the first split building block can be located at position 6 and the second split building block can be located at position 2; the first split building block can be located at position 6 and the second split building block can be located at position 3; the first split building block can be located at position 6 and the second split building block can be located at position 4; the first split building block can be located at position 6 and the second split building block can be located at position 5; the first split building block can be located at position 6 and the second split building block can be located at position 7; the first split building block can be located at position 6 and the second split building block can be located at position 8; the first split building block can be located at position 7 and the second split building block can be located at position 1; the first split building block can be located at position 7 and the second split building block can be located at position 2; the first split building block can be located at position 7 and the second split building block can be located at position 3; the first split building block can be located at position 7 and the second split building block can be located at position 4; the first split building block can be located at position 7 and the second split building block can be located at position 5; the first split building block can be located at position 7 and the second split building block can be located at position 6; the first split building block can be located at position 7 and the second split building block can be located at position 8; the first split building block can be located at position 8 and the second split building block can be located at position 1; the first split building block can be located at position 8 and the second split building block can be located at position 2; the first split building block can be located at position 8 and the second split building block can be located at position 3; the first split building block can be located at position 8 and the second split building block can be located at position 4; the first split building block can be located at position 8 and the second split building block can be located at position 5; the first split building block can be located at position 8 and the second split building block can be located at position 6 or the first split building block can be located at position 8 and the second split building block can be located at position 7.

[0019] In some embodiments, the first and second split building blocks are selected from a Fab region, an ScFv, a VHH, a VH or VL, a cytokine, a split of cytokine, a protein, and / or a split of protein. The estimated molecular weight (MW) can be found in FIGS. 1-3. In some embodiments, the interaction of the first and second split building blocks is impeded due to folding constraints.

[0020] In some embodiments, the split building blocks (e.g., any of the VH / VL pairs described herein that can form an antigen-binding domain but not in a format of a Fab or an ScFv) may bind to each other within the same protein complex (via intra-molecular interactions), thereby generating a functional VH-VL domain. In some embodiments, a VH in a first protein complex may bind to a VL in a second protein complex (via inter-molecular interactions), forming an antigen-binding domain, thereby generating a functional VH-VL domain. In some embodiments, the first and second protein complexes can be any of the protein complexes described herein. In some embodiments, the first and second protein complexes are the same. In some embodiments, the first and second protein complexes are different.

[0021] In one aspect, the disclosure is related to a protein complex, comprising a first polypeptide comprising a heavy chain constant domain 1 (CHI) and a second polypeptide comprising a light chain constant domain (CL), in some embodiments: (i) the first polypeptide does not comprise a heavy chain variable region (VH) at the N-terminus of the CHI, when the second polypeptide comprises a light chain variable region (VL) at the N-terminus of the CL; or (ii) the second polypeptide does not comprise a VL at the N-terminus of the CL, when the first polypeptide comprises a VH at the N-terminus of the CHI.

[0022] In one aspect, the disclosure is related to a protein complex, comprising a first heavy chain variable region (VH1) and a first light chain variable region (VL1) that together form a first antigen-binding domain, wherein the first antigen-binding domain is not a Fab region or an ScFv. In some embodiments, the protein complex further comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2) that together form a second antigen-binding domain, in some embodiments: (i) the second antigen-binding domain is a Fab region or an ScFv; or (ii) the second antigen-binding domain is not a Fab region or an ScFv. In some embodiments, the protein complex further comprises a third heavy chain variable region (VH3) and a third light chain variable region (VL3) that together form a third antigen-binding domain, in some embodiments: (i) the third antigen-binding domain is a Fab region or an ScFv; or (ii) the third antigen-binding domain is not a Fab region or an ScFv.

[0023] In one aspect, the present disclosure provides a protein complex utilizing a Crossbody platform for the construction of bispecific antibodies. This platform incorporates innovative splits of antibody domains designed at various positions within a single antibody framework to reduce toxicity. The protein complex may comprise: (i) a first polypeptide comprising a first light chain variable region (VL1), a light chain constant domain (CL), and a second heavy chain variable region (VH2); (ii) a second polypeptide comprising a first heavy chain variable region (VH1). a heavy chain constant domain 1 (CHI), a first hinge region, a first heavy chain constant domain 2 (CH2) and a first heavy chain constant domain 3 (CH3); (lii) a third polypeptide comprising a second light chain variable region (VL2), a second hinge region, a second CH2 and a second CH3. In some embodiments, the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target. In some embodiments, the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to a second target. In some embodiments, the VL1 and VH1 are covalently linked via a disulfide bond. In some embodiments, the VL1 and VH1 are not covalently linked via a disulfide bond. In some embodiments, the first and second polypeptides are covalently linked via a disulfide bond between the CL and CHI. In some embodiments, the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions.

[0024] In a further aspect, the present disclosure provides a protein complex employing the Crossbody platform to construct bispecific antibodies with splits designed at various positions within a single antibody. The protein complex may comprise: (i) a first polypeptide comprising a first light chain variable region (VL1) and a first CL; (ii) a second polypeptide comprising a first heavy chain variable region (VH1), a first CHI, a first hinge region, a first CH2. a first CH3, and a second heavy chain variable region (VH2); (iii) a third polypeptide comprising a third heavy chain variable region (VH3), a second CHI, a second hinge region, a second CH2, a second CH3, and a second light chain variable region (VL2); (iv) a fourth polypeptide comprising a third light chain variable region (VL3) and a second CL. In some embodiments, the VL1 and VH1 associate to form a first antigen-domain capable of binding to a first target. In some embodiments, the VL2 and VH2 associate to form a second antigenbinding domain capable of binding to a second target. In some embodiments, the VL3 and VH3 associate to form a third antigen-domain capable of binding to the first target, optionally the VL3 is identical to the VL1, and the VH3 is identical to the VH1. In some embodiments, the VL1 and VH1 are covalently linked via a disulfide bond. In some embodiments, the VL1 and VH1 are not covalently linked via a disulfide bond. In some embodiments, the VL2 and VH2 are covalently linked via a disulfide bond. In some embodiments, the VL2 and VH2 are not covalently linked via a disulfide bond. In some embodiments, the VL3 and VH3 are covalently linked via a disulfide bond. In some embodiments, the VL3 and VH3 are not covalently linked via a disulfide bond. In some embodiments, the first and second polypeptides are covalently linked via a disulfide bond between the first CL and the first CHI. In some embodiments, the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions. In some embodiments, the third and fourth polypeptides are covalently linked via a disulfide bond between the second CHI and the second CL.

[0025] In a further aspect, the present disclosure provides a protein complex employing the Crossbody platform to construct bispecific antibodies with splits designed at various positions within a single antibody. The protein complex may comprise: (i) a first polypeptide comprising a first light chain variable region (VL1), a CL, and a second heavy chain variable region (VH2); (ii) a second polypeptide comprising a first heavy chain variable region (VH1), a CHI, a first hinge region, a first CH2, a first CH3; (iii) a third polypeptide comprising a third heavy chain variable region (VH3), a T cell receptor variable alpha region (TCR Va), a second hinge region, a second CH2, a second CH3; (iv) a fourth polypeptide comprising a third light chain variable region (VL3) and a T cell receptor variable beta region (TCR Vb), and a second light chain variable region (VL2). In some embodiments, the VL1 and VH1 associate to form a first antigen-domain capable of binding to a first target. In some embodiments, the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to a second target. In some embodiments, the VL3 and VH3 associate to form a third antigen-domain capable of binding to the first target, optionally the VL3 is identical to the VL1, and the VH3 is identical to the VH1. In some embodiments, the VL1 and VH1 are covalently linked via a disulfide bond. In some embodiments, the VL1 and VH1 are not covalently linked via a disulfide bond. In some embodiments, the VL2 and VH2 are covalently linked via a disulfide bond. In some embodiments, the VL2 and VH2 are not covalently linked via a disulfide bond. In some embodiments, the VL3 and VH3 are covalently linked via a disulfide bond. In some embodiments, the VL3 and VH3 are not covalently linked via a disulfide bond. In some embodiments, the first and second polypeptides are covalently linked via a disulfide bond between the CL and the CHI. In some embodiments, the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions. In some embodiments, the third and fourth polypeptides are covalently linked via a disulfide bond between the TCR Va and the TCR Vb.

[0026] In some embodiments, the positioning of the splits in the second target antibody (VH2 and VL2) can vary within the protein complex (e.g., any of the positions 1-8 in FIG. 3), allowing for flexibility in the arrangement of VH2 and VL2 in the first, second, third, and / or fourth polypeptides to construct novel formats.

[0027] In some embodiments, the number of polypeptides can be adjusted to create new formats, offering versatility in the design of the protein complex.

[0028] In some embodiments, additional disulfide bonds can be introduced at various positions within the first and second polypeptides to construct new formats and enhance the stability of the protein complex. In some embodiments, additional disulfide bonds can be introduced at various positions within the third and fourth polypeptides to construct new formats and enhance the stability of the protein complex. In one aspect, the present disclosure further provides a protein complex using the Crossbody platform to construct trispecific antibodies with splits designed at various positions within a single antibody. The protein complex may comprise: (i) a first polypeptide comprising a first light chain variable region (VL1), a CL, and a second heavy chain variable region (VH2); (ii) a second polypeptide comprising a first heavy chain variable region (VH1), a CHI, a first hinge region, a first CH2 and a first CH3; (iii) a third polypeptide comprising a single-chain variable fragment (ScFv), a second light chain variable region (VL2), a second hinge region, a second CH2 and a second CH3. In some embodiments, the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target. In some embodiments, the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to a second target. In some embodiments, the ScFv is capable of binding to a third target. In some embodiments, the VL1 and VH1 are covalently linked via a disulfide bond. In some embodiments, the VL1 and VH1 are not covalently linked via a disulfide bond. In some embodiments, the first and second polypeptides are covalently linked via a disulfide bond between the CL and CHI. In some embodiments, the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions.

[0029] In some embodiments, the first target may be the same as the third target.

[0030] Additionally, new formats can be constructed by introducing additional disulfide bonds at various positions in the first and second polypeptides to enhance stability of the protein complex. In some embodiments, the protein complex described herein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) non-native disulfide bonds between the first and second polypeptides that can enhance stability of the protein complex.

[0031] In one aspect, the present disclosure provides a protein complex utilizing the Crossbody platform to construct trispecific antibodies or mixtures of different building blocks designed at various positions within a single antibody. The protein complex may comprise: (i) a first polypeptide comprising a first light chain variable region (VL1), a CL, a second heavy chain variable region (VH2), and a third heavy chain variable region (VH3); (ii) a second polypeptide comprising a first heavy chain variable region (VH1), a CHI, a first hinge region, a first CH2, and a first CH3; (iii) a third polypeptide comprising a third light chain variable region (VL3), a second light chain variable region (VL2), a second hinge region, a second CH2, and a second CH3. In some embodiments, the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target. In some embodiments, the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to a second target. In some embodiments, the VL3 and VH3 associate to form a third antigen- binding domain capable of binding to a third target. In some embodiments, the VL1 and VH1 are covalently linked via a disulfide bond. In some embodiments, the VL1 and VH1 are not covalently linked via a disulfide bond. In some embodiments, the first and second polypeptides are covalently linked via a disulfide bond between the CL and CHI. In some embodiments, the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions.

[0032] In some embodiments, the first target is selected for its low expression level and high tumor specificity. For example, this target may direct the antibody primarily to the tumor, ensuring that the antibody is preferentially bound to tumor cells, enhancing its efficacy in targeting the cancer. In some embodiments, the second target is a tumor-associated antigen with high expression but lower tumor specificity. For example, the design of this target includes concealment strategies to minimize on -target / off-tumor toxicity and to enhance the overall potency of the multi-specific antibody; the concealment approach aims to reduce potential adverse effects while improving the therapeutic effect). In some embodiments, the third target is a T cell target. For example, the T cell target may be included with concealment strategies to minimize toxicity. In addition, this target could be either a specific T cell antigen or, wherein both the second and third targets may be T cell antigens with advanced concealment techniques to further reduce the risk of toxicity. In some embodiments, the protein complex described herein comprises one or more non-native disulfide bonds between the first and second polypeptides that can enhance stability of the protein complex. In some embodiments, the number of polypeptides can be adjusted to create new formats, offering versatility in antibody design. In some embodiments, additional disulfide bonds can be introduced at various positions within the first and second polypeptides to construct new formats and enhance the stability of the protein complex.

[0033] In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct trispecific antibodies with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The protein complex may comprise: (i) a first polypeptide comprising a first light chain variable region (VL1), a CL, a third heavy chain variable region (VH3), and a second heavy chain variable region (VH2); (ii) a second polypeptide comprising a first heavy chain variable region (VH1), a CHI, a first hinge region, a first CH2, and a first CH3; (iii) a third polypeptide comprising a third light chain variable region (VL3), a second light chain variable region (VL2). a second hinge region, a second CH2, and a second CH3. In some embodiments, the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target. In some embodiments, the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to a second target. In some embodiments, the VL3 and VH3 associate to form a third antigen-binding domain capable of binding to a third target. In some embodiments, the VL1 and VH1 are covalently linked via a disulfide bond. In some embodiments, the VL1 and VH1 are not covalently linked via a disulfide bond. In some embodiments, the first and second polypeptides are covalently linked via a disulfide bond between the CL and CHI. In some embodiments, the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions.

[0034] In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct trispecific antibodies with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The protein complex may comprise: (i) a first polypeptide comprising a second heavy chain variable region (VH2), a third heavy chain variable region (VH3), a first light chain variable region (VL1), and a CL; (ii) a second polypeptide comprising a first heavy chain variable region (VH1), a CHI, a first hinge region, a first CH2, and a first CH3; (iii) a third polypeptide comprising a third light chain variable region (VL3), a second light chain variable region (VL2), a second hinge region, a second CH2, and a second CH3. In some embodiments, the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target. In some embodiments, the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to a second target. In some embodiments, the VL3 and VH3 associate to form a third antigen-binding domain capable of binding to a third target. In some embodiments, the VL1 and VH1 are covalently linked via a disulfide bond. In some embodiments, the VL1 and VH1 are not covalently linked via a disulfide bond. In some embodiments, the first and second polypeptides are covalently linked via a disulfide bond between the CL and CHI. In some embodiments, the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions.

[0035] In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct trispecific antibodies with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The protein complex may comprise: (i) a first polypeptide comprising a first light chain variable region (VL1), a CL, and a second heavy chain variable region (VH2); (ii) a second polypeptide comprising a first heavy chain variable region (VH1), a CHI, a first hinge region, a first CH2, and a first CH3; (iii) a third polypeptide comprising a second light chain variable region (VL2), a single-chain variable fragment (ScFv), a second hinge region, a second CH2, and a second CH3. In some embodiments, the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target. In some embodiments, the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to a second target. In some embodiments, the ScFv is capable of binding to a third target. In some embodiments, the VL1 and VH1 are covalently linked via a disulfide bond. In some embodiments, the VL1 and VH1 are not covalently linked via a disulfide bond. In some embodiments, the first and second polypeptides are covalently linked via a disulfide bond between the CL and CHI. In some embodiments, the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions.

[0036] In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct trispecific antibodies with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The protein complex may comprise: (i) a first polypeptide comprising a first light chain variable region (VL1), a second heavy chain variable region (VH2), a CL, and a third heavy chain variable region (VH3); (ii) a second polypeptide comprising a second light chain variable region (VL2), a first heavy chain variable region (VH1), a CHI, a first hinge region, a first CH2, and a first CH3; (iii) a third polypeptide comprising a third light chain variable region (VL3), a second hinge region, a second CH2, and a second CH3. In some embodiments, the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target. In some embodiments, the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to a second target. In some embodiments, the VL3 and VH3 associate to form a third antigen-binding domain capable of binding to a third target. In some embodiments, the VL1 and VH1 are covalently linked via a disulfide bond. In some embodiments, the VL1 and VH1 are not covalently linked via a disulfide bond. In some embodiments, the first and second polypeptides are covalently linked via a disulfide bond between the CL and CHI. In some embodiments, the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions.

[0037] In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct tetra-specific antibodies with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The protein complex may comprise: (i) a first polypeptide comprising a second heavy chain variable region (VH2), a first light chain variable region (VL1). a first CL, and a third heavy chain variable region (VH3); (ii) a second polypeptide comprising a first heavy chain variable region (VH1), a first CHI, a first hinge region, a first CH2, and a first CH3; (iii) a third polypeptide comprising a fourth heavy chain variable region (VH4), a second CHI, a second hinge region, a second CH2. and a second CH3; (iv) a fourth polypeptide comprising a second light chain variable region (VL2), a fourth light chain variable region (VL4), a second CL, and a third light chain variable region (VL3). In some embodiments, the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target. In some embodiments, the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to a second target. In some embodiments, the VL3 and VH3 associate to form a third antigen-binding domain capable of binding to a third target. In some embodiments, the VL4 and VH4 associate to form a fourth antigen-binding domain capable of binding to a fourth target. In some embodiments, the VL1 and VH1 are covalently linked via a disulfide bond. In some embodiments, the VL1 and VH1 are not covalently linked via a disulfide bond. In some embodiments, the first and second polypeptides are covalently linked via a disulfide bond between the first CL and the first CHI. In some embodiments, the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions. In some embodiments, the third and fourth polypeptides are covalently linked via a disulfide bond between the second CHI and the second CL.

[0038] In some embodiments, the positioning of the antibody splits (e.g., VH2 and VL2; or VH3 and VL3) can vary within the protein complex (e.g., any of the positions 1-8 in FIG. 3), allowing for flexibility in the arrangement of VH2 and VL2, and / or VH3 and VL3. in the first, second, third, and / or fourth polypeptides to construct novel formats.

[0039] In some embodiments, for multi-specific antibody designs, the approach typically involves several targets: First Target: this target can be chosen for its low expression level and strong tumor specificity. It directs the antibody primarily to the tumor. Second Target: this is a tumor-associated target with high expression but lower tumor specificity. This target can be designed with concealment to minimize on-target / off-tumor toxicity and enhance the potency of the multi-specific antibody. Third Target: this target can be a T cell target and is designed with concealment to minimize toxicity. In some embodiments, both the second and third targets may be T cell targets with concealment strategies to further reduce toxicity.

[0040] In some embodiments, the number of polypeptides can be adjusted to create new formats, offering versatility in the design of the protein complex. In some embodiments, additional disulfide bonds can be introduced at various positions within the first and second polypeptides to construct new formats and enhance the stability of the antibody. In some embodiments, additional disulfide bonds can be introduced at various positions within the third and fourth polypeptides to construct new formats and enhance the stability of the antibody.

[0041] In some embodiments, the splits of the second antibody (e.g., VH2 and VL2) and third antibody (e.g., VH3 and VL3) can be a mixture of different building blocks, such as: VH, VL, ScFv, Fab, and Diabody; and the mixture of different building blocks at the positions of each antigen in the first, second, third, and / or fourth polypeptides can be changed to construct new formats.

[0042] In some embodiments, the fourth target and the first target are the same target. In some embodiments, the third target and the second target are the same target. In some embodiments, the first target and the second target are the same target. In some embodiments, the first target and the third target are the same target.

[0043] In one aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct multi-specific checkpoint antibodies with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The protein complex may comprise: (i) a first polypeptide comprising a first light chain variable region (VL1) and a first CL; (ii) a second polypeptide comprising a first heavy chain variable region (VH1), a first CHI, a first hinge region, a first CH2, a first CH3, and a second heavy chain variable region (VH2); (iii) a third polypeptide comprising a second light chain variable region (VL2), a third heavy chain variable region (VH3), a second CHI. a second hinge region, a second CH2. and a second CH3; (iv) a fourth polypeptide comprising a third light chain variable region (VL3) and a second CL. In some embodiments, the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target. In some embodiments, the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to a second target. In some embodiments, the VL3 and VH3 associate to form a third antigen-domain capable of binding to the first target, optionally the VL3 is identical to the VL1, and the VH3 is identical to the VH1. In some embodiments, the VL1 and VH1 are covalently linked via a disulfide bond. In some embodiments, the VL1 and VH1 are not covalently linked via a disulfide bond. In some embodiments, the first and second polypeptides are covalently linked via a disulfide bond between the first CL and the first CHI. In some embodiments, the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions. In some embodiments, the third and fourth polypeptides are covalently linked via a disulfide bond between the second CHI and the second CL. In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct multi-specific checkpoint antibodies with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The protein complex may comprise: (i) a first polypeptide comprising a second heavy chain variable region (VH2), a first light chain variable region (VL1), a first CL, and a second light chain variable region (VL2); (ii) a second polypeptide comprising a first heavy chain variable region (VH1), a first CHI, a first hinge region, a first CH2, and a first CH3; (iii) a third polypeptide comprising a third heavy chain variable region (VH3), a second CHI, a second hinge region, a second CH2, and a second CH3; (iv) a fourth polypeptide comprising a fourth heavy chain variable region (VH4), a third light chain variable region (VL3), a second CL. and a fourth light chain variable region (VL4). In some embodiments, the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target. In some embodiments, the VL2 and VH2 associate to form a second antigenbinding domain capable of binding to a second target. In some embodiments, the VL3 and VH3 associate to form a third antigen-binding domain capable of binding to the first target, optionally the VL3 is identical to the VL1, and the VH3 is identical to the VH1. In some embodiments, the VL4 and VH4 associate to form a fourth antigen-binding domain capable of binding to the second target, optionally the VL4 is identical to the VL2, and the VH4 is identical to the VH2. In some embodiments, the VL1 and VH1 are covalently linked via a disulfide bond. In some embodiments, the VL1 and VH1 are not covalently linked via a disulfide bond. In some embodiments, the VL3 and VH3 are covalently linked via a disulfide bond. In some embodiments, the VL3 and VH3 are not covalently linked via a disulfide bond. In some embodiments, the first and second polypeptides are covalently linked via a disulfide bond between the first CL and the first CHI. In some embodiments, the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions. In some embodiments, the third and fourth polypeptides are covalently linked via a disulfide bond between the second CHI and the second CL.

[0044] In some embodiments, the positioning of the antibody splits (e.g., VH2 and VL2, or VH4 and VL4) can vary within the protein complex (e.g., any of the positions 1-8 in FIG. 3), allowing for flexibility in the arrangement of VH2 and VL2, and / or VH4 and VL4, in the first, second, third, and / or fourth polypeptides to construct novel formats.

[0045] In some embodiments, the number of polypeptides can be adjusted to create new formats, offering versatility in the design of the protein complex. In some embodiments, additional disulfide bonds can be introduced at various positions within the first and second polypeptides to construct new formats and enhance the stability of the antibody. In some embodiments, additional disulfide bonds can be introduced at various positions within the third and fourth polypeptides to construct new formats and enhance the stability of the protein complex.

[0046] In some embodiments, the splits of the second antibody (e.g., VH2 and VL2) and fourth antibody (e.g., VH4 and VL4) can be a mixture of different building blocks, such as: VH, VL, ScFv, Fab, and Diabody; and the mixture of different building blocks at the positions of each antigen in the first, second, third, and / or fourth polypeptides can be changed to construct new formats.

[0047] In one aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct antibody-cytokines with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The protein complex may comprise: (i) a first polypeptide comprising a first light chain variable region (VL1), a CL. and a first portion of a cytokine; (ii) a second polypeptide comprising a first heavy chain variable region (VH1), a CHI, a first hinge region, a first CH2, and a first CH3; (iii) a third polypeptide comprising a second portion of the cytokine, a second hinge region, a second CH2, and a second CH3. In some embodiments, the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target. In some embodiments, the first and second portions of the cytokine associate to form a functional cytokine. In some embodiments, the VL1 and VH1 are covalently linked via a disulfide bond. In some embodiments, the VL1 and VH1 are not covalently linked via a disulfide bond. In some embodiments, the first and second polypeptides are covalently linked via a disulfide bond between the CL and CHI. In some embodiments, the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions.

[0048] In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct antibody-cytokines with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The protein complex may comprise: (i) a first polypeptide comprising a first light chain variable region (VL1), a first CL, and a first portion of a first cytokine; (ii) a second polypeptide comprising a first heavy chain variable region (VH1), a first CHI, a first hinge region, a first CH2, a first CH3, and a second portion of the first cytokine; (iii) a third polypeptide comprising a second heavy chain variable region (VH2). a second CHI, a second hinge region, a second CH2, a second CH3, and a second portion of a second cytokine; (iv) a fourth polypeptide comprising a second light chain variable region (VL2), a second CL, and a first portion of the second cytokine. In some embodiments, the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target. In some embodiments, the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to the first target, optionally the VL2 is identical to the VL1, and the VH2 is identical to the VH1. In some embodiments, the first and second portions of the first cytokine associate to form a functional first cytokine. In some embodiments, the first and second portions of the second cytokine associate to form a functional second cytokine, optionally the first and second cytokines are identical. In some embodiments, the VL1 and VH1 are covalently linked via a disulfide bond. In some embodiments, the VL1 and VH1 are not covalently linked via a disulfide bond. In some embodiments, the first and second polypeptides are covalently linked via a disulfide bond between the first CL and the first CHI. In some embodiments, the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions. In some embodiments, the third and fourth polypeptides are covalently linked via a disulfide bond between the second CHI and the second CL.

[0049] In some embodiments, the positioning of the splits of the cytokine can vary within the protein complex (e.g., any of the positions 1-8 in FIG. 3), allowing for flexibility in the arrangement of the split of cytokine in the first, second, third, and / or fourth polypeptides to construct novel formats.

[0050] In some embodiments, the number of polypeptides can be adjusted to create new formats, offering versatility in the design of protein complex.

[0051] In some embodiments, additional disulfide bonds can be introduced at various positions within the first and second polypeptides to construct new formats and enhance the stability of the protein complex. In some embodiments, additional disulfide bonds can be introduced at various positions within the third and fourth polypeptides to construct new formats and enhance the stability of the protein complex.

[0052] In one aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct multi-specific probody with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The protein complex may comprise: (i) a first polypeptide comprising a second light chain variable region (VL2), a first heavy chain variable region (VH1), a first light chain variable region (VL1), a second heavy chain variable region (VH2), a first hinge region, a first CH2, and a first CH3; (ii) a second polypeptide comprising a third heavy chain variable region (VH3), a CHI, a second hinge region, a second CH2, and a second CH3; (iii) a third polypeptide comprising a blocking mask, a linker with a cleavage site, a third light chain variable region (VL3), and a CL. In some embodiments, the VL1 and VH1 associate to form a first antigen-binding domain capable of binding a first target. In some embodiments, the VL2 and VH2 associate to form a second antigen-binding domain capable of binding a second target. In some embodiments, the VL3 and VH3 associate to form a third antigen-binding domain capable of binding a third target. In some embodiments, the VL3 and VH3 are covalently linked via a disulfide bond. In some embodiments, the VL3 and VH3 are not covalently linked via a disulfide bond. In some embodiments, the first and second polypeptides are covalently linked via a disulfide bond between the first and second hinge regions. In some embodiments, the second and third polypeptides are covalently linked via a disulfide bond between the CHI and CL.

[0053] In some embodiments, the first target is selected for its low expression level and strong tumor specificity. For example, this target may direct the antibody primarily to the tumor, its specific targeting ensures that the antibody preferentially binds to tumor cells. In some embodiments, the second target is a tumor-associated antigen with high expression but lower tumor specificity. For example, this target is designed with a blocking mask featuring a cleavage site. In some embodiments, the blocking mask helps minimize on-target / off-tumor toxicity by concealing the target until the antibody reaches the tumor environment, the cleavage site allows for activation and effective binding to the second target once inside the tumor, thereby enhancing the potency of the multi-specific antibody. In some embodiments, the third target is a T cell antigen. For example, the T cell antigen may be included with concealment strategies to minimize potential toxicity, this target is designed to avoid nonspecific activation or toxicity in non-tumor tissues, thereby reducing the risk of adverse effects.

[0054] In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct multi-specific probody with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The protein complex may comprise: (i) a first polypeptide comprising a first CL, a first heavy chain variable region (VH1), a first light chain variable region (VL1), a first CHI, a first hinge region, a first CH2, a first CH3, and a second heavy chain variable region (VH2); (ii) a second polypeptide comprising a third heavy chain variable region (VH3), a second CHI, a second hinge region, a second CH2, a second CH3, and a second light chain variable region (VL2); (iii) a third polypeptide comprising a blocking mask, a linker with a cleavage site, a third light chain variable region (VL3), and a second CL. In some embodiments, the VL1 and VH1 associate to form a first antigen-binding domain capable of binding a first target. In some embodiments, the VL2 and VH2 associate to form a second antigen-binding domain capable of binding a second target. In some embodiments, the VL3 and VH3 associate to form a third antigen-binding domain capable of binding a third target. In some embodiments, the VL3 and VH3 are covalently linked via a disulfide bond. In some embodiments, the VL3 and VH3 are not covalently linked via a disulfide bond. In some embodiments, the first CL and the first CHI are covalently linked via a disulfide bond. In some embodiments, the first and second polypeptides are covalently linked via a disulfide bond between the first and second hinge regions. In some embodiments, the second and third polypeptides are covalently linked via a disulfide bond between the second CHI and the second CL.

[0055] In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct multi-specific probody with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The protein complex may comprise: (i) a first polypeptide comprising a single-chain variable fragment (ScFv), a first hinge region, a first CH2, a first CH3, and a second heavy chain variable region (VH2); (ii) a second polypeptide comprising a third heavy chain variable region (VH3), a CHI, a second hinge region, a second CH2, a second CH3, and a second light chain variable region (VL2); (iii) a third polypeptide comprising a blocking mask, a linker with a cleavage site, a third light chain variable region (VL3), and a CL. In some embodiments, the ScFv is capable of binding to a first target. In some embodiments, the VL2 and VH2 associate to form a first antigen-binding domain capable of binding a second target. In some embodiments, the VL3 and VH3 associate to form a second antigen-binding domain capable of binding a third target. In some embodiments, the VL3 and VH3 are covalently linked via a disulfide bond. In some embodiments, the VL3 and VH3 are not covalently linked via a disulfide bond. In some embodiments, the first and second polypeptides are covalently linked via a disulfide bond between the first and second hinge regions. In some embodiments, the second and third polypeptides are covalently linked via a disulfide bond between the CHI and CL.

[0056] In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct multi-specific probody with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The protein complex may comprise: (i) a first polypeptide comprising a first light chain variable region (VL1), a CL, and a second heavy chain variable region (VH2); (ii) a second polypeptide comprising a first heavy chain variable region (VH1), a CHI, a first hinge region, a first CH2, and a first CH3; (iii) a third polypeptide comprising a blocking mask, a linker with a cleavage site, a second light chain variable region (VL2), a single-chain variable fragment (ScFv), a second hinge region, a second CH2, and a second CH3. In some embodiments, the VL1 and VH1 associate to form a first antigen-binding domain capable of binding a first target. In some embodiments, the VL2 and VH2 associate to form a second antigen-binding domain capable of binding a second target. In some embodiments, the ScFv is capable of binding to a third target. In some embodiments, the VL1 and VH1 are covalently linked via a disulfide bond. In some embodiments, the VL1 and VH1 are not covalently linked via a disulfide bond. In some embodiments, the first and second polypeptides are covalently linked via a disulfide bond between the CL and CHI. In some embodiments, the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions.

[0057] In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct multi-specific probody with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The protein complex may comprise: (i) a first polypeptide comprising a blocking mask, a linker with a cleavage site, a first light chain variable region (VL1), a CL, and a second heavy chain variable region (VH2); (ii) a second polypeptide comprising a first heavy chain variable region (VH1), a CHI, a first hinge region, a first CH2, and a first CH3; (iii) a third polypeptide comprising a single-chain variable fragment (ScFv), a second light chain variable region (VL2), a second hinge region, a second CH2, and a second CH3. In some embodiments, the VL1 and VH1 associate to form a first antigen-binding domain capable of binding a first target. In some embodiments, the VL2 and VH2 associate to form a second antigen-binding domain capable of binding a second target. In some embodiments, the ScFv is capable of binding to a third target. In some embodiments, the VL1 and VH1 are covalently linked via a disulfide bond. In some embodiments, the VL1 and VH1 are not covalently linked via a disulfide bond. In some embodiments, the first and second polypeptides are covalently linked via a disulfide bond between the CL and CHI. In some embodiments, the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions.

[0058] In some embodiments, the positioning of the antibody splits (e.g., VH2 and VL2) can vary within the protein complex (e.g., any of the positions 1-8 in FIG. 2), allowing for flexibility in the arrangement of VH2 and VL2 in the first, second, and / or third polypeptides to construct novel formats.

[0059] In some embodiments, for multi-specific probody designs, the approach typically involves several targets: First Target: this target can be chosen for its low expression level and strong tumor specificity. It may direct the antibody primarily to the tumor. Second Target: this can be a tumor-associated target with high expression but lower tumor specificity. This target may be designed with blocking mask with cleavage site to minimize on-target / off-tumor toxicity and enhance the potency of the multi-specific antibody. Third Target: this target can be a T cell target and is designed with concealment to minimize toxicity.

[0060] In some embodiments, the number of polypeptides can be adjusted to create new formats, offering versatility in antibody design. In some embodiments, additional disulfide bonds can be introduced at various positions within the first and second polypeptides to construct new formats and enhance the stability of the antibody.

[0061] In some embodiments, the splits of the second and third antibodies can be a mixture of different building blocks, such as: VH, VL, ScFv, Fab, split of cytokine, and Diabody; and the mixture of different building blocks at the positions of each antigen in the first, second, and / or third polypeptides can be changed to construct new formats.

[0062] In one aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct multi-specific antibody with splits of antibody domains (like only VH or VL) or mixtures of different building blocks designed at various positions within a single antibody. The protein complex may comprise: (i) a first polypeptide comprising a first light chain variable region (VL1), a CL, and a second heavy chain variable region (VH2); (ii) a second polypeptide comprising a first heavy chain variable region (VH1), a CHI, a first hinge region, a first CH2, and a first CH3; (iii) a third polypeptide comprising a third heavy chain variable region (VH3), a second hinge region, a second CH2, and a second CH3. In some embodiments, the VL1 and VH1 associate to form a first antigen-binding domain capable of binding a first target. In some embodiments, the VH2 and VH3 associate to form a second antigen-binding domain capable of binding a second target, optionally the VH2 and VH3 are identical. In some embodiments, the VL1 and VH1 are covalently linked via a disulfide bond. In some embodiments, the VL1 and VH1 are not covalently linked via a disulfide bond. In some embodiments, the first and second polypeptides are covalently linked via a disulfide bond between the CL and CHI. In some embodiments, the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions. In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct multi-specific antibody with splits of antibody domains (like only VH or VL) or mixtures of different building blocks designed at various positions within a single antibody. The protein complex may comprise: (i) a first polypeptide comprising a first light chain variable region (VL1), a CL, and a second light chain variable region (VL2); (ii) a second polypeptide comprising a first heavy chain variable region (VH1). a CHI. a first hinge region, a first CH2, and a first CH3; (iii) a third polypeptide comprising a second hinge region, a second CH2, a second CH3, and a third light chain variable region (VL3). In some embodiments, the VL1 and VH1 associate to form a first antigen-binding domain capable of binding a first target. In some embodiments, the VL2 and VL3 associate to form a second antigen-binding domain capable of binding a second target, optionally the VL2 and VL3 are identical. In some embodiments, the VL1 and VH1 are covalently linked via a disulfide bond. In some embodiments, the VL1 and VH1 are not covalently linked via a disulfide bond. In some embodiments, the first and second polypeptides are covalently linked via a disulfide bond between the CL and CHI. In some embodiments, the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions.

[0063] In one aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct tetra-specific antibodies with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The protein complex may comprise: (i) a first polypeptide comprising a first light chain variable region (VL1), a CL, a second heavy chain variable region (VH2) and a third heavy chain variable region (VH3); (ii) a second polypeptide comprising a first heavy chain variable region (VH1), a CHI, a first hinge region, a first CH2, and a first CH3; (iii) a third polypeptide comprising a single-chain variable fragment (ScFv), optionally a linker with a cleavage site, a second light chain variable region (VL2), a second hinge region, a second CH2, a second CH3; and a third light chain variable region (VL3). In some embodiments, the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target. In some embodiments, the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to a second target. In some embodiments, the VL3 and VH3 associate to form a third antigenbinding domain capable of binding to a third target. In some embodiments, the ScFv comprises a fourth light chain variable region (VL4) and a fourth heavy chain variable region (VH4). In some embodiments, the ScFv is capable of binding to a fourth target. In some embodiments, the VL4 is identical to the VL1, and the VH4 is identical to the VH1. In some embodiments, the VL1 and VH1 are covalently linked via a disulfide bond. In some embodiments, the VL1 and VH1 are not covalently linked via a disulfide bond. In some embodiments, the first and second polypeptides are covalently linked via a disulfide bond between the CL and the CHI. In some embodiments, the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions. In some embodiments, the first and fourth targets are the same.

[0064] In some embodiments, the positioning of the antibody splits (e.g., VH2 and VL2) can vary within the protein complex (e.g., any of the positions 1-8 in FIG. 2), allowing for flexibility in the arrangement of VH2 and VL2 in the first, second, and / or third polypeptides to construct novel formats.

[0065] In some embodiments, for multi-specific probody designs, the approach typically involves several targets: First Target: this target can be chosen for its low expression level and strong tumor specificity. It may direct the antibody primarily to the tumor. Second Target: this can be a tumor-associated target with high expression but lower tumor specificity. This target may be designed with blocking mask with cleavage site to minimize on-target / off-tumor toxicity and enhance the potency of the multi-specific antibody. Third Target: this target can be aT cell target and is designed with concealment to minimize toxicity.

[0066] In some embodiments, the number of polypeptides can be adjusted to create new formats, offering versatility in antibody design. In some embodiments, additional disulfide bonds can be introduced at various positions within the first and second polypeptides to construct new formats and enhance the stability of the antibody.

[0067] In some embodiments, the splits of the second and third antibodies can be a mixture of different building blocks, such as: VH, VL, ScFv, Fab, split of cytokine, and Diabody; and the mixture of different building blocks at the positions of each antigen in the first, second, and / or third polypeptides can be changed to construct new formats.

[0068] In one aspect, the disclosure is related to a nucleic acid comprising a polynucleotide encoding the protein complex described herein. In some embodiments, the nucleic acid is a DNA (e.g., cDNA) or RNA (e.g., mRNA). In one aspect, the disclosure is related to a vector comprising one or more of the nucleic acids described herein. In one aspect, the disclosure is related to a cell comprising the vector described herein. In some embodiments, the cell is a CHO cell. In one aspect, the disclosure is related to a cell comprising one or more of the nucleic acids described herein.

[0069] In one aspect, the disclosure is related to a method of producing a protein complex, the method comprising (a) culturing the cell described herein under conditions sufficient for the cell to produce the protein complex; and (b) collecting the protein complex produced by the cell.

[0070] In one aspect, the disclosure is related to a protein conjugate comprising the protein complex described herein, covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.

[0071] In one aspect, the disclosure is related to a method of treating a subject having a disease or disorder, the method comprising administering a therapeutically elfective amount of a composition comprising the protein complex or the protein conjugate described herein, to the subject. In some embodiments, the subject has a cancer, an autoimmune disease, a hematopoietic diseases, or a metabolic disease.

[0072] In one aspect, the disclosure is related to a method of decreasing the rate of tumor growth, the method comprising contacting a tumor cell with an effective amount of a composition comprising the protein complex or the protein conjugate described herein.

[0073] In one aspect, the disclosure is related to a method of killing a tumor cell, the method comprising contacting a tumor cell with an effective amount of a composition comprising the protein complex or the protein conjugate described herein.

[0074] In one aspect, the disclosure is related to a pharmaceutical composition comprising the protein complex described herein and a pharmaceutically acceptable carrier.

[0075] In some embodiments, the positions of the VH and VL of a VH-VL pair (e.g., the pair of VH 1 / VL1, the pair of VH2 / VL2) can be varied within the protein complex (e.g., any of the protein complexes described herein). In some embodiments, the VH and VL in the first, second, third, and / or fourth polypeptides (e.g., any of the first, second, third, and / or fourth polypeptides described herein) can be repositioned to create new formats of the protein complex. In some embodiments, the number of polypeptides can be varied to create new formats of the protein complex. In some embodiments, the protein complex may include 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 polypeptides (e.g., a combination of any of the polypeptides described herein)

[0076] In some embodiments, additional disulfide bonds (e g., non-native disulfide bonds) can be introduced at specific positions in the first, second, third, and / or fourth polypeptides described herein to create new formats and enhance stability of the protein complex. For example, the protein complex described herein may includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-native disulfide bonds.

[0077] As used herein, the term “"non-native disulfide bond" refers to a disulfide bond that does not naturally exist in a wild-type protein. In some embodiments, the non-native disulfide bond is formed by two cysteine residues, wherein at least one of them is a mutation. In some embodiments, two of them are mutations. In some embodiments, at least one or two cysteine residues are introduced by insertion. In some embodiments, a deletion changes the distance between two existing cysteine residues, which then forms a disulfide bond that does not exist in a wild-ty pe protein.

[0078] In some embodiments, any one or more (e.g., 1, 2. 3, or 4) of the first, second, third, and fourth polypeptides described herein are independently linked at its C-terminus to a hinge region of IgGl, IgG2, IgG3, or IgG4.

[0079] In some embodiments, the positioning of the antibody splits (e.g., any of the VHs or VLs described herein) can vary' within the protein complex (e.g., a single antibody), allowing for flexibility in arranging the VH and VL across the first, second, third, and / or fourth polypeptides to create novel formats. The antibody splits may include combinations such as VH only, VL only, VH + VH, or VL + VL.

[0080] In some embodiments, the number of polypeptides can be adjusted to create new formats, offering versatility in the design of the protein complex.

[0081] In some embodiments, additional disulfide bonds can be introduced at various positions within the first and second polypeptides to construct new formats and enhance the stability of the protein complex.

[0082] In some embodiments, the splits of the second and / or third antibodies can be a mixture of different building blocks, such as: VH. VL, ScFv, Fab, split of cytokine, and Diabody; and the mixture of different building blocks at the positions of each antigen in the first, second, and third polypeptides can be changed to construct new formats.

[0083] In some embodiments, inside the FR of ScFv, Diabody, and Fab formats have different peptide linkers, wherein the linkage of VH, VL, ScFv. Fab, split of cytokine, Diabody, and Fc parts is through different peptide linkers. In some embodiments, the peptide linkers each independently comprise different amino acid sequences. In some embodiments, additional disulfide bonds can be added to help increase stability in Fab, Diabody, and ScFv formats, with cysteine mutations on the FR of VH and VL to form disulfide bonds.

[0084] In some embodiments, the FR of VH, VL, ScFv, Fab, split of cytokine, Diabody, and Fc parts can be extended and / or mixed to construct new formats. In some embodiments, the FR of VH, VL, ScFv, Fab, split of cytokine, Diabody, and Fc parts can be put at the N terminus and / or C terminus of the heavy chain and / or light chain to construct new formats.

[0085] In some embodiments, either of the first polypeptide and the second polypeptide is independently linked at its C terminus to a hinge region of IgGl, IgG2, IgG3, or IgG4. In another aspect, the present disclosure provides a protein complex comprising a dimer of the protein complex provided herein, with each unit of the dimer connected via the hinge region.

[0086] In some embodiments, either of the Fab, ScFv, and Diabody polypeptide is independently linked at its C-terminus to an Fc region. In some embodiments, either of the first polypeptide and the second polypeptide is independently linked at its C-terminus to an albumin or a PEG.

[0087] In some embodiments, the CH2-CH3 domain of the second polypeptide and the CH2-CH3 domain of the third polypeptide are different. In some embodiments, the second polypeptide and the third polypeptide are engineered through modification to the CH3 domain interface with different mutations on each domain. In some embodiments, multispecific antibody constructs are designed based on knobs-into-holes or Fc structure. The CH3 domain is linked with knobs-into-holes technology, and the CH3 position of the hole or knob can be exchanged to construct new formats. The CH3 can also use regular Fc, without knobs-into-holes.

[0088] In another aspect, the present disclosure provides a method for treating a subject in need of treatment using an antibody provided herein.

[0089] In some embodiments, the treatment results in a sustained response in the individual after cessation of the treatment.

[0090] In some embodiments, the immunotherapeutic is administered continuously or intermittently.

[0091] In some embodiments, the individual has cancer, including colorectal cancer, melanoma, non-small cell lung cancer, ovarian cancer, breast cancer, pancreatic cancer, a hematological malignant tumor, and renal cell carcinoma, as well as autoimmune diseases, hematopoietic diseases, metabolic diseases, etc.

[0092] In some embodiments, the antibody is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbi tally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.

[0093] In some embodiments, the therapeutic combination or pharmaceutical composition of the present disclosure further comprises an effective amount of an additional therapeutic agent, such as an anticancer agent.

[0094] In some embodiments, the anticancer agent is an antimetabolite, an inhibitor of topoisomerase I and II, an alkylating agent, a microtubule inhibitor, an antiandrogen agent, a GnRH modulator, or mixtures thereof. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent selected from the group consisting of tamoxifen, raloxifene, anastrozole, exemestane, letrozole, imatinib, paclitaxel, cyclophosphamide, lovastatin, minosine, gemcitabine, cytarabine, 5-fluorouracil, methotrexate, docetaxel, goserelin, vincristine, vinblastine, nocodazole, teniposide, etoposide, gemcitabine, epothilone, vinorelbine, camptothecin, daunorubicin, actinomycin D, mitoxantrone, acridine, doxorubicin, epirubicin, or idarubicin.

[0095] In another aspect, the present disclosure provides a method for treating a disease condition in a subject that is in need of such treatment, comprising administering to the subject the therapeutic combination or pharmaceutical composition provided herein.

[0096] In some embodiments, the disease condition is a tumor. In some embodiments, the disease condition comprises abnormal cell proliferation.

[0097] In some embodiments, the abnormal cell proliferation comprises a pre-cancerous lesion. In some embodiments, the abnormal proliferation is of cancer cells.

[0098] In some embodiments, the cancer is selected from the group consisting of: breast cancer, colorectal cancer, diffuse large B-cell lymphoma, endometrial cancer, follicular lymphoma, gastric cancer, glioblastoma, head and neck cancer, hepatocellular cancer, lung cancer, melanoma, multiple myeloma, ovarian cancer, pancreatic cancer, prostate cancer, and renal cell carcinoma.

[0099] In a further aspect, the present disclosure provides a kit that contains the therapeutic combination provided herein, and optionally with instructions.

[0100] 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 to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0101] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0102] DESCRIPTION OF DRAWINGS

[0103] The novel features of the invention are detailed in the appended claims. A clearer understanding of these features and their advantages can be gained from the following detailed description, which outlines illustrative embodiments of the invention and includes the accompanying drawings.

[0104] FIG. 1 diagrammatically illustrates the structure of IgG-Fc and identifies the four positions where building blocks can be incorporated. These building blocks may include Fab, ScFv, VHH, VH, VL, cytokines, cytokine splits, proteins, split of proteins, or combinations of these components.

[0105] FIG. 2 diagrammatically illustrates the structure of monovalent Fab-IgG-Fc and highlights the six positions available for incorporating building blocks. These building blocks can include Fab, ScFv, VHH, VH, VL, cytokines, cytokine splits, proteins, split of proteins, or combinations of these components.

[0106] FIG. 3 diagrammatically illustrates the structure of bivalent Fab-IgG-Fc and identifies the eight positions where building blocks can be incorporated. These building blocks may include Fab, ScFv, VHH, VH, VL, cytokines, cytokine splits, proteins, split of proteins, or combinations of these components.

[0107] FIG. 4 diagrammatically illustrates the structure of Fab-IgG-Fc for bispecific antibodies, showing the various positions on a single antibody where building blocks can be designed.

[0108] FIG. 5 shows the results of a cell binding assay for bispecific antibodies using the Crossbody platform. The assay illustrates the binding of Crossbody constructs to HT29 cells and PBMCs as target cells.

[0109] FIG. 6 presents the results of a cell killing assay for bispecific antibodies with the Crossbody platform. This assay demonstrates the antibody-mediated cytotoxic effects of resting PBMCs on HT29 cells, depicted as the absolute counts of remaining live cells after 48 hours of co-incubation with the antibodies.

[0110] FIG. 7 diagrammatically illustrates the structure of Fab-IgG-Fc for trispecific antibodies, showing the various positions on a single antibody where building blocks can be designed.

[0111] FIG. 8 shows the results of a cell binding assay for trispecific antibodies using the Crossbody platform. The assay illustrates the binding of Crossbody constructs to MKN45, HepG2, HT29, and MDA-MB-231 cells.

[0112] FIG. 9 presents the results of a cell binding assay for trispecific antibodies with the Crossbody platform. This assay demonstrates the binding of Crossbody constructs to LS174T, Lovo cells, and PBMCs. FIG. 10 depicts the results of a cell killing assay for trispecific antibodies using the Crossbody platform. The assay shows the antibody-mediated cytotoxic effects of resting PBMCs on MKN45, HepG2, HT29, and MDA-MB-231 cells, with the remaining live cells counted after 48 hours of co-incubation.

[0113] FIG. 11 shows the results of a cell killing assay for trispecific antibodies using the Crossbody platform. The assay illustrates the cytotoxic effects of Crossbody constructs on LS174T and Lovo cells. Antibody-mediated effects on tumor cells are depicted as absolute counts of remaining live cells after 48 hours of co-incubation with resting PBMCs.

[0114] FIG. 12 presents the results of a cell killing assay for trispecific antibodies with the Crossbody platform. This assay demonstrates the cytotoxic effects of Crossbody constructs on HepG2 and MDA-MB-231 cells. The results are shown as absolute counts of remaining live cells after 48 hours of co-incubation with resting PBMCs.

[0115] FIG. 13 depicts the efficacy of trispecific antibodies in an hPBMC / NSG mouse model bearing the HT29 tumor xenograft.

[0116] FIG. 14 illustrates the efficacy of trispecific antibodies in an hPBMC / NSG mouse model bearing the MKN45 tumor xenograft.

[0117] FIG. 15 diagrammatically illustrates the structure of Fab-IgG-Fc for trispecific antibodies, showing the various positions on a single antibody where building blocks can be designed.

[0118] FIG. 16 shows the results of a cell binding assay for trispecific antibodies using the Crossbody platform. This assay illustrates the binding of Crossbody constructs to HT29, MDA-MB-231, HPAC, and PBMC cells.

[0119] FIG. 17 shows the results of a cell binding assay for trispecific antibodies using the Crossbody platform. This assay illustrates the binding of Crossbody constructs to HT29, MDA-MB-231, HPAC, and PBMC cells.

[0120] FIG. 18 depicts the results of a cell killing assay for trispecific antibodies using the Crossbody platform. This assay shows the antibody-mediated cytotoxic effects of resting PBMCs on HT29 and MDA-MB-231 cells, with results represented as absolute counts of remaining live cells after 48 hours of co-incubation.

[0121] FIG. 19 illustrates the results of a cell killing assay for trispecific antibodies with the Crossbody platform. The assay highlights the antibody-mediated cytotoxic effects of resting PBMCs on HPAC cells, shown as absolute counts of remaining live cells after 48 hours of co-incubation. FIG. 20 diagrammatically illustrates the structure of Fab-IgG-Fc for trispecific antibodies, showing the various positions on a single antibody where building blocks can be designed.

[0122] FIG. 21 shows the results of a cell binding assay for trispecific antibodies using the Crossbody platform. The assay illustrates the binding of Crossbody constructs to HT29 and MCF7 cells.

[0123] FIG. 22 presents the results of a cell binding assay for trispecific antibodies with the Crossbody platform. This assay demonstrates the binding of Crossbody constructs to PBMCs.

[0124] FIG. 23 depicts the results of a cell killing assay for trispecific antibodies using the Crossbody platform. The assay shows the antibody-mediated cytotoxic effects of resting PBMCs on HT29 and MCF7 cells, with results represented as absolute counts of remaining live cells after 48 hours of co-incubation.

[0125] FIG. 24 diagrammatically illustrates the structure of Fab-IgG-Fc for trispecific antibodies, showing the various positions on a single antibody where building blocks can be designed.

[0126] FIG. 25 presents result from both a cell binding assay and a cell killing assay for trispecific antibodies using the Crossbody platform. The cell binding assay illustrates the interaction of Crossbody constructs with HT29, MCF7, and PBMC cells. The cell killing assay demonstrates the cytotoxic effects of these constructs on HT29 cells.

[0127] FIG. 26 diagrammatically illustrates the structure of Fab-IgG-Fc for trispecific antibodies, showing the various positions on a single antibody where building blocks can be designed.

[0128] FIG. 27 shows the results of a cell binding assay for trispecific antibodies using the Crossbody platform. The assay illustrates the binding of Crossbody constructs to MCF7 cells.

[0129] FIG. 28 presents the results of a cell binding assay for trispecific antibodies with the Crossbody platform, demonstrating the binding of Crossbody constructs to PBMCs.

[0130] FIG. 29 depicts the results of a cell killing assay for trispecific antibodies using the Crossbody platform. This assay shows the antibody -mediated cytotoxic effects of resting PBMCs on HT29 cells, with results represented as absolute counts of remaining live cells after 48 hours of co-incubation.

[0131] FIG. 30 illustrates the results of a cell killing assay for trispecific antibodies with the Crossbody platform. The assay demonstrates the cytotoxic effects of resting PBMCs on MCF7 cells, with remaining live cells counted after 48 hours of co-incubation. FIG. 31 diagrammatically illustrates the structure of Fab-IgG-Fc for trispecific antibodies, showing the various positions on a single antibody where building blocks can be designed.

[0132] FIG. 32 presents result from both a cell binding assay and a cell killing assay for trispecific antibodies using the Crossbody platform. The cell binding assay shows the interaction of Crossbody constructs with MCF7 and PBMC cells. The cell killing assay demonstrates the cytotoxic effects of these constructs on HT29 and MCF7 cells.

[0133] FIG. 33 shows the results of a cell binding assay for trispecific antibodies with the Crossbody platform. This assay illustrates the binding of Crossbody constructs to HT29, MCF7, and PBMC cells.

[0134] FIG. 34 depicts the results of a cell killing assay for trispecific antibodies using the Crossbody platform. The assay highlights the antibody -mediated cytotoxic effects of resting PBMCs on HT29 and MCF7 cells, with results shown as absolute counts of remaining live cells after 48 hours of co-incubation.

[0135] FIG. 35 presents the efficacy results of trispecific antibodies in hPBMC / NSG mice bearing the MKN45 tumor xenograft model.

[0136] FIG. 36 shows the body weight measurements of hPBMC / NSG mice bearing the MKN45 tumor xenograft model following treatment with trispecific antibodies.

[0137] FIG. 37 depicts the efficacy results of trispecific antibodies in hPBMC / NSG mice bearing the HT29 tumor xenograft model.

[0138] FIG. 38 illustrates the body weight measurements of hPBMC / NSG mice bearing the HT29 tumor xenograft model after treatment with trispecific antibodies.

[0139] FIG. 39 diagrammatically illustrates the structure of Fab-IgG-Fc for multi-specific checkpoint antibodies, showing the various positions on a single antibody where building blocks can be designed.

[0140] FIG. 40 shows the results of a cell binding assay for multi-specific checkpoint antibodies using the Crossbody platform. This assay illustrates the binding of Crossbody constructs to HPAC and PBMC cells.

[0141] FIG. 41 presents the results of a cell killing assay for multi-specific checkpoint antibodies using the Crossbody platform, demonstrating the cytotoxic effects on HPAC cells. This figure also includes efficacy and body weight data for multi-specific checkpoint antibodies in hCD3 / hCD28 mice bearing the B16F10 tumor xenograft model. FIG. 42 diagrammatically illustrates the structure of Fab-IgG-Fc for multi-specific antibody-cytokine, showing the various positions on a single antibody where building blocks can be designed.

[0142] FIG. 43 presents the results of a cell killing assay for multi-specific antibody-cytokine constructs using the Crossbody platform. This assay demonstrates the cytotoxic effects of these constructs on HPAC cells, with the antibody-mediated effects of resting NK cells shown as absolute counts of remaining live cells after 48 hours of co-incubation.

[0143] FIG. 44 depicts the results of a HEK BLUE IL-2 assay for multi-specific antibodycytokine constructs using the Crossbody platform. This assay illustrates the activity of these constructs on hPD-1 cells.

[0144] FIG. 45 diagrammatically illustrates the structure of Fab-IgG-Fc for multi-specific antibody-cytokine, showing the various positions on one antibody where building blocks can be designed.

[0145] FIG. 46 shows the results of a HEK BLUE IL-2 assay for multi-specific antibodycytokine constructs using the Crossbody platform. The assay demonstrates the activity of these constructs using HPAC cells as target cells.

[0146] FIG. 47 diagrammatically illustrates the structure of Fab-IgG-Fc for multi-specific costimulatory antibodies, showing the various positions on a single antibody where building blocks can be designed.

[0147] FIG. 48 shows the results of a cell binding assay for multi-specific co-stimulatory antibodies using the Crossbody platform. This assay illustrates the binding of Crossbody constructs to HPAC and PBMC cells.

[0148] FIG. 49 presents the results of a cell killing assay for multi-specific co-stimulatory antibodies with the Crossbody platform. The assay demonstrates the cytotoxic effects of Crossbody constructs on HT29, HPAC, and MCF7 cells.

[0149] FIG. 50 depicts the cytokine release results from a cell killing assay of multi-specific co-stimulatory antibodies using the Crossbody platform. This assay shows the cytokine release when HPAC cells are used as target cells.

[0150] FIG. 51 illustrates the results of a cell killing assay for multi-specific co-stimulatory antibodies using the Crossbody platform. The assay shows the cytotoxic effects on MDA-MB-231 and HPAC cells.

[0151] FIG. 52 diagrammatically illustrates the structure of Fab-IgG-Fc for multi-specific probody, showing the various positions on a single antibody where building blocks can be designed. FIG. 53 shows the results of a cell binding assay for multi-specific probody using the Crossbody platform. This assay illustrates the binding of Crossbody constructs to HT29, MCF7, and PBMC cells.

[0152] FIG. 54 presents the results from a cell binding assay for multi-specific probody with the Crossbody platform, highlighting the interaction of these constructs with HT29 and PBMC cells.

[0153] FIG. 55 depicts the results of a cell killing assay for multi-specific probody using the Crossbody platform. This assay demonstrates the cytotoxic effects of these constructs on HT29 and MCF7 cells.

[0154] FIG. 56 illustrates the results of a cell killing assay for multi-specific probody with the Crossbody platform, showing the effects on HT29, MCF7, HPAC, and MKN45 cells.

[0155] FIG. 57 presents the results from a cell killing assay for multi-specific probody using the Crossbody platform, focusing on HT29 and MCF7 cells.

[0156] FIG. 58 depicts the results of a cell killing assay for multi-specific probody with the Crossbody platform, also targeting HT29 and MCF7 cells.

[0157] FIG. 59 shows the results of a cell killing assay for multi-specific probody using the Crossbody platform, with a focus on HT29 and HPAC cells.

[0158] FIG. 60 diagrammatically illustrates the structure of Fab-IgG-Fc for multi-specific novel antibody, showing the various positions on a single antibody where building blocks can be designed.

[0159] FIG. 61 shows the results of a cell binding assay for multi-specific novel antibodies using the Crossbody platform. This assay illustrates the binding of Crossbody constructs to PBMC cells.

[0160] FIG. 62 presents the results of a cell killing assay for multi-specific novel antibodies with the Crossbody platform, demonstrating the cytotoxic effects on HPAC cells.

[0161] FIG. 63 depicts the efficacy and body weight results of trispecific antibodies in human PBMC / NSG mice bearing the MKN45 tumor xenograft model.

[0162] FIG. 64 diagrammatically illustrates the structure of Fab-IgG-Fc for multi-specific ADCs, showing the various positions on a single antibody where building blocks can be designed.

[0163] FIG. 65 presents result from a cell binding assay for ADCs using the Crossbody platform with HPAC and Huh7 cell.

[0164] FIG. 66 presents result from a cell internalization assay and a cell killing assay for ADCs using the Crossbody platform with HPAC and Huh7 cell. FIG. 67 diagrammatically illustrates the structure of Fab-IgG-Fc for multi-specific antibodies, showing the various positions on a single antibody where building blocks can be designed.

[0165] FIG. 68 presents the results from a cell binding assay for multi-specific antibody with the Crossbody platform, highlighting the interaction of these constructs with HPAC, MCF7 and Jurkat cells.

[0166] FIG. 69 presents the results from a cell binding assay for multi-specific antibody with the Crossbody platform, highlighting the interaction of these constructs with HPAC and Jurkat cells.

[0167] FIG. 70 presents the results from a cell binding assay for multi-specific antibody with the Crossbody platform, highlighting the interaction of these constructs with HPAC, MCF7 and PBMC cells.

[0168] FIG. 71 depicts the results of a cell killing assay for multi-specific antibody using the Crossbody platform. This assay demonstrates the cytotoxic effects of these constructs on HPAC and MCF7 cells.

[0169] FIG. 72 depicts the results of a cell killing assay for multi-specific antibody using the Crossbody platform. This assay demonstrates the cytotoxic effects of these constructs on HPAC and MCF7 cells.

[0170] FIG. 73 presents the results from a cell binding assay for multi-specific antibody with the Crossbody platform, highlighting the interaction of these constructs with HPAC, MCF7, Huh7 and Jurkat cells.

[0171] FIG. 74 depicts the results of a cell killing assay for multi-specific antibody using the Crossbody platform. This assay demonstrates the cytotoxic effects of these constructs on HPAC, MCF7 and Huh7 cells.

[0172] FIG. 75 presents the efficacy results of multi-specific antibodies in hPBMC / NSG mice bearing the HPAC tumor xenograft model.

[0173] FIG. 76 shows the body weight measurements of hPBMC / NSG mice bearing the HPAC tumor xenograft model following treatment with multi-specific antibodies.

[0174] FIG. 77 presents the PK results of multi-specific antibodies in hPBMC / NSG mice bearing the HPAC tumor xenograft model.

[0175] FIG. 78 depicts the binding results of a Elisa assay for ROR1 antibody with human and mouse ROR1 protein.

[0176] FIG. 79 presents the safety study results of multi-specific antibodies in human EpCam and CD3 mice model. FIG. 80 diagrammatically illustrates the structure of Fab-IgG-Fc for multi-specific antibodies, showing the various positions on a single antibody where building blocks can be designed.

[0177] FIG. 81 presents the results from a cell binding assay for multi-specific antibody with the Crossbody platform, highlighting the interaction of these constructs with Raji and Jurkat cells.

[0178] FIG. 82 depicts the results of a Raji cell killing assay, a PBMC assay and the cytokine release results from a PBMC assay for multi-specific antibody using the Crossbody platform. This assay demonstrates the cytotoxic effects and the cytokine release level of these constructs on Raji cells and B cell in PBMC.

[0179] DETAILED DESCRIPTION

[0180] The disclosures described herein are not limited to the specific methods and experimental conditions detailed, which may vary. The terminology used is intended to describe particular embodiments and is not meant to be restrictive; the scope of the disclosure is defined by the claims. The following sections describe various aspects of the disclosure with example applications for illustration. It should be understood that numerous specific details, relationships, and methods are provided to offer a comprehensive understanding of the disclosure. Those skilled in the relevant art will recognize that the disclosure can be practiced without one or more of these specific details or using alternative methods. The disclosure is not constrained by the illustrated sequence of acts or events, as these may occur in different orders or concurrently.

[0181] Additionally, not all illustrated acts or events are necessary to implement the methodology according to the present disclosure.

[0182] The terms “about’' or “approximately" refer to a range of acceptable variation for a particular value, as determined by one skilled in the art. This acceptable error range depends on factors such as the measurement method and the limitations of the measurement system. For example, “about” may encompass within 1 or more than 1 standard deviation, depending on standard practices. Alternatively, “about"’ can indicate a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. In the context of biological systems or processes, “about” can mean within an order of magnitude, preferably within a 5-fold range, and more preferably within a 2-fold range of the value. Unless otherwise specified, the term “about” should be understood to mean within these acceptable error ranges for the particular value. The terminology used herein is intended to describe specific embodiments and is not meant to limit the disclosure. The singular forms “a,” “an,” and “the” are understood to include their plural forms unless the context clearly indicates otherwise. Additionally, terms such as “including,” “includes,” “having,” “has,” and “with,” or variations thereof, are intended to be inclusive and should be interpreted in a manner similar to the term “comprising.”

[0183] I. Definitions and Abbreviations

[0184] Unless otherwise defined, all technical and scientific terms used herein generally have meanings commonly understood by those skilled in the relevant art. The nomenclature and laboratory procedures described, including those related to cell culture, molecular genetics, organic chemistry, nucleic acid chemistry, and hybridization, are well-known and commonly used in the field. Standard techniques are employed for nucleic acid and peptide synthesis, as well as for chemical syntheses and analyses. These techniques are typically carried out according to conventional methods described in various general references provided throughout this document. The nomenclature and procedures used in analytical chemistry and organic synthesis are also those commonly practiced in the art, with standard techniques or their modifications applied as appropriate.

[0185] References in the specification to “some embodiments,” “an embodiment,” “one embodiment.” or “other embodiments” indicate that a particular feature, structure, or characteristic is included in at least some of the embodiments described, but not necessarily all of them.

[0186] Although various features of the disclosure may be described within the context of a single embodiment, these features may also be provided individually or in any suitable combination. Conversely, while the disclosure may be described in separate embodiments for clarity, it can also be implemented within a single embodiment.

[0187] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to a linear sequence of amino acid residues linked by peptide bonds. This includes proteins, polypeptides, oligopeptides, peptides, and their fragments. Such proteins may be composed of naturally occurring and / or synthetic (e g., modified or non-naturally occurring) amino acids. Therefore, “amino acid” or “peptide residue” encompasses both naturally occurring and synthetic amino acids. These terms also include fusion proteins, such as those with heterologous amino acid sequences, leader sequences (both homologous and heterologous), or N-terminal methionine residues; immunologically tagged proteins; fusion proteins with detectable partners (e.g., fluorescent proteins, P-galactosidase, luciferase, etc ); and similar constructs. Additionally, a dash at the beginning or end of an amino acid sequence indicates either a peptide bond to another sequence or a covalent bond to a carboxyl or hydroxyl end group. The absence of a dash does not imply that such peptide bonds or covalent bonds are absent, as it is conventional to omit such details in amino acid sequence representations.

[0188] The terms “conjugated” and “joining” generally refer to a chemical linkage, whether covalent or non-covalent, that associates one molecule with a second molecule.

[0189] The term “nucleic acid” refers to either DNA or RNA, or molecules containing deoxy ribonucleotides and / or ribonucleotides. Nucleic acids may be naturally occurring or synthetically produced and include analogs of naturally occurring polynucleotides where one or more nucleotides are modified from their natural forms.

[0190] As used herein, ranges and amounts may be expressed as “about” a particular value or range. The term “about” includes the exact amount as well. For example, “about 5 pL” encompasses both “about 5 pL” and exactly “5 pL.” Generally, “about” includes amounts that fall within the range of experimental error.

[0191] The terms “potent” or “potency” in the context of a compound refer to its ability or capacity to exhibit a desired activity.

[0192] The term “purified” means that a compound of interest has been separated from other components present in nature or introduced during manufacture, resulting in an enriched form of the compound.

[0193] The terms “antigen” and “epitope” are used interchangeably to refer to the part of a molecule (e.g., a polypeptide) specifically recognized by an immune system component, such as an antibody. The term “antigen” includes antigenic epitopes, which are fragments of an antigen that are recognized as antigenic.

[0194] The term “concentration,” when used in the context of a molecule such as a peptide fragment, refers to the amount of the molecule present in a given volume. In some embodiments, concentration is expressed as molar concentration, indicating the number of moles of the molecule present per unit volume of solution.

[0195] A typical full-length antibody structural unit is known to consist of a tetramer. Each tetramer comprises two identical pairs of polypeptide chains: one pair consisting of a "light" chain (approximately 25 kD) and a "heavy" chain (approximately 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The terms "variable light chain" or “light chain variable region’' (VL) and "variable heavy chain" or “heavy chain variable region” (VH) refer to the light and heavy chains, respectively. The phrase "heavy chain" or "immunoglobulin heavy chain" encompasses an immunoglobulin heavy chain constant region sequence from any organism and, unless otherwise specified, includes a heavy chain variable domain. Heavy chain variable domains ty pically include three CDRs (complementarity-determining regions) and four FR (framework) regions. Fragments of heavy chains can include CDRs, CDRs and FRs, or combinations thereof. A ty pical heavy chain includes, following the variable domain (from N-terminal to C-terminal), a CHI domain, a hinge region, a CH2 domain, and a CH3 domain. A functional fragment of a heavy chain is one that can specifically recognize an antigen, be expressed and secreted from a cell, and comprises at least one CDR. A heavy chain immunoglobulin single variable domain refers to a domain that can function and express in the absence of a cognate light chain variable domain. The term "light chain" includes an immunoglobulin light chain variable region (VL or a functional fragment thereof) and an immunoglobulin constant domain (CL or a functional fragment thereof) from any organism. Unless otherwise specified, it may include light chains selected from human kappa, lambda, or combinations thereof.

[0196] An “antigen-binding site” or “binding portion” refers to the part of an antibody molecule or its fragment that engages in antigen binding. This site is formed by amino acid residues from the N-terminal variable regions of the heavy chain (VH) and light chain (VL). Within these variable regions, there are three highly divergent stretches known as “hypervariable regions,” which are interspersed between more conserved sequences called “framework regions'’ or “FRs.” Therefore, the term “FR” refers to the amino acid sequences naturally situated between and adjacent to the hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of both the light chain and heavy chain are arranged in three-dimensional space to create an antigen-binding “surface” that mediates the recognition and binding of the target antigen. These hypervariable regions are specifically termed “complementarity determining regions” or “CDRs,” and they are primarily responsible for interacting with an epitope of the antigen.

[0197] The term “antibody” encompasses both polyclonal and monoclonal antibodies, including any class of interest such as IgG, IgM, and their subclasses. It also includes hybrid antibodies, altered antibodies, F(ab')2 fragments, F(ab) molecules, Fv fragments, single chain variable fragments (ScFv) displayed on phage, single chain antibodies, single domain antibodies, diabodies, chimeric antibodies, humanized antibodies, and fragments thereof. In some embodiments, antibody fragments may be functional, exhibiting immunological binding properties similar to the parent antibody. The antibodies described herein can be labeled with detectable markers such as radioisotopes, enzymes that produce detectable products, fluorescent proteins, and the like. Labels suitable for in vivo imaging are of particular interest. Additionally, antibodies may be conjugated to other moieties, such as cytotoxic molecules or members of specific binding pairs.

[0198] In this disclosure, antibodies and their fragments include specific "framework regions" (FRs). “ScFv” refers to Single-chain Variable fragments. ‘Tab” denotes Fragment antigen-binding. “Dia” indicates a polypeptide structure with the following formats: CL, linker, VH, linker, VL. linker, CHI; or VL2, linker, VHL linker, VL1, linker, VH2.

[0199] The term “monoclonal antibody” refers to an antibody composition that consists of a homogeneous population of antibodies. This term includes whole antibody molecules, as well as various fragments and derivatives, such as Fab molecules, F(ab')2 fragments, Fv fragments, single-chain variable fragments (ScFv) displayed on phage, and fusion proteins comprising an antigen-binding portion of an antibody fused to a non-antibody protein. These molecules retain the immunological binding properties of the parent monoclonal antibody. Methods for producing and screening both polyclonal and monoclonal antibodies are well-established in the art.

[0200] Antibodies and fragments thereof according to the present disclosure include bispecific antibodies and their fragments. Bispecific antibodies are engineered to have two distinct antigen-binding sites, allowing them to bind to at least two different epitopes. These antibodies can have binding specificities for multiple antigens. Additionally, bispecific antibodies and their fragments can be in the form of heteroantibodies. Heteroantibodies consist of two or more antibodies or antibody fragments (e.g., Fab) that are linked together, each with a different specificity.

[0201] Antibody conjugates are also provided. These conjugates consist of an antibody from the present disclosure coupled with an additional agent. The agent may be selected from therapeutic agents, imaging agents, labeling agents, or any other agent useful for therapeutic and / or labeling purposes.

[0202] Conservative substitutions may be introduced at any position within a predetermined peptide or its fragments. However, non-conservative substitutions can also be desirable, particularly when they lead to the creation of functionally equivalent peptide fragments. Nonconservative substitutions may involve significant changes in polarity, electric charge, or steric bulk, while still maintaining the functional properties of the derivative or variant peptide.

[0203] The terms “derivative” and “variant” refer to any compound or antibody that has a structure or sequence derived from those described herein and is sufficiently similar to the disclosed compounds or antibodies. Based on this similarity, such derivatives or variants are expected to exhibit the same or similar activities and utilities as the original compounds or antibodies. These terms are often used interchangeably with “functional equivalent.” Modifications to create a derivative or variant can include additions, deletions, or substitutions of one or more amino acid residues. A functional equivalent or its fragment may also contain conservative amino acid substitutions. A “conservative amino acid substitution” is the replacement of an amino acid with another that has similar properties. Groups of conservative amino acids are well-known in art.

[0204] The “percentage of sequence identity” is determined by comparing two optimally aligned sequences within a comparison window. This comparison may involve introducing gaps (additions or deletions) to achieve optimal alignment between the sequences. To calculate the percentage of sequence identity: Align the Sequences: Align the two sequences such that they are optimally matched. This alignment may require introducing gaps to accommodate differences. Identify Matches: Determine the number of positions where the nucleic acid bases or amino acid residues are identical in both sequences. Calculate Percentage: Count the number of matched positions. Divide this number by the total number of positions in the comparison window (including gaps). Multiply the result by 100 to obtain the percentage of sequence identify. This percentage reflects the degree of similarity between the two sequences over the specified window.

[0205] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. 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 identities for the test sequences relative to the reference sequence, based on the program parameters.

[0206] The terms “identical” or percent “identify” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identify, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identify’ over a specified region, e.g., of the entire polypeptide sequences or individual domains of the polypeptides), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.’' This definition also refers to the complement of a test sequence. Optionally, the identity exists over a region that is at least about 5 to 50 nucleotides or polypeptide sequences in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides or polypeptide sequences in length.

[0207] “Cell(s) of interest” or “target cell(s)” used herein interchangeably refers to a cell or cells where one or more signaling pathways are intended to modulate. In some embodiments, the target cell(s) includes, but not limited to, a cancer cell(s). In some other embodiments, the target cell(s) includes immune effector cells such as natural killer cell(s), T cell(s), dendritic cell(s) and macrophage(s). A “cancer cell” as used herein refers to a cell exhibiting a neoplastic cellular phenotype, which may be characterized by one or more of, for example, abnormal cell growth, abnormal cellular proliferation, loss of density dependent growth inhibition, anchorage independent growth potential, ability to promote tumor growth and / or development in an immunocompromised non-human animal model, and / or any appropriate indicator of cellular transformation. “Cancer cell” may be used interchangeably herein with “tumor cell” or “cancerous cell”, and encompasses cancer cells of a solid tumor, a semi-solid tumor, a primary tumor, a metastatic tumor, and the like.

[0208] The term “effective amount” of a composition as provided herein is intended to mean a non-lethal but sufficient amount of the composition to provide the desired utility. For instance, for eliciting a favorable response in a cell(s) of interest (“target cell(s)”) such as modulating a signaling pathway, the effective amount of an (active, effective, potent or functional) antibody is the amount which results in notable and substantial change in the level of the activity of the signaling pathway, including downregulation and upregulation of the signaling pathway, when compared to use of no antibody or a control (inactive, ineffective, or non-functional) antibody. The measurement of changes in the level of the activity’ of the signaling pathway can be done by a variety of methods known in art. In another example, for eliciting a favorable response in a subject to treat a disease (e.g., cancer), the effective amount is the amount which reduces, eliminates, or diminishes the symptoms associated with the disorder, e.g., so as to provide for control of cancer metastasis, to eliminate cancer cells, and / or the like. As well be understood by a person having ordinary skill in the art, the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition or disease that is being treated, the particular composition used, its mode of administration, and the like. Thus, it is not possible to specify an exact '‘effective amount.” However, an appropriate effective amount may be determined by one of ordinary skills in the art using only routine experimentation.

[0209] By “treatment” in the context of disease or condition is meant that at least an amelioration of the symptoms associated with the condition afflicting an individual is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with the condition (e g., cancer) being treated. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g.. prevented from happening, or stopped, e.g., terminated, such that the host no longer suffers from the condition, or at least the symptoms that characterize the condition. Thus, treatment includes: (i) prevention, that is, reducing the risk of development of clinical symptoms, including causing the clinical symptoms not to develop, e.g., preventing disease progression to a harmful state; (ii) inhibition, that is, arresting the development or further development of clinical symptoms, e.g., mitigating or completely inhibiting an active disease, e.g., so as to decrease tumor load, which decrease can include elimination of detectable cancerous cells, or so as to protect against disease caused by bacterial infection, which protection can include elimination of detectable bacterial cells; and / or (iii) relief, that is, causing the regression of clinical symptoms.

[0210] The terms “individual” or “subject” are intended to cover humans, mammals, and other animals. The terms “individual” or “subject” are used interchangeably herein to refer to any mammalian subject to whom antibodies or fragments thereof in the present disclosure is subjected. The term “pharmaceutically acceptable excipient” as used herein refers to any suitable substance which provides a pharmaceutically acceptable compound for administration of a compound(s) of interest to a subject. “Pharmaceutically acceptable excipient” can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers.

[0211] As used herein, the term “pharmaceutically acceptable salts” refers to salts that retain the biological effectiveness and properties of the compounds of this disclosure and, which are not biologically or otherwise undesirable. In many cases, the compounds of the present disclosure are capable of forming acid and / or base salts by virtue of the presence of amino and / or carboxyl groups or groups similar thereto (e.g., phenol or hydroxamic acid).

[0212] Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. The pharmaceutically acceptable salts of the present disclosure can be synthesized from a parent compound, a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or the like), or by reacting free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two. Generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred, where practicable.

[0213] As used herein, the term “therapeutic combination” or “combination” refers to a combination of one or more active drug substances, i.e., compounds having a therapeutic utility. Typically, each such compound in the therapeutic combinations of the present disclosure will be present in a pharmaceutical composition comprising that compound and a pharmaceutically acceptable carrier. The compounds in a therapeutic combination of the present disclosure may be administered simultaneously or separately, as part of a regimen.

[0214] As used herein, the term “pharmaceutically acceptable carrier / excipient” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. Except in so far as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0215] As used herein, the term “protein complex” or “protein construct” refers to a complex having one or more polypeptides. In some embodiments, the protein complex has two or more polypeptides, wherein the polypeptides can associate with each other, forming a dimer or a multimer.

[0216] The protein complexes (e.g., antibodies) provided herein comprising three or more immunoglobulin heavy chain single variable domain binding regions are provided, as are methods for making them, nucleic acid constructs, and cell lines for making them.

[0217] The protein complexes (e.g.. antibodies) provided herein comprising one. two, or three or more immunoglobulin light chain single variable domains are provided, as are methods for making them, nucleic acid constructs, and cells lines for making them.

[0218] The protein complexes (e.g., antibodies) provided herein comprising novel formats and combinations of cognate heavy and light chain variable domains are provided, as are methods for making them, nucleic acid constructs, and cell lines for making them.

[0219] The protein complexes (e.g., antibodies) provided herein in various embodiments with various combinations of one or more of immunoglobulin heavy chain single variable domains, immunoglobulin light chain single variable domains, and cognate heavy and light chain variable domains are also provided, as well as methods for making them, nucleic acid constructs, and cell lines for making them.

[0220] The protein complexes (e.g., antibodies) provided herein are generally multivalent, indicating that they comprise two or more binding moieties. The two or more binding moieties may exhibit different specificities. Thus, embodiments of multivalent antigenbinding antibodies include multi-specific antigen-binding antibodies.

[0221] II. Compositions

[0222] In general, the present disclosure provides a crossbody platform that utilizes fragments (splits) of antibodies, cytokines, or other types of building blocks, strategically placed at various positions on a single antibody to create multi-specific antibody constructs. This design reduces toxicity, increases efficacy, and optimizes the number of polypeptide chains. It also enhances the yield, purity, and stability of multi-specific antibodies while improving their function for therapeutic applications, such as immunotherapy’ and antibodydrug conjugates (ADCs). The Crossbody platform can be efficiently expressed and purified using traditional methods. The antibodies demonstrated high potency both in vitro and in vivo, a broad therapeutic window, a favorable balance between efficacy and toxicity, and a longer half-life compared to most other multi-specific antibodies. In some embodiments, the protein complex (e.g., any of the protein complexes described herein) is an antibody or antigen-binding fragment thereof.

[0223] In some embodiments, the positioning of the antibody splits (VH and VL) can vary within a single antibody, allowing for flexibility in the arrangement of VH and VL domains in the first, second, and third polypeptides to construct novel formats.

[0224] In some embodiments, the number of polypeptides can be adjusted to create new7formats, offering versatility in antibody design.

[0225] In some embodiments, additional disulfide bonds can be introduced at various positions within the first and second polypeptides to construct new formats and enhance the stability of the antibody.

[0226] In some embodiments, the splits of the second and third antibodies can be a mixture of different building blocks, such as VH, VL, ScFv, Fab, splits of cytokines, and Diabodies; the mixture of different building blocks in the positions of each antigen in the first, second, and third polypeptides can be changed to construct new formats.

[0227] In some embodiments, multi-specific antibody constructs are designed based on knobs-into-holes or Fc structures. CH3 is linked with knobs-into-holes technology, and the CH3 position of the hole or knob can be exchanged to construct new formats. CH3 can also utilize a regular Fc without knobs-into-holes.

[0228] In general, the antibodies provided herein (Crossbody) resemble classic antibodies. They are more stable, possess a wide therapeutic window and a good balance between efficacy and toxicity, have a longer half-life, and are easier for downstream purification.

[0229] The antibodies provided herein have the following advantages: (1) they retain properties such as avidity, affinity, and potency of multi-specific antibodies (monoclonal antibodies, bispecific antibodies, trispecific antibodies, and tetraspecific antibodies, etc.); (2) they exhibit high stability and reduced aggregation; (3) they are easier to express and purify compared to other multi-specific antibodies; and (4) their structure is similar to native IgG, resulting in decreased immunogenicity.

[0230] A. Bispecific Antibody

[0231] In one aspect, the present disclosure provides antibodies, including bispecific antibodies utilizing the Crossbody platform. In one aspect, the present disclosure provides a protein complex utilizing a Crossbody platform for the construction of bispecific antibodies. This platform incorporates innovative splits of antibody domains designed at various positions within a single antibody framework. These antibodies can simultaneously recognize two antigens and are developed through “knob-into-hole’' technology. The protein complex comprises: (i) a first polypeptide that includes a first light chain variable region (VL1) that binds to a first target, a constant light chain (CL), and a second heavy chain variable region (VH2) that binds to a second target; (ii) a second polypeptide consisting of a first heavy chain variable region (VH1) that binds to the first target, along with a constant heavy chain 1 (CHI), a hinge region, a first CH2 and a first CH3 domain of IgG; (iii) a third polypeptide that includes a second light chain variable region (VL2) that binds to the second target, a hinge region, and a second CH2and a second CH3 domain of IgG.

[0232] Various Formats of Crossbody

[0233] The antibodies provided herein can have various formats or structures.

[0234] In some embodiments, the VH and VL positions of each antigen in the heavy and light chains are as follows: (i) a first polypeptide comprising VL1, CL, and VH2; (ii) a second polypeptide comprising VH1, CHI, CH2, and CH3; and (iii) a third polypeptide comprising VL2, CH2, and CH3.

[0235] In some embodiments, the first polypeptide has the following structure fromN-terminus to C-terminus: VL1-CL-VH2.

[0236] In some embodiments, the second polypeptide has the following structure from N-terminus to C-terminus: VH1-CH1-CH2-CH3.

[0237] In some embodiments, the third polypeptide has the following structure from N-terminus to C-terminus: VL2-CH2-CH3.

[0238] In some embodiments, the VH and VL positions of each antigen in the heavy and light chains can be altered to construct new formats, such as: (i) a first polypeptide comprising VH2-VL1-CL; (ii) a second polypeptide comprising VH1, VL2, CH2, and CH3; and (iii) a third polypeptide comprising VL2, CH2, and CH3.

[0239] In some embodiments, the first polypeptide has the following structure from N-tenninus to C-terminus: VH2-VL1-CL.

[0240] In some embodiments, the second polypeptide has the following structure from N-terminus to C-terminus: VH1-VL2-CH2-CH3. In some embodiments, the third polypeptide has the following structure fromN-terminus to C-terminus: VL2-CH2-CH3.

[0241] The antibodies provided herein generally comprise a covalent link to connect the polypeptides.

[0242] In some embodiments, VL1 and VH1 are covalently linked via a disulfide bond. In some embodiments, the framework region (FR) of VL1 and the FR of VH1 are covalently linked via the aforementioned disulfide bond.

[0243] In some embodiments, VL2 and VH2 are covalently linked via a disulfide bond. In some embodiments, the FR of VL2 and the FR of VH2 are covalently linked via the aforementioned disulfide bond.

[0244] In some embodiments, additional disulfide bond positions can be located at both VH2 / VL2 and VH 1 / VLI in the first and second polypeptides to construct new formats.

[0245] In some embodiments, the positioning of the splits in the second target antibody (VH and VL) can vary within a single antibody, allowing for flexibility in the arrangement of VH and VL domains in the first, second, and third polypeptides to construct novel formats.

[0246] In some embodiments, the number of polypeptides can be changed to construct new formats. In some embodiments, additional targets can be added to construct multi-specific antibodies.

[0247] In some embodiments, position -100 of VL1 (Kabat) and position -44 of VH1 (Kabat) are substituted with cysteine. In some embodiments, for example, VL1:... GXG... to... GGG... and VH 1:... GLEW... to... CLEW.... In some embodiments, contemplated pairs of amino acids to be selected are: VH44-VL100, VH105-VL43, VH105-VL42, VH44-VL101, VH106-VL43, VH104-VL43, VH44-VL99, VH45-VL98, VH46-VL98, VH103-VL43, VH103-VL44, VH103-VL45.

[0248] In some embodiments, position -43 of VL1 (according to Kabat definition) and position -105 of VH1 (according to Kabat definition) are substituted with cysteine. In some embodiments, the VL and VH (e.g., any of the VH / VL pairs described herein) are covalently-linked via a disulfide bond, which is formed by a non-native disulfide bond formed by a cysteine at position 43 in the VL1 (according to Kabat numbering) and a cysteine at position 105 in the VH1 (according to Kabat numbering).

[0249] In some embodiments, either the first polypeptide or the second polypeptide is independently linked at its C-terminus to a hinge region of IgGl, IgG2, IgG3, or IgG4. In some embodiments, either the first polypeptide or the second polypeptide is independently linked at its C-terminus to a hinge region and CH2-CH3 of IgGl, IgG2, IgG3, IgG4, or IgA, to form a classic antibody -like homodimer.

[0250] In a further aspect, the present disclosure provides a protein complex employing the Crossbody platform to construct bispecific antibodies with splits designed at various positions within a single antibody. The antibody comprises: (i) a first polypeptide that includes a first light chain variable region (VL1) that binds to a first target and a first CL; (ii) a second polypeptide that includes a first heavy chain variable region (VH1) that binds to the first target, along with a first CHI, a first hinge region, a first CH2 and a first CH3 domain of IgG, and a second heavy chain variable region (VH2) that binds to a second target; (iii) a third polypeptide that includes a third heavy chain variable region (VH3) that binds to the first target, along with a second CHI, a second hinge region, a second CH2 and a second CH3 domain of IgG, and a second light chain variable region (VL2) that binds to the second target; and (iv) a fourth polypeptide that includes a third light chain variable region (VL3) that binds to the first target and a second CL. In some embodiments, the VH3 is identical to the VHL and the VL3 is identical to the VL1.

[0251] In a further aspect, the present disclosure provides a protein complex employing the Crossbody platform to construct bispecific antibodies with splits designed at various positions within a single antibody. The antibody comprises: (i) a first polypeptide that includes a first light chain variable region (VL1) that binds to a first target, a CL. and a second heavy chain variable region (VH2) that binds to a second target; (ii) a second polypeptide that includes a first heavy chain variable region (VH1) that binds to the first target, along with a CHI, a first hinge region, a first CH2 and a first CH3 domain of IgG; (iii) a third polypeptide that includes a third heavy chain variable region (VH3) that binds to the first target, along with a TCR Va, a second hinge region, a second CH2 and a second CH3 domain of IgG; and (iv) a fourth polypeptide that includes a third light chain variable region (VL3) that binds to the first target, a TCR Vb, and a second light chain variable region (VL2) that binds to a second target. In some embodiments, the VH3 is identical to the VHL and the VL3 is identical to the VL1.

[0252] In some embodiments, the CH3 domain in the polypeptide containing the Fc segment is modified into a “knob’’ structure, while the CH3 domain of the heave chain of a traditional antibody is modified into a “hole” structure, then co-expressed with a light chain (LC) from a traditional antibody. This combination enables the construction of Crossbody. Point mutations at multiple sites are engaged and screened for further optimization, determining the structure and construction method of Crossbody. Through the addition of a third antigenrecognizing functional domain, we achieve either an AAB (2:1) or ABC (1:1:1) configuration (where A, B, and C each represent a target of selection) in target binding, resulting in different mechanisms of action (MO A) and pharmacokinetic properties.

[0253] The AAB (2:1) type of construction consists of two pairs of VH1-VL1 targeting antigen A and one pair of VH2-VL2 targeting antigen B. Bi-covalent interaction between CD3 antibodies and T cells induces T cell apoptosis and significantly increases clinical cytokine release syndrome (CRS) risk due to the large release of cytokines. Thus, to lower CRS risk, single valent interaction for CD3 antibodies is always adopted when constructing T cell-engaged antibodies. The bi-valent interaction for the other antigen increases the antibody’s avidity for the antigen and results in two advantages: (1) the antibody can recognize antigens with low abundance when a single-chain antibody has high affinity for the antigen, and (2) the antibody is highly selective when interacting with antigens, binding only to those with high abundance and not to those with low abundance when a single-chain antibody has low affinity for the antigen.

[0254] In some embodiments, the first polypeptide is linked via a hinge region of its C terminus to the N terminus of an Fc region. The hinge region comprises a hinge from IgGl, IgG2, IgG3, IgG4, or IgA. The Fc region comprises CH2 and CH3 of IgGl, IgG2, IgG3, IgG4, or IgA.

[0255] In some embodiments, the third target and the first target are the same target.

[0256] In some embodiments, the third target and the second target are the same target. In some embodiments, the first target and the second target are the same target.

[0257] In some embodiments, the CH2-CH3 domain of the second polypeptide and the CH2-CH3 domain of the third polypeptide are different.

[0258] In some embodiments, the second polypeptide and the third polypeptide are engineered through modification to the CH3 domain interface with different mutations on each domain.

[0259] B. Trispeciflc Antibody and Other Multi-specific Antibodies

[0260] In one aspect, the present disclosure provides antibodies, such as trispecific antibodies utilizing the Crossbody platform.

[0261] In one aspect, the present disclosure further provides a protein complex using the Crossbody platform to construct trispecific antibodies with splits designed at various positions within a single antibody. The trispecific antibody comprises: (i) a first polypeptide that includes a first light chain variable region (VL1) that binds to a first target, a CL, and a second heavy chain variable region (VH2) that binds to a second target; (ii) a second polypeptide that includes a first heavy chain variable region (VH1) that binds to the first target, along with a CHI, a first hinge region, a first CH2 and a first CH3 domain of IgG; and (iii) a third polypeptide that includes a single-chain variable fragment (ScFv) that binds to a third target, a second light chain variable region (VL2) that binds to the second target, a second hinge region, a second CH2 and a second CH3 domain of IgG.

[0262] In one aspect, the present disclosure provides a protein complex utilizing the Crossbody platform to construct trispecific antibodies or mixtures of different building blocks designed at various positions within a single antibody. The trispecific antibody comprises: (i) a first polypeptide that includes a first light chain variable region (VL1) that binds to a first target, a CL, a second heavy chain variable region (VH2) that binds to a second target, and a third heavy chain variable region (VH3) that binds to a third target; (ii) a second polypeptide that includes a first heavy chain variable region (VH1) binding to the first target, along with a CHI. a first hinge region, a first CH2 and a first CH3 domain of IgG; and (iii) a third polypeptide that includes a third light chain variable region (VL3) that binds to the third target, a second hinge region, a second CH2 and a second CH3 domain of IgG, and a second light chain variable region (VL2) that binds to the second target.

[0263] In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct trispecific antibodies with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The antibody comprises: (i) a first polypeptide that includes a first light chain variable region (VL1) that binds to a first target, a constant light chain (CL), a third heavy chain variable region (VH3) that binds to a third target, and a second heavy chain variable region (VH2) that binds to a second target; (ii) a second polypeptide that includes a first heavy chain variable region (VH1) that binds to the first target, along with a CHI, a first hinge region, a first CH2 and a first CH3 domain of IgG; and (iii) a third polypeptide that includes a third light chain variable region (VL3) that binds to the third target, a second hinge region, a second CH2 and a second CH3 domain of IgG, and a second light chain variable region (VL2) that binds to the second target.

[0264] In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct trispecific antibodies with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The antibody comprises: (i) a first polypeptide that includes a second heavy chain variable region (VH2) that binds to a second target, a third heavy chain variable region (VH3) that binds to a third target, a first light chain variable region (VL1) that binds to a first target, and a CL; (ii) a second polypeptide that includes a first heavy chain variable region (VH1) that binds to the first target, along with a CHI, a first hinge region, a first CH2 and a first CH3 domain of IgG; and (iii) a third polypeptide that includes a third light chain variable region (VL3) that binds to the third target, a second hinge region, a second CH2 and a second CH3 domain of IgG, and a second light chain variable region (VL2) that binds to the second target.

[0265] In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct trispecific antibodies with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The antibody comprises: (i) a first polypeptide that includes a first light chain variable region (VL1) that binds to a first target, a CL, and a second heavy chain variable region (VH2) that binds to a second target; (ii) a second polypeptide that includes a first heavy chain variable region (VH1) that binds to the first target, along with a CHI, a first hinge region, a first CH2 and a first CH3 domain of IgG; and (iii) a third polypeptide that includes a second light chain variable region (VL2) that binds to the second target, a single-chain variable fragment (ScFv) that binds to the third target, a second hinge region, a second CH2 and a second CH3 domain of IgG.

[0266] In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct trispecific antibodies with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The antibody comprises: (i) a first polypeptide that includes a first light chain variable region (VL1) that binds to a first target, a second heavy chain variable region (VH2) that binds to a second target, a CL, and a third heavy chain variable region (VH3) that binds to a third target; (ii) a second polypeptide that includes a second light chain variable region (VL2) that binds to the second target, a first heavy chain variable region (VH1) that binds to the first target, a CHI, a first hinge region, a first CH2 and a first CH3 domain of IgG; and (iii) a third polypeptide that includes a third light chain variable region (VL3) that binds to the third target, a second hinge region, a second CH2 and a second CH3 domain of IgG.

[0267] In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct tetra-specific antibodies with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The antibody comprises: (i) a first polypeptide that includes a second heavy chain variable region (VH2) that binds to a second target, a first light chain variable region (VL1) that binds to a first target, a first CL, and a third heavy chain variable region (VH3) that binds to a third target; (ii) a second polypeptide comprising a first heavy chain variable region (VH1) that binds to the first target, along with a first CHI, a first hinge region, a first CH2 and a first CH3 domain of IgG; (iii) a third polypeptide comprising a fourth heavy chain variable region (VH4) that binds to a fourth target, a second CHI, a second hinge region, a second CH2 and a second CH3 domain of IgG; and (iv) a fourth polypeptide comprising a second light chain variable region (VL2) that binds to the second target, a fourth light chain variable region (VL4) that binds to the fourth target, a second CL, and a third light chain variable region (VL3) that binds to the third target.

[0268] In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct tetra-specific antibodies with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The antibody comprises: (i) a first polypeptide that includes a first light chain variable region (VL1) that binds to a first target, a CL, a second heavy chain variable region (VH2) that binds to a second target, and a third heavy chain variable region (VH3) that binds to a third target; (ii) a second polypeptide that includes a first heavy chain variable region (VH1) that binds to the first target, along with a CHI, a first hinge region, a first CH2 and a first CH3 domain of IgG; and (iii) a third polypeptide that includes a ScFv that binds to the fourth target, a third light chain variable region (VL3) that binds to the third target, a second hinge region, a second CH2 and a second CH3 domain of IgG, and a second light chain variable region (VL2) that binds to the second target.

[0269] In some embodiments, the VH and VL position of each antigen in heavy and light chain are like: (i) a first polypeptide comprising VL1, CL, VH2, and VH3; (ii) a second polypeptide comprising VH1, CHI, CH2, and CH3; and (iii) a third polypeptide comprising VL3, CH2, CH3, and VL2.

[0270] In some embodiments, the first polypeptide has the following structure from N terminus to C terminus: VL1-CL-VH2-VH3.

[0271] In some embodiments, the second polypeptide has the following structure from N terminus to C terminus: VH1-CH1-CH2-CH3.

[0272] In some embodiments, the third polypeptide has the following structure from N terminus to C terminus: VL3-CH2-CH3-VL2.

[0273] In some embodiments, the VH and VL position of each antigen in heavy and light chain can be changed to construct new formats, like: (i) a first polypeptide comprising VL1- CL-VH3-VH2: and (ii) a second polypeptide comprising VH1, VL2, CH2, and CH3; and (iii) a third polypeptide comprising VL3. CH2, CH3, and VL2.

[0274] In some embodiments, the first polypeptide has the following structure from N terminus to C terminus: VL1-CL-VH3-VH2.

[0275] In some embodiments, the second polypeptide has the following structure from N terminus to C terminus: VH1-VL2-CH2-CH3.

[0276] In some embodiments, the third polypeptide has the following structure from N terminus to C terminus: VL3-CH2-CH3-VL2.

[0277] The antibodies provided herein generally comprise a covalent link to link the polypeptides.

[0278] In some embodiments, the VL1 and the VH1 are covalently linked via a disulfide bond.

[0279] In some embodiments, the framework region (FR) of the VL1 and FR of the VH1 are covalently linked via said disulfide bond.

[0280] In some embodiments, the VL2 and the VH2 are covalently linked via a disulfide bond.

[0281] In some embodiments, the framework region (FR) of the VL2 and FR of the VH2 are covalently linked via said disulfide bond.

[0282] In some embodiments, the additional disulfide bond positions can be at both VH2 / VL2 and VH 1 / VL1 in the first and second polypeptide to construct new formats.

[0283] In some embodiments, the positioning of the antibody splits (VH and VL) can vary within a single antibody, allowing for flexibility in the arrangement of VH and VL domains in the first, second, and third polypeptides to construct novel formats.

[0284] In some embodiments, for multi-specific antibody designs, the approach typically involves several targets: First Target: this target is chosen for its low expression level and strong tumor specificity. It directs the antibody primarily to the tumor. Second Target: this is a tumor- associated target with high expression but lower tumor specificity. This target is designed with concealment to minimize on-target / off-tumor toxicity and enhance the potency of the multi-specific antibody. Third Target: this target is a T cell target and is designed with concealment to minimize toxicity. In some embodiments, both the second and third targets may be T cell targets with concealment strategies to further reduce toxicity7.

[0285] In some embodiments, the number of polypeptides can be adjusted to create new formats, offering versatility in antibody design; in some embodiments, additional targets can be added to construct multi-specific antibodies; in some embodiments, additional disulfide bonds can be introduced at various positions within the first and second polypeptides to construct new formats and enhance the stability of the antibody.

[0286] In some embodiments, the splits of the second and third antibodies can be a mixture of different building blocks, such as: VH, VL, ScFv, Fab, and Diabody; and the mixture of different building blocks at the positions of each antigen in the first, second, and third polypeptides can be changed to construct new formats.

[0287] In some embodiments, the fourth target and the first target are the same target. In some embodiments, the third target and the second target are the same target. In some embodiments, the first target and the second target are the same target. In some embodiments, the first target and the third target are the same target.

[0288] C. Checkpoint Antibody

[0289] In one aspect, the present disclosure provides antibodies, such as multi-specific checkpoint antibodies with the Crossbody platform.

[0290] In one aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct multi-specific checkpoint antibodies with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The antibody comprises: (i) a first polypeptide that includes a first light chain variable region (VL1) that binds to a first target, a CL, and a second heavy chain variable region (VH2) that binds to a second target; (ii) a second polypeptide that includes a first heavy chain variable region (VH1 ) that binds to the first target, a CH 1, a first hinge region, a first CH2 and a first CH3 domain of IgG; and (iii) a third polypeptide that includes a second light chain variable region (VL2) that binds to the second target, a second hinge region, a second CH2 and a second CH3 domain of IgG.

[0291] In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct multi-specific checkpoint antibodies with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The antibody comprises: (i) a first polypeptide that includes a first light chain variable region (VL1) that binds to a first target and a first CL; (ii) a second polypeptide that includes a first heavy chain variable region (VH1) that binds to the first target, a first CHI, a first hinge region, a first CH2 and a first CH3 domain of IgG, and a second heavy chain variable region (VH2) that binds to a second target; (iii) a third polypeptide that includes a third heavy chain variable region (VH3) that binds to the first target, a second CHI, a second hinge region, a second CH2 and a second CH3 domain of IgG, and a second light chain variable region (VL2) that binds to the second target; and (iv) a fourth polypeptide that includes a third light chain variable region (VL3) that binds to the first target and a second CL.

[0292] In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct multi-specific checkpoint antibodies with splits of antibodydomains or mixtures of different building blocks designed at various positions within a single antibody. The antibody comprises: (i) a first polypeptide that includes a second heavy chain variable region (VH2) that binds to a second target, a first light chain variable region (VL1) that binds to a first target, a CL, and a second light chain variable region (VL2) that binds to the second target; and (ii) a second polypeptide that includes a first heavy- chain variable region (VH1) that binds to the first target, a CHI, a hinge region, a CH2 and aCH3 domain of IgG.

[0293] In some embodiments, the positioning of the antibody splits (VH and VL) can vary within a single antibody, allowing for flexibility in the arrangement of VH and VL domains in the first, second, and third polypeptides to construct novel formats.

[0294] In some embodiments, the number of polypeptides can be adjusted to create new formats, offering versatility in antibody design; in some embodiments, additional targets can be added to construct multi-specific antibodies.

[0295] In some embodiments, additional disulfide bonds can be introduced at various positions within the first and second polypeptides to construct new formats and enhance the stability of the antibody.

[0296] In some embodiments, the splits of the second and third antibodies can be a mixture of different building blocks, like: VH, VL, ScFv, Fab, and Diabody; and the mixture of different building blocks at the position of each antigen in the first, second, and third polypeptide can be changed to construct new formats.

[0297] D. Antibody-Cytokine

[0298] In one aspect, the present disclosure provides antibodies, such as multi-specific antibody-cytokine with the Crossbody platform.

[0299] In one aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct antibody-cytokines with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The antibody comprises: (i) a first polypeptide that includes a first light chain variable region (VL1) that binds to a first target, a CL, and a split of cytokine-A; (ii) a second polypeptide that includes a first heavy chain variable region (VH1) that binds to the first target, a CHI, a first hinge region, a first CH2 and a first CH3 domain of IgG; and (iii) a third polypeptide that includes a split of cytokine-B, a second hinge region, a second CH2 and a second CH3 domain of IgG.

[0300] In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct antibody-cytokines with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The antibody comprises: (i) a first polypeptide that includes a first light chain variable region (VL1) that binds to a first target, a CL, and a split of cytokine- A; and (ii) a second polypeptide that includes a first heavy7chain variable region (VH1) that binds to the first target, a CHI, a hinge region, a CH2 and a CH3 domain of IgG, and a split of cytokine-B.

[0301] In some embodiments, the positioning of the splits of the cytokine can vary within a single antibody, allowing for flexibility in the arrangement of the split of cytokine in the first, second, and third polypeptides to construct novel formats.

[0302] In some embodiments, the number of polypeptides can be adjusted to create new formats, offering versatility in antibody design; in some embodiments, additional targets can be added to construct multi-specific antibodies.

[0303] In some embodiments, additional disulfide bonds can be introduced at various positions within the first and second polypeptides to construct new formats and enhance the stability of the antibody.

[0304] In some embodiments, the splits of the second and third antibodies can be a mixture of different building blocks, such as: a split of cytokine, VH, VL, ScFv, Fab, and Diabody; and the mixture of different building blocks at the positions of each antigen in the first, second, and third polypeptides can be changed to construct new formats.

[0305] E. Probody

[0306] In one aspect, the present disclosure provides antibodies, such as multi-specific probody with the Crossbody platform.

[0307] In one aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct multi-specific probody with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The antibody comprises: (i) a first polypeptide that includes a second light chain variable region (VL2) that binds to a second target, a first heavy chain variable region (VH1) that binds to a first target, a first light chain variable region (VL1) that binds to the first target, and a second heavy chain variable region (VH2) that binds to the second target, a first hinge region, a first CH2 and a first CH3 domain of IgG; (ii) a second polypeptide that includes a third heavy chain variable region (VH3) that binds to a third target and a CHI. a second hinge region, a second CH2 and a second CH3 domain of IgG; and (iii) a third polypeptide that includes a blocking mask, a linker with a cleavage site, a third light chain variable region (VL3) that binds to the third target, and a CL.

[0308] In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct multi-specific probody with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The antibody comprises: (i) a first polypeptide that includes a first CL, a first heavy chain variable region (VH1) that binds to a first target, a first light chain variable region (VL1) that binds to the first target, a first CHI, a first hinge region, a first CH2 and a first CH3 domain of IgG, and a second heavy chain variable region (VH2) that binds to a second target; (ii) a second polypeptide that includes a third heavy chain variable region (VH3) that binds to the third target and a second CHI, a second hinge region, a second CH2 and a second CH3 domain of IgG, and a second light chain variable region (VL2) that binds to the second target; and (iii) a third polypeptide that includes a blocking mask, a linker with a cleavage site, a third light chain variable region (VL3) that binds to the third target, and a second CL.

[0309] In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct multi-specific probody with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The antibody comprises: (i) a first polypeptide that includes a ScFv that binds to a first target, a first hinge region, a first CH2 and a first CH3 domain of IgG, and a second heavy chain variable region (VH2) that binds to a second target; (ii) a second polypeptide that includes a third heavy chain variable region (VH3) that binds to a third target, a CHI, a second hinge region, a second CH2 and a second CH3 domain of IgG, and a second light chain variable region (VL2) that binds to the second target; and (iii) a third polypeptide that includes a blocking mask, a linker with a cleavage site, a third light chain variable region (VL3) that binds to the third target, and a CL.

[0310] In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct multi-specific probody with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The antibody comprises: (i) a first polypeptide that includes a first light chain variable region (VL1) that binds to a first target, a CL, and a second heavy chain variable region (VH2) that binds to a second target; (ii) a second polypeptide that includes a first heavy chain variable region (VH1) that binds to the first target, a CHI, a first hinge region, a first CH2 and a first CH3 domain of IgG. a blocking mask, a linker w ith a cleavage site, and a second light chain variable region (VL2) that binds to the second target, and (iii) a third polypeptide that includes a ScFv that binds to a third target, a second hinge region, a second CH2 and a second CH3 domain of IgG.

[0311] In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct multi-specific probody with splits of antibody domains or mixtures of different building blocks designed at various positions within a single antibody. The antibody comprises: (i) a first polypeptide that includes a blocking mask, a linker with a cleavage site, a first hinge region, a first light chain variable region (VL1) that binds to a first target, a first CH2 and a first CH3 domain of IgG. and a second heavy chain variable region (VH2) that binds to a second target; (ii) a second polypeptide that includes a first heavy chain variable region (VH1) that binds to the first target, a CHI, a second hinge region, a second CH2 and a second CH3 domain of IgG; and (iii) a third polypeptide that includes a ScFv that binds to a third target, a second light chain variable region (VL2) that binds to the second target, a third hinge region, a third CH2 and a third CH3 domain of IgG.

[0312] In some embodiments, the positioning of the antibody splits (VH and VL) can vary within a single antibody, allowing for flexibility' in the arrangement of VH and VL domains in the first, second, and third polypeptides to construct novel formats.

[0313] In some embodiments, for multi-specific probody designs, the approach typically involves several targets: First Target: This target is chosen for its low' expression level and strong tumor specificity'. It directs the antibody primarily to the tumor. Second Target: This is a tumor-associated target with high expression but lower tumor specificity. This target is designed with blocking mask with cleavage site to minimize on-target / off-tumor toxicity and enhance the potency of the multi-specific antibody. Third Target: This target is aT cell target and is designed with concealment to minimize toxicity.

[0314] In some embodiments, the number of polypeptides can be adjusted to create new formats, offering versatility in antibody design; in some embodiments, additional targets can be added to construct multi-specific antibodies; in some embodiments, additional disulfide bonds can be introduced at various positions within the first and second polypeptides to construct new' formats and enhance the stability' of the antibody.

[0315] In some embodiments, the splits of the second and third antibodies can be a mixture of different building blocks, such as: VH, VL. ScFv, Fab. split of cytokine, and Diabody; and the mixture of different building blocks at the positions of each antigen in the first, second, and third polypeptides can be changed to construct new formats.

[0316] F. Novel Antibody

[0317] In one aspect, the present disclosure provides antibodies, such as multi-specific novel antibody with the Crossbody platform.

[0318] In one aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct multi-specific probody with splits of antibody domains (like only VH or VL) or mixtures of different building blocks designed at various positions within a single antibody. The antibody comprises: (i) a first polypeptide that includes a first light chain variable region (VL1) that binds to a first target, a CL, and a second heavy chain variable region (VH2) that binds to a second target; (ii) a second polypeptide that includes a first heavy chain variable region (VH1) that binds to the first target, a CHI, a first hinge region, a first CH2 and a first CH3 domain of IgG; and (iii) a third polypeptide that comprises a second heavy chain variable region (VH2) that binds to the second target, a second hinge region, a second CH2 and a second CH3 domain of IgG.

[0319] In a further aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct multi-specific probody with splits of antibody domains (like only VH or VL) or mixtures of different building blocks designed at various positions within a single antibody. The antibody comprises: (i) a first polypeptide that includes a first light chain variable region (VL1) that binds to a first target, a CL, and a second light chain variable region (VL2) that binds to a second target; (ii) a second polypeptide that includes a first heavy chain variable region (VH1) that binds to the first target, a CHI, a first hinge region, a first CH2 and a first CH3 domain of IgG; and (iii) a third polypeptide that includes a second CH2 and a second CH3 domain of IgG and a second light chain variable region (VL2) that binds to the second target.

[0320] In some embodiments, the positioning of the antibody splits (VH or VL) can vary' within a single antibody, allowing for flexibility in arranging the VH and VL domains across the first, second, and third polypeptides to create novel formats. The antibody splits may include combinations such as VH only, VL only, VH + VH, or VL + VL.

[0321] In some embodiments, the number of polypeptides can be adjusted to create new' formats, offering versatility' in antibody design; in some embodiments, additional targets can be added to construct multi-specific antibodies. In some embodiments, additional disulfide bonds can be introduced at various positions within the first and second polypeptides to construct new formats and enhance the stability of the antibody.

[0322] In some embodiments, the splits of the second and third antibodies can be a mixture of different building blocks, such as: VH, VL, ScFv, Fab, split of cytokine, and Diabody; and the mixture of different building blocks at the positions of each antigen in the first, second, and third polypeptides can be changed to construct new formats.

[0323] G. Antibody Drug Conjugates

[0324] In one aspect, the present disclosure provides antibodies, such as multi-specific Antibody-drug conjugates with the Crossbody platform.

[0325] In one aspect, the present disclosure provides a protein complex utilizing a Crossbody platform to construct multi-specific probody with splits of antibody domains (like only VH or VL) or mixtures of different building blocks designed at various positions within a single antibody. The antibody comprises: (i) a first polypeptide that includes a first light chain variable region (VL1) that binds to a first target, a CL, and a second heavy chain variable region (VH2) that binds to a second target; (ii) a second polypeptide that includes a first heavy chain variable region (VH1) that binds to the first target, a CHI, a first hinge region, a first CH2 and a first CH3 domain of IgG; and (iii) a third polypeptide that includes a second light chain variable region (VL2) that binds to a second target, a second hinge region, a second CH2 and a second CH3 domain of IgG.

[0326] In some embodiments, the positioning of the antibody splits (VH or VL) can vary' within a single antibody, allowing for flexibility in arranging the VH and VL domains across the first, second, and third polypeptides to create novel formats.

[0327] In some embodiments, the number of polypeptides can be adjusted to create new formats, offering versatility in antibody design; in some embodiments, additional targets can be added to construct multi-specific antibodies.

[0328] In some embodiments, additional disulfide bonds can be introduced at various positions within the first and second polypeptides to construct new formats and enhance the stability of the antibody.

[0329] In some embodiments, the splits of the second and third antibodies can be a mixture of different building blocks, such as: VH, VL, ScFv, Fab, split of cytokine, and Diabody; and the mixture of different building blocks at the positions of each antigen in the first, second, and third polypeptides can be changed to construct new formats. H. Disulfide Stabilized Antibody Molecules

[0330] In some embodiments, position -100 of the VL1 (Kabat) and position -44 of the VH1 (Kabat) are substituted with cystine. In some embodiments, like, VL1:... GXG... to

[0331] ... GGG... and VH1:... GLEW... to... CLEW.... In some embodiments, contemplated pairs of amino acids to be selected are: VH44-VL100, VH105-VL43, VH105-VL42. VH44-VL101, VH106-VL43. VH104-VL43. VH44-VL99, VH45-VL98, VH46-VL98, VH103-VL43, VH103-VL44, VH103-VL45. Details can be found in WO2019120245A1, which is incorporated herein by reference in its entirety.

[0332] In one embodiments, the first and / or the second and / or the third antigen binding site is disulfide stabilized by introduction of cysteine residues independently of each other at the following positions to form a disulfide bond between the VH and VL domains (numbering according to Kabat): VH at position 44, and VL at position 100, VH at position 105, and VL at position 43, or VH at position 101, and VL at position 100. Details can be found, e.g., in EP 3 704 146 Bl, which is incorporated herein by reference in its entirety.

[0333] In one embodiments, the disulfide bond between a variable region pair, for example VH1 and VL1 and / or VH2 and VL2 is between (unless the context indicates otherwise Kabat numbering is employed in the list below). Wherever reference is made to Kabat numbering the relevant reference is Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA:

[0334] I. VH37+VL95C see for example Protein Science 6, 781-788 Zhu et al (1997); 2. VH44+VL100 see for example; Biochemistry 33 5451-5459 Reiter et al (1994); or Journal of Biological Chemistry Vol. 269 No. 28 pp. 18327-18331 Reiter et al (1994); or Protein Engineering, vol. 10 no. 12 pp. 1453-1459 Rajagopal et al (1997);

[0335] 3. VH44+VL105 see for example J Biochem. 118, 825-831 Luo et al (1995); 4. VH45+VL87 see for example Protein Science 6, 781-788 Zhu et al (1997); 5. VH55+VL101 see for example FEBS Letters 377 135-139 Young et al (1995); 6. VH100+VL50 see for example Biochemistry 29 1362-1367 Glockshuber et al (1990);

[0336] 7. VH100b+VL49;

[0337] 8. VH98+VL 46 see for example Protein Science 6, 781-788 Zhu et al (1997); 9. VH101+VL46;

[0338] 10. VH105+VL43 see for example; Proc. Natl. Acad. Sci. USA Vol. 90 pp. 7538-7542 Brinkmann et al (1993); or Proteins 19, 35-47 Jung et al (1994), or 11. VH106+VL57 see for example FEBS Letters 377 135-139 Young et al (1995); 12. or a position corresponding thereto in variable region pair located in the molecule.

[0339] The amino acid pairs listed above are in the positions conducive to replacement by cysteines such that disulfide bonds can be formed. Cysteines can be engineered into these desired positions by known techniques. In one embodiment therefore an engineered cysteine according to the present disclosure refers to where the naturally occurring residue at a given amino acid position has been replaced with a cysteine residue. (Disulfide stabilized DVD-Ig molecules)

[0340] I. TCR and Antigen-Binding Fragment Thereof

[0341] T cells are a type of lymphocyte which typically develops in the thymus gland and plays a central role in the immune response. It plays an important role in the "adaptive immune response. " T cells can be distinguished from other lymphocytes by the presence of a T-cell receptor on the cell surface. Differentiated T cells have an important role in controlling the immune response. CD8+ T cells, also known as "killer cells", are cytotoxic. Once they recognize a target cell, they are able to directly kill the target cell (e.g., virus -infected cells or cancer cells). CD8+ T cells can also produce cytokines and recruit other cells (e.g., macrophages and natural killer (NK) cells) to mount an immune response. CD4+ T cells, also known as "helper cells", can indirectly kill target cells, e.g.. by facilitating maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete cytokines that regulate or assist the immune response. Regulatory T cells are important for tolerance, thereby preventing or inhibiting autoimmune response. The major role of regulatory T cells is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress autoreactive T cells that escaped the process of negative selection in the thymus.

[0342] T cells play an important role in cancer immunity where antigens from the cancer cells are taken up and presented on the cell surface of special immune cells called antigen-presenting cells (APCs) so that other immune cells can recognize the antigens of interest. In the lymph nodes, the APCs activate the T-cells and activate them to recognize the tumor cells. The activated T-cells can then travel via the blood vessels to reach the tumor, infiltrate it, recognize the cancer cells and kill them. The activation of T cells requires T cell receptors (TCRs). A “T cell receptor” or “TCR” is a molecule that contains a variable a (or alpha) and b (or beta) chains (also known as TCRa and TCRβ, respectively) or a variable g (or gamma) and d (or delta) chains (also known as TCRy and TCRδ, respectively), or antigen-binding portions thereof, and which is capable of specifically binding to an antigen, e.g., a peptide antigen or peptide epitope bound to an major histocompatibility complex (MHC) molecule.

[0343] The present disclosure provides aT cell receptor (TCR) or antigen-binding fragment thereof, and binding molecules derived from TCR. In some embodiments, the TCR is in the a.p form. TCRs that exist in a(3 and yS forms are generally structurally similar, but T cells expressing them may have distinct anatomical locations or functions. Generally, a TCR is found on the surface of T cells (or T lymphocytes) where it is generally responsible for recognizing antigens, such as peptides bound to major histocompatibility complex (MHC) molecules.

[0344] In some embodiments, the TCR is an intact or full-length TCR, such as a TCR containing the a chain and b chain. In some embodiments, the TCR is an antigen-binding portion that is less than a full- length TCR but that binds to a specific peptide bound in an MHC molecule, such as binds to an MHC-peptide complex. In some cases, an antigenbinding portion or fragment of a TCR can contain only a portion of the structural domains of a full-length or intact TCR, but yet is able to bind the peptide epitope, such as MHC-peptide complex, to which the full TCR binds. In some cases, an antigen-binding portion contains the variable domains of a TCR, such as variable a (Va or Va) chain and variable b (Vb or V[3) chain of a TCR, or antigen -binding fragments thereof sufficient to form a binding site for binding to a specific MHC-peptide complex.

[0345] The a-chain and / or b-chain of a TCR also can contain a constant domain, a transmembrane domain and / or a short cytoplasmic tail. In some aspects, each chain (e.g. alpha or beta) of the TCR can possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C -terminal end. In some embodiments, a TCR, for example via the cytoplasmic tail, is associated with invariant proteins of the CD3 complex involved in mediating signal transduction. In some cases, the structure allows the TCR to associate with other molecules like CD3 and subunits thereof. For example, a TCR containing constant domains with a transmembrane region may anchor the protein in the cell membrane and associate with invariant subunits of the CD3 signaling apparatus or complex. The intracellular tails of CD3 signaling subunits (e.g. CD3y, CD35, CD3e and CD3z chains) contain one or more immunoreceptor tyrosine-based activation motif or ITAM and generally are involved in the signaling capacity of the TCR complex.

[0346] The exact locus of a domain or region can vary depending on the particular structural or homology modeling or other features used to describe a particular domain. In some cases, the specific domain (e.g. variable or constant) can be several amino acids (such as one, two, three or four) longer or shorter. In some aspects, residues of a TCR are known or can be identified according to the International Immunogenetics Information System (IMGT) numbering system (see e.g. www.imgt.org; Lefranc et al. "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains." Developmental & Comparative Immunology 27.1 (2003): 55-77.). The structures and variations of TCR are known in the art, and are described, e.g., in WO 2019 / 195486, which is incorporated herein by reference in its entirety.

[0347] J. Disease Specific Target

[0348] In general, the target (e.g., the first target) is a disease-specific target.

[0349] As used herein, a “targef ’ refers to an antigen (such as a tumor antigen or cell-specific biomarker like a protein) or an epitope of an antigen.

[0350] The disease-specific target may include a tumor target, immune cell target, neoantigen, or disease-specific receptor.

[0351] In some embodiments, the disease-specific target is selected from the disease markers, cytokines, or chemokines listed in Table 1.

[0352] TABLE 1 Target List

[0353] Receptors Cytokines Chemokines Disease Markers

[0354] PDL1 IL-4 CCL1 HER2

[0355] PDL2 IL- 16 CCL2 HER3

[0356] CTLA4 TGFbeta CCL3 CEA

[0357] KIR IL-1 CCL4 Muc-1

[0358] IDO-1 IL-6 CCL5 GPCR3

[0359] 4-1BB IL-10 CCL6 Alpha fetoprotein (AFP)

[0360] OX40L IL-12 CCL7 CA15-3

[0361] LAG3 IL- 18 CCL8 CA27-29

[0362] CD47 IL- 17 CCL9 CAI 9-9

[0363] CD80 IL- 15 CCL10 CA-125

[0364] CD86 IL13 CCL11 Calcitonin

[0365] B7RP1 IL-23 CCL12 Calretinin

[0366] B7-H3 IL21 CCL13 Carcinoembryonic antigen

[0367] HVEM IL-32 CCL14 CD34

[0368] CD137L IL-9 CCL15 CD99MIC 2

[0369] CD70 IL28 CCL16 CD117

[0370] GAL9 Leptin CCL17 Chromogranin

[0371] CD4 IL9 CCL18 TRK TIM3 IFN CCL19 Cytokcratin (various types: TP A, TPS, Cyfra21-1)

[0372] TIM4 BAFF CCL20 Desmin

[0373] Adenosine receptor Oncostatin CCL21 Epithelial membrane antigen (EMA) TAM VEGF CCL22 Factor VIII, CD31 FL 1

[0374] Vista CCL23 Glial fibrillary acidic protein (GFAP) BTLA Type I CCL24 Gross cystic disease fluid protein (GCDFP- IFNs 15)

[0375] HLA-G TNF CCL25 HMB-45

[0376] IDO-2 RANKL CCL26 Human chorionic gonadotropin (hCG) ARG1 NGF CCL27 immunoglobulin

[0377] GCP3 CSF CCL28 inhibin

[0378] Trop-2 TNF-alpha CXCL1 keratin (various types)

[0379] Claudin CD30L CXCL2 lymphocyte marker (various types) FOXO CD40L CXCL3 MART-1 (Melan-A)

[0380] BCMA CD27L CXCL4 Myo DI

[0381] TRK TNFSF10 CXCL5 muscle- specific actin (MSA)

[0382] Her2 IL-2 CXCL6 neurofilament

[0383] Her3 BMP CXCL7 neuron- specific enolase (NSE) EGFR GDF CXCL8 placental alkaline phosphatase (FLAP) GITR GDNF CXCL9 prostate-specific antigen (PSA) PD1 CXCL10 PTPRC (CD45)

[0384] CD3 CXCL 11 S 100 protein

[0385] CD8 CXCL 12 smooth muscle actin (SMA)

[0386] CD 16 CXCL 13 synaptophysin

[0387] CD19 CXCL14 thymidine kinase

[0388] CD20 CXCL 15 thyroglobulin (Tg)

[0389] CD21 CXCL 16 thyroid transcription factor- 1 (TTF- 1 ) CD22 CXCL17 Tumor M2-PK

[0390] CD23 FAM19 vimentin

[0391] CD24 CA-125

[0392] CD27

[0393] CD33 Epithelial tumor antigen (ET A) CD38 Tyrosinase

[0394] CD40 Melanoma-associated antigen (MAGE) CD32 abnormal products of ras, p53 CD64

[0395] CD 123

[0396] CCR1

[0397] CCR2

[0398] CCR3

[0399] CCR4

[0400] CCR5

[0401] CCR6

[0402] CCR7

[0403] CCR8

[0404] CXCR1

[0405] CXCR2

[0406] CXCR3

[0407] CXCR4

[0408] CXCR5

[0409] CXCR6

[0410] CXCR7

[0411] CD116 / GM-CSFR

[0412] CD131 / CSFR2B / JL3RB / IL5RB

[0413] CD115 / MC SF R / CSF1R

[0414] CD114 / G-CSFR

[0415] BMP receptor

[0416] GDNF receptor

[0417] TGF-beta receptor FcRn

[0418] DR IL6R

[0419] IT,

[0420] GPCR MUC1

[0421] prostate stem cell antigen

[0422] prostate membrane antigen

[0423] Mesothelin

[0424] CD47

[0425] FGFR1

[0426] KLB CD93

[0427] Aug

[0428] ROR

[0429] EpCam

[0430] In certain embodiments, a target is a tumor marker. A tumor marker can be an antigen present in a tumor but not in normal organs, tissues, or cells. It can also be an antigen that is more prevalent in a tumor compared to normal tissues or in malignant cancer cells compared to normal cells.

[0431] As used herein, a “tumor antigen” refers to an antigenic substance produced by tumor cells that triggers an immune response in the host. Normal proteins in the body generally do not provoke an immune response due to self-tolerance, a process in which self-reacting cytotoxic T lymphocytes (CTLs) and autoantibody -producing B lymphocytes are eliminated in primary lymphatic tissues (e.g., bone marrow) and secondary lymphatic tissues (e.g., thymus for T-cells and spleen / lymph nodes for B cells). Consequently, proteins that are not exposed to the immune system can trigger an immune response. This includes normal proteins that are sequestered from the immune system, produced in very small amounts, expressed only during specific developmental stages, or modified due to mutations.

[0432] In some embodiments of the disclosure, a marker is a tumor marker. This marker maybe a polypeptide expressed at higher levels in dividing cells compared to non-dividing cells.

[0433] In some embodiments, the target is preferentially expressed in tumor tissues and / or cells relative to normal tissues and / or cells.

[0434] In some embodiments, the tumor antigen (or tumor target) is selected from the group consisting of: CD2, CD19. CD20, CD22, CD27, CD33. CD37, CD38, CD40, CD44, CD47, CD52, CD56, CD70, CD79, and CD137.

[0435] Antibodies or other drugs that speci fical ly target these tumor antigens can interfere with and regulate the signaling pathways involved in tumor cell behavior. They can directly block these pathways to inhibit tumor cell growth or induce apoptosis. To date, numerous targeted drugs have been approved for the treatment of solid tumors and hematological malignancies, and many targeted therapies are available for hematological malignancies.

[0436] In some embodiments, the tumor antigen (or tumor target) is selected from the group consisting of: 4-1BB, 5T4, AGS-5, AGS-16, Angiopoietin 1, Angiopoietin 2, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, Carcinoembryonic Antigen, CD93, CTLA4, Cripto, ED-B, ErbBl, ErbB2, ErbB3, ErbB4, EGFL7, EpCam, EphA2, EphA3, EphB2, FAP, Fibronectin, Folate Receptor, Ganglioside GM3, GD2, Glucocorticoid-Induced Tumor Necrosis Factor Receptor (GITR), gplOO, gpA33, GPNMB, ICOS, IGF1R, Integrin av, Integrin avp>. KIR, LAG-3, Lewis Y Antigen, Mesothelin, c-MET, MN Carbonic Anhydrase IX, MUCL MUC16, Nectin-4. NKG2D, NOTCH, 0X40, OX40L, PD-1, PDL1, PSCA, PSMA. RANKL. RORL R0R2. SLC44A4, Syndecan-1, TACI, TAG-72, Tenascin, TIM3, TRAILR1, TRAILR2, VEGFR-1, VEGFR-2, VEGFR-3, and their variants. These variants include various mutants or polymorphisms known in the art and / or naturally occurring.

[0437] In some embodiments, the disease-specific target is selected from cancer- or tumor-associated antigens, including CD30, CD33, PSMA, Mesothelin, CD44, CD73, CD38, Mucin 1 (MUC1), Mucin 2 (MUC2), and Mucin 16 (CA-125).

[0438] In some embodiments, the disease-specific target is selected from antigens that are overexpressed in cancer cells, including intercellular adhesion molecule 1 (ICAM-1), ephrin type-A receptor 2 (EphA2), ephrin type-A receptor 3 (EphA3), ephrin type-A receptor 4 (EphA4), or activated leukocyte cell adhesion molecule (ALCAM).

[0439] In some embodiments, the disease-specific target is selected from CD30, CD33, Carcinoembryonic Antigen (CEA), mesothelin, cathepsin G, CD44, CD73, CD38, Mucl, Mucl6, preferentially expressed antigen of melanoma (PRAME). CD52, EpCAM, gpA33. Mucins, TAG-72, carbonic anhydrase IX, PSMA, folate binding protein, gangliosides, Lewis-Y, ICAM-1, EphA2, or ALCAM.

[0440] In some embodiments, the disease-specific target is selected from CD30, CD33, Carcinoembryonic Antigen (CEA), mesothelin, cathepsin G, CD44, CD73, CD38, Mucl, Muc2, Mucl6, PRAME, CD52, EpCAM, gpA33, Mucins, tumor-associated glycoprotein 72 (TAG-72), carbonic anhydrase IX, PSMA, folate binding protein, gangliosides, Lewis-Y, immature laminin receptor, BING-4, calcium-activated chloride channel 2 (CaCC), gplOO, synovial sarcoma X breakpoint 2 (SSX-2), or SAP-I.

[0441] In some embodiments, the immune regulatory function target is selected from one of the receptors listed in Table 1. The immune regulatory function target may be a checkpoint receptor, a regulator ' cytokine receptor, or similar.

[0442] Generally, one of the antigens (e.g., the second antigen) is an immune regulatory function target related to the disease target.

[0443] The single domain of the present disclosure specifically binds to a target.

[0444] In some embodiments, the immune regulatory function target is involved in NK cell activation or inhibition and is selected from CD 16, CD38, NKG2D, NKG2A, NKp46, or Killer-cell immunoglobulin-like receptors (KIRs).

[0445] In some embodiments, the immune regulatory' function target is related to the checkpoint inhibitory' pathway (active in T cells) and is selected from PD-1, CTLA-4, or TIM-3.

[0446] By "‘specifically binds’’ or ‘‘preferably binds,” it is meant that the binding between two binding partners (e.g., between a targeting moiety7and its binding partner) is selective and distinguishable from unwanted or non-specific interactions. For instance, the ability' of an antigen-binding moiety’ to bind to a specific antigenic determinant can be measured using techniques such as enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance. The terms “anti-[antigen] antibody” and “an antibody that binds to [antigen]” refer to an antibody capable of binding to the antigen with sufficient affinity’ for diagnostic and / or therapeutic purposes. In some embodiments, the binding of an anti-[antigen] antibody to an unrelated protein is less than about 10% of its binding to the target antigen, as measured by techniques such as radioimmunoassay (RIA).

[0447] In some embodiments, the antigen-binding moiety' has a dissociation constant (KD) of <100 pM, <10 pM, <1 pM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10-4M or less, e.g., from 10-4M to 10-12M, e.g., from 10-9M to 10-13M), with a preferred range being from 10-4M to 10-6M.

[0448] By “target” or “marker,” it is meant any entity- that can specifically bind to a particular therapeutic agent, such as Her2 / Neu. In some embodiments, targets are specifically-associated with one or more particular cell or tissue types, disease states, or developmental stages. For example, a cell type-specific marker is typically expressed at levels at least 2-fold higher in that cell type compared to a reference population of cells. In some embodiments, the marker may be present at levels that are at least 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, or even 1,000-fold greater than its average expression in a reference population. Detection or measurement of a cell type-specific marker may enable the differentiation of the target cell t pe from many, most, or all other cell types. In some embodiments, a target can include a protein, carbohydrate, lipid, and / or nucleic acid as described herein.

[0449] In some embodiments, the targeted therapeutic comprises an antibody or a functional fragment thereof.

[0450] The term “antibody” is used broadly to include various antibody structures, such as monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g.. bispecific antibodies), and antibody fragments, provided they exhibit the desired antigen-binding activity and include an Fc region or a region equivalent to the Fc region of an immunoglobulin. The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used interchangeably to refer to antibodies with a structure similar to a native antibody or with heavy chains that contain an Fc region.

[0451] The term “immunoglobulin” or “antibody” refers to a full-length immunoglobulin molecule (e.g., an IgG antibody) or an immunologically active portion of an immunoglobulin, such as an antibody fragment. Antibodies and antibody fragments may be conjugated or otherwise modified. Examples include IgGl, IgG2a, IgG3, IgG4 (and its subforms), as well as IgA isotypes.

[0452] An “antibody fragment” refers to a molecule that includes a portion of an intact antibody capable of binding to the same antigen as the intact antibody. Examples of antibody fragments include Fv, Fab. Fab', Fab'-SH, F(ab')2. diabodies, linear antibodies, single-chain antibody molecules (e.g., ScFv). single-domain antibodies, VH, VL, cytokines, cytokine splits, proteins, split of proteins, or combinations of these components, and multi-specific antibodies derived from these fragments. Antibody fragments can be produced through various techniques, including proteolytic digestion of intact antibodies or recombinant expression in host cells (e.g., E. coli or phage).

[0453] “Native antibodies” refer to naturally occurring immunoglobulin molecules with diverse structures. For example, native IgG antibodies are heterotetrameric glycoproteins with a molecular weight of approximately 150,000 daltons. They are composed of two identical light chains and two identical heavy chains, linked by disulfide bonds. Each heavy chain includes a variable region (VH), also known as a variable heavy domain, followed by three constant domains (CHI, CH2, and CH3), collectively termed the heavy chain constant region. Each light chain features a variable region (VL), also called a variable light domain, followed by a constant light domain (CL), known as the light chain constant region. Light chains are classified as either kappa (K) or lambda ( / .) based on the amino acid sequence of their constant domains. The “variable region'’ or “variable domain” of an antibody refers to the part of the heavy or light chain involved in antigen binding. The variable domains of both the heavy chain (VH) and light chain (VL) typically have similar structures, each comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain may be sufficient to confer antigen-binding specificity-.

[0454] The term “antigen-binding domain” refers to the part of an antibody that specifically binds to and is complementary to a part or the entirety of an antigen. This domain may be provided by one or more antibody variable domains, also known as antibody variable regions. Specifically, an antigen-binding domain includes both an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0455] The antibody described herein may be a chimeric antibody, a humanized antibody, a human antibody, or an antibody fusion protein.

[0456] A “human antibody” is an antibody derived from transgenic mice that have been engineered to produce human antibodies in response to antigenic challenge. In this process, human heavy and light chain gene segments are introduced into strains of mice with targeted disruptions of their endogenous heavy and light chain loci. These transgenic mice can then produce human antibodies specific for human antigens, which can be harvested and used to create human antibody-secreting hybridomas.

[0457] A “humanized antibody” is a recombinant protein in which the complementarity-determining regions (CDRs) from an antibody of one species, such as a rodent, are grafted onto human heavy and light chain variable domains. The constant domains of the antibody molecule are derived from human antibodies. In some embodiments, specific residues in the framework regions of the humanized antibody, particularly those near the CDR sequences, may be modified (e.g., replaced with corresponding residues from the original rodent or another species).

[0458] A “chimeric antibody” is a recombinant protein that includes the variable domains of both the heavy and light antibody chains, incorporating the CDRs from an antibody of one species (preferably a rodent, more preferably a murine antibody), while the constant domains are derived from human antibodies. For veterinary applications, the constant domains of the chimeric antibody may come from other species, such as subhuman primates, cats, or dogs.

[0459] An “antibody fusion protein” is a recombinantly-produced antigen-binding molecule where two or more segments from natural antibodies, single-chain antibodies, or antibody fragments, with the same or different specificities, are linked. A fusion protein contains at least one specific binding site. The valency of the fusion protein refers to the total number of binding arms or sites it has for antigens or epitopes, such as monovalent, bivalent, trivalent, or multi -valent. Multivalency enhances the avidity of binding to an antigen or multiple antigens. Specificity denotes the variety of antigens or epitopes the fusion protein can bind, such as monospecific, bispecific, trispecific, or multi-specific. For example, a natural antibody like IgG is bivalent because it has two binding arms but is monospecific because it binds to one type of antigen or epitope. A monospecific, multivalent fusion protein has multiple binding sites for the same antigen or epitope, such as a monospecific diabody with two binding sites for the same antigen. The fusion protein may include a combination of different antibody components, multiple copies of the same component, or an additional therapeutic agent.

[0460] In certain embodiments, the antibodies of the present disclosure may comprise a single-domain antibody or fragment that specifically binds to one or more targets (e.g., antigens) associated with organs, tissues, cells, extracellular matrix components, and / or intracellular compartments. In some embodiments, the compounds include a targeting moiety that specifically binds to targets associated with a particular organ or organ system. In other embodiments, the compounds feature a nucleus-targeting moiety that specifically binds to one or more intracellular targets (e.g., organelles, intracellular proteins). Additionally, some compounds include a targeting moiety that specifically binds to targets associated with diseased organs, tissues, cells, extracellular matrix components, and / or intracellular compartments. In certain cases, the targeting moiety may specifically bind to targets associated with particular cell types (e.g., endothelial cells, cancer cells, malignant cells, prostate cancer cells).

[0461] The term “target"’ or “marker” refers to any entity capable of specifically binding to a particular targeting moiety. Targets may be specifically associated with one or more particular cell or tissue types, disease states, or developmental stages. For example, a cell type-specific marker is typically expressed at levels at least 2-fold greater in that cell type compared to a reference population of cells. In some embodiments, the marker is expressed at levels at least 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, or 1,000-fold greater than its average expression in the reference population. Detection or measurement of a cell type-specific marker may enable differentiation of the cell type or ty pes of interest from many, most, or all other types. Targets may include proteins, carbohydrates, lipids, and / or nucleic acids, as described herein.

[0462] A substance is considered “targeted” for the purposes described herein if it specifically binds to a nucleic acid targeting moiety. In some embodiments, a nucleic acid targeting moiety binds specifically to a target under stringent conditions. An inventive complex or compound is considered "targeted’' if the targeting moiety specifically binds to a target, thereby facilitating the delivery of the entire complex or compound to a specific organ, tissue, cell, extracellular matrix component, and / or intracellular compartment.

[0463] In some embodiments, a target may be a marker that is exclusively or primarily associated with one or a few cell types, diseases, or developmental stages. A cell typespecific marker is typically expressed at levels at least 2-fold greater in that cell type compared to a reference population, which may consist, for example, of a mixture containing cells from multiple (e.g., 5-10 or more) tissues or organs in approximately equal amounts. In some embodiments, the cell type-specific marker is present at levels at least 3 -fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold. 9-fold, 10-fold, 50-fold, 100-fold, or 1,000-fold greater than its average expression in the reference population. Detection or measurement of a cell typespecific marker may enable the distinction of the cell type or types of interest from many, most, or all other cell types.

[0464] In some embodiments, antibodies in accordance with the present disclosure comprise a domain antibody or fragment that binds to a target specific to one or more particular tissue types (e g., liver tissue vs. prostate tissue). In some embodiments, compounds in accordance with the present disclosure comprise a domain that binds to a target specific to one or more particular cell types (e.g., T cells vs. B cells). In some embodiments, antibodies in accordance with the present disclosure comprise a domain that binds to a target specific to one or more particular disease states (e.g., tumor cells vs. healthy cells). In some embodiments, compounds in accordance with the present disclosure comprise a targeting moiety that binds to a target specific to one or more particular developmental stages (e.g., stem cells vs. differentiated cells).

[0465] Numerous markers are known in the art, including typical markers such as cell surface proteins and receptors. Exemplary receptors include, but are not limited to, the transferrin receptor, LDL receptor, growth factor receptors (e.g., epidermal growth factor receptor family members such as EGFR, Her2, Her3, Her4), vascular endothelial growth factor receptors, cytokine receptors, cell adhesion molecules, integrins, selectins, and CD molecules. The marker can be a molecule present exclusively or in higher amounts on malignant cells, such as a tumor antigen.

[0466] In some embodiments, a target comprises a protein, carbohydrate, lipid, and / or nucleic acid. For instance, a target can be a protein or a characteristic portion thereof, such as a tumor marker, integrin, cell surface receptor, transmembrane protein, intercellular protein, ion channel, membrane transporter protein, enzyme, antibody, chimeric protein, or glycoprotein. Similarly, a target can be a carbohydrate or a characteristic portion thereof, such as a glycoprotein, sugar (e.g., monosaccharide, disaccharide, polysaccharide), or glycocalyx (the carbohydrate-rich peripheral zone on the outside surface of most eukaryotic cells). A target may also comprise a lipid or a characteristic portion thereof, such as an oil, fatty acid, glyceride, hormone, steroid (e.g., cholesterol, bile acid), vitamin (e.g., vitamin E), phospholipid, sphingolipid, or lipoprotein. Additionally, a target can be a nucleic acid or a characteristic portion thereof, such as DNA, RNA, modified DNA, modified RNA, or any combination thereof.

[0467] The binding of a targeting moiety to a tumor cell can be measured using assays known in the art.

[0468] In some embodiments, the binding domain binds specifically or preferentially to a tumor cell in comparison to a non-tumor cell.

[0469] In some embodiments, the binding domain is capable of binding specifically or preferentially to a tumor antigen in comparison to a non-tumor antigen.

[0470] In some embodiments, the tumor cell may be from a carcinoma, sarcoma, lymphoma, myeloma, or central nervous system cancer.

[0471] In some embodiments, the targeting moiety7comprises folic acid or a derivative thereof.

[0472] In certain specific embodiments, a target is a tumor marker. A tumor marker is an antigen that is present in a tumor but not in normal organs, tissues, and / or cells. In some embodiments, a tumor marker is an antigen that is more prevalent in a tumor than in normal organs, tissues, and / or cells. Additionally, in some embodiments, a tumor marker is an antigen that is more prevalent in malignant cancer cells compared to normal cells.

[0473] Data indicates that the expression of folate receptor (FR) in tumor cells is 20 to 200 times higher than in normal cells. The expression rates of FR in various malignant tumors are as follows: 82% in ovarian cancer, 66% in non-small cell lung cancer, 64% in kidney cancer, 34% in colon cancer, and 29% in breast cancer. The expression of FR and the degree of malignancy of epithelial tumor invasion and metastasis are positively correlated. Folate (FA) enters cells through FR-mediated endocytosis. FA forms complexes with drugs via its carboxyl group, facilitating drug entry into the cells. Under acidic conditions (pH 5), FR dissociates from the FA, allowing FA to release the drugs into the cytoplasm.

[0474] In some embodiments, the targeting moiety can be a Fab, Fab', F(ab')2, single domain antibody, T and Abs dimer, Fv, ScFv, dsFv, ds-ScFv, Fd, linear antibody, minibody, diabody, bispecific antibody fragment, bibody, tribody, sc-diabody, kappa ( ) body, BiTE, DVD-Ig, SIP, SMIP. DART, Dia, VHH, VH, VL. cytokines, cytokine splits, proteins, split of proteins, or combinations of these components, or an antibody analogue comprising one or more CDRs.

[0475] In some embodiments, the targeting moiety may include extracellular domains (ECDs) or soluble forms of PD-1, PDL-1, CTLA4, CD47, BTLA, KIR, TIM3, 4-1BB, and LAG3. It may also comprise the full length or partial forms of surface ligands such as Amphiregulin, Betacellulin, EGF, Ephrin, Epigen, Epiregulin, IGF, Neuregulin, TGF, TRAIL, Angiopoietin 1, Angiopoietin 2, CD93, or VEGF.

[0476] In some embodiments, the targeting moiety is an antibody or antibody fragment selected based on its specificity for an antigen expressed on a target cell or at a target site of interest. Various tumor-specific or disease-specific antigens have been identified, and antibodies targeting these antigens have been utilized or proposed for the treatment of tumors or other diseases.

[0477] The antibodies that are known in the art can be used in the compounds of the disclosure, in particular for the treatment of the disease with which the target antigen is associated. Examples of target antigens (and their associated diseases) to which an antibodylinker-drug conjugate of the disclosure can be targeted include: CD2, CD19, CD20, CD22, CD27, CD33, CD37. CD38, CD40, CD44, CD47. CD52, CD56, CD70, CD79, CD137, 4-1BB. 5T4, AGS-5. AGS-16. Angiopoietin 1, Angiopoietin 2, CD93, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, Carcinoembryonic antigen, CTLA4, Cripto, ED-B, ErbBl, ErbB2, ErbB3, ErbB4, EGFL7, EpCam, EphA2, EphA3, EphB2, FAP, Fibronectin, Folate Receptor, Ganglioside GM3, GD2, glucocorticoid-induced tumor necrosis factor receptor (GITR), gplOO, gpA33, GPNMB, ICOS, IGF1R, Integrin av, Integrin otvp, KIR, LAG-3, Lewis Y, Mesothelin, c-MET, MN Carbonic anhydrase IX, MUC1, MUC16, Nectin-4, NKGD2, NOTCH, 0X40, OX40L, PD-1, PDL1, PSCA, PSMA, RANKL, R0R1, R0R2, SLC44A4, Syndecan-1, TACI, TAG-72, Tenascin, TIM3, TRAILR1, TRAILR2, VEGFR-1, VEGFR-2, VEGFR-3.

[0478] Clinically, this system can be utilized to selectively deliver drugs to tumor cells. Folate acid (FA), due to its small molecular weight, non-immunogenicity, high stability, and low cost of synthesis, has become a popular choice for targeting molecules in drug delivery systems for cancer treatment. The chemical coupling between the drug and the carrier is straightforward, making FA a promising candidate for constructing effective drug delivery systems. For instance, EC145, a folate-conjugated chemotherapy drug currently in clinical trials, has shown effectiveness in targeting and attacking cancer cells.

[0479] All antibody formats described are derived from the heavy and light chains of an IgG antibody. These antibodies can be produced using well-established methods in the art.

[0480] Typically, the process involves constructing expression cassettes for the heavy and light chain genes, co-transfecting these genes into an appropriate cell system to generate recombinant antibodies, and selecting stable, high-yielding cell clones. The final antibody product is produced through cell fermentation under cGMP conditions.

[0481] The present disclosure also encompasses pharmaceutical formulations that include a compound of the disclosure or a pharmaceutically acceptable salt thereof, combined with one or more pharmaceutically acceptable carriers.

[0482] The terms “administering” or “administration” are intended to cover all methods of delivering a compound to its intended site of action, whether directly or indirectly.

[0483] The compounds described herein, including those formulated with pharmaceutically acceptable carriers, such as addition salts or hydrates, can be administered to a patient via various routes or modes of administration. These routes include, but are not limited to, inhalation, transdermal, oral, rectal, transmucosal, intestinal, and parenteral methods, such as intramuscular, subcutaneous, and intravenous injections. Preferably, compounds comprising an antibody or antibody fragment as the targeting moiety are administered parenterally, with intravenous administration being the most preferred method.

[0484] The compounds described, or their pharmaceutically acceptable salts and / or hydrates, may be administered alone, in combination with other compounds of the disclosure, or as part of a therapeutic cocktail with other agents. The choice of additional therapeutic agents for coadministration will depend on the specific condition being treated.

[0485] When administered to a patient undergoing cancer treatment, the compounds may be provided in combination with anti-cancer agents and / or supplementary potentiating agents. Additionally, the compounds may be included in formulations with agents designed to manage the side effects of radiation therapy, such as anti-emetics and radiation protectants.

[0486] The active compounds of the disclosure may be administered directly or incorporated into a pharmaceutical composition. Such compositions typically consist of the active compound(s) mixed with one or more pharmaceutically acceptable carriers, excipients, or diluents. These pharmaceutical compositions are formulated in a conventional manner using appropriate carriers and auxiliaries that facilitate the processing of the active compounds into usable pharmaceutical preparations. The specific formulation will depend on the chosen route of administration.

[0487] In some embodiments, the pharmaceutical composition of the present disclosure may include an additional therapeutic agent.

[0488] In certain embodiments, the additional anticancer agent may be selected from antimetabolites, inhibitors of topoisomerase I and II, alkylating agents, microtubule inhibitors, antiandrogen agents, GNRH modulators, or a combination thereof.

[0489] In some embodiments, the additional therapeutic agent is an anticancer agent.

[0490] In some embodiments, the additional therapeutic agent is a chemotherapeutic agent. As used herein, a "chemotherapeutic agent" refers to a chemical compound employed in cancer treatment. Examples include, but are not limited to: Gemcitabine. Irinotecan.

[0491] Doxorubicin, 5-Fluorouracil, Cytosine arabinoside (Ara-C), Cyclophosphamide, Thiotepa, Busulfan, Cytoxan, TAXOL, Methotrexate, Cisplatin, Melphalan, Vinblastine, and Carboplatin.

[0492] In some embodiments, the secondary chemotherapeutic agent may be selected from the group consisting of tamoxifen, raloxifene, anastrozole, exemestane, letrozole, imatinib, paclitaxel, cyclophosphamide, lovastatin, minocycline, gemcitabine, cytarabine, 5-fluorouracil, methotrexate, docetaxel, goserelin, vincristine, vinblastine, nocodazole, teniposide, etoposide, epothilone, vinorelbine. camptothecin, daunorubicin, actinomycin D, mitoxantrone, acridine, doxorubicin, epirubicin. or idarubicin.

[0493] In another aspect, the present disclosure provides kits containing the therapeutic combinations described herein, along with instructions for use. The kit may also include a container and optionally one or more vials, test tubes, flasks, bottles, or syringes. Other kit formats will be evident to those skilled in the art and fall within the scope of the present disclosure.

[0494] In a further aspect, the present disclosure provides a method for treating a disease condition in a subject in need thereof, comprising administering to the subject a therapeutic combination or pharmaceutical composition containing a therapeutically effective amount of the compound of the present disclosure or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0495] In addition to the compositions and constructs described above, the present disclosure also provides a number of uses of the combinations of the disclosure. Uses of the combinations of the current disclosure include killing or inhibiting the growth, proliferation or replication of a tumor cell or cancer cell, treating cancer, treating a pre-cancerous condition, preventing the multiplication of a tumor cell or cancer cell, preventing cancer, preventing the multiplication of a cell that expresses an auto-immune antibody. These uses comprise administering to an animal such as a mammal or a human in need thereof an effective amount of a compound of the present disclosure.

[0496] For the purposes of this disclosure, "cancer" refers to the pathological condition in humans characterized by unregulated cell proliferation. Examples include, but are not limited to: carcinoma, lymphoma, blastoma, and leukemia. More specific examples of cancers include, but are not limited to: lung cancer (both small cell and non-small cell), breast cancer, prostate cancer, carcinoid tumor, bladder cancer, gastric cancer, pancreatic cancer, liver cancer (hepatocellular), hepatoblastoma, colorectal cancer, head and neck squamous cell carcinoma, esophageal cancer, ovarian cancer, cervical cancer, endometrial cancer, mesothelioma, melanoma, sarcoma (including osteosarcoma, liposarcoma), thyroid cancer, desmoid tumors, chronic myelogenous leukemia (CML), and acute myeloid leukemia (AML).

[0497] The combination of the present disclosure is useful for treating diseases, such as cancer, in subjects, including humans. Combinations and methods for treating tumors involve administering to a subject a composition in a pharmaceutically acceptable manner, with a pharmaceutically effective amount of a composition of the present disclosure.

[0498] As used herein, a "therapeutically effective amount" refers to the quantity of a compound that is effective to "treat" a disorder in a subject or mammal. In the context of cancer, this therapeutically effective amount may result in one or more of the following outcomes: a reduction in the number of cancer cells, a decrease in tumor size, inhibition of cancer cell infiltration into peripheral organs, prevention of tumor metastasis, attenuation of tumor growth, and / or alleviation of one or more symptoms associated with the cancer.

[0499] As used herein, "inhibiting," "treating," or "treatment" refers to reduction, therapeutic intervention, and prophylactic or preventative measures aimed at reducing or preventing the pathological disorder or condition. For example, after administering a compound of the present disclosure, a cancer patient may experience a reduction in tumor size. "Treatment" or "treating" encompasses (1) inhibiting a disease in a subject who is experiencing or displaying the pathology or symptoms of the disease, (2) ameliorating a disease in a subject with the pathology or symptoms, and / or (3) achieving any measurable decrease in the disease in a subject or patient showing the pathology or symptoms. If a compound of the present disclosure can prevent growth and / or kill cancer cells, it may exhibit cytostatic and / or cytotoxic effects. In some embodiments, the disease condition is a tumor or cancer. Examples of cancers or tumors include, but are not limited to, stomach, colon, rectal, liver, pancreatic, lung, breast, cervix uteri, corpus uteri, ovary, testis, bladder, renal, brain / CNS, head and neck, throat, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, leukemia, melanoma, non-melanoma skin cancer, acute lymphocytic leukemia, acute myelogenous leukemia, Ewing's sarcoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, Wilms' tumor, neuroblastoma, hairy cell leukemia, mouth / pharynx, oesophagus, larynx, kidney cancer, or lymphoma.

[0500] "Administration 'in combination with' one or more further therapeutic agents" includes both simultaneous (concurrent) and sequential administration in any order. The term “pharmaceutical combination” refers to a product obtained by mixing or combining active ingredients and encompasses both fixed and non-fixed combinations of the active ingredients. A "fixed combination" means that the active ingredients and a co-agent are administered simultaneously in the form of a single dosage unit. A "non-fixed combination" refers to the administration of active ingredients and a co-agent as separate entities, either simultaneously, concurrently, or sequentially, without specific time constraints, ensuring that therapeutically effective levels of the active ingredients are achieved in the patient’s body. This approach also applies to cocktail therapies, such as the administration of three or more active ingredients.

[0501] In some embodiments, the disease condition includes abnormal cell proliferation, such as a pre-cancerous lesion.

[0502] The compounds of the present disclosure can target a variety of cancers or tumors, including but not limited to lung cancer, colon cancer, prostate cancer, lymphoma, melanoma, breast cancer, ovarian cancer, testicular cancer, CNS cancer, renal cancer, kidney cancer, pancreatic cancer, stomach cancer, oral cancer, nasal cancer, cervical cancer, and leukemia. The choice of targeting moiety in the compound can be tailored to specifically bind to tumor-specific antigens. Examples of such targeting moieties include anti-Her2 for breast cancer, anti-CD20 for lymphoma, anti-PSMA for prostate cancer, and anti-CD30 for various lymphomas, including non-Hodgkin's lymphoma.

[0503] The disclosure is particularly useful for treating cancer and inhibiting tumor cell proliferation. This includes tumors, metastasis, or any condition characterized by uncontrolled cell growth. The drug-ligand complex delivers the activating moiety specifically to tumor or cancer cells. The targeting moiety binds to a cancer-cell-associated antigen, allowing internalization via receptor-mediated endocytosis. The antigen may be attached to the tumor cell or be an extracellular matrix protein associated with it. Inside the cell, the linker is cleaved by tumor-cell-associated proteases, releasing the activating moiety. This moiety then induces or enhances immune activity against the tumor. Alternatively, the activating moiety can be cleaved in the tumor microenvironment, with the drug subsequently penetrating the cell.

[0504] The disclosure targets abnormal cell proliferation, specifically cancer cells. Examples of cancers include breast cancer, colorectal cancer, diffuse large B-cell lymphoma, endometrial cancer, follicular lymphoma, gastric cancer, glioblastoma, head and neck cancer, hepatocellular cancer, lung cancer, melanoma, multiple myeloma, ovarian cancer, pancreatic cancer, prostate cancer, and renal cell carcinoma.

[0505] The disclosure also provides compounds or pharmaceutical compositions for use as medicaments to kill, inhibit, or delay the proliferation of tumor or cancer cells.

[0506] In some embodiments, a compound is administered to kill a cell or to retard or stop tumor growth. Effective administration should reduce the cell growth rate by at least 10%, with preferred reductions of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or complete cessation.

[0507] Pharmaceutical compositions must contain the active ingredient in a therapeutically effective amount, which depends on the condition being treated. Determining the effective amount is a routine task for those skilled in the art, guided by the details provided.

[0508] Effective doses for humans can be extrapolated from animal models. Dosages are adjusted based on observed effects, such as cellular inhibition, to ensure the proper therapeutic concentration is achieved.

[0509] Effective amounts of a compound can be determined from cell culture assays. The goal is to reach plasma concentrations that inhibit cell growth or division by at least 25%. Preferred concentrations induce higher levels of inhibition, such as 30%, 50%, 75%, or even 90% or more. Monitoring cellular activity helps in adjusting dosages to achieve the desired therapeutic effect.

[0510] Characteristics of Protein Complexes

[0511] The protein complex described herein can include an Fc of an antibody. These antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgEl, IgE2). In some embodiments, the Fc region is derived from human IgG (e.g.. IgGl, IgG2, IgG3. or IgG4). In some embodiments, the Fc region is an IgG4 Fc region (e.g., human IgG4 Fc region). In some embodiments, the protein complex described herein is linked to the Fc region through an antibody hinge region (e.g., IgG. IgE hinge region). In addition, the Fc region can be modified to provide desired effector functions or serum half-life.

[0512] In some embodiments, the protein complex described herein can increase immune response, activity or number of immune cells (e.g., myeloid cells, macrophages, dendritic cells, antigen presenting cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, or 20 folds.

[0513] In some implementations, the protein complex described herein can bind to a target (e.g., any of the targets described herein) with a dissociation rate (koff) of less than 0.1 s'1, less than 0.01 s'1, less than 0.001 s'1, less than 0.0001 s'1, or less than 0.00001 s'1. In some embodiments, the dissociation rate (koff) is greater than 0.01 s'1, greater than 0.001 s'1, greater than 0.0001 s'1, greater than 0.00001 s'1, or greater than 0.000001 s'1. In some embodiments, kinetic association rates (kon) is greater than 1 x 102 / Ms, greater than 1 x 103 / Ms, greater than 1 x 104 / Ms, greater than 1 x 105 / Ms, or greater than 1 x 106 / Ms. In some embodiments, kinetic association rates (kon) is less than 1 x 103 / Ms, less than 1 x 106 / Ms, or less than 1 x 107 / Ms. Affinities can be deduced from the quotient of the kinetic rate constants (KD=koff / kon). In some embodiments, KD is less than 1 x 10'6M, less than 1 x 10'7M, less than 1 x 10'8M, less than 1 x 10'9M, or less than 1 x 10'10M. In some embodiments, the KD is less than 300 nM, 200 nM, 100 nM, 50nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM. 2 nM, 1 nM, 900 pM, 800 pM, 700 pM. 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM. In some embodiments, KD is greater than 1 x 10'7M, greater than 1 x 10'8M, greater than 1 x 10'9M, greater than 1 x 10'10M, greater than 1 x 10'11M, or greater than 1 x 10'12M.

[0514] General techniques for measuring the affinity include, e.g., ELISA, RIA, and surface plasmon resonance (SPR).

[0515] In some embodiments, thermal stabilities are determined. The protein complex described herein can have a Tm greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73. 74. 75. 76, 77, 78, 79, 80, 81, 82, 83, 84, 85. 86. 87. 88. 89. 90, 91, 92, 93, 94, or 95 °C. In some embodiments, Tm is less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C.

[0516] In some embodiments, the protein complex described herein has a tumor growth inhibition percentage (TGI%) that is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the protein complex described herein has a tumor growth inhibition percentage that is less than 60%. 70%. 80%. 90%. 100%, 110%, 120%. 130%, 140%, 150%.

[0517] 160%, 170%, 180%, 190%, or 200%. The TGI% can be determined, e.g., at 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, or 30 days after the treatment starts, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the treatment starts. As used herein, the tumor growth inhibition percentage (TGI%) is calculated using the following formula:

[0518] TGI (%) = [l-(Ti-T0) / (Vi-V0)]xl00

[0519] Ti is the average tumor volume in the treatment group on day i. TO is the average tumor volume in the treatment group on day zero. Vi is the average tumor volume in the control group on day i. VO is the average tumor volume in the control group on day zero.

[0520] In some embodiments, the protein complex described herein has a functional Fc. In some embodiments, the Fc is from human IgGl. human IgG2, human IgG3, or human IgG4. In some embodiments, effector function of a functional Fc is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, effector function of a functional Fc is phagocytosis. In some embodiments, effector function of a functional Fc is ADCC and phagocytosis. In some embodiments, the protein constructs as described herein have an Fc region without effector function. In some embodiments, the Fc is a human IgG4 Fc. In some embodiments, the Fc does not have a functional Fc region. For example, the Fc region has LALA mutations (L234A and L235A mutations in EU numbering), or LALA-PG mutations (L234A, L235A, P329G mutations in EU numbering).

[0521] Some other modifications to the Fc region can be made. For example, a cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric fusion protein thus generated may have any increased half-life in vitro and / or in vivo.

[0522] In some embodiments, the IgG4 has S228P mutation (EU numbering). The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange.

[0523] In some embodiments, Fc regions are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such Fc region composition may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues; or position 314 in Kabat numbering); however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in Fc region sequences. Such fucosylation variants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region can be further engineered to replace the Asparagine at position 297 with Alanine (N297A).

[0524] In some embodiments, the main peak of HPLC-SEC accounts for at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the protein complex described herein after purification by protein A-based affinity chromatography and / or size-exclusive chromatography.

[0525] In some embodiments, the protein complex described herein does not induce cytokine storm in human. In some embodiments, the protein complex described herein is not a superagonist. Details of cytokine storm and superagonist can be found, e.g., in Shimabukuro-Vomhagen, A. et al. "Cytokine release syndrome." Journal for ImmunoTherapy of Cancer 6.1 (2018): 1-14, which is incorporated herein by reference in its entirety.

[0526] In some embodiments, the protein complex described herein can inhibit tumor growth. In some embodiments, the protein complex (e.g., any of the protein complexes described herein) can significantly inhibit tumor growth as compared the vehicle control. In some embodiments, the protein complex described herein can inhibit tumor growth with a TGI value that is at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold, as compared to that of a reference antibody or a reference protein in a mouse xenograft model. In some embodiments, the tumor cells are subcutaneously inoculated in immunocompromised or immunodeficient mice to generate the xenograft model. In some embodiments, the tumor volume of the mice can be analyzed 1. 2, 3, 4, 5, 6, 7, 8. 9, 10, 11, 12, 13, 14. 15. 16, 17, 18, 19, or 20 days post inoculation. In some embodiments, the TGI value of mice treated with the protein complex described herein can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%.

[0527] Methods of Making Protein Complexes Variants of the protein complexes described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding a polypeptide or a part thereof or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequences.

[0528] Screening can be performed to increase binding affinity of the antigen-binding domains. Any combination of deletions, insertions, and / or combinations can be made to arrive at a variant that has increased binding affinity for the target. The amino acid changes introduced into the variant can also alter or introduce new post-translational modifications into the polypeptide, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence such that a different sugar is attached by enzymes present in a cell), or introducing new glycosylation sites.

[0529] Sequences of the antigen-binding domains described herein can be derived from any species of animal, including mammals. Non-limiting examples of binding domain variants include sequences derived from humans, primates, e.g., monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas), chicken, goats, and rodents (e.g.. rats, mice, hamsters, and rabbits).

[0530] The present disclosure also provides recombinant vectors (e.g., an expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein), host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and / or a vector comprising the polynucleotide), and the production of recombinant polypeptides or fragments thereof by recombinant techniques.

[0531] As used herein, a ‘“vector” is any construct capable of delivering one or more polynucleotide(s) of interest to a host cell when the vector is introduced to the host cell. An “expression vector” is capable to deliver and express one or more polynucleotide(s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and / or a poly-A tail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector. A vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., with recombinant virus). Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant virus), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0532] In some implementations, a polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) is introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus, or may use a replication defective virus. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be '‘naked.” The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads that are efficiently transported into the cells.

[0533] For expression, the DNA insert comprising a polypeptide-encoding polynucleotide disclosed herein can be operatively linked to an appropriate promoter (e.g., a heterologous promoter), such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters are known to the skilled artisan. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. The expression constructs can further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs may include a translation initiating at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.

[0534] As indicated, the expression vectors can include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukary otic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HEK293 cells; and plant cells. Appropriate culture mediums and conditions for the host cells described herein are known in the art.

[0535] Non-limiting vectors for use in bacteria include pQE70. pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.

[0536] Non-limiting bacterial promoters suitable for use include the E. coli lad and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV), and metallothionein promoters, such as the mouse metallothionein-I promoter.

[0537] In the yeast Saccharomyces cerevisiae. a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used.

[0538] Introduction of the construct into the host cell can be affected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety7.

[0539] Transcription of DNA encoding a polypeptide of the present disclosure by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity7of a promoter in a given host cel l-ty pe. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0540] For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. The signals may be endogenous to the polypeptide, or they may be heterologous signals.

[0541] The polypeptides can be expressed in a modified form, such as a fusion protein (e.g., a GST-fusion) or with a histidine-tag, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.

[0542] Methods of Treatment

[0543] The protein constructs or polypeptides of the present disclosure can be used for various therapeutic purposes.

[0544] In one aspect, the disclosure provides methods for treating a cancer in a subject, methods of reducing the rate of the increase of volume of a tumor in a subject over time, methods of reducing the risk of developing a metastasis, or methods of reducing the risk of developing an additional metastasis in a subject. In some embodiments, the treatment can halt, slow, retard, or inhibit progression of a cancer. In some embodiments, the treatment can result in the reduction of in the number, severity, and / or duration of one or more symptoms of the cancer in a subject.

[0545] In one aspect, the disclosure features methods that include administering a therapeutically effective amount of protein constructs or polypeptides disclosed herein to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, a cancer), e.g., breast cancer (e.g., triple-negative breast cancer), carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thy roid cancer, bladder cancer, urethral cancer, or hematologic malignancy. In some embodiments, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung carcinoma (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone- refractory prostate cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is squamous cell carcinoma of the head and neck (SCCHN), renal cell carcinoma (RCC), triple-negative breast cancer (TNBC), or colorectal carcinoma. In some embodiments, the cancer is melanoma, pancreatic carcinoma, mesothelioma, hematological malignancies, especially Non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia, or advanced solid tumors. In some embodiments, the compositions and methods disclosed herein can be used for treatment of patients at risk for a cancer. Patients with cancer can be identified with various methods known in the art.

[0546] An effective amount can be administered in one or more administrations. By way of example, an effective amount of the protein constructs or the polypeptides is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and / or delay progression of a cancer in a patient or is an amount sufficient to ameliorate, stop, stabilize, reverse, slow and / or delay proliferation of a cell (e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line)) in vitro. As is understood in the art, an effective amount may vary', depending on, inter alia, patient history' as well as other factors such as the type (and / or dosage) of the protein constructs or the polypeptides used.

[0547] Effective amounts and schedules for administering the protein constructs or the polypeptides, the polynucleotides encoding the protein constructs or the polypeptides, and / or compositions disclosed herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the protein constructs or the polypeptides, the polynucleotides, and / or compositions disclosed herein, the route of administration, the particular ty pe of polynucleotides, and / or compositions disclosed herein used and other drugs being administered to the mammal.

[0548] A typical daily dosage of an effective amount of the protein constructs and / or the polypeptides is 0.1 mg / kg to 100 mg / kg (mg per kg of patient weight). In some embodiments, the dosage can be less than 100 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or 0.1 mg / kg. In some embodiments, the dosage can be greater than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg. 6 mg / kg. 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or 0.1 mg / kg. In some embodiments, the dosage is about 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, or 1 mg / kg. In some embodiments, the dosage is about 1 to 10 mg / kg, about 1 to 5 mg / kg, or about 2 to 5 mg / kg.

[0549] In any of the methods described herein, the protein constructs or the polypeptides can be administered to the subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day).

[0550] In some embodiments, the one or more additional therapeutic agents can be administered to the subject prior to, or after administering the protein constructs or the polypeptides. In some embodiments, the one or more additional therapeutic agents are administered to the subject such that there is an overlap in the bioactive period of the one or more additional therapeutic agents and the protein constructs or the polypeptides in the subject.

[0551] In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of B-Raf, an EGFR inhibitor, an inhibitor of a MEK, an inhibitor of ERK, an inhibitor of K-Ras, an inhibitor of c-Met, an inhibitor of anaplastic lymphoma kinase (ALK), an inhibitor of a phosphatidylinositol 3-kinase (PI3K), an inhibitor of an Akt, an inhibitor of mTOR, a dual PI3K / mT0R inhibitor, an inhibitor of Bruton's tyrosine kinase (BTK). and an inhibitor of Isocitrate dehydrogenase 1 (IDH1) and / or Isocitrate dehydrogenase 2 (IDH2). In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine 2, 3 -di oxygenase- 1) (IDO1) (e.g., epacadostat).

[0552] In some embodiments, the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of HER3, an inhibitor of LSD 1, an inhibitor of MDM2, an inhibitor of BCL2, an inhibitor of CHK1, an inhibitor of activated hedgehog signaling pathway, and an agent that selectively degrades the estrogen receptor.

[0553] In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta. Zykadia, Sutent, temsirolimus, axitinib, everolimus. sorafenib, Vbtrient, Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, an Hsp90 inhibitor, Ad-GM-CSF, Temazolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

[0554] In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, an IL- 10 antagonist, an IL-4 antagonist, an IL- 13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a treatment targeting CX3CL1, a treatment targeting CXCL9, a treatment targeting CXCL10, a treatment targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, and a Selectin agonist.

[0555] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to the subject.

[0556] In some embodiments, the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-PD-L2 antibody, an anti-SIRPa antibody, an anti-CD47 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, or an anti-GITR antibody. In some embodiments, the additional therapeutic agent is an anti-CD20 antibody (e.g., rituximab) or an anti- EGF receptor antibody (e.g., cetuximab).

[0557] Pharmaceutical Compositions and Routes of Administration

[0558] Also provided herein are pharmaceutical compositions that contain the protein constructs, or the polypeptides described herein. The pharmaceutical compositions can be formulated in any manner known in the art.

[0559] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g.. intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The compositions can include a sterile diluent (e.g., sterile water or saline), a fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates, citrates, or phosphates, and isotonic agents, such as sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride), or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. Preparations of the compositions can be formulated and enclosed in ampules, disposable syringes, or multiple dose vials. Where required (as in, for example, injectable formulations), proper fluidity can be maintained by, for example, the use of a coating, such as lecithin, or a surfactant. Absorption of the agents can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid).

[0560] Compositions containing the protein constructs, or the polypeptides described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage).

[0561] Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of administration chosen. For injection, the agents can be formulated in aqueous solutions, preferably in physiologically compatible buffers to reduce discomfort at the site of injection. The solution can contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the protein constructs or the polypeptides can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0562] Toxicity' and therapeutic efficacy of compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). One can, for example, determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population): the therapeutic index being the ratio of LD50: ED50. Agents that exhibit high therapeutic indices are preferred. Where an agent exhibits an undesirable side effect, care should be taken to minimize potential damage (i.e., reduce unwanted side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0563] Exemplary' doses include milligram or microgram amounts of any of the protein constructs or the polypeptides described herein per kilogram of the subject's weight (e.g., about 1 pg / kg to about 500 mg / kg; about 100 pg / kg to about 500 mg / kg; about 100 pg / kg to about 50 mg / kg; about 10 pg / kg to about 5 mg / kg; about 1 pg / kg to about 0.5 mg / kg; about 1 pg / kg to about 50 pg / kg; about 1 mg / kg to about 10 mg / kg; or about 1 mg / kg to about 5 mg / kg). While these doses cover a broad range, one of ordinary skill in the art will understand that therapeutic agents can vary in their potency, and effective amounts can be determined by methods known in the art. Typically, relatively low doses are administered at first, and the attending health care professional or veterinary professional (in the case of therapeutic application) or a researcher (when still working at the development stage) can subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject will depend upon a variety of factors including the activity7of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life in vivo. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. The disclosure also provides methods of manufacturing the protein constructs or the polypeptides for various uses as described herein.

[0564] EXAMPLES

[0565] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0566] Example 1.

[0567] 1.1 Bispecific Antibody Construction

[0568] For constructing the Crossbody molecule used in bi-specific antibodies, the variable heavy chain region (VH) and the corresponding variable light chain region (VL) for anti-R0R1 are derived from a humanized antibody. Similarly, the VH and VL regions for anti-CD3 are derived from the humanized IgG antibody SP34. The bi-specific antibody constructs were designed using either the Fc region or a knobs-into-holes (KIH) structure.

[0569] Tables 2, 3 and 4 summarize the information and sequences related to the bi-specific antibody molecule developed during the invention process.

[0570] TABLE 2

[0571]

[0572]

[0573] *VHR: VH of ROR1 Ab; VLR: VL ofRORl Ab; 34H: VH of CD3 Ab SP34; 34L: VL of CD3 Ab SP34.

[0574] TABLE 3

[0575]

[0576] TABLE 4

[0577]

[0578]

[0579]

[0580]

[0581]

[0582]

[0583] Heavy and light chain fragments were generated by gene synthesis through GeneArt AG (Thermo Fisher Scientific, Regensburg, Germany). The synthesized constructs were then cloned into the pCDNA3.4 vector. DNA for transfection were prepared using Maxipreps and subsequently transfected into the ExpiCHO expression system according to the manufacturer’s instructions. The protein was purified using a Protein A resin column, followed by size exclusion chromatography (SEC) to obtain a single peak.

[0584] As shown in FIG. 4, Antibody #B.l was used as a model system. This antibody was designed with three chains: VHR-CH1-CH2-CH3H, VLR-LC-34H, and 34L-CH2-CH3K. The SP34-derived VH and VL regions were separated and positioned differently to reduce toxicity while maintaining the functionality of the CD3 antibody within a single molecule. The heavy chain is linked using knob-into-hole technology7, allowing for flexibility in the positioning of the VH and VL regions, as well as the hole and knob of the heavy chain.

[0585] 1.2 Cell Binding Assays

[0586] To assess whether the multi-specific antibody binds to the cell surface, FACS analysis was performed. Various tumor cell lines and the CD3-positive Jurkat cell line were used as target cells. Cells were generally detached using TrypLE reagents to preserve the integrity of surface antigens. After dissociation and washing in PBS, 1 x 105target cells were seeded into a 96-well plate. The multi-specific antibody and control antibody were prepared at concentrations of 100 nM and then diluted 3-fold or 4-fold. These antibodies were incubated with the cells for 1 hour at 4°C. Following incubation, cells were washed with FACS wash buffer and then incubated with PE-conjugated goat anti-human IgG, Fc fragment-specific antibody (1:200 dilution in FACS wash buffer) for 20 minutes at 4°C. Mean fluorescence intensity (MFI) was measured using the flow7cytometry7, and results were analyzed with GraphPad software.

[0587] Table 5 shows the cell binding data for bispecific antibody constructs using HT29 cells (ROR1+) and human PBMCs (CD3+) as target cells.

[0588] TABLE 5 HT29 PBMC

[0589] Emax EC50 (nM) Emax EC50 (nM)

[0590] Ab #B.l _ 19862 _ 2.614 _ 6390 _ 138.2

[0591] Ab #B.2 18548 3.299 2901 165.7

[0592] Ab #B.3 17789 2.007 2850 53.33

[0593] Ab #B.4 _ 18293 _ 2.065 _ 1694 _ 8.596

[0594] Ab #B.5 20368 2.845 6120 43.71

[0595] Ab #B.6 _ 17458 _ 3.396 _ 6457 _ 7.229

[0596] Ab #B.7 18135 3.45 2598 11.7

[0597] Ab #B.8 18768 2.843 1567 198.4

[0598] Ab #B.9 16803 3.162 1478 224.6

[0599] Ab #B.10 18778 3.242 18660 62.66

[0600] Ab #B.ll _ 18651 _ 3.594 _ 16259 _ 189 _

[0601] Ab #B.12 32408 1.081 4294 266.8

[0602] Ab #B.13 19381 3.89 17112 10.2

[0603] Antibodies #B.l to #B.12 are ROR1-CD3 bispecific antibodies with different positions of the SP34 VH and VL regions. As shown in FIG. 5, most ROR1-CD3 bispecific antibodies exhibited similar binding to HT29 cells, with the exception of B.12, which is a bivalent ROR1 antibody and showed better binding.

[0604] For CD3 binding, the following observations were made. Antibody #B.10, with SP34 VH and VL at positions 2+5 / 6, showed binding similar to that of the SP34 ScFv (strong binding). Antibodies with SP34 VH and VL at positions 2+1 (#B.l), 1+5 (#B.5), and 2+3 / 4 (#B.6 / 10) exhibited reduced binding compared to SP34 ScFv (#B.13) (medium binding). Antibodies with SP34 VH and VL at positions 1+3 / 4 / 6 (#B.2 / 3 / 4), 3+4 (#B.7 / 12), and 4+5 / 6 (#B.8 / 9) showed weak binding compared to SP34 ScFv (#B.13) (low binding).

[0605] 1.3 Cell Killing Assays

[0606] For T cell engager antibodies, assessing T cell-mediated lysis activity against tumor cells is crucial for evaluating their in vitro potency and specificity. We conducted PBMC killing assays against various tumor cells to measure the antibody-mediated killing effect. PBMCs were used as effector cells in these assays. The protocol is described below.

[0607] Briefly, target cells were detached using TrypLE™, and 10,000 cells per well were seeded in a 96-well flat-bottom opaque plate. Resting or activated human PBMCs were added to each well at effector-to-target (E / T) ratios of 20:1 or 10:1. Antibodies, along with appropriate control antibodies, were added to the co-culture system in serial 10-fold dilutions starting from 15 or 30 nM. Target cell killing was assessed after 48 hours at 37°C for resting human PBMCs, by evaluating luciferase signaling. The luciferase signal was measured as the average relative light units (RLU) from each sample well. Maximum luciferase signals, representing target cell viability, were obtained by incubating target cells with effector cells in the absence of trispecific antibodies. The percentage of viability was calculated as RLU (antibody) / RLU (no antibody control) x 100, and the EC 50 values were determined using Prism software.

[0608] Table 6 presents the killing results of bispecific antibody constructs using HT29 cells (R0R1+) as target cells.

[0609] TABLE 6

[0610] HT29

[0611] Emax EC50 (nM)

[0612] Ab #B.1 10.25 0.02885

[0613] Ab #B.2 20.32 0.1753

[0614] Ab #B.3 37.65 0.06968

[0615] Ab #B.4 23.84 0.04473

[0616] Ab #B.5 30.87 0.02217

[0617] Ab #B.6 24.33 0.01513

[0618] Ab #B.7 36.06 0.1423

[0619] Ab #B.8 24.42 0.107

[0620] Ab #B.9 _ 37.82 _ 0.01729

[0621] Ab #B.10 _ 42.22 _ 0.04851

[0622] Ab #B.11 21.57 0.02223

[0623] Ab #B.12 12.15 0.009085

[0624] Ab #B.13 17.03 0.01769

[0625] For CD3 engager antibodies, evaluating T cell lysis activity against tumor cells is crucial for assessing their in vitro potency and specificity. PBMC killing assays were performed to test these activities. As shown in FIG.6, anti-RORl-CD3 bispecific antibodies demonstrated T cell-dependent cellular cytotoxicity (TDCC) against ROR1 -positive HT29 cells. The potency of HT29 cell killing by ROR1-CD3 bispecific antibodies correlates with their binding to CD3.

[0626] For antibodies with strong and medium CD3 binding, such as SP34 VH and VL at positions 2+5 / 6 (antibody #B.10), 2+1 (antibody #B.1), 1+5 (antibody #B.5), and 2+3 / 4 (antibody #B.6 / 10), the HT29 cell killing potency is similar to that of the SP34 ScFv (antibody #B.13). In contrast, for antibodies with weak CD3 binding, including SP34 VH and VL at positions 1 +3 / 4 / 6 (antibody #B.2 / 3 / 4), 3+4 (antibody #B.7 / 12), and 4+5 / 6 (antibody #B.8 / 9), the HT29 cell killing potency is reduced compared to SP34 ScFv (antibody #B.13), although they still exhibit some killing activity. Example 2

[0627] 2.1 Antibody Construction

[0628] For the construction of the Crossbody molecule used in the trispecific antibody, the anti-EpCam variable heavy chain (VH) and corresponding variable light chain (VL) regions are derived from the IgG antibody Solitomab. The anti-CD3 VH and VL regions are sourced from the humanized IgG antibody SP34. The anti-RORl / CEA / EGFR / GPC3 VH and VL regions are derived from a humanized antibody. The trispecific antibody constructs were designed using a knobs-into-holes structure. In this design, the SP34 VH and VL regions were split and positioned differently with a concealed arrangement to minimize toxicity.

[0629] Tables 7, 8 and 9 summarize the information and sequences for the trispecific antibody molecule developed during the invention process.

[0630] TABLE 7

[0631]

[0632]

[0633] *VHE: VH of EpCam Ab; VLE: VL of EpCam Ab; 34H: VH of CD3 Ab SP34; 34L: VL of CD3 Ab SP34;

[0634] VHR: VH of ROR1 Ab; VLR: VL of ROR1 Ab; VLG: VL of GPC3 Ab; VHG: VH of GPC3 Ab; EP: EpCam; ER: EGFR.

[0635] TABLE 8

[0636]

[0637] TABLE 9

[0638]

[0639]

[0640]

[0641]

[0642]

[0643]

[0644] Heavy and light chain fragments were generated by gene synthesis through GeneArt AG (Thermo Fisher Scientific, Regensburg, Germany). The synthesized constructs were cloned into the pCDNA3.4 vector. DNA for transfection was prepared using Maxipreps and then transfected into the ExpiCHO expression system according to the manufacturer’s instructions. The protein was purified using a Protein A resin column, followed by size exclusion chromatography (SEC) to obtain a single peak.

[0645] As shown in FIG. 7, using Antibody #T.3 as a model system, the antibody was designed with three chains: VHE-CH1-CH2-CH3H, VLE-LC-34H. and EGFR ScFv 34L-CH2-CH3K. The SP34-derived VH and VL regions were split, separated, and positioned differently to reduce toxicity while maintaining the CD3 antibody function within a single molecule. The heavy chain is linked using knob-into-hole technology, allowing for flexibility in the positioning of the VH and VL regions as well as the hole and knob of the heavy chain.

[0646] 2.2 Cell Binding Assays

[0647] Tables 10 and 11 present the cell binding data for antibody constructs using MKN45, HepG2, HT29, MDA-MB-231, Lovo cells, and human PBMCs as target cells. TABLE 10

[0648] _ MKN45 _ HepG2 _ HT29 _ MDA-MB-231

[0649] Emax EC50 Emax EC50 Emax EC50 Emax EC50 _ (nM) _ (nM) _ (nM) _ (nM) Ab #T.l 448485 10.05 145415 12.92 160830 179 73271 91.44 Ab #T.2 792790 2.919 146714 7.724 120370 8.823 88423 31.89 Ab #T.3 815719 3.159 221234 3.069 109597 0.5926 855756 9.81

[0650] TABLE 11

[0651] Lovo PBMC

[0652] Emax EC50 (nM) Emax EC50 (nM)

[0653] Ab #T.l 31162 12.03 10595 16.22

[0654] Ab #T.2 60292 3.529 N / A N / A

[0655] Ab #T.3 78600 0.3637 5084 20.70

[0656] Antibodies #T.1 to #T.3 include the EpCam-CD3 bispecific antibody, the EpCam-CEA-CD3 trispecific antibody, and the EpCam-EGFR-CD3 trispecific antibody. These antibodies feature split SP34 VH and VL regions, which were separated and designed at different positions. As shown in FIGS. 8-9, the three antibodies exhibited different binding profiles with various tumor cells, correlating with the expression levels of EpCam, CEA. and EGFR.

[0657] For CD3 binding, SP34 VH and VL at position 1+2, with an ScFv on the N-terminal of the SP34 VL, showed weaker binding compared to SP34 ScFv (antibody #B.13), indicating low binding. The ScFv on the N-terminal of SP34 VL may obstruct the T cell binding of the SP34 antibody.

[0658] 2.3 Cell Killing Assays

[0659] We conducted PBMC killing assays against various tumor cells to assess antibody-mediated killing effects. PBMCs were used as effector cells in these assays.

[0660] Table 12 presents the killing results of antibody constructs using MKN45, HepG2, and HT29 cells as target cells.

[0661] TABLE 12

[0662] MKN45 HepG2 HT29

[0663] Emax EC50 (nM) Emax EC50 (nM) Emax EC50 (nM) Ab #T.l 10.3 0.01681 7.574 0.3457 22.54 0.1206

[0664] Ab #T.2 9.368 0.001024 1.064 0.141 27.16 0.03818 Ab #T.3 2.094 0.000115 1.829 0.00204 17.73 0.000349 Table 13 presents the killing results of antibody constructs using MDA-MB-231 and Lovo cells as target cells.

[0665] TABLE 13

[0666] MDA-MB-231 Lovo

[0667] Emax EC50 (nM) Emax EC50 (nM)

[0668] Ab #T.l 5.943 0.2799 3.363 0.3135

[0669] Ab #T.2 10.86 0.1234 14.13 0.08351

[0670] Ab #T.3 5.249 0.000122 5.457 0.000632

[0671] As shown in FIGS. 10-11, the tumor cell killing potency of the three antibodies correlates with their binding to tumor cells. The EpCam-CEA-CD3 trispecific antibody exhibited greater killing potency compared to the EpCam-CD3 bispecific antibody in CEA-positive MKN45 cells. Additionally, the EpCam-EGFR-CD3 trispecific antibody demonstrated superior killing potency compared to both the EpCam-CD3 bispecific antibody and the EpCam-CEA-CD3 trispecific antibody in EGFR-positive (CEA-negative) HepG2, HT29, MDA-MB-231, and Lovo cells.

[0672] Additional constructs were generated using different tumor-associated antigens (TAAs) including EpCam. RORL CEA, EGFR. and GPC3 antibodies, and were tested for their killing potency.

[0673] Table 14 presents the killing results of antibody constructs using HepG2 and MDA-MB-231 cells as target cells.

[0674] TABLE 14

[0675] HepG2 MDA-MB-231

[0676] Emax EC50 (nM) Emax EC50 (nM)

[0677] Ab #T.4 13.19 0.005206 5.296 0.8473

[0678] Ab #T.5 2.238 _ 0.09476 3.655 _ 0.01172 Ab #T.6 23.3 4.205 4.554 0.001273 Ab #T.7 2.243 0.00026 28.68 2.387

[0679] Ab #T.8 2.824 0.01227 16.49 5.646

[0680] Ab #T.9 5.94 0.02464 17.18 1.545

[0681] Ab #T.10 17.85 0.0335 35.58 8.72

[0682] Ab #T.11 8.819 0.000767 8.562 0.005761 Ab #T.12 14.84 0.05172 88.25 3.033 As shown in FIG. 12, nine antibodies demonstrate varying tumor cell killing potency, which correlates with their binding to tumor cells. For HepG2 cells (GPC3+++, EpCam+++. EGFR+, R0R1 / CEA negative), the killing potency is ranked as follows: GPC3-34H-Fc / EP ScFv-34L-Fc (antibody #T.7) > GPC3-34H-Fc / ER ScFv-34L-Fc (antibody #T.l 1) > GPC3-34H-Fc / ROR1 ScFv-34L-Fc (antibody #T.8) > GPC3-34H-Fc / CEA ScFv-34L-Fc (antibody #T.1O) > GPC3-34H-Fc / 34L-Fc (antibody #T.12). For EpCam-positive cells: EP-34H-Fc / EP ScFv-34L-Fc (antibody #T.4) > EP-34H-Fc / RORl ScFv-34L-Fc (antibody #T.9) > R0R1-34H-Fc / EP ScFv-34L-Fc (antibody #T.5) > ROR1-34H-Fc / ROR1 ScFv-34L-Fc (antibody #T.6)

[0683] For MDA-MB-231 cells (EGFR+++, R0R1++, EpCam+, GPC3 / CEA negative), the killing potency of the antibodies is ranked as follows: ROR1-34H-Fc / ROR1 ScFv-34L-Fc (antibody #T.6) > R0R1 -34H-Fc / EP ScFv-34L-Fc (antibody #T.5) > EP-34H-Fc / RORl ScFv-34L-Fc (antibody #T.9) > EP-34H-Fc / EP ScFv-34L-Fc (antibody #T.4). For antibodies targeting GPC3: GPC3-34H-Fc / ER ScFv-34L-Fc (antibody #T.ll) > GPC3-34H-Fc / RORl ScFv-34L-Fc (antibody #T.8) > GPC3-34H-Fc / EP ScFv-34L-Fc (antibody #T.7) > GPC3-34H-Fc / CEA ScFv-34L-Fc (antibody #T.1O) > GPC3-34H-Fc / 34L-Fc (antibody #T.12)

[0684] 2.4 In Vivo Tumor Xenograft Model Study

[0685] An in vivo study was conducted using a humanized PBMC / NCG model to evaluate the efficacy of multi-specific antibodies in various formats, following Institutional Animal Care and Use Committee (IACUC) guidelines. Briefly, HT29 or MKN45 tumor cells (1 x 107) were injected subcutaneously into the rear flank of NSG mice. On Day 7, human PBMCs (1 x 107) were administered intravenously. Antibodies (3 mg / mL) or vehicle controls were injected intraperitoneally (i.p.) into the corresponding groups twice a week for 3 weeks. Tumor size was measured twice per week.

[0686] Table 15 presents the efficacy results of Crossbody constructs in HT29 cell tumor treatment using the human PBMC / NSG mice xenograft model.

[0687] TABLE 15

[0688] Group TV(mm3)

[0689] Vehicle 1060.2

[0690]

[0691] Ab #T.1 242.9 ± 90.5 %

[0692] Ab #T.2 316.2 ± 8E0 %

[0693] Ab #T.3 243.2 92.5 \ Table 16 presents the efficacy results of Crossbody constructs in MKN45 cell tumor treatment using the human PBMC / NSG mice xenograft model.

[0694] TABLE 16

[0695] Group TV(mm³)

[0696] Vehicle 1014.4 ±

[0697]

[0698] _ _

[0699] Ab#T.l 5.7 109.2 75%

[0700] Ab #T.2 30.8 107.7 25%

[0701] Ab #T.3 0 110.2 100%

[0702] As shown in FIGS. 13-14, the in vivo tumor cell killing potency of the three antibodies correlates with their in vitro killing potency to tumor cells.

[0703] Example 3

[0704] 3.1 Antibody Construction

[0705] In the construction of the Crossbody molecule for the trispecific antibody, the anti-EpCam and anti-RORl variable heavy chain (VH) and variable light chain (VL) regions are derived from humanized antibodies, while the anti-CD3 VH and VL regions are derived from the humanized IgG antibody SP34. The trispecific antibody constructs were designed using either an Fc or a knobs-into-holes structure.

[0706] In this design, R0R1 was chosen as the primary tumor target due to its low expression level and strong tumor specificity, directing the antibody to the tumor first. EpCam was selected as the secondary' target due to its high expression but lower tumor specificity', which helps to reduce on-target / off-tumor toxicity associated with EpCam and enhances the potency of the trispecific antibody. CD3. as the T cell target, also features a concealed design to minimize toxicity.

[0707] Tables 17, 18 and 19 summarize the information and sequences of the trispecific antibody molecules developed during the course of this study.

[0708] TABLE 17

[0709]

[0710]

[0711] *VHE or EPH: VH of EpCam Ab; VLE or EPL: VL of EpCam Ab; 34H: VH of CD3 Ab SP34; 34L: VL of CD3 Ab SP34; VHR: VH of ROR1 Ab; VLR: VL of ROR1 Ab; VHP: VH of PDL1 Ab; VLP: VL of PDL1 Ab; EP: EpCam.

[0712] TABLE 18

[0713]

[0714]

[0715] TABLE 19

[0716]

[0717]

[0718]

[0719]

[0720]

[0721]

[0722]

[0723] Heavy and light chain fragments were synthesized by GeneArt AG (Thermo Fisher Scientific, Regensburg, Germany). The synthesized constructs were cloned into the pCDNA3.4 vector, and DNA for transfection was prepared using Maxi-preps. These were then transfected into the ExpiCHO expression system following the manufacturer’s instructions. The protein was purified using a Protein A resin column, followed by size exclusion chromatography (SEC) to achieve a single peak.

[0724] As shown in FIG. 15, using Antibody #T.13 as a model system, the antibody was designed with three chains: VHR-CH1-CH2-CH3H, VLR-LC-34H-EPH, and EPL-34L-CH2-CH3K. In this design, the variable heavy chain (VH) and variable light chain (VL) of SP34 were split and positioned differently to reduce toxicity. EpCam was included as the secondary, concealed tumor target (high expression but low tumor specificity) to minimize EpCam-induced on-target / off-tumor toxicity and enhance the potency of the trispecific antibody. Despite these splits, the SP34 and EpCam antibody components are within a single molecule to maintain the functionality of CD3 and EpCam targeting. The heavy chain utilizes knob-into-hole technology, allowing for exchange of VH and VL, as well as the hole and knob positions. This design enables the VH and VL of SP34 and EpCam antibodies to be repositioned within the Fab-IgG-Fc framework to create new antibody candidates.

[0725] 3.2 Cell Binding Assays Table 20 shows the cell binding information for antibody constructs tested with HT29, MDA-MB-231. HPAC cells, and human PBMCs as target cells.

[0726] TABLE 20

[0727] HT29 MDA-MB-231 HPAC PBMC Emax EC5O Emax EC5O Emax EC5O Emax EC50 (nM) (nM) (nM) (nM)

[0728] Ab #T.13 69642 1.479 66454 1.437 723534 8.956 8770 7.218 Ab #T.14 43165 0.9561 69570 1.626 589982 5.949 6107 228.6 Ab #T.15 105190 2.669 97014 0.7108 1431155 19.38 5777 42.14 Ab #T.16 49471 0.3833 75635 0.888 620256 9.573 10421 5.996 Ab #T.17 44605 0.4534 61186 0.8917 300786 2.643 8345 2.496 Ab #T.18 49305 0.4428 69454 0.8332 596344 30.43 5119 2.486 Ab #T.19 22953 3.207 46619 3.965 99204 5.39 4950 3.607 Ab #T.20 60899 0.9753 61449 0.8223 433789 6.677 8677 5.681 Ab #T.21 16767 4.079 46729 4.642 101163 2.319 8530 7.494 Ab #7.22 45429 0.3881 75439 0.6663 525700 5.423 3827 10.58 Ab #T.23 17600 1.928 42765 2.074 109411 3.79 3559 5.928 Ab #T.24 22021 1.796 44803 2.257 131180 2.014 3648 1.839 Ab #T.25 30265 9.491 96314 21.07 223261 15.52 2395 N / A Ab #7.26 23334 8.305 84346 18.7 223585 13.55 2909 5.994 Ab #T.27 105242 1.038 112544 0.5792 1343776 9.306 3607 7.943 Ab #B.l 19862 2.614 49795 3.849 104311 3.235 9791 5.391

[0729] Antibodies #T.13 to #T.27 are RORl-EpCam-CD3 trispecific antibodies with split SP34 and EpCam VH and VL regions, which were separated and designed at different positions. As shown in FIGS. 16-17, the antibodies exhibited varying binding levels related to the different positions of the EpCam splits. Specifically, antibodies T.13, #T.15, #T.2O, and #T.27 demonstrated strong binding to tumor cells. Antibodies #T.14, #T.16, #T.17, #T.18, and #T.22 showed medium binding levels. In contrast, antibodies #T.19, #T.21. #T.23, #T.24, #T.25, and #T.26 exhibited weak binding to tumor cells, similar to antibody #B.l.

[0730] For PBMC binding, antibodies #T.13, #T.16, #T.17, #T.2O, and #T.21 exhibited strong binding to T cells. Antibodies #T,14, #T.15, #T.18, and #T.19 showed medium binding levels with T cells. In contrast, antibodies #T.22, #T.23, #T.24. #T.25, #T.26, and #T.27 displayed weak binding to T cells.

[0731] 3.3 Cell Killing Assays

[0732] We performed PBMC killing assays to evaluate the antibody-mediated killing effect on different tumor cells, using PBMCs as effector cells. Table 21 shows the results of these killing assays for antibody constructs tested against HT29, MDA-MB-231. and HPAC cells as target cells.

[0733] TABLE 21

[0734] HT29 MDA-MB-231 HPAC Emax EC50 (nM) Emax EC50 (nM) Emax EC50 (nM) Ab #T.13 26.75 0.2447 91.56 0.004661 83.93 0.1168 Ab #T.14 32.58 0.01731 47.22 3.874 26.52 5.32 Ab #T.15 4.034 0.000318 3.703 0.000123 5.583 0.000492 Ab #T.16 3.268 0.000696 11.37 0.000117 9.975 0.00133 Ab #T.17 8.213 0.005103 14.47 0.0105 9.254 0.004413 Ab #T.18 2.319 0.000352 6.554 9.86E-05 4.321 0.000572 Ab #T.19 18.92 0.006738 36.66 0.00698 24.33 0.08304 Ab #T.20 0.9814 0.000627 3.699 6.21E-05 0.9512 0.000394 Ab #T.21 4.272 0.105 20.32 0.1753 16.24 0.1666 Ab #T.22 20 0.01418 30.87 0.02217 29.68 0.005389 Ab #T.23 25.89 0.1015 64.53 1.02E+14 40.04 0.7044 Ab #T.24 8.908 0.004843 11.92 0.000121 4.087 0.00233 Ab #T.25 2.174 0.008051 8.832 0.000139 2.611 0.006038 Ab #T.26 1.029 0.02215 15.03 7.02E-05 1.218 0.02607 Ab #T.27 5.202 0.00227 5.343 8.63E-05 7.324 0.006071 Ab #B.l 0.5777 0.0221 10.25 0.02885 5.901 0.02545

[0735] As shown in FIGS. 18-19, some antibodies demonstrated tumor cell killing potency related to tumor cell binding, while others did not exhibit killing potency due to structural design limitations. For MDA-MB-231 cells (R0R1++, EpCam+), HT29 cells (R0R1+, EpCam+++), and HPAC cells (R0R1+, EpCam++), antibodies ROR1-34HEPH-Fc / 34LEPL-Fc (#T.13) and ROR1-EPH34H-Fc / EPL34L-Fc (#T.14) showed poor killing potency. This may be due to their inability to form functional EpCam or SP34 molecules with the VH and VL regions.

[0736] In contrast, antibodies ROR1-34HEPH-Fc / EPL34L-Fc (#T.15) and ROR1-EPH34H-Fc / 34LEPL-Fc (#T.16) exhibited strong killing potency across all three tumor cell lines. This suggests that the configuration w ith AblVH-CHl-CH2-CH3 (hole), AblVL-LC-Ab2VH-Ab3VH, and Ab3VL-Ab2VL-CH2-CH3 (knob) achieves optimal functional activity.

[0737] Additionally, ROR1-34HEPH-Fc / EPL34L-Fc (#T.15) and ROR1-EPH34H-Fc / 34LEPL-Fc (#T.16) show ed significantly stronger killing potency compared to ROR1-34H-Fc / 34L-Fc (#B.1), indicating that the VH and VL regions of the EpCam antibody work effectively together to enhance tumor binding and killing potency. For the killing potency of RORl-EPH-Fc / 34ScFv-EPL-Fc (antibody #T.17), R0R1-EPH-Fc / EPL-34ScFv-Fc (antibody #T.18), and RORl-Fc / EPL-34ScFv-Fc (antibody #T.19), the order of potency is as follows: RORl-EPH-Fc / EPL-34ScFv-Fc (antibody #T.18) > RORl-EPH-Fc / 34ScFv-EPL-Fc (antibody #T.17) > RORl-Fc / EPL-34ScFv-Fc (antibody #T.19). The addition of the EpCam antibody significantly enhances the killing potency of RORl-EPH-Fc / EPL-34ScFv-Fc (antibody #T.18) compared to RORl-Fc / EPL-34ScFv-Fc (antibody #T.19).

[0738] For RORl-34Dia-EPH-Fc / EPL-Fc (antibody #T.2O) and RORl-34Dia-EPH-Fc / 34L-Fc (antibody #T.21), the addition of the EpCam antibody also improved the killing potency, with RORl-34Dia-EPH-Fc / EPL-Fc (antibody #T.2O) showing significantly stronger potency than RORl-34Dia-EPH-Fc / 34L-Fc (antibody #T.21).

[0739] Regarding ROR1-EPH-Fc-34H / EPL-Fc-34L (antibody #T.22), ROR1-Fc-34H / EPL-Fc-34L (antibody #T.23), and ROR1-EPH-Fc-34H / 34L-Fc-EPL (antibody #T.24), the killing potency follows this order: ROR1-EPH-Fc-34H / 34L-Fc-EPL (antibody & T.24) > R0R1-EPH-Fc-34H / EPL-Fc-34L (antibody #T.22) > ROR1-Fc-34H / EPL-Fc-34L (antibody #T.23). The reduced potency of antibodies #T.22 and #T.23 is likely due to the longer distance between the TAA and SP34. However, the addition of the EpCam antibody still enhanced the killing potency of antibody #T.22 compared to antibody #T.23.

[0740] For EPHROR1-34H-Fc / 34LPDL1EPL-Fc (antibody #T.25), EPHROR1-34H-Fc / EPLPDL134L-Fc (antibody #T.26), and EPHROR1-34H-Fc / EPL-34L-Fc (antibody #T.27), all three antibodies demonstrated similar killing potency, which was superior to that of ROR1-34H-Fc / 34L-Fc (antibody #B.1).

[0741] 3.4 Summary

[0742] Table 22 presents a comprehensive summary of the binding and killing potency for Antibodies #T.13 through #T.27.

[0743] TABLE 22

[0744]

[0745]

[0746] negative; “+ to ++++” potency increasing.

[0747] Example 4

[0748] 4.1 Antibody Construction

[0749] In constructing the Crossbody molecule for trispecific antibodies, the anti-EpCam and anti-RORl variable heavy chain (VH) and variable light chain (VL) regions are derived from humanized antibodies, while the anti-CD3 variable heavy chain (VH) and variable light chain (VL) regions come from the humanized IgG antibody SP34. The trispecific antibody constructs were designed using either an Fc or knobs-into-holes structure.

[0750] In this design, R0R1 serves as the primary tumor target due to its low expression and strong tumor specificity, guiding the antibody to the tumor. EpCam is the secondary, less specific tumor target, included to reduce EpCam-induced on-target / off-tumor toxicity and enhance the overall potency of the trispecific antibody. CD3, the T cell target, also features a concealment design to further minimize toxicity.

[0751] Tables 23 and 24 provide a comprehensive summary of the information, and sequences related to the trispecific antibody molecules developed during the invention process.

[0752] TABLE 23

[0753]

[0754]

[0755] *VHE or EPH: VH of EpCam Ab; VLE or EPL: VL of EpCam Ab; 34H: VH of CD3 Ab SP34; 34L: VL of CD3 Ab SP34; VHR: VH of ROR1 Ab; VT. R: VI, of ROR1 Ab; EP: EpCam

[0756] TABLE 24

[0757]

[0758]

[0759]

[0760]

[0761]

[0762] Heavy and light chain fragments were generated by gene synthesis from GeneArt AG (Thermo Fisher Scientific, Regensburg, Germany). The synthesized constructs were then cloned into the pCDNA3.4 vector. DNA for transfection was prepared using Maxi-preps and transfected into the ExpiCHO expression system following the manufacturer's instructions. The protein was purified using a Protein A resin column, followed by size exclusion chromatography (SEC) to achieve a single peak.

[0763] 4.2 Cell Binding Assays

[0764] Table 25 shows the cell binding information for antibody constructs using HT29 (R0R1+, EpCam+++), MCF7 (R0R1-, EpCam+++), and human PBMC as target cells.

[0765] TABLE 25

[0766] _ HT29 _ MCF7 _ PBMC _

[0767] Emax EC50 (nM) Emax EC50 (nM) Emax EC50 (nM) Ab #T.28 88125 0.9768 2843645 16.77 8361 97.18

[0768] Ab #T.29 58372 0.5373 2030414 15.93 12164 48.67 Ab#T.3O 58820 1.973 1811367 7.058 9961 33.19

[0769] Ab #T.31 _ 31659 0.6089 820670 3.521 7297 48.33

[0770] Ab #T.32 44005 0.3921 1419247 18.61 6432 207.8

[0771] Ab #T.33 39303 0.2608 1450881 27.1 3575 12.06

[0772] Ab #T.34 140587 1.122 1654493 1.479 3509 7.207

[0773] Ab #T.35 56379 3.681 1692533 27.29 6553 126.7

[0774] Ab #T.36 84918 2.812 1596259 11.31 2700 102.2

[0775] Ab #T.37 18696 2.628 994576 11.86 8792 55.8

[0776] Ab #B.l 17446 3.995 351360 N / A 12132 43.46 Antibody #T.28 to T.37 are R0Rl-EpCam-CD3 trispecific antibodies with different designs for the split of SP34 and EpCam variable heavy (VH) and light (VL) chains (FIG.

[0777] 20). As shown in FIGS.21-22, these antibodies exhibited varying binding levels related to the specific positions of the EpCam splits.

[0778] For HT29 cell (ROR1+, EpCam+++), antibody #T.28, & T.34 and #T.36 showed strong binding level with the tumor cells, antibody #T.29, #T.3O and #T.35 showed medium binding level with the tumor cells, antibody #T.31, #T.32, #T.33 and #T.37 showed weak binding with the tumor cells (similar as #B.1).

[0779] For MCF7 cells (ROR1-, EpCam+++), antibody #T.28, #T.29, #T.3O, T.32, #T.33, #T.34, #T.35 and #T.36 showed strong binding level with the tumor cells, antibody #T.31, and #T.37 showed medium binding level with the tumor cells, antibody #B.1 didn’t show binding with the tumor cells.

[0780] For PBMC binding, Antibody #T.28, #T.29, #T.3O, #T.37 and #B.l showed strong binding level with the T cells, Antibody #T.31, #T.32 and #T.35 showed medium binding level with the T cells, Antibody #T.33, #T.34 and #T.36 showed weak binding with the T cells.

[0781] 4.3 Cell Killing Assays

[0782] We performed PBMC killing assays against various tumor cells to evaluate the antibody-mediated killing effects. In these assays. PBMCs served as effector cells.

[0783] Table 26 shows the killing results of antibody constructs using HT29 and MCF7 cells as target cells.

[0784] TABLE 26

[0785] HT29 MCF7

[0786] Emax EC5O (nM) Emax EC5O (nM) Ab #T.28 23.42 0.3395 22.37 0.4733

[0787] Ab #T.29 2335 0.0014 11.07 0.0028 Ab #T.3O 77.87 N / A 18.05 6.748

[0788] Ab #T.31 14.07 3.764 2.084 1.407

[0789] Ab #T.32 18.32 0.0053 6.193 0.036

[0790] Ab #T.33 38.38 3.12 8.765 3.214

[0791] Ab #T.34 8.486 0.8608 6.211 0.2432 Ab #T.35 38.14 24.63 91.16 0.1072

[0792] Ab #T.36 18.26 0.0122 19.12 0.8246

[0793] Ab #T.37 12.08 0.0063 9.332 0.0213

[0794] Ab #B.1 15.38 0.2594 53.31 0.3068 As shown in FIG. 23, the tumor cell killing potency of some antibodies correlates with tumor cell binding, while others did not exhibit effective killing due to structural design issues. For HT29 cells (R0R1+, EpCam+++) and MCF7 cells (R0R1-, EpCam+++), antibodies ROR1-34LEPH-Fc / 34HEPL-Fc (antibody #T.3O) and ROR1-34LEPH-Fc / 34HEPL-Fc (antibody #T.31) showed poor killing potency, possibly because they cannot form a functional EpCam or SP34 molecule with VH and VL. In contrast, ROR1-EPH34L-Fc / 34HEPL-Fc (antibody #T.29) demonstrated strong killing potency against both tumor cell lines, suggesting that an antibody configuration with AblVH-CHl-CH2-CH3 hole, AblVL-LC-Ab2VH-Ab3VH, and Ab3VL-Ab2VL-CH2-CH3 knob is effective for achieving the desired potency.

[0795] For the ROR1-EPH-Fc-34H / EPL-34L-Fc (antibody #T.32) and RORl-EPH-Fc-34H / 34L-EPL-Fc (antibody #T.33) constructs, only ROR1-EPH-Fc-34H / EPL-34L-Fc (antibody T.32) exhibited strong killing potency, while ROR1-EPH-Fc-34H / 34L-EPL-Fc (antibody #T.33) did not. This suggests that an antibody configuration with the TAAL-34L-CH2-CH3 knob structure is more effective, whereas the 34L-TAAL-CH2-CH3 knob structure may be less effective. ForHT29 cell killing, ROR1-EPH-Fc-34H / EPL-34L-Fc (antibody #T.32) demonstrated greater killing potency compared to ROR1-34H-Fc / 34L-Fc (antibody #B.1).

[0796] For the killing potency of EPH-ROR1-34H-Fc / EPL-34L-Fc (antibody #T.34) and EPH-ROR1-34H-Fc / 34L-EPL-Fc (antibody #T.35). it was observed that only EPH-R0R1-34H-Fc / EPL-34L-Fc (antibody #T.34) demonstrated medium killing potency. In contrast, EPH-ROR1-34H-Fc / 34L-EPL-Fc (antibody #T.35) did not show this level of efficacy. This suggests that the antibody with the TAAL-34L-CH2-CH3 knob structure may have the appropriate potency function, whereas the 34L-TAAL-CH2-CH3 knob structure might be less effective.

[0797] Regarding HT29 cell killing, EPH-ROR1-34H-Fc / EPL-34L-Fc (antibody #T.34) exhibited similar killing potency to ROR1-34H-Fc / 34L-Fc (antibody #B.1).

[0798] For the killing potency of EPH-ROR1-34H-Fc / EPL-Fc-34L (antibody #T.36) and EPH-ROR1-34H-Fc / 34L-Fc-EPL (antibody #T.37), both antibodies demonstrated strong killing effects. EPH-ROR1-34H-Fc / 34L-Fc-EPL (antibody #T.37) exhibited significantly stronger killing potency compared to ROR1-34H-Fc / 34L-Fc (antibody #B.1). This indicates that the VH and VL domains of the EpCam antibody function effectively together to enhance tumor binding and killing potency. EPH-ROR1-34H-Fc / EPL-Fc-34L (antibody #T.36) showed strong binding to tumor cells with minimal binding to T cells. Despite this, it still had greater killing potency than ROR1-34H-Fc / 34L-Fc (antibody #B.1). Additionally, EPH-ROR1-34H-Fc / EPL-Fc-34L (antibody T.36) demonstrated better killing potency against HT29 cells (R0R1+, EpCam+++) compared to MCF7 cells (R0R1-, EpCam+++). The split of SP34 at positions 1 and 4 in EPH-ROR1-34H-Fc / EPL-Fc-34L (antibody #T.36) was found to reduce T cell binding while still maintaining good killing potency.

[0799] 4.4 Summary

[0800] Table 27 summarize the binding and killing potency of Antibodies #T.28 through #T.37.

[0801] TABLE 27

[0802]

[0803] negative; “+ to ++++” potency increasing.

[0804] Example 5

[0805] 5.1 Antibody Construction

[0806] In the construction of the Crossbody molecule for the trispecific antibody, the variable heavy chain (VH) and the corresponding variable light chain (VL) regions for anti-EpCam and R0R1 are derived from humanized antibodies. The variable heavy chain (VH) and variable light chain (VL) regions for anti-CD3 are sourced from the humanized IgG antibody SP34. The trispecific antibody constructs were designed using a knobs-into-holes approach.

[0807] In this design, R0R1 serves as the primary tumor target due to its low expression level and strong tumor specificity, directing the antibody to the tumor first. EpCam acts as the secondary and concealed tumor target, characterized by high expression but lower tumor specificity. This strategy helps minimizing EpCam-induced on-target / off- tumor toxicity and enhances the potency of the trispecific antibody. CD3 is the T cell target and is also designed to be concealed to reduce potential toxicity.

[0808] Tables 28 and 29 provide a summary of the information and sequences for the trispecific antibody molecule developed during the invention process.

[0809] TABLE 28

[0810]

[0811] *VIIE or EPII: VII of EpCam Ab; VLE or EPL: VL of EpCam Ab; 3411: VII of CD3 Ab SP34; 34L: VL of CD3 Ab SP34; VHR: VH of ROR1 Ab; VLR: VL of ROR1 Ab; EP: EpCam.

[0812] TABLE 29

[0813]

[0814]

[0815]

[0816] Heavy and light chain fragments were synthesized by GeneArt AG (Thermo Fisher Scientific, Regensburg, Germany). These constructs were then cloned into a pCDNA3.4 vector. DNA preparations for transfection were performed using Maxi-preps and subsequently transfected into the Expi CHO expression system according to the manufacturer's instructions. The protein was purified using a Protein A resin column, followed by size exclusion chromatography (SEC) to obtain a single peak.

[0817] 5.2 Cell Binding Assays

[0818] Table 30 presents the cell binding data for antibody constructs using HT29 cells (R0R1+. EpCam+++), MCF7 cells (ROR1-, EpCam+++), and human PBMCs as target cells.

[0819] TABLE 30

[0820] HT29 MCF7 PBMC Emax EC50 (nM) Emax EC50 (nM) Emax EC50 (nM)

[0821]

[0822] Ab #T.38 58201 8.701 241461 438.6 39078 136.7 Ab #T.39 275343 2.936 560794 10.28 1533 2.361 Ab #T.4O 55579 1.981 106509 160.2 16997 277.1 Ab #T.41 333375 5.868 732442 16.25 2955 188.6 Ab #T.42 50714 2.293 27011 N / A 7641 149.7 Ab #T.43 46844 7.524 15336 N / A 13845 22.49

[0823] Antibodies #T.38 to #T.43 are RORl-EpCam-CD3 trispecific antibodies with variations in the positions of the SP34 and EpCam splits, with the VH and VL regions separated and designed at different locations (FIG. 24). As shown in FIG. 25, these antibodies exhibited varying binding levels depending on the positioning of the EpCam splits.

[0824] For HT29 cells (ROR1+, EpCam+++), antibodies #T.39 and #T.41 demonstrated strong binding, whereas antibodies #T.38, #T.4O, #T.42, and #T.43 exhibited weak binding. For MCF7 cells (ROR1-, EpCam+++), antibodies #T.38, #T.4O, #T.42, and #T.43 also showed weak binding levels.

[0825] For PBMC binding, antibodies #T.38 and #T.43 exhibited strong binding to T cells, antibodies #T.4O and #T.42 showed medium binding levels, while antibodies #T.39 and #T.41 demonstrated weak binding.

[0826] 5.3 Cell Killing Assays We conducted PBMC killing assays against various tumor cells, assessing the antibody-mediated killing effect on target cells with PBMCs as effector cells.

[0827] Table 31 presents the results of these assays using HT29 cells as the target cells.

[0828] TABLE 31

[0829] HT29

[0830] Emax EC5O (nM)

[0831] Ab #T.38 _ 16.13 _ 0.02995 _

[0832] Ab #T.39 15.39 0.00571

[0833] Ab #T.4O 18.54 0.00397

[0834] Ab #T.41 24.64 0.00301

[0835] Ab #T.42 16.11 0.0082

[0836] Ab #T.43 19.64 0.5986

[0837] Ab #B.l 26.24 0.1961

[0838] As shown in FIG. 25, the antibodies ROR1-34EI-Eph-Fc / EPL-Fc-34L (antibody #T.39), ROR1-34H-Eph-Fc / 34L-Fc-EPL (antibody #T.4O), RORl-Eph34H-Fc / EPL-Fc-34L (antibody #T.41), and RORl-Eph34H-Fc / 34L-Fc-EPL (antibody #T.42) exhibited similar, stronger killing potency compared to ROR1-34H-Fc / 34L-Fc (antibody #B.1). This suggests that antibodies incorporating the 34VL-CH2-CH3-EPVL knob structure may effectively conceal EpCam binding while providing stronger killing potency. In contrast, antibodies with the EPVL-CH2-CH3-34VL knob structure may still exhibit excessive EpCam binding, potentially leading to on- target / off-tumor toxicity.

[0839] For RORl-34-Dia-EPH-Fc / Fc-EPL (antibody #T.38), the killing potency was stronger compared to ROR1-34H-Fc / 34L-Fc (antibody #B.1). In contrast, EPH-RORl-EPL-Fc-34H / 34L-Fc (antibody #T.43) did not demonstrate better killing potency than ROR1-34H-Fc / 34L-Fc (antibody #B.1). This suggests that the VH and VL regions of the EpCam antibody at positions 1 and 5 may not function optimally.

[0840] 5.4 Summary

[0841] Table 32 summarizes the binding and killing potency of Antibodies #T.38 through #T.44.

[0842] TABLE 32

[0843]

[0844]

[0845] negative; “+ to ++++” potency increasing

[0846] Example 6

[0847] 6.1 Antibody Construction

[0848] In constructing the Crossbody molecule for the trispecific antibody, the variable heavy chain (VH) and variable light chain (VL) regions for anti-EpCam and anti-RORl are derived from humanized antibodies, while those for anti-CD3 are derived from the humanized IgG antibody SP34. The trispecific antibody constructs were designed using either an Fc or knobs-into-holes approach.

[0849] In this design, ROR1 serves as the primary tumor target due to its low expression level and strong tumor specificity, guiding the antibody to the tumor initially. EpCam acts as the secondary' and concealed tumor target, characterized by high expression but lower tumor specificity, which helps reduce EpCam-induced on-target / off- tumor toxicity and enhances the potency of the trispecific antibody. CD3 is the T cell target and is also designed to be concealed to minimize toxicity.

[0850] Tables 33 and 34 summarize the information and sequences for the trispecific antibody molecule developed during the invention process.

[0851] TABLE 33

[0852]

[0853]

[0854] *VHE or EPH: VH of EpCam Ab; VLE or EPL: VL of EpCam Ab; 34H: VH of CD3 Ab SP34; 34L: VL of CD3 Ab SP34; VHR: VH of ROR1 Ab; VLR: VL of ROR1 Ab; EP: EpCam.

[0855] TABLE 34

[0856]

[0857]

[0858]

[0859]

[0860]

[0861]

[0862]

[0863] Heavy and light chain fragments were generated through gene synthesis by GeneArt AG (Thermo Fisher Scientific. Regensburg, Germany). The synthesized constructs were then cloned into the pCDNA3.4 vector. DNA for transfection was prepared using Maxipreps and subsequently transfected into the ExpiCHO expression system according to the manufacturer’s instructions. The protein was purified using a Protein A resin column, followed by size exclusion chromatography (SEC) to obtain a single peak.

[0864] 6.2 Cell Binding Assays

[0865] Table 35 shows the cell binding data for antibody constructs tested on MCF7 cells (ROR1-, EpCam+++) and human PBMCs as target cells.

[0866] TABLE 35

[0867] MCF7 PBMC

[0868] _ Emax _ EC50 (nM) Emax _ EC5O (nM)

[0869] Ab #T.44 152058 20.17 3231 91.18

[0870] Ab #T.45 30914 134 10051 237.7

[0871] Ab #T.46 130744 58.77 2380 41.94

[0872] Ab #T.47 174148 18.49 1855 30.88

[0873] Ab #T.48 _ 147749 _ 43.67 _ 15043 _ 7.09 _

[0874] Ab #T.49 168876 32.43 13422 48.42

[0875] Ab #T.5O _ 54371 _ 11.2 _ 27576 _ 7.149 _

[0876] Ab #T.51 162612 51.51 15027 48.4

[0877] Ab #T.52 13526 22.96 13134 22.91

[0878] Ab #T.53 _ 90513 _ 31.29 _ 5947 _ 60.1 _

[0879] Ab #T.54 128865 8.117 19489 7.164

[0880] Ab #T.55 262314 16.87 1716 37.21

[0881] Ab #T.56 444523 33.45 2096 34.23

[0882] Ab #T.57 533935 3.738 35857 87.14

[0883] Ab #T.58 _ 486869 _ 11.59 _ 7829 _ 50.1 _

[0884] Ab #B.l 31627 41.37 10364 50.27 Antibodies #T.44 to #T.58 are R0R1 -EpCam-CD3 trispecific antibodies, with the SP34 and EpCam domains, as well as the VH and VL regions, positioned differently (FIG.

[0885] 26). As show n in FIG.27, the antibodies exhibited varying levels of binding related to the different positions of the EpCam domains. For MCF7 cells (R0R1-, EpCam+++), antibodies #T.55, #T.56, #T.57, and #T,58 demonstrated strong binding to the tumor cells. Antibodies #T.44, #T.46, #T.47, #T.48, #T.49, #T.51. #T.53, and #T.54 showed medium binding levels, while antibodies #T.45, #T.5O. and #T.52 exhibited weak binding levels to the tumor cells.

[0886] For PBMC binding (FIG.28), antibodies #T.45, #T.48, #T.49, #T.5O, #T.51, #T.52, #T.54, #T.57, and #B.l showed strong binding to T cells. Antibodies #T.53 and #T.58 exhibited medium binding levels, while antibodies #T.44, #T.46, #T.47, #T.55. and #T.56 showed weak binding to T cells.

[0887] 6.3 Cell Killing Assays

[0888] We conducted PBMC killing assays against various tumor cells, using PBMCs as the effector cells.

[0889] Table 36 presents the killing results of antibody constructs targeting HT29 and MCF7 cells.

[0890] TABLE 36

[0891] HT29 MCF7

[0892] Emax EC50 (nM) Emax EC50 (nM)

[0893] Ab #T.44 28.17 0.02868 8.831 0.07877

[0894] Ab #T.45 19.69 0.08938 10.61 3.781

[0895] Ab #T.46 65.68 1.9 28.9 37.91

[0896] Ab #T.47 18.45 0.00306 9.387 0.00720

[0897] Ab #T.48 12.01 0.00573 4.575 0.1661

[0898] Ab#T.49 12.22 0.00136 14.05 6.00554

[0899] Ab #T.50 12.75 0.00144 27.61 0.00083

[0900] Ab #T.51 11.62 0.2037 13.77 0.3006

[0901] Ab #T.52 56 3.065 43.45 N / A

[0902] Ab #T.53 16.42 0.01452 8.135 1.008

[0903] Ab #T.54 22.13 0.00831 11.29 0.01024

[0904] Ab #T.55 11.84 0.2165 3.636 0.9724

[0905] Ab #T.56 14.84 0.03101 22 0.02414

[0906] Ab #T.57 14.58 0.02472 10.72 0.00045

[0907] Ab #T.58 11.22 0.00273 9.407 0.00422

[0908] Ab#B.i 11.57 6.07553 52.3 0.5219

[0909] As shown in FIGS.29-30, some antibodies exhibited tumor cell killing potency that correlates with their binding to tumor cells, while others did not demonstrate killing efficacy due to structural design issues. For HT29 (R0R1+, EpCam+++) and MCF7 (R0R1-, EpCam+++) cells, antibody #T.44 (ROR1-34H-Fc-EPH / EPL-Fc-34L) did not show superior killing potency compared to antibody #B.l (ROR1-34H-Fc / 34L-Fc). It appears that the positioning of the VH and VL domains in the SP34 antibody at positions 1 and 4 may reduce CD3 binding and killing potency. Similarly, antibody #T.45 (ROR1-34H-Fc-EPH / 34L-Fc-EPL) did not demonstrate better killing potency than antibody #B.1. This suggests that the positioning of the VH and VL domains in the EpCam antibody at positions 3 and 4 may be suboptimal for its intended function.

[0910] For the killing potency of ROR1-34H-Fc-EPH / 34L-EPL-Fc (antibody #T.46) and ROR1-34H-Fc-EPH / EPL-34L-Fc (antibody #T.47), only antibody #T.47 exhibited strong killing potency. In contrast, antibody #T.46 did not show similar efficacy. This suggests that the antibody with the TAAL-34L-CH2-CH3 knob structure (antibody #T.47) has the optimal potency function, while the 34L-TAAL-CH2-CH3 knob structure (antibody #T.46) may be less effective. ForHT29 cell killing, antibody #T.47 demonstrated stronger killing potency-compared to antibody #B.l (ROR1-34H-Fc / 34L-Fc).

[0911] For the killing potency of RORl-Fc-EPH / 34ScFv-EPL-Fc (antibody #T.48) and RORl-Fc-EPH / EPL-34ScFv-Fc (antibody T.49), both antibodies exhibited stronger killing potency compared to ROR1-34H-Fc / 34L-Fc (antibody #B.1). However, the VH and VL domains of the EpCam antibody at positions 2 and 3 may still cause excessive EpCam binding, potentially leading to on-target / off-tumor toxicity.

[0912] ROR1-EPLEPH-Fc / 34L34H-Fc (antibody #T.5O) is a novel design where the VH and VL domains of EpCam and SP34 are linked directly without a linker, in contrast to the ScFv structure that includes a short linker between VH and VL. This design results in very weak binding to the EpCam target but very strong binding to T cells. In terms of killing potency, antibody #T.5O demonstrated much stronger activity compared to ROR1-34H-Fc / 34L-Fc (antibody #B.1). However, the direct linkage of VH and VL in the SP34 antibody may cause excessive T cell binding, potentially leading to T cell toxicity.

[0913] For the killing potency of EPH-ROR1-EPL-Fc-34H / Fc-34L (antibody #T.5I) and 34L / EPH-ROR1-EPL-Fc / 34H-Fc (antibody #T.52), antibody #T.51 showed similar killing potency to ROR1-34H-Fc / 34L-Fc (antibody #B.1), while antibody #T.52 exhibited very weak killing potency. This suggests that the antibody with the EPH-VLR-LC-EPL structure does not enhance potency effectively.

[0914] For the killing potency of EPH34H-ROR1-Fc / Fc-EPL34L (antibody #T.53) and 34HEPH-ROR1-Fc / Fc-EPL34L (antibody #T.54), both antibodies demonstrated better killing potency against HT29 cells compared to ROR1-34H-Fc / 34L-Fc (antibody

[0915]

[0916] However, EPH34H-ROR1-Fc / Fc-EPL34L (antibody #T.53) showed weak killing potency against MCF7 cells.

[0917] For the killing potency of EPH / 34H-ROR1-34L-Fc / EPL-Fc (antibody #T.55) and EPH-ROR1-34H-Fc / EPL-Fc-34L (antibody #T.56), antibody #T.56 demonstrated better killing potency compared to ROR1-34H-Fc / 34L-Fc (antibody #B.1). In contrast, antibody #T.55 showed similar killing potency to ROR1-34H-Fc / 34L-Fc. This suggests that the positioning of the VH and VL domains in the SP34 antibody at positions 1 and 4 may diminish CD3 binding and overall killing potency.

[0918] For the killing potency of 34L / EPH34H-ROR1-Fc / EPL-Fc (antibody #T.57) and 34L / ROR1-34HEPH-Fc / EPL-Fc (antibody #T.58), both antibodies demonstrated better killing potency compared to ROR1-34H-Fc / 34L-Fc (antibody #B.1).

[0919] 6.4 Summary

[0920] Table 37 provides a summary of the binding and killing potency for antibodies #T.44 through #T.58.

[0921] TABLE 37

[0922]

[0923] negative; “+ to ++++” potency increasing.

[0924] Example 7

[0925] 7.1 Antibody Construction

[0926] In the construction of the Crossbody molecule for trispecific antibodies, the variable heavy chain (VH) and corresponding variable light chain (VL) regions for anti-EpCam and anti-RORl are derived from humanized antibodies, while the VH and VL regions for anti-CD3 are sourced from the humanized IgG antibody SP34. The trispecific antibody constructs were designed using either an Fc or knobs-into-holes structure.

[0927] In these designs, R0R1 serves as the primary tumor target due to its low expression level and strong tumor specificity, directing the antibody to the tumor. EpCam is used as the secondary tumor target; it has high expression but low tumor specificity, which helps reduce EpCam-induced on-target / off-tumor toxicity’ and enhances the potency of the trispecific antibody. CD3, the T cell target, is also included in a concealed design to minimize toxicity.

[0928] Tables 38 and 39 summarize the information and sequences for the trispecific antibody molecule developed during this study.

[0929] TABLE 38

[0930]

[0931] *VHE or EPH: VH of EpCam Ab; VLE or EPL: VL of EpCam Ab; 34H: VH of CD3 Ab SP34; 34L: VL of CD3 Ab SP34; VHR: VH of ROR1 Ab; VLR: VL of ROR1 Ab; EP: EpCam. TABLE 39

[0932]

[0933]

[0934]

[0935] Heavy and light chain fragments were generated through gene synthesis by GeneArt AG (Thermo Fisher Scientific, Regensburg, Germany). The synthesized constructs were then cloned into the pCDNA3.4 vector. DNA for transfection was prepared using Maxipreps and subsequently transfected into the ExpiCHO expression system according to the manufacturer’s instructions. The protein was purified using a Protein A resin column, followed by size exclusion chromatography (SEC) to obtain a single peak.

[0936] 7.2 Cell Binding Assays

[0937] Table 40 presents the cell binding data for antibody constructs tested on MCF7 cells (ROR1-, EpCam+++) and human PBMCs. TABLE 40

[0938] _ MCF7 _ PBMC _

[0939] Emax EC50 (nM) Emax EC50 (nM) Ab #T.59 164480 1432 6127 64.69

[0940] Ab #T.60 96373 97.86 1468 N / A

[0941] Ab #T.61 30896 35.98 1466 34.43

[0942] Ab #T.62 _ 110435 179.1 _ 7186 _ 63.18 _

[0943] Ab #T.63 240955 203.6 3294 68.7

[0944] Ab #T.64 59775 116.1 21386 6.197

[0945] Ab #T.65 193679 25.04 2221 79.15

[0946] Antibodies #T.59 to #T.64 are RORl-EpCam-CD3 trispecific antibodies with SP34 and / or EpCam splits. The VH and VL domains are separated and positioned differently in e...

Claims

1. WHAT TS CLAIMED IS:

1. A protein complex, comprising3.a first split building block,4.a second split building block, and5.an Fc region,6.wherein the first and second split building blocks are located at any two of the positions 1-4 in FIG. 1, any two of the positions 1-6 in FIG. 2, or any two of the positions 1-8 in FIG. 3.

2. A protein complex, comprising a first heavy chain variable region (VH1) and a first light chain variable region (VL1) that together form a first antigen-binding domain, wherein the first antigen-binding domain is not a Fab region or an ScFv.

3. The protein complex of claim 2, further comprising a second heavy chain variable region (VH2) and a second light chain variable region (VL2) that together form a second antigenbinding domain, wherein:9.(i) the second antigen-binding domain is a Fab region or an ScFv; or10.(ii) the second antigen-binding domain is not a Fab region or an ScFv.

4. The protein complex of claim 3, further comprising a third heavy chain variable region (VH3) and a second light chain variable region (VL3) that together form a third antigenbinding domain, wherein:12.(i) the third antigen-binding domain is a Fab region or an ScFv; or13.(ii) the third antigen-binding domain is not a Fab region or an ScFv.

5. A protein complex, comprising:15.(i) a first polypeptide comprising a first light chain variable region (VL1), a light chain constant domain (CL), and a second heavy chain variable region (VH2);16.(ii) a second polypeptide comprising a first heavy chain variable region (VH1), a heavy chain constant domain 1 (CHI), a first hinge region, a first heavy chain constant domain 2 (CH2) and a first heavy chain constant domain 3 (CH3); (iii) a third polypeptide comprising a second light chain variable region (VL2), a second hinge region, a second CH2 and a second CH3;17.wherein the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target;18.wherein the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to a second target;19.optionally wherein the VL1 and VH1 are covalently linked via a disulfide bond; wherein the first and second polypeptides are covalently linked via a disulfide bond between the CL and CHI; and20.wherein the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions.

6. A protein complex, comprising:22.(i) a first polypeptide comprising a first light chain variable region (VL1) and a first CL; (ii) a second polypeptide comprising a first heavy chain variable region (VH1), a first CHI, a first hinge region, a first CH2, a first CH3, and a second heavy chain variable region (VH2); (iii) a third polypeptide comprising a third heavy chain variable region (VH3), a second CHI, a second hinge region, a second CH2, a second CH3, and a second light chain variable region (VL2);23.(iv) a fourth polypeptide comprising a third light chain variable region (VL3) and a second CL;24.wherein the VL1 and VH1 associate to form a first antigen-domain capable of binding to a first target;25.wherein the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to a second target;26.wherein the VL3 and VH3 associate to form a third antigen-domain capable of binding to the first target, optionally the VL3 is identical to the VL1, and the VH3 is identical to the VH1; optionally wherein the VL1 and VH1 are covalently linked via a disulfide bond; optionally wherein the VL2 and VH2 are covalently linked via a disulfide bond; optionally wherein the VL3 and VH3 are covalently linked via a disulfide bond; wherein the first and second polypeptides are covalently linked via a disulfide bond between the first CL and the first CHI;27.wherein the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions; and28.wherein the third and fourth polypeptides are covalently linked via a disulfide bond between the second CHI and the second CL.

7. A protein complex, comprising:30.(i) a first polypeptide comprising a first light chain variable region (VL1), a CL and a second heavy chain variable region (VH2);31.(ii) a second polypeptide comprising a first heavy chain variable region (VH1), a CHI, a first hinge region, a first CH2 and a first CH3;32.(iii) a third polypeptide comprising a third heavy chain variable region (VH3), a T cell receptor variable alpha region (TCR Va), a second hinge region, a second CH2 and a second CH3;33.(iv) a fourth polypeptide comprising a third light chain variable region (VL3), a T cell receptor variable beta region (TCR Vb) and a second light chain variable region (VL2); wherein the VL1 and VH1 associate to form a first antigen-domain capable of binding to a first target;34.wherein the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to a second target;35.wherein the VL3 and VH3 associate to form a third antigen-domain capable of binding to the first target, optionally the VL3 is identical to the VL1, and the VH3 is identical to the VH1; optionally wherein the VL1 and VH1 are covalently linked via a disulfide bond; optionally wherein the VL2 and VH2 are covalently linked via a disulfide bond; optionally wherein the VL3 and VH3 are covalently linked via a disulfide bond; wherein the first and second polypeptides are covalently linked via a disulfide bond between the CL and the CHI;36.wherein the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions; and wherein the third and fourth polypeptides are covalently linked via a disulfide bond between the TCR Va and the TCR Vb.

8. A protein complex comprising:38.(i) a first polypeptide comprising a first light chain variable region (VL1), a CL, and a second heavy chain variable region (VH2);39.(ii) a second polypeptide comprising a first heavy chain variable region (VH1), a CHI, a first hinge region, a first CH2 and a first CH3;40.(iii) a third polypeptide comprising a single-chain variable fragment (ScFv), a second light chain variable region (VL2), a second hinge region, a second CH2 and a second CH3; wherein the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target;41.wherein the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to a second target;42.wherein the ScFv is capable of binding to a third target;43.optionally wherein the VL1 and VH1 are covalently linked via a disulfide bond; wherein the first and second polypeptides are covalently linked via a disulfide bond between the CL and CHI; and44.wherein the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions.

9. The protein complex of claim 8, wherein the first target is the same as the third target;46.wherein the protein complex comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nonnative disulfide bonds between the first and second polypeptides that can enhance stability of the protein complex.

10. A protein complex comprising:48.(i) a first polypeptide comprising a first light chain variable region (VL1), a CL, a second heavy chain variable region (VH2), and a third heavy chain variable region (VH3);49.(ii) a second polypeptide comprising a first heavy chain variable region (VH1), a CHI, a first hinge region, a first CH2, and a first CH3; (iii) a third polypeptide comprising a third light chain variable region (VL3), a second light chain variable region (VL2), a second hinge region, a second CH2, and a second CH3; wherein the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target;50.wherein the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to a second target;51.wherein the VL3 and VH3 associate to form a third antigen-binding domain capable of binding to a third target;52.optionally wherein the VL1 and VH1 are covalently linked via a disulfide bond; wherein the first and second polypeptides are covalently linked via a disulfide bond between the CL and CHI; and53.wherein the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions.

11. The protein complex of claim 10, wherein the first target is selected for its low expression level and high tumor specificity; wherein the second target is a tumor-associated antigen with high expression but lower tumor specificity; wherein the third target is a T cell target; wherein the protein complex comprises one or more non-native disulfide bonds between the first and second polypeptides that can enhance stability of the protein complex.

12. A protein complex comprising:56.(i) a first polypeptide comprising a first light chain variable region (VL1), a CL, a third heavy chain variable region (VH3), and a second heavy chain variable region (VH2);57.(ii) a second polypeptide comprising a first heavy chain variable region (VH1), a CHI, a first hinge region, a first CH2, and a first CH3;58.(iii) a third polypeptide comprising a third light chain variable region (VL3), a second light chain variable region (VL2), a second hinge region, a second CH2, and a second CH3; wherein the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target;59.wherein the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to a second target; wherein the VL3 and VH3 associate to form a third antigen-binding domain capable of binding to a third target;60.optionally wherein the VL1 and VH1 are covalently linked via a disulfide bond; wherein the first and second polypeptides are covalently linked via a disulfide bond between the CL and CHI; and61.wherein the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions.

13. A protein complex comprising:63.(i) a first polypeptide comprising a second heavy chain variable region (VH2), a third heavy chain variable region (VH3), a first light chain variable region (VL1), and a CL;64.(ii) a second polypeptide comprising a first heavy chain variable region (VH1), a CHI, a first hinge region, a first CH2, and a first CH3;65.(iii) a third polypeptide comprising a third light chain variable region (VL3), a second light chain variable region (VL2), a second hinge region, a second CH2, and a second CH3; wherein the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target;66.wherein the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to a second target;67.wherein the VL3 and VH3 associate to form a third antigen-binding domain capable of binding to a third target;68.optionally wherein the VL1 and VH1 are covalently linked via a disulfide bond; wherein the first and second polypeptides are covalently linked via a disulfide bond between the CL and CHI; and69.wherein the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions.

14. A protein complex comprising:71.(i) a first polypeptide comprising a first light chain variable region (VL1), a CL, and a second heavy chain variable region (VH2); (ii) a second polypeptide comprising a first heavy chain variable region (VH1), a CHI, a first hinge region, a first CH2, and a first CH3;72.(iii) a third polypeptide comprising a second light chain variable region (VL2), a single-chain variable fragment (ScFv), a second hinge region, a second CH2, and a second CH3; wherein the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target;73.wherein the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to a second target;74.wherein the ScFv is capable of binding to a third target;75.optionally wherein the VL1 and VH1 are covalently linked via a disulfide bond; wherein the first and second polypeptides are covalently linked via a disulfide bond between the CL and CHI; and76.wherein the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions.

15. A protein complex comprising:78.(i) a first polypeptide comprising a first light chain variable region (VL1), a second heavy chain variable region (VH2), a CL, and a third heavy chain variable region (VH3);79.(ii) a second polypeptide comprising a second light chain variable region (VL2), a first heavy chain variable region (VH1), a CHI, a first hinge region, a first CH2, and a first CH3;80.(iii) a third polypeptide comprising a third light chain variable region (VL3), a second hinge region, a second CH2, and a second CH3;81.wherein the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target;82.wherein the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to a second target;83.wherein the VL3 and VH3 associate to form a third antigen-binding domain capable of binding to a third target;84.optionally wherein the VL1 and VH1 are covalently linked via a disulfide bond; wherein the first and second polypeptides are covalently linked via a disulfide bond between the CL and CHI; and wherein the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions.

16. A protein complex comprising:86.(i) a first polypeptide comprising a second heavy chain variable region (VH2), a first light chain variable region (VL1), a first CL, and a third heavy chain variable region (VH3);87.(ii) a second polypeptide comprising a first heavy chain variable region (VH1), a first CHI, a first hinge region, a first CH2, and a first CH3;88.(iii) a third polypeptide comprising a fourth heavy chain variable region (VH4), a second CHI, a second hinge region, a second CH2, and a second CH3;89.(iv) a fourth polypeptide comprising a second light chain variable region (VL2), a fourth light chain variable region (VL4), a second CL, and a third light chain variable region (VL3); wherein the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target;90.wherein the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to a second target;91.wherein the VL3 and VH3 associate to form a third antigen-binding domain capable of binding to a third target;92.wherein the VL4 and VH4 associate to form a fourth antigen-binding domain capable of binding to a fourth target;93.optionally wherein the VL1 and VH1 are covalently linked via a disulfide bond; wherein the first and second polypeptides are covalently linked via a disulfide bond between the first CL and the first CHI;94.wherein the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions; and95.wherein the third and fourth polypeptides are covalently linked via a disulfide bond between the second CHI and the second CL.

17. A protein complex comprising:97.(i) a first polypeptide comprising a first light chain variable region (VL1) and a first CL; (ii) a second polypeptide comprising a first heavy chain variable region (VH1), a first CHI, a first hinge region, a first CH2, a first CH3, and a second heavy chain variable region (VH2); (iii) a third polypeptide comprising a second light chain variable region (VL2), a third heavy chain variable region (VH3), a second CHI, a second hinge region, a second CH2, and a second CH3;98.(iv) a fourth polypeptide comprising a third light chain variable region (VL3) and a second CL;99.wherein the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target;100.wherein the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to a second target;101.wherein the VL3 and VH3 associate to form a third antigen-domain capable of binding to the first target, optionally the VL3 is identical to the VL1, and the VH3 is identical to the VH1; optionally wherein the VL1 and VH1 are covalently linked via a disulfide bond; wherein the first and second polypeptides are covalently linked via a disulfide bond between the first CL and the first CHI;102.wherein the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions; and103.wherein the third and fourth polypeptides are covalently linked via a disulfide bond between the second CHI and the second CL.

18. A protein complex comprising:105.(i) a first polypeptide comprising a second heavy chain variable region (VH2), a first light chain variable region (VL1), a first CL, and a second light chain variable region (VL2); (ii) a second polypeptide comprising a first heavy chain variable region (VH1), a first CHI, a first hinge region, a first CH2, and a first CH3;106.(iii) a third polypeptide comprising a third heavy chain variable region (VH3), a second CHI, a second hinge region, a second CH2, and a second CH3;107.(iv) a fourth polypeptide comprising a fourth heavy chain variable region (VH4), a third light chain variable region (VL3), a second CL, and a fourth light chain variable region (VL4); wherein the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target;108.wherein the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to a second target;109.wherein the VL3 and VH3 associate to form a third antigen-binding domain capable of binding to the first target, optionally the VL3 is identical to the VL1, and the VH3 is identical to the VH1;110.wherein the VL4 and VH4 associate to form a fourth antigen-binding domain capable of binding to the second target, optionally the VL4 is identical to the VL2, and the VH4 is identical to the VH2;111.optionally wherein the VL1 and VH1 are covalently linked via a disulfide bond; optionally wherein the VL3 and VH3 are covalently linked via a disulfide bond; wherein the first and second polypeptides are covalently linked via a disulfide bond between the first CL and the first CHI;112.wherein the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions; and113.wherein the third and fourth polypeptides are covalently linked via a disulfide bond between the second CHI and the second CL.

19. A protein complex comprising:115.(i) a first polypeptide comprising a first light chain variable region (VL1), a CL, and a first portion of a cytokine;116.(ii) a second polypeptide comprising a first heavy chain variable region (VH1), a CHI, a first hinge region, a first CH2, and a first CH3;117.(iii) a third polypeptide comprising a second portion of the cytokine, a second hinge region, a second CH2, and a second CH3;118.wherein the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target;119.wherein the first and second portions of the cytokine associate to form a functional cytokine; optionally wherein the VL1 and VH1 are covalently linked via a disulfide bond; wherein the first and second polypeptides are covalently linked via a disulfide bond between the CL and CHI; and120.wherein the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions.

20. A protein complex comprising:122.(i) a first polypeptide comprising a first light chain variable region (VL1), a first CL, and a first portion of a first cytokine;123.(ii) a second polypeptide comprising a first heavy chain variable region (VH1), a first CHI, a first hinge region, a first CH2, a first CH3, and a second portion of the first cytokine;124.(iii) a third polypeptide comprising a second heavy chain variable region (VH2), a second CHI, a second hinge region, a second CH2, a second CH3, and a second portion of a second cytokine;125.(iv) a fourth polypeptide comprising a second light chain variable region (VL2), a second CL, and a first portion of the second cytokine;126.wherein the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target;127.wherein the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to the first target, optionally the VL2 is identical to the VL1, and the VH2 is identical to the VH1;128.wherein the first and second portions of the first cytokine associate to form a functional first cytokine;129.wherein the first and second portions of the second cytokine associate to form a functional second cytokine, optionally the first and second cytokines are identical;130.optionally wherein the VL1 and VH1 are covalently linked via a disulfide bond; wherein the first and second polypeptides are covalently linked via a disulfide bond between the first CL and the first CHI;131.wherein the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions; and132.wherein the third and fourth polypeptides are covalently linked via a disulfide bond between the second CHI and the second CL.

21. A protein complex comprising:133.(i) a first polypeptide comprising a second light chain variable region (VL2), a first heavy chain variable region (VH1), a first light chain variable region (VL1), a second heavy chain variable region (VH2), a first hinge region, a first CH2, and a first CH3;134.(ii) a second polypeptide comprising a third heavy chain variable region (VH3), a CHI, a second hinge region, a second CH2, and a second CH3;135.(iii) a third polypeptide comprising a blocking mask, a linker with a cleavage site, a third light chain variable region (VL3), and a CL;136.wherein the VL1 and VH1 associate to form a first antigen-binding domain capable of binding a first target;137.wherein the VL2 and VH2 associate to form a second antigen-binding domain capable of binding a second target;138.wherein the VL3 and VH3 associate to form a third antigen-binding domain capable of binding a third target;139.optionally wherein the VL3 and VH3 are covalently linked via a disulfide bond; wherein the first and second polypeptides are covalently linked via a disulfide bond between the first and second hinge regions; and140.wherein the second and third polypeptides are covalently linked via a disulfide bond between the CHI and CL.

22. The antibody of claim 21, wherein the first target is selected for its low expression level and strong tumor specificity; wherein the second target is a tumor-associated antigen with high expression but lower tumor specificity; wherein the third target is a T cell antigen.

23. A protein complex comprising:143.(i) a first polypeptide comprising a first CL, a first heavy chain variable region (VH1), a first light chain variable region (VL1), a first CHI, a first hinge region, a first CH2, a first CH3, and a second heavy chain variable region (VH2); (ii) a second polypeptide comprising a third heavy chain variable region (VH3), a second CHI, a second hinge region, a second CH2, a second CH3, and a second light chain variable region (VL2);144.(iii) a third polypeptide comprising a blocking mask, a linker with a cleavage site, a third light chain variable region (VL3), and a second CL;145.wherein the VL1 and VH1 associate to form a first antigen-binding domain capable of binding a first target;146.wherein the VL2 and VH2 associate to form a second antigen-binding domain capable of binding a second target;147.wherein the VL3 and VH3 associate to form a third antigen-binding domain capable of binding a third target;148.optionally wherein the VL3 and VH3 are covalently linked via a disulfide bond; wherein the first CL and the first CHI are covalently linked via a disulfide bond; wherein the first and second polypeptides are covalently linked via a disulfide bond between the first and second hinge regions; and149.wherein the second and third polypeptides are covalently linked via a disulfide bond between the second CHI and the second CL.

24. A protein complex comprising:151.(i) a first polypeptide comprising a single-chain variable fragment (ScFv), a first hinge region, a first CH2, a first CH3, and a second heavy chain variable region (VH2);152.(ii) a second polypeptide comprising a third heavy chain variable region (VH3), a CHI, a second hinge region, a second CH2, a second CH3, and a second light chain variable region (VL2);153.(iii) a third polypeptide comprising a blocking mask, a linker with a cleavage site, a third light chain variable region (VL3), and a CL;154.wherein the ScFv is capable of binding to a first target;155.wherein the VL2 and VH2 associate to form a first antigen-binding domain capable of binding a second target;156.wherein the VL3 and VH3 associate to form a second antigen-binding domain capable of binding a third target; optionally wherein the VL3 and VH3 are covalently linked via a disulfide bond; wherein the first and second polypeptides are covalently linked via a disulfide bond between the first and second hinge regions; and157.wherein the second and third polypeptides are covalently linked via a disulfide bond between the CHI and CL.

25. A protein complex comprising:159.(i) a first polypeptide comprising a first light chain variable region (VL1), a CL, and a second heavy chain variable region (VH2);160.(ii) a second polypeptide comprising a first heavy chain variable region (VH1), a CHI, a first hinge region, a first CH2, and a first CH3;161.(iii) a third polypeptide comprising a blocking mask, a linker with a cleavage site, a second light chain variable region (VL2), a single-chain variable fragment (ScFv), a second hinge region, a second CH2, and a second CH3;162.wherein the VL1 and VH1 associate to form a first antigen-binding domain capable of binding a first target;163.wherein the VL2 and VH2 associate to form a second antigen-binding domain capable of binding a second target;164.wherein the ScFv is capable of binding to a third target;165.optionally wherein the VL1 and VH1 are covalently linked via a disulfide bond; wherein the first and second polypeptides are covalently linked via a disulfide bond between the CL and CHI; and166.wherein the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions.

26. A protein complex comprising:168.(i) a first polypeptide comprising a blocking mask, a linker with a cleavage site, a first light chain variable region (VL1), a CL, and a second heavy chain variable region (VH2);169.(ii) a second polypeptide comprising a first heavy chain variable region (VH1), a CHI, a first hinge region, a first CH2, and a first CH3; (iii) a third polypeptide comprising a single-chain variable fragment (ScFv), a second light chain variable region (VL2), a second hinge region, a second CH2, and a second CH3; wherein the VL1 and VH1 associate to form a first antigen-binding domain capable of binding a first target;170.wherein the VL2 and VH2 associate to form a second antigen-binding domain capable of binding a second target;171.wherein the ScFv is capable of binding to a third target;172.optionally wherein the VL1 and VH1 are covalently linked via a disulfide bond; wherein the first and second polypeptides are covalently linked via a disulfide bond between the CL and CHI; and173.wherein the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions.

27. A protein complex comprising:175.(i) a first polypeptide comprising a first light chain variable region (VL1), a CL, and a second heavy chain variable region (VH2);176.(ii) a second polypeptide comprising a first heavy chain variable region (VH1), a CHI, a first hinge region, a first CH2, and a first CH3;177.(iii) a third polypeptide comprising a third heavy chain variable region (VH3), a second hinge region, a second CH2, and a second CH3;178.wherein the VL1 and VH1 associate to form a first antigen-binding domain capable of binding a first target;179.optionally wherein the VH2 and VH3 associate to form a second antigen-binding domain capable of binding a second target, optionally the VH2 and VH3 are identical; optionally wherein the VL1 and VH1 are covalently linked via a disulfide bond; wherein the first and second polypeptides are covalently linked via a disulfide bond between the CL and CHI; and180.wherein the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions.

28. A protein complex comprising:(i) a first polypeptide comprising a first light chain variable region (VL1), a CL, and a second light chain variable region (VL2);182.(ii) a second polypeptide comprising a first heavy chain variable region (VH1), a CHI, a first hinge region, a first CH2, and a first CH3;183.(iii) a third polypeptide comprising a second hinge region, a second CH2, a second CH3, and a third light chain variable region (VL3);184.wherein the VL1 and VH1 associate to form a first antigen-binding domain capable of binding a first target;185.optionally wherein the VL2 and VL3 associate to form a second antigen-binding domain capable of binding a second target, optionally the VL2 and VL3 are identical; optionally wherein the VL1 and VH1 are covalently linked via a disulfide bond; wherein the first and second polypeptides are covalently linked via a disulfide bond between the CL and CHI; and186.wherein the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions.

29. A protein complex comprising:188.(i) a first polypeptide comprising a first light chain variable region (VL1), a CL, a second heavy chain variable region (VH2) and a third heavy chain variable region (VH3);189.(ii) a second polypeptide comprising a first heavy chain variable region (VH1), a CHI, a first hinge region, a first CH2, and a first CH3;190.(iii) a third polypeptide comprising a single-chain variable fragment (ScFv), a second light chain variable region (VL2), a second hinge region, a second CH2, a second CH3 and a third light chain variable region (VL3);191.wherein the VL1 and VH1 associate to form a first antigen-binding domain capable of binding to a first target;192.wherein the VL2 and VH2 associate to form a second antigen-binding domain capable of binding to a second target;193.wherein the VL3 and VH3 associate to form a second antigen-binding domain capable of binding to a third target; wherein the ScFv comprises a fourth light chain variable region (VL4) and a fourth heavy chain variable region (VH4), wherein the ScFv is capable of binding to a fourth target, optionally the VL4 is identical to the VL1, and the VH4 is identical to the VH1; optionally wherein the VL1 and VH1 are covalently linked via a disulfide bond; wherein the first and second polypeptides are covalently linked via a disulfide bond between the CL and CHI; and194.wherein the second and third polypeptides are covalently linked via a disulfide bond between the first and second hinge regions.

30. A nucleic acid comprising a polynucleotide encoding the protein complex of any one of claims 1-29.

31. The nucleic acid of claim 30, wherein the nucleic acid is a DNA (e.g., cDNA) or RNA (e.g., mRNA).

32. A vector comprising one or more of the nucleic acids of claim 30 or 31.

33. A cell comprising the vector of claim 32.

34. The cell of claim 33, wherein the cell is a CHO cell.

35. A cell comprising one or more of the nucleic acids of claim 30 or 31.

36. A method of producing a protein complex, the method comprising202.(a) culturing the cell of any one of claims 33-35 under conditions sufficient for the cell to produce the protein complex; and203.(b) collecting the protein complex produced by the cell.

37. A protein conjugate comprising the protein complex of any one of claims 1-29, covalently bound to a therapeutic agent.

38. The protein conjugate of claim 37, wherein the therapeutic agent is a cytotoxic or cytostatic agent.

39. A method of treating a subject having a disease or disorder, the method comprising administering a therapeutically effective amount of a composition comprising the protein complex of any one of claims 1-29, or the protein conjugate of claims 37 or 38, to the subject.

40. The method of claim 39, wherein the subject has a cancer, an autoimmune disease, a hematopoietic diseases, or a metabolic disease.

41. A method of decreasing the rate of tumor growth, the method comprising208.contacting a tumor cell with an effective amount of a composition comprising the protein complex of any one of claims 1-29, or the protein conjugate of claim 37 or 38.

42. A method of killing a tumor cell, the method comprising210.contacting a tumor cell with an effective amount of a composition comprising the protein complex of any one of claims 1-29, or the protein conjugate of claim 37 or 38.

43. A pharmaceutical composition comprising the protein complex of any one of claims 1-29, and a pharmaceutically acceptable carrier.

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