DLL3 antigen binding constructs

Abstract

Provided herein are components of antigen binding constructs that include antibodies and fragments thereof, such as microantibodies and cys-diantibodies, that bind to a target molecule, e.g., DLL3. In some embodiments, these components are novel complementarity determining region (CDR) sequences and / or sequences related to CDR sequences and / or parts of CDR sequences. In some embodiments, these components are novel framework region (FR) sequences and / or sequences related to FR sequences and / or parts of FR sequences. These CDR and FR sequences can provide a variety of benefits. Also provided herein are antigen binding constructs (e.g., microantibodies, cys-diantibodies, etc.) comprising one or more CDR or FR sequences or subsequences provided herein.

Description

[0001] Incorporation by reference of any priority application

[0002] Any and all applications that identify foreign or domestic priority claims in the Application Data Sheet filed with the present application are incorporated herein by reference pursuant to 37 CFR 1.57.

[0003] Sequence Listing Reference

[0004] The present application is submitted with a sequence listing in electronic format. The sequence listing is provided as a file titled Seqlist_IGNAB059WO.xml, which was created and last modified on June 30, 2023, and has a size of 155593 bytes. The information in the electronic sequence listing is incorporated herein by reference in its entirety. Technical Field

[0005] Embodiments herein relate to antigen-binding constructs, minibodies and cys-diabodies. Specifically, DLL3-specific antigen-binding constructs, minibodies and cys-diabodies. Background Art

[0006] Delta-like protein 3 (DLL3) is an inhibitory protein of the Notch signaling pathway. DLL3 is expressed on the cell surface of small cell lung cancer (SCLC) and other high-grade endocrine tumors. Summary of the invention

[0007] Embodiments herein relate to antigen binding constructs, minibodies and cys-diabodies. Some embodiments are DLL3-specific antigen binding constructs, minibodies and cys-diabodies. Some embodiments herein relate to therapeutic agents, formulations and compositions comprising D113-specific antigen binding constructs, minibodies and cys-diabodies. Some embodiments herein relate to methods of treating DLL3-related diseases or disorders using the antigen binding constructs, minibodies and cys-diabodies disclosed herein.

[0008] Provided herein are antigen-binding constructs comprising: a light chain variable (V L ) domain: LCDR1 as SEQ ID NO: 15; LCDR2 as any one of SEQ ID NO: 19, 21 or 23; LCDR3 as SEQ ID NO: 25; and / or a heavy chain variable (V H ) domains: HCDR1 as SEQ ID NO: 27; HCDR2 as SEQ ID NO: 29; and HCDR3 as SEQ ID NO: 31.

[0009] Provided herein are minibodies that bind to DLL3, the minibodies comprising: a single chain variable fragment (scFv) that binds to DLL3; a hinge-extension domain comprising a hinge region; and an IgG C H 3 sequences, the scFv comprising a heavy chain variable (V H ) domain connected to the light chain variable (V L ) domain, the V L The domain comprises: LCDR1 as SEQ ID NO: 15; LCDR2 as any one of SEQ ID NO: 19, 21 or 23; LCDR3 as SEQ ID NO: 25; and the V H The domain comprises: HCDR1 as SEQ ID NO:27; HCDR2 as SEQ ID NO:29; HCDR3 as SEQ ID NO:31.

[0010] Provided herein are cys-diabodies that bind to DLL3, the cys-diabodies comprising a polypeptide comprising: a single chain variable fragment (scFv) comprising a heavy chain variable (V H ) domain connected to the light chain variable (V L ) domain; and said V L The domain comprises: LCDR1 as SEQ ID NO: 15; LCDR2 as any one of SEQ ID NO: 19, 21 or 23; LCDR3 as SEQ ID NO: 25; and the V H The domain comprises: HCDR1 as SEQ ID NO:27; HCDR2 as SEQ ID NO:29; and HCDR3 as SEQ ID NO:31.

[0011] According to any one of the above embodiments of the antigen-binding construct, minibody or cys-diabody, wherein the V L The domain further comprises LFR2 shown in SEQ ID NO:35 or 37.

[0012] According to any one of the above embodiments of the antigen-binding construct, miniantibody or cys-diabody; wherein the V L The domain comprises the same V as in SEQ ID NO: 48 L The domains have an amino acid sequence that is at least 90% identical; and wherein the antigen-binding construct, minibody or cys-diabody comprises a Y50D and / or Y55N mutation (Kabat).

[0013] According to any one of the above embodiments of the antigen-binding construct, miniantibody or cys-diabody; wherein the V L The domain comprises the same V as in SEQ ID NO: 48 L The domains have an amino acid sequence that is at least 90% identical; and wherein the antigen-binding construct, minibody or cys-diabody comprises an A43S and / or Q45R mutation (Kabat).

[0014] According to any one of the above embodiments of the antigen-binding construct, miniantibody or cys-diabody; wherein the V L The domain comprises the same V as in SEQ ID NO: 49 L The domains have an amino acid sequence that is at least 90% identical, and wherein the antigen-binding construct, minibody or cys-diabody comprises a Y50D and / or Y55N mutation (Kabat).

[0015] According to any one of the above embodiments of the antigen-binding construct, miniantibody or cys-diabody; wherein the V L The domain comprises the same V as in SEQ ID NO: 49 L The domains have an amino acid sequence that is at least 90% identical; and wherein the antigen-binding construct, minibody or cys-diabody comprises an A43S and / or Q45R mutation (Kabat).

[0016] An antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments; wherein the antigen-binding construct, minibody or cys-diabody further comprises a signal peptide as SEQ ID NO: 45.

[0017] An antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments; wherein the antigen-binding construct, minibody or cys-diabody comprises a linker that is any one of SEQ ID NOs: 39-44.

[0018] An antigen-binding construct or minibody according to any one of the above embodiments; wherein said C H The 3 domain is IgG C H 3 domains.

[0019] An antigen-binding construct or minibody according to any one of the above embodiments; wherein the IgG C H 3 domains are IgG1, IgG2, IgG3 or IgG4 C H 3 domains.

[0020] An antigen-binding construct or minibody according to any one of the above embodiments; wherein the IgG C H The 3 domain comprises an IgG1 C sequence as any one of SEQ ID NOs: 65-68 H 3 domains.

[0021] An antigen-binding construct or minibody according to any one of the above embodiments; wherein the IgG C H The 3 domain comprises an IgG1 C sequence as any one of SEQ ID NOs: 69-71. H 3 domains.

[0022] An antigen-binding construct or minibody according to any one of the above embodiments; wherein the IgG C H The 3 domain comprises an IgG1 C sequence as any one of SEQ ID NOs: 72-82. H 3 domains.

[0023] An antigen-binding construct or minibody according to any one of the above embodiments; wherein the IgG C H The 3 domain comprises an IgG1 C sequence as any one of SEQ ID NOs: 83-85. H 3 domains.

[0024] The antigen-binding construct according to any one of the above embodiments, wherein the antigen-binding construct is an antibody.

[0025] The antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the antigen-binding construct, minibody or cys-diabody specifically binds to DLL3.

[0026] The antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments further comprises a detectable marker.

[0027] The antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the detectable marker is a fluorescent detectable marker.

[0028] An antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the detectable marker comprises a phototherapy compatible dye.

[0029] An antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the detectable marker is compatible with use using boron neutron capture therapy (BNCT).

[0030] The antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the detectable marker is a radiolabel.

[0031] The antigen binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the detectable marker is an alpha emitter radiolabel.

[0032] The antigen binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the detectable marker is a beta emitter radiolabel.

[0033] The antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the detectable marker is a positron-emitter radiolabel.

[0034] The antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the detectable marker is a gamma-emitter radiolabel.

[0035] The antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the detectable marker comprises lutetium-177.

[0036] The antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the detectable marker is suitable for use in Auger electron spectroscopy.

[0037] The antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the detectable marker comprises an isotope.

[0038] The antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the detectable marker comprises a bioluminescent compound.

[0039] The antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the detectable marker comprises a chemiluminescent compound.

[0040] The antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the detectable marker comprises an enzyme.

[0041] According to any one of the above embodiments of the antigen-binding construct, minibody or cys-diabody, wherein the detectable marker comprises a metal chelator.

[0042] The antigen-binding construct, minibody or cys-diabody according to any of the above embodiments further comprises a therapeutic agent.

[0043] An antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the therapeutic agent comprises a therapeutic isotope or ion.

[0044] An antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the therapeutic agent is a radiolabel.

[0045] An antigen binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the therapeutic agent is an alpha emitter radiolabel.

[0046] An antigen binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the therapeutic agent is a beta emitter radiolabel.

[0047] An antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the therapeutic agent is a positron emitter radiolabel.

[0048] An antigen binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the therapeutic agent is a gamma emitter radiolabel.

[0049] The antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the therapeutic agent comprises lutetium-177.

[0050] An antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the therapeutic agent comprises a phototherapy-compatible dye.

[0051] The antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the therapeutic agent comprises boron.

[0052] The antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the therapeutic agent is compatible with use with boron neutron capture therapy (BNCT).

[0053] An antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the detectable marker and / or therapeutic agent comprises a toxic payload.

[0054] The antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the antigen-binding construct, minibody or cys-diabody is bispecific.

[0055] The antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the antigen-binding construct, minibody or cys-diabody comprises a monovalent scFv.

[0056] According to any one of the above embodiments of the antigen-binding construct, miniantibody or cys-diabody, from the N-terminus to the C-terminus of the polypeptide is V L , V H .

[0057] According to any one of the above embodiments of the antigen-binding construct, miniantibody or cys-diabody, the order of the variable domains is V from the N-terminus to the C-terminus of the polypeptide. H , V L .

[0058] An antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the minibody is a humanized antigen-binding construct, minibody or cys-diabody.

[0059] An antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the humanized antigen-binding construct, minibody or cys-diabody comprises: LCDR1 as SEQ ID NO: 15; LCDR2 as any one of SEQ ID NO: 19, 21 or 23; LCDR3 as SEQ ID NO: 25; HCDR1 as SEQ ID NO: 27; HCDR2 as SEQ ID NO: 29; and HCDR3 as SEQ ID NO: 31.

[0060] According to any one of the above embodiments of the antigen-binding construct, minibody or cys-diabody, wherein the V L The domain also comprises LFR2 of LFR2 shown in SEQ ID NO:35 or 37.

[0061] An antigen-binding construct, miniantibody or cys-diabody according to any one of the above embodiments, wherein the antigen-binding construct, miniantibody or cys-diabody has an improved expression yield in mammalian cells compared to an antigen-binding construct, miniantibody or cys-diabody comprising SEQ ID NO: 1.

[0062] An antigen-binding construct, miniantibody or cys-diabody according to any one of the above embodiments, wherein the antigen-binding construct, miniantibody or cys-diabody has an expression yield of about 75% to 300%, 75% to 200%, 75% to 150% or 75% to 100% compared to an antigen-binding construct, miniantibody or cys-diabody comprising SEQ ID NO: 1.

[0063] An antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the antigen-binding construct, minibody or cys-diabody has an expression yield of about 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 150%, 175%, 200%, 250% or 300% compared to an antigen-binding construct, minibody or cys-diabody comprising SEQ ID NO: 1, or a percentage within the range defined by any two of the above values ​​(e.g., about 75-300%, about 80-250%, about 100%-300%, about 110%-250%, about 120%-200%, etc.).

[0064] An antigen-binding construct, miniantibody or cys-diabody according to any one of the above embodiments, wherein the antigen-binding construct, miniantibody or cys-diabody accumulates to a detectable level in the subject's blood, liver, kidney, spleen, lung, muscle, bone, heart, stomach, small intestine, large intestine, pancreas, tumor or any combination thereof.

[0065] According to any one of the above embodiments of the antigen-binding construct, minibody or cys-diabody, wherein the subject is a mammal.

[0066] An antigen-binding construct, minibody or cys-diabody according to any of the above embodiments, wherein the antigen-binding construct, minibody or cys-diabody is detectable at, about or at most about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30% or 35% ID / g, or a percentage within the range defined by any two of the above values.

[0067] An antigen-binding construct, minibody or cys-diabody according to any of the above embodiments, wherein the antigen-binding construct, minibody or cys-diabody is detectable at between about 0.1% to 35%, 0.1% to 30%, 1% to 35%, 1% to 30%, 5% to 35% or 5% to 30% ID / g.

[0068] An antigen-binding construct, miniantibody or cys-diabody according to any one of the above embodiments, wherein the antigen-binding construct, miniantibody or cys-diabody comprises LFR2 as shown in SEQ ID NO: 35 or 37; and wherein the antigen-binding construct, miniantibody or cys-diabody has improved biodistribution compared to an antigen-binding construct, miniantibody or cys-diabody comprising LFR2 as shown in SEQ ID NO: 33.

[0069] The antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the KD of the antigen-binding construct, minibody or cys-diabody is less than about 1×10 -10 M.

[0070] The antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments, wherein the KD of the antigen-binding construct, minibody or cys-diabody is less than about 1×10 -12 M.

[0071] According to any one of the above embodiments, the antigen-binding construct, minibody or cys-diabody, wherein the EC of the antigen-binding construct, minibody or cys-diabody 50 is, is about, or is at most 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.20 nM, or an EC 50 Within the range defined by any two of the above values.

[0072] According to any one of the above embodiments, the antigen-binding construct, minibody or cys-diabody, wherein the EC of the antigen-binding construct, minibody or cys-diabody 50Between about 0.001 to 0.20 nM, 0.001 to 0.17 nM, 0.001 to 0.12 nM, 0.01 to 0.20 nM, 0.01 to 0.17 nM, 0.01 to 0.12 nM, 0.05 to 0.20 nM, 0.05 to 0.17 nM, 0.05 to 0.15 nM, 0.05 to 0.12 nM, 0.08 to 0.20 nM, 0.08 to 0.17 nM, or 0.08 to 0.12 nM.

[0073] According to any one of the above embodiments, the antigen-binding construct, minibody or cys-diabody, wherein the EC of the antigen-binding construct, minibody or cys-diabody 50 Up to about 10 nM.

[0074] Provided herein are nucleic acids encoding an antigen binding construct, minibody or cys-diabody according to any of the above embodiments.

[0075] The nucleic acid according to any one of the above embodiments, wherein the nucleic acid is any one of SEQ ID NOs: 51-65.

[0076] Provided herein are cell lines that produce an antigen-binding construct, minibody or cys-diabody according to any of the above embodiments.

[0077] Provided herein is a kit comprising: an antigen-binding construct, a minibody or a cys-diabody according to any one of the above embodiments; and a chelator, wherein the chelator allows for the incorporation of a detectable marker.

[0078] Provided herein are kits comprising: an antigen binding construct, minibody or cys-diabody according to any one of the above embodiments; and a chelator, wherein the chelator allows for the incorporation of a therapeutic isotope or agent.

[0079] Provided herein is a kit comprising: an antigen binding construct, a minibody or a cys-diabody according to any one of the above embodiments; and a linker, wherein the linker allows for the incorporation of a detectable marker.

[0080] Provided herein are kits comprising: an antigen binding construct, a minibody or a cys-diabody according to any one of the above embodiments; and a linker, wherein the linker allows for the incorporation of a therapeutic isotope or agent.

[0081] Provided herein are kits comprising: an antigen-binding construct, a minibody or a cys-diabody according to any one of the above embodiments; and a detectable marker.

[0082] Provided herein is a method for detecting the presence or absence of DLL3, the method comprising: applying the antigen-binding construct, minibody or cys-diabody according to any one of the above embodiments to a sample; and detecting the presence or absence of the antigen-binding construct, thereby detecting the presence or absence of DLL3.

[0083] The method according to any one of the above embodiments, wherein the antigen binding construct, minibody or cys-diabody is conjugated to a detectable marker.

[0084] The method according to any one of the above embodiments, wherein applying the antigen-binding construct, minibody or cys-diabody comprises administering the antigen-binding construct to a subject.

[0085] The method according to any one of the above embodiments, wherein detecting the binding or absence of binding of the antigen-binding construct, miniantibody or cys-diabody to DLL3 comprises at least one of positron emission tomography, single-photon emission computed tomography or fluorescent tomography.

[0086] According to the method of any one of the above embodiments, the method further comprises applying a second antigen-binding construct to the sample, wherein the second antigen-binding construct specifically binds to the antigen-binding construct.

[0087] The method according to any one of the above embodiments, wherein the antigen-binding construct, minibody or cys-diabody is incubated with the sample for no longer than 20 hours.

[0088] The method according to any one of the above embodiments, wherein the antigen-binding construct, minibody or cys-diabody is incubated with the sample for no longer than 6 hours.

[0089] The method according to any one of the above embodiments, wherein the antigen-binding construct, miniantibody or cys-diabody is administered to a host, and wherein a first amount of the antigen-binding construct, miniantibody or cys-diabody is unbound from DLL3, and a second amount of the antigen-binding construct, miniantibody or cys-diabody is bound to DLL3, wherein at least about 80% of the first amount of the antigen-binding construct, miniantibody or cys-diabody is eliminated in no more than 12 hours.

[0090] Provided herein are methods of targeting a therapeutic agent to DLL3, the method comprising administering to a subject an antigen binding construct, minibody or cys-diabody according to any one of the preceding claims, wherein the antigen binding construct is conjugated to the therapeutic agent.

[0091] Provided herein are therapeutic compositions targeting DLL3, wherein the therapeutic composition comprises: an antigen-binding construct comprising: a light chain variable (V L ) domain: LCDR1 as SEQ ID NO: 15; LCDR2 as any one of SEQ ID NO: 19, 21 or 23; LCDR3 as SEQ ID NO: 25; and a heavy chain variable (V H ) domains: HCDR1 being the HCDR1 of SEQ ID NO: 27; HCDR2 being the HCDR2 of SEQ ID NO: 29; HCDR3 being the HCDR3 of SEQ ID NO: 31; and a therapeutic agent, a toxic payload, and / or a detectable marker.

[0092] Provided herein is a therapeutic composition targeting DLL3, wherein the therapeutic composition comprises: a miniantibody that binds to DLL3, the miniantibody comprising: a single chain variable fragment (scFv) that binds to DLL3; a hinge-extension domain comprising an IgG1 hinge region; an IgG C H 3 sequence; and a therapeutic agent, a toxic payload and / or a detectable marker, the scFv comprising a heavy chain variable (V H ) domain connected to the light chain variable (V L ) domain, the V L The domain comprises: LCDR1 as SEQ ID NO: 15; LCDR2 as any one of SEQ ID NO: 19, 21 or 23; LCDR3 as SEQ ID NO: 25, and the V H The domain comprises: HCDR1 as SEQ ID NO:27; HCDR2 as SEQ ID NO:29; HCDR3 as SEQ ID NO:31.

[0093] Provided herein are therapeutic compositions targeting DLL3, wherein the therapeutic composition comprises: a cys-diabody that binds to DLL3; and a therapeutic agent, a toxic payload and / or a detectable marker, the cys-diabody comprising a polypeptide comprising: a single-chain variable fragment (scFv) comprising a light chain variable (Vy) domain connected to a heavy chain variable (Vy) domain; the Vy domain comprising: LCDR1 as SEQ ID NO: 15; LCDR2 as SEQ ID NO: 19, 21 or 23; LCDR3 as SEQ ID NO: 25; the Vy domain comprising: HCDR1 as SEQ ID NO: 27; HCDR2 as SEQ ID NO: 29; HCDR3 as SEQ ID NO: 31.

[0094] According to any one of the above embodiments, the therapeutic composition, wherein the detectable marker is a radiolabel.

[0095]

[00136] According to any one of the above embodiments, the detectable marker is an alpha emitter radiolabel.

[0096]

[00136] According to any one of the above embodiments, the detectable marker is a beta emitter radiolabel.

[0097] The therapeutic composition according to any of the above embodiments, wherein the detectable marker is a positron emitter radiolabel.

[0098] According to any one of the above embodiments, the therapeutic composition, wherein the detectable marker comprises Lutetium-177. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Figure 1 Some embodiments of polypeptide sequences of antigen-binding minibodies are shown (SEQ ID NOs: 1-10).

[0100] Figure 2 Some embodiments of polypeptide sequences of antigen-binding cys-diabodies are shown (SEQ ID NOs: 11-14).

[0101] Figure 3 LCDR (SEQ ID NO: 15-26), HCDR (SEQ ID NO: 27-32), LFR (SEQ ID NO: 33-38), linker (SEQ ID NO: 39-44), signal peptide (SEQ ID NO: 45-46), parent V H (SEQ ID NO: 47) and parent V LSome embodiments of the polypeptide sequence of (SEQ ID NO: 48).

[0102] Figure 4 Some embodiments of annotated polypeptide sequences of antigen-binding minibodies are shown.

[0103] Figure 5 Some embodiments of variable region alignments of antigen-binding minibody polypeptide sequences and lovalpituzumab polypeptide sequences are shown.

[0104] Figure 6 An embodiment of the polypeptide sequence of human-DLL3 is shown (SEQ ID NO: 50).

[0105] Figure 7 is a bar graph showing an embodiment of minibody expression levels in mammalian cells.

[0106] Figure 8 EC of antigen-binding miniantibodies was shown by ELISA 50 A series of figures and corresponding tables of some embodiments of the invention are defined.

[0107] Fig. 9 is a series of graphs and corresponding tables showing some embodiments of the determination of equilibrium dissociation constants for antigen-binding minibodies.

[0108] Fig.10 EC of antigen-binding microantibodies was shown by flow cytometry 50 A series of diagrams of some embodiments of the invention are described.

[0109] Fig.11 is a bar graph showing some embodiments of the in vivo biodistribution of antigen-binding minibodies in a tumor-bearing mouse model.

[0110] Fig.12 is a bar graph showing some embodiments of the in vivo biodistribution of antigen-binding minibodies and cys-diabodies.

[0111] Fig.13 Some embodiments of nucleic acid sequences of antigen binding constructs, minibodies and cys-diabodies are shown (SEQ ID NOs: 51-64).

[0112] Fig.14 Shows C H 3. Some embodiments of polypeptide sequences. (SEQ ID NO: 65-85). DETAILED DESCRIPTION

[0113] Antigen binding constructs are described herein, including antibodies and fragments thereof, such as miniantibodies and cys-diabodies. In some embodiments, these are bound to target molecules, for example, DLL3. In some embodiments, these components are novel complementary determining region (CDR) sequences and / or sequences related to CDR sequences and / or parts of CDR sequences. In some embodiments, these components are novel framework region (FR) sequences and / or sequences related to FR sequences and / or parts of FR sequences. These CDR and FR sequences can provide a variety of benefits and improvements to this area. Antigen binding constructs (such as miniantibodies, cys-diabodies, etc.) are also provided herein, and the antigen binding constructs include one or more CDR or FR sequences or subsequences provided herein.

[0114] In some embodiments, antigen binding constructs can be used to target therapeutic agents to cells expressing target molecules. In some embodiments, methods of detecting the presence or absence of target molecules (or "targets") using antigen binding constructs (including antibodies, minibodies, and cys-diabodies) are provided. In some embodiments, methods of using antigen binding constructs for therapeutic purposes are provided.

[0115] In some embodiments, antigen-binding constructs, such as miniantibodies and cys-diabodies, can have excellent pharmacokinetic properties for faster diagnostic imaging while maintaining the binding specificity and affinity of the parental antibody. Current technology utilizes full-length antibody imaging, which usually requires significantly longer time (~7-8 days after injection) to produce high-contrast images due to the slow serum clearance of complete antibodies. Some embodiments of antigen-binding constructs, miniantibodies and cys-diabodies provided herein provide the opportunity for imaging on the same day or the next day.

[0116] In some embodiments, the antigen binding construct is used for diagnosis. When labeled with an appropriate radionuclide (e.g., positron emitters iodine-124, copper-64, fluorine-18, gallium-68, and / or zirconium-89 for PET imaging) or a fluorophore (for fluorescence imaging) or an infrared dye for optical imaging, the antibody fragment can be used for preclinical imaging as shown herein and for clinical imaging of patients. These antigen binding constructs can also be used as potential SPECT imaging agents by simply changing the radiolabel to a single-photon emitting radionuclide, such as indium-111, iodine-123, technetium-99M, and lutetium-177.

[0117] In some embodiments, the antigen binding constructs can be clinical imaging agents (PET / SPECT) in humans. Therefore, in some embodiments, the antigen binding constructs can be used for targeted diagnostic testing of these conditions. In some embodiments, the antigen binding constructs can be used as therapeutic agents.

[0118] definition

[0119] In view of this disclosure, all terms have the ordinary and customary meanings as understood by those of ordinary skill in the art.

[0120] The term "antigen-binding construct" includes all kinds of antibodies, including binding fragments thereof. Also included are constructs containing 1, 2, 3, 4, 5 and / or 6 CDRs. In some embodiments, a tandem scFv can be provided, which can provide two arms with bivalent binding. In some embodiments, these CDRs can be distributed between their appropriate framework regions in conventional antibodies. In some embodiments, the CDRs may be contained in the heavy chain and / or light chain variable region. In some embodiments, the CDRs may be in the heavy chain and / or light chain. In some embodiments, the CDRs may be in a single peptide chain. Unless otherwise specified herein, the antigen-binding constructs described herein bind to the specified target molecule. The term "target" or "target molecule" refers to the protein to which the antigen-binding construct binds. Examples of target proteins are known in the art and include, for example, DLL3 ( Figure 6 ; SEQ ID NO: 50).

[0121] The term "antibody" includes, but is not limited to, genetically engineered or other modified forms of immunoglobulins, such as intracellular antibodies, chimeric antibodies, fully human antibodies, humanized antibodies, antibody fragments, single-chain variable fragments (scFv) and heterologous conjugated antibodies (e.g., bispecific antibodies, double antibodies, three antibodies, four antibodies and nanobodies, etc.). The term "antibody" includes microantibodies and double antibodies. The term "antibody" includes polypeptides of the immunoglobulin family or polypeptides comprising immunoglobulin fragments, which can bind to corresponding antigens in a non-covalent, reversible and specific manner. Exemplary antibody structural units include tetramers. In some embodiments, a full-length antibody can be composed of two identical pairs of polypeptide chains, each pair having a "light" chain and a "heavy" chain (connected by a disulfide bond). The recognized immunoglobulin genes include κ, λ, α, γ, δ, ε, hinge and μ constant region genes, as well as a large number of immunoglobulin variable region genes. For full-length chains, light chains are classified as κ or λ. For full-length chains, heavy chains are classified as γ, μ, α, δ, or ε, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. The N-terminus of each chain defines a variable region of up to about 149 or more amino acids that is primarily responsible for antigen recognition. The term variable light chain (V L ) and variable heavy chain (V H ) refer to these regions of the light chain and heavy chain, respectively. As used in this application, "antibody" covers all variants of antibodies and fragments thereof. In addition, the term "antibody" includes immunoglobulins of camelid origin, such as single heavy chain antibodies and (Nanobodies). Thus, full-length antibodies, chimeric antibodies, humanized antibodies, single-chain antibodies (scFv), Fab, Fab', and multimeric forms of these fragments with the same binding specificity (e.g., F(ab')2), scFv-FC, single domain fragments (e.g., ), peptide body, -FC, minibodies and diabodies fall within the scope of this concept.In some embodiments, the antibody specifically binds to a desired target.

[0122] The terms "complementarity determining domain" or "complementarity determining region" ("CDR") are interchangeable L and V H CDR is the target molecule binding site of the antibody chain, which has specificity for this target molecule. In some embodiments, in each V L and / or V H There are three CDRs (CDR1-3, numbered sequentially from the N-terminus) in the variable domain, which constitute about 15-20% of the variable domain. The CDRs are structurally complementary to the target molecule epitope and are therefore directly responsible for binding specificity. L or V H The remaining stretch, the so-called FR, shows little variation in amino acid sequence (Kuby, Immunology, 4th edition, Chapter 4. WH Freeman & Co., New York, 2000).

[0123] The positions of CDRs and framework regions can be determined using a variety of definitions well known in the art, for example, Kabat (Wu, TT et al., "An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity," J. Exp. Med., Vol. 132, No. 2, pp. 211-250, 1970; Kabat, EA et al., "Sequences of Proteins of Immunological Interest," 5th Edition, NIH Publication No. 91-3242, Bethesda, MD, 1991); Chothia (Chothia C. et al., "Canonical structures for the hypervariable regions of immunoglobulins," J. Mol. Biol., Vol. 196, No. 4, pp. 901-917, 1987; Chothia C. et al., "Conformations of immunoglobulin hypervariable regions," Nature, Vol. 342, No. 6252, pp. 877-883, 1989; Chothia C. et al., "Structural repertoire of the human VH segments," J. Mol. Biol., Vol. 227, No. 3, pp. 799-817, 1992; Al-Lazikani B. et al., "Standard conformations for the canonical structures of immunoglobulins," J. Mol. Biol., Vol. 273, No. 4, pp. 927-748, 1997), ImMunoGeneTics database (IMGT) (on the World Wide Web: imgt.org / ) (Giudicelli, V. et al., "IMGT / LIGM-DB, the "Comprehensive database of immunoglobulin and T cell receptor nucleotide sequences," Nucleic Acids Res., Vol. 34 (Database Issue), pp. D781 - D784, 2006; Lefranc, M.P. et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V - like domains," Dev. Comp. Immunol., Vol. 27, No. 1, pp. 55 - 77, 2003; Brochet, X. et al., "IMGT / V - QUEST: the highly customized and integrated system for IG and TR standardized V - J and V - D - J sequence analysis," Nucleic Acids Res., Vol. 36 (Web Server Issue), pp. W503 - 508, 2008); AbM (Martin, A.C. et al., "Modeling antibody hypervariable loops: a combined algorithm," Proc. Natl. Acad. Sci. U.S.A., Vol. 86, No. 23, pp. 9268 - 9272, 1989); AHo (Honegger A, Plückthun A. Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool. J Mol Biol. (2001) 309:657 - 70); Gelfand (Gelfand IM, Kister a E. Analysis of the relation between the sequence and secondary and three - dimensional structures of immunoglobulin molecules. Proc Natl Acad Sci USA.(1995) 92: 10884 / 8); North (North B, Lehmann A, Dunbrack RLJ. A new clustering of antibody CDR loop conformations. J Mol Biol. (2011) 406: 228 / 56); contact definition (MacCallum, RM et al., "Antibody-antigen interactions: contact analysis and binding site topography," J. Mol. Biol., Vol. 262, No. 5, pp. 732-745, 1996), and / or automatic modeling and analysis tools (Honegger, A. et al., accessible on the World Wide Web at bioc.uzh.ch / plueckthun / antibody / Numbering / ). In some embodiments, the polypeptides are numbered from the start of the polypeptide signal sequence. In some embodiments, the polypeptides are numbered from the start of the polypeptide and do not include the signal sequence. .

[0124] As used herein, "antibody variable light chain" or "antibody variable heavy chain" refers to a variable chain comprising V L or V H Endogenous V L Encoded by gene segments V (variable) and J (joining), and the endogenous V H Encoded by V, D (diversity) and J. V L or V H Each of the CDRs and framework regions includes a CDR region and a framework region. In the present application, the antibody variable light chain and / or the antibody variable heavy chain may sometimes be collectively referred to as an "antibody chain". These terms cover antibody chains that do not disrupt V L or V H In some embodiments, the antibody chain of the mutation of the basic structure of the antibody chain of the present invention is considered, as those skilled in the art will easily recognize. In some embodiments, full-length heavy chain and / or light chain are considered. In some embodiments, only the variable region of the heavy chain and / or light chain is considered.

[0125] The term "hinge" refers to an antigen-binding construct such as an antibody, minibody, scFv-Fc or -At least a portion of the hinge region of Fc. The hinge region may include a combination of an upper hinge, a core (or middle) hinge, and a lower hinge region. In some embodiments, the hinge is defined according to any antibody hinge definition. Natural IgG1, IgG2, and IgG4 antibodies have a hinge region having 12-15 amino acids. IgG3 has an extended hinge region having 62 amino acids, including 21 prolines and 11 cysteines. It is inferred from crystallographic studies that the functional hinge region of naturally occurring antibodies extends from amino acid residues 216-237 of the IgG1 H chain (EU numbering; ref.12) and includes a small segment of the N-terminus of the CH2 domain in the lower hinge, wherein the lower hinge is the N-terminus of the CH2 domain. The hinge can be divided into three regions; "upper hinge", "core", and "lower hinge".

[0126] When the hinge (or its sub-portion) is modified, the term "artificial" or "non-natural" means that the sequence in question does not exist in nature in the specified state. In the context of the present invention, the hinges have been changed from their natural state so that their sequences are no longer those present in wild-type antibodies. As will be appreciated by those skilled in the art, miniantibodies do not occur naturally in nature, and therefore no constructs that are miniantibody constructs are found in nature. This also applies to at least some constructs present in and / or incorporated into the sequences of any hinge sequence table (e.g., Table 4) provided herein. In some embodiments, any hinge sub-portion or complete hinge sequence can be a human common hinge sequence, as long as the sequence (or the resulting combination of hinges) does not exist in nature.

[0127] The term "complete hinge region" or "complete hinge region" means that the entire upper hinge region, core hinge region and lower hinge region exist as a single construct. The upper region, core region and lower region can be located next to each other, or other residues can be added between these regions or at these regional N-terminals or C-terminals. In some embodiments, the natural lower hinge can be replaced by an extension sequence. In some embodiments, the natural lower hinge can be combined with an extension sequence. In some embodiments, an extension sequence or other sequence groups can be added after the upper sequence and / or core sequence.

[0128] The phrase "effective hinge region" means that a portion of at least one of the upper hinge region, the core hinge region, and the lower hinge region is present in sufficient amounts to render the hinge region effective for its intended purpose. Thus, the phrase encompasses variants of the hinge region and fragments of various hinge regions. In some embodiments, the function of the hinge region is one or more of the following: to bind the scFv to the C HThe three domains are connected to provide flexibility and spacing for the two scFvs to properly bind to the target, connect the two half molecules together, provide overall stability to the molecule, and / or provide a site for site-specific conjugation due to its solvent exposure. In some embodiments, the hinge should be close to native to reduce potential immunogenicity. In some embodiments, the upper hinge provides flexibility to the scFv (starting at residue 216 in native IgG), the middle hinge provides stability, and the lower hinge mediates C H 3 flexibility (starting at residue 231 in native IgG).

[0129] The term "upper hinge" refers to the first portion of the hinge starting at the end of the scFv. The upper hinge includes amino acids from the end of the scFv up to (but not including) the first cysteine ​​residue in the core hinge. As described above, the term "effective upper hinge" means that there is sufficient sequence to allow the portion to function as an upper hinge; the term encompasses functional variants and fragments of the indicated hinge portion.

[0130] The term "core hinge" refers to the second portion of the hinge region, which is located at the C-terminus of the upper hinge. The core hinge contains interchain disulfide bridges and a high content of proline. As described above, the term "effective core hinge" means that sufficient sequence is present to allow the portion to function as a core hinge; the term encompasses functional variants and fragments of the specified hinge portion.

[0131] The term "lower hinge" refers to the third portion of the hinge region, which is located at the C-terminus of the core hinge. In the context of a minibody or antibody fragment, the lower hinge is attached to the C H 3 domains. As described above, the term "effective lower hinge" means that there is enough sequence to allow the part to function as a lower hinge; the term covers functional variants and fragments of the hinge part specified. The term "lower hinge" as used herein can cover a variety of amino acid sequences, including naturally occurring IgG lower hinge sequences and artificial extension sequences that replace each other or combinations thereof provided herein. In some embodiments, a variety of extensions can be considered to be all lower hinge regions or their replacements.

[0132] Antibodies can exist as intact immunoglobulins or as multiple fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide bonds in the hinge region to produce F(ab)'2, which is a dimer of Fab', which itself is bound to V by disulfide bonds. H -C H 1 light chain (V L -C L). F(ab)'2 can be reduced under mild conditions to destroy the disulfide bonds in the hinge region, thereby converting the F(ab)'2 dimer into a Fab' monomer. The Fab' monomer is a Fab with a portion of the hinge region. (Paul, WE, "Fundamental Immunology," 3rd edition, New York: Raven Press, 1993). Although various antibody fragments are defined based on the digestion of intact antibodies, it will be appreciated by those skilled in the art that these fragments can be synthesized de novo by chemical methods or by using recombinant DNA methods. Therefore, the term "antibody" as used herein also includes antibody fragments produced by modifying intact antibodies, or antibody fragments synthesized de novo using recombinant DNA methods (e.g., single-chain Fv), or antibody fragments identified using phage display libraries (see, e.g., McCafferty, J. et al., "Phage antibodies: filamentous phage displaying antibody variable domains," Nature, Vol. 348, No. 66301, pp. 552-554, 1990).For the preparation of monoclonal or polyclonal antibodies, any technique known in the art can be used (see, e.g., Kohler, G. et al., “Continuous cultures of fused cells secreting antibody of predefined specificity,” Nature, Vol. 256, No. 5517, pp. 495-497, 1975; Kozbor, D. et al., “The production of monoclonal antibodies from human lymphocytes,” Immunology Today, Vol. 4, No. 3, pp. 72-79, 1983; Cole, et al., “Monoclonal Antibodies and Cancer Therapy,” Alan R. Liss, Inc., pp. 77-96, 1985; Wang, S., “Advances in the production of human monoclonal antibodies,” Antibody Technology Journal, Vol. 1, pp. 1-4, 2011; Sharon, J. et al., “Recombinant polyclonal antibodies for cancer therapy,” J. Cell Biochem., Vol. 96, No. 2, pp. 305-313, 2005; Haurum, JS, "Recombinant polyclonal antibodies: thenext generation of antibody therapeutics?", Drug Discov. Today, Vol. 11, No. 13-14, pp. 655-660, 2006). The technology used to produce single-chain antibodies (U.S. Pat. No. 4,946,778) can be applied to produce antibodies against the polypeptides disclosed herein. In addition, transgenic mice or other organisms, such as other mammals, can be used to express fully human monoclonal antibodies.In addition, Escherichia coli (E. coli) or yeast can be used to express and produce recombinant antibodies and antibody fragments (Simmons LC, Reilly D., Klimowski L., Shantha Raju T., Meng G., Sims P., Hong K., Shields RL, Damico LA, Rancatore P., Yansura DG; Expression of full-length immunoglobulins in Escherichia coli: rapid and efficient production of aglycosylated antibodies, J Immunol Methods. 2002 May 1; 263(1-2): 133-47; Kulagina N, Besseau S, Godon C, Goldman GH, Papon N., Courdavault V., Yeasts as biopharmaceutical production platforms; Front. Fungal Biol., 22 September 2021). Alternatively, phage display and yeast display technologies can be used to identify high affinity binders for selected antigens (see, e.g., McCafferty et al., supra; Marks, JD et al., "By-passing immunization: building high affinity human antibodies by chain shuffling," Biotechnology (NY), Vol. 10, No. 7, pp. 779-783, 1992; Feldhaus MJ, Siegel RW, Yeast display of antibody fragments: a discovery and characterization platform, J Immunol Methods, 2004 Jul; 290 (1-2): 69-80). Alternatively, antibodies can be produced from human hosts by B cell screening technology (Pedrioli A., Oxenius A., Single B cell technologies for monoclonal antibody discovery, Trends in Immunology, (2021) Vol. 42, No. 12, pp. 1143-1158).In addition, antibodies can be derived from immunization of camelids or screening of camelid phage libraries (Harmsen MM, DeHaard HJ, Properties, production, and applications of camelid single-domain antibody fragments, Appl Microbiol Biotechnol. 2007; 77 (1): 13-22). Alternatively, antibodies can be obtained from computer screening simulations by deep learning and artificial intelligence algorithms (Graves J., Byerly J., Priego E., Makkapati N., Vince Parish S., Brenda Medellin and Monica Berrondo, A Review of Deep Learning Methods for Antibodies, Antibodies 2020, 9, 12).

[0133] Methods for humanizing or primatizing non-human antibodies are well known in the art. Usually, humanized antibodies have one or more amino acid residues introduced therein from non-human sources. These non-human amino acid residues are commonly referred to as import residues, which are usually collected from the import variable domains. In some embodiments, the terms "donor" and "acceptor" sequences can be used. Basically, the method described in Winter and colleagues (see, e.g., Jones, PT et al., "Replacing the complementarity-determining regions in a human antibody with those from a mouse," Nature, Vol. 321, No. 6069, pp. 522-525, 1986; Riechmann, L. et al., "Reshaping human antibodies for therapy," Nature, Vol. 332, No. 6162, pp. 323-327, 1988; Verhoeyen, M. et al., "Reshaping human antibodies: grafting anantilysozyme activity," Science, Vol. 239, No. 4847, pp. 1534-1536, 1988; Presta, LG, "Antibody Engineering", Curr. Op. Struct. Biol., Vol. 2, No. 4, pp. 593-596, 1992), by replacing rodent CDR or CDR sequence with the corresponding human antibody sequence for humanization. Therefore, these humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), in which substantially less than the entire human variable domain is replaced by the corresponding sequence from a non-human species. In fact, humanized antibodies are usually human antibodies in which some complementary determining region ("CDR") residues and possibly some framework ("FR") residues are replaced by residues from analogous sites in rodent antibodies.

[0134] The term "Fc region" or "Fc domain" or "Fc" refers to the C-terminal region of an immunoglobulin heavy chain. The "Fc region" can be a native sequence Fc region or a variant Fc region (e.g., a variant having one or more mutations that reduce effector function, such as FcγR binding and / or binding to the Fc neocortex (FcRn), etc.). The Fc region of an immunoglobulin (e.g., IgG) generally comprises two constant domains, C H 2 and C H3. As described herein, the Fc region may include a hinge region or sequence. The Fc region may exist in dimer or monomer form. In some embodiments, the Fc region is a human Fc region or a variant thereof.

[0135] A "chimeric antibody" is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and / or species, or to an entirely different molecule, e.g., enzymes, toxins, hormones, growth factors and drugs, which confers new properties to the chimeric antibody; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigenic specificity.

[0136] The term "antibody fragment" includes, but is not limited to, one or more antigen-binding fragments of an antibody, alone or in combination with other molecules, including, but not limited to, Fab', F(ab')2, Fab, Fv, rIgG (reduced IgG), scFv fragments, scFv-Fc, single domain fragments (e.g., or single domain fragments), peptibodies, - Fc, minibodies and diabodies. The term "scFv" refers to a single-chain Fv ("fragment variable") antibody, in which the variable domains of the heavy and light chains of a conventional diabody have been linked to form one chain.

[0137] When a CDR or FR (or a subpart thereof) is modified, the term "artificial" or "non-natural" means that the sequence in question does not exist in nature in the specified state. In the context of the present invention, the CDR or FR has been changed from its natural state so that their sequences are no longer those present in a wild-type antibody. As will be appreciated by those skilled in the art, minibodies and cys-diabodies do not occur naturally in nature, and therefore no constructs are found in nature that are minibodies or cys-diabody constructs. This also applies to at least some of the constructs present in and / or incorporated into the sequences of any CDR or FR sequence table provided herein. In some embodiments, a figure or table, such as Figure 1 Any CDR or FR sequence in Table 1 or Table 2 may be an artificial CDR or FR sequence, as long as the sequence (or the resulting combination of CDR or FR) does not exist in nature.

[0138] "Miniantibodies" are a form of antibody that has a smaller molecular weight than full-length antibodies while maintaining bivalent binding properties to antigens. Due to its smaller size, the absence of the CH2 domain that binds to Fc-γ and FcRn receptors, and the absence of glycosylation, miniantibodies have faster systemic clearance and potentially enhanced permeability when targeting tumor tissues. Due to its strong targeting and rapid clearance capabilities, miniantibodies are beneficial for diagnostic imaging and the delivery of radioactive payloads, and the prolonged circulation time of the payload may lead to poor patient dose administration or dose determination. In some embodiments, due to the above-mentioned characteristics, such as tumor penetration and faster clearance, it can also be advantageous for the delivery of cytotoxic payloads. "Miniantibodies" as described herein encompass homodimers in which each monomer is bound to a human IgG C by a hinge sequence. H In some embodiments, the miniantibody is a bivalent or bispecific, covalently bound homodimer (homodimer) of ∼80 kDa. In some embodiments, each monomer (half molecule) is connected to the corresponding light chain variable (V) by a Gly-Ser-rich linker sequence of about 15-18 amino acids. L ) domain of the heavy chain variable (V H ) domain composition.

[0139] A "diabody" comprises a first polypeptide chain comprising a heavy chain variable domain (V H ), the heavy chain variable domain and the light chain variable domain (V L ) connected by a peptide linker (V H -V L or V L -V H ), the peptide linker does not allow pairing between the two domains on the first polypeptide chain (e.g., the peptide linker is too short to allow pairing), the second polypeptide chain comprising a heavy chain variable domain (V H ), the heavy chain variable domain (V H ) and the light chain variable domain (V L ) connected by a peptide linker (V H- V L or V L -V H), the peptide linker does not allow pairing between the two domains on the second polypeptide chain (e.g., the peptide linker is too short to allow pairing). Without being limited by theory, short bonding can force chain pairing between the complementary domains of the first and second polypeptide chains and promote the assembly of dimeric molecules with two functional antigen binding sites. Therefore, the peptide linker can be any suitable length that promotes such assembly, for example, between 5 and 20 amino acids in length. "Cys-diabody" refers to a diabody in which the monomer chains are covalently linked by disulfide bonds. In some embodiments, a "cys-diabody" is a diabody with one or more than one C-terminal cysteine.

[0140] The term "extension sequence" (e.g., in the context of a diabody) refers to a sequence that connects the first V H Domain to the second V H Domain or linker first V L To the second V L The region of the variable domain. The extension sequence can be connected to the domain through the C-terminus of each domain. In some embodiments, the extension sequence is connected to the domain by a covalent bond. In some embodiments, the extension sequence will include one or more cysteines, thereby allowing one or more disulfide bonds to be formed between two such extension sequences. Non-limiting examples of extension sequences include -(Gly)2-(Cys). In some embodiments, the extension sequence includes 1, 2, 3 or more cysteines per monomer chain. Although the extension sequence will be toward the C-terminus of the construct, it does not need to be the absolute last amino acid in the variable domain. That is, the extension sequence can be located at the C-terminus slightly closer to the N-terminus (slightly towards the N-terminus of the C-terminus). For example, the extension sequence can be placed within 10 amino acids of the C-terminus of the monomer. Similarly, other sequences can be set between the native C-terminus and the beginning of the extension sequence. The extension sequence can connect the V to the C-terminus through a disulfide bond. H Connect to V H Or V L Connect to V L In some embodiments, the extension sequence comprises GGCPPCPPC (SEQ ID NO: 93).

[0141] As used herein, "pharmaceutically acceptable" has a simple and conventional meaning as understood in accordance with the specification, and refers to carriers, excipients and / or stabilizers that are non-toxic or have an acceptable level of toxicity to cells or mammals exposed thereto at the doses and concentrations used. As used herein, "pharmaceutically acceptable", "diluent", "excipient" and / or "carrier" have a simple and conventional meaning as understood in accordance with the specification, and are intended to include any and all solvents, dispersion media (dispersion media), coatings (paints), antibacterial or antifungal agents, isotonic agents or absorption delaying agents that are compatible with administration to humans, primates, cats, dogs or other vertebrate hosts. Typically, pharmaceutically acceptable diluents, excipients and / or carriers are approved by federal, state government regulatory agencies or other regulatory agencies, or listed in the U.S. Pharmacopeia or other recognized pharmacopeias for use in animals, including humans and non-human mammals, such as cats and dogs. The term diluent, excipient and / or "carrier" can represent a diluent, adjuvant, excipient or vehicle used together with a pharmaceutical composition. These pharmaceutical diluents, excipients and / or carriers that can be incorporated into any one or more compositions described herein include sterile liquids, such as water and oil, including those oils of petroleum, animal, plant or synthetic origin. Water, saline solutions or glucose and glycerol aqueous solutions can be used as liquid diluents, excipients and / or carriers. Suitable pharmaceutical diluents and / or excipients (which can be incorporated into any one or more compositions described herein) also include starch, glucose, lactose, sucrose, gelatin, malt (malt), rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol (glycol), water or ethanol. Physiologically acceptable carriers may also include one or more of the following: antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids, carbohydrates such as glucose, mannose or dextrin, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming counterions such as sodium, and nonionic surfactants such as Polyethylene glycol (PEG), Or preservatives, such as essential oils, methyl paraben, propyl paraben or sodium salt of parabens. In some embodiments, the preservative is bronidiol. If necessary, the composition may also contain a small amount of wetting agent, bulking agent, emulsifier or pH buffer. These compositions can take the form of solutions, suspensions, emulsions, sustained release preparations, etc. The preparation should be suitable for the form of administration.

[0142] Other excipients with desired properties include, but are not limited to, preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizers, detergents, surfactants, chelating agents, antioxidants, alcohols, ketones, aldehydes, ethylenediaminetetraacetic acid (EDTA), citric acid, salts, sodium chloride, sodium bicarbonate, sodium phosphate, sodium borate, sodium citrate, potassium chloride, potassium phosphate, magnesium sulfate sugar, dextrose, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80, sodium deoxycholate, sodium taurodeoxycholate, magnesium stearate, octylphenol ethoxylate, benzethonium chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, urea or vitamins or any combination thereof. In some embodiments, the formulation includes at least one agent that acts to reduce radiolysis (also referred to as a "radioprotectant") or a renal protective agent. Non-limiting examples of radiolysis reducing agents include gentisic acid, acetylcholine, AET, ACE inhibitors, acteoside, alpha-tocopherolacetate, amifostine, ascorbic acid, aspirin, atorvastatin, beta-carotene, Bowman-Birk protease inhibitors, caffeic acid, captopril, carbaminoylcholine, carvacrol, celecoxib, coenzyme Q10, COX2 inhibitors / NSAIDs, curcumin, cysteine, cysteamine, cystamine, dendrodine analogs, dithiolthione, dopamine, enalapril, epigallocatechin-3-gal epigallocatechin-3-gallate, epinephrine, 17-β-estradiol, GANRA-5, genistein, green tea extract, growth factor, guanylate, halofuginone, Hmg-CoA reductase inhibitors (statins), heroin, histamine, ibuprofen, inapoyl-E-glucoside, isoflavones, isofraxidin, kukoamine A, lactoferrinamifostine, lipoic acidacid), lovastatin, luteolin-7-O-(2-apiosyl)-glucoside, 2-mercaptoethylguanidine, melatonin, methacholine, morphine, N-acetylcysteine, oltipraz, palifermin, phenethyl esters, polyphenols, pravastatin, protease inhibitors, quercetin-3-O-rhamnoside-7-O-glucoside O-rhamnoside-7-O-glucoside), quercetin-3-O-rhamnoside, ramipril, resveratrol, rutin, serotonin, simvastatin, sodium ascorbate, superoxide dismutase, TGF-signaling inhibitor, tocopherol, vitamin C, vitamin E, watermelon juice, black grape juice and thiols, such as glutathione. Renal protective agents include free lysine, arginine, probenecid, succinylated gelatin and other compositions. Some excipients can be in residual amounts or contaminants from the manufacturing process, including but not limited to serum, albumin, ovalbumin, antibiotics, inactivators, formaldehyde, glutaraldehyde, β-propiolactone, gelatin, cell debris, nucleic acids, peptides, amino acids or growth medium components or any combination thereof. The amount of excipient in the composition can be, about, at least, at least about, not more than or not more than about: 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w / w percentage, or any weight percentage within the range limited by any two of the above numbers.

[0143] As used herein, "carrier" has its simple and conventional meaning as understood in light of the present specification, and refers to a compound, particle, solid, semisolid, liquid or diluent that facilitates the passage, delivery and / or introduction of a compound into cells, tissues and / or organs of the body.

[0144] As used herein, "diluent" has a simple and conventional meaning as understood in accordance with this specification, and refers to a component in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable. For example, a diluent can be used to increase the volume of an effective drug that is too small for production and / or administration. It can also be a liquid used to dissolve a drug that is administered by injection, ingestion, or inhalation. A common form of diluent in the art is a buffered aqueous solution, such as, but not limited to, a phosphate buffered saline that simulates the composition of human blood.

[0145] The term "target molecule dependent disorder" or "target molecule associated disorder" includes any disorder in which the target molecule plays a role in the disorder itself. In some embodiments, this means overexpression of the target molecule. In some embodiments, the disorder may include any disorder discussed herein. In some embodiments, the disorder may be any disorder in which there is a target molecule that can be targeted by binding, the binding of which will result in the detection and / or treatment of the disorder.

[0146] The term "treating" a condition may refer to preventing the condition, slowing the onset and / or rate of development of the condition, reducing the risk of the condition, preventing and / or delaying the development of symptoms associated with the condition, alleviating or terminating symptoms associated with the condition, producing complete or partial regression of the condition, or some combination thereof. The term "preventing" does not require an absolute prohibition of a condition or disease. Examples of conditions or diseases include fibrosis, cancer, tumors and neoplasms, autoimmune diseases, cardiovascular, neurodegenerative, metabolic and endocrine conditions, inflammatory, immune, genetic conditions, infectious conditions, hematology, congenital conditions, musculoskeletal, oral and gastrointestinal, renal and urogenital conditions, reproductive conditions, respiratory conditions, skin and / or epithelial, and conditions of controversial or unknown etiology.

[0147] As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and refers to all precancerous and cancerous cells and tissues. As mentioned herein, the terms "cancer," "cancerous," "cell proliferative disorders," "proliferative disorders," and "tumor" are not mutually exclusive. The term "neoplasia" encompasses the term tumor.

[0148] The term "cancer" or "cancerous" refers to or describes the physiological condition in mammals that is generally characterized by unrestrained cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of these cancers include lung cancer, including small cell lung cancer, non-small cell lung cancer, and lung adenocarcinoma with neuroendocrine features; neuroendocrine prostate cancer, melanoma, glioma, low grade glioma and glioblastoma, medullary thyroid cancer, carcinoid tumors, neuroendocrine tumors in the pancreas, bladder cancer, testicular cancer, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), neuroendocrine neoplasms, such as neuroendocrine tumors of unknown primary, small intestine, carotid body, adrenal gland, colorectal gynecological organs, of the lining of the stomach, intestine, bladder, urethra, liver, breast, colon, rectum, colorectal, endometrial or uterine, salivary gland, kidney or renal cancer, prostate, vulva, thyroid, bone, anal, thyroid gland, stomach, melanoma, multiple myeloma and B-cell lymphoma, brain cancer, and head and neck cancer and related metastases. The term cancer includes adult and pediatric solid cancers. In some embodiments, the cancer can be a solid tumor. In some embodiments, the cancer is a highly fibrotic tumor or cancer. In some embodiments, the cancer is desmoplasia.

[0149] A "therapeutically effective amount" or "therapeutically effective dose" is an amount that produces a desired therapeutic effect in a subject, such as preventing, treating a target condition, delaying the onset of a disorder and / or symptom, and / or alleviating symptoms associated with the condition. This amount will vary according to a variety of factors, including, but not limited to, the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, biodistribution, and bioavailability), the subject's physiological condition (including age, sex, disease type and stage, general physical condition, responsiveness to a given dose, and drug type), the nature of the carrier available for one or more drugs in the formulation, and / or the route of administration. In light of this disclosure, a person skilled in the art of clinical and pharmacological science will be able to determine a therapeutically effective amount by routine experimentation, such as by monitoring the subject's response to the administration of the compound and adjusting the dose accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy 21st Edition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005.

[0150] "Label," "detectable label," or "detectable marker" are used interchangeably herein and refer to a detectable compound or composition that is conjugated directly to an antibody or is indirectly associated with an antibody to produce a "labeled" antibody. The label itself may be detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.

[0151] The term "payload" refers to an atom or molecule or other entity that is bound (covalently or otherwise) to an antigen-binding construct. For example, it includes labels or markers for diagnostics, and toxins, cytotoxic agents, chemotherapeutic agents for various therapies. In some embodiments, the payload participates as a chelating agent to connect the antigen-binding construct to a molecule or atom to be delivered by the antigen-binding construct or co-localized by the antigen-binding construct.

[0152] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and / or causes cell death or destruction. The term is intended to include non-radioactive isotopes (ADCs), radioactive isotopes (e.g., 177 Lu, 225 Ac, 67 Cu, 227 Th, 211 At 131 I. 125 I. 90 Y.186 Re, 188 Re, 153 Sm, 212 Bi, 213 Bi, 32 P. 149 Tb, 161 Tb, 212 Pb, and radioactive isotopes of Lu), chemotherapeutic agents (as defined elsewhere herein). Other cytotoxic agents are described below. Tumoricidal agents result in the destruction of tumor cells.

[0153] "Toxin" is any substance that can have an adverse effect on cell growth or proliferation. Non-radioactive payloads include those commonly used in antibody drug conjugates (ADC) and fragment drug conjugates (FDC), such as toxins belonging to the family of auristatins, maytansines, maytansinoids, calicheamicins, duocarymycins, pyrrolobenzodiazepines dimers, and amatoxins.

[0154] "Therapeutic ions" refer to charged particles that are useful in treating a condition associated with a target molecule. Examples of therapeutic ions include 18 F. 18 F-FAC, 32 P. 33 P. 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y. 90 Y. 89 Sr. 89 Zr, 94 Tc, 94 Tc, 99 mTc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I. 124 I. 125I. 131 I. 142 Pr, 143 Pr, 149 Pm, 149 Tb, 153 Sm, 154-158 Gd, 161 Tb, 166 Dy, 166 Ho, 169 2. 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra, 227 Th and 225 Ac. These are also options for therapeutic agents.

[0155] A "chemotherapeutic agent" is a compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN TM Cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquinone, meturedopa, and uredopa; ethyleneimines and methylamelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL TM ); β-lapachone; lapachol; colchicine; betulinic acid; camptothecin (including the synthetic analogue topotecan (HYCAMTIN TM )、CPT-11(irinotecan, CAMPTOSAR TM), acetylcamptothecin, scopolamine, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride, oxide hydrochloride), melphalan, nebixin, cholesteryl phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma ll and calicheamicin omega ll (see, e.g., Agnew, Chem Intl. Ed. Engl., 33:183-186 (1994)); danemycins, including danemycin A; esperamicins;and the neocarzinostatin chromophore and the related chromoprotein enediyne antibiotic chromophores. chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN™ doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid (mycophenolic acid), acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, daunorubicin; metabolic inhibition antagonists, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as caprotestosterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenal agents, such as aminoglutethimide, mitotane, trilostane;Folic acid supplements, such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate acetate; epothilone; etoglu; gallium nitrate; hydroxyurea; lentinan; lonidainine; matanycins, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-acetylhydrazine; procarbazine; PSK.RTM. polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, baculocin A and anguidine); ethyl carbamates; vindesine (ELDISINETM, FILDESINTM); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; cytarabine ("Ara-C"); thiotepa;Taxoids, for example, TAXOL.RTM. paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE™, a cremophor-free albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE™ docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (GEMZARTM); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine (VELBANTM); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVINTM); oxaliplatin; folinic acid; vinorelbine (NAVELBINETM); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitors (TOPOIsomerase inhibitors inhibitor)RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODATM); any of the above-mentioned drug-usable salts, acids or derivatives; and combinations of two or more of the above, such as CHOP (abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone) and FOLFOX (abbreviation for the treatment regimen of oxaliplatin (ELOXATINTM) combined with 5-FU and folinic acid). These are also options for therapeutic agents. ;

[0156] "Radiotherapy" refers to treatment using radiation or radioisotopes for therapeutic purposes. It includes radiation therapy intended to have abscopal effect, as described in Yang Liu, Yinping Dong, Li Kong, Fang Shi, Hui Zhu & Jinming Yu; "Abscopal effect of radiotherapy combined with immune checkpoint inhibitors"; Journal of Hematology & Oncology Volume 11, Article Number: 104 (2018); and Melek Tugce Yilmaz, Aysenur Elmali, and Gozde Yazici; "Abscopal Effect, From Myth to Reality: From Radiation Oncologists' Perspective"; Cureus. 2019Jan; 11(1).

[0157] The terms "subject", "patient" and "individual" interchangeably refer to the entity being examined and / or treated. The term "mammal" is used in its common biological sense. Thus, it specifically includes, but is not limited to, primates, including great apes (chimpanzees, apes, monkeys), humans, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice or guinea pigs.

[0158] The term "co-administration" refers to the administration of two active agents in the blood of an individual or a sample to be tested. Combination co-administration or sequential delivery of active agents. "Combination" refers to the delivery of two (or more) different compositions to a subject during the subject's illness, for example, after the subject is diagnosed or selected as suffering from the illness and before the illness is cured or eliminated, two or more compositions are delivered. In some embodiments, the subject is selected to receive any one or more compositions described herein by diagnostic analysis or clinical evaluation or both. In some embodiments, when the delivery of the second therapy is started, the delivery of a therapy is still occurring, so that there is overlap. This is sometimes referred to as "simultaneous" or "accompanied" or "simultaneous delivery" in this article. In other embodiments, a therapy delivery is terminated before the delivery of other therapies begins. This is sometimes referred to as "continuous" or "sequential delivery" in this article. In embodiments of any one case, due to the combined administration, the therapy is more effective. For example, compared with the observation of the administration of the second therapy in the absence of the first therapy, or compared with the similar situation observed using the first therapy, the second therapy is more effective, for example, the same effect is observed with less second therapy, or the second therapy alleviates symptoms to a greater extent. In some embodiments, delivery results in a reduction in symptoms or other parameters associated with a condition that is greater than the reduction observed by delivery of one therapy in the absence of the other therapy. The effects of the two therapies may be partially additive, fully additive, or greater than additive (e.g., synergistic). Delivery may be such that the effects of the first therapy delivered are still detectable when the second therapy is delivered.

[0159] When used in the context of describing the interaction between an antigen (e.g., a protein) and an antibody or an antibody-derived binding agent, the phrase "specific (or selective) binding" refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biological products, for example, in a biological sample, such as blood, serum, plasma, or a tissue sample. Thus, under specified immunoassay conditions, in some embodiments, an antibody or binding agent with a specific binding specificity binds to a specific antigen at least twice as much as the background, and does not substantially bind to other antigens present in the sample in significant amounts. Specific binding to an antibody or binding agent under these conditions may require the selection of an antibody or reagent for its specificity for a particular protein. A variety of immunoassay formats can be used to select antibodies that are specifically immunoreactive to a particular protein. For example, solid phase ELISA immunoassays are commonly used to select antibodies that are specifically immunoreactive to a protein (for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity, see, for example, Harlow, E. & Lane D., "Using Antibodies, A Laboratory Manual," Cold Spring Harbor Laboratory Press, 1998). Typically, a specific or selective binding reaction will produce a signal that is at least two times greater than background, and more typically at least 10 to 100 times greater than background.

[0160] The term "equilibrium dissociation constant (K D , M)” refers to the dissociation rate constant (k d ,time -1 ) divided by the binding rate constant (k a ,time -1 M -1 The equilibrium dissociation constant can be measured using any method known in the art. The equilibrium dissociation constant of the antibodies disclosed herein is generally less than (i.e., binds better) about 10 -7 or 10 -8 M, for example, is less than about 10 -9 M or 10 -10 M, in some embodiments, is less than about 10 -11 M, 10 -12 M or 10 -13 M.

[0161] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of the corresponding naturally occurring amino acid, and to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0162] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologous genes, SNPs, and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon replacement can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed bases and / or deoxyinosine residues (Batzer, MA et al., "Enhanced evolutionary PCR using oligonucleotides with inosine at the 3'-terminus," Nucleic Acid Res., Vol. 19, No. 18, p. 5081, 1991; Ohtsuka, E. et al., "An alternative approach to deoxyoligonucleotides as hybridization probes by insertion of deoxyinosine at ambiguous codon positions," J. Biol. Chem., Vol. 260, No. 5, pp. 2605-2608, 1985; Rossolini, GM et al., "Use of deoxyinosine-containing primers vs degenerate primers for polymerase chain reaction based on ambiguous sequence information,” Mol. Cell. Probes, Vol. 8, No. 2, pp. 91-98, 1994).

[0163] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are subsequently modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as naturally occurring amino acids, e.g., α-carbon bound to hydrogen, carboxyl, amino, and R groups, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. These analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to compounds having a structure different from the general chemical structure of an amino acid, but functioning in a manner similar to naturally occurring amino acids.

[0164] The term "conservatively modified variants" is applicable to amino acid and nucleic acid sequences. For a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids encoding identical or substantially identical amino acid sequences, or refer to situations in which the nucleic acid does not encode an amino acid sequence as substantially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at each position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. This nucleic acid change is a "silent change," which is a class of conservatively modified changes. In this article, each nucleic acid sequence encoding a polypeptide also describes each possible silent change of the nucleic acid. Those skilled in the art will recognize that each codon in the nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to produce functionally identical molecules. Therefore, each silent change of the nucleic acid encoding a polypeptide is implicit in each of the described sequences.

[0165] With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide or protein sequence that alter, add or delete a single amino acid or a small percentage of amino acids in the coded sequence are "conservatively modified variants," wherein such alterations result in replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables that provide functionally similar amino acids are well known in the art. These conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs and alleles of the present disclosure.

[0166] The following eight groups contain amino acids that are conservatively substituted for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, e.g., Creighton, TE, "Proteins-Structures and Molecular Properties," WH Freeman & Co. Ltd., 1984).

[0167] The term "percentage of sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e., gaps) compared to a reference sequence (e.g., a polypeptide of the present disclosure), and for optimal alignment of the two sequences, the polynucleotide sequence does not include additions or deletions. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue exists in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window and multiplying the result by 100 to obtain the percentage of sequence identity.

[0168] In the context of two or more nucleic acids or polypeptide sequences, the term "identical" or percentage "identity" refers to that two or more sequences or subsequences are identical sequences. When using one of the following sequence comparison algorithms or by manual alignment and visual inspection to compare and compare the maximum correspondence on the comparison window or specified region as measured, if the two sequences have the same amino acid residues or nucleotides of the specified percentage (for example, on the specified region, or when not specified, on the entire sequence of the reference sequence, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity), then the two sequences are "substantially identical". Some embodiments provided herein provide polypeptides or polynucleotides substantially identical to the polypeptides or polynucleotides illustrated herein, respectively. Optionally, identity is present in a region of at least about 15, 25 or 50 nucleotides in length, or more preferably in a region of 100 to 500 or 1000 or more nucleotides in length, or in the full length of the reference sequence. For amino acid sequences, identity or substantial identity can exist over a region of at least 5, 10, 15, or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75, or 100 amino acids in length, optionally at least about 150, 200, or 250 amino acids in length, or the full length of the reference sequence. For shorter amino acid sequences, e.g., amino acid sequences of 20 or fewer amino acids, in some embodiments, substantial identity exists when one or two amino acid residues are conservatively substituted according to conservative substitutions as defined herein.

[0169] In some embodiments, the percentage identity is in the CDR and / or FR regions specified herein. In these cases, the percentage identity of CDR or FR can be identified separately from the rest of the protein or nucleic acid sequence. Therefore, two CDRs or FRs can have the same amino acid residues or nucleotides of the specified percentage (e.g., in the specified region, or when not specified, in the entire sequence of the reference sequence, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity), while allowing the rest of the protein to keep 100% identical with the comparison protein, while also allowing the rest of the protein to have the change of the specified percentage identity.

[0170] For sequence comparison, usually a sequence is used as a reference sequence, and the test sequence is compared thereto. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, and if necessary, subsequence coordinates are specified, and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. Then, based on the program parameters, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence.

[0171] As used herein, a "comparison window" includes reference to a segment having a number of adjacent positions selected from any one of: 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, wherein after the two sequences are optimally aligned, a sequence can be compared to a reference sequence having the same number of adjacent positions. Methods for comparing sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed by, for example, the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, the homology alignment algorithm of Needleman, SB et al., "A general method applicable to the search for similarities in the amino acid sequence of two proteins," J. Mol. Biol., Vol. 48, No. 3, pp. 443-453, 1970, the search similarity method of Pearson, WR et al., "Improved tools for biological sequence comparison," Proc. Natl. Acad. Sci. USA, Vol. 85, No. 8, pp. 2444-2448, 1988, by computerized implementations of these algorithms (e.g., Genetics Computer Group, 575 Science Dr., Madison, Wis. Wisconsin Genetics software package) or by manual alignment and visual inspection (see, e.g., Ausubel, FM et al., Current Protocols in Molecular Biology, Supplement, 1995).

[0172] Two examples of algorithms suitable for determining percentages of sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul, SF et al., "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res., Vol. 25, No. 17, pp. 3389-3402, 1977 and Altschul, SF et al., "Basic local alignment search tool," J. Mol. Biol., Vol. 215, No. 3, pp. 403-410, 1990, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy a positive threshold score T when aligned with a word of the same length in the database sequence. T is called the neighborhood word score threshold (Altschul, SF et al., as above). These initial neighborhood word hits serve as seeds for starting searches to find longer HSPs containing them. Word hits are extended in both directions along each sequence until the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (a reward score for a pair of matching residues; always>0) and N (a penalty score for mismatched residues; always<0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The expansion of word hits in each direction stops when the following occurs: the cumulative alignment score drops from its maximum achieved value by a number X; the cumulative score becomes zero or lower due to the accumulation of one or more negative score residue alignments; or the end of any sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expected value (E) of 10, M=5, N=-4 and two chain comparisons as default.For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, Henikoff, S. et al., “Amino acid substitution matrices from protein blocks,” Proc. Natl. Acad. Sci. USA, Vol. 89, No. 22, pp. 10915-10919, 1992), alignments (B) 50, expectation (E) 10, M=5, N=-4, and a comparison of both chains.

[0173] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin, S. et al., "Applications and statistics for multiple high-scoring segments inmolecular sequences," Proc. Natl. Acad. Sci. USA, Vol. 90, No. 12, pp. 5873-5787, 1993). A similarity metric provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match occurs by chance between two nucleotide or amino acid sequences. For example, if the minimum sum probability in a comparison of a test nucleic acid to a reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001, the nucleic acid is considered similar to the reference sequence.

[0174] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, in some embodiments, for example, when the two peptides differ only in conservative substitutions, a polypeptide is generally substantially identical to a second polypeptide. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complementary sequences hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the sequences can be amplified using the same primers.

[0175] Antigen Binding Constructs

[0176] In this context, it will be appreciated that sequences within CDRs and / or FRs may have particular relevance for a variety of antigen-binding constructs. In some embodiments, the values ​​of CDRs and / or FRs may be particularly high, such as in minibodies or diabodies, for example, in cys-diabody schemes.

[0177] Some aspects of the present disclosure relate to antigen-binding constructs. In some embodiments, the antigen-binding construct comprises: a light chain variable (VL ) domain: LCDR1 as SEQ ID NO: 15; LCDR2 as any one of SEQ ID NO: 19, 21 or 23; LCDR3 as SEQ ID NO: 25; and / or a heavy chain variable (V H ) domain: HCDR1 as SEQ ID NO: 27; HCDR2 as SEQ ID NO: 29; and HCDR3 as SEQ ID NO: 31. In some embodiments, the V L comprising: LCDR1 as LCDR1 in any one of SEQ ID NOs: 1-14, 90 and 91; LCDR2 as LCDR2 in any one of SEQ ID NOs: 1-14, 90 and 91; LCDR3 as LCDR3 in any one of SEQ ID NOs: 1-14, 90 and 91; and the V H comprising: a HCDR1 that is a HCDR1 in any one of SEQ ID NOs: 1-14, 87, and 88; a HCDR2 that is a HCDR2 in any one of SEQ ID NOs: 1-14, 87, and 88; and a HCDR3 that is a HCDR3 in any one of SEQ ID NOs: 1-14, 87, and 88. In some embodiments, the antigen-binding construct comprises: a heavy chain variable (V H ) domains: HCDR1 as SEQ ID NO: 27; HCDR2 as SEQ ID NO: 29; and HCDR3 as SEQ ID NO: 31. In some embodiments, the antigen-binding construct comprises: a heavy chain variable (V H ) domains: HCDR1 being the HCDR1 in any one of SEQ ID NOs: 1-14, 87 and 88; HCDR2 being the HCDR2 in any one of SEQ ID NOs: 1-14, 87 and 88; and HCDR3 being the HCDR3 in any one of SEQ ID NOs: 1-14, 87 and 88.

[0178] In some embodiments, the antigen binding construct comprises a miniantibody. In some embodiments, a miniantibody that binds to DLL3 is provided, wherein the miniantibody comprises: a single chain variable fragment (scFv) that binds to DLL3. In some embodiments, the scFv comprises a single chain variable fragment (V H ) domain of the light chain variable (V L ) domain. LThe domain comprises: LCDR1 as SEQ ID NO: 15; LCDR2 as any one of SEQ ID NO: 19, 21 or 23; and LCDR3 as SEQ ID NO: 25. In some embodiments, the V H The V domain comprises: HCDR1 as SEQ ID NO: 27; HCDR2 as SEQ ID NO: 29; HCDR3 as SEQ ID NO: 31. In some embodiments, the V L comprising: LCDR1 as LCDR1 in any one of SEQ ID NOs: 1-14, 90 and 91; LCDR2 as LCDR2 in any one of SEQ ID NOs: 1-14, 90 and 91; LCDR3 as LCDR3 in any one of SEQ ID NOs: 1-14, 90 and 91; and the V H comprising: a HCDR1 that is the HCDR1 in any one of SEQ ID NOs: 1-14, 87, and 88; a HCDR2 that is the HCDR2 in any one of SEQ ID NOs: 1-14, 87, and 88; and a HCDR3 that is the HCDR3 in any one of SEQ ID NOs: 1-14, 87, and 88. In some embodiments, the minibody comprises: a heavy chain variable (V H ) domains: HCDR1 as SEQ ID NO: 27; HCDR2 as SEQ ID NO: 29; and HCDR3 as SEQ ID NO: 31. In some embodiments, the minibody comprises: a heavy chain variable (V H ) domain: HCDR1 as HCDR1 in any one of SEQ ID NOs: 1-14, 87 and 88; HCDR2 as HCDR2 in any one of SEQ ID NOs: 1-14, 87 and 88; and HCDR3 as HCDR3 in any one of SEQ ID NOs: 1-14, 87 and 88. The antigen-binding construct further comprises a hinge-extension domain comprising an IgG hinge region, and an IgG C H 3 sequences.

[0179] In some embodiments, the antigen binding construct comprises a cys-diabody. In some embodiments, a cys-diabody that binds to DLL3 is provided, wherein the cys-diabody comprises a polypeptide comprising: a single chain variable fragment (scFv) comprising a heavy chain variable (V H ) domain of the light chain variable (V L ) domain. In some embodiments, the V LThe domain comprises: LCDR1 as SEQ ID NO: 15; LCDR2 as any one of SEQ ID NO: 19, 21 or 23; and LCDR3 as SEQ ID NO: 25. In some embodiments, the V H The V domain comprises: HCDR1 as SEQ ID NO: 27; HCDR2 as SEQ ID NO: 29; and HCDR3 as SEQ ID NO: 31. In some embodiments, the V L comprising: LCDR1 as LCDR1 in any one of SEQ ID NOs: 1-14, 90 and 91; LCDR2 as LCDR2 in any one of SEQ ID NOs: 1-14, 90 and 91; LCDR3 as LCDR3 in any one of SEQ ID NOs: 1-14, 90 and 91; and the V H comprising: a HCDR1 that is a HCDR1 in any one of SEQ ID NOs: 1-14, 87, and 88; a HCDR2 that is a HCDR2 in any one of SEQ ID NOs: 1-14, 87, and 88; and a HCDR3 that is a HCDR3 in any one of SEQ ID NOs: 1-14, 87, and 88. In some embodiments, the V L The domain comprises: LCDR1 as SEQ ID NO: 15; LCDR2 as any one of SEQ ID NO: 19, 21 or 23; and LCDR3 as SEQ ID NO: 25, and the V H The domain comprises: HCDR1 as SEQ ID NO: 27; HCDR2 as SEQ ID NO: 29; and HCDR3 as SEQ ID NO: 31. In some embodiments, the cys-diabody comprises: a heavy chain variable (V H ) domains: HCDR1 as SEQ ID NO: 27; HCDR2 as SEQ ID NO: 29; and HCDR3 as SEQ ID NO: 31. In some embodiments, the cys-diabody comprises: a heavy chain variable (V H ) domains: HCDR1 being the HCDR1 in any one of SEQ ID NOs: 1-14, 87 and 88; HCDR2 being the HCDR2 in any one of SEQ ID NOs: 1-14, 87 and 88; and HCDR3 being the HCDR3 in any one of SEQ ID NOs: 1-14, 87 and 88.

[0180] Figure 1Some embodiments of polypeptide sequences of antigen-binding minibodies are shown (SEQ ID NOs: 1-10).

[0181] In some embodiments, the antigen-binding construct, minibody or cys-diabody comprises CDRs that are the CDRs in Table 1.

[0182] Table 1

[0183]

[0184]

[0185] Figure 4 Some embodiments of annotated polypeptide sequences of antigen-binding minibodies are shown.

[0186] Figure 5 Some embodiments of the variable region alignment of the polypeptide sequence of the antigen-binding minibody and the lovastatin polypeptide sequence are shown. In some embodiments, an antigen-binding construct, minibody or cys-diabody is provided, which includes a sequence having 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 3, wherein V L Residue number 50 is Y. In some embodiments, an antigen-binding construct, minibody or cys-diabody is provided, which includes a sequence having 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 3, wherein V L Residue number 55 is N.

[0187] Figure 6 An embodiment of the polypeptide sequence of human-DLL3 is shown. (SEQ ID NO: 50). Figure 6 In some embodiments, the antigen binding construct, minibody and / or cys-diabody binds to a protein having an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to DLL3 as SEQ ID NO: 50 (with or without a signal peptide). In some embodiments, the antigen binding construct, minibody and / or cys-diabody binds to a protein having an amino acid sequence as set forth in SEQ ID NO: 50 without a signal peptide.

[0188] In some embodiments, the V L The domain further comprises LFR2 as shown in SEQ ID NO: 35 or 37. In some embodiments, the V LThe domain comprises the same V as in SEQ ID NO: 48 L The domains have an amino acid sequence that is at least 90% identical; and wherein the antigen-binding construct, minibody or cys-diabody comprises a Y50D and / or Y55N mutation, as numbered according to the numbering in SEQ ID NO: 1 (Kabat).

[0189] In some embodiments, the V L The domain further comprises LFR2 as shown in SEQ ID NO: 35 or 37. In some embodiments, the V L The domain comprises the V in SEQ ID NO: 49 L The domains have an amino acid sequence that is at least 90% identical; and wherein the antigen-binding construct, minibody or cys-diabody comprises a Y50D and / or Y55N mutation, as numbered according to the numbering in SEQ ID NO: 1 (Kabat).

[0190] In some embodiments, the V L The domain comprises the same V as in SEQ ID NO: 48 L In some embodiments, the V domain has an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical, wherein residue 43 is S, as numbered according to the numbering in SEQ ID NO: 1 (Kabat). L The domain comprises the same V as in SEQ ID NO: 48 L The domain has an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical, wherein residue 45 is R, as numbered according to the numbering in SEQ ID NO: 1 (Kabat).

[0191] In some embodiments, the V L The domain comprises the V in SEQ ID NO: 49 L In some embodiments, the V domain has an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical, wherein residue 43 is S, as numbered according to the numbering in SEQ ID NO: 1 (Kabat). L The domain comprises the V in SEQ ID NO: 49 LThe domain has an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical, wherein residue 45 is R, as numbered according to the numbering in SEQ ID NO: 1 (Kabat).

[0192] Figure 2 Some embodiments of polypeptide sequences of antigen-binding cys-diabodies are shown. (SEQ ID NOs: 11-14). In some embodiments, the cys-diabodies have at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs: 11-14. In some embodiments, the cys-diabodies have at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 11. In some embodiments, the cys-diabodies have at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 12. In some embodiments, the cys-diabody has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 13. In some embodiments, the cys-diabody has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 14.

[0193] In some embodiments, the antigen binding construct, minibody or cys-diabody comprises a FR that is a FR in Table 2. In some embodiments, the FR has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with a FR that is any one of SEQ ID NOs: 33-38. In some embodiments, the FR has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with a FR that is SEQ ID NO: 33. In some embodiments, the FR has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with a FR that is SEQ ID NO: 34. In some embodiments, the FR has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the FR that is SEQ ID NO: 35. In some embodiments, the FR has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the FR that is SEQ ID NO: 36. In some embodiments, the FR has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the FR that is SEQ ID NO: 37. In some embodiments, the FR has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the FR of SEQ ID NO:38.

[0194] Table 2

[0195]

[0196] Figure 3 LCDR (SEQ ID NO: 15-26), HCDR (SEQ ID NO: 27-32), LFR (SEQ ID NO: 33-38), linker (SEQ ID NO: 39-44), parent V H (SEQ ID NO: 47) and parent V L Some non-limiting embodiments of the polypeptide sequence of (SEQ ID NO: 48).

[0197] In some embodiments, antigen-binding constructs, miniantibodies or cys-diabodies have surface-exposed, positively charged amino acid destruction clusters, and compared with the original constructs with surface-exposed, positively charged amino acid original clusters, have enhanced biodistribution and / or pharmacokinetics, and the constructs with the destruction clusters are derived (derived) from the original constructs (e.g., only by destroying the original clusters in the original antibodies, while other sequence-based features can remain the same). In some embodiments, compared with the original constructs with intact positively charged amino acid clusters, antigen-binding constructs, miniantibodies or cys-diabodies have reduced renal uptake when administered to a subject. In some embodiments, the antigen-binding construct has a molecular weight of 15-160 kDa. In some embodiments, the antigen-binding construct has a molecular weight of about 50-80 kDa. In some embodiments, for antigen-binding constructs with a molecular weight of ~50-80 kDa (as dimers), such as miniantibodies or cys-diabodies, changes in biodistribution due to the destruction of the clusters are easily observed. Typically, the original antigen-binding construct is modified by disrupting the positively charged amino acid clusters to maintain at least the same binding specificity and binding affinity as the original antigen-binding construct while enhancing biodistribution and / or pharmacokinetics.

[0198] In some embodiments, the antigen-binding construct, minibody or cys-diabody is a variant comprising at least one disrupted surface-exposed positively charged amino acid cluster, the original cluster having at least two (e.g., 2, 3, 4, 5, 6 or more) surface-exposed positively charged amino acids within about 30 angstroms of each other, wherein the variant is different from the original antigen-binding construct comprising the original cluster by replacing at least one surface-exposed positively charged amino acid of the original cluster with one or more negatively charged or uncharged amino acids, whereby the original positively charged cluster is disrupted (compared to the original). In some embodiments, the original cluster has at least two (e.g., 2, 3, 4, 5, 6 or more) surface-exposed positively charged amino acids, which are within or about 30, 25, 20, 15, 10, 8 or 5 angstroms of each other, or within a distance within a range defined by any two of the above values ​​(e.g., 5-30 angstroms, 5-25 angstroms, 5-15 angstroms, 10-20 angstroms, etc.). In some embodiments, the variant antigen-binding construct exhibits reduced renal uptake when compared to the original antigen-binding construct (eg, when the variant antigen-binding construct is radiolabeled and administered to a subject).

[0199] In some embodiments, an antigen-binding construct, minibody, or cys-diabody comprises at least one disrupted cluster of surface-exposed positively charged amino acids, the original cluster having at least two (e.g., 2, 3, 4, 5, 6, or more) surface-exposed positively charged amino acids within about 30 angstroms of each other, wherein the variant differs from the original binding construct comprising the original cluster by replacing at least one surface-exposed positively charged amino acid of the original cluster with one or more negatively charged or uncharged amino acids, whereby the positively charged cluster is disrupted (compared to the binding construct). In some embodiments, an antigen-binding construct, minibody, or cys-diabody comprises a V having a variant framework region 2 (FR2): L, the variant framework region 2 (FR2) has been modified from an original construct comprising the original sequence X1X2X3X4X5X6X7 (SEQ ID NO: 92), wherein X1 is a positively charged amino acid (e.g., lysine or arginine), wherein X2, X3, X5, and X6 are each independently any negatively charged or uncharged amino acid, and wherein X4 and X7 are each independently any amino acid, provided that at least one is a positively charged amino acid, wherein the sequence is outside any CDR of the antigen-binding construct, and wherein at least one of X4 and X7, which are positively charged amino acids, is surface exposed and is part of the original cluster. In some embodiments, X1 is part of the original cluster. In some embodiments, X1 and at least one of X4 and X7, which are positively charged amino acids, are part of the original cluster. In some embodiments, all positively charged amino acids of the original sequence are part of the original cluster. In some embodiments, X2, X3, X5, and X6 are each independently any uncharged amino acid. Antigen binding constructs, such as variant antigen binding constructs, can include the original sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 92), wherein at least one surface-exposed positively charged amino acid of the cluster is replaced with a negatively charged or uncharged amino acid that disrupts the original cluster. In some embodiments, the negatively charged or uncharged amino acid used to replace at least one surface-exposed, positively charged amino acid of the original cluster is glutamine (e.g., a K to Q or R to Q substitution). In some embodiments, the framework region 2 (FR2) of the original antigen-binding construct includes at least one of the following original sequences: KX2X3KX5X6K (SEQ ID NO: 143), wherein X2, X3, X5 and X6 are independently any negatively charged or uncharged amino acids; KX2X3X4X5X6R (SEQ ID NO: 144), wherein X2, X3, X4, X5 and X6 are independently any negatively charged or uncharged amino acids; or KX2X3X4X5X6K (SEQ ID NO: 145), wherein X2, X3, X4, X5 and X6 are independently any negatively charged or uncharged amino acids.In some embodiments, the framework region 2 (FR2) of the original antigen-binding construct includes at least one of the following sequences: KPGKAPK (SEQ ID NO: 94), KPGQAPR (SEQ ID NO: 95), KPEKAPK (SEQ ID NO: 96), KPGKVPK (SEQ ID NO: 97), KPGQPPR (SEQ ID NO: 98), KPGQSPR (SEQ ID NO: 99), KPGLAPR (SEQ ID NO: 100) or KPGQPPK (SEQ ID NO: 101), corresponding to X1X2X3X4X5X6X7 (SEQ ID NO: 92). In some embodiments, the original sequence is KPGKAPK (SEQ ID NO: 94) or KPGQAPR (SEQ ID NO: 95), corresponding to X1X2X3X4X5X6X7 (SEQ ID NO: 92). In some embodiments, the antigen binding construct, minibody or cys-diabody includes at least one of KPGQAPR (SEQ ID NO: 95), KPGQAPK (SEQ ID NO: 146), KPGQAPQ (SEQ ID NO: 111), KPGQSPQ (SEQ ID NO: 110) and QQKPGQSPQ (SEQ ID NO: 147), for example, in V. L FR2.

[0200] In some embodiments, the antigen-binding construct, minibody or cys-diabody further comprises a signal peptide that is the signal peptide in SEQ ID NO:45.

[0201] In some embodiments, the antigen-binding construct, minibody or cys-diabody comprises a linker as any one of SEQ ID NOs: 39-44 (e.g., V of the antigen-binding construct is connected to L Domain and V H In some embodiments, the antigen-binding construct, minibody or cys-diabody comprises a linker that is a linker in Table 3.

[0202] Table 3

[0203]

[0204]

[0205] Table A shows the antigen-binding constructs (e.g., minibodies, scFv-Fc, -Fc) of some non-limiting embodiments of hinge sequences. In some embodiments, the antigen binding construct (e.g., miniantibodies, scFv-Fc, -Fc) includes a hinge region having any of the sequences listed in Table A. In some embodiments, the antigen binding construct (e.g., miniantibodies, scFv-Fc, -Fc) comprises a hinge region having an upper hinge, a core hinge and a lower hinge, wherein the upper hinge, the core hinge and the lower hinge comprise the sequence of any one of the upper hinge, the core hinge and the lower hinge as shown in Table A, respectively. In some embodiments, the antigen binding construct (e.g., miniantibody, scFv-Fc, -Fc) comprises a hinge region having a sequence of any one of the complete hinge sequences shown in Table A. In some embodiments, the antigen-binding construct comprises an upper hinge having the sequence EPKSSDKTHT (SEQ ID NO: 102). In some embodiments, the antigen-binding construct comprises an upper hinge having the sequence EPGSSDGTHT (SEQ ID NO: 103). In some embodiments, the antigen-binding construct comprises a core hinge having the sequence CPPCPPC (SEQ ID NO: 104). In some embodiments, the antigen-binding construct comprises a core hinge having the sequence CPPCP (SEQ ID NO: 105). In some embodiments, the antigen-binding construct comprises a lower hinge having at least one of the following sequences: APELLGGP (SEQ ID NO: 106), GGGSSGGGSG (SEQ ID NO: 107), and APPVAGP (SEQ ID NO: 109).

[0206] Table A

[0207]

[0208]

[0209] Fig.14 Shows C H 3 Some non-limiting embodiments of polypeptide sequences (SEQ ID NOs: 65-85).

[0210] In some embodiments, the antigen-binding construct, minibody or cys-diabody comprises a C as in Table 4 H 3 domain C H 3 domain. In some embodiments, the C H The 3 domain is IgG C H 3 domains. In some embodiments, IgG C HThe 3 domains are IgG1, IgG2, IgG3 and / or IgG4 C H 3 domains. In some embodiments, C H 3 domains contain germline C H 3 domain. In some embodiments, the C H The 3 domains comprise one or more allotypes. In some embodiments, the IgG1 C H 3 domain is any one of SEQ ID No.65-68. In some embodiments, C H 3 domain and C as any one of SEQ ID NOs: 65-68 H In some embodiments, the C H 3 domain and C as SEQ ID NO: 65 H The hinge region has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the C3 domain of SEQ ID NO: 66. H In some embodiments, the C H 3 domain and C as SEQ ID NO: 67 H In some embodiments, the C H 3 domain and C as SEQ ID NO: 68 H In some embodiments, the C H The 3 domain is IgG2 C H 3 domains. In some embodiments, IgG2 C H 3 domain is any one of SEQ ID No.69-71. In some embodiments, C H 3 domain and C as any one of SEQ ID NOs: 69-71 H In some embodiments, the CH 3 domain and C as SEQ ID NO: 69 H In some embodiments, the C H 3 domain and C as SEQ ID NO: 70 H In some embodiments, the C H 3 domain and C as SEQ ID NO: 71 H In some embodiments, the hinge region is an IgG3 C domain. H 3 domains. In some embodiments, IgG3 C H 3 domain is any one of SEQ ID No.72-82. In some embodiments, C H 3 domain and C as any one of SEQ ID NOs: 72-82 H In some embodiments, the C H 3 domain and C as SEQ ID NO: 72 H In some embodiments, the C H 3 domain and C as SEQ ID NO: 73 H In some embodiments, the C H 3 domain and C as SEQ ID NO: 74 H In some embodiments, the C H 3 domain and C as SEQ ID NO: 75 HIn some embodiments, the C H 3 domain and C as SEQ ID NO: 76 H In some embodiments, the C H 3 domain and C as SEQ ID NO: 77 H In some embodiments, the C H 3 domain and C as SEQ ID NO: 78 H In some embodiments, the C H 3 domain and C as SEQ ID NO: 79 H In some embodiments, the C H 3 domain and C as SEQ ID NO: 80 H In some embodiments, the C H 3 domain and C as SEQ ID NO: 81 H In some embodiments, the C H 3 domain and C as SEQ ID NO: 82 H In some embodiments, the C H The 3 domain is IgG2 C H 3 domains. In some embodiments, IgG2C H 3 domain is any one of SEQ ID No.83-85. In some embodiments, C H3 domain and C as any one of SEQ ID NOs: 83-85 H In some embodiments, the C H 3 domain and C as SEQ ID NO: 83 H In some embodiments, the C H 3 domain and C as SEQ ID NO: 84 H In some embodiments, the C H 3 domain and C as SEQ ID NO: 85 H The 3 domains have at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0211] Table 4.

[0212]

[0213]

[0214]

[0215] In some embodiments, the antigen binding construct comprises an Fc region (e.g., scFv-Fc, -Fc). In some embodiments, the antigen-binding construct comprises an amino acid sequence identical to the amino acid sequence of a native or naturally occurring Fc region (e.g., a human IgG1 Fc region). In some embodiments, the Fc region comprises the amino acid sequence shown in SEQ ID NO: 109 as shown below.

[0216]

[0217] In some embodiments, the Fc region comprises an amino acid sequence that is at least 70, 80, 90, 95, 96, 97, 98, 99, or about 100% identical to SEQ ID NO: 109, or a percentage within a range defined by any two of the above values ​​(e.g., 80-100%, 85-97%, 85-95%, 90-100%, etc.). In some embodiments, the Fc region comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) mutations (e.g., substitutions) that alter (e.g., increase or decrease) effector function (e.g., FcγR binding) and / or binding to an Fc neonatal receptor (FcRn). In some embodiments, the Fc region comprises the amino acid sequence set forth in SEQ ID NO: 109, wherein one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) mutations (e.g., substitutions) alter (e.g., increase or decrease) effector function (e.g., FcγR binding) and / or binding to the Fc neonatal receptor (FcRn). In some embodiments, the Fc region comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) mutations (e.g., substitutions) that reduce effector function (e.g., FcγR binding) and / or binding to the Fc neonatal receptor (FcRn). In some embodiments, the Fc region comprises the amino acid sequence set forth in SEQ ID NO: 109, wherein one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) mutations (e.g., substitutions) reduce effector function (e.g., FcγR binding) and / or binding to the Fc neonatal receptor (FcRn). In some embodiments, the one or more mutations that reduce FcRn binding are mutations (e.g., substitutions) of any one or more (e.g., 1, 2, or all 3) of I253, H310, and H435 (EU numbering). In some embodiments, the one or more mutations that reduce binding to FcRn are any one or more (e.g., 1, 2, or all 3) of I253A, H310A, and H435A (EU numbering). In some embodiments, the Fc region includes one or more (e.g., 1, 2, or all 3) of I253A, H310A, and H435A (EU numbering). In some embodiments, the one or more mutations that reduce binding to FcγR are mutations (e.g., substitutions) of N297 (EU numbering). In some embodiments, the one or more mutations that reduce binding to Fc neoreceptors are N297Q, N297A, or N297G (EU numbering). In some embodiments, the Fc region includes N297Q (EU numbering).In some embodiments, the one or more mutations that reduce Fc effector function are mutations (e.g., substitutions) of any one or more of the following: L234, L235, G236, G237, P238, H268, K322, L328, P329, A330, P331. In some embodiments, the one or more mutations that reduce Fc effector function are any one or more of the following: L234A, L235A, G236R, G237A, P238S, H268A, K322A, L328R, P329G, A330S, P331S. Other non-limiting examples of known mutations that alter Fc effector function can be found in: Wilkinson and Hale (2022) Systematic analysis of the varied designs of 819 therapeutic antibodies and Fc fusion proteins assigned international nonproprietary names. MAbs. 2022 Jan-Dec; 14(1): 2123299. doi: 10.1080 / 19420862.2022.2123299. In some embodiments, the Fc region does not include a C-terminal lysine.

[0218] In some embodiments, the antigen binding construct is a scFv-Fc. In some embodiments, the scFv-Fc comprises any of the scFvs that bind to DLL3 (e.g., as described herein, having a heavy chain variable (V H ) domain connected to the light chain variable (V L ) domain), hinge domain, and Fc region. In some embodiments, the scFv-Fc includes any of the scFvs that bind to DLL3 (e.g., as described herein, having a heavy chain variable (V H ) domain connected to the light chain variable (V L ) domain), as described herein, the scFv is connected to the Fc region via a hinge domain. In some embodiments, the hinge domain is any suitable hinge domain as described herein (e.g., Table A). In some embodiments, the antigen binding construct is -Fc (or a single domain fragment fused to an Fc region). In some embodiments, -Fc comprises a variable domain (VHH) having any one or more of HCDR1, HCDR2 and HCDR3 sequences, as described herein. In some embodiments, -Fc comprises a variable domain (VHH) having HCDR1 as SEQ ID NO: 27; HCDR2 as SEQ ID NO: 29; and HCDR3 as SEQ ID NO: 31. In some embodiments, -Fc comprises a variable domain (VHH) having a HCDR1 that is a HCDR1 in any one of SEQ ID NOs: 1-14, 87, and 88; a HCDR2 that is a HCDR2 in any one of SEQ ID NOs: 1-14, 87, and 88; and a HCDR3 that is a HCDR3 in any one of SEQ ID NOs: 1-14, 87, and 88. In some embodiments, -Fc comprises a V region connected to an Fc region via a hinge domain as described herein H domain, the V H The domain has any one or more (eg, any 1, 2, or all 3) of the HCDR1, HCDR2, and HCDR3 sequences as described herein.

[0219] In some embodiments, antigen-binding constructs include at least one modification. Exemplary modifications include but are not limited to antigen-binding constructs modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, conjugation with metal chelators, fluorescent or infrared dyes, or conjugation with toxins, proteolytic cleavage, and connection with cell ligands or other proteins. Any of a variety of chemical modifications can be carried out by known techniques, including but not limited to the metabolic synthesis of amine-containing amino acids, cystine coupling, specific chemical cleavage, acetylation, and tunicamycin. In some embodiments, the derivative may contain one or more non-natural amino acids.

[0220] In some embodiments, the antigen-binding construct is conjugated to another substance to form an anti-target conjugate. The conjugate described herein (conjugate, conjugate) can be prepared by a known method of connecting the antigen-binding construct to a lipid, carbohydrate, protein or other atoms and molecules. In some embodiments, the conjugate is formed by using a site-specific conjugation of a suitable bonding or bond. Site-specific conjugation is more likely to retain the binding activity of the antigen-binding construct. The substance can be conjugated or connected to the hinge region of the reduced antigen-binding construct through thioether bond formation. In some embodiments, tyrosine conjugation can be used. Other bonding or bonds for forming a conjugate may include but are not limited to covalent bonds, non-covalent bonds, disulfide bonds, hydrazone bonds, ester bonds, amide bonds and amino bonds, imino bonds, thiosemicarbazone bonds (thiosemicarbazone linkage), semicarbazone bonds (semicarbazone linkage), oxime bonds and carbon-carbon bonds. In some embodiments, cysteine ​​or other connection aspects do not need to be included in the antigen-binding construct.

[0221] In some embodiments, the antigen binding construct, minibody and / or cys-diabody are conjugated to a chemotherapeutic agent. Chemotherapeutic agents typically have cytotoxic or cytostatic properties and may include alkylating agents, antimetabolites, antitumor antibiotics, local isomerase inhibitors, mitotic inhibitors, hormone therapy (hormonal therapeutic agents), targeted therapy (targeted therapeutic agents) and immunotherapy (immunotherapeutic agents). In some embodiments, the chemotherapeutic agent that can be used as a detectable marker according to embodiments of the present disclosure is selected from those chemotherapeutic agents defined elsewhere herein.

[0222] In some embodiments, the antigen binding construct, minibody and / or cys-diabody are conjugated to a toxin. Toxins that can be used according to embodiments of the present disclosure include, but are not limited to, auristatin E, auristatin F, dolastatin 10, dolastatin 15, combretastatin and its analogs, maytansinoid, calicheamicin, alpha-amanitin, pyrrolobenzodiazepine dimer, epothilone, duocartoxin and its analogs, tubulysin D, basillistatins, ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcus enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin and Pseudomonas endotoxin.

[0223] In some embodiments, the antigen binding construct, miniantibody and / or cys-diabody are conjugated to a radioactive label. In some embodiments, the radioactive label includes iodine-131, a beta-emitter or an alpha-emitter, such as yttrium-90, copper-67, terbium-149, terbium-161, lutetium-177, astatine-211, lead-212, bismuth-212, actinium-225, bismuth-213 or 227-thorium, a positron emitter, such as zirconium-89, copper-64, gallium-68, fluorine-18, indium-111, iodine-124. In some embodiments, the radioactive label includes terbium-149, terbium-161 or lead-212. In some embodiments, the antigen binding construct, miniantibody and / or cys-diabody are conjugated to a gamma emitter. In some embodiments, the radioactive label comprises a gamma emitter.

[0224] In some embodiments, the antigen binding construct, minibody and / or cys-diabody is conjugated to a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator or an enzyme. In some embodiments, at least one payload comprises 18 F. 18 F-FAC, 32 P. 33 P. 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y. 90 Y. 89 Sr. 89 Zr, 94 Tc, 94 Tc, 99 mTc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I. 124 I. 125 I. 131 I. 142 Pr, 143 Pr, 149 Pm, 149 Tb, 153 Sm, 154-158 Gd, 161 Tb,166 Dy, 166 Ho, 169 2. 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra, 227 Th and 225 Ac or any combination thereof. In some embodiments, at least one payload comprises 149 Tb, 161 Tb or 212 Pb.

[0225] In some embodiments, the antigen binding construct is an antibody (~140-170 kDa), a multispecific antibody (~100 kDa and larger), a one-armed antibody or one-armed antibody (~110 kDa and smaller), a modified antibody, etc. In some embodiments, the antigen binding construct, minibody, or cys-diabody specifically binds to DLL3. In some embodiments, the antigen binding construct, minibody, or cys-diabody is bispecific. In some embodiments, the antigen binding construct, minibody, or cys-diabody is bivalent. In some embodiments, the antigen binding construct, minibody, or cys-diabody comprises a monovalent scFv. In some embodiments, the antigen binding construct is a scFv-Fc (e.g., a scFv fused to an Fc). In some embodiments, the antigen binding construct is -Fc (e.g., fused to Fc (or single domain fragments)).

[0226] In some embodiments, the order of the variable domains of the antigen-binding construct, minibody or cys-diabody (from the N-terminus to the C-terminus of the polypeptide) is V L , V H In some embodiments, the variable domains are in the order of V from the N-terminus to the C-terminus of the polypeptide. H , V L .

[0227] In some embodiments, the antigen-binding construct, miniantibody or cys-diabody is a humanized antigen-binding construct, miniantibody or cys-diabody. Figure 1-6 The antigen-binding construct, minibody and / or cys-diabody of any one of the above can be reconstituted from the antigen-binding construct, minibody or cys-diabody into an antigen-binding construct, minibody and / or cys-diabody. Figure 1 The minibodies shown can be reconstituted as antigen-binding constructs or cys-diabodies. Figure 2 The cys-diabodies shown can be reconstituted as antigen binding constructs or minibodies. For example, Figure 1 and 2 The minibodies or cys-diabodies shown in can be reconstituted into scFv-Fc.

[0228] In some embodiments, the miniantibody that binds to DLL3 comprises: a single chain variable fragment (scFv) that binds to DLL3; a hinge region; an IgG C H 3 sequence; and a therapeutic agent, a toxic payload and / or a detectable marker, the scFv comprising a heavy chain variable (V H ) domain of the light chain variable (V L ) domain, the V L The domain comprises: LCDR1 as SEQ ID NO: 15; LCDR2 as any one of SEQ ID NO: 19, 21 or 23; LCDR3 as SEQ ID NO: 25, wherein the V H The domain comprises: HCDR1 as SEQ ID NO:27; HCDR2 as SEQ ID NO:29; HCDR3 as SEQ ID NO:31.

[0229] In some embodiments, a cys-diabody that binds to DLL3 comprises: a polypeptide comprising: a single-chain variable fragment (scFv); an extended sequence; and a therapeutic agent, a toxic payload, and / or a detectable marker, wherein the single-chain variable fragment comprises a heavy chain variable (V H ) domain connected to the light chain variable (V L ) domain; said V L The domain comprises: LCDR1 of LCDR1 shown in SEQ ID NO: 15; LCDR2 of LCDR2 shown in SEQ ID NO: 19, 21 or 23; LCDR3 of LCDR3 shown in SEQ ID NO: 25; V HThe domain comprises: HCDR1 of HCDR1 shown in SEQ ID NO:27; HCDR2 of HCDR2 shown in SEQ ID NO:29; and HCDR3 of HCDR3 shown in SEQ ID NO:31.

[0230] In some embodiments, the scFv-Fc that binds to DLL3 comprises: a single chain variable fragment (scFv) that binds to DLL3; a hinge region; an Fc region; and a therapeutic agent, a toxic payload, and / or a detectable marker, wherein the scFv comprises a heavy chain variable (V H ) domain connected to the light chain variable (V L ) domain, the V L The domain comprises: LCDR1 as SEQ ID NO: 15; LCDR2 as any one of SEQ ID NO: 19, 21 or 23; LCDR3 as SEQ ID NO: 25, wherein the V H The domain comprises: HCDR1 as SEQ ID NO:27; HCDR2 as SEQ ID NO:29; HCDR3 as SEQ ID NO:31.

[0231] In some embodiments, the humanized antigen-binding construct, minibody or cys-diabody comprises: LCDR1 of LCDR1 shown in SEQ ID NO: 15; LCDR2 of LCDR2 shown in any one of SEQ ID NO: 19, 21 or 23; LCDR3 of LCDR3 shown in SEQ ID NO: 25; HCDR1 of HCDR1 shown in SEQ ID NO: 27; HCDR2 of HCDR2 shown in SEQ ID NO: 29; and HCDR3 of HCDR3 shown in SEQ ID NO: 31.

[0232] In some embodiments, the V L The domain comprises or further comprises LFR2 of LFR2 shown in SEQ ID NO:35 or 37.

[0233] nature

[0234] Figure 7 is a bar graph showing an embodiment of minibody expression yields in Expi293 mammalian cells.

[0235] In some embodiments, the expression yield of the antigen-binding construct, miniantibody and / or cys-diabody is determined using ultraviolet-visible spectroscopy (UV-Vis or UV / Vis). In some embodiments, the expression yield of the antigen-binding construct, miniantibody and / or cys-diabody is determined using chromatographic techniques. In some embodiments, the expression yield of the antigen-binding construct, miniantibody and / or cys-diabody is determined using biofilm interferometry. In some embodiments, the expression yield of the antigen-binding construct, miniantibody or cys-diabody is about or at least 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 110, 120, 125, 150, 175, 200, 250, 300, 350, 400, 450 or 500 mg / L, or the yield is within the range defined by any two of the above values. For example, in some embodiments, the expression yield of the antigen-binding construct, miniantibody or cys-diabody is between about 1 to 500 mg / L, 1 to 350 mg / L, 1 to 200 mg / L, 1 to 100 mg / L, 1 to 50 mg / L, 1 to 25 mg / L, 5 to 500 mg / L, 5 to 350 mg / L, 5 to 200 mg / L, 5 to 100 mg / L, 5 to 50 mg / L, 5 to 25 mg / L, 25 to 500 mg / L, 25 to 350 mg / L, 25 to 20 mg / L, 25 to 100 mg / L or 25 to 50 mg / L. In some embodiments, the expression yield of the antigen-binding construct, miniantibody and / or cys-diabody is determined relative to the expression yield of the parent antigen-binding construct, miniantibody and / or cys-diabody. For example, in some embodiments, the expression yield of the antigen-binding construct, miniantibody or cys-diabody is about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times higher than the expression yield of the parent antigen-binding construct, miniantibody and / or cys-diabody, or an increase in the range defined by any two of the above values. For example, in some embodiments, the expression yield of the antigen-binding construct, miniantibody and / or cys-diabody is 1 to 10 times, 1 to 8 times, 1 to 5 times, 1 to 3 times, 3 to 10 times, 3 to 8 times, 3 to 5 times, 5 to 10 times or 5 to 8 times higher than the expression yield of the parent antigen-binding construct, miniantibody and / or cys-diabody.

[0236] Fig.11 is a bar graph showing some embodiments of the in vivo biodistribution of antigen-binding minibodies in a tumor-bearing mouse model.

[0237] In some embodiments, the antigen-binding construct, minibody or cys-diabody accumulates to detectable levels in the subject's blood, liver, kidney, spleen, lung, muscle, bone, heart, stomach, small intestine, large intestine, pancreas, tumor, or any combination thereof. In some embodiments, the antigen-binding construct is preferentially distributed to the subject's blood, liver, kidney, spleen, lung, muscle, bone, heart, stomach, small intestine, large intestine, pancreas, tumor, or any combination thereof.

[0238] In some embodiments, the antigen binding construct, minibody or cys-diabody is detectable at, about, up to, or up to about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30% or 35% ID / g, or a percentage within the range defined by any two of the above values. For example, in some embodiments, the antigen binding construct, minibody or cys-diabody is detectable at about 0.1% to 35%, 0.1% to 30%, 1% to 35%, 1% to 30%, 5% to 35% or 5% to 30% ID / g.

[0239] In some embodiments, the antigen-binding construct, minibody and / or cys-diabody is distributed to the blood, liver, kidney, spleen, lung, muscle, bone, heart, stomach, small intestine, large intestine, pancreas, tumor or any combination thereof. In some embodiments, the antigen-binding construct, minibody and / or cys-diabody is distributed to the blood, liver, kidney, spleen, lung, muscle, bone, heart, stomach, small intestine, large intestine, pancreas, tumor or any combination thereof at or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95% or 100% ID / g or a percentage within the range defined by any two of the above values. For example, in some embodiments, the antigen binding constructs, minibodies and / or cys-diabodies are distributed to the blood, liver, kidney, spleen, lung, muscle, bone, heart, stomach, small intestine, large intestine, pancreas, tumor, or any combination thereof at an ID / g of between about 1% to 100%, 1% to 75%, 1% to 50%, 1% to 30%, 1% to 25%, 5% to 100%, 5% to 75%, 5% to 50%, 5% to 30%, 5% to 25%, 10% to 100%, 10% to 75%, 10% to 50%, 10% to 30%, or 10% to 25%.

[0240] Fig.12 is a bar graph showing some embodiments of the in vivo biodistribution of antigen-binding minibodies.

[0241] In some embodiments, the antigen-binding construct, minibody and / or cys-diabody is distributed to the blood, liver, kidney, spleen, lung, muscle, bone, heart, stomach, small intestine, large intestine, pancreas, tumor, or any combination thereof. In some embodiments, the antigen-binding construct, minibody and / or cys-diabody is distributed to the blood, liver, kidney, spleen, lung, muscle, bone, heart, stomach, small intestine, large intestine, pancreas, tumor, or any combination thereof at or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95% or 100%, ID / g or a percentage within the range defined by any two of the above values. For example, in some embodiments, the antigen binding constructs, minibodies and / or cys-diabodies are distributed to the blood, liver, kidney, spleen, lung, muscle, bone, heart, stomach, small intestine, large intestine, pancreas, tumor, or any combination thereof at an ID / g of between about 1% to 100%, 1% to 75%, 1% to 50%, 1% to 30%, 1% to 25%, 5% to 100%, 5% to 75%, 5% to 50%, 5% to 30%, 5% to 25%, 10% to 100%, 10% to 75%, 10% to 50%, 10% to 30%, or 10% to 25%.

[0242] In some embodiments, the antigen-binding construct, minibody or cys-diabody comprises LFR2 as SEQ ID NO: 35 or 37; and has improved biodistribution compared to an antigen-binding construct, minibody or cys-diabody comprising LFR2 as SEQ ID NO: 33.

[0243] Fig. 9 is a graph showing some embodiments of determination of equilibrium dissociation constants for antigen binding minibodies.

[0244] In some embodiments, the KD of the antigen-binding construct, minibody, or cys-diabody (eg, KD for binding to a DLL3 protein) is less than about 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M, 10 -14 M or 10 -15M, or KD is within the range defined by any two of the above values. For example, in some embodiments, the KD of the antigen-binding construct, minibody or cys-diabody (e.g., KD for binding to an antigen) is about 10 -5 M to 10 -15 M, 10 -5 M to 10 -12 M, 10 -5 M to 10 -10 M, 10 -7 M to 10 -15 M, 10 -7 M to 10 -12 M, 10 -7 M to 10 -10 M, 10 -10 M to 10 -15 M or 10 -10 M to 10 -12 In some embodiments, the KD of an antibody dissolved in a buffer is determined. In some embodiments, the buffer is a phosphate buffer. If desired, any suitable option, such as biofilm interferometry, can be used to determine the KD.

[0245] Figure 8 EC showing antigen binding of miniantibodies by ELISA 50 Figures of some embodiments of the determination.

[0246] Fig.10 The EC of the antigen-binding miniantibodies was shown by flow cytometry 50 Figures of some embodiments of the invention are identified.

[0247] In some embodiments, the EC of the antigen-binding construct, minibody or cys-diabody 50 (For example, ECs that bind to DLL3 protein, or cells expressing DLL3 protein 50 ) is, is about, is at most or is less than 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19 or 0.20 nM, or EC 50 In the range defined by any two of the above values. For example, in some embodiments, the EC of an antigen-binding construct, minibody, or cys-diabody is 50 (e.g., ECs that bind to antigens or cells expressing antigens 50) is between about 0.001 to 0.20 nM, 0.001 to 0.17 nM, 0.001 to 0.12 nM, 0.01 to 0.20 nM, 0.01 to 0.17 nM, 0.01 to 0.12 nM, 0.05 to 0.20 nM, 0.05 to 0.17 nM, 0.05 to 0.15 nM, 0.05 to 0.12 nM, 0.08 to 0.20 nM, 0.08 to 0.17 nM, or 0.08 to 0.12 nM. In some embodiments, the EC of the antigen-binding construct, minibody, or cys-diabody is between about 0.001 to 0.20 nM, 0.001 to 0.17 nM, 0.01 to 0.12 nM, 0.05 to 0.20 nM, 0.05 to 0.17 nM, 0.05 to 0.15 nM, 0.05 to 0.12 nM. 50 (e.g., ECs that bind to DLL3 protein or cells expressing DLL3 protein 50 ) is up to about 10 nM. If desired, any suitable selection method may be used, for example, using ELISA or flow cytometry to determine EC 50 .

[0248] Therapeutic agents and compositions

[0249] In some embodiments, pharmaceutical composition can also include a pharmaceutically available carrier. Pharmaceutically available carrier can be pharmaceutically available material, composition or vehicle, which participates in carrying or transporting the compound of interest from a tissue, organ or part of health to another tissue, organ or part of health. For example, carrier can be a liquid or solid filler, diluent, excipient, solvent or encapsulating material or some combination thereof. Every kind of component of the carrier is "pharmaceutically available" because it is compatible with other ingredients of the preparation. It is also suitable for contacting any tissue, organ or body part that it may encounter, which means that ideally, it will not have significant toxicity, irritation, allergic reaction, immunogenicity or any other serious risk of complications that exceed its therapeutic benefit.

[0250] In some embodiments, the therapeutic composition comprises: an antigen-binding construct; and a therapeutic agent, a toxic payload, and / or a detectable marker, wherein the antigen-binding construct comprises: a light chain variable (V L ) domain: LCDR1 as SEQ ID NO: 15; LCDR2 as any one of SEQ ID NO: 19, 21 or 23; LCDR3 as SEQ ID NO: 25; and a heavy chain variable (V H ) domains: HCDR1 as SEQ ID NO: 27; HCDR2 as SEQ ID NO: 29; HCDR3 as SEQ ID NO: 31. In some embodiments, the antigen-binding construct is a minibody, which further includes a hinge region (e.g., any of the hinge regions described herein) and an IgG C H 3 sequences (e.g., CH 3 sequences). In some embodiments, the antigen-binding construct is a cys-diabody, which further includes an extension sequence (eg, GGC or GGCPPCPPC (SEQ ID NO: 93)) at the C-terminus of the monomer.

[0251] In some embodiments, the therapeutic composition comprises: a mini-antibody that binds to DLL3; a hinge-extension domain comprising an IgG1 hinge region; an IgG C H 3 sequence; and a therapeutic agent, a toxic payload and / or a detectable marker, the miniantibody comprising: a single chain variable fragment (scFv) that binds to DLL3, the scFv comprising a heavy chain variable (V H ) domain of the light chain variable (V L ) domain, the V L The domain comprises: LCDR1 as SEQ ID NO: 15; LCDR2 as any one of SEQ ID NO: 19, 21 or 23; LCDR3 as SEQ ID NO: 25, and the V H The domain comprises: HCDR1 as SEQ ID NO: 27; HCDR2 as SEQ ID NO: 29; HCDR3 as SEQ ID NO: 31. In some embodiments, the therapeutic composition comprises: a mini-antibody that binds to DLL3; a hinge region; an IgG C H 3 sequence; and a therapeutic agent, a toxic payload and / or a detectable marker, the miniantibody comprising: a single chain variable fragment (scFv) that binds to DLL3, the scFv comprising a heavy chain variable (V H ) domain of the light chain variable (V L ) domain, the V L The domain comprises: LCDR1 as SEQ ID NO: 15; LCDR2 as any one of SEQ ID NO: 19, 21 or 23; LCDR3 as SEQ ID NO: 25, and the V H The domain comprises: HCDR1 as SEQ ID NO:27; HCDR2 as SEQ ID NO:29; HCDR3 as SEQ ID NO:31.

[0252] In some embodiments, the therapeutic composition comprises: a cys-diabody that binds to DLL3; and a therapeutic agent, a toxic payload, and / or a detectable marker, wherein the cys-diabody comprises a polypeptide comprising: a single-chain variable fragment (scFv) comprising a heavy chain variable (V H ) domain connected to the light chain variable (VL ) domain; said V L The domain comprises: LCDR1 as LCDR1 of SEQ ID NO: 15; LCDR2 as LCDR2 of SEQ ID NO: 19, 21 or 23; LCDR3 as LCDR3 of SEQ ID NO: 25; the V H The domain comprises: HCDR1 of HCDR1 as set forth in SEQ ID NO: 27; HCDR2 of HCDR2 as set forth in SEQ ID NO: 29; and HCDR3 of HCDR3 as set forth in SEQ ID NO: 31. In some embodiments, the therapeutic composition comprises: a cys-diabody that binds to DLL3; an extension sequence; and a therapeutic agent, a toxic payload, and / or a detectable marker, the cys-diabody comprising a polypeptide comprising: a single-chain variable fragment (scFv) comprising a heavy chain variable (V H ) domain connected to the light chain variable (V L ) domain; said V L The domain comprises: LCDR1 of LCDR1 shown in SEQ ID NO: 15; LCDR2 of LCDR2 shown in SEQ ID NO: 19, 21 or 23; LCDR3 of LCDR3 shown in SEQ ID NO: 25; V H The domain comprises: HCDR1 of HCDR1 shown in SEQ ID NO:27; HCDR2 of HCDR2 shown in SEQ ID NO:29; and HCDR3 of HCDR3 shown in SEQ ID NO:31.

[0253] In some embodiments, the therapeutic composition comprises one or more of the miniantibody schemes provided in any of the Figures and / or Tables (eg, Table 5).

[0254] In some embodiments, the therapeutic composition comprises one or more of the cys-diabody regimens provided in any of the Figures and / or Tables (eg, Table 6).

[0255] In some embodiments, the therapeutic composition comprises an antigen binding construct, minibody, cys-diabody, VL, VH, CDR, FR, linker, signal peptide, or any combination thereof, which has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one or more of the antigen binding constructs, minibody, cys-diabody, VL, VH, CDR, FR, linker, signal peptides set forth in SEQ ID NO: 1-48. In some embodiments, the therapeutic composition comprises a minibody that is any one of SEQ ID NO: 1-10. In some embodiments, the therapeutic composition comprises a minibody that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a minibody that is any one of SEQ ID NO: 1-10. In some embodiments, the therapeutic composition comprises a cys-diabody that is any one of SEQ ID NOs: 11-14. In some embodiments, the therapeutic composition comprises a cys-diabody that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a cys-diabody that is any one of SEQ ID NOs: 11-14.

[0256] In some embodiments, the therapeutic composition comprises a LCDR that is any one of SEQ ID NOs: 15-26. In some embodiments, the therapeutic composition comprises a LCDR that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a LCDR that is any one of SEQ ID NOs: 15-26. In some embodiments, the therapeutic composition comprises a HCDR that is any one of SEQ ID NOs: 27-32. In some embodiments, the therapeutic composition comprises a HCDR that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a HCDR that is any one of SEQ ID NOs: 27-32. In some embodiments, the therapeutic composition comprises a FR that is any one of SEQ ID NOs: 33-38. In some embodiments, the therapeutic composition comprises a FR having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a FR that is any one of SEQ ID NOs: 33-38. In some embodiments, the therapeutic composition comprises a linker that is any one of SEQ ID NOs: 39-44. In some embodiments, the therapeutic composition comprises a linker that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a linker that is any one of SEQ ID NOs: 39-44. In some embodiments, the therapeutic composition comprises a signal peptide that is any one of SEQ ID NOs: 45-46. In some embodiments, the therapeutic composition comprises a signal peptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a signal peptide that is any one of SEQ ID NOs: 45-46. In some embodiments, the therapeutic composition comprises a VH that is SEQ ID NO: 47. In some embodiments, the therapeutic composition comprises a minibody that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a VH that is SEQ ID NO: 47. In some embodiments, the therapeutic composition comprises a VL that is SEQ ID NO: 48.In some embodiments, the therapeutic composition comprises a VL having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the VL of any one of SEQ ID NO:48.

[0257] Medicine or therapeutic composition described herein can be applied by any suitable route of administration.Route of administration can refer to any route of administration known in the art, including but not limited to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, transdermal (e.g., topical cream or ointment, patch) or vaginal administration. "Transdermal" administration can be completed using topical cream or ointment or by transdermal patch. "Parenteral" refers to the route of administration usually relevant to injection, including suborbital, infusion, intra-arterial, intra-capsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intracranial, intraventricular, intrauterine, intravenous, subarachnoid, subcapsular (subcapsular), subcutaneous, sublingual, transmucosal or transtracheal administration. In some embodiments, antigen-binding constructs can be delivered in surgery as topical administration during intervention or resection.

[0258] In some embodiments, antigen binding constructs, miniantibodies or cys-diabodies are conjugated to therapeutic agents. "Therapeutic agent" as used herein refers to atoms, molecules or compounds useful in treating a disease related to a target molecule. Examples of therapeutic agents include, but are not limited to, drugs, chemotherapeutics, therapeutic antibodies and antibody fragments, toxins, radioisotopes, enzymes (e.g., enzymes that cut prodrugs into cytotoxic agents at antigen binding construct binding sites), nucleases, hormones, immunomodulators, antisense oligonucleotides, chelating agents, boron compounds, photoactive agents and dyes, elastin-like polypeptides such as PLGA and nanoparticles. Examples of disease include those diseases associated with one or more target molecules.

[0259] Chemotherapeutic agents generally have cytotoxic or cytostatic properties and may include alkylating agents, antimetabolites, antitumor antibiotics, local isomerase inhibitors, mitotic inhibitors, hormone therapy (hormonal therapeutic agents), targeted therapy (targeted therapeutic agents) and immunotherapy (immunotherapeutic agents). In some embodiments, chemotherapeutic agents that can be used as detectable markers according to embodiments of the present disclosure include, but are not limited to, 13-cis-retinoic acid, 2-chlorodeoxyadenosine, 5-azacitidine, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine, actinomycin-D, adriamycin, aldesleukin, alemtuzumab, alitretinoin, all-transretinoic acid, acid), interferon alpha, altretamine, methotrexate, amifostine, anagrelide, anastrozole, arabinosylcytosine, arsenic trioxide, amsacrine, aminocamptothecin, aminoglutethimide, asparaginase, azacytidine, BCG, bendamustine, bevacizumab, bexarotene, bicalutamide, bortezomib, bleomycin, busulfan, calcium leucovorin, citrovorum factor, capecitabine, canertinib, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, cladribine, cortisone, cyclophosphamide, cytarabine, darbepoetin alfa alfa), dasatinib, daunomycin, decitabine, denileukin diftitox, dexamethasone, dexamethasone sodium phosphate (dexasone), dexrazoxane, dactinomycin, daunorubicin, decarbazine, docetaxel, doxorubicin, doxifluridine, eniluracil, epirubicin, epoetin alfa, erlotinib, everolimus, exemestane, estramustine, etoposide, filgrastim, fluoxymesterone, fulvestrant, flavopiridol, floxuridine, fludarabine, fluorouracil, flutamide, gefitinib, gemcitabine, gemtuzumab ozogamicin ozogamicin), goserelin, granulocyte-colony stimulating factor, granulocyte-macrophage-colony stimulating factor, hexamethonium, hydrocortisone hydroxyureahydroxyurea), ibritumomab, interferon alpha, interleukin-2, interleukin-11, isotretinoin, ixabepilone, idarubicin, imatinib mesylate, ifosfamide, irinotecan, lapatinib, lenalidomide, letrozole, leucovorin, leuprolide, liposomal cytarabine Ara-C), lomustine, nitrogen mustard, megestrol acetate, melphalan, mercaptopurine, mesna, methotrexate, methylprednisolone, mitomycin C, mitotane, mitoxantrone, nelarabine, nilutamide, octreotide, oprelvekin, oxaliplatin, paclitaxel, pamidronate, pemetrezole, panitumumab, PEG interferon, pegaspargase, pegfilgrastim, PEG-L-asparaginase, pentostatin, plicamycin, prednisolone, prednisone, procarbazine, raloxifene, rituximab, romiplostim ( romiplostim), ralitrexed, sapacitabine, sargramostim, satraplatin, sorafenib, sunitinib, semustine, streptozocin, tamoxifen, tegafur, tegafur-uracil, temsirolimus, temozolomide, teniposide, thalidomide, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, trimetrexate, alrubicin, vincristine, vinblastine, vindesine, vinorelbine, vorinostat, or zoledronic acid.

[0260] Toxins that can be used according to embodiments of the present disclosure include, but are not limited to, auristatin E, auristatin F, dolestadin 10, dolestadin 15, combretastatin and its analogs, matanamicin, calicheamicin, α-amanitin, pyrrolobenzodiazepine dimer, epothilone, duocartoxin and its analogs, tubulysin D, basillistatins, ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, American pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin and Pseudomonas endotoxin.

[0261] In some embodiments, the nanoparticles are used as drug carriers in therapeutic applications, which, when conjugated to antigen-binding constructs, minibodies or cys-diabodies, deliver chemotherapeutic agents, hormonal therapeutic agents, radiotherapeutic agents, toxins, or any other cytotoxic or anti-cancer agents known in the art to cancer cells that overexpress the target on the cell surface.

[0262] In some embodiments, the antigen-binding constructs, miniantibodies or cys-diabodies described herein may be further conjugated to one or more immunotherapeutics. In some embodiments, the antigen-binding constructs, miniantibodies or cys-diabodies described herein may be co-administered with one or more immunotherapeutics. For example, in some embodiments, the antigen-binding constructs, miniantibodies or cys-diabodies described herein may be further conjugated or co-administered with anti-PD1 and anti-PD-L1 binding agents, anti-CTLA4 agents and multi-specific agents (including but not limited to anti-CTLA-4 / B7-1 / B7-2). Other immunotherapies include checkpoint inhibitors such as pililumab (Yervoy), pembrolizumab (Keytruda), nivolumab (Opdivo), atezolizumab (Tecentriq), avelumab (Bavencio) and durvalumab (Imfinzi). Immunotherapy also includes tremelimumab and pidilizumab. Small molecule immunotherapies are also under development, including: BMS-1001, BMS-1116, CA-170, CA-327, Imiquimod, Resiquimod, 852A, VTX-2337, ADU-S100, MK-1454, Ibrutinib, 3AC, Idelalis, IPI-549, Epacadostat, AT-38, CPI-444, Vipadenant, Preladenant, PBF, AZD4635, Galuniseritib, OTX015 / MK-8628, CPI-0610 (see Kerr and Chisolm (2019) The Journal of Immunology, 2019, 202: 11-19).

[0263] In some embodiments, the antigen-binding constructs, miniantibodies or cys-diabodies described herein may be further conjugated to one or more renal protective agents and / or radioprotectants. In some embodiments, the antigen-binding constructs, miniantibodies or cys-diabodies described herein may be co-administered with one or more renal protective agents and / or radioprotectants. In some embodiments, the radioprotectant comprises free lysine, free arginine, probenecid, succinylated gelatin, 2,4-dinitrophenol, caffeine, ibuprofen, ascorbic acid, caffeic acid, aspirin, carnosine, minocycline, catechin, 4'-O-methylcatechin, 4-phenylbutyric acid, 14937-32-7, lithium chloride, cyclosporine, epoprostenol, fullerene, fumarate, gallic acid, metformin, tformin), indirubin, kaempferol, sodium butyrate, spermidine, vitamin D3 (cholecalciferol), avobenzone, octinoxate, titanium dioxide, tempol, gusperimus, irsogladine, tetrachlorodecaoxide, calcium alginate, gusperimus hydrochloride hydrochloride), sodium salt of D-galacturonic acid, cystamine hydrobromide, cystaphos, N-acetyl-Ser-Asp-Lys-Pro, D08Eai, lycopene, veraresol, U-74389G maleate, trans-2-hexenyl salicylate, 3-benzylidenecamphor, irsogladine maleate, sodium alginate, Gsh-Mee, triethylenediamine diacetate, cystamine hydrochloride, amifostine, glutathione reduced ethyl ester ester), homocysteine ​​thiolactone, 132316-35-9, hydroquinone, 13258-59-8, adeturon, unii-S8Kc806H2T, nitrogen oxide, 2-ethyl-3-hydroxy-6-methylpyridine, S-ethyl glutathione, (8S,10S,13S,14S,17S)-17-[2-[4-(2,6-dipyrrolidin-1-ylpyrimidin-4-yl)piperazin-1-yl]acetyl]-10,13-dimethyl-6,7,8,12,14,15,16,17-octahydrocyclopenta[A]phenanthrene-3-one, trilazad mesylate, Wr 1065, posphonol, amifostine, 1,4-diazabicyclo[2.2.2]octane, gusperimus trihydrochloride, tributyl phosphate, sulfolane, deuterium oxide, benzobarbital, 6-methyluracil, homosalate, cystamine dihydrochloride, 2-(2-aminoethyl)isothiourea dihydrobromide, tempol-H, fenofibrate, cystamine, β-aminoethylisothiourea, zingerone, aeol 10150, artemisinin, 16,16-dimethyl prostaglandin E2, theaflavins, succinic acid, andrographis, balsalazide, N-adenylyl-L-phenylalanine, chlorophyllin, nacu, dieckol, eckol, diltiazem, edaravone, rhepo, ferulic acid, glutathione, glycyrrhizic acid, hesperidin, indomethacin, misoprostol, morin, myricetin, tiopronin, naringin, recilisib sodium sodium), probucol, hydroxyethylrutoside, cystaphos, sulfasalazine, sucralfate, chugai, silymarin, flucloxacillin, cyproterone, cysteamine, thymol, mercaptoethanol, desogestrel, cellobiose, kanamycin, sodium alginate, acetylcysteine, zinc oxide, cystaphos, 10-hydroxy-2-nonanoic acid, teduglutide, turmeric, troxerutin, trichostatin A, selegiline, lycopene, nitrendipine, glycerol, 21245-02-3, rosmarinic acid, simvastatin, flavonoids of Rhus versicolor, sirolimus, oxybenzone, ascophyllum, ecamsule, Fsfjezphmoqyqb-Oasotcbpsa-N, Sczygarxjqtndn-Ikfjuqjosa-N, Snzlftjkilzfkf-Uhfffaoysa-M, alginate, alginic acid, potassium salt, potassium alginate, U-74389G, calcium alginate, Hoechst 33342 (trihydrochloride), polydatin, lipoic acid, enalapril, amifostine, ursolic acid, Unii-Sm5Yj88Ltu, carbonyl cyanide m-chlorophenylhydrazone, genistein, resveratrol, (-)-epigallocatechin gallate, ellagic acid, begaine, valproic acid, pentoxifylline, gum arabic, melatonin, trehalose, actiside, scopolamine, atorvastatin, carvacrol, isoflavone, palifermin, cysteine, vitamin E, heparin, chondroitin sulfate, AET, M40403, EUK-189, EUK-207, vitamin C, vitamin A, lipoic acid, beta-carotene, L-selenomethionine and / or Q10. ,

[0264] Any antigen binding construct, minibody or cys-diabody described herein can be further conjugated with one or more other therapeutic agents, detectable markers, nanoparticles, carriers or combinations thereof. For example, the antigen binding construct can be radiolabeled with iodine-131 and conjugated with a lipid carrier so that the anti-target molecule-lipid conjugate forms micelles. Micelles can incorporate one or more therapeutic agents, isotopes, ions and / or detectable markers.

[0265] In some embodiments, antigen binding constructs, miniantibodies or cys-diabodies are conjugated to therapeutic agents. Although these antigen binding constructs may have a shorter circulation half-life than full-length antibodies, in some embodiments, these forms may exhibit improved tumor penetration based on their smaller size and are therapeutically effective when properly equipped with cytotoxic drugs or radioisotopes. In some embodiments, antigen binding constructs, miniantibodies or cys-diabodies, drug conjugate methods may be used. In some embodiments, treatment methods include radioimmunotherapy by connecting appropriate radiolabels, such as iodine-131, β-emitters or α-emitters, such as yttrium-90, lutetium-177, copper-67, terbium-149, terbium-161, astatine-211, lead-212, bismuth-212, actinium-225, bismuth-213 and 227-thorium, which can deliver cell damage and death to target tissues. In some embodiments, treatment with these fragments armed with cytotoxic drugs or radionuclides results in less non-specific toxicity because they will be cleared from the body more quickly. In some embodiments, the radiolabel comprises terbium-149, terbium-161, or lead-212.

[0266] In some embodiments, the marker and / or therapeutic agent comprises 18 F. 18 F-FAC, 32 P. 33 P. 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y. 90 Y. 89 Sr. 89 Zr, 94 Tc, 94 Tc, 99 mTc, 99 Mo,105 Pd, 105 Rh, 111 Ag, 111 In, 123 I. 124 I. 125 I. 131 I. 142 Pr, 143 Pr, 149 Pm, 149 Tb, 153 Sm, 154-158 Gd, 161 Tb, 166 Dy, 166 Ho, 169 2. 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra, 227 Th and 225 Ac or any combination thereof.

[0267] In some embodiments, the antigen binding construct, minibody or cys-diabody can be linked to a therapeutic agent for a disease associated with expression of the target molecule.

[0268] In some embodiments, the target molecule antigen binding construct is used as a stand-alone agent (eg, the antigen binding construct in the absence of a therapeutic agent) in the treatment of a disease associated with target molecule expression.

[0269] Methods for detecting the presence or absence of target molecules

[0270] The antigen binding construct, minibody or cys-diabody can be used to detect the presence or absence of a target molecule in vivo and / or in vitro. Thus, some embodiments include methods of detecting the presence or absence of a target.

[0271] In some embodiments, the antigen binding construct, minibody or cys-diabody is labeled with a detectable marker. The marker can be, for example, a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator or an enzyme. In some embodiments, the detectable marker comprises a phototherapy dye. In some embodiments, the detectable marker comprises boron. In some embodiments, the detectable marker comprises a marker compatible with boron neutron capture therapy (BNCT). In some embodiments, the detectable marker is suitable for use in Auger electron spectroscopy. In some embodiments, the detectable marker comprises a payload. In some embodiments, at least one payload is selected from 18 F. 18 F-FAC, 32 P. 33 P. 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y. 90 Y. 89 Sr. 89 Zr, 94 Tc, 94 Tc, 99 mTc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I. 124 I. 125 I. 131 I. 142 Pr, 143 Pr, 149 Pm, 149 Tb, 153 Sm, 154-158 Gd, 161 Tb, 166 Dy, 166 Ho, 169 2. 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198Au, 199 Au, 211 At 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra, 227 Th and 225 Ac or any combination thereof. In some embodiments, the marker is a radioactive label. A variety of radionucleotides can be used as imaging labels, including without limitation 211 At 131 I. 125 I. 90 Y. 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P. 212 Pb, 89 Zr, 64 Cu, 18 F. 68 G. 124 I, and radioactive isotopes of Lu, etc. Those skilled in the art will be aware of other radionuclides that are particularly suitable for use in the present disclosure.

[0272] The method can kill cancer cells. In some embodiments, the construct recognizes and binds to the DLL3 protein. In other embodiments, the method further comprises administering radiation therapy to the chemotherapeutic drug. In some embodiments, the subject is also treated with hormone ablation therapy or hormone antagonist therapy.

[0273] The method may include applying an antigen binding construct, a miniantibody or a cys-diabody to a subject. In some embodiments, an antigen binding construct, a miniantibody or a cys-diabody is applied to a sample. The method may include detecting the binding or absence of the antigen binding construct, miniantibody or cys-diabody to a target molecule. In some embodiments, any target molecule may be detected by the selection provided herein. In some embodiments, the target molecule to be detected is DLL3. In some embodiments, one or more miniantibody schemes provided in the accompanying drawings and / or tables (such as Table 5) are used to detect target molecules.

[0274] Table 5 provides embodiments that are compositions and useful in the methods described herein:

[0275] Table 5

[0276]

[0277]

[0278]

[0279]

[0280] In some embodiments, any construct provided herein can be used without a signal peptide or may not include a signal peptide (e.g., the underlined original sequence in Table 5 or 6). In some embodiments, the signal sequence is SEQ ID NO: 45. In some embodiments, the signal sequence is located immediately upstream of the antigen binding construct, minibody or cys-diabody. In some embodiments, Figure 3 Residue 20 of the signal peptide numbered (Kabat) is identical to Figure 1 The signal sequence is directly adjacent to (immediately adjacent to) residue number 1 of the antigen-binding construct, miniantibody and / or cys-diabody of numbering (Kabat). In any and every embodiment provided herein, the signal sequence is optional and may or may not be included in any antigen-binding construct, miniantibody and / or cys-diabody disclosed herein. In some embodiments, one or more cys-diabody schemes provided in the accompanying drawings and / or tables (such as Table 6) are used to detect target molecules.

[0281] Table 6

[0282]

[0283]

[0284] Provided herein are methods for detecting the presence or absence of a target molecule. It will be understood that the following methods can be performed in any order, and / or can be optionally repeated and / or eliminated, and other steps can be optionally added to the method. In some embodiments, an antigen-binding construct, a mini-antibody or a cys-diabody as described herein can be applied to a sample. In some embodiments, an optional cleaning can be performed. Optionally, a second antigen-binding construct can be applied to a sample. An optional cleaning can be performed. In some embodiments, the binding or absence of the antigen-binding construct, mini-antibody or cys-diabody to a target molecule can be detected.

[0285] In some embodiments, antigen-binding constructs, miniantibodies or cys-diabodies as described herein can be applied to samples in vivo. The antigen-binding construct can be applied to a subject. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal, for example, a rat, a mouse, a guinea pig, a hamster, a rabbit, a dog, a cat, a cow, a horse, a goat, a sheep, a donkey, a pig, a monkey or an ape. In some embodiments, the antigen-binding construct is infused into the subject. In some embodiments, the infusion is intravenous. In some embodiments, the infusion is intraperitoneal. In some embodiments, the antigen-binding construct, miniantibody or cys-diabodies are applied topically or locally (such as in the case of interventional or surgical application) to the subject. In some embodiments, a capsule containing the antigen-binding construct, miniantibody or cys-diabodies is applied to the subject, for example, orally or intraperitoneally. In some embodiments, an antigen-binding construct, miniantibody or cys-diabodies are selected to reduce the risk of immunogenic responses in the subject. For example, for human subjects, the antigen-binding construct, miniantibody or cys-diabody can be humanized as described herein. In some embodiments, after the antigen-binding construct, miniantibody or cys-diabody is applied in vivo, a sample or a portion of the sample is removed from the host. In some embodiments, as described herein, the antigen-binding construct, miniantibody or cys-diabody is applied in vivo, incubated in vivo for a period of time, and the sample is removed for in vitro analysis, for example, as described herein, in vitro detection of antigen-binding constructs, miniantibodies or cys-diabodies that bind or do not bind to a target molecule.

[0286] In some embodiments, the antigen-binding construct, miniantibody or cys-diabody is applied to a sample in vitro. In some embodiments, the sample is freshly collected from a subject, such as a biopsy. In some embodiments, the sample is cultivated after being harvested from the subject. In some embodiments, the sample is fixed. In some embodiments, the sample includes a whole organ and / or tissue. In some embodiments, the sample includes one or more intact cells (whole cells). In some embodiments, the sample is from a cell extract, such as a lysate. In some embodiments, the antigen-binding construct in the solution is added to the sample solution. In some embodiments, the antigen-binding construct, miniantibody or cys-diabody in the solution is added to a sample (such as a lyophilized sample) that does not contain a solution, thereby reconstructing the sample. In some embodiments, the lyophilized antigen-binding construct, miniantibody or cys-diabody is added to a sample containing a solution, thereby reconstructing the antigen-binding construct, miniantibody or cys-diabody.

[0287] In some embodiments, antigen-binding construct, miniantibody or cys-diabody are optionally cultivated with sample. The antigen-binding construct, miniantibody or cys-diabody can be cultivated for no more than about 14 days, for example, no more than about 14 days, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day, or no more than about 23 hours, for example, no more than about 23 hours, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5, 0.25 or 0.1 hour, or a period of time within the range defined by any two of the above values. In some embodiments, cultivation is carried out in a subject to which antigen-binding construct, miniantibody or cys-diabody has been applied. In some embodiments, cultivation is carried out in an incubator. In some embodiments, the incubator is maintained at a fixed temperature, such as about 21°C, room temperature, 25°C, 29°C, 34°C, 37°C, or 40°C.

[0288] In some embodiments, antigen-binding constructs, miniantibodies or cys-diabodies that are not bound to the target are optionally removed from the sample. In some embodiments, the sample is cleaned. Cleaning the sample may include removing a solution containing unbound antigen-binding constructs, miniantibodies or cys-diabodies, and adding a solution, such as a buffer solution, that does not contain antigen-binding constructs, miniantibodies or cys-diabodies. In some embodiments, the in vitro sample is cleaned, such as by aspirating, pipetting, pumping out or discharging a solution containing unbound antigen-binding constructs, miniantibodies or cys-diabodies, and adding a solution that does not contain antigen-binding constructs, miniantibodies or cys-diabodies. In some embodiments, the in vivo sample is cleaned, such as by applying a solution that does not contain antigen-binding constructs, miniantibodies or cys-diabodies to the subject, or by cleaning a surface local antigen-binding construct application site. In some embodiments, cleaning is performed at least twice, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 times. In some embodiments, at least about 50% of unbound antibody is removed from the sample after one or more washes, e.g., at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more unbound antibody.

[0289] In some embodiments, unbound antigen-binding constructs, miniantibodies or cys-diabodies are removed from the sample. After the antigen-binding construct, miniantibody or cys-diabodies are applied to the sample, the antigen-binding construct bound to the target reaches equilibrium with the antigen-binding construct not bound to the target, so that at a certain time after the application of the antigen-binding construct, the amount of the antigen-binding construct bound to the target does not increase substantially. After this, at least a portion of the antigen-binding constructs, miniantibodies or cys-diabodies not bound to the target can be eliminated. In some embodiments, unbound antigen-binding constructs, miniantibodies or cys-diabodies are eliminated by the metabolism or other body processes of the subject to which the antigen-binding construct, miniantibody or cys-diabodies are delivered. In some embodiments, unbound antigen-binding constructs, miniantibodies or cys-diabodies are eliminated by adding agents that destroy unbound antigen-binding constructs, miniantibodies or cys-diabodies or destabilize them, such as proteases or neutralizing antibodies. In some embodiments, at least about 30% of the applied antigen-binding construct is eliminated, such as at least about 30%, 40%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.9% is eliminated 1 day after applying the antigen-binding construct, minibody or cys-diabody. In some embodiments, at least about 40% of the applied antigen-binding construct, minibody or cys-diabody is eliminated, such as at least about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.9% is eliminated 2 days after applying the antigen-binding construct, minibody or cys-diabody.

[0290] In some embodiments, the presence or absence of a target molecule is detected. The presence or absence of a target can be detected based on the presence or absence of an antigen binding construct in a sample. For example, after the antigen binding construct is removed and / or eliminated from a sample by washing and / or metabolic elimination, the remaining antigen binding construct in the sample can indicate the presence of the target, while the absence of the antigen binding construct in the sample can indicate the absence of the target.

[0291] In some embodiments, the antigen binding construct comprises a detectable marker as described herein. Thus, the presence of the antigen binding construct can be inferred by detecting the detectable marker.

[0292] In some embodiments, the second antigen-binding construct, miniantibody or cys-diabody is used to detect the antigen-binding construct, miniantibody or cys-diabody. The second antigen-binding construct, miniantibody or cys-diabody can be specifically bound to the antigen-binding construct. For example, the second antigen-binding construct, miniantibody or cys-diabody can include polyclonal or monoclonal antibodies, diabodies, miniantibodies, etc. for the host type of the antibody or for the antigen-binding construct, miniantibody or cys-diabody itself. The second antigen-binding construct, miniantibody or cys-diabody can be conjugated with a detectable marker described herein. The second antigen-binding construct, miniantibody or cys-diabody can be applied to a sample. In some embodiments, in a manner substantially the same as the antigen-binding construct, miniantibody or cys-diabody, the second antigen-binding construct, miniantibody or cys-diabody is applied to a sample. For example, if the antigen-binding construct, miniantibody or cys-diabody is infused into a subject, the second antigen-binding construct, miniantibody or cys-diabody can also be infused into a subject.

[0293] In some embodiments, the binding or absence of binding of an antigen-binding construct, miniantibody, or cys-diabody is detected by at least one of positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), computed tomography (CT), or fluorescence emission detection. PET may include, but is not limited to, small animal PET imaging. In some embodiments, the binding or absence of binding of an antigen-binding construct, miniantibody, or cys-diabody is detected by two or more forms of imaging. In some embodiments, detection may be by near infrared (NIR) and / or Cerenkov.

[0294] In some embodiments, any combination of imaging modalities is possible, including, for example, PET+CR, SPECT+CT, PET+MRI, PET+NIR, PET+SPECT, etc. "PET" is a diagnostic technique that can be used to observe the function and metabolism of human organs and tissues at the molecular level. For PET, positron radiopharmaceuticals (e.g., 18F-FDG) can be injected into the human body. If FDG is used, since the metabolism of fluorodeoxyglucose (FDG) is similar to that of glucose, FDG will accumulate in cells that digest glucose. Positrons emitted by 18F decay and electrons in tissues will undergo annihilation reactions to produce two γ-photons with the same energy and opposite directions. The detector array surrounding the human body can detect two photons using a coincidence measurement technique and determine the position information of the positron. Then, a tomographic image of the positron in the human body can be constructed by processing the position information using image reconstruction software. In some cases, immune-PET can be used, in which a label (e.g., 18F) is connected or combined with an antigen binding construct. In these embodiments, the distribution of the antigen binding construct can be monitored, which will depend on the binding properties and distribution characteristics of the antigen binding construct. For example, if a CD8-directed miniantibody is used, PET can be used to monitor the distribution of CD8 molecules throughout the host system. PET systems are known in the art and include, for example, U.S. Patent Publication Nos. 20170357015, 20170153337, 20150196266, 20150087974, 20120318988, and 20090159804, the entire contents of each of which are incorporated herein by reference for their description of PET and its uses.

[0295] In some embodiments, imaging is performed by PET scanning, PET / CT scanning, or SPECT scanning. In some embodiments, imaging is performed by photoacoustics, optical probes, MR imaging, magnetic nanoparticles for imaging, spectroscopy, and / or any other standard imaging method. In some embodiments, at least one optical probe is coupled to at least one metal chelator. In some embodiments, optical imaging is used to assist surgery. In some embodiments, photodynamic therapy is used. In some embodiments, photodynamic therapy is used to assist surgery. In some embodiments, infrared fluorescence is used to assist surgery. In some embodiments, photodynamic therapy is used for theranostics.

[0296] Nucleic Acids

[0297] In some embodiments, the polypeptides of antigen-binding constructs, miniantibodies or cys-diabodies can be encoded by nucleic acids and expressed in vivo or in vitro, or these peptides can be chemically synthesized. Therefore, in some embodiments, nucleic acids encoding antigen-binding constructs, miniantibodies or cys-diabodies are provided. In some embodiments, the nucleic acid encodes a part or monomer of an antigen-binding construct, miniantibody or cys-diabodies. In some embodiments, the nucleic acid encodes two or more monomers, such as at least 2 monomers. The nucleic acid encoding multiple monomers can include a nucleic acid cleavage site between at least two monomers, can encode a transcription or translation start site between two or more monomers, and / or can encode a proteolytic target site between two or more monomers. In some embodiments, the nucleic acid encodes an antigen-binding construct, a miniantibody, a cys-diabodies, VL, VH, CDR, FR, a linker, a signal peptide or any combination thereof. In some embodiments, the nucleic acid encodes an antigen-binding construct, a minibody, a cys-diabody, a VL, a VH, a CDR, a FR, a linker, a signal peptide, or any combination thereof, which has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to an antigen-binding construct, a minibody, a cys-diabody, a VL, a VH, a CDR, a FR, a linker, a signal peptide as set forth in any one of SEQ ID NOs: 1-48. In some embodiments, the nucleic acid encodes a minibody as any one of SEQ ID NOs: 1-10. In some embodiments, the nucleic acid encodes a minibody having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a minibody as any one of SEQ ID NOs: 1-10. In some embodiments, the minibody encoding nucleic acid is a nucleic acid having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid that is any one of SEQ ID NOs: 51-58 or 64-65. In some embodiments, the nucleic acid encodes a cys-diabody that is any one of SEQ ID NOs: 11-14. In some embodiments, the nucleic acid encodes a cys-diabody that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a cys-diabody that is any one of SEQ ID NOs: 11-14. In some embodiments, the cys-diabody encoding nucleic acid is a nucleic acid having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a nucleic acid that is any one of SEQ ID NOs: 59-63.In some embodiments, the nucleic acid encodes a LCDR that is any one of SEQ ID NOs: 15-26. In some embodiments, the nucleic acid encodes a LCDR that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a LCDR that is any one of SEQ ID NOs: 15-26. In some embodiments, the nucleic acid encodes a HCDR that is any one of SEQ ID NOs: 27-32. In some embodiments, the nucleic acid encodes a HCDR that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a HCDR that is any one of SEQ ID NOs: 27-32. In some embodiments, the nucleic acid encodes a FR that is any one of SEQ ID NOs: 33-38. In some embodiments, the nucleic acid encodes a FR having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a FR that is any one of SEQ ID NOs: 33-38. In some embodiments, the nucleic acid encodes a linker that is any one of SEQ ID NOs: 39-44. In some embodiments, the nucleic acid encodes a linker that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a linker that is any one of SEQ ID NOs: 39-44. In some embodiments, the nucleic acid encodes a signal peptide that is any one of SEQ ID NOs: 45-46. In some embodiments, the nucleic acid encodes a signal peptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a signal peptide that is any one of SEQ ID NOs: 45-46. In some embodiments, the nucleic acid encodes a VH that is SEQ ID NO: 47. In some embodiments, the nucleic acid encodes a minibody having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a VH that is SEQ ID NO: 47. In some embodiments, the nucleic acid encodes a VL that is SEQ ID NO: 48. In some embodiments, the nucleic acid encodes a VL that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a VL that is any one of SEQ ID NOs: 48.

[0298] In some embodiments, the expression vector contains a nucleic acid encoding an antigen-binding construct, a minibody, or a cys-diabody as disclosed herein. In some embodiments, the expression vector comprises pcDNA3.1 for mammalian expression. TM / myc-His(-)Version A vector (Invitrogen, Inc.) or its variant. The pcDNA3.1 expression vector has a CMV promoter for mammalian expression and mammalian (neomycin) and bacterial (ampicillin) selection markers. In some embodiments, the expression vector comprises a plasmid. In some embodiments, the vector comprises a viral vector, such as a retrovirus or adenovirus vector. In embodiments, the vector comprises a cosmid, a YAC or a BAC. In some embodiments, the expression vector encodes an antigen-binding construct, a miniantibody, a cys-diabody, a VL, a VH, a CDR, a FR, a linker, a signal peptide or any combination thereof. In some embodiments, the expression vector encodes an antigen-binding construct, a minibody, a cys-diabody, a VL, a VH, a CDR, a FR, a linker, a signal peptide, or any combination thereof, which has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to an antigen-binding construct, a minibody, a cys-diabody, a VL, a VH, a CDR, a FR, a linker, a signal peptide as set forth in any one of SEQ ID NOs: 1-48. In some embodiments, the expression vector encodes a minibody as any one of SEQ ID NOs: 1-10. In some embodiments, the expression vector encodes a minibody as any one of SEQ ID NOs: 1-10 having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the expression vector encodes a cys-diabody as any one of SEQ ID NOs: 11-14. In some embodiments, the expression vector encodes a cys-diabody having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a cys-diabody as any one of SEQ ID NOs: 11-14. In some embodiments, the expression vector encodes a LCDR as any one of SEQ ID NOs: 15-26. In some embodiments, the expression vector encodes a LCDR having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a LCDR as any one of SEQ ID NOs: 15-26. In some embodiments, the expression vector encodes a HCDR as any one of SEQ ID NOs: 27-32. In some embodiments, the expression vector encodes a HCDR having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a HCDR that is any one of SEQ ID NOs: 27-32.In some embodiments, the expression vector encodes a FR that is any one of SEQ ID NOs: 33-38. In some embodiments, the expression vector encodes a FR that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a FR that is any one of SEQ ID NOs: 33-38. In some embodiments, the expression vector encodes a linker that is any one of SEQ ID NOs: 39-44. In some embodiments, the expression vector encodes a linker that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a linker that is any one of SEQ ID NOs: 39-44. In some embodiments, the expression vector encodes a signal peptide that is any one of SEQ ID NOs: 45-46. In some embodiments, the expression vector encodes a signal peptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the signal peptide of any one of SEQ ID NOs: 45-46. In some embodiments, the expression vector encodes a VH as SEQ ID NO: 47. In some embodiments, the nucleic acid encodes a minibody having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the VH as SEQ ID NO: 47. In some embodiments, the expression vector encodes a VL as SEQ ID NO: 48. In some embodiments, the expression vector encodes a VL having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the VL as any one of SEQ ID NOs: 48. In any of the embodiments provided herein, for some embodiments, the amino acid sequence may not include a signal peptide.

[0299] Fig.14 Some embodiments of nucleic acid sequences of antigen-binding constructs, minibodies and / or cys-diabodies are shown. (SEQ ID NOs: 51-64). In some embodiments, the nucleic acid binding construct and / or minibodies are antigen-binding constructs and / or minibodies in Table 6. In some embodiments, the expression vector encodes an antigen-binding construct and / or minibodies that are antigen-binding constructs and / or minibodies in Table 7.

[0300] Table 7

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308] In some embodiments, the nucleic acid binding construct and / or miniantibody is an antigen binding construct and / or miniantibody in Table 7. In some embodiments, the expression vector encodes an antigen binding construct and / or cys-diabody, which is an antigen binding construct and / or cys-diabody in Table 8.

[0309] Table 8

[0310]

[0311]

[0312]

[0313] cell

[0314] In some embodiments, a cell line expressing at least one of the antigen-binding constructs, miniantibodies or cys-diabodies described herein is provided. In some embodiments, a mammalian cell line (e.g., CHO-K1, ExpiCHO, HEK-293, 293f, Expi293 cell line) is an expression system for producing antigen-binding constructs, miniantibodies, cys-diabodies or other antibodies as described herein. In some embodiments, the antigen-binding constructs, miniantibodies, cys-diabodies and other antibodies or antibody fragments described herein are non-glycosylated and do not require a mammalian expression system because these post-translational modifications are not required. Therefore, in some embodiments, one or more of a variety of mammalian or non-mammalian expression systems are used to produce antigen-binding constructs, miniantibodies and cys-diabodies described herein, including but not limited to mammalian expression systems (e.g., CHO-K1 cells), bacterial expression systems (e.g., Escherichia coli (E. coli), Bacillus subtilis (B. subtilis)), yeast expression systems (e.g., Pichia (Pichia), Saccharomyces cerevisiae (S. cerevisiae)) or any other known expression systems. Other systems may include insect cells and / or plant cells.

[0315] In some embodiments, the cell expresses an antigen binding construct, a minibody, a cys-diabody, VL, VH, CDR, FR, linker, signal peptide, or any combination thereof. In some embodiments, the cell expresses an antigen binding construct, a minibody, a cys-diabody, VL, VH, CDR, FR, linker, signal peptide, or any combination thereof, which has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with an antigen binding construct, a minibody, a cys-diabody, VL, VH, CDR, FR, linker, signal peptide as shown in any one of SEQ ID NOs: 1-48. In some embodiments, the cell expresses a minibody as any one of SEQ ID NOs: 1-10. In some embodiments, the cells express a minibody having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a minibody that is any one of SEQ ID NOs: 1-10. In some embodiments, the cells express a cys-diabody that is any one of SEQ ID NOs: 11-14. In some embodiments, the cells express a cys-diabody that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a cys-diabody that is any one of SEQ ID NOs: 11-14. In some embodiments, the cells express a LCDR that is any one of SEQ ID NOs: 15-26. In some embodiments, the expression vector encodes a LCDR having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a LCDR as any one of SEQ ID NOs: 15-26. In some embodiments, the cell expresses a HCDR as any one of SEQ ID NOs: 27-32. In some embodiments, the cell expresses a HCDR having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a HCDR as any one of SEQ ID NOs: 27-32. In some embodiments, the cell expresses a FR as any one of SEQ ID NOs: 33-38. In some embodiments, the cells express a FR having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a FR that is any one of SEQ ID NOs: 33-38. In some embodiments, the cells express a linker that is any one of SEQ ID NOs: 39-44.In some embodiments, the cells express a linker having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a linker that is any one of SEQ ID NOs: 39-44. In some embodiments, the cells express a signal peptide that is any one of SEQ ID NOs: 45-46. In some embodiments, the cells express a signal peptide that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a signal peptide that is any one of SEQ ID NOs: 45-46. In some embodiments, the cells express a VH that is SEQ ID NO: 47. In some embodiments, the cells express a minibody having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a VH that is SEQ ID NO: 47. In some embodiments, the cells express a VL that is SEQ ID NO: 48. In some embodiments, the cells express a VL that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a VL that is any one of SEQ ID NO: 48.

[0316] Reagent test kit

[0317] In some embodiments, a kit is provided. In some embodiments, the kit includes an antigen-binding construct, a mini-antibody, a cys-diabody, or any combination thereof, as described herein. In some embodiments, the kit includes a nucleic acid encoding an antigen-binding construct as described herein. In some embodiments, the kit includes a cell line that produces an antigen-binding construct as described herein. In some embodiments, the kit includes a detectable marker as described herein. In some embodiments, the kit includes a therapeutic agent as described herein. In some embodiments, the kit includes a buffer. In some embodiments, the kit includes a positive control, such as a target-specific cell or fragment thereof. In some embodiments, the kit includes a negative control, such as a surface or solution that is substantially free of a target. In some embodiments, the kit includes packaging. In some embodiments, the kit includes instructions. In some embodiments, the kit includes: an antigen-binding construct, a mini-antibody, or a cys-diabody according to any one of the above embodiments; and a chelator, wherein the chelator allows the incorporation of a detectable marker. In some embodiments, the kit comprises: an antigen-binding construct, a minibody, or a cys-diabody according to any one of the above embodiments; and a chelator, wherein the chelator allows for the incorporation of a therapeutic isotope. In some embodiments, the kit comprises: an antigen-binding construct, a minibody, or a cys-diabody according to any one of the above embodiments; and a linker, wherein the linker allows for the incorporation of a detectable marker. In some embodiments, the kit comprises: an antigen-binding construct, a minibody, or a cys-diabody according to any one of the above embodiments; and a linker, wherein the linker allows for the incorporation of a therapeutic isotope.

[0318] Other embodiments of the present disclosure are provided in the following numbering scheme (setting).

[0319] 1. An antigen-binding construct, comprising:

[0320] Light chain variable (V L ) domain, light chain variable (V L ) domain contains:

[0321] LCDR1 as SEQ ID NO: 15;

[0322] LCDR2 as any one of SEQ ID NO: 19, 21 or 23;

[0323] LCDR3 being SEQ ID NO: 25; and

[0324] Heavy chain variable (V H) domain, the heavy chain variable (V H ) domain contains:

[0325] HCDR1 as SEQ ID NO: 27;

[0326] HCDR2 as SEQ ID NO: 29; and

[0327] HCDR3 as SEQ ID NO:31.

[0328] 2. A miniantibody that binds to DLL3, the miniantibody comprising:

[0329] A single-chain variable fragment (scFv) that binds to DLL3, the scFv comprising a heavy chain variable (V H ) domain connected to the light chain variable (V L ) domain, the V L The domains include:

[0330] LCDR1 as SEQ ID NO: 15;

[0331] LCDR2 as any one of SEQ ID NO: 19, 21 or 23;

[0332] LCDR3 being SEQ ID NO: 25; and

[0333] The V H The domains include:

[0334] HCDR1 as SEQ ID NO: 27;

[0335] HCDR2 as SEQ ID NO: 29;

[0336] HCDR3 as SEQ ID NO: 31;

[0337] a hinge-extension domain comprising the hinge region; and

[0338] IgG C H 3 sequences.

[0339] 3. A cys-diabody that binds to DLL3, the cys-diabody comprising a polypeptide comprising:

[0340] A single chain variable fragment (scFv) comprising a heavy chain variable (V H ) domain connected to the light chain variable (V L ) domain; and

[0341] The V L The domains include:

[0342] LCDR1 as SEQ ID NO: 15;

[0343] LCDR2 as any one of SEQ ID NO: 19, 21 or 23;

[0344] LCDR3 being SEQ ID NO: 25; and

[0345] The V H The domains include:

[0346] HCDR1 as SEQ ID NO: 27;

[0347] HCDR2 as SEQ ID NO: 29; and

[0348] HCDR3 as SEQ ID NO:31.

[0349] 4. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the V L The domain further comprises LFR2 shown in SEQ ID NO:35 or 37.

[0350] 5. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the V L The domain comprises the same V as in SEQ ID NO: 48 L The domains have an amino acid sequence that is at least 90% identical, and wherein the antigen-binding construct, minibody or cys-diabody comprises a Y50D and / or Y55N mutation (Kabat).

[0351] 6. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the V L The domain comprises the same V as in SEQ ID NO: 48 L The domains have an amino acid sequence that is at least 90% identical, and wherein the antigen-binding construct, minibody or cys-diabody comprises an A43S and / or Q45R mutation (Kabat).

[0352] 7. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the V L The domain comprises the V in SEQ ID NO: 49 L The domains have an amino acid sequence that is at least 90% identical, and wherein the antigen-binding construct, minibody or cys-diabody comprises a Y50D and / or Y55N mutation (Kabat).

[0353] 8. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the V L The domain comprises the same V as in SEQ ID NO: 49 L The domains have an amino acid sequence that is at least 90% identical, and wherein the antigen-binding construct, minibody or cys-diabody comprises an A43S and / or Q45R mutation (Kabat).

[0354] 9. The antigen-binding construct, minibody or cys-diabody according to any one of the above schemes, wherein the antigen-binding construct, minibody or cys-diabody further comprises a signal peptide as SEQ ID NO: 45.

[0355] 10. The antigen-binding construct, minibody or cys-diabody according to any one of the above schemes, wherein the antigen-binding construct, minibody or cys-diabody comprises a linker as any one of SEQ ID NOs: 39-44.

[0356] 11. An antigen-binding construct or miniantibody according to any one of the above schemes, wherein the C H The 3 domain is IgG C H 3 domains.

[0357] 12. An antigen-binding construct or miniantibody according to any one of the above schemes, wherein the IgG C H 3 domains are IgG1, IgG2, IgG3 or IgG4 C H 3 domains.

[0358] 13. An antigen-binding construct or miniantibody according to any one of the above schemes, wherein the IgG C H The 3 domain comprises an IgG1 C sequence as any one of SEQ ID NOs: 65-68 H 3 domains.

[0359] 14. An antigen-binding construct or miniantibody according to any one of the above schemes, wherein the IgG C H The 3 domain comprises an IgG1 C sequence as any one of SEQ ID NOs: 69-71. H 3 domains.

[0360] 15. An antigen-binding construct or miniantibody according to any one of the above schemes, wherein the IgG C H The 3 domain comprises an IgG1 C sequence as any one of SEQ ID NOs: 72-82. H 3 domains.

[0361] 16. An antigen-binding construct or miniantibody according to any one of the above schemes, wherein the IgG C H The 3 domain comprises an IgG1 C sequence as any one of SEQ ID NOs: 83-85. H 3 domains.

[0362] 17. The antigen-binding construct of claim 1, wherein the antigen-binding construct is an antibody.

[0363] 18. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the antigen-binding construct, miniantibody or cys-diabody specifically binds to DLL3.

[0364] 19. The antigen-binding construct, minibody or cys-diabody according to any one of the above schemes, further comprising a detectable marker.

[0365] 20. An antigen-binding construct, minibody or cys-diabody according to any one of the above schemes, wherein the detectable marker is a fluorescent detectable marker.

[0366] 21. An antigen-binding construct, minibody or cys-diabody according to any one of the above schemes, wherein the detectable marker comprises a phototherapy-compatible dye.

[0367] 22. An antigen-binding construct, minibody or cys-diabody according to any one of the above schemes, wherein the detectable marker comprises boron.

[0368] 23. An antigen-binding construct, minibody or cys-diabody according to any one of the above schemes, wherein the detectable marker is compatible with use using boron neutron capture therapy (BNCT).

[0369] 24. An antigen-binding construct, minibody or cys-diabody according to any one of the above schemes, wherein the detectable marker is a radioactive label.

[0370] 25. An antigen-binding construct, minibody or cys-diabody according to any one of the above schemes, wherein the detectable marker is an alpha-emitter radiolabel.

[0371] 26. An antigen-binding construct, minibody or cys-diabody according to any one of the above schemes, wherein the detectable marker is a beta-emitter radiolabel.

[0372] 27. An antigen-binding construct, minibody or cys-diabody according to any one of the above schemes, wherein the detectable marker is a positron emitter radiolabel.

[0373] 28. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the detectable marker comprises lutetium-177.

[0374] 29. An antigen-binding construct, minibody or cys-diabody according to any one of the above schemes, wherein the detectable marker is a gamma-emitter radiolabel.

[0375] 30. An antigen-binding construct, minibody or cys-diabody according to any one of the above schemes, wherein the detectable marker is suitable for use in Auger electron spectroscopy.

[0376] 31. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the detectable marker comprises an isotope.

[0377] 32. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the detectable marker comprises a bioluminescent compound.

[0378] 33. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the detectable marker comprises a chemiluminescent compound.

[0379] 34. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the detectable marker comprises an enzyme.

[0380] 35. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the detectable marker comprises a metal chelator.

[0381] 36. An antigen-binding construct, minibody or cys-diabody according to any one of the above schemes, further comprising a therapeutic agent.

[0382] 37. An antigen-binding construct, miniantibody or cys-diabody according to any of the above schemes, wherein the therapeutic agent comprises a therapeutic isotope or ion.

[0383] 38. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the therapeutic agent is a radiolabeled agent.

[0384] 39. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the therapeutic agent is an alpha-emitter radiolabeled.

[0385] 40. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the therapeutic agent is a beta-emitter radiolabel.

[0386] 41. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the therapeutic agent is a positron emitter radiolabeled.

[0387] 42. An antigen-binding construct, miniantibody or cys-diabody according to any of the above schemes, wherein the therapeutic agent is a gamma-emitter radiolabeled.

[0388] 43. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the therapeutic agent comprises lutetium-177.

[0389] 44. An antigen-binding construct, miniantibody or cys-diabody according to any of the above schemes, wherein the therapeutic agent comprises a phototherapy-compatible dye.

[0390] 45. An antigen-binding construct, miniantibody or cys-diabody according to any of the above schemes, wherein the therapeutic agent comprises boron.

[0391] 46. ​​An antigen-binding construct, minibody or cys-diabody according to any of the above schemes, wherein the therapeutic agent is compatible with use using boron neutron capture therapy (BNCT).

[0392] 47. An antigen-binding construct, miniantibody or cys-diabody according to any of the above schemes, wherein the therapeutic agent and / or detectable marker comprises a toxic payload.

[0393] 48. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the antigen-binding construct, miniantibody or cys-diabody is dual-specific.

[0394] 49. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the antigen-binding construct, miniantibody or cys-diabody comprises a monovalent scFv.

[0395] 50. The antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein from the N-terminus to the C-terminus of the polypeptide is V L , V H .

[0396] 51. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the order of the variable domains is V from the N-terminus to the C-terminus of the polypeptide. H , V L .

[0397] 52. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the miniantibody is a humanized antigen-binding construct, miniantibody or cys-diabody.

[0398] 53. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the humanized antigen-binding construct, miniantibody or cys-diabody comprises:

[0399] LCDR1 as SEQ ID NO: 15;

[0400] LCDR2 as any one of SEQ ID NO: 19, 21 or 23;

[0401] LCDR3 as SEQ ID NO: 25;

[0402] HCDR1 as SEQ ID NO: 27;

[0403] HCDR2 as SEQ ID NO: 29; and

[0404] HCDR3 as SEQ ID NO:31.

[0405] 54. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the antigen-binding construct, miniantibody or cys-diabody has an improved expression yield in mammalian cells compared to an antigen-binding construct, miniantibody or cys-diabody comprising SEQ ID NO: 1.

[0406] 55. An antigen-binding construct, miniantibody or cys-diabody according to any one of schemes 1-53, wherein the antigen-binding construct, miniantibody or cys-diabody has an expression yield of about 75% to 300%, 75% to 200%, 75% to 150% or 75% to 100% compared to an antigen-binding construct, miniantibody or cys-diabody comprising SEQ ID NO: 1.

[0407] 56. An antigen-binding construct, minibody or cys-diabody according to any one of schemes 1-53, wherein the antigen-binding construct, minibody or cys-diabody has an expression yield of about 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 150%, 175%, 200%, 250% or 300% compared to an antigen-binding construct, minibody or cys-diabody comprising SEQ ID NO: 1.

[0408] 57. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the antigen-binding construct, miniantibody or cys-diabody accumulates to a detectable level in the subject's blood, liver, kidney, spleen, lung, muscle, bone, heart, stomach, small intestine, large intestine, pancreas, tumor or any combination thereof.

[0409] 58. The antigen-binding construct, miniantibody or cys-diabody of claim 57, wherein the subject is a mammal.

[0410] 59. The antigen-binding construct, minibody or cys-diabody of claim 57 or 58, wherein the antigen-binding construct, minibody or cys-diabody is detectable at up to about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30% or 35% ID / g.

[0411] 60. The antigen-binding construct, minibody or cys-diabody of claim 57 or 58, wherein the antigen-binding construct, minibody or cys-diabody is detectable at between about 0.1% to 35%, 0.1% to 30%, 1% to 35%, 1% to 30%, 5% to 35% or 5% to 30% ID / g.

[0412] 61. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the antigen-binding construct, miniantibody or cys-diabody comprises LFR2 of LFR2 shown in SEQ ID NO: 35 or 37; and wherein the antigen-binding construct, miniantibody or cys-diabody has improved biodistribution compared to an antigen-binding construct, miniantibody or cys-diabody comprising LFR2 as LFR2 shown in SEQ ID NO: 33.

[0413] 62. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the KD of the antigen-binding construct, miniantibody or cys-diabody is less than about 1×10-10 M.

[0414] 63. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the KD of the antigen-binding construct, miniantibody or cys-diabody is less than about 1×10 -12 M.

[0415] 64. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the EC of the antigen-binding construct, miniantibody or cys-diabody is 50 is about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19 or 0.20 nM.

[0416] 65. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the EC of the antigen-binding construct, miniantibody or cys-diabody 50 Between about 0.001 to 0.20 nM, 0.001 to 0.17 nM, 0.001 to 0.12 nM, 0.01 to 0.20 nM, 0.01 to 0.17 nM, 0.01 to 0.12 nM, 0.05 to 0.20 nM, 0.05 to 0.17 nM. 0.05 to 0.15 nM, 0.05 to 0.12 nM, 0.08 to 0.20 nM, 0.08 to 0.17 nM or 0.08 to 0.12 nM.

[0417] 66. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, wherein the EC of the antigen-binding construct, miniantibody or cys-diabody is 50 Up to about 10 nM.

[0418] 67. A nucleic acid encoding an antigen-binding construct, a miniantibody or a cys-diabody according to any one of the above schemes

[0419] 68. A cell line that produces an antigen-binding construct, a minibody or a cys-diabody according to any one of the above schemes.

[0420] 69. A nucleic acid according to any one of the above schemes, wherein the nucleic acid is any one of SEQ ID NOs: 51-65.

[0421] 70. A cell line that produces an antigen-binding construct, a minibody or a cys-diabody according to any one of the above schemes.

[0422] 71. A kit, comprising:

[0423] An antigen-binding construct, minibody or cys-diabody according to any one of the above schemes; and

[0424] Detectable markers.

[0425] 72. A kit comprising: an antigen-binding construct, a minibody or a cys-diabody according to any one of the above schemes; and a chelator, wherein the chelator allows the incorporation of a detectable marker.

[0426] 73. A kit comprising: an antigen-binding construct, a minibody or a cys-diabody according to any one of the above schemes; and a chelator, wherein the chelator allows the incorporation of a therapeutic isotope.

[0427] 74. A kit comprising: an antigen-binding construct, a minibody or a cys-diabody according to any one of the above schemes; and a linker, wherein the linker allows the incorporation of a detectable marker.

[0428] 75. A kit comprising: an antigen-binding construct, a minibody or a cys-diabody according to any one of the above schemes; and a linker, wherein the linker allows the incorporation of a therapeutic isotope.

[0429] 76. A kit comprising: an antigen-binding construct, a minibody or a cys-diabody according to any one of the above schemes; and a detectable marker.

[0430] 77. A method of detecting the presence or absence of DLL3, the method comprising:

[0431] applying an antigen-binding construct, minibody or cys-diabody according to any one of the above schemes to a sample; and

[0432] The presence or absence of the antigen binding construct is detected, thereby detecting the presence or absence of DLL3.

[0433] 78. A method according to scheme 77, wherein the antigen-binding construct, miniantibody or cys-diabody is conjugated to a detectable marker.

[0434] 79. The method of claim 77 or 78, wherein applying the antigen-binding construct, miniantibody or cys-diabody comprises administering the antigen-binding construct to a subject.

[0435] 80. A method according to any one of schemes 77-79, wherein detecting the binding or absence of binding of the antigen-binding construct, miniantibody or cys-diabody to DLL3 comprises at least one of positron emission tomography or single photon emission computed tomography.

[0436] 81. The method of any one of schemes 77-80, further comprising applying a second antigen-binding construct to the sample, wherein the second antigen-binding construct specifically binds to the antigen-binding construct.

[0437] 82. A method according to any one of schemes 77-81, wherein the antigen-binding construct, miniantibody or cys-diabody is incubated with the sample for no more than 20 hours.

[0438] 83. A method according to any one of schemes 77-82, wherein the antigen-binding construct, miniantibody or cys-diabody is incubated with the sample for no more than 6 hours.

[0439] 84. The method of any one of schemes 77-83, wherein the antigen-binding construct, miniantibody or cys-diabody is administered to a host, and wherein a first amount of the antigen-binding construct, miniantibody or cys-diabody is debound from DLL3, and a second amount of the antigen-binding construct, miniantibody or cys-diabody is bound to DLL3, wherein at least about 80% of the first amount of the antigen-binding construct, miniantibody or cys-diabody is eliminated in no more than 12 hours.

[0440] 85. A method of targeting a therapeutic agent to DLL3, the method comprising administering to a subject an antigen binding construct, minibody or cys-diabody according to any one of the above schemes, wherein the antigen binding construct is conjugated to a therapeutic agent.

[0441] 86. A therapeutic composition targeting DLL3, wherein the therapeutic composition comprises:

[0442] An antigen-binding construct comprising:

[0443] Light chain variable (V L ) domain, light chain variable (V L ) domain contains:

[0444] LCDR1 as SEQ ID NO: 15;

[0445] LCDR2 as any one of SEQ ID NO: 19, 21 or 23;

[0446] LCDR3 being SEQ ID NO: 25; and

[0447] Heavy chain variable (V H ) domain, the heavy chain variable (V H ) domain contains:

[0448] HCDR1 being the HCDR1 of SEQ ID NO: 27;

[0449] HCDR2 being the HCDR2 of SEQ ID NO: 29;

[0450] A HCDR3 that is the HCDR3 of SEQ ID NO: 31; and

[0451] Therapeutic agents, toxic payloads and / or detectable markers.

[0452] 87. A therapeutic composition targeting DLL3, wherein the therapeutic composition comprises:

[0453] A minibody that binds to DLL3, the minibody comprising:

[0454] A single-chain variable fragment (scFv) that binds to DLL3, the scFv comprising a heavy chain variable (V H ) domain connected to the light chain variable (V L ) domain, the V L The domains include:

[0455] LCDR1 as SEQ ID NO: 15;

[0456] LCDR2 as any one of SEQ ID NO: 19, 21 or 23;

[0457] LCDR3 being SEQ ID NO: 25, and

[0458] The V H The domains include:

[0459] HCDR1 as SEQ ID NO: 27;

[0460] HCDR2 as SEQ ID NO: 29;

[0461] HCDR3 as SEQ ID NO: 31;

[0462] a hinge-extension domain comprising the hinge region of IgG1;

[0463] IgG C H 3 sequences; and

[0464] Therapeutic agents, toxic payloads and / or detectable markers.

[0465] 88. A therapeutic composition targeting DLL3, wherein the therapeutic composition comprises:

[0466] A cys-diabody that binds to DLL3, the cys-diabody comprising a polypeptide comprising:

[0467] A single chain variable fragment (scFv) comprising a heavy chain variable (V H ) domain connected to the light chain variable (V L ) domain;

[0468] The V L The domains include:

[0469] LCDR1 as SEQ ID NO: 15;

[0470] LCDR2 being SEQ ID NO: 19, 21 or 23;

[0471] LCDR3 as SEQ ID NO: 25;

[0472] The V H The domains include:

[0473] HCDR1 as SEQ ID NO: 27;

[0474] HCDR2 as SEQ ID NO: 29;

[0475] HCDR3 being SEQ ID NO: 31; and

[0476] Therapeutic agents, toxic payloads and / or detectable markers.

[0477] 89. A therapeutic composition according to any of the above schemes, wherein the detectable marker is a radioactive label.

[0478] 90. A therapeutic composition according to any of the above schemes, wherein the detectable marker is an alpha emitter radiolabel.

[0479] 91. A therapeutic composition according to any of the above schemes, wherein the detectable marker is a beta emitter radiolabel.

[0480] 92. A therapeutic composition according to any of the above schemes, wherein the detectable marker is a positron emitter radiolabel.

[0481] 93. A therapeutic composition according to any of the above schemes, wherein the detectable marker comprises Lutetium-177.

[0482] 94. An antigen-binding construct, miniantibody or cys-diabody according to any one of the above schemes, or a composition according to any one of the above schemes, or a kit according to any one of the above schemes, or a method according to any one of the above schemes, wherein the therapeutic agent comprises terbium-149, terbium-161 or lead-212.

[0483] 95. An antigen-binding construct according to any one of the above schemes, or a composition according to any one of the above schemes, or a kit according to any one of the above schemes, or a method according to any one of the above schemes, wherein the antigen-binding construct is a scFv-Fc, and the scFv-Fc comprises:

[0484] A single-chain variable fragment (scFv) that binds to DLL3, the scFv comprising a heavy chain variable (V H ) domain connected to the light chain variable (V L ) domain;

[0485] a hinge domain; and

[0486] Fc region.

[0487] 96. An antigen binding construct that binds DLL3, wherein the antigen binding construct is a construct that binds DLL3 -Fc and comprises a heavy chain variable (V H ) domain.

[0488] 97. The antigen-binding construct according to claim 96, wherein the heavy chain variable (V H ) domain contains:

[0489] HCDR1 as SEQ ID NO: 27;

[0490] HCDR2 as SEQ ID NO: 29; and

[0491] HCDR3 as SEQ ID NO:31.

[0492] The following examples further define the embodiments of the present disclosure. It should be understood that these examples are provided by way of illustration only. Based on the above discussion and these examples, those skilled in the art can determine the essential characteristics of the present disclosure, and without departing from its spirit and scope, various changes and modifications can be made to the embodiments of the present disclosure to adapt them to various uses and conditions. Therefore, in addition to those shown and described herein, various modifications of the embodiments of the present disclosure will be apparent to those skilled in the art based on the above description. These modifications are also intended to be within the scope of the appended claims. The disclosure of each reference described herein is incorporated herein by reference in its entirety and for the disclosure referenced herein.

[0493] Example

[0494] Example 1: Isolation and identification of novel antigen-binding constructs

[0495] Isolation of a series of novel antigen-binding constructs as described herein can also be performed using any standard method known to those skilled in the art.

[0496] As disclosed herein, a series of antigen-binding constructs specific for DLL3 (SEQ ID NOs: 1-46) were designed and isolated. Each antigen-binding construct is human or murine and is an antibody, minibody or cys-diabody. In one example, the variable region from the parent antibody is converted into an scFv and grafted into a minibody or cys-diabody framework. In another example, the variable region from the parent antibody is tested for immunogenic epitopes by computer methods known in the art, and is converted into an scFv and grafted into a minibody or cys-diabody framework. In addition, each construct comprises a heavy chain and a light chain having a HCDR1 set forth in SEQ ID NO: 27, a HCDR2 set forth in SEQ ID NO: 29, a HCDR3 set forth in SEQ ID NO: 31, a LCDR1 set forth in SEQ ID NO: 15, a LCDR2 set forth in SEQ ID NO: 19, 21 or 23, and a LCDR3 set forth in SEQ ID NO: 25. The sequence of each antigen-binding construct is different; for example, some contain serine at position 43 of VL, arginine at position 45 of VL, aspartic acid at position 50 of VL, and / or asparagine (Kabat) at position 55 of VL. The sequence changes of each antigen-binding construct are selected for improved characteristics compared to the antigen-binding construct without the change. For example, the changes in the antigen-binding constructs result in improved antigen-binding kinetics, as measured by the dissociation constant (K off ) is shown by the flattening or lowering of the

[0497] These antigen binding constructs were then transfected into Expi293 using a lipofectamine transfection protocol (Thermo Exp293 kit). TM The vector contains a cleavable signal peptide as shown in SEQ ID NO: 45. In other alternatives, the sequence of the antigen binding construct can be inserted into any cell line using any conventional method, such as electroporation.

[0498] The expression of the constructs was measured compared to the parental miniantibodies IAB57M1-1 and IAB57M2-2. Figure 7 As shown, the novel antigen-binding construct disclosed herein is in Expi293 TM The expression yield in mammalian cells was up to 4-fold higher than that of the parental minibody.

[0499] The binding kinetics of the novel antigen-binding constructs were also determined by biofilm interferometry (Table 9) ( Fig. 9 ).

[0500] Table 9: Binding kinetics of antigen binding constructs

[0501] Construct KD(M) Ka(1 / Ms) Kd(1 / s) IAB57M1-3 4.00E-9 5.988E+5 2.39E-3 IAB57M2-4 4.84E-9 5.81E+5 2.81E-3 IAB57M2-6 <1E-12 7.85E+5 <1E-7 IAB57M1-1 3.33E-9 8.38E+5 2.79E-3 IAB57M2-2 3.07E-9 6.98E+5 2.14E-3

[0502] As disclosed herein, the half maximal effective concentration (EC 50 ). 50 (Table 10) Figure 8 The novel antigen-binding construct disclosed herein has high affinity for tumor cells in SHP-77 and NCI-H82 cell lines, and its EC 50 Between 0.01-0.1nM ( Fig.10 ).

[0503] Table 10: EC of antigen binding constructs 50

[0504] Construct <![CDATA[EC 50 (nM)]]> IAB57M1-3 0.1089 IAB57M1-7 0.1113 IAB57M2-4 0.1179 IAB57M2-6 0.1614 IAB57M1-5 0.1250 IAB57M2-2 0.1197 IAB57M1-1 0.09457 IAB57M2-8 0.1231

[0505] The binding affinity of the novel antigen binding constructs to purified human / cynomolgus monkey / dog / mouse / rat or minipig DLL3 antigens was evaluated using ELISA. For this assay, a 96-well ELISA plate was coated with DLL3 antigen diluted to a concentration of 2 mg / mL in coating buffer (0.05M sodium carbonate-sodium bicarbonate buffer, pH 9.6). 100 μL of this coating solution was added to each well of the plate and incubated overnight at 4°C. The plate was washed 3 times with 300 μL of wash buffer (PBS containing 0.05% Tween-20) per well. After the last wash, the plate was blocked by adding 200 μL of blocking buffer (PBS containing 1% BSA) heated to room temperature to each well. The plate was then incubated at room temperature for 1 hour with gentle agitation. After blocking, the plate was washed 3 times with 300 μL of wash buffer per well. Dilution samples of each miniantibody were generated by diluting the starting sample 2.5× at 100 ng / mL, or 250 ng / mL, or 1000 ng / mL, or 3000 ng / mL in 100 μL sample buffer (PBS containing 1% BSA). The diluted samples were transferred to the assay plate and incubated at room temperature for 1 hour with gentle agitation. The plate was then washed 3 times with 300 μL of wash buffer per well.

[0506] Detection was performed by adding 100 μL of appropriately diluted HRP-labeled detection secondary antibody and incubating the plate for 1 hour at room temperature with gentle agitation. The plate was then washed 3 times with 300 μL of wash buffer per well. For color development, 100 μL of TMB substrate reagent preheated to room temperature was added to the plate and incubated in the dark at room temperature for 15 min. The reaction was then terminated by adding 100 μL of 650 nm stop solution to the plate and mixing at room temperature for 1 min with gentle agitation. The plate was recorded by reading the OD at 650 nm in a microplate reader within 20 min after the termination reaction.

[0507] As disclosed herein, the binding kinetics of the novel antigen binding constructs to purified human / cynomolgus monkey / dog / mouse / rat or minipig DLL3 antigens were evaluated using biomembrane interferometry (Table 9) ( Fig. 9 ). His-tagged DLL3 antigen was diluted at 10 μg / mL in phosphate buffered saline (PBS) and loaded onto the Ni-NTA tip until saturated. The binding rate (K) was recorded on an Octet BLItz instrument. on ) and dissociation rate (K off ), and the dissociation constant K DDilution samples of the antigen binding construct were prepared at concentrations between 100 nM and 0.1 nM in PBS at a 2.5× dilution and binding curves were recorded at each concentration. Dissociation rate curves were recorded by immersing the tip in PBS.

[0508] As disclosed herein, flow cytometry was used to evaluate the binding of the construct to the cellular target. In 180 μl FACS buffer (PBS 2% FBS), starting from a concentration of 100 nM, 2.5× serial dilutions of the miniantibody stock were generated. The cells were added to Corning V-bottom polypropylene cell culture plates at 200,000 cells / well. The cells were centrifuged at 100 rpm for 3 minutes and the supernatant was discarded. The miniantibody dilution series was transferred to a plate containing a cell pellet and incubated at 4°C for 1 hr. The plate was then washed 3 times with 200 μl / well of FACS buffer, and the supernatant was discarded after each wash by centrifugation at 1000 rpm. 50 μl / well of the detection antibody R&D AlexaFluor-647 goat anti-human antibody (H+L) was added at a dilution of 1:100 in FACS buffer and incubated at 4°C for 1 hr. The plate was washed 3 times with 200 μl / well of FACS buffer, and the supernatant was discarded after each wash by centrifugation at 1000 rpm. The cells were then fixed by adding 100 μL of 1% paraformaldehyde in PBS and incubating at room temperature for 15 minutes. 100 μl of PBS was added to each well, and the plate was analyzed by flow cytometry using the allophycocyanin fluorescence setting.

[0509] Humanized V in IAB57M1-3 L The germline sequence of IGVK3-15*01 was used. L The antibody has a framework region 2 (FR2) sequence (WYQQKPGQAPRLLI (SEQ ID NO: 70) (according to North)) that contains a cluster of two arginines at positions 39 and 45 (IMGT numbering). Molecular modeling of the surface charge using an Adaptive Poisson-Boltzmann Solver identified a localized patch of surface-exposed positive charge formed by the two arginines. Without being bound by theory, it is hypothesized that this localized patch of positive charge may lead to accumulation of the minibody in the kidney, resulting in too rapid clearance of the minibody and potential kidney damage.

[0510] To test this, R45 was replaced with glutamine (Q), as Figure 1 Therefore, the V LKPGQAPR (SEQ ID NO: 95) in FR2 was changed to KPGQAPQ (SEQ ID NO: 111) to prepare IAB57M1-13. Figure 1 As shown in FIG. 1 , IAB57M1-14 was prepared by further replacing the alanine (A) at position 43 with serine (S). L KPGQAPR (SEQ ID NO: 95) in FR2 was further changed to KPGQSPQ (SEQ ID NO: 110) in IAB57M1-14.

[0511] The in vivo biodistribution of 89Zr-deferoxamine-IAB57M1-3 in NCr nude mice bearing SHP77 and NCI-H82 tumors was evaluated. Fig.11 The in vivo biodistribution of 89-Zr-Df-IAB57M1-3, 89-Zr-Df-IAB57M1-13, and 89-Zr-Df-IAB57M1-14 was also evaluated ( Fig.12 ). In both cases, the M1-3 antigen-binding construct was relatively highly distributed in the liver and kidneys. In contrast, M1-13 and M1-14 showed improved biodistribution, as shown by reduced accumulation of these minibodies in the kidneys. The antigen-binding constructs were also distributed in vivo in the blood, spleen, lungs, muscle, bone, heart, stomach, large intestine, small intestine, pancreas, and tumors.

[0512] Example 2: Use of an antigen binding construct for treating a subject in need thereof

[0513] The methods of using the novel antigen-binding constructs described herein as medicaments for use in a subject in need thereof can confer benefit on the subject.

[0514] As disclosed herein, a pharmaceutical composition is prepared comprising a novel antigen-binding construct against DLL3, alone or in combination with a chemotherapeutic or immuno-oncology drug, and a pharmaceutically acceptable carrier. In other alternatives, the dose is a radioactive dose. In other alternatives, the dose is not fixed. In other alternatives, the dose is provided as a divided dose. In other alternatives, the dose is measured in mCi or MBq. In other alternatives, the composition may have more than one antigen-binding construct. In other alternatives, the antigen-binding construct may be an antibody, a mini-antibody, a cys-diabody, or another combination thereof. In other alternatives, the antigen-binding construct may be present in the composition at any pharmaceutically effective concentration, for example, at about 0.01 mg / kg to about 25 mg / kg. In other alternatives, the immuno-oncology drug may be omitted from the composition. In other alternatives, the immuno-oncology drug can be replaced by one or more small molecules, therapeutic agents or antigen-binding constructs that are effective in treating the disease, such as antibodies, chemotherapeutic drugs, DNA repair inhibitors, alkylating agents, metabolic inhibitors, radiosensitizers, anti-tumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, nitrosoureas, corticosteroids, anti-angiogenic agents, apoptosis inducers, anti-microtubule agents, vinca alkaloids, taxanes, anthracyclines, anti-androgens, VEGF pathway inhibitors, VEGF pathway inhibitors, MAPK / Ras / Raf pathway inhibitors and EGFR pathway inhibitors.

[0515] The composition of the present invention can be used in the combination therapy with other agents to achieve the improved treatment outcome to the subject.Other agents that can be used in combination include therapeutic agents, immunotherapeutics, chemotherapeutics, radiation protectants and renal protectants.Useful immunotherapeutics in the combination with the present invention include atezolizumab (Tecentriq), Avelumab (Avelumab) (Bavencio), Dostarlizumab (Dostarlizumab) (Jemperli), Durvalumab (Imfinzi), Pulimumab (Yervoy), Nivolumab (Opdivo), Pembrolizumab (Keytruda) and other immunotherapeutics in clinical development.

[0516] As disclosed herein, the composition is administered to a human subject at a pharmaceutically acceptable dose to treat SCLC. In other alternatives, the disease can be other neuroendocrine tumors, such as neuroendocrine prostate cancer, glioblastoma, fibrosis, carcinoma, tumor, solid tumor, autoimmune disease, cardiovascular disease, metabolic disease, bone cancer, osteosarcoma, breast cancer, carcinoid, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, epithelial ovarian cancer, esophageal cancer, gastric cancer, gastrointestinal cancer, glioma, head and neck cancer, hepatocellular carcinoma, renal cancer, leukemia, liver cancer, lung cancer, lymphoma, medullary thyroid carcinoma, melanoma, non-small cell lung cancer, osteosarcoma, oral squamous cell carcinoma, oral cancer, ovarian carcinoma, ovarian cancer, pancreatic adenocarcinoma, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, gastric cancer, testicular cancer, thyroid cancer, urothelial carcinoma or any other disease associated with altered DLL3 expression. In other alternatives, the composition is administered to a subject to suppress or ameliorate disease.

[0517] After administration, the disease is monitored by imaging the relevant cells, tissues or organs in the subject using the methods listed in the following examples. As observed by imaging, after the composition is administered to the subject, the cancer cells are destroyed. In other alternatives, the cancer cells are inhibited, improved, damaged, killed or induced to apoptosis in response to contact with the composition.

[0518] Example 3: Use of antigen binding constructs for diagnosis

[0519] The use of the novel antigen-binding constructs described herein in methods of diagnosis thereof can confer benefit on a subject suspected of having a disease.

[0520] As disclosed herein, novel antigen binding constructs are used to image tissues of human subjects suspected of having cancer. In other alternatives, antigen binding constructs can be used to image cells, cultured cell lines, tissue sections, organs, organ sections, multiple tissues, multiple organs, or entire subjects. In other alternatives, subjects can be screened for tumors, fibrosis, autoimmune diseases, cardiovascular diseases, or any other disease or abnormality associated with DLL3. In other alternatives, the subject can be any mammal, including mice, rats, dogs, miniature pigs, and non-human primates.

[0521] The antigen binding construct is administered to epithelial tissue of the subject and screened for binding to DLL3. The presence of DLL3 in the subject is assessed by in vivo diagnostic medical imaging methods, such as positron emission tomography (PET) or single photon emission computed tomography (SPECT). Binding of the antigen binding construct to DLL3 indicates a high likelihood of cancer being present in the tissue. In other alternatives, the absence of binding of the antigen binding construct to DLL3 indicates a low likelihood of cancer being present in the tissue. In other alternatives, binding of the antigen binding construct to DLL3 is determined by a payload added to the antigen binding construct, such as 18 F. 18 F-FAC, 32 P. 33 P. 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y. 90 Y. 89 Sr. 89 Zr, 94 Tc, 94 Tc, 99 mTc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I. 124 I. 125 I. 131 I. 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154-158 Gd, 161 Tb, 166 Dy, 166 Ho, 169 2. 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra, 227 Th or 225 Ac. In a further alternative, binding of the antigen binding construct to DLL3 is determined by PET scanning.

[0522] Example 4: Use of antigen binding constructs to reduce uptake in the kidney

[0523] The methods of modifying antigen-binding constructs described herein can confer upon a subject the benefit of reduced renal uptake of radiolabeled minibodies.

[0524] Minibodies are prepared by humanization of mouse hybridomas as disclosed herein. In some embodiments, humanization of antibodies is based on germline: IGHV1-69*01 / IGHV1-39*01.

[0525] A positively charged patch was identified on the germline IGKV1-39*01 that is responsible for the accumulation of the miniantibody in the kidney. As disclosed herein, the patch is located in the sequence KPGKAPK containing three charged lysine residues. In some embodiments, mutations on framework 2 of the germline were identified that reduced the uptake of radiolabeled miniantibodies by the kidney.

[0526] As disclosed herein, several human germlines have a pattern of 3 positively charged residues on framework 2. In some embodiments, the positively charged residues are lysine (K) and / or arginine (R). In some embodiments, germline IGKV1-39*01 is modified where framework 2 is replaced with a similar sequence from a germline that does not contain this charge pattern. A non-limiting example of such a replacement is framework 2 from IGKV3-20*01 or IGKV2-28*01.

[0527] As used herein, section headings are only for organizational purposes and should not be considered as limiting the subject matter in any way. All documents and similar materials cited in the present application, including but not limited to patents, patent applications, papers, books, discussions and Internet web pages are clearly incorporated herein by reference in their entirety for any purpose, including the disclosure specifically referenced herein. When the definition of terms in the reference introduced seems to be different from the definition provided in the present teaching, the definition provided in the present teaching should be used as the standard. It will be understood that there is an implicit "about" before the temperature, concentration, time, etc. discussed in the present teaching, so that small and non-substantial deviations are within the scope of the present teaching in this article.

[0528] Although the present disclosure has been provided in the context of certain embodiments and examples, it will be understood by those skilled in the art that the present disclosure extends beyond the specifically disclosed embodiments and extends to other alternative embodiments and / or uses of the present disclosure and obvious modifications and equivalent forms thereof. In addition, although some variations of the present disclosure have been shown and described in detail, other modifications within the scope of the present disclosure will be apparent to those skilled in the art based on the present disclosure. It is also contemplated that various combinations or sub-combinations of the specific features and aspects of the embodiments may be performed and are still within the scope of the present disclosure. It should be understood that the various features and aspects of the disclosed embodiments may be combined or replaced with each other to form different modes or embodiments of the present disclosure. Therefore, it is intended that the scope of the present disclosure set forth herein should not be limited by the specifically disclosed embodiments described above.

[0529] It should be understood, however, that this detailed description, while indicating preferred embodiments of the present disclosure, is provided by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will be apparent to those skilled in the art.

[0530] The terminology used in the description provided herein is not intended to be interpreted in any limited or restrictive manner. Instead, the terminology is used only in conjunction with the detailed description of embodiments of the systems, methods, and related components. Furthermore, embodiments may include a number of novel features, no single feature of which is solely responsible for its desirable attributes or is considered essential to practicing the disclosure described herein.

Claims

1. An antigen-binding construct, comprising: Light chain variable (V L ) domain, the light chain variable (V L ) domain contains: LCDR1 as SEQ ID NO: 15; LCDR2 as any one of SEQ ID NO: 19, 21 or 23; LCDR3 being SEQ ID NO: 25; and Heavy chain variable (V H ) domain, the heavy chain variable (V H ) domain contains: HCDR1 as SEQ ID NO: 27; HCDR2 as SEQ ID NO: 29; and HCDR3 as SEQ ID NO:

31.

2. A miniantibody that binds to DLL3, the miniantibody comprising: A single-chain variable fragment (scFv) that binds to DLL3, the scFv comprising a heavy chain variable (V H ) domain connected to the light chain variable (V L ) domain, the V L The domains include: LCDR1 as SEQ ID NO: 15; LCDR2 as any one of SEQ ID NO: 19, 21 or 23; LCDR3 being SEQ ID NO: 25; and The V H The domains include: HCDR1 as SEQ ID NO: 27; HCDR2 as SEQ ID NO: 29; HCDR3 as SEQ ID NO: 31; a hinge-extension domain comprising the hinge region; and IgG C H 3 sequences.

3. A cys-diabody that binds to DLL3, the cys-diabody comprising a polypeptide comprising: A single chain variable fragment (scFv) comprising a heavy chain variable (V H ) domain connected to the light chain variable (V L ) domain; and The V L The domains include: LCDR1 as SEQ ID NO: 15; LCDR2 as any one of SEQ ID NO: 19, 21 or 23; LCDR3 being SEQ ID NO: 25; and The V H The domains include: HCDR1 as SEQ ID NO: 27; HCDR2 as SEQ ID NO: 29; and HCDR3 as SEQ ID NO:

31.

4. The antigen-binding construct, miniantibody or cys-diabody according to any one of the preceding claims, wherein the V L The domain further comprises LFR2 shown in SEQ ID NO:35 or 37.

5. The antigen-binding construct, miniantibody or cys-diabody according to any one of the preceding claims, wherein the V L The domain comprises the same V as in SEQ ID NO: 48 L The domains have an amino acid sequence that is at least 90% identical, and wherein the antigen-binding construct, minibody or cys-diabody comprises a Y50D and / or Y55N mutation (Kabat).

6. The antigen-binding construct, miniantibody or cys-diabody according to any one of the preceding claims, wherein the V L The domain comprises the same V as in SEQ ID NO: 48 L The domains have an amino acid sequence that is at least 90% identical, and wherein the antigen-binding construct, minibody or cys-diabody comprises an A43S and / or Q45R mutation (Kabat).

7. The antigen-binding construct, miniantibody or cys-diabody according to any one of the preceding claims, wherein the V L The domain comprises the same V as in SEQ ID NO: 49 L The domains have an amino acid sequence that is at least 90% identical, and wherein the antigen-binding construct, minibody or cys-diabody comprises a Y50D and / or Y55N mutation (Kabat).

8. The antigen-binding construct, miniantibody or cys-diabody according to any one of the preceding claims, wherein the V L The domain comprises the same V as in SEQ ID NO: 49 L The domains have an amino acid sequence that is at least 90% identical, and wherein the antigen-binding construct, minibody or cys-diabody comprises an A43S and / or Q45R mutation (Kabat).

9. The antigen-binding construct, minibody or cys-diabody according to any one of the preceding claims, wherein the antigen-binding construct, minibody or cys-diabody further comprises a signal peptide as SEQ ID NO:

45.

10. The antigen-binding construct, minibody or cys-diabody according to any one of the preceding claims, wherein the antigen-binding construct, minibody or cys-diabody comprises a linker that is any one of SEQ ID NOs: 39-44.

11. The antigen-binding construct or minibody according to any one of the preceding claims, wherein the C H The 3 domain is IgG C H 3 domains.

12. The antigen-binding construct or minibody according to any one of the preceding claims, wherein the IgG C H 3 domains are IgG1, IgG2, IgG3 or IgG4 C H 3 domains.

13. The antigen-binding construct or minibody according to any one of the preceding claims, wherein the IgG C H The 3 domain comprises an IgG1C as any one of SEQ ID NOs: 65-68 H 3 domains.

14. The antigen-binding construct or minibody according to any one of the preceding claims, wherein the IgG C H The 3 domain comprises an IgG1C as any one of SEQ ID NOs: 69-71 H 3 domains.

15. The antigen-binding construct or minibody according to any one of the preceding claims, wherein the IgG C H The 3 domain comprises an IgG1C as any one of SEQ ID NOs: 72-82 H 3 domains.

16. The antigen-binding construct or minibody according to any one of the preceding claims, wherein the IgG C H The 3 domain comprises an IgG1C as any one of SEQ ID NOs: 83-85 H 3 domains.

17. The antigen-binding construct of claim 1, wherein the antigen-binding construct is an antibody.

18. The antigen-binding construct, minibody or cys-diabody of any of the preceding claims, wherein the antigen-binding construct, minibody or cys-diabody specifically binds to DLL3.

19. The antigen-binding construct, minibody or cys-diabody according to any one of the preceding claims, further comprising a detectable marker.

20. The antigen binding construct, minibody or cys-diabody of any preceding claim, wherein the detectable marker is a fluorescent detectable marker.

21. The antigen-binding construct, minibody or cys-diabody of any of the preceding claims, wherein the detectable marker comprises a phototherapy compatible dye.

22. The antigen-binding construct, minibody or cys-diabody of any of the preceding claims, wherein the detectable marker comprises boron.

23. The antigen binding construct, minibody or cys-diabody of any preceding claim, wherein the detectable marker is compatible with use with boron neutron capture therapy (BNCT).

24. The antigen binding construct, minibody or cys-diabody of any of the preceding claims, wherein the detectable marker is a radiolabel.

25. The antigen binding construct, minibody or cys-diabody of any of the preceding claims, wherein the detectable marker is an alpha emitter radiolabel.

26. The antigen binding construct, minibody or cys-diabody of any of the preceding claims, wherein the detectable marker is a beta emitter radiolabel.

27. The antigen binding construct, minibody or cys-diabody of any of the preceding claims, wherein the detectable marker is a positron emitter radiolabel.

28. The antigen-binding construct, minibody or cys-diabody of any of the preceding claims, wherein the detectable marker comprises Lutetium-177.

29. The antigen binding construct, minibody or cys-diabody of any of the preceding claims, wherein the detectable marker is a gamma emitter radiolabel.

30. The antigen-binding construct, minibody or cys-diabody of any preceding claim, wherein the detectable marker is suitable for use with Auger electron spectroscopy.

31. The antigen-binding construct, minibody or cys-diabody of any of the preceding claims, wherein the detectable marker comprises an isotope.

32. The antigen-binding construct, minibody or cys-diabody of any of the preceding claims, wherein the detectable marker comprises a bioluminescent compound.

33. The antigen-binding construct, minibody or cys-diabody of any of the preceding claims, wherein the detectable marker comprises a chemiluminescent compound.

34. The antigen-binding construct, minibody or cys-diabody of any of the preceding claims, wherein the detectable marker comprises an enzyme.

35. The antigen-binding construct, minibody or cys-diabody of any of the preceding claims, wherein the detectable marker comprises a metal chelator.

36. The antigen-binding construct, minibody or cys-diabody of any of the preceding claims, further comprising a therapeutic agent.

37. The antigen-binding construct, minibody or cys-diabody of any of the preceding claims, wherein the therapeutic agent comprises a therapeutic isotope or ion.

38. The antigen binding construct, minibody or cys-diabody of any of the preceding claims, wherein the therapeutic agent is a radiolabel.

39. The antigen binding construct, minibody or cys-diabody of any of the preceding claims, wherein the therapeutic agent is an alpha emitter radiolabel.

40. The antigen binding construct, minibody or cys-diabody of any of the preceding claims, wherein the therapeutic agent is a beta emitter radiolabel.

41. The antigen binding construct, minibody or cys-diabody of any of the preceding claims, wherein the therapeutic agent is a positron emitter radiolabel.

42. The antigen binding construct, minibody or cys-diabody of any of the preceding claims, wherein the therapeutic agent is a gamma emitter radiolabel.

43. The antigen-binding construct, minibody or cys-diabody of any of the preceding claims, wherein the therapeutic agent comprises Lutetium-177.

44. The antigen-binding construct, minibody or cys-diabody of any of the preceding claims, wherein the therapeutic agent comprises a phototherapy-compatible dye.

45. The antigen-binding construct, minibody or cys-diabody of any of the preceding claims, wherein the therapeutic agent comprises boron.

46. ​​The antigen binding construct, minibody or cys-diabody of any of the preceding claims, wherein the therapeutic agent is compatible with use with boron neutron capture therapy (BNCT).

47. The antigen-binding construct, minibody or cys-diabody of any of the preceding claims, wherein the therapeutic agent and / or detectable marker comprises a toxic payload.

48. The antigen-binding construct, minibody or cys-diabody of any of the preceding claims, wherein the antigen-binding construct, minibody or cys-diabody is bispecific.

49. The antigen-binding construct, minibody or cys-diabody of any of the preceding claims, wherein the antigen-binding construct, minibody or cys-diabody comprises a monovalent scFv.

50. The antigen-binding construct, minibody or cys-diabody according to any one of the preceding claims, wherein from the N-terminus to the C-terminus of the polypeptide is V L 、V H .

51. An antigen-binding construct, minibody or cys-diabody according to any of the preceding claims, wherein the order of the variable domains from the N-terminus to the C-terminus of the polypeptide is V H 、V L .

52. The antigen-binding construct, minibody or cys-diabody of any of the preceding claims, wherein the minibody is a humanized antigen-binding construct, minibody or cys-diabody.

53. The antigen-binding construct, minibody or cys-diabody of any of the preceding claims, wherein the humanized antigen-binding construct, minibody or cys-diabody comprises: LCDR1 as SEQ ID NO: 15; LCDR2 as any one of SEQ ID NO: 19, 21 or 23; LCDR3 as SEQ ID NO: 25; HCDR1 as SEQ ID NO: 27; HCDR2 as SEQ ID NO: 29; and HCDR3 as SEQ ID NO:

31.

54. An antigen-binding construct, minibody or cys-diabody according to any of the preceding claims, wherein the antigen-binding construct, minibody or cys-diabody has an increased expression yield in mammalian cells compared to an antigen-binding construct, minibody or cys-diabody comprising SEQ ID NO:

1.

55. The antigen-binding construct, minibody or cys-diabody of any one of claims 1-53, wherein the antigen-binding construct, minibody or cys-diabody has an expression yield of about 75% to 300%, 75% to 200%, 75% to 150% or 75% to 100% compared to an antigen-binding construct, minibody or cys-diabody comprising SEQ ID NO:

1.

56. The antigen-binding construct, minibody or cys-diabody of any one of claims 1-53, wherein the antigen-binding construct, minibody or cys-diabody has an expression yield of about 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 150%, 175%, 200%, 250% or 300% compared to an antigen-binding construct, minibody or cys-diabody comprising SEQ ID NO:

1.

57. An antigen-binding construct, minibody or cys-diabody according to any of the preceding claims, wherein the antigen-binding construct, minibody or cys-diabody accumulates to detectable levels in the blood, liver, kidney, spleen, lung, muscle, bone, heart, stomach, small intestine, large intestine, pancreas, tumor or any combination thereof of a subject.

58. The antigen-binding construct, minibody or cys-diabody of claim 57, wherein the subject is a mammal.

59. The antigen-binding construct, minibody or cys-diabody of claim 57 or 58, wherein the antigen-binding construct, minibody or cys-diabody is detectable at up to about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30% or 35% ID / g.

60. The antigen-binding construct, minibody or cys-diabody of claim 57 or 58, wherein the antigen-binding construct, minibody or cys-diabody is detectable at between about 0.1% to 35%, 0.1% to 30%, 1% to 35%, 1% to 30%, 5% to 35% or 5% to 30% ID / g.

61. An antigen-binding construct, miniantibody or cys-diabody according to any of the preceding claims, wherein the antigen-binding construct, miniantibody or cys-diabody comprises an LFR2 that is LFR2 as shown in SEQ ID NO: 35 or 37; and wherein the antigen-binding construct, miniantibody or cys-diabody has improved biodistribution compared to an antigen-binding construct, miniantibody or cys-diabody comprising an LFR2 that is LFR2 as shown in SEQ ID NO:

33.

62. The antigen-binding construct, minibody or cys-diabody of any of the preceding claims, wherein the antigen-binding construct, minibody or cys-diabody has a KD of less than about 1×10 -10 M.

63. The antigen-binding construct, minibody or cys-diabody of any of the preceding claims, wherein the KD of the antigen-binding construct, minibody or cys-diabody is less than about 1×10 -12 M.

64. An antigen-binding construct, minibody or cys-diabody according to any of the preceding claims, wherein the EC of the antigen-binding construct, minibody or cys-diabody is 50 is about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19 or 0.20 nM.

65. An antigen-binding construct, minibody or cys-diabody according to any of the preceding claims, wherein the EC of the antigen-binding construct, minibody or cys-diabody is 50 Between about 0.001 to 0.20 nM, 0.001 to 0.17 nM, 0.001 to 0.12 nM, 0.01 to 0.20 nM, 0.01 to 0.17 nM, 0.01 to 0.12 nM, 0.05 to 0.20 nM, 0.05 to 0.17 nM, 0.05 to 0.15 nM, 0.05 to 0.12 nM, 0.08 to 0.20 nM, 0.08 to 0.17 nM, or 0.08 to 0.12 nM.

66. An antigen-binding construct, minibody or cys-diabody according to any of the preceding claims, wherein the EC of the antigen-binding construct, minibody or cys-diabody is 50 Up to about 10 nM.

67. A nucleic acid encoding the antigen-binding construct, minibody or cys-diabody of any of the preceding claims.

68. A cell line that produces the antigen-binding construct, minibody or cys-diabody of any of the preceding claims.

69. The nucleic acid of any one of the preceding claims, wherein the nucleic acid is any one of SEQ ID NOs: 51-65.

70. A cell line that produces the antigen-binding construct, minibody or cys-diabody of any of the preceding claims.

71. A kit comprising: The antigen-binding construct, minibody or cys-diabody of any of the preceding claims; and Detectable markers.

72. A kit comprising: the antigen binding construct, minibody or cys-diabody of any of the preceding claims; and a chelator, wherein the chelator allows for incorporation of a detectable marker.

73. A kit comprising: an antigen binding construct, minibody or cys-diabody as claimed in any one of the preceding claims; and a chelator, wherein the chelator allows for the incorporation of a therapeutic isotope.

74. A kit comprising: the antigen binding construct, minibody or cys-diabody of any of the preceding claims; and a linker, wherein the linker allows for incorporation of a detectable marker.

75. A kit comprising: the antigen binding construct, minibody or cys-diabody of any of the preceding claims; and a linker, wherein the linker allows for incorporation of a therapeutic isotope.

76. A kit comprising: the antigen-binding construct, minibody or cys-diabody of any one of the preceding claims; and a detectable marker.

77. A method of detecting the presence or absence of a DLL3, the method comprising: applying the antigen-binding construct, minibody or cys-diabody of any of the preceding claims to a sample; as well as The presence or absence of the antigen binding construct is detected, thereby detecting the presence or absence of DLL3.

78. The method of claim 77, wherein the antigen binding construct, minibody or cys-diabody is conjugated to a detectable marker.

79. The method of claim 77 or 78, wherein applying the antigen-binding construct, minibody or cys-diabody comprises administering the antigen-binding construct to a subject.

80. The method of any one of claims 77-79, wherein detecting the presence or absence of binding of the antigen binding construct, minibody or cys-diabody to DLL3 comprises at least one of positron emission tomography or single photon emission computed tomography.

81. The method of any one of claims 77-80, further comprising applying a second antigen binding construct to the sample, wherein the second antigen binding construct specifically binds to the antigen binding construct.

82. The method of any one of claims 77-81, wherein the antigen binding construct, minibody or cys-diabody is incubated with the sample for no longer than 20 hours.

83. The method of any one of claims 77-82, wherein the antigen binding construct, minibody or cys-diabody is incubated with the sample for no longer than 6 hours.

84. The method of any one of claims 77-83, wherein the antigen-binding construct, minibody or cys-diabody is administered to a host, and wherein a first amount of the antigen-binding construct, minibody or cys-diabody is unbound from DLL3, and a second amount of the antigen-binding construct, minibody or cys-diabody is bound to DLL3, wherein at least about 80% of the first amount of the antigen-binding construct, minibody or cys-diabody is eliminated in no more than 12 hours.

85. A method of targeting a therapeutic agent to DLL3, the method comprising administering to a subject the antigen binding construct, minibody or cys-diabody of any of the preceding claims, wherein the antigen binding construct is conjugated to the therapeutic agent.

86. A therapeutic composition targeting DLL3, wherein the therapeutic composition comprises: An antigen-binding construct comprising: Light chain variable (V L ) domain, the light chain variable (V L ) domain contains: LCDR1 as SEQ ID NO: 15; LCDR2 as any one of SEQ ID NO: 19, 21 or 23; LCDR3 being SEQ ID NO: 25; and Heavy chain variable (V H ) domain, the heavy chain variable (V H ) domain contains: HCDR1 being the HCDR1 of SEQ ID NO: 27; HCDR2 being the HCDR2 of SEQ ID NO: 29; A HCDR3 that is the HCDR3 of SEQ ID NO: 31; and Therapeutic agents, toxic payloads and / or detectable markers.

87. A therapeutic composition targeting DLL3, wherein the therapeutic composition comprises: A minibody that binds to DLL3, the minibody comprising: A single-chain variable fragment (scFv) that binds to DLL3, the scFv comprising a heavy chain variable (V H ) domain connected to the light chain variable (V L ) domain, the V L The domains include: LCDR1 as SEQ ID NO: 15; LCDR2 as any one of SEQ ID NO: 19, 21 or 23; LCDR3 being SEQ ID NO: 25, and The V H The domains include: HCDR1 as SEQ ID NO: 27; HCDR2 as SEQ ID NO: 29; HCDR3 as SEQ ID NO: 31; a hinge-extension domain comprising the hinge region of IgG1; IgG C H 3 sequences; and Therapeutic agents, toxic payloads and / or detectable markers.

88. A therapeutic composition targeting DLL3, wherein the therapeutic composition comprises: A cys-diabody that binds to DLL3, the cys-diabody comprising a polypeptide comprising: A single chain variable fragment (scFv) comprising a heavy chain variable (V H ) domain connected to the light chain variable (V L ) domain; The V L The domains include: LCDR1 as SEQ ID NO: 15; LCDR2 being SEQ ID NO: 19, 21 or 23; LCDR3 as SEQ ID NO: 25; The V H The domains include: HCDR1 as SEQ ID NO: 27; HCDR2 as SEQ ID NO: 29; HCDR3 being SEQ ID NO: 31; and Therapeutic agents, toxic payloads and / or detectable markers.

89. The therapeutic composition of any of the preceding claims, wherein the detectable marker is a radiolabel.

90. The therapeutic composition of any of the preceding claims, wherein the detectable marker is an alpha emitter radiolabel.

91. The therapeutic composition of any of the preceding claims, wherein the detectable marker is a beta emitter radiolabel.

92. The therapeutic composition of any of the preceding claims, wherein the detectable marker is a positron emitter radiolabel.

93. The therapeutic composition of any of the preceding claims, wherein the detectable marker comprises Lutetium-177.

94. An antigen-binding construct, miniantibody or cys-diabody according to any of the preceding claims, or a composition according to any of the preceding claims, or a kit according to any of the preceding claims, or a method according to any of the preceding claims, wherein the therapeutic agent comprises terbium-149, terbium-161 or lead-212.

95. The antigen-binding construct of any of the preceding claims, or the composition of any of the preceding claims, or the kit of any of the preceding claims, or the method of any of the preceding claims, wherein the antigen-binding construct is a scFv-Fc, the scFv-Fc comprising: A single-chain variable fragment (scFv) that binds to DLL3, the scFv comprising a heavy chain variable (V H ) domain connected to the light chain variable (V L ) domain; a hinge domain; and Fc region.

96. An antigen binding construct that binds DLL3, wherein the antigen binding construct is a -Fc and comprises a heavy chain variable (V H ) domain.

97. The antigen-binding construct of claim 96, wherein the heavy chain variable (V H ) domain contains: HCDR1 as SEQ ID NO: 27; HCDR2 as SEQ ID NO: 29; and HCDR3 as SEQ ID NO:31.

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