Multivalent multispecific constructs for targeted payload delivery

Abstract

The present disclosure In particular relates to multivalent, multispecific constructs having two or more antigen-binding polypeptides and conjugated therapeutically effective payloads, and methods of using the constructs to treat a disease or disorder.

Description

Technical Field

[0001] This disclosure especially This relates to multivalent, multispecific constructs coupled with therapeutic payloads, and the use of said constructs in the treatment of diseases or conditions.

[0002] Cross-references to related applications

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 562,832, filed March 8, 2024, the entire contents of which are incorporated herein by reference in their entirety.

[0004] Description of XML files submitted electronically

[0005] This application contains a sequence list, which is submitted electronically in XML file format and incorporated herein by reference in its entirety. The XML file was created on February 18, 2025, named "SDL-001PC_136782-5001_Sequence_Listing", and has a size of 25,313 bytes. Background Technology

[0006] Current methods for targeted payload delivery to specific cells are not efficient enough to fully realize the benefits of such approaches. Using existing formats, such as IgG antibody-drug conjugates, targeted payload delivery is highly dependent on target expression. In the context of cancer, few tumor-associated antigens (TAAs) have sufficiently high expression levels to achieve efficient, specific payload delivery for optimal biological effects and clinical outcomes. For example, antibody-drug conjugates targeting Her2+ overexpression (such as trastuzumab dxd) have achieved near-80% response rates and near-2-year durable response durations in cancer patients with high antigen expression (<25% of breast cancer, <2% of lung cancer). However, no other TAA expression level approaches Her2. Other approved ADCs targeting low-expressing TAAs (such as Trop2) have not shown benefits approaching those of Her2. Generally, current methods are limited by payload internalization efficiency, which is in turn affected by low efficacy and toxicity. In view of this, there is an urgent need for targeted payload delivery methods that can specifically and efficiently deliver large numbers of payloads to target cells, such as cancer cells.

[0007] IgG-based methods are limited by their valence state, which significantly restricts their ability to activate target cell internalization mechanisms. Higher valence methods (such as IgM antibodies or engineered hexabodies) are limited by short pharmacokinetic (PK), large size, difficulty in achieving ordered and reproducible aggregation of one or more targets, and control over geometry. Two-site formats allow binding of different domains on the target, leading to cross-linking and the formation of numerous bound target clusters on the target cell surface. However, this often occurs best under optimal conditions, including high target expression, which is itself a limiting factor for targets other than Her2. Such formats are also limited by poor aggregation control and reproducibility across different patients, resulting in poor therapeutic indexes and limited multispecific targeting capabilities. Darpins, nanobodies, and bicycles are small and inexpensive to produce, but they suffer from short half-lives, low valence, lack of controllable geometry for aggregation, limited multispecific capabilities, and poor target aggregation. In summary, current methods for targeted payload delivery struggle to deliver sufficient payloads to target cells to achieve optimal biological and clinical benefits.

[0008] There remains a need in the field for novel approaches that can achieve both dense clusters of the same type of tightly bound cell surface molecules (multivalence) and dense clusters of multiple different targets (multispecificity) to maximize cell internalization and delivery of targeted therapeutic agents. Summary of the Invention

[0009] In terms of aspects and implementation methods, this document provides a multivalent and multispecific construct comprising two or more antigen-binding peptides and a conjugated therapeutic payload, wherein each of the two or more antigen-binding peptides comprises three or more antigen-binding domains, wherein each of the three or more antigen-binding domains of each of the two or more antigen-binding peptides is capable of binding to a respective target on its target cell, and wherein the construct is capable of simultaneously binding to four or more targets on the surface of the target cell.

[0010] In terms of aspects and implementation methods, this document provides a multivalent and multispecific construct comprising two or more antigen-binding peptides and a conjugated therapeutic payload, wherein each of the two or more antigen-binding peptides comprises three or more antigen-binding domains, wherein each of the three or more antigen-binding domains of each of the two or more antigen-binding peptides is capable of binding to a respective target on its target cell, and wherein the construct is capable of simultaneously binding to four or more targets on the surface of the target cell.

[0011] In aspects and implementations, the construct comprises: (a) a therapeutic payload; and (b) two or more antigen-binding peptides targeting two or more different targets, each antigen-binding peptide comprising, from the N-terminus to the C-terminus, a first antigen-binding domain (ABD C), a first adapter (L1), a second antigen-binding domain (ABD B), a second adapter (L2), a third antigen-binding domain (ABD A), an optional third adapter (L3), and a scaffold region; wherein L1 attaches ABD C to ABD B, L2 attaches ABDB to ABD A, and L3 optionally attaches ABD A to the scaffold region, such that each antigen-binding peptide comprises, from the N-terminus to the C-terminus, (i) ABD C – L1 – ABD B – L2 – ABD A – scaffold region or (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region; and wherein the scaffold region comprises a loop, a central protein anchor, or IgG Fc or a portion thereof or a modified form thereof.

[0012] In aspects and embodiments, the scaffold region comprises IgG Fc or a portion thereof or a modified form thereof, wherein the construct comprises two antigen-binding peptides, wherein each of the two antigen-binding peptides further comprises (i) ABD C – L1 – ABD B – L2 – ABD A arranged from N-terminus to C-terminus, such that each antigen-binding peptide comprises ABD C – L1 – ABD B – L2 – ABD A – scaffold region – ABDA – L2 – ABD B – L1 – ABD C, or (ii) ABD C – L1 – ABD B – L2 – ABD A – L3, such that each antigen-binding peptide comprises ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region – L3 – ABD A – L2 – ABD B – L1 – ABD C, wherein the N-terminal and C-terminal ABD C domains are the same or different, wherein the N-terminal and C-terminal ABD C domains are identical. The B structural domains may be the same or different, and the N-terminal and C-terminal A structural domains may be the same or different. Similarly, the N-terminal and C-terminal L1 connectors may be the same or different, the N-terminal and C-terminal L2 connectors may be the same or different, the N-terminal and C-terminal L3 connectors may be the same or different, and the N-terminal and C-terminal hinges may be the same or different.

[0013] In aspects and implementations, the scaffold region comprises a rigid polypeptide; wherein the construct comprises 'n' antigen-binding polypeptides; wherein each of the antigen-binding polypeptides further comprises a fourth linker (L4), wherein L4 attaches the scaffold region to ABD C; and wherein the scaffold region further comprises a clasp domain, the clasp domain attaching the first antigen-binding polypeptide and the nth antigen-binding polypeptide to form a closed loop, wherein n is 3-12.

[0014] In terms of aspects and implementation methods, this document provides a multivalent and multispecific construct comprising: (a) a therapeutic payload; and (b) two antigen-binding peptides targeting two or more different targets, each antigen-binding peptide comprising, from the N-terminus to the C-terminus, a first antigen-binding domain (ABD C), a first linker (L1), a second antigen-binding domain (ABDB), a second linker (L2), a third antigen-binding domain (ABD A), an optional third linker (L3), a scaffold region, an optional second L3, a second ABD A, a second L2, a second ABD B, a second L1, and a second ABD C; wherein L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, and L3 optionally attaches ABD A to the scaffold region, such that each antigen-binding peptide comprises, from the N-terminus to the C-terminus, (i) ABD C – L1 – ABD B – L2 – ABD A – scaffold region – ABD A – L2 – ABD B – L1 – ABD C or (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region – L3 – ABD A – L2 – ABD B – L1 – ABD C; and wherein the scaffold region contains IgG Fc or a portion thereof or a modified form thereof, wherein the N-terminal and C-terminal ABD C domains are the same or different, wherein the N-terminal and C-terminal ABD B domains are the same or different, and wherein the N-terminal and C-terminal ABD A domains are the same or different.

[0015] In terms of aspects and implementation methods, this document provides a multivalent and multispecific construct comprising: (a) a therapeutic payload; and (b) two antigen-binding peptides targeting two or more different targets, each antigen-binding peptide comprising, from its N-terminus to its C-terminus, a first antigen-binding domain (ABD C), a first adapter (L1), a second antigen-binding domain (ABDB), a second adapter (L2), a third antigen-binding domain (ABD A), an optional third adapter (L3), and a scaffold region; wherein L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, and L3 optionally attaches ABD A to the scaffold region, such that each antigen-binding peptide comprises, from its N-terminus to its C-terminus, (i) ABD C – L1 – ABD B – L2 – ABD A – scaffold region or (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region; and wherein the scaffold region comprises IgGFc or a portion thereof or a modified form thereof.

[0016] In terms of aspects and implementation methods, this document provides a multivalent and multispecific construct comprising: (a) a therapeutic payload; and (b) four antigen-binding peptides targeting two or more different targets, each antigen-binding peptide comprising, from the N-terminus to the C-terminus, a first antigen-binding domain (ABD C), a first linker (L1), a second antigen-binding domain (ABDB), a second linker (L2), a third antigen-binding domain (ABD A), an optional third linker (L3), and a scaffold region; wherein L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, and L3 optionally attaches ABD A to the scaffold region, such that each antigen-binding peptide comprises, from the N-terminus to the C-terminus, (i) ABD C – L1 – ABD B – L2 – ABD A – scaffold region or (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region; and wherein the scaffold region comprises a central protein anchor, optionally wherein the central protein anchor comprises an FcRn binding domain.

[0017] In terms of aspects and implementation methods, this document provides a multivalent and multispecific construct comprising: (a) a therapeutic payload; and (b) 'n' antigen-binding peptides targeting two or more distinct targets, each antigen-binding peptide comprising, from N-terminus to C-terminus, a first antigen-binding domain (ABD C), a first linker (L1), a second antigen-binding domain (ABDB), a second linker (L2), a third antigen-binding domain (ABD A), an optional third linker (L3), a scaffold region, and a fourth linker (L4); wherein L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, L3 optionally attaches ABD A to the scaffold region, and L4 attaches the scaffold region to ABD C, such that each antigen-binding peptide comprises, from N-terminus to C-terminus, (i) ABD C – L1 – ABD B – L2 – ABD A – scaffold region – L4 or (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region – L4; wherein the scaffold region contains a rigid polypeptide; wherein the construct further contains a clasp domain that attaches a first antigen-binding polypeptide and an nth antigen-binding polypeptide to form a closed loop; and wherein n is 3-12.

[0018] In aspects and embodiments, ABD C, ABD B, and / or ABD A are VHH, VNAR, Fab, ScFv, nanobodies, biantibodies, synthetic microbodies, ligands, or their binding fragments. In aspects and embodiments, ABD C, ABD B, and / or ABD A are conditional binders, optionally wherein the binding of ABD C, ABD B, and / or ABD A to their respective targets depends on pH, calcium, or ATP levels. In aspects and embodiments, ABD C, ABD B, and / or ABD A target specific cell types, optionally wherein ABDC, ABD B, and / or ABD A target cancer cells or immune cells. In aspects and embodiments, ABD C, ABD B, and / or ABDA promote the internalization of the multivalent multispecific construct by target cells, optionally by promoting lysosomal transport of the multivalent multispecific construct by target cells, optionally wherein lysosomal transport is clathrin-mediated endocytosis or receptor-mediated endocytosis. In aspects and embodiments, ABD C, ABD B, and / or ABD A promote the chemotherapeutic sensitivity of target cells, and / or wherein ABD C, ABD B, and / or ABD A promotes apoptosis or necrosis of target cells. In aspects and embodiments, ABD C, ABD B, and / or ABD A comprises at least one half-life-extending molecule, optionally wherein the half-life-extending molecule is an FcRn binding domain, albumin, PEG, XTEN, or human serum albumin (HSA) binding domain.

[0019] In terms of aspects and implementation methods, the stent region contains IgG C H 2-C H 3 or a fragment thereof, optionally including IgG C H 2-C H 3 further includes a second hinge area, optionally wherein the support area includes hinge-C H 2-C H 3-Hinge.

[0020] In aspects and implementations, ABD C, ABD B, ABD A, L1, L2, L3, L4, hinge region, second hinge region and / or scaffold region contain at least one site for conjugating payload, optionally at least one of which contains Cys, Tyr or Lys residues or engineered glycans or tags for site-specific conjugation.

[0021] In aspects and embodiments, ABD C binds to a first target molecule, ABD B binds to a second target molecule, and / or ABD A binds to a third target molecule, optionally wherein N-terminal ABD C binds to the first target molecule, N-terminal ABD B binds to the second target molecule, N-terminal ABD A binds to the third target molecule, C-terminal ABD C binds to a fourth target molecule, C-terminal ABD B binds to a fifth target molecule, and / or C-terminal ABD A binds to a sixth target molecule. In aspects and embodiments, ABD C binds to a first epitope on the first target molecule, ABD B binds to a second epitope on the first target molecule, and / or ABD A binds to a third epitope on the first target molecule.

[0022] In aspects and embodiments, L1, L2, L3, and / or L4 are approximately 15-30 aa in length. In aspects and embodiments, L1, L2, L3, and / or L4 comprise one or more of Gly, Ser, Thr, Pro, Arg, Phe, Glu, Cys, Asn, Ala, Asp, and Gln residues, optionally wherein L1, L2, L3, and / or L4 primarily comprise Gly, Ser, and Thr residues. In aspects and embodiments, L1, L2, L3, and / or L4 primarily comprise Gly and Ser residues. In aspects and embodiments, L1, L2, L3, and / or L4 comprise (Gly-Ser) residues. n (Gly4-Ser) n (SEQ ID NO: 1), (Gly3-Ser) n(SEQ ID NO: 2), Gly-Gly-Ser, Gly-Gly-Ser-Gly (SEQ ID NO: 3), Gly-Gly-Gly-Gly (SEQ ID NO: 4), Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 5), Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 6), Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 7), Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 8), Gly-Ser-Gly-Ser-Gly-Ser (SEQ ID NO: 9), (Gly4-Ser)3 (SEQ ID NO: 10), (Gly2-Ser)3 (SEQ ID NO: 11), (Gly2-Ser)2 (SEQ ID NO: 12), ferritin E-helix, (Glu-Ala-Ala-Ala-Lys) 1-3 (SEQ ID NO: 13), (Ala-Pro) n (SEQ ID NO: 14), IgG C H 1. IgG C H 2-C H 3. Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 15) or Gly-Gly-Ser-Asn-Gly-Ser-Gly-Gly-Ser (SEQ ID NO: 28).

[0023] In aspects and embodiments, the scaffold region comprises a rigid protein. In aspects and embodiments, the scaffold region comprises a portion for extending PK, optionally wherein the portion for extending PK is an FcRn binding domain, IgG C H 2-C H 3. Human serum albumin, human transferrin, or fragments thereof.

[0024] In terms of aspects and implementation methods, the multivalent multispecific construct (i) delivers the payload to target cells more efficiently than monomers or single-specific binders containing only one copy of ABD C, ABD B, or ABD A, (ii) is internalized by target cells at a higher rate than monomers or single-specific binders containing only one copy of ABD C, ABD B, or ABD A, and / or (iii) has improved specificity for binding to and / or killing tumor cells than monomers or single-specific binders containing only one copy of ABD C, ABD B, or ABD A.

[0025] In terms of aspects and implementation methods, this document provides a nucleic acid that encodes the multivalent and multispecific construct described herein.

[0026] In terms of aspects and implementation methods, this document provides a host cell containing the nucleic acids described herein.

[0027] In terms of aspects and implementation methods, this document provides a pharmaceutical composition comprising the multivalent and multispecific construct described herein, the nucleic acid described herein, or the host cell described herein.

[0028] In terms of aspects and implementation methods, this document provides a method for treating a disease or condition in a subject of need, optionally wherein the disease or condition is cancer, the method comprising administering the multivalent multispecific construct described herein, the nucleic acid described herein, the host cell described herein, or the pharmaceutical composition described herein.

[0029] In the aspects and implementation plan, cancer is a solid tumor, optionally including NSCLC, ovarian cancer, breast cancer, pancreatic cancer, colorectal cancer, or gastric cancer.

[0030] In the context and implementation plan, the cancer is a blood cancer, optionally including leukemia, lymphoma, or multiple myeloma. Attached Figure Description

[0031] Figure 1Non-limiting examples of the multivalent, multispecific constructs described herein, having two antigen-binding peptides targeting two or more different targets, are shown. Each antigen-binding peptide comprises two copies of antigen-binding domains (ABDs) A, B, and C, two copies of linkers L1, L2, and optionally L3, and a scaffold region arranged from the N-terminus to the C-terminus as (i) ABD C – L1 – ABD B – L2 – ABD A – scaffold region – ABD A – L2 – ABD B – L1 – ABD C or (ii) ABDC – L1 – ABD B – L2 – ABD A – L3 – scaffold region – L3 – ABD A – L2 – ABD B – L1 – ABDC. It should be noted that the features described (e.g., “rigid,” “planar,” etc.) are for illustrative purposes only.

[0032] Figure 2 Non-limiting examples of the multivalent, multispecific constructs described herein, having two antigen-binding peptides targeting two or more different targets, are shown. Each antigen-binding peptide comprises antigen-binding domains (ABDs) A, B, and C, linkers L1, L2, and optionally L3, and a scaffold region arranged from the N-terminus to the C-terminus as (i) ABD C – L1 – ABD B – L2 – ABD A – scaffold region or (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region. It should be noted that the features described (e.g., “rigid,” “planar,” etc.) are for illustrative purposes only.

[0033] Figure 3 Non-limiting examples of the multivalent, multispecific constructs described herein are shown, each containing four antigen-binding peptides targeting two or more different targets. Each antigen-binding peptide comprises antigen-binding domains (ABDs) A, B, and C, linkers L1, L2, and optionally L3, and a scaffold region arranged from the N-terminus to the C-terminus as (i) ABD C – L1 – ABD B – L2 – ABD A – scaffold region or (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region. It should be noted that dimensions and other characteristics (e.g., “rigid,” “planar,” etc.) are for illustrative purposes only.

[0034] Figure 4Non-limiting examples of the multivalent multispecific constructs described herein are shown, each containing 'n' antigen-binding peptides targeting two or more different targets. Each antigen-binding peptide comprises antigen-binding domains (ABDs) A, B, and C, linkers L1, L2, optionally L3 and L4, and a scaffold region arranged from the N-terminus to the C-terminus as (i) ABD C – L1 – ABD B – L2 – ABD A – scaffold region – L4 or (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region – L4, and a clasp domain that attaches the first antigen-binding peptide and the nth antigen-binding peptide to form a closed loop. It should be noted that binding affinity and other characteristics (e.g., “rigid,” “angled,” “flexible,” “strong binding,” “single specificity,” etc.) are used for illustration only. It should also be noted that functional language (e.g., “increasing the half-life of a molecule by rescuing the protein from intracellular degradation,” “allowing multispecificity,” etc.) is used for illustration only and is not intended to be theoretically binding.

[0035] Figure 5A and Figure 5B The binding and functional activity of the multivalent construct described herein, which includes ABD targeting OX40, are illustrated.

[0036] Figure 6A The expression assembly and quality of Y6 and X12 constructs containing VHH binding subunits targeting OX40, PD-L1, and 41BB are shown.

[0037] Figure 6B The activity of the multivalent and multispecific construct described herein, containing ABD targeting OX40, PD-L1, and 4-1BB, in the OX40 activation reporter gene assay is demonstrated.

[0038] Figure 7 The activities of the multivalent multispecific construct described herein, comprising ABD targeting OX40, PD-L1 and 4-1BB, and the hybrid multispecific construct described herein, comprising a 41BB fusion peptide trimer and an ABD (Y12 format) targeting CD38, are shown.

[0039] Figure 8A Biological conjugation of multivalent constructs with a second C-terminal hinge to achieve DAR equivalent to IgG – illustrated here schematically with VHH-Fc as an example.

[0040] Figure 8B The activity of the multivalent construct described herein, which contains an ABD targeting OX40 and has different connectors in the VHH interphase, is demonstrated.

[0041] Figure 8CExpressions of the constructs described herein, which include different joint-hinge combinations, are shown.

[0042] Figure 9 The internalization assay results of the multivalent construct described herein, which includes ABD targeting BCMA, are shown.

[0043] Figure 10 The results of internalization assays for the multivalent construct described herein, which includes ABD targeting GPRC5D, are shown.

[0044] Figure 11 The results of internalization assays for the multivalent construct described herein, which includes CD38-targeting ABD, are shown.

[0045] Figure 12 The internalization assay results of the described multivalent and multispecific construct containing ABD targeting BCMA, CD38, and GPRC5D are shown.

[0046] Figure 13 Cell killing assays are shown for the multivalent and multispecific constructs described herein, including ABDs targeting CD38, GPRC5D, and / or BCMA.

[0047] Figure 14 Activity assays of the described multivalent and multispecific constructs of ABD containing GLP-1 and / or GCG receptors are shown.

[0048] Figure 15 The internalization rates of various X12 and Y6 constructs on OX40-expressing cell lines are shown. Detailed Implementation

[0049] This disclosure is partly based on the finding that multifunctional and multivalent platform technologies can be used to integrate multiple targets and signals, thereby enabling the precise delivery of highly effective therapeutic payloads with enhanced functional capabilities. While not wishing to be bound by theory, it is believed that multiple simultaneous contacts of the multivalent and multispecific constructs described herein with multiple targets on the surface of target cells increase their functional effects and / or internalization efficiency.

[0050] Multivalent and multispecific constructs

[0051] In terms of aspects and implementation methods, this document provides a multivalent and multispecific construct comprising two or more antigen-binding peptides and a conjugated therapeutic payload, wherein each of the two or more antigen-binding peptides comprises three or more antigen-binding domains, wherein each of the three or more antigen-binding domains of each of the two or more antigen-binding peptides is capable of binding to a respective target on its target cell, and wherein the construct is capable of simultaneously binding to four or more targets on the surface of the target cell.

[0052] In embodiments, the construct of the present invention is multivalent and has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 binding domains. In embodiments, the construct of the present invention is multispecific and targets 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 targets. In the embodiments, the construct of the present invention is multivalent and has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 binding domains, and the construct of the present invention is multispecific and targets 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 targets.

[0053] In one embodiment, the construct of the present invention has two binding domains and targets two targets. In another embodiment, the construct of the present invention has three binding domains and targets two targets. In another embodiment, the construct of the present invention has four binding domains and targets two targets. In another embodiment, the construct of the present invention has five binding domains and targets two targets. In another embodiment, the construct of the present invention has six binding domains and targets two targets. In another embodiment, the construct of the present invention has seven binding domains and targets two targets. In another embodiment, the construct of the present invention has eight binding domains and targets two targets. In another embodiment, the construct of the present invention has nine binding domains and targets two targets. In another embodiment, the construct of the present invention has ten binding domains and targets two targets. In another embodiment, the construct of the present invention has eleven binding domains and targets two targets. In another embodiment, the construct of the present invention has twelve binding domains and targets two targets.

[0054] In one embodiment, the construct of the present invention has two binding domains and targets three targets. In another embodiment, the construct of the present invention has two binding domains and targets four targets. In another embodiment, the construct of the present invention has two binding domains and targets five targets. In another embodiment, the construct of the present invention has two binding domains and targets six targets. In another embodiment, the construct of the present invention has two binding domains and targets seven targets. In another embodiment, the construct of the present invention has two binding domains and targets eight targets. In another embodiment, the construct of the present invention has two binding domains and targets nine targets. In another embodiment, the construct of the present invention has two binding domains and targets ten targets. In another embodiment, the construct of the present invention has two binding domains and targets eleven targets. In another embodiment, the construct of the present invention has two binding domains and targets twelve targets.

[0055] In one embodiment, the construct of the present invention has two binding domains and targets three targets. In another embodiment, the construct of the present invention has three binding domains and targets three targets. In another embodiment, the construct of the present invention has four binding domains and targets three targets. In another embodiment, the construct of the present invention has five binding domains and targets three targets. In another embodiment, the construct of the present invention has six binding domains and targets three targets. In another embodiment, the construct of the present invention has seven binding domains and targets three targets. In another embodiment, the construct of the present invention has eight binding domains and targets three targets. In another embodiment, the construct of the present invention has nine binding domains and targets three targets. In another embodiment, the construct of the present invention has ten binding domains and targets three targets. In another embodiment, the construct of the present invention has eleven binding domains and targets three targets. In another embodiment, the construct of the present invention has twelve binding domains and targets three targets.

[0056] In one embodiment, the construct of the present invention has three binding domains and targets three targets. In another embodiment, the construct of the present invention has three binding domains and targets four targets. In another embodiment, the construct of the present invention has three binding domains and targets five targets. In another embodiment, the construct of the present invention has three binding domains and targets six targets. In another embodiment, the construct of the present invention has three binding domains and targets seven targets. In another embodiment, the construct of the present invention has three binding domains and targets eight targets. In another embodiment, the construct of the present invention has three binding domains and targets nine targets. In another embodiment, the construct of the present invention has three binding domains and targets ten targets. In another embodiment, the construct of the present invention has three binding domains and targets eleven targets. In another embodiment, the construct of the present invention has three binding domains and targets twelve targets.

[0057] In one embodiment, the construct of the present invention has two binding domains and targets four targets. In another embodiment, the construct of the present invention has three binding domains and targets four targets. In another embodiment, the construct of the present invention has four binding domains and targets four targets. In another embodiment, the construct of the present invention has five binding domains and targets four targets. In another embodiment, the construct of the present invention has six binding domains and targets four targets. In another embodiment, the construct of the present invention has seven binding domains and targets four targets. In another embodiment, the construct of the present invention has eight binding domains and targets four targets. In another embodiment, the construct of the present invention has nine binding domains and targets four targets. In another embodiment, the construct of the present invention has ten binding domains and targets four targets. In another embodiment, the construct of the present invention has eleven binding domains and targets four targets. In another embodiment, the construct of the present invention has twelve binding domains and targets four targets.

[0058] In one embodiment, the construct of the present invention has four binding domains and targets three targets. In another embodiment, the construct of the present invention has four binding domains and targets four targets. In another embodiment, the construct of the present invention has four binding domains and targets five targets. In another embodiment, the construct of the present invention has four binding domains and targets six targets. In another embodiment, the construct of the present invention has four binding domains and targets seven targets. In another embodiment, the construct of the present invention has four binding domains and targets eight targets. In another embodiment, the construct of the present invention has four binding domains and targets nine targets. In another embodiment, the construct of the present invention has four binding domains and targets ten targets. In another embodiment, the construct of the present invention has four binding domains and targets eleven targets. In another embodiment, the construct of the present invention has four binding domains and targets twelve targets.

[0059] In aspects and embodiments, the construct binds to four or more targets on the surface of target cells within a 20-30 nm radius. While not wishing to be bound by theory, it is believed that this binding allows for increased internalization through multiple target internalization mechanisms, thus maximizing payload internalization. In aspects and embodiments, the multivalent multispecific construct includes a scaffold region containing a rigid protein. In aspects and embodiments, the multivalent multispecific construct includes a scaffold region containing IgG C. H 2-C H 3 or fragments thereof, for FcRn binding and elongation of the PK. In aspects and embodiments, the multivalent multispecific construct comprises an FcRn binding domain or portion thereof for FcRn binding and elongation of the PK. In aspects and embodiments, the multivalent multispecific construct comprises a linker between each of three or more antigen-binding domains having a rigid-to-flexible gradient from the center to the distal end, wherein the central linker is rigid and the distal linker is highly flexible. While not wishing to be bound by theory, it is believed that this linker configuration allows the construct to maintain a central planar interaction surface, thereby maximizing the density of aggregated targets, while allowing the edges to adapt to the target geometry.

[0060] In terms of aspects and implementation methods, the multivalent multispecific construct comprises (a) a therapeutic payload; and (b) two or more antigen-binding peptides targeting two or more different targets, each antigen-binding peptide comprising, from the N-terminus to the C-terminus, a first antigen-binding domain (ABD C), a first linker (L1), a second antigen-binding domain (ABD B), a second linker (L2), a third antigen-binding domain (ABD A), an optional third linker (L3), and a scaffold region; wherein L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, and L3 optionally attaches ABD A to the scaffold region, such that each antigen-binding peptide comprises, from the N-terminus to the C-terminus, a first antigen-binding domain (ABD C), a second antigen-binding domain (ABD B), a second linker (L2), a third antigen-binding domain (ABD A), an optional third linker (L3), and a scaffold region; such that each antigen-binding peptide comprises, from the N-terminus to the C-terminus, a first antigen-binding domain (ABD C), a second antigen-binding domain (ABD B), a third antigen-binding domain (ABD A), an optional third linker (L3), and a scaffold region.

[0061] (i) ABD C – L1 – ABD B – L2 – ABD A – Support area or

[0062] (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – Support area; Furthermore, the scaffold region contains a loop, a central protein anchor, or IgG Fc or a portion thereof or a modified form.

[0063] This permutation may be referred to herein as "Y6". In aspects and implementations, ABD C and L1 are absent. This permutation may be referred to herein as "Y4". In aspects and implementations, ABD C, L1, ABD B, and L2 are absent. This permutation may be referred to herein as "Y2".

[0064] In aspects and embodiments, the scaffold region comprises IgG Fc or a portion thereof or a modified form thereof. In aspects and embodiments, the scaffold region comprises IgG C. H 2-C H 3. In aspects and implementation schemes, the support area includes hinge-C H 2-C H 3. In aspects and implementation schemes, the support area includes hinge-C H 2-C H 3-Hinge. In aspects and implementations, the support region includes a chemical core bonded using click chemistry.

[0065] In aspects and embodiments, the scaffold region comprises IgG Fc or a portion thereof or a modified form thereof, and the multivalent multispecific construct comprises two antigen-binding peptides of the antigen-binding polypeptides. In aspects and embodiments, each of the two antigen-binding peptides further comprises, arranged from the N-terminus to the C-terminus,

[0066] (i) ABD A – L2 – ABD B – L1 – ABD C, such that each antigen-binding polypeptide contains an ABD C – L1 – ABD B – L2 – ABD A – scaffold region – ABD A – L2 – ABD B – L1 – ABD C arranged from the N-terminus to the C-terminus, or

[0067] (ii) L3 – ABD A – L2 – ABD B – L1 – ABD C, such that each antigen-binding polypeptide contains ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region – L3 – ABD A – L2 – ABD B – L1 – ABD C arranged from the N-terminus to the C-terminus.

[0068] This arrangement may be referred to herein as "X12". In aspects and embodiments, the N-terminal and C-terminal ABD C domains are identical. In aspects and embodiments, the N-terminal and C-terminal ABD C domains are different. In aspects and embodiments, the N-terminal and C-terminal ABD C domains bind to the same target. In aspects and embodiments, the N-terminal and C-terminal ABD C domains bind to different targets. In aspects and embodiments, the N-terminal and C-terminal ABD B domains are identical. In aspects and embodiments, the N-terminal and C-terminal ABD B domains are different. In aspects and embodiments, the N-terminal and C-terminal ABD B domains bind to the same target. In aspects and embodiments, the N-terminal and C-terminal ABD B domains bind to different targets. In aspects and embodiments, the N-terminal and C-terminal ABD A domains are identical. In aspects and embodiments, the N-terminal and C-terminal ABD A domains are different. In aspects and embodiments, the N-terminal and C-terminal ABD A domains bind to the same target. In aspects and embodiments, the N-terminal and C-terminal ABD A domains bind to different targets. In the aspects and implementation methods, the N-terminal ABD C binds to the first target molecule, the N-terminal ABD B binds to the second target molecule, the N-terminal ABD A binds to the third target molecule, the C-terminal ABD C binds to the fourth target molecule, the C-terminal ABD B binds to the fifth target molecule, and / or the C-terminal ABD A binds to the sixth target molecule.

[0069] In this aspect and implementation, ABD C and L1 are not present. This permutation may be referred to herein as "X8". In this aspect and implementation, ABD C, L1, ABD B, and L2 are not present. This permutation may be referred to herein as "X4".

[0070] In aspects and embodiments, one or more of ABD A, L2, ABD B, L1, and ABD C are absent. For example, in aspects and embodiments, each of the two antigen-binding polypeptides comprises an array of [missing information - likely a sequence of numbers] arranged from the N-terminus to the C-terminus.

[0071] (i) ABD C – L1 – ABD B – L2 – ABD A – Scaffold Area – ABD A – L2 – ABD B; (ii) ABD C – L1 – ABD B – L2 – ABD A – Support area – ABD A; (iii) ABD B – L2 – ABD A – stent area – ABD A – L2 – ABD B – L1 – ABD C; (iv) ABD A – stent area – ABD A – L2 – ABD B – L1 – ABD C; (v) ABD B – L2 – ABD A – Scaffold Area – ABD A – L2 – ABD B; (vi) ABD B – L2 – ABD A – Scaffold Area – ABD A; (vii) ABD A – Support area – ABD A – L2 – ABD B; (viii) ABD C – L1 – ABD B – L2 – ABD A – L3 – Scaffold Area – L3 – ABD A – L2 – ABD B; (ix) ABD C – L1 – ABD B – L2 – ABD A – L3 – Support area – L3 – ABD A; (x) ABD B – L2 – ABD A – L3 – Support area – L3 – ABD A – L2 – ABD B – L1 – ABD C; (xi) ABD A – L3 – Support area – L3 – ABD A – L2 – ABD B – L1 – ABD C; (xii) ABD B – L2 – ABD A – L3 – Scaffold Area – L3 – ABD A – L2 – ABD B; (xiii) ABD B – L2 – ABD A – L3 – Support area – L3 – ABD A; or (xiv) ABD A – L3 – Stent area – L3 – ABD A – L2 – ABD B.

[0072] These arrangements may be referred to as "asymmetrical" in this article.

[0073] In aspects and implementations, the scaffold region includes a central protein anchor, optionally wherein the central protein anchor includes an FcRn binding domain, and the construct includes four antigen-binding peptides among the antigen-binding peptides.

[0074] In aspects and embodiments, the scaffold region comprises a rigid polypeptide; wherein the construct comprises 'n' antigen-binding polypeptides; wherein each antigen-binding polypeptide further comprises a fourth linker (L4), wherein the L4 attaches the scaffold region to ABD C; and wherein the scaffold region further comprises a clasp domain, the clasp domain attaching the first antigen-binding polypeptide and the nth antigen-binding polypeptide to form a closed loop, wherein n is 3-12. In aspects and embodiments, the scaffold region comprises an angled π-α helical framework. In aspects and embodiments, the clasp domain comprises an IgM tail.

[0075] In terms of aspects and implementation methods, this document provides a multivalent and multispecific construct comprising (a) a therapeutic payload; and (b) two antigen-binding peptides targeting two or more different targets, each antigen-binding peptide comprising, from the N-terminus to the C-terminus, a first antigen-binding domain (ABD C), a first linker (L1), a second antigen-binding domain (ABD B), a second linker (L2), a third antigen-binding domain (ABD A), an optional third linker (L3), a scaffold region, an optional second L3, a second ABD A, a second L2, a second ABD B, a second L1, and a second ABD C; wherein L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, and L3 optionally attaches ABD A to the scaffold region, such that each antigen-binding peptide comprises, from the N-terminus to the C-terminus, a first antigen-binding domain (ABD C), a second linker (L1), a second linker (L2), a second linker (L2), a second linker (L1), and a second linker (L2); wherein L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, and L3 optionally attaches ABD A to the scaffold region, such that each antigen-binding peptide comprises, from the N-terminus to the C-terminus, a first linker (L1), a second linker (L2), a second linker (L3), and a second linker (L3).

[0076] (i) ABD C – L1 – ABD B – L2 – ABD A – Support area – ABD A – L2 – ABD B – L1 – ABD C or

[0077] (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – Scaffold Area – L3 – ABD A – L2 –ABD B – L1 – ABD C; Furthermore, the scaffold region contains IgG Fc or a portion thereof or a modified form thereof.

[0078] In some aspects and implementations, the N-terminal and C-terminal ABD C domains are identical. In some aspects and implementations, the N-terminal and C-terminal ABD C domains are different. In some aspects and implementations, the N-terminal and C-terminal ABD C domains bind to the same target. In some aspects and implementations, the N-terminal and C-terminal ABD C domains bind to different targets. In some aspects and implementations, the N-terminal and C-terminal ABD B domains are identical. In some aspects and implementations, the N-terminal and C-terminal ABD B domains are different. In some aspects and implementations, the N-terminal and C-terminal ABD B domains bind to the same target. In some aspects and implementations, the N-terminal and C-terminal ABD B domains bind to different targets. In some aspects and implementations, the N-terminal and C-terminal ABD A domains are identical. In some aspects and implementations, the N-terminal and C-terminal ABD A domains are different. In some aspects and implementations, the N-terminal and C-terminal ABD A domains bind to the same target. In some aspects and implementations, the N-terminal and C-terminal ABD A domains bind to different targets. In the aspects and implementation methods, the N-terminal ABD C binds to the first target molecule, the N-terminal ABD B binds to the second target molecule, the N-terminal ABD A binds to the third target molecule, the C-terminal ABD C binds to the fourth target molecule, the C-terminal ABD B binds to the fifth target molecule, and / or the C-terminal ABD A binds to the sixth target molecule.

[0079] In aspects and embodiments, one or more of ABD A, L2, ABD B, L1, and ABD C are absent. For example, in aspects and embodiments, each of the two antigen-binding polypeptides comprises an array of [missing information - likely a sequence of numbers] arranged from the N-terminus to the C-terminus.

[0080] (i) ABD C – L1 – ABD B – L2 – ABD A – Scaffold Area – ABD A – L2 – ABD B; (ii) ABD C – L1 – ABD B – L2 – ABD A – Support area – ABD A; (iii) ABD B – L2 – ABD A – stent area – ABD A – L2 – ABD B – L1 – ABD C; (iv) ABD A – stent area – ABD A – L2 – ABD B – L1 – ABD C; (v) ABD B – L2 – ABD A – Scaffold Area – ABD A – L2 – ABD B; (vi) ABD B – L2 – ABD A – Scaffold Area – ABD A; (vii) ABD A – Support area – ABD A – L2 – ABD B; (viii) ABD C – L1 – ABD B – L2 – ABD A – L3 – Scaffold Area – L3 – ABD A – L2 – ABD B; (ix) ABD C – L1 – ABD B – L2 – ABD A – L3 – Support area – L3 – ABD A; (x) ABD B – L2 – ABD A – L3 – Support area – L3 – ABD A – L2 – ABD B – L1 – ABD C; (xi) ABD A – L3 – Support area – L3 – ABD A – L2 – ABD B – L1 – ABD C; (xii) ABD B – L2 – ABD A – L3 – Scaffold Area – L3 – ABD A – L2 – ABD B; (xiii) ABD B – L2 – ABD A – L3 – Support area – L3 – ABD A; or (xiv) ABD A – L3 – Stent area – L3 – ABD A – L2 – ABD B.

[0081] In terms of aspects and implementation methods, this document provides a multivalent and multispecific construct comprising (a) a therapeutic payload; and (b) two antigen-binding peptides targeting two or more different targets, each antigen-binding peptide comprising, from the N-terminus to the C-terminus, a first antigen-binding domain (ABD C), a first linker (L1), a second antigen-binding domain (ABD B), a second linker (L2), a third antigen-binding domain (ABD A), an optional third linker (L3), and a scaffold region; wherein L1 attaches ABDC to ABD B, L2 attaches ABD B to ABD A, and L3 optionally attaches ABD A to the scaffold region, such that each antigen-binding peptide comprises, from the N-terminus to the C-terminus, a first antigen-binding domain (ABD C), a second antigen-binding domain (ABD B), a second linker (L2), a third antigen-binding domain (ABD A), an optional third linker (L3), and a scaffold region; wherein L1 attaches ABDC to ABD B, L2 attaches ABD B to ABD A, and L3 optionally attaches ABD A to the scaffold region, such that each antigen-binding peptide comprises, from the N-terminus to the C-terminus, a first antigen-binding domain (ABD B), a second antigen-binding domain (ABD B), a third antigen-binding domain (ABD A), an optional third linker (L3), and a scaffold region.

[0082] (i) ABD C – L1 – ABD B – L2 – ABD A – Support area or

[0083] (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – Support area; Furthermore, the scaffold region contains IgG Fc or a portion thereof or a modified form thereof.

[0084] In terms of aspects and implementation methods, this document provides a multispecific construct comprising (a) a therapeutic payload; and (b) four antigen-binding peptides targeting two or more distinct targets, each antigen-binding peptide comprising, from the N-terminus to the C-terminus, a first antigen-binding domain (ABD C), a first linker (L1), a second antigen-binding domain (ABD B), a second linker (L2), a third antigen-binding domain (ABD A), an optional third linker (L3), and a scaffold region; wherein L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, and L3 optionally attaches ABD A to the scaffold region, such that each antigen-binding peptide comprises, from the N-terminus to the C-terminus, a first antigen-binding domain (ABD C), a second antigen-binding domain (ABD B), a second linker (L2), a third antigen-binding domain (ABD A), an optional third linker (L3), and a scaffold region; wherein L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, and L3 optionally attaches ABD A to the scaffold region, such that each antigen-binding peptide comprises, from the N-terminus to the C-terminus, a first antigen-binding domain (ABD B), a second antigen-binding domain (ABD B), a third antigen-binding domain (ABD A), and a third antigen-binding domain (ABD A), ... A), a third antigen-binding domain (ABD A

[0085] (i) ABD C – L1 – ABD B – L2 – ABD A – Support area or

[0086] (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – Support area; Furthermore, the scaffold region contains a central protein anchor, which optionally contains an FcRn binding domain.

[0087] In terms of aspects and implementation methods, this document provides a multispecific construct comprising (a) a therapeutic payload; and (b) 'n' antigen-binding peptides targeting two or more distinct targets, each antigen-binding peptide comprising, from N-terminus to C-terminus, a first antigen-binding domain (ABD C), a first linker (L1), a second antigen-binding domain (ABD B), a second linker (L2), a third antigen-binding domain (ABD A), optionally a third linker (L3), a scaffold region, and a fourth linker (L4); wherein L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, L3 optionally attaches ABD A to the scaffold region, and L4 attaches the scaffold region to ABD C, such that each antigen-binding peptide comprises, arranged from N-terminus to C-terminus,

[0088] (i) ABD C – L1 – ABD B – L2 – ABD A – Support area – L4 or

[0089] (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – Support area – L4; The scaffold region contains a rigid polypeptide; the construct further contains a clasp domain that attaches a first antigen-binding polypeptide and an nth antigen-binding polypeptide to form a closed loop; and n is 3-12.

[0090] Antigen-binding domain

[0091] In aspects and embodiments, the multivalent multispecific constructs provided herein comprise two or more antigen-binding peptides, each of which comprises three or more antigen-binding domains. As used herein, the term "antigen-binding domain" refers to a peptide, peptide ligand, modified peptide, non-natural peptide, polypeptide, or a group of related polypeptides capable of specifically binding to a target molecule. In some embodiments, an "antigen-binding domain" is the minimum peptide ligand sequence of a natural or synthetic peptide ligand that specifically binds to the antigen bound by the ligand. In some embodiments, an "antigen-binding domain" is the minimum sequence of an antibody that specifically binds to the antigen bound by the antibody. In aspects and embodiments, the multivalent multispecific constructs provided herein comprise two or more antigen-binding peptides, each of which comprises one or more of a first antigen-binding domain (ABD C), a second antigen-binding domain (ABD B), and a third antigen-binding domain (ABD A). In aspects and embodiments, the multivalent multispecific constructs provided herein can bind to two or more targets simultaneously. In terms of aspects and implementation schemes, the simultaneous inclusion of the meaning that there are no geometric or other constraints prevents two or more ABDs present in a multivalent multispecific construct from simultaneously functionally interacting with their respective targets.

[0092] In aspects and embodiments, ABD C, ABD B, and / or ABD A comprise any antigen-binding domain or format known in the art. Non-limiting examples of antigen-binding domains include ligands, extracellular domains and engineered protein-binding motifs, VHH, VNAR, Fab, ScFv, nanobodies, biantibodies, synthetic microantibodies, or any binding fragment thereof. In aspects and embodiments, ABD C, ABD B, and / or ABD A comprise VHH or nanobodies or binding fragments thereof. In aspects and embodiments, ABD C, ABD B, and / or ABD A comprise a VH domain or a binding fragment thereof. In aspects and embodiments, ABD C, ABD B, and / or ABD A comprise a VNAR or a binding fragment thereof. In aspects and embodiments, ABD C, ABD B, and / or ABD A comprise a CDR1-HV2-HV4-CDR3 domain or a binding fragment thereof. In aspects and embodiments, ABD C, ABD B, and / or ABD A comprise Fab or a binding fragment thereof. In aspects and embodiments, ABD C, ABD B, and / or ABD A contain a VH-CH1-CL-VL domain or a binding fragment thereof. In aspects and embodiments, ABD C, ABD B, and / or ABD A contain ScFv or a binding fragment thereof. In aspects and embodiments, ABD C, ABD B, and / or ABD A contain a VH-VL domain or a binding fragment thereof. In aspects and embodiments, ABD C, ABD B, and / or ABD A contain a biantibody or a binding fragment thereof. In aspects and embodiments, ABD C, ABD B, and / or ABD A contain 2x VH-VL domains or a binding fragment thereof. In aspects and embodiments, ABD C, ABD B, and / or ABD A contain a synthetic microantibody or a binding fragment thereof. In aspects and embodiments, ABD C, ABD B, and / or ABD A contain 2x VH-VL-CH3 domains or a binding fragment thereof. In aspects and embodiments, ABD C, ABD B, and / or ABD A contain an extracellular domain or a binding fragment thereof. In aspects and implementations, ABD C, ABD B, and / or ABDA contain a ligand or a binding fragment thereof. In aspects and implementations, ABD C, ABD B, and / or ABD A contain a BCMA ligand.

[0093] In aspects and implementations, ABD C is combined with OX40, ABD B with PDL1, and ABD A with 41BB. In aspects and implementations, ABD C is combined with OX40, ABD B with 41BB, and ABD A with PDL1.

[0094] In aspects and embodiments, ABD C, ABD B, and / or ABD A contain a HER2 ligand. In aspects and embodiments, ABD C, ABD B, and / or ABD A contain a HER2 ligand containing the sequence KCCYSL (SEQ ID NO: 27). In aspects and embodiments, ABD C, ABD B, and / or ABD A contain a 41BB ligand. In aspects and embodiments, ABD C, ABD B, and / or ABD A contain a trimeric 41BB ligand. In aspects and embodiments, ABD C, ABD B, and / or ABD A contain BAFF. In aspects and embodiments, ABD C, ABD B, and / or ABD A contain APRIL. In aspects and embodiments, ABDC, ABD B, and / or ABD A are immunoglobulin-based agents. In aspects and embodiments, ABD C, ABD B, and / or ABDA are non-immunoglobulin-based agents.

[0095] In aspects and implementations, ABD C, ABD B, and / or ABD A are conditional binders. As used herein, the term "conditional binder" is a binder that preferentially binds under certain biological conditions, including but not limited to pH levels, ATP levels, calcium levels, efficient binding only when bound by one, two, three, or four other binders, and protease levels.

[0096] In aspects and implementations, the binding of ABD C, ABD B, and / or ABD A to their respective targets depends on pH. In aspects and implementations, ABD C, ABD B, and / or ABD A preferentially bind to their respective targets under acidic pH conditions. In aspects and implementations, ABD C, ABD B, and / or ABD A preferentially bind to their respective targets at pH values ​​of about 6.5 or below, about 6.4 or below, about 6.3 or below, about 6.2 or below, about 6.1 or below, about 6.0 or below, about 5.9 or below, about 5.8 or below, about 5.7 or below, about 5.6 or below, or about 5.5 or below. In aspects and implementations, ABD C, ABD B, and / or ABD A preferentially bind to their respective targets under neutral pH conditions. In aspects and implementations, ABD C, ABD B, and / or ABD A preferentially bind to their respective targets at pH values ​​of about 6.5 to about 7.5, about 6.6 to about 7.5, about 6.7 to about 7.5, about 6.8 to about 7.5, about 6.9 to about 7.5, about 7.0 to about 7.5, about 7.1 to about 7.5, about 7.2 to about 7.5, about 7.3 to about 7.5, or about 7.4 to about 7.5. In aspects and implementations, ABD C, ABD B, and / or ABD A preferentially bind to their respective targets under alkaline pH conditions. In aspects and implementations, ABD C, ABD B, and / or ABD A preferentially bind to their respective targets at pH values ​​of about 7.8 or higher, 7.9 or higher, 8.0 or higher, 8.1 or higher, 8.2 or higher, 8.3 or higher, 8.4 or higher, or 8.5 or higher.

[0097] In aspects and embodiments, the binding of ABD C, ABD B, and / or ABD A to their respective targets depends on the calcium ion concentration. In aspects and embodiments, ABD C, ABD B, and / or ABD A preferentially bind to their respective targets at low calcium ion concentrations. In aspects and embodiments, ABD C, ABD B, and / or ABD A preferentially bind to their respective targets at calcium ion concentrations of about 9 μM CaCl2 or less, about 8 μM CaCl2 or less, about 7 μM CaCl2 or less, about 6 μM CaCl2 or less, about 5 μM CaCl2 or less, about 4 μM CaCl2 or less, about 3 μM CaCl2 or less, about 2 μM CaCl2 or less, or about 1 μM CaCl2 or less. In aspects and embodiments, ABD C, ABD B, and / or ABD A preferentially bind to their respective targets at high calcium ion concentrations. In aspects and implementation methods, ABD C, ABD B and / or ABD A preferentially bind to their respective targets at calcium ion concentrations of about 1 mM CaCl2 or more, about 1.2 mM CaCl2 or more, about 1.4 mM CaCl2 or more, about 1.6 mM CaCl2 or more, about 1.8 mM CaCl2 or more, about 2 mM CaCl2 or more, about 2.2 mM CaCl2 or more, about 2.4 mM CaCl2 or more, about 2.6 mM CaCl2 or more, about 2.8 mM CaCl2 or more, about 3 mM CaCl2 or more, about 3.5 mM CaCl2 or more, about 4 mM CaCl2 or more, about 4.5 mM CaCl2 or more, or about 5 mM CaCl2 or more.

[0098] In aspects and embodiments, the binding of ABD C, ABD B, and / or ABD A to their respective targets depends on the protease level and / or activity. In aspects and embodiments, ABD C, ABD B, and / or ABD A comprises a masking moiety. In aspects and embodiments, the masking moiety is a peptide capable of binding to an ABD or a functional fragment thereof or otherwise exhibiting an affinity for said ABD or a functional fragment thereof, such that the masking moiety blocks, blocks, inhibits (e.g., reduces), or otherwise prevents (e.g., masks) the binding of ABD to its target. In aspects and embodiments, a proteolytically cleavable linker connects the masking moiety to ABD C, ABD B, and / or ABD A. In aspects and embodiments, cleavage of the proteolytically cleavable linker by a protease exposes the ABD, enabling it to bind to its target.

[0099] In aspects and implementations, the binding of ABD C, ABD B, and / or ABD A to their respective targets depends on extracellular ATP levels. In aspects and implementations, ABD C, ABD B, and / or ABD A preferentially bind to their respective targets at low extracellular ATP levels. In aspects and implementations, ABD C, ABD B, and / or ABD A preferentially bind to their respective targets at extracellular ATP levels of approximately 50 nM ATP or less, approximately 40 nM ATP or less, approximately 30 nM ATP or less, approximately 20 nM ATP or less, approximately 15 nM ATP or less, or approximately 10 nM ATP or less. In aspects and implementations, ABD C, ABD B, and / or ABD A preferentially bind to their respective targets at high extracellular ATP levels. In terms of aspects and implementation methods, ABD C, ABDB and / or ABD A preferentially bind to their respective targets at extracellular ATP levels of about 10 μM ATP or more, about 20 μM ATP or more, about 30 μM ATP or more, about 40 μM ATP or more, about 50 μM ATP or more, about 60 μM ATP or more, about 70 μM ATP or more, about 80 μM ATP or more, about 90 μM ATP or more, or about 100 μM ATP or more.

[0100] In aspects and implementations, the multivalent and multispecific constructs provided herein can be used to target their therapeutic payloads to any desired eukaryotic cell, referred to herein as a “target cell.” As used herein, a “target cell” means any cell expressing one or more antigens recognized by ABD C, ABD B, and / or ABD A. In aspects and implementations, ABD C, ABD B, and / or ABD A target a specific cell type. In aspects and implementations, ABD C, ABD B, and / or ABD A target mammalian cells (e.g., rodent, mouse, rat, ungulate, cow, sheep, pig, horse, camel, rabbit, canine (dog), feline (cat), primate, non-human primate, or human cells). In aspects and implementation schemes, ABD C, ABD B, and / or ABD A target bone marrow cells, hematopoietic stem cells (HSCs), long-term HSCs, short-term HSCs, hematopoietic stem cells and progenitor cells (HSPCs), peripheral blood monocytes (PBMCs), myeloid progenitor cells, lymphoid progenitor cells, T cells, B cells, NKT cells, NK cells, dendritic cells, monocytes, granulocytes, erythrocytes, megakaryocytes, mast cells, basophils, eosinophils, neutrophils, macrophages, erythroid progenitor cells (e.g., HUDEP cells), megakaryocyte-erythroid progenitor cells (MEP), common myeloid progenitor cells (CMP), pluripotent progenitor cells (MPP), long-term HSCs (LT-HSCs), endothelial cells, neurons, astrocytes, pancreatic cells, pancreatic β-islet cells, myocytes, skeletal muscle cells, cardiomyocytes, hepatocytes, adipocytes, intestinal cells, colon cells, and gastric cells. In aspects and implementation schemes, ABD C, ABD B, and / or ABD A target diseased cells. In some aspects and implementations, ABD C, ABD B, and / or ABD A target cancer cells. In some aspects and implementations, ABD C, ABD B, and / or ABD A target solid cancer cells. In some aspects and implementations, ABD C, ABD B, and / or ABD A target hematologic malignancies. In some aspects and implementations, ABD C, ABD B, and / or ABD A target pathogen-infected cells, including but not limited to cells infected by bacteria, fungi, or viruses. In some aspects and implementations, ABD C, ABD B, and / or ABD A target cancer cells, nerve cells, stem cells, regenerative medicine cells, or immune cells.

[0101] In aspects and embodiments, ABD C, ABD B, and / or ABD A promote the internalization of the multivalent multispecific construct by target cells. In aspects and embodiments, ABD C, ABD B, and / or ABD A promote the internalization of the multivalent multispecific construct by target cells through promoting lysosomal transport of the multivalent multispecific construct to target cells. In aspects and embodiments, ABD C, ABD B, and / or ABD A promote the internalization of the multivalent multispecific construct by target cells through promoting clathrin-mediated endocytosis of the multivalent multispecific construct to target cells. In aspects and embodiments, ABD C, ABD B, and / or ABD A promote the internalization of the multivalent multispecific construct by target cells through promoting receptor-mediated endocytosis of the multivalent multispecific construct to target cells. In aspects and embodiments, ABD C, ABD B, and / or ABD A promote the internalization of the multivalent multispecific construct by target cells through binding to one or more antigens selected from the IGF2R, CASP9 domain, endosome degradation subdomain, and recycling subdomain.

[0102] In aspects and implementation methods, ABD C, ABD B, and / or ABD A promote the chemotherapy sensitivity of target cells. In aspects and implementation methods, ABD C, ABD B, and / or ABD A promote the chemotherapy sensitivity of target cells by binding to one or more antigens selected from apoptosis targets, chemotherapy efflux pumps, PGP, MRP, BCRP / ABCG2, MDR, and microRNAs.

[0103] In aspects and implementation methods, ABD C, ABD B, and / or ABD A promote apoptosis or necrosis of target cells. In aspects and implementation methods, ABD C, ABD B, and / or ABD A promote apoptosis or necrosis of target cells by binding to one or more antigens selected from DR5, DR4, FAS, TNFR1, and IAP antagonists.

[0104] In aspects and implementations, ABD C, ABD B, and / or ABD A promote the stimulation of immune cells, for example, by binding to one or more of PD1, PDL1, CTLA4, LAG3, 41BB, and OX40. In aspects and implementations, ABD C, ABD B, and / or ABD A promote rapid internalization and lysosomal localization to target cells, for example, by binding to one or more of CD63, PRLR, and APLP2. In aspects and implementations, ABD C, ABD B, and / or ABD A bind to and isolate soluble factors, such as VEGF, TGFβ, etc.

[0105] In aspects and embodiments, ABD C, ABD B, and / or ABD A comprise at least one half-life-extending molecule. As used herein, a "half-life-extending molecule" means a molecule that, when (e.g., covalently) attached to a second molecule, extends the half-life of the second molecule. Non-limiting examples of half-life-extending molecules include Fc domains, modified Fc domains carrying mutations (such as YTE, mutations reducing FcγR binding, and / or mutations reducing FcγRIII binding, etc.) to further extend the in vivo half-life, human serum albumin (HSA), HSA-binding molecules, polyethylene glycol (PEG), polypropylene glycol (PPG), FcRn-binding domains, and XTEN. In aspects and embodiments, ABD C, ABD B, and / or ABD A and / or the scaffold comprises an FcRn-binding domain. In aspects and embodiments, ABD C, ABD B, and / or ABD A and / or the scaffold comprises albumin. In aspects and embodiments, ABD C, ABD B, and / or ABD A and / or the scaffold comprises human serum albumin (HSA). In aspects and embodiments, ABD C, ABD B, and / or ABD A and / or the scaffold comprises PEG. In aspects and embodiments, ABD C, ABD B, and / or ABD A and / or the scaffold comprises XTEN. In aspects and embodiments, ABD C, ABD B, and / or ABD A and / or the scaffold comprises an HSA-binding domain. In aspects and embodiments, ABD C, ABD B, and / or ABD A and / or the scaffold comprises PPG. In aspects and embodiments, ABD C, ABD B, and / or ABD A and / or the scaffold comprises an Fc domain. In aspects and embodiments, ABD C, ABD B, and / or ABD A and / or the scaffold comprises an Fc domain modified with a YTE mutation. In aspects and embodiments, ABD C, ABD B, and / or ABD A and / or the scaffold comprises an Fc domain modified with a mutation that reduces FcγR binding. In aspects and implementations, ABD C, ABD B and / or ABD A and / or the scaffold comprises an Fc domain modified with a mutation that reduces FcγRIII binding.

[0106] In aspects and embodiments, ABD C, ABD B, and / or ABD A, and / or the scaffold and / or the connector, comprise at least one site for conjugating the payload. In aspects and embodiments, ABD C, ABD B, and / or ABD A, and / or the scaffold and / or the connector, comprise one or more Cys residues that can be used for conjugating the payload. In aspects and embodiments, ABD C, ABD B, and / or ABD A, and / or the scaffold and / or the connector, comprise one or more Lys residues that can be used for conjugating the payload. In aspects and embodiments, ABD C, ABD B, and / or ABD A, and / or the scaffold and / or the connector, comprise one or more Tyr residues that can be used for conjugating the payload. In aspects and embodiments, L1, L2, L3, and / or L4 comprise one or more residues or glycans for site-specific conjugation with the payload.

[0107] In some aspects and embodiments, ABD C binds to a first target molecule, ABD B binds to a second target molecule, and ABD A binds to a third target molecule. In some aspects and embodiments, ABD C and ABD B bind to the first target molecule, and ABD A binds to the second target molecule. In some aspects and embodiments, ABD C and ABD A bind to the first target molecule, and ABD B binds to the second target molecule. In some aspects and embodiments, ABD B and ABD A bind to the first target molecule, and ABD C binds to the second target molecule. In some aspects and embodiments, ABD C, ABD B, and ABD A bind to the first target molecule.

[0108] In the aspects and implementation schemes, ABD C binds to a first epitope on the first target molecule, ABD B binds to a second epitope on the first target molecule, and / or ABD A binds to a third epitope on the first target molecule.

[0109] In aspects and embodiments, ABD A is used as a primary anchor for binding to target cells. As used herein, "primary anchor" refers to the target molecule most highly and / or most frequently expressed on the target cell of two or more target molecules of a multivalent, multispecific construct. In aspects and embodiments, ABD A binds to a target molecule that is expressed more and / or more frequently on the target cell than the target molecules bound by ABD B and / or ABD C. While not wishing to be bound by theory, it is believed that ABD A used as a primary anchor for binding to target cells will preferentially consider the strongest potential target antigen, i.e., the target molecule expressed more and / or more frequently on the target cell. In aspects and embodiments, ABDA used as a primary anchor for binding to target cells enables ABD B and / or ABD C to conditionally bind to their respective targets when ABD A binds to its target cell. In aspects and embodiments, ABD C is used as a secondary anchor for binding to target cells. As used herein, a “secondary anchor” refers to a target molecule expressed at the second highest and / or second most frequently on target cells of two or more target molecules of a multivalent, multispecific construct. In aspects and embodiments, ABD C binds to a target molecule that is expressed at a lower and / or more frequently on target cells than a target molecule bound by ABD A, but at a higher and / or more frequently on target cells than a target molecule bound by ABD B. In aspects and embodiments, ABD C used as a secondary anchor for binding to target cells enables ABD B to conditionally bind to its target when ABD C binds to its target cell. In aspects and embodiments, ABD B can bind substantially stably to its target only when ABD A binds to its target and / or ABD C binds to its target.

[0110] In aspects and embodiments, ABD C is used as a primary anchor for binding to target cells. As used herein, "primary anchor" refers to the target molecule most highly and / or most frequently expressed on target cells of two or more target molecules of a multivalent, multispecific construct. In aspects and embodiments, ABD C binds to target molecules that are expressed more and / or more frequently on target cells than the target molecules bound by ABD B and / or ABD A. While not wishing to be bound by theory, it is believed that in some cases, ABD C used as a primary anchor for binding to target cells will preferentially consider the strongest potential target antigen, i.e., the target molecule expressed more and / or more frequently on target cells. In aspects and embodiments, ABD C used as a primary anchor for binding to target cells enables ABD B and / or ABD A to conditionally bind to their respective targets when ABD C binds to its target cells. In aspects and embodiments, ABD A or ABD B is used as a secondary anchor for binding to target cells. As used herein, a “secondary anchor” refers to a target molecule expressed at the second highest and / or second most frequently on target cells of two or more target molecules of a multivalent, multispecific construct. In aspects and embodiments, ABD A and / or B bind to target molecules that are expressed at lower and / or more frequently on target cells than target molecules bound by ABD C, but at higher and / or more frequently on target cells than target molecules bound by ABD B or A. In aspects and embodiments, ABD A or B used as a secondary anchor for binding to target cells enables the remaining ABD B (or A) to conditionally bind to its target when ABD C binds to its target cell. In aspects and embodiments, ABD B (or A) can bind substantially stably to its target only when ABD A binds to its target and / or ABD C binds to its target.

[0111] Cellular targets

[0112] From the aspects and implementation methods, the multivalent and multispecific constructs provided herein can be directed to any desired cellular target. Non-limiting examples of cellular targets include NaPi2b, Trop2, MUC1, Ly6G6D, Claudin18.2, B7H3, BCMA, CD22, Her2, EGFR, PDL1, CEA, DLL3, CD47, Her3, FolRa, DR5, mesothelin, CEACAM5, CD38, CD19, ALPL2, PRLR, integrin, CD20, FcRn, FcRH5, GPRC5D, Her2, Her3, OX40, 41BB, and CD79b.

[0113] In aspects and implementation schemes, ABD A combines with one or more of NaPi2b, Trop2, MUC1, Ly6G6D, Claudin18.2, B7H3, BCMA, CD22, Her2, EGFR, PDL1, CEA, FolRa, DR5, mesothelin, CEACAM5, CD38, CD19, ALPL2, PRLR, integrin, CD20, FcRn, FcRH5, GPRC5D, Her2, Her3, OX40, 41BB, and CD79b. In terms of aspects and implementation methods, ABD B combines with one or more of NaPi2b, Trop2, MUC1, Ly6G6D, Claudin18.2, B7H3, BCMA, CD22, Her2, EGFR, PDL1, CEA, FolRa, DR5, mesothelin, CEACAM5, CD38, CD19, ALPL2, PRLR, integrin, CD20, FcRn, FcRH5, GPRC5D, Her2, Her3, OX40, 41BB, and CD79b. In terms of aspects and implementation methods, ABD C binds to one or more of NaPi2b, Trop2, MUC1, Ly6G6D, Claudin18.2, B7H3, BCMA, CD22, Her2, EGFR, PDL1, CEA, FolRa, DR5, mesothelin, CEACAM5, CD38, CD19, ALPL2, PRLR, integrin, CD20, FcRn, FcRH5, GPRC5D, Her2, Her3, OX40, 41BB, and CD79b.

[0114] In terms of aspects and implementation schemes, one of ABD C, ABD B and ABD A combines Her2, and one of ABD C, ABD B and ABD A combines Trop2 and / or Her3.

[0115] In aspects and implementation schemes, ABD C binds to NaPi2b, ABD B binds to MUC1, and ABD A binds to mesothelin. In aspects and implementation schemes, the multivalent multispecific construct is suitable for the treatment of ovarian cancer or NSCLC.

[0116] In aspects and implementation schemes, ABD C binds to NaPi2b, ABD B binds to MUC1, and ABD A binds to FolRa. In aspects and implementation schemes, the multivalent multispecific construct is suitable for the treatment of ovarian cancer or NSCLC.

[0117] In aspects and implementation schemes, ABD C binds to B7H3, ABD B binds to PDL1, and ABD A binds to Trop2. In aspects and implementation schemes, the multivalent multispecific construct is suitable for the treatment of NSCLC.

[0118] In aspects and implementation schemes, ABD C binds to B7H3, ABD B binds to EGFR, and ABD A binds to Trop2. In aspects and implementation schemes, the multivalent multispecific construct is suitable for the treatment of NSCLC.

[0119] In aspects and implementation schemes, ABD C binds to B7H3, ABD B binds to EGFR, and ABD A binds to Trop2. In aspects and implementation schemes, the multivalent multispecific construct is suitable for the treatment of NSCLC.

[0120] In aspects and implementations, ABD C binds to DLL3, ABD B binds to B7H3, and ABD A binds to Trop2. In aspects and implementations, the multivalent multispecific construct is suitable for the treatment of SCLC.

[0121] In aspects and implementations, ABD C binds to Ly6G6D, ABD B binds to CEACAM5, and ABD A binds to Claudin18.2. In aspects and implementations, the multivalent, multispecific construct is suitable for treating GI cancers.

[0122] In aspects and implementation schemes, ABD C binds to BCMA, ABD B binds to CD38, and ABD A binds to FcRH5. In aspects and implementation schemes, the multivalent multispecific construct is suitable for the treatment of myeloma.

[0123] In aspects and implementation schemes, ABD C binds to BCMA, ABD B binds to CD38, and ABD A binds to GPRC5D. In aspects and implementation schemes, the multivalent multispecific construct is suitable for the treatment of myeloma.

[0124] In aspects and implementation schemes, ABD C binds to CD22, ABD B binds to CD19, and ABD A binds to CD79b. In aspects and implementation schemes, the multivalent, multispecific construct is suitable for the treatment of NHL or autoimmune diseases.

[0125] In aspects and implementation schemes, ABD C binds to Her2, ABD B binds to MUC1, and ABD A binds to Trop2. In aspects and implementation schemes, the multivalent multispecific construct is suitable for the treatment of breast cancer.

[0126] In aspects and implementation schemes, ABD C binds to GPRC5D, and ABD A binds to BCMA. In aspects and implementation schemes, the multivalent multispecific construct is suitable for the treatment of myeloma.

[0127] In aspects and implementation schemes, ABD C binds to CD22, and ABD A binds to CD79b. In aspects and implementation schemes, the multivalent multispecific construct is suitable for the treatment of B-cell malignancies.

[0128] In aspects and implementations, ABD C is combined with OX40, ABD B with PDL1, and ABD A with 41BB. In aspects and implementations, ABD C is combined with OX40, ABD B with 41BB, and ABD A with PDL1.

[0129] In aspects and embodiments, ABD C contains a 41BB ligand, and ABD B and / or ABD A contains a HER2 ligand. In aspects and embodiments, ABD C contains a trimeric 41BB ligand, and ABD B and ABD A each contain a HER2 ligand. In aspects and embodiments, ABD C contains a trimeric 41BB ligand, and ABD B and ABD A each contain a HER2 ligand containing the sequence KCCYSL (SEQ ID NO: 27).

[0130] connector

[0131] In aspects and embodiments, the multivalent multispecific constructs described herein include linkers between antigen-binding domains and linkers between antigen-binding domains and scaffold regions. As used herein, the term "linker" refers to a molecule that connects two other molecules or portions. In aspects and embodiments, the multivalent multispecific constructs described herein include a first linker (L1), a second linker (L2), an optional third linker (L3), and an optional fourth linker (L4). In aspects and embodiments, L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, and ABD A attaches to the scaffold region, such that each antigen-binding polypeptide includes an ABD C – L1 – ABD B – L2 – ABD A – scaffold region arranged from the N-terminus to the C-terminus. In aspects and embodiments, L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, and L3 attaches ABD A to the scaffold region, such that each antigen-binding polypeptide comprises, from the N-terminus to the C-terminus, ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region. In aspects and embodiments, each antigen-binding polypeptide comprises, from the N-terminus to the C-terminus, ABD C – L1 – ABD B – L2 – ABD A – scaffold region – ABD A – L2 – ABD B – L1 – ABD C. In aspects and embodiments, each antigen-binding polypeptide comprises, from the N-terminus to the C-terminus, ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region – L3 – ABD A – L2 – ABD B – L1 – ABD C. In aspects and embodiments, L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, ABD A attaches to the scaffold region, and L4 attaches the scaffold region to ABD C, such that each antigen-binding polypeptide comprises ABD C – L1 – ABD B – L2 – ABD A – scaffold region – L4 arranged from N-terminus to C-terminus. In aspects and embodiments, L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, L3 attaches ABD A to the scaffold region, and L4 attaches the scaffold region to ABD C, such that each antigen-binding polypeptide comprises ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region – L4 arranged from N-terminus to C-terminus.

[0132] In the aspects and implementation schemes, the lengths of L1, L2, L3 and / or L4 are approximately 4-50 aa, approximately 10-45 aa, approximately 15-40 aa, approximately 15-35 aa, or approximately 15-30 aa. In the aspects and implementation schemes, the lengths of L1, L2, L3 and / or L4 are approximately 4 aa, approximately 5 aa, approximately 6 aa, approximately 7 aa, approximately 8 aa, approximately 9 aa, approximately 10 aa, approximately 11 aa, approximately 12 aa, approximately 13 aa, approximately 14 aa, approximately 15 aa, approximately 16 aa, approximately 17 aa, approximately 18 aa, approximately 19 aa, approximately 20 aa, approximately 21 aa, approximately 22 aa, approximately 23 aa, approximately 24 aa, approximately 25 aa, approximately 26 aa, approximately 27 aa, approximately 28 aa, approximately 29 aa, approximately 30 aa, approximately 31 aa, and approximately 32 aa. aa, length approximately 33 aa, length approximately 34 aa, length approximately 35 aa, length approximately 36 aa, length approximately 37 aa, length approximately 38 aa, length approximately 39 aa, length approximately 40 aa, length approximately 41 aa, length approximately 42 aa, length approximately 43 aa, length approximately 44 aa, length approximately 45 aa, length approximately 46 aa, length approximately 47 aa, length approximately 48 aa, length approximately 49 aa, or length approximately 50 aa.

[0133] In aspects and embodiments, L1, L2, L3, and / or L4 comprise one or more of the following residues: Gly, Ser, Thr, Pro, Arg, Phe, Glu, Cys, Asn, Ala, Asp, and Gln. In aspects and embodiments, L1, L2, L3, and / or L4 primarily comprise Gly, Ser, and Thr residues. In aspects and embodiments, L1, L2, L3, and / or L4 primarily comprise Gly and Ser residues.

[0134] In terms of aspects and implementation schemes, L1, L2, L3, and / or L4 include (Gly-Ser). n (Gly4-Ser) n (SEQ ID NO: 1), (Gly3-Ser) n(SEQ ID NO: 2), Gly-Gly-Ser, Gly-Gly-Ser-Gly (SEQ ID NO: 3), Gly-Gly-Gly-Gly (SEQ ID NO: 4), Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 5), Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 6), Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 7), Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 8), Gly-Ser-Gly-Ser-Gly-Ser (SEQ ID NO: 9), (Gly4-Ser)3 (SEQ IDNO: 10), (Gly2-Ser)3 (SEQ ID NO: 11), (Gly2-Ser)2 (SEQ ID NO: 12), ferritin E-helix, (Glu-Ala-Ala-Ala-Lys) 1-3 (SEQ ID NO: 13), (Ala-Pro) n (SEQ ID NO: 14), IgG C H 1. IgGC H 2-C H 3 or Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 15).

[0135] In aspects and embodiments, L1, L2, L3, and / or L4 are hydrophilic. Hydrophobic values ​​are determined according to methods known in the art. In aspects and embodiments, hydrophobic values ​​are calculated using the normalized hydrophobicity scale of Eisenberg, Ann. Rev. Biochem. 53:595-623, 1984. In aspects and embodiments, L1, L2, L3, and / or L4 have a hydrophobicity of about 0.4 to about 0.8, about 0.5 to about 0.7, or about 0.6 to about 0.65. In aspects and embodiments, L1, L2, L3, and / or L4 have a hydrophobicity of about 0.4, about 0.45, about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, or about 0.8.

[0136] In aspects and implementation methods, L1, L2, L3 and / or L4 are generally non-immunogenic. As used herein, “non-immunogenic” means a characteristic of the composition that, when administered to mammals such as humans, does not contribute to the development of humoral (antibody) and / or cellular immune responses.

[0137] In aspects and implementation methods, L1, L2, L3 and / or L4 are substantially non-cleavable. As used herein, a “non-cleavable adapter” means an adapter whose release of the therapeutic payload is independent of, for example, differential properties between plasma and certain cytoplasmic compartments, or independent of whether the adapter has physical properties that allow for enzymatic or chemical cleavage.

[0138] In aspects and embodiments, L1, L2, L3, and / or L4 are generally cleavable. As used herein, a "cleavable linker" refers to a linker capable of linking two or more molecules and then cleaving upon exposure to an agent. Cleavable linkers may include chemically or enzymatically unstable or degradable bonds. In aspects and embodiments, cleavable linkers may rely on intracellular processes to release the therapeutic payload, such as reduction of cytoplasm, exposure to acidic conditions in lysosomes, or cleavage by specific intracellular proteases or other enzymes. In aspects and embodiments, cleavable linkers incorporate one or more chemically or enzymatically cleavable bonds, while the remainder of the linker is non-cleavable. Such linkers may also be sensitive to their environment or signals, which may lead to changes in their biophysical properties.

[0139] In aspects and implementations, the lengths of L1, L2, L3, and / or L4 are approximately 4-30 nm, approximately 5-25 nm, approximately 5-20 nm, approximately 5-15 nm, or approximately 5-10 nm. In aspects and implementations, the lengths of L1, L2, L3, and / or L4 are approximately 5 nm, approximately 6 nm, approximately 7 nm, approximately 8 nm, approximately 9 nm, approximately 10 nm, approximately 12 nm, approximately 14 nm, approximately 16 nm, approximately 18 nm, approximately 20 nm, approximately 22 nm, approximately 24 nm, approximately 26 nm, approximately 28 nm, or approximately 30 nm.

[0140] In aspects and embodiments, L2 and / or L3 are rigid linkers. As used herein, a “rigid” linker refers to a molecule that adopts a relatively well-defined conformation when in solution. In aspects and embodiments, a rigid linker is a linker having a specific secondary and / or tertiary structure in solution. In aspects and embodiments, rigid linkers include aromatic molecules (see, for example, U.S. Patent No. 6,096,875 or U.S. Patent No. 5,948,648), proline-rich peptide linkers, or peptide linkers having a non-flexible helical structure. Rigid linkers are described, for example, in Chen et al. (2013) Adv. Drug Deliv. Rev. 65(10):1357-1369; US 2010 / 0158823 and US 2009 / 10221477, each of which is incorporated herein by reference in its entirety. In aspects and embodiments, rigid linkers comprise a helical structure. In aspects and embodiments, rigid linkers are proline-rich. In aspects and embodiments, L3 has a length of about 5-15 nm, about 6-4 nm, or about 7-12 nm. In aspects and embodiments, L3 comprises an α-helix.

[0141] In aspects and embodiments, L1 is a flexible linker. As used herein, a “flexible linker” refers to a hydrocarbon or peptide linker that does not have a fixed structure (secondary or tertiary structure) in solution. In aspects and embodiments, flexible linkers include hydrocarbon linkers and peptide linkers composed of small nonpolar (e.g., Gly) and / or polar (e.g., Ser or Thr) amino acid residues. In aspects and embodiments, flexible linkers contain simple amino acids (e.g., amino acids with simple side chains (e.g., H, CH3, or CH2OH)) because these amino acids lack branched side chains, thereby providing greater flexibility (e.g., two-dimensional or three-dimensional flexibility) within the linker and within the corresponding peptide composition. In aspects and embodiments, flexible linkers contain additional amino acids (such as Thr and Ala) to maintain flexibility, and polar amino acids (such as Lys and Glu) to improve solubility. Flexible connectors are described, for example, in Chen et al. (2013) Adv. Drug Deliv. Rev. 65(10):1357-1369; US 2012 / 0232021; US2014 / 0079701; WO 1999 / 045132; WO 1994 / 012520 and WO 2001 / 1053480, each of which is incorporated herein by reference in its entirety. In aspects and embodiments, L1 has a length of about 5-15 nm, about 6-4 nm, or about 7-12 nm. In aspects and embodiments, L1 comprises (Gly-Ser) n (Gly4-Ser) n(SEQ ID NO: 1), (Gly3-Ser) n (SEQ ID NO: 2), Gly-Gly-Ser, Gly-Gly-Ser-Gly (SEQ ID NO: 3), Gly-Gly-Gly-Gly (SEQ ID NO: 4), Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 5), Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 6), Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 7), Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 8), Gly-Ser-Gly-Ser-Gly-Ser (SEQ ID NO: 9), (Gly4-Ser)3 (SEQID NO: 10), (Gly2-Ser)3 (SEQ ID NO: 11), (Gly2-Ser)2 (SEQ ID NO: 12), ferritin E-helix, (Glu-Ala-Ala-Ala-Lys) 1-3 (SEQ ID NO: 13), (Ala-Pro) n (SEQ ID NO: 14), IgG C H 1. IgG C H 2-C H 3 or Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 15).

[0142] In aspects and embodiments, L2 is a moderately rigid connector. Any moderately rigid connector known in the art may be used herein. In aspects and embodiments, L2 is about 5-15 nm long, about 6-4 nm long, or about 7-12 nm long. In aspects and embodiments, L2 comprises b2-microglobulin, Zn-a2-glycoprotein, 34-peptide repeat sequences, or non-α-helices with moderate bending and rotational rigidity. In aspects and embodiments, L2 comprises a cleavable connector.

[0143] In the aspects and embodiments, the joint stiffness is determined using modeling techniques known in the art. In the aspects and embodiments, the joint stiffness is determined using electron microscopy techniques known in the art, as well as artificial intelligence and machine learning modeling, to reveal the flexibility of the structure.

[0144] In the aspects and implementation schemes, the joints are arranged in order of increasing rigidity from L4, L3, L2, and L1. While not wishing to be bound by theory, it is believed that this arrangement of joints allows multivalent, multispecific constructs to maintain a central plane interaction surface, thereby maximizing the density of aggregated targets, while allowing the binder at more distal ends to adapt to the target geometry.

[0145] support area

[0146] In terms of aspects and implementation methods, the scaffold region of the multivalent and multispecific constructs described herein contains rigid proteins.

[0147] In terms of aspects and implementation methods, the scaffold region of the multivalent multispecific construct described herein includes a portion for extending PK. Non-limiting examples of portions that can be used to extend PK include FcRn binding domains, IgG C H 2-C H 3. Domains, human serum albumin, and human transferrin. In aspects and implementation methods, the scaffold region of the multivalent multispecific construct described herein includes an FcRn binding domain, IgG C... H 2-C H 3. Human serum albumin, human transferrin, or fragments thereof.

[0148] In aspects and embodiments, the scaffold region comprises IgG Fc or a portion thereof or a modified form thereof. In aspects and embodiments, the IgG Fc is Fc-deficient (Fc-null) or silenced. In aspects and embodiments, the scaffold region comprises a central protein anchor, optionally wherein the central protein anchor comprises an FcRn binding domain. In aspects and embodiments, the scaffold region comprises IgGC. H 2-C H 3 or fragments thereof. In aspects and implementation methods, IgG C H 2-C H 3. It is incorporated into FcRn and extends the PK of the multivalent, multispecific construct. In aspects and embodiments, the scaffold region further includes a second hinge region. In aspects and embodiments, the scaffold region further includes a hinge-C H 2-C H 3-Hinge.

[0149] hinge

[0150] In aspects and embodiments, the multivalent multispecific constructs described herein include hinge regions. In aspects and embodiments, the multivalent multispecific constructs described herein include hinge regions at both the N-terminus and C-terminus of the scaffold. As used herein, the term "hinge region" refers to a short, flexible amino acid sequence. In aspects and embodiments, the hinge region is derived from an IgG sequence or an interdomain region of a transmembrane protein. In aspects and embodiments, the hinge region comprises one or more of the following: DKTHTCPPCPAPELLGG (SEQ ID NO: 16), DKTHTCPPCP (SEQ ID NO: 17), CPPC (SEQ ID NO: 18), GGLLEPAPCPPCTHTKD (SEQ ID NO: 19), PPCPSCPAPEFLGG (SEQ ID NO: 20), PPCPSCP (SEQ ID NO: 21), CPSCP (SEQ ID NO: 22), GGLFEPAPCSPCPP (SEQ ID NO: 23), GGGGDKTHTCPPCPAPELLGG (SEQ ID NO: 24), (G2S)3 (SEQ ID NO: 25), and GGGG (SEQ ID NO: 26). In aspects and embodiments, the first hinge or the second hinge comprises one or more residues or glycans for site-specific conjugation with the payload. While not wishing to be bound by theory, it is believed that the inclusion of a second hinge region at the C-terminus of the scaffold region allows for conjugation with interchain cysteine ​​residues after limited reduction, thereby achieving a higher drug-to-antibody ratio (DAR) that is feasible in the absence of a second hinge.

[0151] Treatment payload

[0152] In aspects and embodiments, the multivalent and multispecific constructs described herein comprise one or more therapeutic payloads. As used herein, the term "therapeutic payload" refers to a molecule that has a biological, cytotoxic, or therapeutic effect in cells. In aspects and embodiments, the therapeutic payload may be an inorganic molecule, an organic molecule, a small molecule, a pharmaceutical compound, a peptide, a polypeptide, a degrading agent, a bioregulator, an immunostimulatory motif, an immunosuppressant, a steroid, an antibiotic, or an antiviral drug. In aspects and embodiments, the therapeutic payload may be a functional nucleic acid, such as an oligonucleotide or a polynucleotide, such as mRNA or siRNA. In aspects and embodiments, the therapeutic payload may be mRNA encoding a functional peptide or polypeptide, such as gene therapy or a gene modification system. In aspects and embodiments, the multivalent and multispecific constructs described herein comprise dual payloads. The multivalent and multispecific constructs described herein comprise multiple payloads.

[0153] In aspects and implementation methods, the therapeutic payload comprises a protein. In aspects and implementation methods, the therapeutic payload comprises an enzyme, an immunostimulant, an immunosuppressant, or a topoisomerase.

[0154] In aspects and implementation methods, the therapeutic payload comprises small molecules. In aspects and implementation methods, the therapeutic payload comprises small molecule inhibitors, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), muscarinic acid, PNU, belotecan, exatecan, pyrrolobenzodiazepines (PBD), ado-trastuzumab emtansine (T-DM1), steroids, chemotherapy, BCL-XL inhibitors, or STING agonists, or other related or combined payloads.

[0155] In some aspects and embodiments, the therapeutic payload comprises an RNA molecule. In some aspects and embodiments, the therapeutic payload comprises an siRNA molecule. In some aspects and embodiments, the therapeutic payload comprises an mRNA molecule. In some aspects and embodiments, the mRNA molecule encodes a therapeutic peptide or protein.

[0156] In some aspects and implementations, the therapeutic payload includes a chemical sensitizer. In some aspects and implementations, the therapeutic payload includes a gene-modifying agent.

[0157] In terms of aspects and implementation methods, the therapeutic payload comprises a beloteccan derivative attached to the stent region with a cathepsin-cleavable linker, a non-cleavable linker, or a partially cleavable linker.

[0158] In aspects and implementations, the multivalent multispecific construct comprises more than one payload. In aspects and implementations, the multivalent multispecific construct has a drug-to-antibody ratio (DAR) of about 1-10, about 2-9, about 2-8, about 3-8, about 4-7, or about 5-6. In implementations, the multivalent multispecific construct has a DAR of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or higher.

[0159] Fusing sites and methods

[0160] In aspects and implementations, the multivalent multispecific constructs described herein comprise a therapeutic payload conjugated to one or more antigen-binding peptides. Conjugation methods are well known in the art, and any known conjugation method may be used herein.

[0161] In aspects and embodiments, the multivalent multispecific constructs described herein comprise a therapeutic payload that is conjugated to one or more antigen-binding peptides via amine bonding at one or more surface-exposed lysine residues, via sulfur bonding at one or more surface-exposed cysteine ​​residues, or via deglycosylation of the primary amide side chain of an antibody or its fragment containing Gln 295. In aspects and embodiments, the therapeutic payload is conjugated to Cys, Tyr, or Lys residues in the scaffold region. In aspects and embodiments, the therapeutic payload is conjugated to Gln 295.

[0162] In terms of aspects and implementation schemes, the payload is conjugated with a multivalent and multispecific construct using TCEP reduction, transglutaminase, random conjugation, polysaccharide conjugation, lysine conjugation, formylglycine synthase (FGE) oxidation, AjiCap technology, or click chemistry.

[0163] In aspects and embodiments, the payload is conjugated to a multivalent multispecific construct using random conjugation via limited reduction of interchain cysteine. In aspects and embodiments, conjugation includes the use of at least one Fc unit having an intact disulfide bond within the hinge region. In aspects and embodiments, conjugation includes partial reduction using TCEP. In aspects and embodiments, conjugation includes conjugation of cysteine ​​with a standard maleimide-based chemical. In embodiments, the DAR of the multivalent multispecific construct is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10.

[0164] In aspects and embodiments, the payload is conjugated to a multivalent multispecific construct using a glycan conjugation of a natural Fc glycan or a glycan engineered at another point. In aspects and embodiments, the payload is conjugated to a multivalent multispecific construct using a glycan conjugation of one or more engineered glycans in a VHH indirector. In embodiments, the DAR of the multivalent multispecific construct is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10.

[0165] In aspects and embodiments, the payload is conjugated to a specific glutamine of the multivalent multispecific construct using bacterial transglutaminase. In aspects and embodiments, the payload is conjugated to the multivalent multispecific construct either by transglutaminase conjugation to natural glutamine (Gln 295) or by insertion of an LLQG tag site.

[0166] In aspects and implementations, the payload is conjugated to specific residues of a multivalent and multispecific construct by inserting a specific CxPxR tag, thereby allowing hydrazino-Pictet-Spengler (HIPS) conjugation based on formylglycine synthase (FGE).

[0167] In aspects and embodiments, the payload uses AjiCap technology to conjugate a specific lysine of the multivalent multispecific construct. In aspects and embodiments, the payload uses a peptide-based Fc binder to perform site-specific conjugation on one or more cysteine ​​residues to conjugate a specific glutamine of the multivalent multispecific construct. In embodiments, the DAR of the multivalent multispecific construct is about 1, about 2, about 3, or about 4.

[0168] Other components and characteristics

[0169] In terms of aspects and implementation schemes, multivalent and multispecific constructs further include one or more enhancement subdomains.

[0170] In aspects and implementations, the multivalent and multispecific construct further comprises one or more FcRn binding domains. In aspects and implementations, the one or more FcRn binding domains comprise IgG FcC H 2-C H 3. Structural domain or VHH structural domain.

[0171] In aspects and embodiments, the diameter of the multivalent multispecific construct is about 5-50 nm, about 10-40 nm, about 20-30 nm, about 20-25 nm, or about 25-30 nm. In aspects and embodiments, the diameter of the multivalent multispecific construct is about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, or about 50 nm.

[0172] In terms of aspects and implementation methods, each antigen-binding polypeptide of the multivalent multispecific construct contains a terminal suitable for covalent or non-covalent cyclization or subunit assembly within the generating cell.

[0173] Cyclation methods are known in the art. See, for example, Hayes et al., “Approaches for peptide and protein cyclisation,” Org Biomol ChemMay 12, 2021; 19(18):3983-4001, the referenced herein is incorporated herein by reference in its entirety. Non-limiting cyclization methods include chemical methods, enzymatic methods, and protein tagging methods. Non-limiting examples of chemical cyclization methods include direct coupling, natural chemical linkage, Syr / Thr linkage, CyClick, traceless Staudinger ligation, KAHA (type I and II), TAMM, Cu-catalyzed azido-yne cycloaddition (CuAAC), strain-promoted azido-yne cycloaddition (SPAAC), cysteine ​​disulfide bonds, orthogonal disulfide pairing, and disulfide binding. Non-limiting examples of enzymatic and protein tagging cyclization methods include Bacillus subtilis protease variants, sorting enzymes, asparagine endopeptidase-butyrylase 1, asparagine endopeptidase-OaAEP1, microbial transglutaminase (Streptomyces moharawae ( S. mobaraensis ()), protein tag – inpeptide, protein tag – SpyTag / SpyCatcher, protein tag – SpyLigase, protein tag – SnoopTag / SnoopCatcher, and protein tag – SnoopLigase. In aspects and embodiments, the multivalent multispecific constructs described herein deliver payloads to target cells more efficiently than monomers or single-specific binders having only one copy of ABD C, ABD B, or ABD A.

[0174] In aspects and implementation methods, the multivalent multispecific constructs described herein are internalized by target cells at a higher rate than monomers or single-specific binders having only one copy of ABD C, ABD B, or ABD A. In implementation methods, even if only a single target of the multivalent multispecific construct is expressed on the target cell, the multivalent multispecific constructs described herein exhibit improved specificity for binding to and / or killing tumor cells compared to monomers or single-specific binders having only one copy of ABD C, ABD B, or ABD A.

[0175] In aspects and embodiments, the multivalent multispecific constructs described herein have improved specificity for binding to target cells compared to monomers or single-specific binders containing only one copy of ABD C, ABD B, or ABD A. In aspects and embodiments, the multivalent multispecific constructs described herein have improved specificity for internalization by target cells compared to monomers or single-specific binders containing only one copy of ABD C, ABD B, or ABD A. In aspects and embodiments, the multivalent multispecific constructs described herein have improved specificity for delivering therapeutic payloads to target cells compared to monomers or single-specific binders containing only one copy of ABD C, ABD B, or ABD A. In aspects and embodiments, the multivalent multispecific constructs described herein have improved specificity for killing cancer cells or tumor cells compared to monomers or single-specific binders containing only one copy of ABD C, ABD B, or ABD A.

[0176] Component evaluation

[0177] In the implementation scheme, assays are performed to evaluate the therapeutic payload delivery (e.g., specificity of binding to target cells, affinity for target cells, internalization efficiency, etc.) and therapeutic payload efficacy (e.g., depending on the payload, cell killing level, cytotoxicity, apoptosis induction, cell stimulation, gene editing, RNAi silencing, and mRNA expression, etc.) of the multivalent and multispecific constructs described herein.

[0178] Methods for evaluating the binding specificity of multivalent and multispecific constructs are known. Non-limiting examples of assays used to evaluate the binding affinity of multivalent and multispecific constructs to their targets and target cells include ELISA assays, surface plasmon resonance assays, Western blot analysis, flow cytometry, and immunohistochemistry.

[0179] Methods for evaluating the in vivo incorporation of multivalent and multispecific constructs are known. Non-limiting examples of assays used to evaluate the internalization of multivalent and multispecific constructs into their targets and target cells include flow cytometry and microscopy.

[0180] Methods for determining whether a multivalent multispecific construct produces cell-inhibiting and / or cytotoxic effects on cells are known. For example, the cytotoxic or inhibitory activity of a multivalent multispecific construct can be measured by, for instance, by exposing mammalian cells expressing the target antigen of the multivalent multispecific construct to a cell culture medium; culturing the cells for a period of approximately 6 hours to approximately 6 days; and measuring cell viability (e.g., using a CellTiter-Glo® (CTG) or MTT cell viability assay). In embodiments, cell-based in vitro assays can also be used to measure the viability (proliferation), cytotoxicity, and apoptosis induction (cysteine ​​activation) of the multivalent multispecific construct.

[0181] In the implementation plan, to determine cytotoxicity, necrosis or apoptosis (programmed cell death) can be measured. Necrosis is typically accompanied by increased plasma membrane permeability, cell swelling, and plasma membrane rupture. Apoptosis can be quantified, for example, by measuring DNA fragmentation. Commercially available spectrophotometric methods for the quantitative in vitro determination of DNA fragmentation are available. Examples of such assays include TUNEL (which detects the incorporation of labeled nucleotides into fragmented DNA) and ELISA-based assays, described in Biochemica (1999) 2:34-7 (Roche Molecular Biochemicals).

[0182] In some embodiments, apoptosis can also be determined by measuring morphological changes in cells. For example, similar to necrosis, loss of plasma membrane integrity can be determined by measuring the uptake of certain dyes (e.g., fluorescent dyes, such as acridine orange or ethidium bromide). Duke and Cohen, Current Protocols in Immunology (Coligan et al., eds. (1992), pp. 3.17.1–3.17.16) describe methods for measuring the number of apoptotic cells. Cells can also be labeled with DNA dyes (e.g., acridine orange, ethidium bromide, or propidium iodide) and chromatin condensation and limbing along the inner nuclear membrane can be observed. In some embodiments, apoptosis can also be determined by screening for caspase activity. In some embodiments, the Caspase-Glo® assay can be used to measure the activities of caspase-3 and caspase-7. In some embodiments, the assay provides luminescent caspase-3 / 7 substrates in reagents optimized for caspase activity, luciferase activity, and cell lysis. In this implementation, adding Caspase-Glo® 3 / 7 reagent in an "add-mix-measure" format may induce cell lysis, followed by caspase cleavage of the substrate and the generation of a "luminescent" signal produced by luciferase. In this implementation, the luminescence is proportional to the present caspase activity and can serve as an indicator of apoptosis. Other morphological changes that can be measured to determine apoptosis include, for example, cytoplasmic condensation, increased membrane bubbling, and cell contraction. In this implementation, identifying any of these effects on cancer cells indicates that the multivalent, multispecific construct may be used for cancer treatment.

[0183] In embodiments, cell viability can be measured, for example, by determining the uptake of dyes such as Neutral Red, Trypan Blue, Crystal Violet, or Alamar™ Blue in cells (see, for example, Page et al. (1993) Intl J Oncology 3:473-6). In such an assay, cells are incubated in a dye-containing medium, washed, and the remaining dye reflecting the cells' uptake of the dye is measured spectrophotometrically. In embodiments, cell viability can also be measured, for example, by quantifying ATP (an indicator of metabolically active cells). In embodiments, the CellTiter-Glo® (CTG) cell viability assay can be used to evaluate the in vitro potency and / or cell viability of prepared ADC or Bcl-xL inhibitor compounds. In this assay, in some embodiments, a single reagent (CellTiter-Glo® reagent) is added directly to cells cultured in a serum-supplemented medium. The addition of the reagent causes cell lysis and produces a luminescent signal proportional to the amount of ATP present. The amount of ATP is proportional to the number of cells present in the culture. In embodiments, cell viability can also be measured, for example, by measuring the reduction of tetrazolium salts. In the embodiments, the MTT cell viability assay can be used to evaluate the in vitro potency and / or cell viability of the prepared ADC or Bcl-xL inhibitor compounds, as described in the examples provided herein. In this assay, in some embodiments, the yellow tetrazolium salt MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) is reduced by metabolically active cells (partly by the action of dehydrogenases) to produce reducing equivalents such as NADH and NADPH. The resulting intracellular purple formazan can then be dissolved and quantified by spectrophotometry.

[0184] Methods for evaluating gene editing, RNAi silencing, and mRNA expression efficiency of multivalent multispecific constructs are known. Non-limiting examples of evaluating gene editing, RNAi silencing, and mRNA expression efficiency of multivalent multispecific constructs include sequencing-based analyses and qPCR analyses.

[0185] Treatment

[0186] In aspects and embodiments, this document provides a method for treating a disease or condition in a subject of need, comprising administering the multivalent, multispecific construct described herein. In aspects and embodiments, the disease or condition is cancer. In aspects and embodiments, the disease or condition is an immune disease. In aspects and embodiments, the disease or condition is an autoimmune disease. In aspects and embodiments, the disease or condition is a neurological disease. In aspects and embodiments, the disease or condition is a cardiovascular disease. In aspects and embodiments, the disease or condition is a skin disease. In aspects and embodiments, the disease or condition is an eye disease. In aspects and embodiments, the disease or condition is an infectious disease. In aspects and embodiments, the disease or condition is one that can benefit from regenerative medicine. In aspects and embodiments, the disease or condition is a genetic disease or condition.

[0187] In the aspects and implementation plans, the disease or symptom is cancer, and said cancer is a solid tumor. In the aspects and implementation plans, the cancer is NSCLC, ovarian cancer, breast cancer, pancreatic cancer, colorectal cancer, or stomach cancer.

[0188] In the aspects and implementations, the disease or symptom is cancer, and said cancer is a blood cancer. In the aspects and implementations, the cancer is leukemia, lymphoma, or multiple myeloma.

[0189] In terms of aspects and implementation methods, this document provides a method for treating ovarian cancer or NSCLC in a subject of need, comprising administering the multivalent multispecific construct described herein, wherein ABD C binds NaPi2b, ABD B binds MUC1, and ABD A binds mesothelin.

[0190] In terms of aspects and implementation methods, this document provides a method for treating NSCLC in a subject of need, comprising administering the multivalent multispecific construct described herein, wherein ABD C binds to B7H3, ABD B binds to PDL1, and ABD A binds to Trop2.

[0191] In terms of aspects and implementation methods, this document provides a method for treating GI cancer in a subject of need, comprising administering the multivalent multispecific construct described herein, wherein ABD C binds to Ly6G6D, ABD B binds to CEACAM5, and ABD A binds to Claudin18.2.

[0192] In terms of aspects and implementation methods, this article provides a method for treating a subject with myeloma in need, comprising administering the multivalent multispecific construct described herein, wherein ABD C binds to BCMA, ABD B binds to CD38, and ABDA binds to FcRH5.

[0193] In terms of aspects and implementation methods, this article provides a method for treating a subject with myeloma in need, comprising administering the multivalent multispecific construct described herein, wherein ABD C binds GPRC5D and ABD A binds BCMA.

[0194] In terms of aspects and implementation methods, this document provides a method for treating a subject with B-cell malignancies, comprising administering the multivalent and multispecific construct described herein, wherein ABD C binds CD22 and ABD A binds CD79b.

[0195] In terms of aspects and implementation methods, this document provides a method for treating a subject in need of NHL or an autoimmune disease, comprising administering the multivalent and multispecific construct described herein, wherein ABD C binds CD22, ABD B binds CD19, and ABD A binds CD79b.

[0196] In terms of aspects and implementation methods, this document provides a method for treating breast cancer in a subject of need, comprising administering the multivalent multispecific construct described herein, wherein ABD C binds Her2, ABD B binds MUC1, and ABDA binds Trop2.

[0197] In terms of aspects and implementation methods, this document provides a method for treating cancer in a subject of need, comprising administering the multivalent multispecific construct described herein, wherein ABD C binds to OX40, ABD B binds to PDL1, and ABD A binds to 41BB.

[0198] In terms of aspects and implementation methods, this document provides a method for treating cancer in a subject of need, comprising administering the multivalent multispecific construct described herein, wherein ABD C binds to OX40, ABD B binds to 41BB, and ABD A binds to PDL1.

[0199] In aspects and embodiments, this document provides a method of treating cancer in a subject of need, comprising administering the multivalent multispecific construct described herein, wherein ABD C comprises a 41BB ligand, and ABD B and / or ABD A comprises a HER2 ligand. In aspects and embodiments, ABD C comprises a trimeric 41BB ligand, and ABD B and ABD A each comprise a HER2 ligand. In aspects and embodiments, ABD C comprises a trimeric 41BB ligand, and ABD B and ABD A each comprise a HER2 ligand containing the sequence KCCYSL (SEQ ID NO: 27). In aspects and embodiments, this document provides a nucleic acid encoding the multivalent multispecific construct described herein. In aspects and embodiments, this document provides a host cell comprising the multivalent multispecific construct described herein. In aspects and embodiments, this document provides a host cell comprising the nucleic acid described herein. In aspects and embodiments, this document provides a pharmaceutical composition comprising the multivalent multispecific construct, nucleic acid, or host cell described herein.

[0200] In terms of aspects and implementation methods, this document provides a method for manufacturing the multivalent multispecific construct described herein, comprising (b) culturing cells expressing the multivalent multispecific construct; and (c) isolating the multivalent multispecific construct from the cultured cells.

[0201] definition

[0202] Generally, the terms used in the claims and specification are intended to be interpreted in the ordinary sense as understood by one of ordinary skill in the art. Some terms are defined below to provide additional clarity. In the event of a conflict between the ordinary sense and the provided definitions, the provided definitions shall prevail.

[0203] Any term not directly defined herein shall be understood to have the meaning commonly associated with it as understood in the art of this invention. Certain terms are discussed herein to provide additional guidance to those skilled in the art in describing compositions, apparatuses, methods, etc., and how to manufacture or use said compositions, apparatuses, methods, etc., of this invention. It should be understood that the same thing may be expressed in more than one way. Therefore, alternative languages ​​and synonyms may be used for any one or more terms discussed herein. Whether a term is elaborated or discussed herein is not significant. Several synonyms or alternative methods, materials, etc., are provided. Unless expressly stated otherwise, listing one or more synonyms or equivalents does not preclude the use of other synonyms or equivalents. Examples of use (including examples of terms) are for illustrative purposes only and do not limit the scope and meaning of the invention herein.

[0204] The term "subject" refers to an individual organism, such as a human or animal. In the implementation, the subject is a mammal (e.g., a human, a non-human primate, or a non-human mammal), a vertebrate, a laboratory animal, livestock, an agricultural animal, or a companion animal. In the implementation, the subject is a human (e.g., a human patient). In the implementation, the subject is a rodent, mouse, rat, hamster, rabbit, dog, cat, cow, goat, sheep, or pig.

[0205] Unless the context clearly indicates otherwise, as used in this specification and the appended claims, the singular forms “a / an” and “the” include a plural of indicators.

[0206] Unless otherwise expressly stated or the context clearly indicates otherwise, as used herein, the term “or” should be understood as inclusive and covers both “or” and “and”. Similarly, the terms “and / or” cover both “or” and “and”.

[0207] Unless otherwise expressly specified or the context clearly indicates otherwise, as used herein, the term “about” shall be understood to be within the normal tolerance range in the art, such as within 2 standard deviations of the mean. “About” shall be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless the context clearly indicates otherwise, all numerical values ​​provided herein are modified by the term “about”.

[0208] The present invention will be further described in the following embodiments, which do not limit the scope of the invention as described in the claims.

[0209] Example

[0210] Example 1: Evaluation of multivalent and multispecific constructs

[0211] Component Design

[0212] In a non-restrictive instance, a multivalent multispecific construct is manufactured having one or more of the following structures.

[0213] ● X-IgG format: ○ Treatment payload; and ○ Two antigen-binding peptides targeting two or more different targets, each antigen-binding peptide comprising, from N-terminus to C-terminus, a first antigen-binding domain (ABD C), a first linker (L1), a second antigen-binding domain (ABD B), a second linker (L2), a third antigen-binding domain (ABD A), an optional third linker (L3), a scaffold region, an optional second L3, a second ABD A, a second L2, a second ABD B, a second L1, and a second ABD C; wherein L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, and L3 optionally attaches ABD A to the scaffold region, such that each antigen-binding peptide comprises, from N-terminus to C-terminus, (i) ABD C – L1 – ABD B – L2 – ABD A – scaffold region – ABD A – L2 – ABD B – L1 – ABD C or (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region – L3 – ABD A – L2 – ABD B – L1 – ABD C; and the scaffold region contains IgG Fc or a portion thereof or a modified form thereof.

[0214] ● Y-IgG format: ○ Treatment payload; and ○ Two antigen-binding peptides targeting two or more different targets, each antigen-binding peptide comprising, from the N-terminus to the C-terminus, a first antigen-binding domain (ABD C), a first adapter (L1), a second antigen-binding domain (ABD B), a second adapter (L2), a third antigen-binding domain (ABD A), an optional third adapter (L3), and a scaffold region; wherein L1 attaches ABDC to ABD B, L2 attaches ABD B to ABD A, and L3 optionally attaches ABD A to the scaffold region, such that each antigen-binding peptide comprises, from the N-terminus to the C-terminus, (i) ABD C – L1 – ABD B – L2 – ABD A – scaffold region or (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region; and wherein the scaffold region comprises IgG Fc or a portion thereof or a modified form thereof.

[0215] ● Multi-pronged approach: ○ Treatment payload; and ○ Four antigen-binding peptides targeting two or more different targets, each antigen-binding peptide comprising, from N-terminus to C-terminus, a first antigen-binding domain (ABD C), a first linker (L1), a second antigen-binding domain (ABD B), a second linker (L2), a third antigen-binding domain (ABD A), an optional third linker (L3), and a scaffold region; wherein L1 attaches ABDC to ABD B, L2 attaches ABD B to ABD A, and L3 optionally attaches ABD A to the scaffold region, such that each antigen-binding peptide comprises, from N-terminus to C-terminus, (i) ABD C – L1 – ABD B – L2 – ABD A – scaffold region or (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region; and wherein the scaffold region comprises a central protein anchor, optionally wherein the central protein anchor comprises an FcRn binding domain.

[0216] ● Ring format: ○ Treatment payload; and ○ 'n' antigen-binding peptides targeting two or more different targets, each antigen-binding peptide comprising, from N-terminus to C-terminus, a first antigen-binding domain (ABD C), a first linker (L1), a second antigen-binding domain (ABDB), a second linker (L2), a third antigen-binding domain (ABD A), an optional third linker (L3), a scaffold region, and a fourth linker (L4); wherein L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, L3 optionally attaches ABD A to the scaffold region, and L4 attaches the scaffold region to ABD C, such that each antigen-binding peptide comprises, from N-terminus to C-terminus, (i) ABD C – L1 – ABD B – L2 – ABD A – scaffold region – L4 or (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region – L4; wherein the scaffold region contains a rigid polypeptide; wherein the construct further contains a clasp domain that attaches a first antigen-binding polypeptide and an nth antigen-binding polypeptide to form a closed loop; and wherein n is 3-12.

[0217] In a non-limiting example, each of ABD A, ABD B, and ABD C independently binds to one of NaPi2b, Trop2, MUC1, Ly6G6D, Claudin18.2, B7H3, BCMA, CD22, Her2, EGFR, PDL1, CEA, FolRa, DR5, mesothelin, CEACAM5, CD38, CD19, ALPL2, PRLR, integrin, CD20, FcRn, FcRH5, GPRC5D, and CD79b.

[0218] Component evaluation

[0219] In non-limiting examples, methods known in the art are used to evaluate the efficacy of the construct in targeted delivery and therapeutic payload. Examples of assays that can be used to evaluate the construct include, but are not limited to, assays for evaluating therapeutic payload delivery (e.g., specificity of binding to target cells, affinity for target cells, internalization efficiency, etc.) and therapeutic payload efficacy (e.g., depending on payload, cell-killing level, cytotoxicity, apoptosis induction, cell stimulation, gene editing, RNAi silencing, and mRNA expression, etc.). Animal models can then be used to evaluate the efficiency of the construct in targeted delivery of therapeutic payloads.

[0220] Example 2: Experimental evaluation of multivalent and multispecific constructs

[0221] Flow cytometry binding assay

[0222] Cells and the primary antibody of interest were prepared in cell culture medium containing 10% heat-inactivated fetal bovine serum and seeded together in 96-well V-type plates (ThermoFisher #277143). Cells and antibody were incubated in the dark at 4°C for 30 min. The plates were washed twice with staining buffer (Invitrogen #00-4222-26) and then resuspended at the manufacturer's recommended concentration in a staining mixture containing a viable immobilizing dye and a fluorescent secondary antibody, and incubated in the dark at 4°C for 30 min. The plates were washed once with staining buffer, and the cells were resuspended in staining buffer before flow cytometry analysis on a BioRad ZE5 analyzer. Flow cytometry data were analyzed using FCS Express software, and median fluorescence intensity values ​​were exported and plotted using Graphpad Prism software.

[0223] OX40 and 41BB activation reporter gene assay

[0224] Activation of OX40 and 41BB was evaluated using Promega's luciferase reporter gene systems (catalog numbers JA2195 and JA2351). Briefly, genetically engineered reporter Jurkat cells were incubated with specific concentrations of the test material in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum on 384-well opaque assay plates (ThermoFisher #164610) at 37°C and 5% CO2 for 6 hours. The plates were then removed from the incubator and allowed to equilibrate to room temperature before luminescent readout using Bio-glo substrate (Promega, catalog number G7940) according to the manufacturer's protocol.

[0225] Antigen Quantification Assay

[0226] Fluorescent antibodies against the antigens of interest and corresponding isotype controls were prepared at saturation concentrations in 1X pH 7.4 phosphate-buffered saline containing immobilizable active dyes. Cells and quantification beads (Bangs Laboratories #815) were incubated with the prepared fluorescent antibodies at 4°C in the dark for 30 min. The assay plate was washed once with staining buffer (Invitrogen #00-4222-26), and the cells were resuspended in staining buffer for flow cytometry analysis on a BioRad ZE5 analyzer. The specific antibody binding capacity (ABC) on the cell lines was determined using a regression analysis template (QuickCal® v. 3.0 data analysis template) provided by Bangs Laboratories.

[0227] Internalization determination was performed using the Alexa Fluor 488 quenching method.

[0228] Cells were prepared in cell culture medium containing 10% heat-inactivated fetal bovine serum and seeded into 96-well V-type plates (ThermoFisher #277143). Antibodies of interest were pre-incubated with Alexa Fluor 488 secondary antibody in the medium at room temperature for at least 10 minutes (primary antibody: AF488 mouse anti-human IgG Fc [Southern Biotech #9040-30] molar ratio 1:1, or primary antibody: alpaca anti-human IgG, recombinant VHH, CoraLite® Plus 488 [Proteintech Group #shuGCL488-2] molar ratio 1:2). The primary antibody:secondary antibody mixture was then added to the seeded cells in the dark at 37°C or 4°C for incubation times of 4 h, 1 h, 30 min, and 15 min. After incubation, all plates were cooled on ice for 10 minutes and kept cool in all subsequent steps. Following the manufacturer's recommendations, the plates were washed with staining buffer and resuspended in a viable fixative dye. For each internalization condition, cells were aliquoted into fresh plates for quenching (each well was resuspended in 50 μL of viable dye, and 25 μL was aliquoted into fresh plates). Plates were incubated at 4°C in the dark for 15 minutes, followed by washing with staining buffer. Before flow cytometry readout on a BioRad ZE5 analyzer (temperature controlled at 4°C before and during readout), cells were resuspended in staining buffer or quenching antibody (Alexa Fluor 488 polyclonal antibody, ThermoFisher #A11094). Antibody internalization was determined by the median fluorescence intensity of the Alexa Fluor 488 channel.

[0229] ADC cytotoxicity assay

[0230] The drug-conjugated secondary antibodies (MORADEC #AH-202AE-50, MORADEC #AH-207DX-50) and the primary antibody of interest were pre-incubated in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum. Titrates of this mixture were prepared by dilution into the medium and incubation at room temperature for 10 minutes. NCI-H929 cells (ATCC CRL-3580) and other cells of interest were prepared at 200,000 / mL in the medium, and 25 μL (5kJ) of cells were aliquoted into each well of a 384-well plate (ThermoFisher #164610). An equal volume of the primary antibody:secondary antibody-drug mixture was aliquoted into the cells, bringing the total well volume to 50 μL. The assay plate was incubated in a cell culture incubator (37°C, 5% CO2) for 72 hours. CellTiter Glo (CTG 2.0, Promega #G9242) was equilibrated to room temperature before being added to the assay plate. A 1:2 CTG:well volume was added to each well, and the plates were incubated in the dark at room temperature for 10–15 minutes. Cell viability was assessed using PromegaGloMax luminescence assay to detect ATP.

[0231] GIP / GLP activation assay using reporter cell lines

[0232] Genetically engineered 293T-CRE-Luc2-GLP1R-T2A-GIPR reporter cells (KYinno KC-3142) and antibodies of interest were prepared in DMEM medium containing 10% heat-inactivated fetal bovine serum. Cells were seeded at 10 kJ / well in 384-well opaque assay plates (ThermoFisher #164610) and allowed to adhere overnight in an incubator (37°C, 5% CO2). Antibody titrants were prepared and added to the overnight-seeded assay plates, and incubated together for 5–6 hours (37°C, 5% CO2). Following the manufacturer's protocol, the Bio-Glo luciferase assay reagent was prepared, equilibrated to room temperature, and then added to the assay plates at a 1:2 volume ratio. The plates were incubated in the dark for 10 minutes, followed by luminescence readout. Cell activation was evaluated using luminescence on a Promega GloMax.

[0233] Site-specific markers of antibodies

[0234] Following the manufacturer's protocol and with minor adjustments based on the scale of the labeling reaction, the Invitrogen SiteClick Antibody Labeling Kit (SiteClick™ Antibody Azide Modification Kit, catalog S10901; SiteClick™ Alexa Fluor™ 488 sDIBO alkyne, catalog S10904; SiteClick™ pHrodo™ iFL RedsDIBO alkyne, catalog S10903) was used to conjugate fluorescent dyes (such as Alexa Fluor 488 and pHrodo Red) to the glycans on the Fc region of IgG antibodies, VHH-Fc, or other Fc fusion proteins. In short, the antibody or Fc fusion protein was first concentrated using a Pierce protein concentrator (Thermo Scientific, catalog 88513), and then the buffer was exchanged for 1X TBS buffer (pH 7.2–7.5) using a Zeba desalting column (Thermo Scientific, catalog A57759). The protein sample was then incubated overnight at 37°C with β-galactosidase to remove terminal galactose residues from the glycan. The next day, UDP-GalNAz, GalT enzyme, Tris buffer, and the buffer additives supplied with the kit were added to the protein sample, and the reaction was carried out overnight at 30°C. The sample was then purified using a Zeba desalting column, followed by mixing with sDIBO alkyne conjugation reagent and incubating overnight at room temperature. Excess dye was removed using a Zeba desalting column or a dye removal column, and the degree of labeling was determined based on absorbance measured at a specific wavelength, according to the SiteClick kit manual.

[0235] Figure 5 to Figure 15 The results of characterizing the multivalent and multivalent multispecific constructs described in this paper using these methods are depicted.

[0236] like Figure 5A and Figure 5B As shown, increased multivalentity enhances binding and functional activation. Data demonstrate that the multivalent constructs described herein, having four (Y4 format) or six ABDs (Y6 format), exhibit increased binding (Y2, similar to IgG) compared to constructs with only two ABDs (Y2, similar to IgG). Figure 5A ) and functional activity ( Figure 5B ).

[0237] like Figure 6A and Figure 6BAs shown, multivalent and multispecific constructs can be generated with high titers and purity. As described herein, constructs with 12 ABDs (X12 format) exhibit increased activity compared to constructs with only two or six ABDs, and the multivalent and multispecific constructs described herein, containing OX40 ABD, PDL-1 ABD, and 4-1BB ABD, demonstrate increased activity in OX40 reporter gene assays when the OX40 ABD is present in an external position.

[0238] like Figure 7 As shown, compared with the constructs evaluated in Figure 6, the hybrid multivalent and multispecific constructs described herein, having two 41BB ligand peptide trimers and two, four, or six CD38 VHHs, exhibited further increased activity.

[0239] like Figures 8A to 8C As shown, the inclusion of a second hinge in the multivalent and multispecific construct described herein allows for the limited reduction post-conjugation of four additional cysteine ​​residues at inter-chain disulfide bonds, and the incorporation of C-terminal hinges with various sequences in the multivalent formulation has no negative impact on the activity in the OX40 activation assay, indicating correct expression and assembly.

[0240] like Figures 9 to 11 As shown, when compared to constructs with only two ABDs, the multivalent constructs described herein with four or six ABDs exhibit a logarithmic increase in internalization, and the increase depends on the target and antigen density on the target cells. The figure shows the results for constructs containing BCMA ABDs (… Figure 9 ); GPRC5D ( Figure 10 ) and CD38 ( Figure 11 The results of internalization measurements of the construct.

[0241] like Figure 12 As shown, the internalization of the triple-targeted BCMA / CD38 / GPRC5D Y6 construct is several log-order higher than that of the Y2 construct.

[0242] like Figure 13 As shown, in a secondary antibody-based cell killing assay, the triple-targeting BCMA / CD38 / GPRC5D Y6 construct demonstrated advantages over the Y2 and IgG-like controls.

[0243] In summary, the multivalent and multispecific constructs described in this paper exhibited significant (>10X) antigen, epitope, and antigen density-dependent increases in binding, function, internalization, and killing.

[0244] Example 3: Evaluation of multivalent and multispecific GLP-1-like peptide constructs

[0245] GLP-1-like peptides and their derivatives have been successfully used to treat diabetes and obesity. Incretin peptides interact with GPCRs, thereby inducing downstream signaling in relevant cells. Due to the close interaction between peptides and homologous GPCRs, fusing peptides with half-life-prolonging features (such as the Fc domain of IgG) typically results in a significant loss of activity relative to native incretins. The ability of multivalent constructs in the format described herein to demonstrate activity in functional assays on relevant reporter cell lines was evaluated.

[0246] like Figure 14 As shown, the Y6 format of the GLP-1-like peptide was able to restore the activity lost during fusion and exhibited activity comparable to that of the free GLP-1 control.

[0247] Example 4: Evaluation of multivalent and multispecific X12 and Y6

[0248] like Figure 15 As shown, in cell lines expressing OX40, the internalization of the X12-based construct is superior to that of Y6.

[0249] By incorporating via reference

[0250] All patents and publications cited in this article are incorporated herein by reference in their entirety.

[0251] The publications discussed herein are provided only for their disclosure prior to the filing date of this application. Nothing herein shall be construed as an admission that the invention is not entitled to precede such publications by virtue of a prior invention.

[0252] As used herein, all headings are for organization purposes only and are not intended to limit this disclosure in any way. The content of any single section is equally applicable to all sections.

[0253] Implementation Plan

[0254] The embodiments listed below provide various additional embodiments of this disclosure, which can be combined in any number and in any combination.

[0255] Implementation Scheme 1. A multivalent and multispecific construct comprising two or more antigen-binding peptides and a conjugated therapeutic payload, wherein each of the two or more antigen-binding peptides comprises three or more antigen-binding domains, wherein each of the three or more antigen-binding domains of each of the two or more antigen-binding peptides is capable of binding to a separate target on its target cell, wherein the construct is capable of simultaneously binding to four or more targets on the surface of the target cell.

[0256] Implementation Scheme 2. A multivalent and multispecific construct as described in Implementation Scheme 1, wherein the construct comprises: (a) Therapeutic payload; and (b) Two or more antigen-binding peptides targeting two or more different targets, each antigen-binding peptide comprising, from the N-terminus to the C-terminus, a first antigen-binding domain (ABD C), a first linker (L1), a second antigen-binding domain (ABD B), a second linker (L2), a third antigen-binding domain (ABD A), an optional third linker (L3), and a scaffold region. Wherein L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, and L3 optionally attaches ABD A to the scaffold region, such that each antigen-binding polypeptide comprises (i) ABD C – L1 – ABD B – L2 – ABD A – scaffold region or (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region arranged from the N-terminus to the C-terminus; and The scaffold region contains a loop, a central protein anchor, or IgG Fc or a portion thereof or a modified form.

[0257] Implementation Scheme 3. A multivalent and multispecific construct as described in Implementation Scheme 2, wherein the scaffold region comprises IgG Fc or a portion thereof or a modified form thereof, and wherein the construct comprises two antigen-binding peptides of an antigen-binding peptide.

[0258] Implementation Scheme 4. The multivalent multispecific construct as described in Implementation Scheme 3, wherein each of the two antigen-binding peptides further comprises (i) ABD A – L2 – ABD B – L1 – ABD C arranged from the N-terminus to the C-terminus, such that each antigen-binding peptide comprises ABD C – L1 – ABD B – L2 – ABD A – scaffold region – ABD A – L2 – ABD B – L1 – ABD C arranged from the N-terminus to the C-terminus, or (ii) L3 – ABD A – L2 – ABD B – L1 – ABD C, such that each antigen-binding peptide comprises ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region – L3 – ABD A – L2 – ABD B – L1 – ABD C arranged from the N-terminus to the C-terminus.

[0259] Implementation Scheme 5. A multivalent and multispecific construct as described in Implementation Scheme 2, wherein the scaffold region includes a central protein anchor, optionally wherein the central protein anchor includes an FcRn binding domain, and wherein the construct includes four antigen-binding peptides of an antigen-binding peptide.

[0260] Implementation Scheme 6. A multivalent and multispecific construct as described in Implementation Scheme 2, wherein the scaffold region comprises a rigid polypeptide; wherein the construct comprises 'n' antigen-binding polypeptides; wherein each of the antigen-binding polypeptides further comprises a fourth linker (L4), wherein the L4 attaches the scaffold region to ABD C; and wherein the scaffold region further comprises a clasp domain, the clasp domain attaching the first antigen-binding polypeptide and the nth antigen-binding polypeptide to form a closed loop, wherein n is 3-12.

[0261] Implementation Scheme 7. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 6, wherein ABD C, ABD B and / or ABD A are VHH, VNAR, Fab, ScFv, nanobody, biantibody, synthetic microbody, ligand or its binding fragment.

[0262] Implementation Scheme 8. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 7, wherein ABD C, ABD B and / or ABD A are conditional binders, optionally wherein the binding of ABD C, ABD B and / or ABD A to their respective targets depends on pH, calcium or ATP levels.

[0263] Implementation Scheme 9. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 8, wherein ABD C, ABD B and / or ABD A target a specific cell type.

[0264] Implementation Scheme 10. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 9, wherein ABD C, ABD B and / or ABD A target cancer cells or immune cells.

[0265] Implementation Scheme 11. A multivalent multispecific construct as described in any one of Implementation Schemes 2 to 10, wherein ABDC, ABD B and / or ABD A promote the internalization of the multivalent multispecific construct by target cells, optionally by promoting lysosomal transport of the multivalent multispecific construct by target cells, optionally wherein lysosomal transport is clathrin-mediated endocytosis or receptor-mediated endocytosis.

[0266] Implementation Scheme 12. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 11, wherein ABDC, ABD B and / or ABD A promote the chemotherapeutic sensitivity of target cells.

[0267] Implementation Scheme 13. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 12, wherein ABDC, ABD B and / or ABD A promote apoptosis or necrosis of target cells.

[0268] Implementation Scheme 14. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 13, wherein ABDC, ABD B and / or ABD A comprises at least one half-life extension molecule, optionally wherein the half-life extension molecule is an FcRn binding domain, albumin, PEG, XTEN or human serum albumin (HSA) binding domain.

[0269] Implementation Scheme 15. A multivalent multispecific construct as described in any one of Implementation Schemes 2 to 14, wherein ABDC, ABD B and / or ABD A contain at least one site for conjugating the payload, and optionally at least one site contains a Cys, Tyr or Lys residue.

[0270] Implementation Scheme 16. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 15, wherein ABD C binds to a first target molecule, ABD B binds to a second target molecule, and / or ABD A binds to a third target molecule.

[0271] Implementation Scheme 17. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 16, wherein ABD C binds to a first epitope on a first target molecule, ABD B binds to a second epitope on a first target molecule, and / or ABD A binds to a third epitope on a first target molecule.

[0272] Implementation Scheme 18. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 17, wherein ABD C is used as a primary anchor for binding to target cells.

[0273] Implementation Scheme 19. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 18, wherein ABD B can bind substantially stably to its target only when ABD C binds to its target and / or ABD A binds to its target.

[0274] Implementation Scheme 20. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 19, wherein ABD A is used as a secondary anchor for binding to target cells.

[0275] Implementation Scheme 21. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 20, wherein ABD C binds to one or more of NaPi2b, Trop2, MUC1, Ly6G6D, Claudin18.2, B7H3, BCMA, CD22, and Her2.

[0276] Implementation Scheme 22. A multivalent multispecific construct as described in any one of Implementation Schemes 2 to 21, wherein ABD B binds to one or more of EGFR, PDL1, CEA, FolRa, DR5, MUC1, mesothelin, Claudin18.2, CEACAM5, CD38, and CD19.

[0277] Implementation Scheme 23. A multivalent multispecific construct as described in any one of Implementation Schemes 2 to 22, wherein ABD A binds to one or more of ALPL2, PRLR, integrin, DR5, CD20, FcRn, EGFR, Trop2, Claudin18.2, FcRH5, and CD79b.

[0278] Implementation Scheme 24. A multivalent multispecific construct as described in any one of Implementation Schemes 2 to 23, wherein one of ABDC, ABD B and ABD A binds Her2, and wherein one of ABD C, ABD B and ABD A binds Trop2.

[0279] Implementation Scheme 25. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 24, wherein ABD C binds NaPi2b, ABD B binds MUC1, and ABD A binds mesothelin.

[0280] Implementation Scheme 26. A multivalent multispecific construct as described in Implementation Scheme 25, wherein the multivalent multispecific construct is suitable for the treatment of ovarian cancer or NSCLC.

[0281] Implementation Scheme 27. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 24, wherein ABD C binds to B7H3, ABD B binds to PDL1, and ABD A binds to Trop2.

[0282] Implementation Scheme 28. A multivalent multispecific construct as described in Implementation Scheme 27, wherein the multivalent multispecific construct is suitable for the treatment of NSCLC.

[0283] Implementation Scheme 29. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 24, wherein ABD C binds to Ly6G6D, ABD B binds to CEACAM5, and ABD A binds to Claudin18.2.

[0284] Implementation Scheme 30. The multivalent multispecific construct as described in Implementation Scheme 29, wherein the multivalent multispecific construct is suitable for treating GI cancers.

[0285] Implementation Scheme 31. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 24, wherein ABD C binds to BCMA, ABD B binds to CD38, and ABD A binds to FcRH5.

[0286] Implementation Scheme 32. The multivalent multispecific construct as described in Implementation Scheme 31, wherein the multivalent multispecific construct is suitable for the treatment of myeloma.

[0287] Implementation Scheme 33. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 24, wherein ABD C binds to CD22, ABD B binds to CD19, and ABD A binds to CD79b.

[0288] Implementation Scheme 34. A multivalent multispecific construct as described in Implementation Scheme 33, wherein the multivalent multispecific construct is suitable for the treatment of NHL or autoimmune diseases.

[0289] Implementation Scheme 35. A multivalent multispecific construct as described in any one of Implementation Schemes 2 to 24, wherein ABD C binds Her2, ABD B binds MUC1, and ABD A binds Trop2.

[0290] Implementation Scheme 36. The multivalent multispecific construct as described in Implementation Scheme 35, wherein the multivalent multispecific construct is suitable for the treatment of breast cancer.

[0291] Implementation Scheme 37. A multivalent multispecific construct as described in any one of Implementation Schemes 2 to 36, wherein the lengths of L1, L2, L3 and / or L4 are about 15-30 aa.

[0292] Implementation Scheme 38. A multivalent multispecific construct as described in any one of Implementation Schemes 2 to 37, wherein L1, L2, L3 and / or L4 comprises one or more of Gly, Ser, Thr, Pro, Arg, Phe, Glu, Cys, Asn, Ala, Asp and Gln residues, optionally wherein L1, L2, L3 and / or L4 primarily comprises Gly, Ser and Thr residues.

[0293] Implementation Scheme 39. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 38, wherein L1, L2, L3 and / or L4 primarily comprise Gly and Ser residues.

[0294] Implementation Scheme 40. A multivalent, multispecific construct as described in Implementation Scheme 39, wherein L1, L2, L3, and / or L4 comprise (Gly-Ser). n (Gly4-Ser) n (SEQ ID NO: 1), (Gly3-Ser)n (SEQ ID NO: 2), Gly-Gly-Ser, Gly-Gly-Ser-Gly (SEQ ID NO: 3), Gly-Gly-Gly-Gly (SEQ ID NO: 4), Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 5), Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 5) NO: 6), Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 7), Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 8), Gly-Ser-Gly-Ser-Gly-Ser (SEQ ID NO: 9), (Gly4-Ser)3 (SEQ ID NO: 10), (Gly2-Ser)3 (SEQ ID NO: 11), (Gly2-Ser)2 (SEQ ID NO: 12), ferritin E-helix, (Glu-Ala-Ala-Ala-Lys) 1-3 (SEQ ID NO:13), (Ala-Pro) n (SEQ ID NO: 14), IgG C H 1. IgG C H 2-C H 3 or Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 15).

[0295] Implementation Scheme 41. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 40, wherein L1, L2, L3 and / or L4 are hydrophilic, and optionally wherein L1, L2, L3 and / or L4 have a hydrophobicity of about 0.6.

[0296] Implementation Scheme 42. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 41, wherein L1, L2, L3 and / or L4 are hydrophilic, optionally wherein L1, L2, L3 and / or L4 have a hydrophobicity of about 0.4 to about 0.8, optionally wherein L1, L2, L3 and / or L4 have a hydrophobicity of about 0.5 to about 0.7, optionally wherein L1, L2, L3 and / or L4 have a hydrophobicity of about 0.6 to about 0.65.

[0297] Implementation Scheme 43. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 42, wherein L1, L2, L3 and / or L4 are substantially non-immunogenic.

[0298] Implementation Scheme 44. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 43, wherein L1, L2, L3 and / or L4 are substantially non-cleavable.

[0299] Implementation Scheme 45. A multivalent multispecific construct as described in any one of Implementation Schemes 2 to 44, wherein L1, L2, L3 and / or L4 are substantially cleavable.

[0300] Implementation Scheme 46. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 45, wherein the lengths of L1, L2, L3 and / or L4 are about 5-10 nm; optionally, the lengths are about 7 nm.

[0301] Implementation Scheme 47. A multivalent multispecific construct as described in any one of Implementation Schemes 2 to 46, wherein L3 is a rigid connector with a length of about 7-12 nm and / or contains an α-helix.

[0302] Implementation Scheme 48. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 47, wherein L2 is a moderately rigid linker with a length of about 7-12 nm, and / or comprises b2-microglobulin, Zn-a2-glycoprotein, 34-peptide repeat sequence or non-α-helix with moderate bending and rotational rigidity, optionally wherein L2 is a cleavable linker.

[0303] Implementation Scheme 49. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 48, wherein L1 is a flexible linker with a length of about 7-12 nm and / or is (Gly-Ser). n (Gly4-Ser) n (SEQ ID NO: 1), (Gly3-Ser) n(SEQ ID NO: 2), Gly-Gly-Ser, Gly-Gly-Ser-Gly (SEQ ID NO: 3), Gly-Gly-Gly-Gly (SEQ ID NO: 4), Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 5), Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 6), Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 7), Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 8), Gly-Ser-Gly-Ser-Gly-Ser (SEQ ID NO: 9), (Gly4-Ser)3 (SEQ ID NO: 10), (Gly2-Ser)3 (SEQ ID NO: 11), (Gly2-Ser)2 (SEQ ID NO: 12), ferritin E-helix, (Glu-Ala-Ala-Ala-Lys) 1-3 (SEQ ID NO: 13), (Ala-Pro) n (SEQ ID NO: 14), IgG C H 1. IgG C H 2-C H 3 or Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 15).

[0304] Implementation scheme 50. A multivalent multispecific construct as described in any one of implementation schemes 2 to 49, wherein the joints are L4, L3, L2 and L1 in order of strongest to weakest rigidity.

[0305] Implementation Scheme 51. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 50, wherein the scaffold region comprises a rigid protein.

[0306] Implementation Scheme 52. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 51, wherein the scaffold region includes a portion for extending PK, optionally wherein the portion for extending PK is an FcRn binding domain, IgG C H 2-C H 3. Human serum albumin, human transferrin, or fragments thereof.

[0307] Implementation Scheme 53. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 52, wherein the scaffold region comprises IgG C H 2-C H 3 or a fragment thereof, optionally including IgG C H 2-C H 3 further includes a second hinge area, optionally wherein the support area includes hinge-C H 2-C H 3-Hinge.

[0308] Implementation Scheme 54. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 53, wherein the payload is conjugated to Cys, Tyr, or Lys residues in the scaffold region.

[0309] Implementation Scheme 55. A multivalent multispecific construct as described in any one of Implementation Schemes 2 to 54, wherein the payload is conjugated to the multivalent multispecific construct using TCEP reduction, transglutaminase, random conjugation, glycan conjugation, lysine conjugation, formylglycine synthase (FGE) oxidation, AjiCap technology, or click chemistry.

[0310] Implementation Scheme 56. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 55, wherein the payload comprises a protein, optionally wherein the protein comprises an enzyme, an immunostimulant, an immunosuppressant, or a topoisomerase.

[0311] Implementation Scheme 57. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 55, wherein the payload comprises a small molecule, optionally comprising a small molecule inhibitor, monomethylaurestatin E (MMAE), monomethylaurestatin F (MMAF), muscarinic acid, PNU, belotecone, essanotecan, pyrrolobenzodiazepines (PBD), trastuzumab emtansine (T-DM1), steroids, chemotherapy, BCL-XL inhibitors, or STING agonists.

[0312] Implementation Scheme 58. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 55, wherein the payload comprises an RNA molecule, optionally wherein the RNA molecule is a siRNA molecule.

[0313] Implementation Scheme 59. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 55, wherein the payload comprises a chemical sensitizer.

[0314] Implementation Scheme 60. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 55, wherein the payload comprises a gene modifier.

[0315] Implementation Scheme 61. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 55, wherein the payload is a beloteccan derivative attached to the scaffold region using a cathepsin-cleavable linker.

[0316] Implementation Scheme 62. A multivalent multispecific construct as described in any one of Implementation Schemes 2 to 61, wherein the multivalent multispecific construct comprises more than one payload, optionally wherein the drug-to-antibody ratio (DAR) is about 2-8.

[0317] Implementation Scheme 63. A multivalent multispecific construct as described in any one of Implementation Schemes 2 to 62, wherein the multivalent multispecific construct further comprises one or more enhancer domains and / or one or more FcRn binding domains, optionally wherein one or more FcRn binding domains comprise IgG FcC H 2-C H 3. Structural domain or VHH structural domain.

[0318] Implementation Scheme 64. The multivalent multispecific construct as described in any one of Implementation Schemes 2 to 63, wherein the diameter of the multivalent multispecific construct is about 5-50 nm, about 10-40 nm, about 20-30 nm, about 20-25 nm or about 25-30 nm.

[0319] Implementation Scheme 65. A multivalent and multispecific construct as described in any one of Implementation Schemes 2 to 64, wherein each antigen-binding polypeptide contains a terminal suitable for covalent or non-covalent cyclization or subunit assembly within the generating cell.

[0320] Implementation Scheme 66. A multivalent multispecific construct as described in any one of Implementation Schemes 2 to 65, wherein the multivalent multispecific construct (i) delivers the payload to target cells more efficiently than a monomer or single-specific binder having only one copy of ABD C, ABD B, or ABD A, (ii) is internalized by target cells at a higher rate than a monomer or single-specific binder having only one copy of ABD C, ABD B, or ABD A, and / or (iii) has improved specificity for binding to and / or killing tumor cells than a monomer or single-specific binder having only one copy of ABD C, ABD B, or ABD A.

[0321] Implementation Scheme 67. A nucleic acid encoding a multivalent and multispecific construct as described in any one of Implementation Schemes 1 to 66.

[0322] Implementation Scheme 68. A host cell comprising the nucleic acid described in Implementation Scheme 67.

[0323] Implementation Scheme 69. A pharmaceutical composition comprising the multivalent and multispecific construct of any one of Implementation Schemes 1 to 66, the nucleic acid of Implementation Scheme 67, or the host cell of Implementation Scheme 68.

[0324] Implementation Scheme 70. A method for treating a disease or condition in a subject in need, optionally wherein the disease or condition is cancer, said method comprising administering a multivalent multispecific construct of any one of Implementation Schemes 1 to 66, a nucleic acid of Implementation Scheme 67, a host cell of Implementation Scheme 68, or a pharmaceutical composition of Implementation Scheme 69.

[0325] Implementation Scheme 71. The method as described in Implementation Scheme 70, wherein the cancer is a solid tumor.

[0326] Implementation Scheme 72. The method described in Implementation Scheme 71, wherein the cancer is NSCLC, ovarian cancer, breast cancer, pancreatic cancer, colorectal cancer, or gastric cancer.

[0327] Implementation Scheme 73. The method as described in Implementation Scheme 70, wherein the cancer is a blood cancer.

[0328] Implementation Scheme 74. The method described in Implementation Scheme 73, wherein the cancer is leukemia, lymphoma, or multiple myeloma.

[0329] Implementation Scheme 75. A method of treating a subject with ovarian cancer or NSCLC, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 2 to 69, wherein ABD C binds NaPi2b, ABD B binds MUC1, and ABD A binds mesothelin.

[0330] Implementation Scheme 76. A method of treating NSCLC in a subject of need, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 2 to 69, wherein ABD C binds to B7H3, ABD B binds to PDL1, and ABD A binds to Trop2.

[0331] Implementation Scheme 77. A method of treating a subject with GI cancer, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 2 to 69, wherein ABD C binds to Ly6G6D, ABD B binds to CEACAM5, and ABD A binds to Claudin18.2.

[0332] Implementation Scheme 78. A method of treating a subject with myeloma in need, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 2 to 69, wherein ABD C binds to BCMA, ABD B binds to CD38, and ABDA binds to FcRH5.

[0333] Implementation Scheme 79. A method for treating a subject with NHL or an autoimmune disease of need, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 2 to 69, wherein ABD C binds CD22, ABD B binds CD19, and ABD A binds CD79b.

[0334] Implementation Scheme 80. A method of treating breast cancer in a subject of need, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 2 to 69, wherein ABD C binds Her2, ABD B binds MUC1, and ABDA binds Trop2.

[0335] Implementation Scheme 81. A multivalent and multispecific construct comprising: (a) Therapeutic payload; and (b) Two antigen-binding peptides targeting two or more different targets, each antigen-binding peptide comprising, from the N-terminus to the C-terminus, a first antigen-binding domain (ABD C), a first linker (L1), a second antigen-binding domain (ABDB), a second linker (L2), a third antigen-binding domain (ABD A), an optional third linker (L3), a scaffold region, an optional second L3, a second ABD A, a second L2, a second ABD B, a second L1, and a second ABD C; Wherein L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, and L3 optionally attaches ABD A to the scaffold region, such that each antigen-binding polypeptide comprises, from the N-terminus to the C-terminus, (i) ABD C – L1 – ABD B – L2 – ABD A – scaffold region – ABD A – L2 – ABD B – L1 – ABD C or (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region – L3 – ABD A – L2 – ABD B – L1 – ABD C; and The scaffold region contains IgG Fc or a portion thereof or a modified form thereof.

[0336] Implementation Scheme 82. The multivalent and multispecific construct as described in Implementation Scheme 81, wherein ABD C, ABD B and / or ABD A are VHH, VNAR, Fab, ScFv, nanobody, biantibody, synthetic microbody, ligand or its binding fragment.

[0337] Implementation Scheme 83. A multivalent and multispecific construct as described in any one of Implementation Schemes 81 to 82, wherein ABDC, ABD B and / or ABD A are conditional binders, optionally wherein the binding of ABD C, ABD B and / or ABD A to their respective targets depends on pH, calcium or ATP levels.

[0338] Implementation Scheme 84. A multivalent and multispecific construct as described in any one of Implementation Schemes 81 to 83, wherein ABDC, ABD B and / or ABD A target a specific cell type.

[0339] Implementation Scheme 85. A multivalent and multispecific construct as described in any one of Implementation Schemes 81 to 84, wherein ABDC, ABD B and / or ABD A target cancer cells or immune cells.

[0340] Implementation Scheme 86. A multivalent multispecific construct as described in any one of Implementation Schemes 81 to 85, wherein ABDC, ABD B and / or ABD A promote the internalization of the multivalent multispecific construct by target cells, optionally by promoting lysosomal transport of the multivalent multispecific construct by target cells, optionally wherein the lysosomal transport is clathrin-mediated endocytosis or receptor-mediated endocytosis.

[0341] Implementation Scheme 87. A multivalent and multispecific construct as described in any one of Implementation Schemes 81 to 86, wherein ABDC, ABD B and / or ABD A promote the chemotherapeutic sensitivity of target cells.

[0342] Implementation Scheme 88. A multivalent and multispecific construct as described in any one of Implementation Schemes 81 to 87, wherein ABDC, ABD B and / or ABD A promote apoptosis or necrosis of target cells.

[0343] Implementation Scheme 89. A multivalent and multispecific construct as described in any one of Implementation Schemes 81 to 88, wherein ABDC, ABD B and / or ABD A comprises at least one half-life extension molecule, optionally wherein the half-life extension molecule is an FcRn binding domain, albumin, PEG, XTEN or human serum albumin (HSA) binding domain.

[0344] Implementation Scheme 90. A multivalent multispecific construct as described in any one of Implementation Schemes 81 to 89, wherein ABDC, ABD B and / or ABD A contain at least one site for conjugating the payload, and optionally at least one site contains a Cys, Tyr or Lys residue.

[0345] Implementation Scheme 91. A multivalent and multispecific construct as described in any one of Implementation Schemes 81 to 90, wherein ABDC binds to a first target molecule, ABD B binds to a second target molecule, and / or ABD A binds to a third target molecule.

[0346] Implementation Scheme 92. A multivalent and multispecific construct as described in any one of Implementation Schemes 81 to 91, wherein ABDC binds to a first epitope on a first target molecule, ABD B binds to a second epitope on a first target molecule, and / or ABDA binds to a third epitope on a first target molecule.

[0347] Implementation Scheme 93. A multivalent and multispecific construct as described in any one of Implementation Schemes 81 to 92, wherein ABDC is used as a primary anchor for binding to target cells.

[0348] Implementation Scheme 94. A multivalent and multispecific construct as described in any one of Implementation Schemes 81 to 93, wherein ABD B can bind substantially stably to its target only when ABD C binds to its target and / or ABD A binds to its target.

[0349] Implementation Scheme 95. A multivalent and multispecific construct as described in any one of Implementation Schemes 81 to 94, wherein ABDA is used as a secondary anchor for binding to target cells.

[0350] Implementation Scheme 96. A multivalent multispecific construct as described in any one of Implementation Schemes 81 to 95, wherein ABDC binds to one or more of NaPi2b, Trop2, MUC1, Ly6G6D, Claudin18.2, B7H3, BCMA, CD22, and Her2.

[0351] Implementation Scheme 97. A multivalent multispecific construct as described in any one of Implementation Schemes 81 to 96, wherein ABDB binds to one or more of EGFR, PDL1, CEA, FolRa, DR5, MUC1, mesothelin, Claudin18.2, CEACAM5, CD38, and CD19.

[0352] Implementation Scheme 98. A multivalent multispecific construct as described in any one of Implementation Schemes 81 to 97, wherein ABDA binds to one or more of ALPL2, PRLR, integrin, DR5, CD20, FcRn, EGFR, Trop2, Claudin18.2, FcRH5, and CD79b.

[0353] Implementation Scheme 99. A multivalent multispecific construct as described in any one of Implementation Schemes 81 to 98, wherein one of ABDC, ABD B and ABD A binds Her2, and wherein one of ABD C, ABD B and ABD A binds Trop2.

[0354] Implementation scheme 100. A multivalent and multispecific construct as described in any one of embodiments 81 to 99, wherein ABDC binds NaPi2b, ABD B binds MUC1, and ABD A binds mesothelin.

[0355] Implementation Scheme 101. A multivalent multispecific construct as described in Implementation Scheme 100, wherein the multivalent multispecific construct is suitable for the treatment of ovarian cancer or NSCLC.

[0356] Implementation scheme 102. A multivalent multispecific construct as described in any one of implementation schemes 81 to 99, wherein ABDC binds to B7H3, ABD B binds to PDL1, and ABD A binds to Trop2.

[0357] Implementation Scheme 103. The multivalent multispecific construct as described in Implementation Scheme 102, wherein the multivalent multispecific construct is suitable for the treatment of NSCLC.

[0358] Implementation scheme 104. A multivalent and multispecific construct as described in any one of implementation schemes 81 to 99, wherein ABDC binds to Ly6G6D, ABD B binds to CEACAM5, and ABD A binds to Claudin18.2.

[0359] Implementation Scheme 105. A multivalent multispecific construct as described in Implementation Scheme 104, wherein the multivalent multispecific construct is suitable for treating GI cancers.

[0360] Implementation scheme 106. A multivalent multispecific construct as described in any one of embodiments 81 to 99, wherein ABDC binds to BCMA, ABD B binds to CD38, and ABD A binds to FcRH5.

[0361] Implementation Scheme 107. The multivalent multispecific construct as described in Implementation Scheme 106, wherein the multivalent multispecific construct is suitable for the treatment of myeloma.

[0362] Implementation scheme 108. A multivalent multispecific construct as described in any one of implementation schemes 81 to 99, wherein ABDC binds CD22, ABD B binds CD19, and ABD A binds CD79b.

[0363] Implementation Scheme 109. A multivalent multispecific construct as described in Implementation Scheme 108, wherein the multivalent multispecific construct is suitable for the treatment of NHL or autoimmune diseases.

[0364] Implementation scheme 110. A multivalent multispecific construct as described in any one of embodiments 81 to 99, wherein ABDC binds Her2, ABD B binds MUC1, and ABD A binds Trop2.

[0365] Implementation Scheme 111. The multivalent multispecific construct as described in Implementation Scheme 110, wherein the multivalent multispecific construct is suitable for the treatment of breast cancer.

[0366] Implementation scheme 112. A multivalent multispecific construct as described in any one of embodiments 81 to 111, wherein the lengths of L1, L2, L3 and / or L4 are about 15-30 aa.

[0367] Implementation Scheme 113. A multivalent multispecific construct as described in any one of Implementation Schemes 81 to 112, wherein L1, L2, L3 and / or L4 comprises one or more of Gly, Ser, Thr, Pro, Arg, Phe, Glu, Cys, Asn, Ala, Asp and Gln residues, optionally wherein L1, L2, L3 and / or L4 primarily comprises Gly, Ser and Thr residues.

[0368] Implementation Scheme 114. A multivalent multispecific construct as described in any one of Implementation Schemes 81 to 113, wherein L1, L2, L3 and / or L4 primarily comprise Gly and Ser residues.

[0369] Implementation Scheme 115. A multivalent, multispecific construct as described in Implementation Scheme 114, wherein L1, L2, L3, and / or L4 comprise (Gly-Ser). n (Gly4-Ser) n (SEQ ID NO: 1), (Gly3-Ser) n(SEQ ID NO: 2), Gly-Gly-Ser, Gly-Gly-Ser-Gly (SEQ ID NO: 3), Gly-Gly-Gly-Gly (SEQ ID NO: 4), Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 5), Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 5) NO: 6), Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 7), Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 8), Gly-Ser-Gly-Ser-Gly-Ser (SEQ ID NO: 9), (Gly4-Ser)3 (SEQ ID NO: 10), (Gly2-Ser)3 (SEQ ID NO: 11), (Gly2-Ser)2 (SEQ ID NO: 12), ferritin E-helix, (Glu-Ala-Ala-Ala-Lys) 1-3 (SEQ ID NO:13), (Ala-Pro) n (SEQ ID NO: 14), IgG C H 1. IgG C H 2-C H 3 or Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 15).

[0370] Implementation Scheme 116. A multivalent multispecific construct as described in any one of Implementation Schemes 81 to 115, wherein L1, L2, L3 and / or L4 are hydrophilic, and optionally wherein L1, L2, L3 and / or L4 have a hydrophobicity of about 0.6.

[0371] Implementation Scheme 117. A multivalent and multispecific construct as described in any one of Implementation Schemes 81 to 116, wherein L1, L2, L3 and / or L4 are hydrophilic, optionally wherein L1, L2, L3 and / or L4 have a hydrophobicity of about 0.4 to about 0.8, optionally wherein L1, L2, L3 and / or L4 have a hydrophobicity of about 0.5 to about 0.7, optionally wherein L1, L2, L3 and / or L4 have a hydrophobicity of about 0.6 to about 0.65.

[0372] Implementation scheme 118. A multivalent and multispecific construct as described in any one of embodiments 81 to 117, wherein L1, L2, L3 and / or L4 are substantially non-immunogenic.

[0373] Implementation Scheme 119. A multivalent multispecific construct as described in any one of Implementation Schemes 81 to 118, wherein L1, L2, L3 and / or L4 are substantially non-cleavable.

[0374] Implementation scheme 120. A multivalent multispecific construct as described in any one of embodiments 81 to 119, wherein L1, L2, L3 and / or L4 are substantially cleavable.

[0375] Implementation Scheme 121. A multivalent multispecific construct as described in any one of Implementation Schemes 81 to 120, wherein the lengths of L1, L2, L3 and / or L4 are about 5-10 nm; optionally, the lengths are about 7 nm.

[0376] Implementation scheme 122. A multivalent multispecific construct as described in any one of embodiments 81 to 121, wherein L3 is a rigid connector with a length of about 7-12 nm and / or contains an α-helix.

[0377] Implementation Scheme 123. A multivalent and multispecific construct as described in any one of Implementation Schemes 81 to 122, wherein L2 is a moderately rigid linker with a length of about 7-12 nm, and / or comprises b2-microglobulin, Zn-a2-glycoprotein, 34-peptide repeat sequence or non-α-helix with moderate bending and rotational rigidity, optionally wherein L2 is a cleavable linker.

[0378] Implementation Scheme 124. A multivalent, multispecific construct as described in any one of Implementation Schemes 81 to 123, wherein L1 is a flexible linker with a length of about 7-12 nm and / or contains (Gly-Ser). n (Gly4-Ser) n (SEQ ID NO: 1), (Gly3-Ser) n(SEQ ID NO: 2), Gly-Gly-Ser, Gly-Gly-Ser-Gly (SEQ ID NO: 3), Gly-Gly-Gly-Gly (SEQ ID NO: 4), Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 5), Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 6), Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 7), Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 8), Gly-Ser-Gly-Ser-Gly-Ser (SEQ ID NO: 9), (Gly4-Ser)3 (SEQ ID NO: 10), (Gly2-Ser)3 (SEQ ID NO: 11), (Gly2-Ser)2 (SEQ ID NO: 12), ferritin E-helix, (Glu-Ala-Ala-Ala-Lys) 1-3 (SEQ ID NO: 13), (Ala-Pro) n (SEQ ID NO: 14), IgG C H 1. IgG C H 2-C H 3 or Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 15).

[0379] Implementation scheme 125. A multivalent multispecific construct as described in any one of implementation schemes 81 to 124, wherein the joints are L4, L3, L2 and L1 in order of strongest to weakest rigidity.

[0380] Implementation Scheme 126. A multivalent and multispecific construct as described in any one of Implementation Schemes 81 to 125, wherein the scaffold region comprises a rigid protein.

[0381] Implementation Scheme 127. A multivalent and multispecific construct as described in any one of Implementation Schemes 81 to 126, wherein the scaffold region includes a portion for extending PK, optionally wherein the portion for extending PK is an FcRn binding domain, IgG C H 2-C H 3. Human serum albumin, human transferrin, or fragments thereof.

[0382] Implementation Scheme 128. A multivalent and multispecific construct as described in any one of Implementation Schemes 81 to 127, wherein the scaffold region comprises IgG C H 2-C H 3 or a fragment thereof, optionally including IgG C H 2-C H 3 further includes a second hinge area, optionally wherein the support area includes hinge-C H 2-C H 3-Hinge.

[0383] Implementation Scheme 129. A multivalent multispecific construct as described in any one of Implementation Schemes 81 to 128, wherein the payload is conjugated to Cys, Tyr, or Lys residues in the scaffold region.

[0384] Implementation Scheme 130. A multivalent multispecific construct as described in any one of Implementation Schemes 81 to 129, wherein the payload is conjugated to the multivalent multispecific construct using TCEP reduction, transglutaminase, random conjugation, glycan conjugation, lysine conjugation, formylglycine synthase (FGE) oxidation, AjiCap technology, or click chemistry.

[0385] Implementation Scheme 131. A multivalent and multispecific construct as described in any one of Implementation Schemes 81 to 130, wherein the payload comprises a protein, optionally wherein the protein comprises an enzyme, an immunostimulant, an immunosuppressant, or a topoisomerase.

[0386] Implementation Scheme 132. A multivalent and multispecific construct as described in any one of Implementation Schemes 81 to 130, wherein the payload comprises a small molecule, optionally wherein the small molecule comprises a small molecule inhibitor, monomethylaurestatin E (MMAE), monomethylaurestatin F (MMAF), muscarinic acid, PNU, beloteccan, essanotecan, pyrrolobenzodiazepines (PBD), trastuzumab emtansine (T-DM1), steroids, chemotherapy, BCL-XL inhibitors, or STING agonists.

[0387] Implementation Scheme 133. A multivalent and multispecific construct as described in any one of Implementation Schemes 81 to 130, wherein the payload comprises an RNA molecule, optionally wherein the RNA molecule is a siRNA molecule.

[0388] Implementation Scheme 134. A multivalent and multispecific construct as described in any one of Implementation Schemes 81 to 130, wherein the payload comprises a chemical sensitizer.

[0389] Implementation Scheme 135. A multivalent and multispecific construct as described in any one of Implementation Schemes 81 to 130, wherein the payload comprises a gene modifier.

[0390] Implementation Scheme 136. A multivalent and multispecific construct as described in any one of Implementation Schemes 81 to 130, wherein the payload is a beloteccan derivative attached to the scaffold region using a cathepsin-cleavable linker.

[0391] Implementation Scheme 137. A multivalent multispecific construct as described in any one of Implementation Schemes 81 to 136, wherein the multivalent multispecific construct comprises more than one payload, optionally wherein the drug-to-antibody ratio (DAR) is about 2-8.

[0392] Implementation Scheme 138. A multivalent multispecific construct as described in any one of Implementation Schemes 81 to 137, wherein the multivalent multispecific construct further comprises one or more enhancer domains and / or one or more FcRn binding domains, optionally wherein one or more FcRn binding domains comprise IgG FcC H 2-C H 3. Structural domain or VHH structural domain.

[0393] Implementation Scheme 139. A multivalent multispecific construct as described in any one of Implementation Schemes 81 to 138, wherein the diameter of the multivalent multispecific construct is about 5-50 nm, about 10-40 nm, about 20-30 nm, about 20-25 nm or about 25-30 nm.

[0394] Implementation Scheme 140. A multivalent and multispecific construct as described in any one of Implementation Schemes 81 to 139, wherein each antigen-binding polypeptide contains a terminal suitable for covalent or non-covalent cyclization or subunit assembly within the generating cell.

[0395] Implementation Scheme 141. A multivalent multispecific construct as described in any one of Implementation Schemes 81 to 140, wherein the multivalent multispecific construct (i) delivers the payload to the target cell more efficiently than a monomer or single-specific binder having only one copy of ABD C, ABD B, or ABD A, (ii) is internalized by the target cell at a higher rate than a monomer or single-specific binder having only one copy of ABD C, ABD B, or ABD A, and / or (iii) has improved specificity for binding to and / or killing tumor cells than a monomer or single-specific binder having only one copy of ABD C, ABD B, or ABD A.

[0396] Implementation scheme 142. A nucleic acid encoding a multivalent and multispecific construct as described in any one of implementation schemes 81 to 141.

[0397] Implementation Scheme 143. A host cell comprising the nucleic acid described in Implementation Scheme 142.

[0398] Implementation Scheme 144. A pharmaceutical composition comprising the multivalent and multispecific construct of any one of Implementation Schemes 81 to 141, the nucleic acid of Implementation Scheme 142, or the host cell of Implementation Scheme 143.

[0399] Implementation Scheme 145. A method for treating a disease or condition in a subject of need, optionally wherein the disease or condition is cancer, said method comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 81 to 141, a nucleic acid as described in Implementation Scheme 142, a host cell as described in Implementation Scheme 143, or a pharmaceutical composition as described in Implementation Scheme 144.

[0400] Implementation scheme 146. The method as described in implementation scheme 145, wherein the cancer is a solid tumor.

[0401] Implementation Scheme 147. The method as described in Implementation Scheme 146, wherein the cancer is NSCLC, ovarian cancer, breast cancer, pancreatic cancer, colorectal cancer, or gastric cancer.

[0402] Implementation scheme 148. The method as described in implementation scheme 145, wherein the cancer is a blood cancer.

[0403] Implementation Scheme 149. The method described in Implementation Scheme 148, wherein the cancer is leukemia, lymphoma, or multiple myeloma.

[0404] Implementation Scheme 150. A method of treating a subject with ovarian cancer or NSCLC, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 81 to 144, wherein ABD C binds NaPi2b, ABD B binds MUC1, and ABD A binds mesothelin.

[0405] Implementation Scheme 151. A method of treating NSCLC in a subject of need, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 81 to 144, wherein ABD C binds to B7H3, ABD B binds to PDL1, and ABDA binds to Trop2.

[0406] Implementation Scheme 152. A method of treating a subject with GI cancer, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 81 to 144, wherein ABD C binds to Ly6G6D, ABD B binds to CEACAM5, and ABD A binds to Claudin18.2.

[0407] Implementation Scheme 153. A method of treating a subject with myeloma in need, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 81 to 144, wherein ABD C binds to BCMA, ABD B binds to CD38, and ABD A binds to FcRH5.

[0408] Implementation Scheme 154. A method of treating a subject with NHL or an autoimmune disease of need, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 81 to 144, wherein ABD C binds to CD22, ABD B binds to CD19, and ABD A binds to CD79b.

[0409] Implementation Scheme 155. A method of treating breast cancer in a subject of need, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 81 to 144, wherein ABD C binds Her2, ABD B binds MUC1, and ABD A binds Trop2.

[0410] Implementation Scheme 156. A multivalent and multispecific construct comprising: (a) Therapeutic payload; and (b) Two antigen-binding peptides targeting two or more different targets, each antigen-binding peptide comprising, from the N-terminus to the C-terminus, a first antigen-binding domain (ABD C), a first linker (L1), a second antigen-binding domain (ABDB), a second linker (L2), a third antigen-binding domain (ABD A), an optional third linker (L3), and a scaffold region. Wherein L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, and L3 optionally attaches ABD A to the scaffold region, such that each antigen-binding polypeptide comprises (i) ABD C – L1 – ABD B – L2 – ABD A – scaffold region or (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region arranged from the N-terminus to the C-terminus; and The scaffold region contains IgG Fc or a portion thereof or a modified form thereof.

[0411] Implementation Scheme 157. A multivalent and multispecific construct as described in Implementation Scheme 156, wherein ABD C, ABD B and / or ABD A are VHH, VNAR, Fab, ScFv, nanobody, biantibody, synthetic microbody, ligand or its binding fragment.

[0412] Implementation Scheme 158. A multivalent and multispecific construct as described in any one of Implementation Schemes 156 to 157, wherein ABD C, ABD B and / or ABD A are conditional binders, optionally wherein the binding of ABD C, ABD B and / or ABD A to their respective targets depends on pH, calcium or ATP levels.

[0413] Implementation Scheme 159. A multivalent and multispecific construct as described in any one of Implementation Schemes 156 to 158, wherein ABD C, ABD B and / or ABD A target a specific cell type.

[0414] Implementation scheme 160. A multivalent and multispecific construct as described in any one of implementation schemes 156 to 159, wherein ABD C, ABD B and / or ABD A target cancer cells or immune cells.

[0415] Implementation Scheme 161. A multivalent multispecific construct as described in any one of Implementation Schemes 156 to 160, wherein ABD C, ABD B and / or ABD A promote the internalization of the multivalent multispecific construct by target cells, optionally by promoting lysosomal transport of the multivalent multispecific construct by target cells, optionally wherein lysosomal transport is clathrin-mediated endocytosis or receptor-mediated endocytosis.

[0416] Implementation Scheme 162. A multivalent and multispecific construct as described in any one of Implementation Schemes 156 to 161, wherein ABD C, ABD B and / or ABD A promote the chemotherapeutic sensitivity of target cells.

[0417] Implementation Scheme 163. A multivalent and multispecific construct as described in any one of Implementation Schemes 156 to 162, wherein ABD C, ABD B and / or ABD A promote apoptosis or necrosis of target cells.

[0418] Implementation Scheme 164. A multivalent and multispecific construct as described in any one of Implementation Schemes 156 to 163, wherein ABD C, ABD B and / or ABD A comprises at least one half-life extension molecule, optionally wherein the half-life extension molecule is an FcRn binding domain, albumin, PEG, XTEN or human serum albumin (HSA) binding domain.

[0419] Implementation Scheme 165. A multivalent multispecific construct as described in any one of Implementation Schemes 156 to 164, wherein ABD C, ABD B and / or ABD A comprises at least one site for conjugating the payload, optionally wherein at least one site comprises a Cys, Tyr or Lys residue.

[0420] Implementation Scheme 166. A multivalent and multispecific construct as described in any one of Implementation Schemes 156 to 165, wherein ABD C binds to a first target molecule, ABD B binds to a second target molecule, and / or ABD A binds to a third target molecule.

[0421] Implementation Scheme 167. A multivalent and multispecific construct as described in any one of Implementation Schemes 156 to 166, wherein ABD C binds to a first epitope on a first target molecule, ABD B binds to a second epitope on a first target molecule, and / or ABD A binds to a third epitope on a first target molecule.

[0422] Implementation scheme 168. A multivalent and multispecific construct as described in any one of implementation schemes 156 to 167, wherein ABD C is used as a primary anchor for binding to target cells.

[0423] Implementation Scheme 169. A multivalent and multispecific construct as described in any one of Implementation Schemes 156 to 168, wherein ABD B can bind substantially stably to its target only when ABD C binds to its target and / or ABD A binds to its target.

[0424] Implementation scheme 170. A multivalent and multispecific construct as described in any one of implementation schemes 156 to 169, wherein ABD A is used as a secondary anchor for binding to target cells.

[0425] Implementation Scheme 171. A multivalent multispecific construct as described in any one of Implementation Schemes 156 to 170, wherein ABD C binds to one or more of NaPi2b, Trop2, MUC1, Ly6G6D, Claudin18.2, B7H3, BCMA, CD22, and Her2.

[0426] Implementation Scheme 172. A multivalent multispecific construct as described in any one of Implementation Schemes 156 to 171, wherein ABD B binds to one or more of EGFR, PDL1, CEA, FolRa, DR5, MUC1, mesothelin, Claudin18.2, CEACAM5, CD38, and CD19.

[0427] Implementation scheme 173. A multivalent multispecific construct as described in any one of implementation schemes 156 to 172, wherein ABD A binds to one or more of ALPL2, PRLR, integrin, DR5, CD20, FcRn, EGFR, Trop2, Claudin18.2, FcRH5 and CD79b.

[0428] Implementation Scheme 174. A multivalent multispecific construct as described in any one of Implementation Schemes 156 to 173, wherein one of ABD C, ABD B and ABD A binds Her2, and wherein one of ABD C, ABD B and ABD A binds Trop2.

[0429] Implementation Scheme 175. A multivalent and multispecific construct as described in any one of Implementation Schemes 156 to 174, wherein ABD C binds NaPi2b, ABD B binds MUC1, and ABD A binds mesothelin.

[0430] Implementation Scheme 176. A multivalent multispecific construct as described in Implementation Scheme 175, wherein the multivalent multispecific construct is suitable for the treatment of ovarian cancer or NSCLC.

[0431] Implementation scheme 177. A multivalent multispecific construct as described in any one of implementation schemes 156 to 174, wherein ABD C binds to B7H3, ABD B binds to PDL1, and ABD A binds to Trop2.

[0432] Implementation Scheme 178. A multivalent multispecific construct as described in Implementation Scheme 177, wherein the multivalent multispecific construct is suitable for the treatment of NSCLC.

[0433] Implementation scheme 179. A multivalent and multispecific construct as described in any one of implementation schemes 156 to 174, wherein ABD C binds to Ly6G6D, ABD B binds to CEACAM5, and ABD A binds to Claudin18.2.

[0434] Implementation scheme 180. The multivalent multispecific construct as described in implementation scheme 179, wherein the multivalent multispecific construct is suitable for treating GI cancers.

[0435] Implementation scheme 181. A multivalent multispecific construct as described in any one of implementation schemes 156 to 174, wherein ABD C binds to BCMA, ABD B binds to CD38, and ABD A binds to FcRH5.

[0436] Implementation Scheme 182. The multivalent multispecific construct as described in Implementation Scheme 181, wherein the multivalent multispecific construct is suitable for the treatment of myeloma.

[0437] Implementation scheme 183. A multivalent multispecific construct as described in any one of implementation schemes 156 to 174, wherein ABD C binds to CD22, ABD B binds to CD19, and ABD A binds to CD79b.

[0438] Implementation Scheme 184. A multivalent multispecific construct as described in Implementation Scheme 183, wherein the multivalent multispecific construct is suitable for the treatment of NHL or autoimmune diseases.

[0439] Implementation scheme 185. A multivalent multispecific construct as described in any one of implementation schemes 156 to 174, wherein ABD C binds Her2, ABD B binds MUC1, and ABD A binds Trop2.

[0440] Implementation Scheme 186. The multivalent multispecific construct as described in Implementation Scheme 185, wherein the multivalent multispecific construct is suitable for the treatment of breast cancer.

[0441] Implementation scheme 187. A multivalent multispecific construct as described in any one of implementation schemes 156 to 186, wherein the lengths of L1, L2, L3 and / or L4 are about 15-30 aa.

[0442] Implementation Scheme 188. A multivalent multispecific construct as described in any one of Implementation Schemes 156 to 187, wherein L1, L2, L3 and / or L4 comprises one or more of the following residues: Gly, Ser, Thr, Pro, Arg, Phe, Glu, Cys, Asn, Ala, Asp and Gln, optionally wherein L1, L2, L3 and / or L4 primarily comprises Gly, Ser and Thr residues.

[0443] Implementation scheme 189. A multivalent multispecific construct as described in any one of implementation schemes 156 to 188, wherein L1, L2, L3 and / or L4 primarily contain Gly and Ser residues.

[0444] Implementation Scheme 190. A multivalent, multispecific construct as described in Implementation Scheme 189, wherein L1, L2, L3, and / or L4 comprise (Gly-Ser). n (Gly4-Ser) n (SEQ ID NO: 1), (Gly3-Ser) n(SEQ ID NO: 2), Gly-Gly-Ser, Gly-Gly-Ser-Gly (SEQ ID NO: 3), Gly-Gly-Gly-Gly (SEQ ID NO: 4), Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 5), Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 5) NO: 6), Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 7), Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 8), Gly-Ser-Gly-Ser-Gly-Ser (SEQ ID NO: 9), (Gly4-Ser)3 (SEQ ID NO: 10), (Gly2-Ser)3 (SEQ ID NO: 11), (Gly2-Ser)2 (SEQ ID NO: 12), ferritin E-helix, (Glu-Ala-Ala-Ala-Lys) 1-3 (SEQ ID NO:13), (Ala-Pro) n (SEQ ID NO: 14), IgG C H 1. IgG C H 2-C H 3 or Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 15).

[0445] Implementation Scheme 191. A multivalent and multispecific construct as described in any one of Implementation Schemes 156 to 190, wherein L1, L2, L3 and / or L4 are hydrophilic, and optionally wherein L1, L2, L3 and / or L4 have a hydrophobicity of about 0.6.

[0446] Implementation Scheme 192. A multivalent and multispecific construct as described in any one of Implementation Schemes 156 to 191, wherein L1, L2, L3 and / or L4 are hydrophilic, optionally wherein L1, L2, L3 and / or L4 have a hydrophobicity of about 0.4 to about 0.8, optionally wherein L1, L2, L3 and / or L4 have a hydrophobicity of about 0.5 to about 0.7, optionally wherein L1, L2, L3 and / or L4 have a hydrophobicity of about 0.6 to about 0.65.

[0447] Implementation scheme 193. A multivalent and multispecific construct as described in any one of implementation schemes 156 to 192, wherein L1, L2, L3 and / or L4 are substantially non-immunogenic.

[0448] Implementation scheme 194. A multivalent multispecific construct as described in any one of implementation schemes 156 to 193, wherein L1, L2, L3 and / or L4 are substantially non-cleavable.

[0449] Implementation Scheme 195. A multivalent multispecific construct as described in any one of Implementation Schemes 156 to 194, wherein L1, L2, L3 and / or L4 are substantially cleavable.

[0450] Implementation Scheme 196. A multivalent and multispecific construct as described in any one of Implementation Schemes 156 to 195, wherein the lengths of L1, L2, L3 and / or L4 are about 5-10 nm; optionally, the lengths are about 7 nm.

[0451] Implementation Scheme 197. A multivalent multispecific construct as described in any one of Implementation Schemes 156 to 196, wherein L3 is a rigid connector with a length of about 7-12 nm and / or contains an α-helix.

[0452] Implementation Scheme 198. A multivalent and multispecific construct as described in any one of Implementation Schemes 156 to 197, wherein L2 is a moderately rigid linker with a length of about 7-12 nm, and / or comprises b2-microglobulin, Zn-a2-glycoprotein, 34-peptide repeat sequence or non-α-helix with moderate bending and rotational rigidity, optionally wherein L2 is a cleavable linker.

[0453] Implementation Scheme 199. A multivalent, multispecific construct as described in any one of Implementation Schemes 156 to 198, wherein L1 is a flexible linker with a length of about 7-12 nm and / or contains (Gly-Ser). n (Gly4-Ser) n (SEQ ID NO:1), (Gly3-Ser) n(SEQ ID NO: 2), Gly-Gly-Ser, Gly-Gly-Ser-Gly (SEQ ID NO: 3), Gly-Gly-Gly-Gly (SEQ ID NO: 4), Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 5), Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 6), Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 7), Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 8), Gly-Ser-Gly-Ser-Gly-Ser (SEQ ID NO: 9), (Gly4-Ser)3 (SEQ ID NO:10), (Gly2-Ser)3 (SEQ ID NO: 11), (Gly2-Ser)2 (SEQ ID NO: 12), ferritin E-helix, (Glu-Ala-Ala-Ala-Lys) 1-3 (SEQ ID NO: 13), (Ala-Pro) n (SEQ ID NO: 14), IgG C H 1. IgG C H 2-C H 3 or Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 15).

[0454] Implementation scheme 200. A multivalent multispecific construct as described in any one of implementation schemes 156 to 199, wherein the joints are L4, L3, L2 and L1 in order of strongest to weakest rigidity.

[0455] Implementation scheme 201. A multivalent and multispecific construct as described in any one of implementation schemes 156 to 200, wherein the scaffold region comprises a rigid protein.

[0456] Implementation Scheme 202. A multivalent and multispecific construct as described in any one of Implementation Schemes 156 to 201, wherein the scaffold region includes a portion for extending PK, optionally wherein the portion for extending PK is an FcRn binding domain, IgGC H 2-C H 3. Human serum albumin, human transferrin, or fragments thereof.

[0457] Implementation Scheme 203. A multivalent and multispecific construct as described in any one of Implementation Schemes 156 to 202, wherein the scaffold region comprises IgG C H 2-C H 3 or a fragment thereof, optionally including IgG C H 2-C H 3 further includes a second hinge area, optionally wherein the support area includes hinge-C H 2-C H 3-Hinge.

[0458] Implementation scheme 204. A multivalent multispecific construct as described in any one of implementation schemes 156 to 203, wherein the payload is conjugated to Cys, Tyr or Lys residues in the scaffold region.

[0459] Implementation Scheme 205. A multivalent multispecific construct as described in any one of Implementation Schemes 156 to 204, wherein the payload is conjugated to the multivalent multispecific construct using TCEP reduction, transglutaminase, random conjugation, glycan conjugation, lysine conjugation, formylglycine synthase (FGE) oxidation, AjiCap technology, or click chemistry.

[0460] Implementation Scheme 206. A multivalent and multispecific construct as described in any one of Implementation Schemes 156 to 205, wherein the payload comprises a protein, optionally wherein the protein comprises an enzyme, an immunostimulant, an immunosuppressant, or a topoisomerase.

[0461] Implementation Scheme 207. A multivalent and multispecific construct as described in any one of Implementation Schemes 156 to 205, wherein the payload comprises a small molecule, optionally wherein the small molecule comprises a small molecule inhibitor, monomethylaurestatin E (MMAE), monomethylaurestatin F (MMAF), muscarinic acid, PNU, beloteccan, ethanotecan, pyrrolobenzodiazepines (PBD), trastuzumab emtansine (T-DM1), steroids, chemotherapy, BCL-XL inhibitors, or STING agonists.

[0462] Implementation Scheme 208. A multivalent and multispecific construct as described in any one of Implementation Schemes 156 to 205, wherein the payload comprises an RNA molecule, optionally wherein the RNA molecule is a siRNA molecule.

[0463] Implementation scheme 209. A multivalent and multispecific construct as described in any one of implementation schemes 156 to 205, wherein the payload comprises a chemical sensitizer.

[0464] Implementation scheme 210. A multivalent and multispecific construct as described in any one of implementation schemes 156 to 205, wherein the payload comprises a gene modifier.

[0465] Implementation Scheme 211. A multivalent and multispecific construct as described in any one of Implementation Schemes 156 to 205, wherein the payload is a beloteccan derivative attached to the scaffold region by a cathepsin-cleavable linker.

[0466] Implementation Scheme 212. A multivalent multispecific construct as described in any one of Implementation Schemes 156 to 211, wherein the multivalent multispecific construct comprises more than one payload, optionally wherein the drug-to-antibody ratio (DAR) is about 2-8.

[0467] Implementation Scheme 213. A multivalent multispecific construct as described in any one of Implementation Schemes 156 to 212, wherein the multivalent multispecific construct further comprises one or more enhancer domains and / or one or more FcRn binding domains, optionally wherein one or more FcRn binding domains comprise IgG FcC H 2-C H 3. Structural domain or VHH structural domain.

[0468] Implementation Scheme 214. A multivalent multispecific construct as described in any one of Implementation Schemes 156 to 213, wherein the diameter of the multivalent multispecific construct is about 5-50 nm, about 10-40 nm, about 20-30 nm, about 20-25 nm or about 25-30 nm.

[0469] Implementation Scheme 215. A multivalent and multispecific construct as described in any one of Implementation Schemes 156 to 214, wherein each antigen-binding polypeptide contains a terminal suitable for covalent or non-covalent cyclization or subunit assembly within the generating cell.

[0470] Implementation Scheme 216. A multivalent multispecific construct as described in any one of Implementation Schemes 156 to 215, wherein the multivalent multispecific construct (i) delivers the payload to the target cell more efficiently than a monomer or single-specific binder having only one copy of ABD C, ABD B, or ABD A, (ii) is internalized by the target cell at a higher rate than a monomer or single-specific binder having only one copy of ABD C, ABD B, or ABD A, and / or (iii) has improved specificity for binding to and / or killing tumor cells than a monomer or single-specific binder having only one copy of ABD C, ABD B, or ABDA.

[0471] Implementation Scheme 217. A nucleic acid encoding a multivalent and multispecific construct as described in any one of Implementation Schemes 156 to 216.

[0472] Implementation Scheme 218. A host cell comprising the nucleic acid described in Implementation Scheme 217.

[0473] Implementation Scheme 219. A pharmaceutical composition comprising the multivalent multispecific construct of any one of Implementation Schemes 156 to 216, the nucleic acid of Implementation Scheme 217, or the host cell of Implementation Scheme 218.

[0474] Implementation Scheme 220. A method for treating a disease or condition in a subject of need, optionally wherein the disease or condition is cancer, said method comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 156 to 216, a nucleic acid as described in Implementation Scheme 217, a host cell as described in Implementation Scheme 218, or a pharmaceutical composition as described in Implementation Scheme 219.

[0475] Implementation Scheme 221. The method as described in Implementation Scheme 220, wherein the cancer is a solid tumor.

[0476] Implementation Scheme 222. The method as described in Implementation Scheme 221, wherein the cancer is NSCLC, ovarian cancer, breast cancer, pancreatic cancer, colorectal cancer, or gastric cancer.

[0477] Implementation scheme 223. The method as described in implementation scheme 220, wherein the cancer is a blood cancer.

[0478] Implementation scheme 224. The method described in implementation scheme 223, wherein the cancer is leukemia, lymphoma, or multiple myeloma.

[0479] Implementation Scheme 225. A method of treating a subject with ovarian cancer or NSCLC, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 156 to 219, wherein ABD C binds NaPi2b, ABD B binds MUC1, and ABD A binds mesothelin.

[0480] Implementation Scheme 226. A method of treating NSCLC in a subject of need, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 156 to 219, wherein ABD C binds to B7H3, ABD B binds to PDL1, and ABD A binds to Trop2.

[0481] Implementation Scheme 227. A method of treating a subject with GI cancer, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 156 to 219, wherein ABD C binds to Ly6G6D, ABD B binds to CEACAM5, and ABD A binds to Claudin18.2.

[0482] Implementation Scheme 228. A method of treating a subject with myeloma in need, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 156 to 219, wherein ABD C binds to BCMA, ABD B binds to CD38, and ABD A binds to FcRH5.

[0483] Implementation Scheme 229. A method for treating a subject with NHL or an autoimmune disease, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 156 to 219, wherein ABD C binds to CD22, ABD B binds to CD19, and ABD A binds to CD79b.

[0484] Implementation Scheme 230. A method of treating breast cancer in a subject of need, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 156 to 219, wherein ABD C binds Her2, ABD B binds MUC1, and ABD A binds Trop2.

[0485] Implementation Scheme 231. A multispecific construct comprising: (a) Therapeutic payload; and (b) Four antigen-binding peptides targeting two or more different targets, each antigen-binding peptide comprising, from the N-terminus to the C-terminus, a first antigen-binding domain (ABD C), a first linker (L1), a second antigen-binding domain (ABDB), a second linker (L2), a third antigen-binding domain (ABD A), an optional third linker (L3), and a scaffold region. Wherein L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, and L3 optionally attaches ABD A to the scaffold region, such that each antigen-binding polypeptide comprises (i) ABD C – L1 – ABD B – L2 – ABD A – scaffold region or (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region arranged from the N-terminus to the C-terminus; and The scaffold region includes a central protein anchor, which optionally includes an FcRn binding domain.

[0486] Implementation Scheme 232. A multivalent and multispecific construct as described in Implementation Scheme 231, wherein ABD C, ABD B and / or ABD A are VHH, VNAR, Fab, ScFv, nanobody, biantibody, synthetic microbody, ligand or its binding fragment.

[0487] Implementation Scheme 233. A multivalent and multispecific construct as described in any one of Implementation Schemes 231 to 232, wherein ABD C, ABD B and / or ABD A are conditional binders, optionally wherein the binding of ABD C, ABD B and / or ABD A to their respective targets depends on pH, calcium or ATP levels.

[0488] Implementation Scheme 234. A multivalent and multispecific construct as described in any one of Implementation Schemes 231 to 233, wherein ABD C, ABD B and / or ABD A target a specific cell type.

[0489] Implementation Scheme 235. A multivalent and multispecific construct as described in any one of Implementation Schemes 231 to 234, wherein ABD C, ABD B and / or ABD A target cancer cells or immune cells.

[0490] Implementation Scheme 236. A multivalent multispecific construct as described in any one of Implementation Schemes 231 to 235, wherein ABD C, ABD B and / or ABD A promote the internalization of the multivalent multispecific construct by target cells, optionally by promoting lysosomal transport of the multivalent multispecific construct by target cells, optionally wherein lysosomal transport is clathrin-mediated endocytosis or receptor-mediated endocytosis.

[0491] Implementation Scheme 237. A multivalent and multispecific construct as described in any one of Implementation Schemes 231 to 236, wherein ABD C, ABD B and / or ABD A promote the chemotherapeutic sensitivity of target cells.

[0492] Implementation Scheme 238. A multivalent and multispecific construct as described in any one of Implementation Schemes 231 to 237, wherein ABD C, ABD B and / or ABD A promote apoptosis or necrosis of target cells.

[0493] Implementation Scheme 239. A multivalent and multispecific construct as described in any one of Implementation Schemes 231 to 238, wherein ABD C, ABD B and / or ABD A comprises at least one half-life extension molecule, optionally wherein the half-life extension molecule is an FcRn binding domain, albumin, PEG, XTEN or human serum albumin (HSA) binding domain.

[0494] Implementation Scheme 240. A multivalent multispecific construct as described in any one of Implementation Schemes 231 to 239, wherein ABD C, ABD B and / or ABD A contains at least one site for conjugating the payload, and optionally at least one site contains a Cys, Tyr or Lys residue.

[0495] Implementation Scheme 241. A multivalent and multispecific construct as described in any one of Implementation Schemes 231 to 240, wherein ABD C binds to a first target molecule, ABD B binds to a second target molecule, and / or ABD A binds to a third target molecule.

[0496] Implementation Scheme 242. A multivalent and multispecific construct as described in any one of Implementation Schemes 231 to 241, wherein ABD C binds to a first epitope on a first target molecule, ABD B binds to a second epitope on a first target molecule, and / or ABD A binds to a third epitope on a first target molecule.

[0497] Implementation Scheme 243. A multivalent and multispecific construct as described in any one of Implementation Schemes 231 to 242, wherein ABD C is used as a primary anchor for binding to target cells.

[0498] Implementation scheme 244. A multivalent and multispecific construct as described in any one of embodiments 231 to 243, wherein ABD B can bind substantially stably to its target only when ABD C binds to its target and / or ABD A binds to its target.

[0499] Implementation Scheme 245. A multivalent and multispecific construct as described in any one of Implementation Schemes 231 to 244, wherein ABD A is used as a secondary anchor for binding to target cells.

[0500] Implementation scheme 246. A multivalent multispecific construct as described in any one of implementation schemes 231 to 245, wherein ABD C binds to one or more of NaPi2b, Trop2, MUC1, Ly6G6D, Claudin18.2, B7H3, BCMA, CD22 and Her2.

[0501] Implementation Scheme 247. A multivalent multispecific construct as described in any one of Implementation Schemes 231 to 246, wherein ABD B binds to one or more of EGFR, PDL1, CEA, FolRa, DR5, MUC1, mesothelin, Claudin18.2, CEACAM5, CD38, and CD19.

[0502] Implementation scheme 248. A multivalent multispecific construct as described in any one of implementation schemes 231 to 247, wherein ABD A binds to one or more of ALPL2, PRLR, integrin, DR5, CD20, FcRn, EGFR, Trop2, Claudin18.2, FcRH5, and CD79b.

[0503] Implementation scheme 249. A multivalent multispecific construct as described in any one of implementation schemes 231 to 248, wherein one of ABD C, ABD B and ABD A binds Her2, and wherein one of ABD C, ABD B and ABD A binds Trop2.

[0504] Implementation scheme 250. A multivalent and multispecific construct as described in any one of implementation schemes 231 to 249, wherein ABD C binds NaPi2b, ABD B binds MUC1, and ABD A binds mesothelin.

[0505] Implementation Scheme 251. A multivalent multispecific construct as described in Implementation Scheme 250, wherein the multivalent multispecific construct is suitable for the treatment of ovarian cancer or NSCLC.

[0506] Implementation scheme 252. A multivalent and multispecific construct as described in any one of implementation schemes 231 to 249, wherein ABD C binds to B7H3, ABD B binds to PDL1, and ABD A binds to Trop2.

[0507] Implementation Scheme 253. A multivalent multispecific construct as described in Implementation Scheme 252, wherein the multivalent multispecific construct is suitable for the treatment of NSCLC.

[0508] Implementation scheme 254. A multivalent and multispecific construct as described in any one of implementation schemes 231 to 249, wherein ABD C binds to Ly6G6D, ABD B binds to CEACAM5, and ABD A binds to Claudin18.2.

[0509] Implementation Scheme 255. A multivalent multispecific construct as described in Implementation Scheme 254, wherein the multivalent multispecific construct is suitable for treating GI cancers.

[0510] Implementation scheme 256. A multivalent multispecific construct as described in any one of implementation schemes 231 to 249, wherein ABD C binds to BCMA, ABD B binds to CD38, and ABD A binds to FcRH5.

[0511] Implementation Scheme 257. A multivalent multispecific construct as described in Implementation Scheme 256, wherein the multivalent multispecific construct is suitable for the treatment of myeloma.

[0512] Implementation scheme 258. A multivalent multispecific construct as described in any one of implementation schemes 231 to 249, wherein ABD C binds to CD22, ABD B binds to CD19, and ABD A binds to CD79b.

[0513] Implementation Scheme 259. A multivalent multispecific construct as described in Implementation Scheme 258, wherein the multivalent multispecific construct is suitable for the treatment of NHL or autoimmune diseases.

[0514] Implementation scheme 260. A multivalent multispecific construct as described in any one of implementation schemes 231 to 249, wherein ABD C binds Her2, ABD B binds MUC1, and ABD A binds Trop2.

[0515] Implementation Scheme 261. The multivalent multispecific construct as described in Implementation Scheme 260, wherein the multivalent multispecific construct is suitable for the treatment of breast cancer.

[0516] Implementation Scheme 262. A multivalent multispecific construct as described in any one of Implementation Schemes 231 to 261, wherein the lengths of L1, L2, L3 and / or L4 are about 15-30 aa.

[0517] Implementation Scheme 263. A multivalent multispecific construct as described in any one of Implementation Schemes 231 to 262, wherein L1, L2, L3 and / or L4 comprises one or more of Gly, Ser, Thr, Pro, Arg, Phe, Glu, Cys, Asn, Ala, Asp and Gln residues, optionally wherein L1, L2, L3 and / or L4 primarily comprises Gly, Ser and Thr residues.

[0518] Implementation Scheme 264. A multivalent multispecific construct as described in any one of Implementation Schemes 231 to 263, wherein L1, L2, L3 and / or L4 primarily comprise Gly and Ser residues.

[0519] Implementation Scheme 265. A multivalent, multispecific construct as described in Implementation Scheme 264, wherein L1, L2, L3, and / or L4 comprise (Gly-Ser). n (Gly4-Ser) n (SEQ ID NO: 1), (Gly3-Ser) n(SEQ ID NO: 2), Gly-Gly-Ser, Gly-Gly-Ser-Gly (SEQ ID NO: 3), Gly-Gly-Gly-Gly (SEQ ID NO: 4), Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 5), Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 5) NO: 6), Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 7), Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 8), Gly-Ser-Gly-Ser-Gly-Ser (SEQ ID NO: 9), (Gly4-Ser)3 (SEQ ID NO: 10), (Gly2-Ser)3 (SEQ ID NO: 11), (Gly2-Ser)2 (SEQ ID NO: 12), ferritin E-helix, (Glu-Ala-Ala-Ala-Lys) 1-3 (SEQ ID NO:13), (Ala-Pro) n (SEQ ID NO: 14), IgG C H 1. IgG C H 2-C H 3 or Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 15).

[0520] Implementation Scheme 266. A multivalent and multispecific construct as described in any one of Implementation Schemes 231 to 265, wherein L1, L2, L3 and / or L4 are hydrophilic, and optionally wherein L1, L2, L3 and / or L4 have a hydrophobicity of about 0.6.

[0521] Implementation Scheme 267. A multivalent and multispecific construct as described in any one of Implementation Schemes 231 to 266, wherein L1, L2, L3 and / or L4 are hydrophilic, optionally wherein L1, L2, L3 and / or L4 have a hydrophobicity of about 0.4 to about 0.8, optionally wherein L1, L2, L3 and / or L4 have a hydrophobicity of about 0.5 to about 0.7, optionally wherein L1, L2, L3 and / or L4 have a hydrophobicity of about 0.6 to about 0.65.

[0522] Implementation scheme 268. A multivalent and multispecific construct as described in any one of implementation schemes 231 to 267, wherein L1, L2, L3 and / or L4 are substantially non-immunogenic.

[0523] Implementation Scheme 269. A multivalent multispecific construct as described in any one of Implementation Schemes 231 to 268, wherein L1, L2, L3 and / or L4 are substantially non-cleavable.

[0524] Implementation Scheme 270. A multivalent multispecific construct as described in any one of Implementation Schemes 231 to 269, wherein L1, L2, L3 and / or L4 are substantially cleavable.

[0525] Implementation Scheme 271. A multivalent multispecific construct as described in any one of Implementation Schemes 231 to 270, wherein the lengths of L1, L2, L3 and / or L4 are about 5-10 nm; optionally, the lengths are about 7 nm.

[0526] Implementation Scheme 272. A multivalent multispecific construct as described in any one of Implementation Schemes 231 to 271, wherein L3 is a rigid connector with a length of about 7-12 nm and / or contains an α-helix.

[0527] Implementation Scheme 273. A multivalent multispecific construct as described in any one of Implementation Schemes 231 to 272, wherein L2 is a moderately rigid linker with a length of about 7-12 nm, and / or comprises b2-microglobulin, Zn-a2-glycoprotein, 34-peptide repeat sequence or non-α-helix with moderate bending and rotational rigidity, optionally wherein L2 is a cleavable linker.

[0528] Implementation Scheme 274. A multivalent multispecific construct as described in any one of Implementation Schemes 231 to 273, wherein L1 is a flexible connector with a length of about 7-12 nm and / or contains (Gly-Ser). n (Gly4-Ser) n (SEQ ID NO:1), (Gly3-Ser) n(SEQ ID NO: 2), Gly-Gly-Ser, Gly-Gly-Ser-Gly (SEQ ID NO: 3), Gly-Gly-Gly-Gly (SEQ ID NO: 4), Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 5), Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 6), Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 7), Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 8), Gly-Ser-Gly-Ser-Gly-Ser (SEQ ID NO: 9), (Gly4-Ser)3 (SEQ ID NO:10), (Gly2-Ser)3 (SEQ ID NO: 11), (Gly2-Ser)2 (SEQ ID NO: 12), ferritin E-helix, (Glu-Ala-Ala-Ala-Lys) 1-3 (SEQ ID NO: 13), (Ala-Pro) n (SEQ ID NO: 14), IgG C H 1. IgG C H 2-C H 3 or Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 15).

[0529] Implementation scheme 275. A multivalent multispecific construct as described in any one of implementation schemes 231 to 274, wherein the joints are L4, L3, L2 and L1 in order of strongest to weakest rigidity.

[0530] Implementation scheme 276. A multivalent and multispecific construct as described in any one of implementation schemes 231 to 275, wherein the scaffold region comprises a rigid protein.

[0531] Implementation Scheme 277. A multivalent and multispecific construct as described in any one of Implementation Schemes 231 to 276, wherein the scaffold region includes a portion for extending PK, optionally wherein the portion for extending PK is an FcRn binding domain, IgGC H 2-C H 3. Human serum albumin, human transferrin, or fragments thereof.

[0532] Implementation Scheme 278. A multivalent and multispecific construct as described in any one of Implementation Schemes 231 to 277, wherein the scaffold region comprises IgG C H 2-C H 3 or a fragment thereof, optionally including IgG C H 2-C H 3 further includes a second hinge area, optionally wherein the support area includes hinge-C H 2-C H 3-Hinge.

[0533] Implementation Scheme 279. A multivalent multispecific construct as described in any one of Implementation Schemes 231 to 278, wherein the payload is conjugated to Cys, Tyr, or Lys residues in the scaffold region.

[0534] Implementation Scheme 280. A multivalent multispecific construct as described in any one of Implementation Schemes 231 to 279, wherein the payload is conjugated to the multivalent multispecific construct using TCEP reduction, transglutaminase, random conjugation, glycan conjugation, lysine conjugation, formylglycine synthase (FGE) oxidation, AjiCap technology, or click chemistry.

[0535] Implementation Scheme 281. A multivalent and multispecific construct as described in any one of Implementation Schemes 231 to 280, wherein the payload comprises a protein, optionally wherein the protein comprises an enzyme, an immunostimulant, an immunosuppressant, or a topoisomerase.

[0536] Implementation Scheme 282. A multivalent and multispecific construct as described in any one of Implementation Schemes 231 to 280, wherein the payload comprises a small molecule, optionally wherein the small molecule comprises a small molecule inhibitor, monomethylaurestatin E (MMAE), monomethylaurestatin F (MMAF), muscarinic acid, PNU, beloteccan, ethanotecan, pyrrolobenzodiazepines (PBD), trastuzumab emtansine (T-DM1), steroids, chemotherapy, BCL-XL inhibitors, or STING agonists.

[0537] Implementation Scheme 283. A multivalent and multispecific construct as described in any one of Implementation Schemes 231 to 280, wherein the payload comprises an RNA molecule, optionally wherein the RNA molecule is a siRNA molecule.

[0538] Implementation scheme 284. A multivalent and multispecific construct as described in any one of implementation schemes 231 to 280, wherein the payload comprises a chemical sensitizer.

[0539] Implementation scheme 285. A multivalent and multispecific construct as described in any one of implementation schemes 231 to 280, wherein the payload comprises a gene modifier.

[0540] Implementation Scheme 286. A multivalent and multispecific construct as described in any one of Implementation Schemes 231 to 280, wherein the payload is a beloteccan derivative attached to the scaffold region using a cathepsin-cleavable linker.

[0541] Implementation Scheme 287. A multivalent multispecific construct as described in any one of Implementation Schemes 231 to 286, wherein the multivalent multispecific construct comprises more than one payload, optionally wherein the drug-to-antibody ratio (DAR) is about 2-8.

[0542] Implementation Scheme 288. A multivalent multispecific construct as described in any one of Implementation Schemes 231 to 287, wherein the multivalent multispecific construct further comprises one or more enhancer domains and / or one or more FcRn binding domains, optionally wherein one or more FcRn binding domains comprise IgG FcC H 2-C H 3. Structural domain or VHH structural domain.

[0543] Implementation Scheme 289. A multivalent multispecific construct as described in any one of Implementation Schemes 231 to 288, wherein the diameter of the multivalent multispecific construct is about 5-50 nm, about 10-40 nm, about 20-30 nm, about 20-25 nm or about 25-30 nm.

[0544] Implementation Scheme 290. A multivalent and multispecific construct as described in any one of Implementation Schemes 231 to 289, wherein each antigen-binding polypeptide contains a terminal suitable for covalent or non-covalent cyclization or subunit assembly within the generating cell.

[0545] Implementation Scheme 291. A multivalent multispecific construct as described in any one of Implementation Schemes 231 to 290, wherein the multivalent multispecific construct (i) delivers the payload to the target cell more efficiently than a monomer or monospecific binder having only one copy of ABD C, ABD B, or ABD A, (ii) is internalized by the target cell at a higher rate than a monomer or monospecific binder having only one copy of ABD C, ABD B, or ABD A, and / or (iii) has improved specificity for binding to and / or killing tumor cells than a monomer or monospecific binder having only one copy of ABD C, ABD B, or ABDA.

[0546] Implementation scheme 292. A nucleic acid encoding a multivalent and multispecific construct as described in any one of implementation schemes 231 to 291.

[0547] Implementation Scheme 293. A host cell comprising the nucleic acid described in Implementation Scheme 292.

[0548] Implementation Scheme 294. A pharmaceutical composition comprising the multivalent multispecific construct of any one of Implementation Schemes 231 to 291, the nucleic acid of Implementation Scheme 292, or the host cell of Implementation Scheme 293.

[0549] Implementation Scheme 295. A method for treating a disease or condition in a subject of need, optionally wherein the disease or condition is cancer, said method comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 231 to 291, a nucleic acid as described in Implementation Scheme 292, a host cell as described in Implementation Scheme 293, or a pharmaceutical composition as described in Implementation Scheme 294.

[0550] Implementation scheme 296. The method as described in implementation scheme 295, wherein the cancer is a solid tumor.

[0551] Implementation Scheme 297. The method described in Implementation Scheme 296, wherein the cancer is NSCLC, ovarian cancer, breast cancer, pancreatic cancer, colorectal cancer, or gastric cancer.

[0552] Implementation scheme 298. The method as described in implementation scheme 295, wherein the cancer is a blood cancer.

[0553] Implementation scheme 299. The method described in implementation scheme 298, wherein the cancer is leukemia, lymphoma, or multiple myeloma.

[0554] Implementation Scheme 300. A method of treating a subject with ovarian cancer or NSCLC, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 231 to 294, wherein ABD C binds NaPi2b, ABD B binds MUC1, and ABD A binds mesothelin.

[0555] Implementation Scheme 301. A method of treating NSCLC in a subject of need, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 231 to 294, wherein ABD C binds to B7H3, ABD B binds to PDL1, and ABD A binds to Trop2.

[0556] Implementation Scheme 302. A method of treating a subject with GI cancer, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 231 to 294, wherein ABD C binds to Ly6G6D, ABD B binds to CEACAM5, and ABD A binds to Claudin18.2.

[0557] Implementation Scheme 303. A method of treating a subject with myeloma in need, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 231 to 294, wherein ABD C binds to BCMA, ABD B binds to CD38, and ABD A binds to FcRH5.

[0558] Implementation Scheme 304. A method for treating a subject with NHL or an autoimmune disease, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 231 to 294, wherein ABD C binds to CD22, ABD B binds to CD19, and ABD A binds to CD79b.

[0559] Implementation Scheme 305. A method of treating breast cancer in a subject of need, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 231 to 294, wherein ABD C binds Her2, ABD B binds MUC1, and ABD A binds Trop2.

[0560] Implementation Scheme 306. A multispecific construct comprising: (a) Therapeutic payload; and (b) n antigen-binding peptides targeting two or more different targets, each antigen-binding peptide comprising, from the N-terminus to the C-terminus, a first antigen-binding domain (ABD C), a first linker (L1), a second antigen-binding domain (ABDB), a second linker (L2), a third antigen-binding domain (ABD A), an optional third linker (L3), a scaffold region, and a fourth linker (L4). Wherein L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, L3 optionally attaches ABD A to the scaffold region, and L4 attaches the scaffold region to ABD C, such that each antigen-binding polypeptide comprises (i) ABD C – L1 – ABD B – L2 – ABD A – scaffold region – L4 or (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region – L4, arranged from N-terminus to C-terminus. The scaffold region contains rigid polypeptides; The construct further includes a clasp domain, which attaches the first antigen-binding polypeptide and the nth antigen-binding polypeptide to form a closed loop; and Where n is 3-12.

[0561] Implementation scheme 307. A multivalent multispecific construct as described in implementation scheme 306, wherein n is 3, 4, 5, 6, 7, 8, 9 or 10.

[0562] Implementation Scheme 308. A multivalent and multispecific construct as described in any one of Implementation Schemes 306 to 307, wherein ABD C, ABD B and / or ABD A are VHH, VNAR, Fab, ScFv, nanobody, biantibody, synthetic microbody, ligand or its binding fragment.

[0563] Implementation Scheme 309. A multivalent and multispecific construct as described in any one of Implementation Schemes 306 to 308, wherein ABD C, ABD B and / or ABD A are conditional binders, optionally wherein the binding of ABD C, ABD B and / or ABD A to their respective targets depends on pH, calcium or ATP levels.

[0564] Implementation Scheme 310. A multivalent and multispecific construct as described in any one of Implementation Schemes 306 to 309, wherein ABD C, ABD B and / or ABD A target a specific cell type.

[0565] Implementation Scheme 311. A multivalent and multispecific construct as described in any one of Implementation Schemes 306 to 310, wherein ABD C, ABD B and / or ABD A target cancer cells or immune cells.

[0566] Implementation Scheme 312. A multivalent multispecific construct as described in any one of Implementation Schemes 306 to 311, wherein ABD C, ABD B and / or ABD A promote the internalization of the multivalent multispecific construct by target cells, optionally by promoting lysosomal transport of the multivalent multispecific construct by target cells, optionally wherein lysosomal transport is clathrin-mediated endocytosis or receptor-mediated endocytosis.

[0567] Implementation Scheme 313. A multivalent and multispecific construct as described in any one of Implementation Schemes 306 to 312, wherein ABD C, ABD B and / or ABD A promote the chemotherapeutic sensitivity of target cells.

[0568] Implementation Scheme 314. A multivalent and multispecific construct as described in any one of Implementation Schemes 306 to 313, wherein ABD C, ABD B and / or ABD A promote apoptosis or necrosis of target cells.

[0569] Implementation Scheme 315. A multivalent and multispecific construct as described in any one of Implementation Schemes 306 to 314, wherein ABD C, ABD B and / or ABD A comprises at least one half-life extension molecule, optionally wherein the half-life extension molecule is an FcRn binding domain, albumin, PEG, XTEN or human serum albumin (HSA) binding domain.

[0570] Implementation Scheme 316. A multivalent multispecific construct as described in any one of Implementation Schemes 306 to 315, wherein ABD C, ABD B and / or ABD A comprises at least one site for conjugating a payload, optionally wherein at least one site comprises a Cys, Tyr or Lys residue.

[0571] Implementation Scheme 317. A multivalent and multispecific construct as described in any one of Implementation Schemes 306 to 316, wherein ABD C binds to a first target molecule, ABD B binds to a second target molecule, and / or ABD A binds to a third target molecule.

[0572] Implementation Scheme 318. A multivalent and multispecific construct as described in any one of Implementation Schemes 306 to 317, wherein ABD C binds to a first epitope on a first target molecule, ABD B binds to a second epitope on a first target molecule, and / or ABD A binds to a third epitope on a first target molecule.

[0573] Implementation Scheme 319. A multivalent and multispecific construct as described in any one of Implementation Schemes 306 to 318, wherein ABD C is used as a primary anchor for binding to target cells.

[0574] Implementation scheme 320. A multivalent and multispecific construct as described in any one of embodiments 306 to 319, wherein ABD B can bind substantially stably to its target only when ABD C binds to its target and / or ABD A binds to its target.

[0575] Implementation Scheme 321. A multivalent and multispecific construct as described in any one of Implementation Schemes 306 to 320, wherein ABD A is used as a secondary anchor for binding to target cells.

[0576] Implementation scheme 322. A multivalent multispecific construct as described in any one of implementation schemes 306 to 321, wherein ABD C binds to one or more of NaPi2b, Trop2, MUC1, Ly6G6D, Claudin18.2, B7H3, BCMA, CD22 and Her2.

[0577] Implementation scheme 323. A multivalent multispecific construct as described in any one of implementation schemes 306 to 322, wherein ABD B binds to one or more of EGFR, PDL1, CEA, FolRa, DR5, MUC1, mesothelin, Claudin18.2, CEACAM5, CD38, and CD19.

[0578] Implementation scheme 324. A multivalent multispecific construct as described in any one of implementation schemes 306 to 323, wherein ABD A binds to one or more of ALPL2, PRLR, integrin, DR5, CD20, FcRn, EGFR, Trop2, Claudin18.2, FcRH5 and CD79b.

[0579] Implementation scheme 325. A multivalent multispecific construct as described in any one of implementation schemes 306 to 324, wherein one of ABD C, ABD B and ABD A binds Her2, and wherein one of ABD C, ABD B and ABD A binds Trop2.

[0580] Implementation scheme 326. A multivalent and multispecific construct as described in any one of embodiments 306 to 325, wherein ABD C binds NaPi2b, ABD B binds MUC1, and ABD A binds mesothelin.

[0581] Implementation Scheme 327. A multivalent multispecific construct as described in Implementation Scheme 326, wherein the multivalent multispecific construct is suitable for the treatment of ovarian cancer or NSCLC.

[0582] Implementation scheme 328. A multivalent multispecific construct as described in any one of implementation schemes 306 to 325, wherein ABD C binds to B7H3, ABD B binds to PDL1, and ABD A binds to Trop2.

[0583] Implementation Scheme 329. A multivalent multispecific construct as described in Implementation Scheme 328, wherein the multivalent multispecific construct is suitable for the treatment of NSCLC.

[0584] Implementation scheme 330. A multivalent and multispecific construct as described in any one of implementation schemes 306 to 325, wherein ABD C binds to Ly6G6D, ABD B binds to CEACAM5, and ABD A binds to Claudin18.2.

[0585] Implementation Scheme 331. The multivalent multispecific construct as described in Implementation Scheme 330, wherein the multivalent multispecific construct is suitable for treating GI cancers.

[0586] Implementation scheme 332. A multivalent multispecific construct as described in any one of implementation schemes 306 to 325, wherein ABD C binds to BCMA, ABD B binds to CD38, and ABD A binds to FcRH5.

[0587] Implementation Scheme 333. The multivalent multispecific construct as described in Implementation Scheme 332, wherein the multivalent multispecific construct is suitable for the treatment of myeloma.

[0588] Implementation scheme 334. A multivalent multispecific construct as described in any one of implementation schemes 306 to 325, wherein ABD C binds to CD22, ABD B binds to CD19, and ABD A binds to CD79b.

[0589] Implementation Scheme 335. A multivalent multispecific construct as described in Implementation Scheme 334, wherein the multivalent multispecific construct is suitable for the treatment of NHL or autoimmune diseases.

[0590] Implementation scheme 336. A multivalent multispecific construct as described in any one of implementation schemes 306 to 325, wherein ABD C binds Her2, ABD B binds MUC1, and ABD A binds Trop2.

[0591] Implementation Scheme 337. The multivalent multispecific construct as described in Implementation Scheme 336, wherein the multivalent multispecific construct is suitable for the treatment of breast cancer.

[0592] Implementation scheme 338. A multivalent multispecific construct as described in any one of implementation schemes 306 to 337, wherein the lengths of L1, L2, L3 and / or L4 are about 15-30 aa.

[0593] Implementation Scheme 339. A multivalent multispecific construct as described in any one of Implementation Schemes 306 to 338, wherein L1, L2, L3 and / or L4 comprises one or more of Gly, Ser, Thr, Pro, Arg, Phe, Glu, Cys, Asn, Ala, Asp and Gln residues, optionally wherein L1, L2, L3 and / or L4 primarily comprises Gly, Ser and Thr residues.

[0594] Implementation scheme 340. A multivalent multispecific construct as described in any one of implementation schemes 306 to 339, wherein L1, L2, L3 and / or L4 primarily comprise Gly and Ser residues.

[0595] Implementation Scheme 341. A multivalent multispecific construct as described in Implementation Scheme 340, wherein L1, L2, L3 and / or L4 comprise (Gly-Ser) n (Gly4-Ser) n (SEQ ID NO: 1), (Gly3-Ser) n(SEQ ID NO: 2), Gly-Gly-Ser, Gly-Gly-Ser-Gly (SEQ ID NO: 3), Gly-Gly-Gly-Gly (SEQ ID NO: 4), Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 5), Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 5) NO: 6), Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 7), Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 8), Gly-Ser-Gly-Ser-Gly-Ser (SEQ ID NO: 9), (Gly4-Ser)3 (SEQ ID NO: 10), (Gly2-Ser)3 (SEQ ID NO: 11), (Gly2-Ser)2 (SEQ ID NO: 12), ferritin E-helix, (Glu-Ala-Ala-Ala-Lys) 1-3 (SEQ ID NO:13), (Ala-Pro) n (SEQ ID NO: 14), IgG C H 1. IgG C H 2-C H 3 or Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 15).

[0596] Implementation Scheme 342. The multivalent multispecific construct as described in any one of Implementation Schemes 306 to 341, wherein L1, L2, L3 and / or L4 are hydrophilic, and optionally wherein L1, L2, L3 and / or L4 have a hydrophobicity of about 0.6.

[0597] Implementation Scheme 343. A multivalent and multispecific construct as described in any one of Implementation Schemes 306 to 342, wherein L1, L2, L3 and / or L4 are hydrophilic, optionally wherein L1, L2, L3 and / or L4 have a hydrophobicity of about 0.4 to about 0.8, optionally wherein L1, L2, L3 and / or L4 have a hydrophobicity of about 0.5 to about 0.7, optionally wherein L1, L2, L3 and / or L4 have a hydrophobicity of about 0.6 to about 0.65.

[0598] Implementation scheme 344. A multivalent and multispecific construct as described in any one of implementation schemes 306 to 343, wherein L1, L2, L3 and / or L4 are substantially non-immunogenic.

[0599] Implementation scheme 345. A multivalent multispecific construct as described in any one of implementation schemes 306 to 344, wherein L1, L2, L3 and / or L4 are substantially non-cleavable.

[0600] Implementation scheme 346. A multivalent multispecific construct as described in any one of embodiments 306 to 345, wherein L1, L2, L3 and / or L4 are substantially cleavable.

[0601] Implementation Scheme 347. A multivalent and multispecific construct as described in any one of Implementation Schemes 306 to 346, wherein the lengths of L1, L2, L3 and / or L4 are about 5-10 nm; optionally, the lengths are about 7 nm.

[0602] Implementation scheme 348. A multivalent multispecific construct as described in any one of embodiments 306 to 347, wherein L3 is a rigid connector with a length of about 7-12 nm and / or contains an α-helix.

[0603] Implementation Scheme 349. A multivalent multispecific construct as described in any one of Implementation Schemes 306 to 348, wherein L2 is a moderately rigid linker with a length of about 7-12 nm, and / or comprises b2-microglobulin, Zn-a2-glycoprotein, 34-peptide repeat sequence or non-α-helix with moderate bending and rotational rigidity, optionally wherein L2 is a cleavable linker.

[0604] Implementation Scheme 350. A multivalent multispecific construct as described in any one of Implementation Schemes 306 to 349, wherein L1 is a flexible linker with a length of about 7-12 nm and / or contains (Gly-Ser). n (Gly4-Ser) n (SEQ ID NO:1), (Gly3-Ser) n(SEQ ID NO: 2), Gly-Gly-Ser, Gly-Gly-Ser-Gly (SEQ ID NO: 3), Gly-Gly-Gly-Gly (SEQ ID NO: 4), Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 5), Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 6), Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 7), Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 8), Gly-Ser-Gly-Ser-Gly-Ser (SEQ ID NO: 9), (Gly4-Ser)3 (SEQ ID NO:10), (Gly2-Ser)3 (SEQ ID NO: 11), (Gly2-Ser)2 (SEQ ID NO: 12), ferritin E-helix, (Glu-Ala-Ala-Ala-Lys) 1-3 (SEQ ID NO: 13), (Ala-Pro) n (SEQ ID NO: 14), IgG C H 1. IgG C H 2-C H 3 or Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 15).

[0605] Implementation scheme 351. A multivalent multispecific construct as described in any one of implementation schemes 306 to 350, wherein the joints are L4, L3, L2 and L1 in order of strongest to weakest rigidity.

[0606] Implementation scheme 352. A multivalent and multispecific construct as described in any one of implementation schemes 306 to 351, wherein the scaffold region comprises a rigid protein.

[0607] Implementation Scheme 353. A multivalent and multispecific construct as described in any one of Implementation Schemes 306 to 352, wherein the scaffold region includes a portion for extending PK, optionally wherein the portion for extending PK is an FcRn binding domain, IgGC H 2-C H 3. Human serum albumin, human transferrin, or fragments thereof.

[0608] Implementation Scheme 354. A multivalent and multispecific construct as described in any one of Implementation Schemes 306 to 353, wherein the scaffold region comprises IgG C H 2-C H 3 or a fragment thereof, optionally including IgG C H 2-C H 3 further includes a second hinge area, optionally wherein the support area includes hinge-C H 2-C H 3-Hinge.

[0609] Implementation scheme 355. A multivalent multispecific construct as described in any one of implementation schemes 306 to 354, wherein the payload is conjugated to Cys, Tyr or Lys residues in the scaffold region.

[0610] Implementation Scheme 356. A multivalent multispecific construct as described in any one of Implementation Schemes 306 to 355, wherein the payload is conjugated to the multivalent multispecific construct using TCEP reduction, transglutaminase, random conjugation, glycan conjugation, lysine conjugation, formylglycine synthase (FGE) oxidation, AjiCap technology, or click chemistry.

[0611] Implementation Scheme 357. A multivalent and multispecific construct as described in any one of Implementation Schemes 306 to 356, wherein the payload comprises a protein, optionally wherein the protein comprises an enzyme, an immunostimulant, an immunosuppressant, or a topoisomerase.

[0612] Implementation Scheme 358. A multivalent and multispecific construct as described in any one of Implementation Schemes 306 to 356, wherein the payload comprises a small molecule, optionally comprising a small molecule inhibitor, monomethylaurestatin E (MMAE), monomethylaurestatin F (MMAF), muscarinic acid, PNU, beloteccan, ethanotecan, pyrrolobenzodiazepines (PBD), trastuzumab emtansine (T-DM1), steroids, chemotherapy, BCL-XL inhibitors, or STING agonists.

[0613] Implementation Scheme 359. A multivalent and multispecific construct as described in any one of Implementation Schemes 306 to 356, wherein the payload comprises an RNA molecule, optionally wherein the RNA molecule is a siRNA molecule.

[0614] Implementation scheme 360. A multivalent and multispecific construct as described in any one of implementation schemes 306 to 356, wherein the payload comprises a chemical sensitizer.

[0615] Implementation scheme 361. A multivalent and multispecific construct as described in any one of implementation schemes 306 to 356, wherein the payload comprises a gene modifier.

[0616] Implementation Scheme 362. The multivalent multispecific construct as described in any one of Implementation Schemes 306 to 356, wherein the payload is a beloteccan derivative attached to the scaffold region using a cathepsin-cleavable linker.

[0617] Implementation Scheme 363. A multivalent multispecific construct as described in any one of Implementation Schemes 306 to 362, wherein the multivalent multispecific construct comprises more than one payload, optionally wherein the drug-to-antibody ratio (DAR) is about 2-8.

[0618] Implementation Scheme 364. A multivalent multispecific construct as described in any one of Implementation Schemes 306 to 363, wherein the multivalent multispecific construct further comprises one or more enhancer domains and / or one or more FcRn binding domains, optionally wherein one or more FcRn binding domains comprise IgG FcC H 2-C H 3. Structural domain or VHH structural domain.

[0619] Implementation scheme 365. The multivalent multispecific construct as described in any one of implementation schemes 306 to 364, wherein the diameter of the multivalent multispecific construct is about 5-50 nm, about 10-40 nm, about 20-30 nm, about 20-25 nm or about 25-30 nm.

[0620] Implementation Scheme 366. A multivalent and multispecific construct as described in any one of Implementation Schemes 306 to 365, wherein each antigen-binding polypeptide contains a terminal suitable for covalent or non-covalent cyclization or subunit assembly within the generating cell.

[0621] Implementation Scheme 367. A multivalent multispecific construct as described in any one of Implementation Schemes 306 to 366, wherein the multivalent multispecific construct (i) delivers the payload to target cells more efficiently than a monomer or single-specific binder having only one copy of ABD C, ABD B, or ABD A, (ii) is internalized by target cells at a higher rate than a monomer or single-specific binder having only one copy of ABD C, ABD B, or ABD A, and / or (iii) has improved specificity for binding to and / or killing tumor cells than a monomer or single-specific binder having only one copy of ABD C, ABD B, or ABDA.

[0622] Implementation scheme 368. A nucleic acid encoding a multivalent and multispecific construct as described in any one of implementation schemes 306 to 367.

[0623] Implementation Scheme 369. A host cell comprising the nucleic acid described in Implementation Scheme 368.

[0624] Implementation Scheme 370. A pharmaceutical composition comprising any one of the multivalent and multispecific constructs of Implementation Schemes 306 to 367, the nucleic acid of Implementation Scheme 368, or the host cell of Implementation Scheme 369.

[0625] Implementation Scheme 371. A method for treating a disease or condition in a subject in need, optionally wherein the disease or condition is cancer, said method comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 306 to 367, a nucleic acid as described in Implementation Scheme 368, a host cell as described in Implementation Scheme 369, or a pharmaceutical composition as described in Implementation Scheme 370.

[0626] Implementation scheme 372. The method as described in implementation scheme 371, wherein the cancer is a solid tumor.

[0627] Implementation scheme 373. The method as described in implementation scheme 372, wherein the cancer is NSCLC, ovarian cancer, breast cancer, pancreatic cancer, colorectal cancer, or gastric cancer.

[0628] Implementation scheme 374. The method as described in implementation scheme 371, wherein the cancer is a blood cancer.

[0629] Implementation scheme 375. The method as described in implementation scheme 374, wherein the cancer is leukemia, lymphoma, or multiple myeloma.

[0630] Implementation Scheme 376. A method of treating a subject with ovarian cancer or NSCLC, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 306 to 370, wherein ABD C binds NaPi2b, ABD B binds MUC1, and ABD A binds mesothelin.

[0631] Implementation Scheme 377. A method of treating NSCLC in a subject of need, comprising administering a multivalent multispecific construct according to any one of Implementation Schemes 306 to 370, wherein ABD C binds to B7H3, ABD B binds to PDL1, and ABD A binds to Trop2.

[0632] Implementation Scheme 378. A method of treating a subject with GI cancer, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 306 to 370, wherein ABD C binds to Ly6G6D, ABD B binds to CEACAM5, and ABD A binds to Claudin18.2.

[0633] Implementation Scheme 379. A method of treating a subject with myeloma in need, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 306 to 370, wherein ABD C binds to BCMA, ABD B binds to CD38, and ABD A binds to FcRH5.

[0634] Implementation Scheme 380. A method of treating a subject with NHL or an autoimmune disease, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 306 to 370, wherein ABD C binds to CD22, ABD B binds to CD19, and ABD A binds to CD79b.

[0635] Implementation Scheme 381. A method of treating breast cancer in a subject of need, comprising administering a multivalent multispecific construct as described in any one of Implementation Schemes 306 to 370, wherein ABD C binds Her2, ABD B binds MUC1, and ABD A binds Trop2.

[0636] Implementation Scheme 382. A method for manufacturing a multivalent multispecific construct according to any one of Implementation Schemes 1 to 66, 81 to 141, 156 to 216, 231 to 291 and 306 to 367, comprising (b) culturing cells expressing the multivalent multispecific construct; and (b) isolating the multivalent multispecific construct from the cultured cells.

[0637] Equivalent solution

[0638] Although the present invention has been disclosed in conjunction with specific embodiments thereof, it should be understood that the invention is capable of other modifications, and this application is intended to cover any variations, uses or alterations that generally follow the principles of the invention, including well-known or commonly used techniques that, although not within the scope of the present invention, fall within the field of the present invention and can be applied to the essential features set forth above and the appended claims.

[0639] Those skilled in the art, using only conventional experiments, will recognize or be able to determine numerous equivalents of the specific embodiments precisely disclosed herein. Such equivalents are intended to be covered within the scope of the appended claims.

Claims

1. A multivalent and multispecific construct comprising two or more antigen-binding peptides and a conjugated therapeutic payload, wherein each of the two or more antigen-binding peptides comprises three or more antigen-binding domains, wherein each of the three or more antigen-binding domains of each of the two or more antigen-binding peptides is capable of binding to a respective target on its target cell, wherein the construct is capable of simultaneously binding to four or more targets on the surface of the target cell.

2. The multivalent and multispecific construct of claim 1, wherein the construct comprises: (a) Therapeutic payload; and (b) Two or more antigen-binding peptides targeting two or more different targets, each of the antigen-binding peptides comprising, from the N-terminus to the C-terminus, a first antigen-binding domain (ABD C), a first linker (L1), a second antigen-binding domain (ABD B), a second linker (L2), a third antigen-binding domain (ABD A), an optional third linker (L3), and a scaffold region. Wherein L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, and L3 optionally attaches ABD A to the scaffold region, such that each antigen-binding polypeptide comprises (i) ABD C – L1 – ABD B – L2 – ABD A – scaffold region or (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region arranged from N-terminus to C-terminus; and The scaffold region comprises a loop, a central protein anchor, or IgG Fc or a portion thereof or a modified form thereof.

3. The multivalent and multispecific construct of claim 2, wherein the scaffold region comprises IgG Fc or a portion thereof or a modified form thereof, wherein the construct comprises two antigen-binding peptides of the antigen-binding peptides, wherein each of the two antigen-binding peptides further comprises (i) ABD A – L2 – ABD B – L1 – ABD C arranged from N-terminus to C-terminus, such that each antigen-binding peptide comprises ABD C – L1 – ABD B – L2 – ABD A – scaffold region – ABD A – L2 – ABD B – L1 – ABD C, or (ii) L3 – ABD A – L2 – ABD B – L1 – ABD C arranged from N-terminus to C-terminus, such that each antigen-binding peptide comprises ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region – L3 – ABD A – L2 – ABD B – L1 – ABD C, wherein the N-terminal and C-terminal ABD C domains are the same or different, wherein the N-terminal and C-terminal ABD C domains are... The B structural domains are the same or different, and the N-terminal and C-terminal ABD structural domains are the same or different.

4. The multivalent and multispecific construct of claim 2, wherein the scaffold region comprises a rigid polypeptide; wherein the construct comprises 'n' of the antigen-binding polypeptides; wherein each of the antigen-binding polypeptides further comprises a fourth linker (L4), wherein L4 attaches the scaffold region to ABD C; and wherein the scaffold region further comprises a clasp domain, the clasp domain attaching the first antigen-binding polypeptide and the nth antigen-binding polypeptide to form a closed loop, wherein n is 3-12.

5. A multivalent and multispecific construct comprising: (a) Therapeutic payload; and (b) Two antigen-binding peptides targeting two or more different targets, each of the antigen-binding peptides comprising, from the N-terminus to the C-terminus, a first antigen-binding domain (ABD C), a first linker (L1), a second antigen-binding domain (ABDB), a second linker (L2), a third antigen-binding domain (ABD A), an optional third linker (L3), a scaffold region, an optional second L3, a second ABD A, a second L2, a second ABD B, a second L1, and a second ABD C; Wherein L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, and L3 optionally attaches ABD A to the scaffold region, such that each antigen-binding polypeptide comprises, from the N-terminus to the C-terminus, (i) ABD C – L1 – ABD B – L2 – ABD A – scaffold region – ABD A – L2 – ABD B – L1 – ABD C or (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region – L3 – ABD A – L2 – ABD B – L1 – ABD C; and The scaffold region contains IgG Fc or a portion thereof or a modified form thereof, wherein the N-terminal and C-terminal ABD C domains are the same or different, wherein the N-terminal and C-terminal ABD B domains are the same or different, and wherein the N-terminal and C-terminal ABD A domains are the same or different.

6. A multivalent and multispecific construct comprising: (a) Therapeutic payload; and (b) Two antigen-binding peptides targeting two or more different targets, each of the antigen-binding peptides comprising, from the N-terminus to the C-terminus, a first antigen-binding domain (ABD C), a first linker (L1), a second antigen-binding domain (ABDB), a second linker (L2), a third antigen-binding domain (ABD A), an optional third linker (L3), and a scaffold region. Wherein L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, and L3 optionally attaches ABD A to the scaffold region, such that each antigen-binding polypeptide comprises (i) ABD C – L1 – ABD B – L2 – ABD A – scaffold region or (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region arranged from N-terminus to C-terminus; and The scaffold region contains IgG Fc or a portion thereof or a modified form thereof.

7. A multivalent and multispecific construct comprising: (a) Therapeutic payload; and (b) Four antigen-binding peptides targeting two or more different targets, each of the antigen-binding peptides comprising, from the N-terminus to the C-terminus, a first antigen-binding domain (ABD C), a first linker (L1), a second antigen-binding domain (ABDB), a second linker (L2), a third antigen-binding domain (ABD A), an optional third linker (L3), and a scaffold region. Wherein L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, and L3 optionally attaches ABD A to the scaffold region, such that each antigen-binding polypeptide comprises (i) ABD C – L1 – ABD B – L2 – ABD A – scaffold region or (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region arranged from N-terminus to C-terminus; and The scaffold region includes a central protein anchor, and optionally the central protein anchor includes an FcRn binding domain.

8. A multivalent and multispecific construct comprising: (a) Therapeutic payload; and (b) n antigen-binding peptides targeting two or more different targets, each of the antigen-binding peptides comprising, from the N-terminus to the C-terminus, a first antigen-binding domain (ABD C), a first linker (L1), a second antigen-binding domain (ABDB), a second linker (L2), a third antigen-binding domain (ABD A), an optional third linker (L3), a scaffold region, and a fourth linker (L4). Wherein L1 attaches ABD C to ABD B, L2 attaches ABD B to ABD A, L3 optionally attaches ABD A to the scaffold region, and L4 attaches the scaffold region to ABD C, such that each antigen-binding polypeptide comprises (i) ABD C – L1 – ABD B – L2 – ABD A – scaffold region – L4 or (ii) ABD C – L1 – ABD B – L2 – ABD A – L3 – scaffold region – L4, arranged from N-terminus to C-terminus. The scaffold region contains a rigid polypeptide; The construct further comprises a clasp domain that attaches a first antigen-binding polypeptide and an nth antigen-binding polypeptide to form a closed loop; and Where n is 3-12.

9. The multivalent and multispecific construct according to any one of claims 2 to 8, wherein ABD C, ABD B and / or ABDA are VHH, VNAR, Fab, ScFv, nanobody, biantibody, synthetic microbody, ligand or its binding fragment.

10. The multivalent and multispecific construct according to any one of claims 2 to 9, wherein ABD C, ABD B and / or ABDA are conditional binders, optionally wherein the binding of ABD C, ABD B and / or ABD A to their respective targets depends on pH, calcium or ATP levels.

11. The multivalent and multispecific construct according to any one of claims 2 to 10, wherein ABD C, ABD B and / or ABD A target a specific cell type, optionally wherein ABD C, ABD B and / or ABD A target cancer cells or immune cells.

12. The multivalent multispecific construct of any one of claims 2 to 11, wherein ABD C, ABD B and / or ABD A promote the internalization of the multivalent multispecific construct by the target cells, optionally by promoting lysosomal transport of the multivalent multispecific construct by the target cells, optionally wherein the lysosomal transport is clathrin-mediated endocytosis or receptor-mediated endocytosis.

13. The multivalent and multispecific construct of any one of claims 2 to 12, wherein ABD C, ABD B and / or ABD A promote the chemotherapeutic sensitivity of the target cells, and / or wherein ABD C, ABD B and / or ABD A promote apoptosis or necrosis of the target cells.

14. The multivalent and multispecific construct according to any one of claims 2 to 13, wherein ABD C, ABD B and / or ABD A comprises at least one half-life extension molecule, optionally wherein the half-life extension molecule is an FcRn binding domain, albumin, PEG, XTEN or human serum albumin (HSA) binding domain.

15. The multivalent and multispecific construct according to any one of claims 2 to 13, wherein the scaffold region comprises IgGC. H 2-C H 3 or a fragment thereof, optionally wherein said IgG C H 2-C H 3 further includes a second hinge area, optionally wherein the support area includes a hinge-C H 2-C H 3-Hinge.

16. The multivalent multispecific construct of any one of claims 2 to 15, wherein ABD C, ABD B, ABD A, L1, L2, L3, L4, hinge region, second hinge region and / or the scaffold region comprises at least one site for conjugating the payload, optionally wherein the at least one site comprises Cys, Tyr or Lys residues or engineered glycans or tags for site-specific conjugation.

17. The multivalent and multispecific construct of any one of claims 2 to 16, wherein ABD C binds to a first target molecule, ABD B binds to a second target molecule, and / or ABD A binds to a third target molecule, optionally wherein the N-terminal ABD C binds to the first target molecule, the N-terminal ABD B binds to the second target molecule, the N-terminal ABD A binds to the third target molecule, the C-terminal ABD C binds to a fourth target molecule, the C-terminal ABD B binds to a fifth target molecule, and / or the C-terminal ABD A binds to a sixth target molecule.

18. The multivalent and multispecific construct according to any one of claims 2 to 17, wherein ABD C binds to a first epitope on the first target molecule, ABD B binds to a second epitope on the first target molecule, and / or ABD A binds to a third epitope on the first target molecule.

19. The multivalent multispecific construct according to any one of claims 2 to 18, wherein the lengths of L1, L2, L3 and / or L4 are about 15-30 aa.

20. The multivalent multispecific construct according to any one of claims 2 to 19, wherein L1, L2, L3 and / or L4 comprises one or more of Gly, Ser, Thr, Pro, Arg, Phe, Glu, Cys, Asn, Ala, Asp and Gln residues, optionally wherein L1, L2, L3 and / or L4 primarily comprises Gly, Ser and Thr residues.

21. The multivalent and multispecific construct according to any one of claims 2 to 20, wherein L1, L2, L3 and / or L4 primarily comprise Gly and Ser residues.

22. The multivalent multispecific construct of claim 21, wherein L1, L2, L3 and / or L4 comprise (Gly-Ser) n , (Gly4-Ser) n (SEQ ID NO: 1), (Gly3-Ser) n (SEQ ID NO: 2), Gly-Gly-Ser, Gly-Gly-Ser-Gly (SEQ ID NO: 3), Gly-Gly-Gly-Gly (SEQ ID NO: 4), Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 5), Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 6), Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 7), Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 8), Gly-Ser-Gly-Ser-Gly-Ser (SEQ ID NO:9), (Gly4-Ser)3 (SEQ ID NO: 10), (Gly2-Ser)3 (SEQ ID NO: 11), (Gly2-Ser)2 (SEQ ID NO: 12), ferritin E helix, (Glu-Ala-Ala-Ala-Lys) 1-3 (SEQ ID NO: 13), (Ala-Pro) n (SEQ ID NO: 14), IgG C H 1, IgG C H 2-C H 3, Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 15) or Gly-Gly-Ser-Asn-Gly-Ser-Gly-Gly-Ser (SEQ ID NO: 28).

23. The multivalent and multispecific construct according to any one of claims 2 to 22, wherein the scaffold region comprises a rigid protein.

24. The multivalent and multispecific construct according to any one of claims 2 to 23, wherein the scaffold region includes a portion for extending PK, optionally wherein the portion for extending PK is an FcRn binding domain, IgG C H 2-C H 3. Human serum albumin, human transferrin, or fragments thereof.

25. The multivalent multispecific construct according to any one of claims 2 to 24, wherein the multivalent multispecific construct (i) delivers the payload to the target cell more efficiently than a monomer or single-specific binder having only one copy of ABD C, ABD B, or ABD A, (ii) is internalized by the target cell at a higher rate than a monomer or single-specific binder having only one copy of ABD C, ABD B, or ABD A, and / or (iii) has improved specificity for binding to and / or killing tumor cells than a monomer or single-specific binder having only one copy of ABD C, ABD B, or ABD A.

26. A nucleic acid encoding a multivalent and multispecific construct according to any one of claims 1 to 25.

27. A host cell comprising the nucleic acid of claim 26.

28. A pharmaceutical composition comprising the multivalent and multispecific construct of any one of claims 1 to 25, the nucleic acid of claim 26, or the host cell of claim 27.

29. A method of treating a disease or condition in a subject in need, optionally wherein the disease or condition is cancer, the method comprising administering a multivalent multispecific construct of any one of claims 1 to 25, a nucleic acid of claim 26, a host cell of claim 27, or a pharmaceutical composition of claim 28.

30. The method of claim 29, wherein the cancer is a solid tumor, optionally wherein the cancer is NSCLC, ovarian cancer, breast cancer, pancreatic cancer, colorectal cancer, or gastric cancer.

31. The method of claim 29, wherein the cancer is a blood cancer, optionally wherein the cancer is leukemia, lymphoma, or multiple myeloma.

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