DLL3 antigen-binding construct

Abstract

Provided herein are components for antigen-binding constructs, including antibodies and fragments thereof, such as minibodies and cys-diabodies, that bind to target molecules, e.g., DLL3. In some embodiments, these components are novel complementarity-determining region (CDR) sequences and / or sequences related to and / or portions of CDR sequences. In some embodiments, these components are novel framework region (FR) sequences and / or sequences related to and / or portions of FR sequences. These CDR and FR sequences may provide various benefits. Also provided herein are antigen-binding constructs (minibodies, cys-diabodies, etc.) that include one or more of the CDR or FR sequences or subsequences provided herein.

Description

[Technical Field]

[0001] Incorporation by reference to any priority application Any and all applications for which a foreign or domestic priority claim is identified in the Application Data Sheet filed with this application are hereby incorporated by reference under 37 CFR 1.57.

[0002] Reference to sequence listing This application has been submitted with an electronic Sequence Listing. The Sequence Listing is provided as a file entitled Seqlist_IGNAB059WO.xml, created and last modified on June 30, 2023, and is 155,593 bytes in size. The information in the electronic Sequence Listing is hereby incorporated by reference in its entirety.

[0003] SUMMARY OF THE INVENTION Embodiments herein relate to antigen-binding constructs, minibodies, and cys-diabodies, particularly DLL3-specific antigen-binding constructs, minibodies, and cys-diabodies. [Background technology]

[0004] 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. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 4,946,778 [Patent Document 2] U.S. Patent No. 4,816,567 [Patent Document 3] U.S. Patent Publication No. 20170357015 [Patent Document 4] U.S. Patent Publication No. 20170153337 [Patent Document 5] U.S. Patent No. 20150196266 [Patent Document 6] U.S. Patent No. 20150087974 [Patent Document 7] U.S. Patent No. 20120318988 [Patent Document 8] U.S. Patent No. 20090159804

Non-licensed literature

[0006]

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[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 DLL3-specific antigen-binding constructs, minibodies, and cys-diabodies. Some embodiments herein relate to methods of treating DLL3-associated diseases or conditions using the antigen-binding constructs, minibodies, and cys-diabodies disclosed herein.

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

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

[0010] A cys-diabody that binds to DLL3, comprising a variable heavy chain (V H ) domains linked to the variable light chain (V L ) domain, L The domain comprises an LCDR1 having SEQ ID NO: 15; an LCDR2 having any one of SEQ ID NOs: 19, 21, or 23; and an LCDR3 having SEQ ID NO: 25, H Provided herein is a cys-diabody comprising a polypeptide comprising a single chain variable fragment (scFv), the domains of which comprise: HCDR1 is SEQ ID NO: 27; HCDR2 is SEQ ID NO: 29; and HCDR3 is SEQ ID NO: 31.

[0011] V L 2. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the domain further comprises an LFR2 of SEQ ID NO: 35 or 37.

[0012] V L The domain is V in SEQ ID NO: 48 L

[0023] The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, comprising an amino acid sequence having at least 90% identity to the domain; and wherein the antigen-binding construct, minibody, or cys-diabody comprises a Y50D and / or Y55N mutation (Kabat).

[0013] V L The domain is V in SEQ ID NO: 48 L

[0023] The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, comprising an amino acid sequence having at least 90% identity to the domain; and wherein the antigen-binding construct, minibody, or cys-diabody comprises an A43S and / or Q45R mutation (Kabat).

[0014] V L The domain is V in SEQ ID NO: 49 L

[0023] The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, comprising an amino acid sequence having at least 90% identity to the domain, wherein the antigen-binding construct, minibody, or cys-diabody comprises a Y50D and / or Y55N mutation (Kabat).

[0015] V L The domain is V in SEQ ID NO: 49 L

[0023] The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, comprising an amino acid sequence having at least 90% identity to the domain; and wherein the antigen-binding construct, minibody, or cys-diabody comprises an A43S and / or Q45R mutation (Kabat).

[0016] 10. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the antigen-binding construct, minibody, or cys-diabody further comprises a signal peptide that is SEQ ID NO:45.

[0017] 10. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the antigen-binding construct, minibody, or cys-diabody comprises a linker that is any one of SEQ ID NOs: 39-44.

[0018] C H 3 domains are IgG C H 10. The antigen-binding construct or minibody of any one of the preceding embodiments, which is three domains.

[0019] IgG C H 3 domains, IgG1, IgG2, IgG3, or IgG4 C H 10. The antigen-binding construct or minibody of any one of the preceding embodiments, which is three domains.

[0020] IgG C H The three domains are IgG1 C, which is any one of SEQ ID NOs: 65 to 68. H10. The antigen-binding construct or minibody of any one of the preceding embodiments, comprising three domains.

[0021] IgG C H The three domains are IgG1 C, which is any one of SEQ ID NOs: 69 to 71. H 10. The antigen-binding construct or minibody of any one of the preceding embodiments, comprising three domains.

[0022] IgG C H The three domains are IgG1 C, which is any one of SEQ ID NOs: 72 to 82. H 10. The antigen-binding construct or minibody of any one of the preceding embodiments, comprising three domains.

[0023] IgG C H The three domains are IgG1 C1 domains having any one of SEQ ID NOs: 83 to 85. H 10. The antigen-binding construct or minibody of any one of the preceding embodiments, comprising three domains.

[0024] 10. The antigen-binding construct of any one of the preceding embodiments, wherein the antigen-binding construct is an antibody.

[0025] 10. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the antigen-binding construct, minibody, or cys-diabody specifically binds to DLL3.

[0026] 10. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, further comprising a detectable marker.

[0027] 10. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the detectable marker is a fluorescently detectable marker.

[0028] 10. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the detectable marker comprises a phototherapy compatible dye.

[0029]

[0023] The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the detectable marker is compatible for use with boron neutron capture therapy (BNCT).

[0030] 10. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the detectable marker is a radioactive label.

[0031] 10. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the detectable marker is an alpha-emitter radiolabel.

[0032] 10. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the detectable marker is a beta-emitter radiolabel.

[0033] 10. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the detectable marker is a positron emitter radiolabel.

[0034] 10. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the detectable marker is a gamma-emitter radiolabel.

[0035] 10. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the detectable marker comprises lutetium-177.

[0036]

[0023] The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the detectable marker is suitable for use with Auger electron spectroscopy.

[0037]

[0023] The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the detectable marker comprises an isotope.

[0038] 10. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the detectable marker comprises a bioluminescent compound.

[0039] 10. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the detectable marker comprises a chemiluminescent compound.

[0040] 10. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the detectable marker comprises an enzyme.

[0041] 10. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the detectable marker comprises a metal chelator.

[0042] 10. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, further comprising a therapeutic agent.

[0043]

[0023] 2. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the therapeutic agent comprises a therapeutic isotope or ion.

[0044]

[0023] The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the therapeutic agent is a radiolabel.

[0045]

[0023] 2. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the therapeutic agent is an alpha-emitter radiolabel.

[0046]

[0023] 2. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the therapeutic agent is a beta-emitter radiolabel.

[0047]

[0023] The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the therapeutic agent is a positron emitter radiolabel.

[0048]

[0023] The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the therapeutic agent is a gamma-emitter radiolabel.

[0049]

[0023] 2. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the therapeutic agent comprises lutetium-177.

[0050]

[0023] 2. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the therapeutic agent comprises a phototherapy compatible dye.

[0051]

[0023] The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the therapeutic agent comprises boron.

[0052]

[0023] The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the therapeutic agent is adapted for use with boron neutron capture therapy (BNCT).

[0053] 10. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the detectable marker and / or therapeutic agent comprises a toxic payload.

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

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

[0056] From the N-terminus to the C-terminus of the polypeptide, V L , V H 2. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein

[0057] The order of variable domains from the N-terminus to the C-terminus of a polypeptide is V H , V L 2. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein

[0058] The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the minibody is a humanized antigen-binding construct, minibody, or cys-diabody.

[0059] The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the humanized antigen-binding construct, minibody, or cys-diabody comprises an LCDR1 that is SEQ ID NO: 15; an LCDR2 that is any one of SEQ ID NOs: 19, 21, or 23; an LCDR3 that is SEQ ID NO: 25; an HCDR1 that is SEQ ID NO: 27; an HCDR2 that is SEQ ID NO: 29; and an HCDR3 that is SEQ ID NO: 31.

[0060] V L 2. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein the domain further comprises an LFR2 of SEQ ID NO: 35 or 37.

[0061] 10. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, 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.

[0062] 10. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, 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.

[0063]

[0023] The antigen-binding construct, minibody, or cys-diabody of any one of the preceding 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%, or a percentage within a range defined by any two of the preceding values (e.g., about 75-300%, about 80-250%, about 100%-300%, about 110%-250%, about 120%-200%, etc.), compared to an antigen-binding construct, minibody, or cys-diabody comprising SEQ ID NO:1.

[0064]

[0023] The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein 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.

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

[0066] The antigen-binding construct, minibody, or cys-diabody may have an activity of about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, or 35% ID / g, 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. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, which is detectable in ID / g or as a percentage within the range defined by any two of the preceding values.

[0067]

[0023] The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, wherein 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.

[0068] 35 or 37; and wherein the antigen-binding construct, minibody, or cys-diabody has improved biodistribution compared to an antigen-binding construct, minibody, or cys-diabody comprising an LFR2 that is the LFR2 of SEQ ID NO: 33.

[0069] The antigen-binding construct, minibody, or cys-diabody is approximately 1 x 10 -10 10. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, having a KD of less than M.

[0070] The antigen-binding construct, minibody, or cys-diabody is approximately 1 x 10 -12 10. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, having a KD of less than M.

[0071] The antigen-binding construct, minibody, or cys-diabody may be administered at a concentration of 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. an EC50 of 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 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 50 or the EC within the region defined by any two of the preceding values 50 2. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, comprising:

[0072] The antigen-binding construct, minibody, or cys-diabody may have an EC of about 0.001-0.20 nM, 0.001-0.17 nM, 0.001-0.12 nM, 0.01-0.20 nM, 0.01-0.17 nM, 0.01-0.12 nM, 0.05-0.20 nM, 0.05-0.17 nM, 0.05-0.15 nM, 0.05-0.12 nM, 0.08-0.20 nM, 0.08-0.17 nM, or 0.08-0.12 nM. 50 2. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, comprising:

[0073] The antigen-binding construct, minibody, or cys-diabody has an EC of up to about 10 nM. 50 2. The antigen-binding construct, minibody, or cys-diabody of any one of the preceding embodiments, comprising:

[0074] Provided herein is a nucleic acid encoding the antigen-binding construct, minibody, or cys-diabody of any of the preceding embodiments.

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

[0076] Provided herein is a cell line producing the antigen-binding construct, minibody, or cys-diabody of any of the preceding embodiments.

[0077] Provided herein is a kit comprising the antigen-binding construct, minibody, or cys-diabody of any of the preceding embodiments; and a chelating agent that allows for the incorporation of a detectable marker.

[0078] Provided herein is a kit comprising the antigen-binding construct, minibody, or cys-diabody of any of the preceding embodiments; and a chelator that allows for the incorporation of a therapeutic isotope or therapeutic agent.

[0079] Provided herein is a kit comprising the antigen-binding construct, minibody, or cys-diabody of any of the preceding embodiments; and a linker that allows for the incorporation of a detectable marker.

[0080] Provided herein is a kit comprising the antigen-binding construct, minibody, or cys-diabody of any of the preceding embodiments; and a linker that allows for the incorporation of a therapeutic isotope or therapeutic agent.

[0081] Provided herein is a kit comprising the antigen-binding construct, minibody, or cys-diabody of any of the preceding embodiments; and a detectable marker.

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

[0083]

[0023] The method of any one of the preceding embodiments, wherein the antigen-binding construct, minibody, or cys-diabody is conjugated to a detectable marker.

[0084]

[0023] 2. The method of any one of the preceding embodiments, wherein applying the antigen-binding construct, minibody, or cys-diabody comprises administering the antigen-binding construct to the subject.

[0085]

[0023] The method of any one of the preceding embodiments, wherein detecting binding or absence of binding of the antigen-binding construct, minibody, or cys-diabody to DLL3 comprises at least one of positron emission tomography, single-photon emission computed tomography, or fluorescence tomography.

[0086] 10. The method of any one of the preceding embodiments, wherein 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]

[0023] The method of any one of the preceding embodiments, wherein the antigen-binding construct, minibody, or cys-diabody is incubated with the sample for no more than 20 hours.

[0088]

[0023] The method of any one of the preceding embodiments, wherein the antigen-binding construct, minibody, or cys-diabody is incubated with the sample for no more than 6 hours.

[0089]

[0023] The method of any one of the preceding embodiments, wherein the antigen-binding construct, minibody, or cys-diabody is administered to a host, wherein a first quantity of the antigen-binding construct, minibody, or cys-diabody is not bound to DLL3, and a second quantity of the antigen-binding construct, minibody, or cys-diabody is bound to DLL3, and wherein at least about 80% of the first quantity of the antigen-binding construct, minibody, or cys-diabody is cleared within 12 hours.

[0090] Provided herein is 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 one of the preceding embodiments, wherein the antigen-binding construct is conjugated to a therapeutic agent.

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

[0092] A therapeutic composition that targets DLL3, the therapeutic composition comprising a minibody that binds to DLL3 and comprises a variable heavy chain (V H ) domains linked to the variable light chain (V L a single-chain variable fragment (scFv) that binds to DLL3 and comprises a V L The domain comprises an LCDR1 having SEQ ID NO: 15; an LCDR2 having any one of SEQ ID NOs: 19, 21, or 23; and an LCDR3 having SEQ ID NO: 25, Ha single chain variable fragment (scFv) comprising domains: HCDR1 which is SEQ ID NO: 27; HCDR2 which is SEQ ID NO: 29; HCDR3 which is SEQ ID NO: 31; a hinge extension domain comprising an IgG1 hinge region; an IgG C H Provided herein are therapeutic compositions comprising a minibody comprising the three sequences; and a therapeutic agent, a toxic payload, and / or a detectable marker.

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

[0094] The therapeutic composition of any of the preceding embodiments, wherein the detectable marker is a radioactive label.

[0095] The therapeutic composition of any of the preceding embodiments, wherein the detectable marker is an alpha-emitter radiolabel.

[0096] The therapeutic composition of any of the preceding embodiments, wherein the detectable marker is a beta-emitter radiolabel.

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

[0098] The therapeutic composition of any of the preceding embodiments, wherein the detectable marker comprises lutetium-177. [Brief explanation of the drawings]

[0099] [Figure 1] 1 shows some embodiments of polypeptide sequences of antigen-binding minibodies (SEQ ID NOs: 1 to 10). [Figure 2] 1 shows some embodiments of polypeptide sequences of antigen-binding cys-diabodies (SEQ ID NOs: 11 to 14). [Figure 3] FIG. 1 shows some embodiments of the polypeptide sequences of LCDR (SEQ ID NOs: 15 to 26), HCDR (SEQ ID NOs: 27 to 32), LFR (SEQ ID NOs: 33 to 38), linker (SEQ ID NOs: 39 to 44), signal peptide (SEQ ID NOs: 45 to 46), parent VH (SEQ ID NO: 47), and parent VL (SEQ ID NO: 48). [Figure 4] FIG. 1 shows some embodiments of annotated polypeptide sequences of antigen-binding minibodies. [Figure 5] FIG. 1 shows some embodiments of variable region alignments of antigen-binding minibody polypeptide sequences and rovalpituzumab polypeptide sequences. [Figure 6] 1 shows an embodiment of the polypeptide sequence of human DLL3 (SEQ ID NO: 50). [Figure 7] 1 is a bar graph showing an embodiment of minibody expression levels in mammalian cells. [Figure 8] 1 is a collection of graphs and corresponding tables showing some embodiments of EC50 determination of antigen-binding minibodies by ELISA. [Figure 9] 1 is a collection of graphs and corresponding tables illustrating some embodiments of equilibrium dissociation constant determination of antigen-binding minibodies. [Figure 10] 1 is a collection of graphs showing some embodiments of EC50 determination of antigen-binding minibodies by flow cytometry. [Figure 11] 1 is a bar graph showing some embodiments of the in vivo biodistribution of antigen-binding minibodies in a tumor-bearing mouse model. [Figure 12]1 is a bar graph showing some embodiments of the in vivo biodistribution of antigen-binding minibodies and cys-diabodies. [Figure 13] 1 shows some embodiments of nucleic acid sequences for antigen-binding constructs, minibodies, and cys-diabodies (SEQ ID NOs: 51-64). [Figure 14] 1 shows some embodiments of CH3 polypeptide sequences (SEQ ID NOs: 65 to 85). DETAILED DESCRIPTION OF THE INVENTION

[0100] Detailed Description Described herein are antigen-binding constructs, including antibodies and fragments thereof, such as minibodies and cys-diabodies. In some embodiments, these bind to target molecules, such as DLL3. In some embodiments, these components are novel complementarity-determining region (CDR) sequences and / or sequences related to and / or portions of CDR sequences. In some embodiments, these components are novel framework region (FR) sequences and / or sequences related to and / or portions of FR sequences. These CDR and FR sequences may provide various benefits and improvements over the prior art. Also provided herein are antigen-binding constructs (minibodies, cys-diabodies, etc.) comprising one or more of the CDR or FR sequences or subsequences provided herein.

[0101] In some embodiments, antigen-binding constructs may be useful for targeting therapeutic agents to cells expressing the target molecule. In some embodiments, methods are provided for detecting the presence or absence of a target molecule (or "target") using antigen-binding constructs (including antibodies, minibodies, and cys-diabodies). In some embodiments, methods are provided for using antigen-binding constructs for therapeutic purposes.

[0102] In some embodiments, antigen-binding constructs such as minibodies and cys-diabodies may possess superior pharmacokinetic properties for more rapid diagnostic imaging while maintaining the binding specificity and affinity of the parent antibody. Current technology utilizes imaging with full-length antibodies, which often requires significantly longer times (approximately 7-8 days after injection) due to the slow serum clearance of intact antibodies, to generate high-construct images. Some embodiments of the antigen-binding constructs, minibodies, and cys-diabodies provided herein offer the opportunity for same-day or next-day imaging.

[0103] In some embodiments, the antigen-binding constructs are intended for diagnostic purposes. When labeled with an appropriate radionuclide (e.g., the 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 fragments can be used for preclinical imaging and clinical imaging in patients as described herein. 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.

[0104] In some embodiments, the antigen-binding constructs can be used as clinical imaging agents (PET / SPECT) in humans. Thus, in some embodiments, the antigen-binding constructs can be used for targeted diagnostic detection of these disorders. In some embodiments, the antigen-binding constructs can be used as therapeutic agents.

[0105] definition All terms have their ordinary and accustomed meaning as understood by one of ordinary skill in the art in light of this disclosure.

[0106] The term "antigen-binding construct" includes all types of antibodies, including binding fragments thereof. Constructs containing one, two, three, four, five, and / or six CDRs are further included. In some embodiments, a tandem scFv may be provided, which can provide two arms with bivalent binding. In some embodiments, these CDRs may be distributed between their appropriate framework regions as in traditional antibodies. In some embodiments, the CDRs may be contained within the heavy and / or light chain variable regions. In some embodiments, the CDRs may be within the heavy and / or light chain. In some embodiments, the CDRs may be within a single peptide chain. Unless otherwise stated herein, the antigen-binding constructs described herein bind to a reference target molecule. The term "target" or "target molecule" refers to a 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).

[0107] The term "antibody" includes genetically engineered or otherwise modified forms of immunoglobulins, such as, but not limited to, intrabodies, chimeric antibodies, fully human antibodies, humanized antibodies, antibody fragments, single-chain variable fragments (scFv), and heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, nanobodies, etc.). The term "antibody" includes minibodies and diabodies. The term "antibody" includes polypeptides comprising a polypeptide of the immunoglobulin family or a fragment of an immunoglobulin that can bind to a corresponding antigen in a noncovalent, reversible, and specific manner. An exemplary antibody structural unit includes a tetramer. In some embodiments, a full-length antibody may be composed of two identical pairs of polypeptide chains (connected through disulfide bonds), each pair having one "light" and one "heavy" chain. Recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, hinge, and mu constant region genes, as well as myriad immunoglobulin variable region genes. With respect to full-length chains, light chains are classified as either kappa or lambda. With respect to full-length chains, heavy chains are classified as gamma, mu, alpha, delta, or epsilon, 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 primarily responsible for antigen recognition. Variable light chains (VLCs) L ) and variable heavy chain (V HThe terms "antibody" and "antibody" refer to these regions of the light and heavy chains, respectively. As used in this application, "antibody" encompasses all variations of antibodies and fragments thereof. The term "antibody" also includes single heavy chain antibodies and camelid-derived immunoglobulins such as nanobodies®. Thus, within this concept are full-length antibodies, chimeric antibodies, humanized antibodies, single chain antibodies (scFv), Fab, Fab', and multimeric versions of these fragments having the same binding specificity (e.g., F(ab')2), scFv-Fc, single domain fragments (e.g., nanobodies®), peptibodies, nanobodies®, nanobody®-Fc, minibodies, and diabodies. In some embodiments, antibodies specifically bind to a desired target.

[0108] The term "complementarity determining domain" or "complementarity determining region ("CDR")" refers to a V L and V H The CDRs are interchangeably referred to as the hypervariable regions of the V. The CDRs are the target molecule binding sites of the antibody chain that have specificity for such target molecule. In some embodiments, each V L and / or V H There are three CDRs (numbered sequentially from the N-terminus) in the variable domain, which make up approximately 15-20% of the V domain. The CDRs are structurally complementary to the epitope of the target molecule and are therefore directly responsible for binding specificity. L or V H The remaining stretches, the so-called FRs, exhibit lower variability in amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4, WH Freeman & Co., New York, 2000).

[0109] The locations of the CDRs and framework regions may be determined according to various well-known definitions in the art, such as those defined by Kabat (Wu, T.T. 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, E.A. et al., "Sequences of Proteins of Immunological Interest," 5th ed., 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," J. Mol. Biol., Vol. 196, No. 4, pp. 901-917, 1987); "Canonical 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 at imgt.org / ) (Giudicelli, V.et al., "IMGT / LIGM-DB, the IMGT® comprehensive database of immunoglobulin and T cell receptor nucleotide sequences," Nucleic Acids Res., Vol. 34 (Database Issue), pp. D781-D784, 2006; Lefranc, MP 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 VJ and VDJ sequence analysis," Nucleic Acids Res., Vol. 36 (Web Server Issue), pp. W503-508, 2008; AbM (Martin, AC et al., "Modeling antibody hypervariable loops: a combined algorithm," Proc. Natl. Acad. Sci. USA, Vol. 86, No. 23, pp. 9268-9272, 1989);AHo(Honegger A, Pluckthun 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 definitions (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 automated modeling and analysis tools (Honegger, A. et al., available on the World Wide Web at bioc.uzh.ch / plueckthun / antibody / Numbering / ). In some embodiments, polypeptides are numbered from the beginning of the polypeptide signal sequence. In some embodiments, polypeptides are numbered according to the beginning of the polypeptide and not including the signal sequence.

[0110] As used herein, an "antibody variable light chain" or an "antibody variable heavy chain" refers to a V L or V H refers to a polypeptide comprising an endogenous V L is encoded by gene segments V (variable) and J (joining), and the endogenous V H is coded by V, D (diversity), and J. V L or V H Each of the V and V chains comprises a CDR and a framework region. In this application, the antibody variable light chain and / or the antibody variable heavy chain may sometimes be collectively referred to as "antibody chains." These terms refer to the V and V chains, as will be readily recognized by those skilled in the art. L or V H In some embodiments, full-length heavy and / or light chains are contemplated. In some embodiments, only the variable regions of the heavy and / or light chains are contemplated.

[0111] The term "hinge" refers to at least a portion of the hinge region of an antigen-binding construct, such as an antibody, minibody, scFv-Fc, or nanobody®-Fc. The hinge region may include a combination of upper hinge, core (or middle) hinge, and lower hinge regions. In some embodiments, the hinge is defined according to any of the antibody hinge definitions. Naturally occurring IgG1, IgG2, and IgG4 antibodies have a hinge region of 12-15 amino acids. IgG3 has an extended hinge region of 62 amino acids, including 21 prolines and 11 cysteines. The functional hinge region of naturally occurring antibodies, as predicted from crystallographic studies, extends from amino acid residues 216-237 (EU numbering; Reference 12) of the IgG1 heavy chain and includes a small segment N-terminal to the CH2 domain in the lower hinge, which is N-terminal to the CH2 domain. The hinge can be divided into three regions: the "upper hinge," the "core," and the "lower hinge."

[0112] The terms "artificial" or "non-naturally occurring" when referring to modifying a hinge (or subparts thereof) indicate that the sequence in question does not occur in nature in the state referred to. In this context, the hinges have been altered from their natural state such that their sequence is no longer found in wild-type antibodies. As will be understood by those skilled in the art, minibodies do not naturally occur in nature, and therefore, any construct that is a minibody construct is also not found in nature. This also applies to at least a portion of constructs and / or constructs incorporating sequences found in any of the tables of hinge sequences provided herein (e.g., Table 4). In some embodiments, either the hinge subpart or the complete hinge sequence can be an artificial hinge sequence, so long as the sequence (or the resulting combination for the hinge) does not occur in nature.

[0113] The term "complete hinge region" or "entire hinge region" refers to the presence of the entire upper, core, and lower hinge regions as a single construct. The upper, core, and lower regions may be located directly adjacent to one another, or additional residues may be added between or N- or C-terminal to the regions. In some embodiments, the native lower hinge may be replaced with an extension sequence. In some embodiments, the native lower hinge may be combined with an extension sequence. In some embodiments, an extension sequence or other set of sequences may be added after the upper and / or core sequence.

[0114] The phrase "effective hinge region" refers to the presence of a sufficient amount of at least a portion of the upper, core, and lower hinge regions to allow the hinge region to be effective for its intended purpose. Thus, the phrase encompasses variants of the hinge region and various fragments of the hinge region. In some embodiments, the function of the hinge region is as follows: H The hinge has one or more of the following functions: linking the three domains, providing flexibility and spacing for proper binding of the two scFvs to the target, linking the two half molecules together, providing overall stability to the molecule, and / or providing a site for site-specific conjugation due to its solvent exposure. In some embodiments, the hinge should be near-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 is C H 3 (starting at residue 231 in native IgG).

[0115] The term "upper hinge" refers to the first part of the hinge, starting from 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 noted above, the term "effective upper hinge" refers to the presence of sufficient sequence to allow the section to function as an upper hinge; the term encompasses functional variants and fragments of the specified hinge section.

[0116] The term "core hinge" refers to the second portion of the hinge region, C-terminal to the upper hinge. The core hinge contains interchain disulfide bridges and a high proline content. As noted above, the term "effective core hinge" refers to the presence of sufficient sequence to allow the section to function as a core hinge; the term encompasses functional variants and fragments of the designated hinge section.

[0117] The term "lower hinge" refers to the third portion of the hinge region, C-terminal to the core hinge. In the context of a minibody or antibody fragment, the lower hinge is the C H The lower hinge connects the three domains. As noted above, the term "effective lower hinge" refers to the presence of sufficient sequence to allow the section to function as a lower hinge; the term encompasses functional variants and fragments of the designated hinge section. As used herein, the term "lower hinge" can encompass various amino acid sequences, including naturally occurring IgG lower hinge sequences and artificial extension sequences, or combinations thereof, as provided herein, in place of each other. In some embodiments, various extensions can be considered to be the lower hinge region in its entirety or as replacements.

[0118] Antibodies can exist as intact immunoglobulins or as several fragments produced by digestion with various peptidases. Thus, for example, pepsin digests antibodies below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab', itself held together by disulfide bonds. H -C H 1-linked light chain (V L -C L). F(ab)'2 can be reduced under mild conditions to disrupt the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into a Fab' monomer. A Fab' monomer is a Fab with part of the hinge region. (Paul, WE, Fundamental Immunology, 3rd ed., New York: Raven Press, 1993). While various antibody fragments are defined in terms of the digestion of intact antibodies, those skilled in the art will appreciate that such fragments can be synthesized chemically or de novo using recombinant DNA methodologies. Thus, the term "antibody," as used herein, also includes antibody fragments produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (e.g., single-chain Fvs) or those 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).Regarding the preparation of monoclonal or polyclonal antibodies, any technique known in the art may be used (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: the next generation of antibody therapeutics?", Drug Discov. Today, Vol. 11, No. 13-14, pp. 655-660, 2006). Techniques for the production of single-chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies against the polypeptides of the present disclosure. Transgenic mice, or other organisms such as other mammals, can also be used to express fully human monoclonal antibodies.Additionally, recombinant antibodies and antibody fragments can be expressed and produced using Escherichia coli (E. coli) or yeast (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., 2021 Sept. 22). Alternatively, phage display and yeast display technologies can be used to identify high affinity binders to a selected antigen (see, e.g., McCafferty et al., supra; Marks, JD et al., "Bypassing 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, July 2004;290(1-2):69-80).Alternatively, antibodies can be produced by B cell screening techniques from human hosts (Pedrioli A., Oxenius A., Single B cell technologies for monoclonal antibody discovery, Trends in Immunology, (2021), Vol. 42, No. 12, pp. 1143-1158). Furthermore, antibodies can be obtained from immunization of camelids or screening of camelid phage libraries (Harmsen MM, De Haard 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 in silico screening simulations using 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).

[0119] Methods for humanizing or primatizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. In some embodiments, the terms "donor" and "acceptor" sequences may be employed. Humanization can be performed essentially according to the method of Winter and colleagues by replacing rodent CDR sequences or one CDR sequence with the corresponding sequences of a human antibody (see, e.g., Jones, P.T. 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 an antilysozyme activity," Science, Vol. 239, No. 4847, pp. 1534-1536, 1988; Presta, L.G., "Antibody engineering," Curr. Op. Struct. Biol., vol. 2, no. 4, pp. 593-596, 1992). Accordingly, such humanized antibodies are chimeric antibodies in which substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species (U.S. Pat. No. 4,816,567). In practice, humanized antibodies are typically human antibodies in which some complementarity-determining region ("CDR") residues and possibly some framework ("FR") residues are substituted by residues from analogous sites in rodent antibodies.

[0120] The terms "Fc region" or "Fc domain" or "Fc" refer to the C-terminal region of an immunoglobulin heavy chain. The "Fc region" may 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 neonatal receptor (FcRn)). The Fc region of an immunoglobulin (e.g., IgG) generally comprises the C-terminal region of an immunoglobulin. H 2 and C H The Fc region comprises two constant domains, Fc1, Fc2, Fc3, Fc4, Fc5, Fc6, Fc7, Fc8, Fc9, Fc10, Fc11, Fc12, Fc13, Fc14, Fc15, Fc16, Fc17, Fc18, Fc19 ...0, Fc11, Fc12, Fc13, F

[0121] A "chimeric antibody" is an antibody molecule in which (a) the antigen-binding site (variable region) has been modified, replaced, or exchanged so that the constant region or a portion thereof is linked to a constant region of a different or altered class, effector function, and / or species, or to an entirely different molecule that confers new properties to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, or drug; or (b) the variable region or a portion thereof has been modified, replaced, or exchanged with a variable region having a different or altered antigen specificity.

[0122] The term "antibody fragment" includes 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., nanobodies® or single domain fragments), peptibodies, nanobodies®, nanobody®-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 traditional two-chain antibody have been joined to form a single chain.

[0123] The term "artificial" or "non-naturally occurring" when referring to modified CDRs or FRs (or subparts thereof) indicates that the sequences in question do not occur in nature in the state referred to. In this context, the CDRs or FRs have been altered from their natural state, such that the sequences are no longer found in wild-type antibodies. As will be understood by those skilled in the art, minibodies and cys-diabodies do not naturally occur in nature, and therefore, any construct that is a minibody or cys-diabody construct is also not found in nature. This also applies to at least some of the constructs and / or constructs incorporating sequences found in any of the tables of CDR or FR sequences provided herein. In some embodiments, any of the CDR or FR sequences in a figure or table, e.g., in Figure 1, Table 1, or Table 2, can be an artificial CDR or FR sequence, so long as the sequence (or the resulting combination for the CDR or FR) does not occur in nature.

[0124] A "minibody" is an antibody format that has a smaller molecular weight than a full-length antibody while maintaining bivalent binding properties to an antigen. Due to its smaller size, the absence of a CH2 domain that binds to Fc-gamma and FcRn receptors, and the absence of glycosylation, minibodies have faster clearance from the body and potentially enhanced penetration when targeting tumor tissue. With strong targeting capabilities combined with rapid clearance, minibodies are advantageous for the delivery of diagnostic imaging and radioactive payloads, where prolonged circulation time can result in harmful patient dosing or dosimetry. In some embodiments, they may also be advantageous for the delivery of cytotoxic payloads due to the above-mentioned features, such as tumor penetration and faster clearance. The "minibody" described herein is a minibody in which each monomer is bound to a human IgG C domain by a hinge sequence. HIn some embodiments, minibodies are bivalent or bispecific, covalently linked homodimers of approximately 80 kDa. In some embodiments, each monomer (half molecule) is linked to its corresponding variable light chain (VL) by a Gly-Ser-rich linker sequence of approximately 15-18 amino acids. L ) domains of the variable heavy chain (V H ) domains.

[0125] A "diabody" is a molecule comprising two light chain variable domains (V) on a first polypeptide chain connected by a peptide linker that does not allow pairing between the two domains on the first polypeptide chain (e.g., the peptide linker is too short to allow pairing). L ) connected to the heavy chain variable domain (V H ) (V H -V L or V L -V H ) on a second polypeptide chain connected by a peptide linker that does not allow pairing between the two domains on the second polypeptide chain (e.g., the peptide linker is too short to allow pairing). L ) linked to a heavy chain variable domain (V H ) (V H -V L or V L -V H and a second polypeptide chain comprising a C-terminal cysteine. Without being limited by theory, a short linkage can force chain pairing between the complementary domains of the first and second polypeptide chains, promoting the association of a dimeric molecule having two functional antigen-binding sites. Thus, the peptide linker can be of any suitable length that promotes such association, e.g., 5 to 20 amino acids in length. A "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 having one or more C-terminal cysteines.

[0126] The term "extension sequence" (e.g., in a diabody context) refers to a sequence that extends the first V H Domain as a second V H domain, or the first V L The second V L The extension sequence represents the region connecting the domains. An extension sequence may connect the domains through the C-terminus of each domain. In some embodiments, the extension sequence connects the domains through a covalent bond. In some embodiments, the extension sequence will contain one or more cysteines that allow one or more disulfide bonds to form between two such extension sequences. A non-limiting example of an extension sequence includes -(Gly)2-(Cys). In some embodiments, the extension sequence contains one, two, three, or more cysteines per monomer chain. The extension sequence will be toward the C-terminus of the construct, but it need not be the absolute last amino acid in the variable domain. That is, the extension sequence may be located slightly N-terminal to the C-terminus. For example, the extension sequence may be placed within 10 amino acids of the C-terminus of the monomer. Similarly, additional sequence may be placed between the natural C-terminus and where the extension sequence begins. The extension sequence will connect the V domains through disulfide bonds. H V H ni or V L V L In some embodiments, the extension sequence comprises GGCPPCPPC (SEQ ID NO: 93).

[0127] As used herein, "pharmaceutically acceptable" has its plain and ordinary meaning as understood in light of the present specification and refers to a carrier, excipient, and / or stabilizer that is non-toxic or has an acceptable level of toxicity to cells or mammals exposed thereto at the dosages and concentrations employed. As used herein, "pharmaceutically acceptable," "diluent," "excipient," and / or "carrier" have their plain and ordinary meaning as understood in light of the present specification and are intended to include any and all solvents, dispersion media, coatings, antibacterial or antifungal agents, isotonic or absorption delaying agents that are compatible with administration to a human, primate, feline, canine, or other vertebrate host. Typically, pharmaceutically acceptable diluents, excipients, and / or carriers are those approved by federal, state, or other regulatory agencies, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia, for use in humans and animals, including non-human mammals such as cats and dogs. The terms diluent, excipient, and / or "carrier" may refer to a diluent, adjuvant, excipient, or vehicle with which a pharmaceutical composition is administered. Such pharmaceutical diluents, excipients, and / or carriers that may be incorporated into any one or more of the compositions described herein include sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin. Water, saline solution, or aqueous dextrose and glycerol solutions may be employed as liquid diluents, excipients, and / or carriers. Suitable pharmaceutical diluents and / or excipients that may be incorporated into any one or more of the compositions described herein also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, 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 preservatives such as TWEEN®, polyethylene glycol (PEG), PLURONICS®, or non-ionic surfactants, essential oils, methylparaben, propylparaben, or sodium salts of parabens. In some embodiments, the preservative is bronidiol. The compositions may also contain minor amounts of wetting agents, bulking agents, emulsifiers, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, sustained-release formulations, etc. The formulation should suit the mode of administration.

[0128] Additional excipients having desirable 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, sugars, 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 combinations thereof. In some embodiments, the formulation includes at least one agent that acts to reduce radiolysis (also known as a "radioprotectant") or is a nephroprotectant.Non-limiting examples of radiolysis reducing agents include gentisic acid, acetylcholine, AET, ACE inhibitors, acteoside, alpha-tocopheryl acetate, amifostine, ascorbic acid, aspirin, atorvastatin, beta-carotene, Bowman-Birk proteinase inhibitors, caffeic acid, captopril, carbaminoylcholine, carvacrol, celecoxib, coenzyme Q10, COX2 inhibitors / NSAIDs, curcumin, cysteine, cysteamine, cystamine, dendrodine analogs, dithiolthiones, dopamine, enalapril, epigallocatechin-3-gallate, epinephrine, 17-beta-estradiol, GANRA-5, genistein, green tea extract, growth factors, guanine nucleotides, halofuginone, Hmg-CoA reductase inhibitors (statins), heroin, histamine, hydroxybenzoates ... Ingredients include amine, ibuprofen, inapoyl-E-glucoside, isoflavones, isofraxidin, kukoamine A, lactoferrin, amifostine, lipoic acid, 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, quercetin-3-O-rhamnoside, ramipril, resveratrol, rutin, serotonin, simvastatin, sodium ascorbate, superoxide dismutase, TGF-signaling inhibitors, tocopherol, vitamin C, vitamin E, watermelon juice, black grape juice, and thiols such as glutathione. Nephroprotectants include free lysine, arginine, probenecid, gelofusin, and other compositions. Some excipients may be residual amounts or contaminants from the manufacturing process, including, but not limited to, serum, albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde, glutaraldehyde, β-propiolactone, gelatin, cell debris, nucleic acids, peptides, amino acids, or growth medium components, or any combination thereof.The amount of excipient was approximately 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. 0%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w / w, at least 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, at least 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,At most 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%, It may be found in the composition in a percentage that is 90%, 95%, 100% w / w, or at most 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, or any weight percentage within the range defined by any two of the foregoing numbers.

[0129] As used herein, "carrier" has its plain and ordinary meaning as understood in light of this specification and refers to a compound, particle, solid, semi-solid, liquid, or diluent that facilitates the passage, delivery, and / or incorporation of a compound into cells, tissues, and / or bodily organs.

[0130] As used herein, the term "diluent" has its plain and ordinary meaning as understood in light of this specification and refers to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable. For example, a diluent may be used to increase the volume of a potent drug whose mass is too small for manufacture and / or administration. It may also be a liquid for dissolving a drug to be administered by injection, oral ingestion, or inhalation. A common form of diluent in the art is a buffered aqueous solution, such as, but not limited to, phosphate-buffered saline, which mimics the composition of human blood.

[0131] The term "target molecule-dependent disorder" or "target molecule-associated disorder" includes any disorder in which a target molecule plays a role in the disorder itself. In some embodiments, this represents overexpression of a target molecule. In some embodiments, the disorder can include any of the disorders discussed herein. In some embodiments, the disorder can be any in which there is a target molecule that can be targeted by binding, which binding will result in detection and / or treatment of the disorder.

[0132] The terms "treating" or "treatment" of a condition can refer to preventing the condition, slowing the onset and / or progression of the condition, reducing the risk of developing the condition, preventing and / or delaying the onset of symptoms associated with the condition, reducing and / or terminating symptoms associated with the condition, causing complete or partial regression of the condition, or some combination thereof. The term "preventing" does not require an absolute prohibition of the disorder or disease. Examples of disorders or diseases include fibrosis, cancer, tumors and neoplasms, autoimmune diseases, cardiovascular, neurodegenerative, metabolic, and endocrine disorders, inflammatory, immune, genetic disorders, infectious disorders, hematological, congenital disorders, musculoskeletal, oral and gastrointestinal, renal and genitourinary disorders, reproductive disorders, respiratory disorders, skin and / or epithelial disorders, and disorders with disputed or unknown etiology.

[0133] As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor," as referred to herein, are not mutually exclusive. The term "neoplasia" encompasses the term tumor.

[0134] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such 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 tumors of unknown primary site, small intestine, carotid body, adrenal gland, colorectal gynecological organs, abdomen, esophagus, gastrointestinal tract, bile duct, nervous system, appendix, liver, anus, thymus, ileocecal junction, head and neck, breast, peritoneum and retroperitoneum, kidney, thyroid, stomach, Neuroendocrine neoplasms, such as neuroendocrine neoplasms of bone; adenocarcinomas, such as adenocarcinoma of the lung and squamous cell carcinoma of the lung; cancer of the peritoneum; hepatocellular carcinoma; gastric or stomach cancer, including gastrointestinal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer; urinary tract cancer; hepatoma; breast cancer; colon cancer; rectal cancer; colorectal cancer; endometrial or uterine cancer; salivary gland cancer; kidney or renal cancer; prostate cancer; vulvar cancer; thyroid cancer; bone cancer; liver cancer; anal cancer; penile cancer; melanoma; multiple myeloma and B-cell lymphoma; brain and head and neck cancer, and associated 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 high-grade fibrosing tumor or carcinoma. In some embodiments, the cancer is desmoplasia.

[0135] A "therapeutically effective amount" or "therapeutically effective dose" is an amount that produces a desired therapeutic effect in a subject, such as preventing or treating a target disease state, delaying the onset of a disorder and / or condition, and / or alleviating symptoms associated with a disease state. This amount will vary depending on a variety of factors, including, but not limited to, the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, biodistribution, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier(s) in the formulation, and / or the route of administration. Those skilled in the clinical and pharmacological arts, given the present disclosure, will be able to determine a therapeutically effective amount by routine experimentation, e.g., by monitoring the subject's response to administration of the compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy, 21st ed., University of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005.

[0136] "Label," "detectable label," or "detectable marker" are used interchangeably herein to refer to a detectable compound or composition that is directly or indirectly conjugated in association with an antibody to produce a "labeled" antibody. The label may be detectable itself (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.

[0137] The term "payload" refers to an atom or molecule or other entity that is attached (covalently or otherwise) to an antigen-binding construct. It includes, for example, labels or markers for diagnostic aspects, as well as toxins, cytotoxic agents, chemotherapeutic agents for various therapies. In some embodiments, the payload refers to a chelator for attaching the antigen-binding construct to a molecule or atom of interest that is to be delivered or co-localized via the antigen-binding construct.

[0138] 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 includes 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 (defined elsewhere herein). Other cytotoxic agents are described below. Tumoricidal agents cause the destruction of tumor cells.

[0139] A "toxin" is any substance that can have a deleterious effect on cell growth or proliferation. Non-radioactive payloads include those commonly used in antibody drug conjugates (ADCs) and fragment drug conjugates (FDCs), such as toxins from the auristatin, maytansine, maytansinoid, calicheamicin, duocarymycin, pyrrolobenzodiazepine dimer, and amatoxin families.

[0140] "Therapeutic ions" refer to electrically charged particles that are useful in treating disorders 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, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 149 Tb, 153 Sm, 154~158 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 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 These also represent therapeutic options.

[0141] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN™ cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (among others). (bulatacin and bulatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL™); beta-lapachone; lapachol; colchicines; betulinic acid; camptothecin (including synthetic analogs topotecan (HYCAMTIN™), CPT-11 (irinotecan, CAMPTOSAR™), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); podophyllotoxin; podophyllinic acid acid); teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbiquine, fenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;Enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma II and calicheamicin omega II (see, e.g., Agnew, Chem Intl. Ed. Engl., 33:183-186 (1994)); dynemicins, including dynemicin A; esperamicins; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, dow Norubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN™ doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, pepromycin, antibiotics such as benzodiazepine, porfiromycin, puromycin, quelamycin, rhodrubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; metabolic inhibitors such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; fludarabine, 6-mercaptopurine, and thiamine Purine analogues such as purines and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine;Diazicon; eflornithine, elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK.R™ polysaccharide complex (JHS Natural Products, Inc., Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine (ELDISINE™, FILDESIN™); dacarbazine; mannommustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, such as TAXOL.R™ paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE™ cremophor-free albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE™ docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (GEMZAR™); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN™); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN™); oxaliplatin; leucovorin; vinorelbine (NAVELBINE™); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; the topoisomerase inhibitor RFS 2000;Treatment options also include difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA™); pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, such as CHOP, an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for the treatment regimen using oxaliplatin (ELOXATIN™) in combination with 5-FU and leucovorin.

[0142] "Radiotherapy" means treatment using radiation or radioisotopes for therapeutic purposes, including radiotherapy intended to have an 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, Vol. 11, Article No.: 104 (2018); and Melek Tugce Yilmaz, Aysenur Elmali, and Gozde Yazici; "Abscopal Effect, From Myth to Reality: From Radiation Oncologists' Perspective"; Cureus. January 2019; 11(1).

[0143] The terms "subject," "patient," and "individual" refer interchangeably to the entity being tested and / or treated. The term "mammal" is used in its ordinary biological sense. Thus, it specifically includes, but is not limited to, primates, including monkeys (chimpanzees, apes, monkeys), humans, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, or guinea pigs.

[0144] The term "co-administering" refers to the administration of two active agents in the blood or sample of an individual being tested. Co-administered active agents may be delivered in combination or sequentially. "In combination" means that two (or more) different compositions are delivered to a subject during the course of the subject's illness with a disorder, for example, two or more compositions are delivered after the subject is diagnosed or selected as having a disorder and before the disorder is cured or eliminated. In some embodiments, a subject is selected by diagnostic analysis or clinical evaluation, or both, to receive any one or more of the compositions described herein. In some embodiments, the delivery of one therapy is still ongoing when the delivery of a second therapy begins, such that there is overlap. This may also be referred to herein as "simultaneous," "concomitant," or "concurrent delivery." In other embodiments, the delivery of one therapy ends before the delivery of the other therapy begins. This may also be referred to herein as "continuous" or "sequential delivery." In either case, the therapies are more effective due to the combined administration. For example, the second therapy may be more effective, e.g., an equivalent effect may be observed using less of the second therapy, or the second therapy may reduce symptoms to a greater extent than would be observed if the second therapy were administered in the absence of the first therapy, or a similar situation may be observed with the first therapy. In some embodiments, delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than would be observed with one therapy delivered in the absence of the other. 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 effect of the first therapy delivered is still detectable at the time the second is delivered.

[0145] The phrase "specifically (or selectively) binds," when used in the context of describing the interaction of an antigen, e.g., a protein, with an antibody or antibody-derived binding agent, refers to a binding reaction that determines the presence of the antigen in a heterogeneous population of proteins and other biologics, e.g., in a biological sample, e.g., blood, serum, plasma, or tissue sample. Thus, under specified immunoassay conditions, in some embodiments, an antibody or binding agent with a particular binding specificity will bind to the particular antigen at least twice background and will not bind substantially in significant amounts to other antigens present in the sample. Specific binding to an antibody or binding agent under such conditions may require that the antibody or agent be selected for its specificity for a particular protein. A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow, E. & Lane D., "Using Antibodies, A Laboratory Manual," Cold Spring Harbor Laboratory Press, 1998, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, a specific or selective binding reaction will produce a signal that is at least 2-fold over background, and more typically at least 10-100-fold over background.

[0146] “Equilibrium dissociation constant (K D The term "association rate constant (k a ,time -1 M -1 ) divided by the dissociation rate constant (k d ,time -1 The equilibrium dissociation constant can be measured using any method known in the art. Antibodies of the present disclosure generally have an equilibrium dissociation constant of about 10 -7 or 10 -8 Less than M (good binding), e.g., about 10 -9 M or 10 -10less than about 10 M, in some embodiments less than about 10 -11 M, 10 -12 M or 10 -13 will have an equilibrium dissociation constant less than M.

[0147] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers.

[0148] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitution can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted 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, pp. 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).

[0149] 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 later modified, such as hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, such as an alpha-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

[0150] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to those nucleic acids that encode identical or essentially identical amino acid sequences, or, if the nucleic acid does not encode an amino acid sequence, essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. Those of skill in the art will recognize that each codon within a nucleic acid (except AUG, which is normally the only codon for methionine, and TGG, which is normally the only codon for tryptophan) can be altered to produce a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0151] 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 number of amino acids in the encoded sequence are "conservatively modified variants," in that the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the present disclosure.

[0152] The following eight groups each contain amino acids that are conservative substitutions for one another: 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", W.H. Freeman & Co. Ltd., 1984).

[0153] The term "percentage of sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (e.g., a polypeptide of the present disclosure) that does not contain additions or deletions relative to the optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to arrive at 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 arrive at the percentage of sequence identity.

[0154] The term "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same sequence. Two sequences are "substantially identical" if they have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over a specified region, or, if not specified, over the entire sequence of the reference sequence), when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Some embodiments provided herein provide polypeptides or polynucleotides that are substantially identical to the polypeptides or polynucleotides exemplified herein, respectively. Optionally, identity exists over a region that is at least about 15, 25, or 50 nucleotides in length, or more preferably over a region that is 100-500 or 1000 or more nucleotides in length, or over the entire length of the reference sequence. With respect to amino acid sequences, identity or substantial identity can exist over a region that is at least about 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 over the entire length of the reference sequence. With respect to 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.

[0155] In some embodiments, the percent identity is over the CDR and / or FR regions referred to herein. In such circumstances, the percent identity of a CDR or FR can be specified separately from the remainder of the protein or nucleic acid sequence. Thus, two CDRs or FRs can have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over a specified region, or, if not specified, over the entire sequence of a reference sequence), while allowing the remainder of the protein to remain 100% identical to the comparison protein, or allowing the remainder of the protein to vary by the specified percent identity.

[0156] For sequence comparison, typically, one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence based on the program parameters.

[0157] As used herein, a "comparison window" refers to any segment of a number of contiguous positions selected from the group consisting of 20 to 600, usually about 50 to about 200, and more usually about 100 to about 150, within which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by 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, by the similarity search 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, or by computer implementations of these algorithms (Wisconsin Genetics software package, Genetics Computer Group, Inc., 575 Science Alignment can be performed using a variety of algorithms, including GAP, BESTFIT, FASTA, and TFASTA (see, for example, Ausubel, FM et al., Current Protocols in Molecular Biology, Supplement, 1995), or by manual alignment and visual inspection (see, for example, Ausubel, FM et al., Current Protocols in Molecular Biology, Supplement, 1995).

[0158] Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described, respectively, in Altschul, S. F. 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, S. F. et al., "Basic local alignment search tool," J. Mol. Biol., Vol. 215, No. 3, pp. 403-410, 1990. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in a query sequence that match or meet a certain positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul, SF et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction is halted when the cumulative alignment score falls by an amount X from its maximum achieved value; when the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments; or when the end of either 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 as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length 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), an alignment (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0159] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin, S. et al., "Applications and statistics for multiple high-scoring segments in molecular sequences," Proc. Natl. Acad. Sci. USA, Vol. 90, No. 12, pp. 5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in the comparison of the test nucleic acid with the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0160] 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 an antibody raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, in some embodiments, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.

[0161] Antigen-binding constructs It is understood herein that sequences within the CDRs and / or FRs may be particularly relevant to various antigen-binding constructs. In some embodiments, the value of the CDRs and / or FRs may be particularly high in minibodies or diabodies, such as cys-diabody configurations.

[0162] Some aspects of the present disclosure relate to antigen-binding constructs. In some embodiments, the antigen-binding construct comprises a variable light chain (VLCDR1) comprising SEQ ID NO: 15; an LCDR2 comprising any one of SEQ ID NOs: 19, 21, or 23; and an LCDR3 comprising SEQ ID NO: 25. L ) domain; and / or a variable heavy chain (V) comprising an HCDR1 that is SEQ ID NO: 27; an HCDR2 that is SEQ ID NO: 29; and an HCDR3 that is SEQ ID NO: 31 H ) domain. In some embodiments, the V L comprises an LCDR1 that is the LCDR1 in any one of SEQ ID NOs: 1 to 14, 90, and 91; an LCDR2 that is the LCDR2 in any one of SEQ ID NOs: 1 to 14, 90, and 91; an LCDR3 that is the LCDR3 in any one of SEQ ID NOs: 1 to 14, 90, and 91; Hcomprises an HCDR1 that is the HCDR1 in any one of SEQ ID NOs: 1-14, 87, and 88; an HCDR2 that is the HCDR2 in any one of SEQ ID NOs: 1-14, 87, and 88; and an HCDR3 that is the HCDR3 in any one of SEQ ID NOs: 1-14, 87, and 88. In some embodiments, the antigen-binding construct comprises a variable heavy chain (VH) comprising an HCDR1 that is SEQ ID NO: 27; an HCDR2 that is SEQ ID NO: 29; and an HCDR3 that is SEQ ID NO: 31. H In some embodiments, the antigen-binding construct comprises a variable heavy chain (VH) domain comprising an HCDR1 that is the HCDR1 in any one of SEQ ID NOs: 1-14, 87, and 88; an HCDR2 that is the HCDR2 in any one of SEQ ID NOs: 1-14, 87, and 88; and an HCDR3 that is the HCDR3 in any one of SEQ ID NOs: 1-14, 87, and 88. H ) domain.

[0163] In some embodiments, the antigen-binding construct comprises a minibody. In some embodiments, a minibody that binds to DLL3 is provided, wherein the minibody comprises a single chain variable fragment (scFv) that binds to DLL3. In some embodiments, the scFv comprises a variable heavy chain (V H ) domains linked to the variable light chain (V L ) domain. V L The domain comprises an LCDR1 that is SEQ ID NO: 15; an LCDR2 that is any one of SEQ ID NOs: 19, 21, or 23; and an LCDR3 that is SEQ ID NO: 25. H The domain comprises an HCDR1 that is SEQ ID NO: 27; an HCDR2 that is SEQ ID NO: 29; and an HCDR3 that is SEQ ID NO: 31. L comprises an LCDR1 that is the LCDR1 in any one of SEQ ID NOs: 1 to 14, 90, and 91; an LCDR2 that is the LCDR2 in any one of SEQ ID NOs: 1 to 14, 90, and 91; an LCDR3 that is the LCDR3 in any one of SEQ ID NOs: 1 to 14, 90, and 91; HIn some embodiments, the minibody comprises a variable heavy chain (VH) comprising an HCDR1 that is HCDR1 in any one of SEQ ID NOs: 1-14, 87, and 88; an HCDR2 that is HCDR2 in any one of SEQ ID NOs: 1-14, 87, and 88; and an HCDR3 that is HCDR3 in any one of SEQ ID NOs: 1-14, 87, and 88. In some embodiments, the minibody comprises a variable heavy chain (VH) comprising an HCDR1 that is SEQ ID NO: 27; an HCDR2 that is SEQ ID NO: 29; and an HCDR3 that is SEQ ID NO: 31. H In some embodiments, the minibody comprises a variable heavy chain (VH) domain comprising an HCDR1 that is the HCDR1 in any one of SEQ ID NOs: 1-14, 87, and 88; an HCDR2 that is the HCDR2 in any one of SEQ ID NOs: 1-14, 87, and 88; and an HCDR3 that is the HCDR3 in any one of SEQ ID NOs: 1-14, 87, and 88. H The antigen-binding construct comprises a hinge extension domain comprising an IgG hinge region, and an IgG C domain. H 3 sequences.

[0164] 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 binds to a variable heavy chain (V H ) domains linked to the variable light chain (V L In some embodiments, the V L The domain comprises an LCDR1 that is SEQ ID NO: 15; an LCDR2 that is any one of SEQ ID NOs: 19, 21, or 23; and an LCDR3 that is SEQ ID NO: 25. H The domain comprises an HCDR1 that is SEQ ID NO: 27; an HCDR2 that is SEQ ID NO: 29; and an HCDR3 that is SEQ ID NO: 31. Lcomprises an LCDR1 that is the LCDR1 in any one of SEQ ID NOs: 1 to 14, 90, and 91; an LCDR2 that is the LCDR2 in any one of SEQ ID NOs: 1 to 14, 90, and 91; an LCDR3 that is the LCDR3 in any one of SEQ ID NOs: 1 to 14, 90, and 91; H comprises an HCDR1 that is the HCDR1 in any one of SEQ ID NOs: 1-14, 87, and 88; an HCDR2 that is the HCDR2 in any one of SEQ ID NOs: 1-14, 87, and 88; and an HCDR3 that is the HCDR3 in any one of SEQ ID NOs: 1-14, 87, and 88. L The domain comprises an LCDR1 that is SEQ ID NO: 15; an LCDR2 that is any one of SEQ ID NOs: 19, 21, or 23; and an LCDR3 that is SEQ ID NO: 25, H The domain comprises an HCDR1 that is SEQ ID NO: 27; an HCDR2 that is SEQ ID NO: 29; and an HCDR3 that is SEQ ID NO: 31. In some embodiments, the cys-diabody comprises a variable heavy chain (VH) comprising an HCDR1 that is SEQ ID NO: 27; an HCDR2 that is SEQ ID NO: 29; and an HCDR3 that is SEQ ID NO: 31. H In some embodiments, the cys-diabody comprises a variable heavy chain (VH) domain that comprises an HCDR1 that is the HCDR1 in any one of SEQ ID NOs: 1-14, 87, and 88; an HCDR2 that is the HCDR2 in any one of SEQ ID NOs: 1-14, 87, and 88; and an HCDR3 that is the HCDR3 in any one of SEQ ID NOs: 1-14, 87, and 88. H ) domain.

[0165] Figure 1 shows some embodiments of polypeptide sequences of antigen-binding minibodies (SEQ ID NOS: 1-10).

[0166] In some embodiments, the antigen-binding construct, minibody, or cys-diabody comprises CDRs that are CDRs in Table 1.

[0167] [Table 1]

[0168] FIG. 4 shows some embodiments of annotated polypeptide sequences of antigen-binding minibodies.

[0169] 5 shows some embodiments of variable region alignments of polypeptide sequences of antigen-binding minibodies and rovalpituzumab polypeptide sequences. In some embodiments, the V L and V is a Y. In some embodiments, the antigen-binding construct, minibody, or cys-diabody comprises a sequence that is 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 3, and L and wherein residue number 55 is N.

[0170] Figure 6 shows an embodiment of the polypeptide sequence of human DLL3 (SEQ ID NO: 50). In Figure 6, the signal peptide is underlined and the transmembrane domain is shown in bold italics. In some embodiments, the antigen-binding constructs, minibodies, and / or cys-diabodies bind to a protein having an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to DLL3 (with or without the signal peptide) that is SEQ ID NO: 50. In some embodiments, the antigen-binding constructs, minibodies, and / or cys-diabodies bind to a protein having the amino acid sequence of SEQ ID NO: 50 without the signal peptide.

[0171] In some embodiments, V L The V domain further comprises an LFR2 of SEQ ID NO: 35 or 37. L The domain is V in SEQ ID NO: 48 Lthe antigen-binding construct, minibody, or cys-diabody comprises an amino acid sequence having at least 90% identity to the domain; the antigen-binding construct, minibody, or cys-diabody comprises a Y50D and / or Y55N mutation numbered according to the numbering (Kabat) in SEQ ID NO: 1.

[0172] In some embodiments, V L The V domain further comprises an LFR2 of SEQ ID NO: 35 or 37. L The domain is V in SEQ ID NO: 49 L the antigen-binding construct, minibody, or cys-diabody comprises an amino acid sequence having at least 90% identity to the domain; the antigen-binding construct, minibody, or cys-diabody comprises a Y50D and / or Y55N mutation numbered according to the numbering (Kabat) in SEQ ID NO: 1.

[0173] In some embodiments, V L The domain is V in SEQ ID NO: 48 L In some embodiments, the V domain comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the V domain, as numbered according to the numbering (Kabat) in SEQ ID NO: 1, where residue 43 is S. L The domain is V in SEQ ID NO: 48 L domain, wherein residue 45 is R, as numbered according to the numbering (Kabat) in SEQ ID NO:1.

[0174] In some embodiments, V L The domain is V in SEQ ID NO: 49 L In some embodiments, the V domain comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the V domain, as numbered according to the numbering (Kabat) in SEQ ID NO: 1, where residue 43 is S. L The domain is V in SEQ ID NO: 49L domain, wherein residue 45 is R, as numbered according to the numbering (Kabat) in SEQ ID NO:1.

[0175] Figure 2 shows some embodiments of polypeptide sequences for antigen-binding cys-diabodies (SEQ ID NOS: 11-14). In some embodiments, the cys-diabody has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOS: 11-14. 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 NOS: 11. 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 NOS: 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.

[0176] 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 to a FR that is any 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 to 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 to 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 that is SEQ ID NO: 38.

[0177] [Table 2]

[0178] FIG. 3 shows LCDRs (SEQ ID NOs: 15 to 26), HCDRs (SEQ ID NOs: 27 to 32), LFRs (SEQ ID NOs: 33 to 38), linkers (SEQ ID NOs: 39 to 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) are shown.

[0179] In some embodiments, an antigen-binding construct, minibody, or cys-diabody has a disrupted cluster of surface-exposed positively charged amino acids and has enhanced biodistribution and / or pharmacokinetics compared to the original construct from which the construct with the disrupted cluster was derived (e.g., by simply disrupting the original cluster in the original antibody, while other sequence-based features may otherwise remain the same). In some embodiments, an antigen-binding construct, minibody, or cys-diabody has reduced renal uptake when administered to a subject compared to the original construct with an intact cluster of positively charged amino acids. 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, altered biodistribution due to disrupted clusters is readily observed for antigen-binding constructs, such as minibodies or cys-diabodies, having a molecular weight of about 50-80 kDa (as a dimer). Generally, modification of the original antigen-binding construct by disrupting clusters of positively charged amino acids will at least maintain the same binding specificity and binding affinity of the original antigen-binding construct while enhancing biodistribution and / or pharmacokinetics.

[0180] In some embodiments, the antigen-binding construct, minibody, or cys-diabody is a variant comprising at least one disrupted cluster of surface-exposed positively charged amino acids, where the original cluster has 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, and the variant differs from the original antigen-binding construct comprising the original cluster by having a substitution of at least one surface-exposed positively charged amino acid of the original cluster with one or more negatively or uncharged amino acids, thereby disrupting the original positive cluster (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 within 30, 25, 20, 15, 10, 8, or 5 angstroms of each other, or within about 30, 25, 20, 15, 10, 8, or 5 angstroms, or within a distance defined by any two of the preceding 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 (e.g., when the variant antigen-binding construct is radiolabeled and administered to a subject).

[0181] In some embodiments, the antigen-binding construct, minibody, or cys-diabody comprises at least one disrupted cluster of surface-exposed positively charged amino acids, wherein the original cluster has 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, and the variant differs from the original binding construct comprising the original cluster by having a substitution of at least one surface-exposed positively charged amino acid of the original cluster with one or more negatively or uncharged amino acids, whereby the positive cluster is disrupted (relative to the binding construct). In some embodiments, the antigen-binding construct, minibody, or cys-diabody comprises a V having a variant framework region 2 (FR2) that has been altered from the original construct comprising the sequence X1X2X3X4X5X6X7 (SEQ ID NO: 92, where X1 is a positively charged amino acid (e.g., lysine or arginine); X2, X3, X5, and X6 are each independently any negatively or uncharged amino acid; and X4 and X7 are each independently any amino acid, provided that at least one is a positively charged amino acid). Lwherein the sequence is outside any CDR of the antigen-binding construct, and at least one of the positively charged amino acids X4 and X7 is surface-exposed and part of the original cluster. In some embodiments, X1 is part of the original cluster. In some embodiments, X1 and at least one of the positively charged amino acids X4 and X7 are part of the original cluster. In some embodiments, all of the 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. An antigen-binding construct, e.g., a variant antigen-binding construct, may comprise an original sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 92) with a substitution of at least one surface-exposed positively charged amino acid of the cluster with a negatively or uncharged amino acid that disrupts the original cluster. In some embodiments, the negatively or uncharged amino acid substituting a surface-exposed positively charged amino acid in at least one of the original clusters is glutamine (e.g., a K to Q or an R to Q substitution). In some embodiments, framework region 2 (FR2) of the original antigen-binding construct comprises at least one of the following original sequences: KX2X3KX5X6K (SEQ ID NO: 143, where X2, X3, X5, and X6 are each independently any negatively or uncharged amino acid; KX2X3X4X5X6R (SEQ ID NO: 144, where X2, X3, X4, X5, and X6 are each independently any negatively or uncharged amino acid; or KX2X3X4X5X6K (SEQ ID NO: 145, where X2, X3, X4, X5, and X6 are each independently any negatively or uncharged amino acid).In some embodiments, framework region 2 (FR2) of the original antigen-binding construct comprises 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), which correspond to X1X2X3X4X5X6X7 (SEQ ID NO:92). In some embodiments, the original sequence is KPGKAPK (SEQ ID NO:94) or KPGQAPR (SEQ ID NO:95), which correspond to X1X2X3X4X5X6X7 (SEQ ID NO:92). In some embodiments, the antigen-binding construct, minibody, or cys-diabody is, for example, V. L FR2 contains 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).

[0182] 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.

[0183] In some embodiments, the antigen-binding construct, minibody, or cys-diabody comprises a linker (e.g., a linker fragment of the V 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.

[0184] [Table 3]

[0185] Table A (Table 4) shows some non-limiting embodiments of hinge sequences for antigen-binding constructs (e.g., minibodies, scFv-Fc, nanobody®-Fc) of the present disclosure. In some embodiments, an antigen-binding construct (e.g., minibody, scFv-Fc, nanobody®-Fc) comprises a hinge region having any one of the sequences specified in Table A (Table 4). In some embodiments, an antigen-binding construct (e.g., minibody, scFv-Fc, nanobody®-Fc) comprises a hinge region having an upper hinge, a core hinge, and a lower hinge, each comprising any one of the sequences of the upper hinge, core hinge, and lower hinge specified in Table A (Table 4). In some embodiments, the antigen-binding construct (e.g., minibody, scFv-Fc, nanobody®-Fc) comprises a hinge region having the sequence of any one of the complete hinge sequences set forth in Table A (Table 4). 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).

[0186] [Table 4]

[0187] Figure 14 shows the C H Some non-limiting embodiments of the three polypeptide sequences (SEQ ID NOs: 65-85) are shown.

[0188] In some embodiments, the antigen-binding construct, minibody, or cys-diabody is selected from the group consisting of C in Table 4. H 3 domains C H In some embodiments, the C H 3 domains are IgG C H In some embodiments, the IgG C H The three domains are IgG1, IgG2, IgG3, and / or IgG4 C H In some embodiments, the C H 3 domain is germline C H In some embodiments, the C H In some embodiments, the IgG1 C3 domain comprises one or more allotypes. H In some embodiments, the C3 domain is any of SEQ ID NOs: 65 to 68. H The C3 domain is any one of SEQ ID NOs: 65 to 68. H In some embodiments, the C domain has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the C domain. H The 3 domain is C, which is SEQ ID NO: 65 H In some embodiments, the hinge region has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the C3 domain, which is SEQ ID NO: 66. H In some embodiments, the C domain has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the C domain. H The 3 domain is C, which is SEQ ID NO: 67 H In some embodiments, the C domain has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the C domain. H The 3 domain is C, which is SEQ ID NO: 68 HIn some embodiments, the C domain has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the C domain. H 3 domain is IgG2 C H In some embodiments, the IgG2 C H In some embodiments, the C3 domain is any of SEQ ID NOs: 69 to 71. H The C3 domain is any one of SEQ ID NOs: 69 to 71. H In some embodiments, the C domain has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the C domain. H The 3 domain is C, which is SEQ ID NO: 69 H In some embodiments, the C domain has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the C domain. H The 3 domain is C, which is SEQ ID NO: 70 H In some embodiments, the C domain has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the C domain. H The 3 domain is C, which is SEQ ID NO: 71 H In some embodiments, the hinge region has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the IgG3 C3 domain. H In some embodiments, the IgG3 C H In some embodiments, the C3 domain is any of SEQ ID NOs: 72 to 82. H The C3 domain is any one of SEQ ID NOs: 72 to 82. H In some embodiments, the C domain has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the C domain. H The 3 domain is C, which is SEQ ID NO: 72 HIn some embodiments, the C domain has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the C domain. H The 3 domain is C, which is SEQ ID NO: 73 H In some embodiments, the C domain has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the C domain. H The 3 domain is C, which is SEQ ID NO: 74 H In some embodiments, the C domain has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the C domain. H The 3 domain is C, which is SEQ ID NO: 75 H In some embodiments, the C domain has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the C domain. H The 3 domain is C, which is SEQ ID NO: 76 H In some embodiments, the C domain has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the C domain. H The 3 domain is C, which is SEQ ID NO: 77 H In some embodiments, the C domain has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the C domain. H The 3 domain is C, which is SEQ ID NO: 78 H In some embodiments, the C domain has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the C domain. H The 3 domain is C, which is SEQ ID NO: 79 H In some embodiments, the C domain has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the C domain. H The 3 domain is C, which is SEQ ID NO: 80 HIn some embodiments, the C domain has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the C domain. H The 3 domain is C, which is SEQ ID NO: 81 H In some embodiments, the C domain has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the C domain. H The 3 domain is C, which is SEQ ID NO: 82 H In some embodiments, the C domain has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the C domain. H 3 domain is IgG2 C H In some embodiments, the IgG2 C H In some embodiments, the C3 domain is any of SEQ ID NOs: 83 to 85. H The C3 domain is any one of SEQ ID NOs: 83 to 85. H In some embodiments, the C domain has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the C domain. H The 3 domain is C, which is SEQ ID NO: 83 H In some embodiments, the C domain has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the C domain. H The 3 domain is C, which is SEQ ID NO: 84 H In some embodiments, the C domain has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the C domain. H The 3 domain is C, which is SEQ ID NO: 85 H The three domains have at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0189] [Table 5A]

[0190] [Table 5B]

[0191] In some embodiments, the antigen-binding construct comprises an Fc region (e.g., scFv-Fc, nanobody®-Fc). In some embodiments, the antigen-binding construct comprises an amino acid sequence identical to that 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 of SEQ ID NO: 109, as set forth below.

[0192] [ka]

[0193] 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, or within a range defined by any two of the preceding values (e.g., 80-100%, 85-97%, 85-95%, 90-100%, etc.), to SEQ ID NO: 109. 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 the Fc neonatal receptor (FcRn). In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 109 with 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 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 decrease 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 of SEQ ID NO: 109 with 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 one or more mutations that reduce FcRn binding are mutations (e.g., substitutions) at any one or more (e.g., one, two, or all three) of I253, H310, and H435 (EU numbering). In some embodiments, the one or more mutations that reduce FcRn binding are mutations (e.g., substitutions) at any one or more (e.g., one, two, or all three) of I253A, H310A, and H435A (EU numbering). In some embodiments, the Fc region comprises one or more (eg, one, two, or all three) of I253A, H310A, and H435A (EU numbering).In some embodiments, the one or more mutations that reduce binding to an FcγR are mutations (e.g., substitutions) at N297 (EU numbering). In some embodiments, the one or more mutations that reduce binding to an Fc neonatal receptor are N297Q, N297A, or N297G (EU numbering). In some embodiments, the Fc region comprises N297Q (EU numbering). In some embodiments, the one or more mutations that reduce Fc effector function are mutations (e.g., substitutions) at any one or more of 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 L234A, L235A, G236R, G237A, P238S, H268A, K322A, L328R, P329G, A330S, and P331S. Further 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.

[0194] In some embodiments, the antigen-binding construct is an scFv-Fc. In some embodiments, the scFv-Fc comprises a variable heavy chain (VH) that binds to DLL3, e.g., a variable heavy chain (VH) as described herein. H ) domains linked to the variable light chain (V LIn some embodiments, the scFv-Fc comprises any one of an scFv having a variable heavy chain (V) domain, a hinge domain, and an Fc region that binds to DLL3, e.g., a variable heavy chain (V) as described herein, linked to an Fc region via a hinge domain as described herein. H ) domains linked to the variable light chain (V L ) domain. In some embodiments, the hinge domain is any suitable hinge domain described herein (e.g., Table A (Table 4)). In some embodiments, the antigen-binding construct is nanobody®-Fc (or a single domain fragment fused to an Fc region). In some embodiments, nanobody®-Fc comprises a variable domain (VHH) having any one or more of the HCDR1, HCDR2, and HCDR3 sequences described herein. In some embodiments, nanobody®-Fc comprises a variable domain (VHH) having an HCDR1 that is SEQ ID NO: 27; an HCDR2 that is SEQ ID NO: 29; and an HCDR3 that is SEQ ID NO: 31. In some embodiments, nanobody®-Fc comprises a variable domain (VHH) having an HCDR1 that is the HCDR1 in any one of SEQ ID NOs: 1-14, 87, and 88; an HCDR2 that is the HCDR2 in any one of SEQ ID NOs: 1-14, 87, and 88; and an HCDR3 that is the HCDR3 in any one of SEQ ID NOs: 1-14, 87, and 88. In some embodiments, nanobody®-Fc comprises a VHH having any one or more (e.g., any one, two, or all three) of the HCDR1, HCDR2, and HCDR3 sequences described herein, linked to an Fc region via a hinge domain as described herein. H Includes the domain.

[0195] In some embodiments, the antigen-binding construct comprises at least one modification. Exemplary modifications include, but are not limited to, antigen-binding constructs modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, conjugation to metal chelators, fluorescent or infrared dyes, or conjugation to toxins, proteolytic cleavage, and linkage to cellular ligands or other proteins. Any of a number of chemical modifications can be performed by known techniques, including, but not limited to, amine coupling to amine-containing amino acids, cystine coupling, specific chemical cleavage, acetylation, and metabolic synthesis of tunicamycin. In some embodiments, the derivative may contain one or more unnatural amino acids.

[0196] In some embodiments, the antigen-binding construct is conjugated to another substance to form an anti-target conjugate. The conjugates described herein can be prepared by known methods for linking antigen-binding constructs to lipids, carbohydrates, proteins, or other atoms and molecules. In some embodiments, the conjugate is formed by site-specific conjugation using an appropriate linkage or bond. Site-specific conjugation is likely to preserve the binding activity of the antigen-binding construct. The substance can be conjugated or attached to the hinge region of the reduced antigen-binding construct via thioether bond formation. In some embodiments, tyrosine conjugation can be employed. Other linkages or bonds used to form the conjugate can include, but are not limited to, covalent bonds, non-covalent bonds, disulfide bonds, hydrazone bonds, ester bonds, amide and amino bonds, imino bonds, thiosemicarbazone bonds, semicarbazone bonds, oxime bonds, and carbon-carbon bonds. In some embodiments, cysteines or other linking modes need not be included in the antigen-binding construct.

[0197] In some embodiments, the antigen-binding construct, minibody, and / or cys-diabody is conjugated to a chemotherapeutic agent. Chemotherapeutic agents are often cytotoxic or cytostatic in nature and may include alkylating agents, antimetabolites, antitumor antibiotics, topoisomerase inhibitors, antimitotic agents, hormonal therapies, targeted therapeutic agents, and immunotherapeutic agents. In some embodiments, chemotherapeutic agents that may be used as detectable markers according to embodiments of the present disclosure are selected from among those chemotherapeutic agents defined elsewhere herein.

[0198] In some embodiments, the antigen-binding construct, minibody, and / or cys-diabody is conjugated to a toxin. Toxins that may be used in accordance with embodiments of the present disclosure include, but are not limited to, auristatin E, auristatin F, dolastatin 10, dolastatin 15, combretastatins and their analogs, maytansinoids, calicheamicin, alpha-amanitin, pyrrolobenzodiazepine dimers, epothilones, duocarmycins and their analogs, tubulysin D, basilistatins, ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.

[0199] In some embodiments, the antigen-binding construct, minibody, and / or cys-diabody is conjugated to a radioactive label. In some embodiments, the radioactive label comprises 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 thorium-227, a positron-emitter such as zirconium-89, copper-64, gallium-68, fluorine-18, indium-111, or iodine-124. In some embodiments, the radioactive label comprises terbium-149, terbium-161, or lead-212. In some embodiments, the antigen-binding construct, minibody, and / or cys-diabody is conjugated to a gamma-emitter. In some embodiments, the radiolabel comprises a gamma emitter.

[0200] 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. 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 Er, 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 In some embodiments, the at least one payload comprises: 149 Tb, 161 Tb, or 212 Contains Pb.

[0201] In some embodiments, the antigen-binding construct is an antibody (about 140-170 kDa), a multispecific antibody (about 100 kDa or greater), a single-arm or mono-arm antibody (about 110 kDa or less), 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 an scFv-Fc (e.g., an scFv fused to an Fc). In some embodiments, the antigen-binding construct is a nanobody®-Fc (e.g., a nanobody® (or single domain fragment) fused to an Fc).

[0202] In some embodiments, the order of the variable domains from the N-terminus to the C-terminus of the antigen-binding construct, minibody, or cys-diabody is, from the N-terminus to the C-terminus of the polypeptide, V L , V H In some embodiments, the order of the variable domains from N-terminus to C-terminus of the polypeptide is V H , V L is.

[0203] In some embodiments, the antigen-binding construct, minibody, or cys-diabody is a humanized antigen-binding construct, minibody, or cys-diabody. In some embodiments, the antigen-binding construct, minibody, and / or cys-diabody of any of Figures 1-6 can be reformatted from an antigen-binding construct, minibody, or cys-diabody to an antigen-binding construct, minibody, and / or cys-diabody. For example, the minibody presented in Figure 1 can be reformatted into an antigen-binding construct or cys-diabody. The cys-diabody presented in Figure 2 can be reformatted into an antigen-binding construct or minibody. For example, the minibody or cys-diabody presented in Figures 1 and 2 can be reformatted into an scFv-Fc.

[0204] In some embodiments, a minibody that binds to DLL3 comprises a variable heavy chain (V H ) domains linked to the variable light chain (V L a single-chain variable fragment (scFv) that binds to DLL3 and comprises a V L The domain comprises an LCDR1 having SEQ ID NO: 15; an LCDR2 having any one of SEQ ID NOs: 19, 21, or 23; and an LCDR3 having SEQ ID NO: 25, H A single chain variable fragment (scFv) comprising domains: HCDR1, which is SEQ ID NO: 27; HCDR2, which is SEQ ID NO: 29; HCDR3, which is SEQ ID NO: 31; hinge region; IgG C H 3 sequence; and a therapeutic agent, a toxic payload, and / or a detectable marker.

[0205] In some embodiments, the cys-diabody that binds to DLL3 comprises a variable heavy chain (V H ) domains linked to the variable light chain (V L ) domain, L The domain comprises an LCDR1 of SEQ ID NO: 15; an LCDR2 of SEQ ID NO: 19, 21, or 23; an LCDR3 of SEQ ID NO: 25; HThe domains include a single-chain variable fragment (scFv) comprising HCDR1 of HCDR1 of SEQ ID NO: 27; HCDR2 of HCDR2 of SEQ ID NO: 29; HCDR3 of HCDR3 of SEQ ID NO: 31; an extension sequence; and a polypeptide comprising a therapeutic agent, a toxic payload, and / or a detectable marker.

[0206] In some embodiments, the scFv-Fc that binds to DLL3 comprises a variable heavy chain (V H ) domains linked to the variable light chain (V L a single-chain variable fragment (scFv) that binds to DLL3 and comprises a V L The domain comprises an LCDR1 having SEQ ID NO: 15; an LCDR2 having any one of SEQ ID NOs: 19, 21, or 23; and an LCDR3 having SEQ ID NO: 25, H The domains comprise a single-chain variable fragment (scFv) comprising HCDR1 which is SEQ ID NO: 27; HCDR2 which is SEQ ID NO: 29; HCDR3 which is SEQ ID NO: 31; a hinge region; an Fc region; and a therapeutic agent, a toxic payload, and / or a detectable marker.

[0207] In some embodiments, the humanized antigen-binding construct, minibody, or cys-diabody comprises an LCDR1 of SEQ ID NO: 15; an LCDR2 of any one of SEQ ID NOs: 19, 21, or 23; an LCDR3 of an LCDR3 of SEQ ID NO: 25; an HCDR1 of an HCDR1 of SEQ ID NO: 27; an HCDR2 of an HCDR2 of SEQ ID NO: 29; and an HCDR3 of an HCDR3 of SEQ ID NO: 31.

[0208] In some embodiments, V L The domain comprises or further comprises the LFR2 of SEQ ID NO: 35 or 37.

[0209] characteristics FIG. 7 is a bar graph showing an embodiment of minibody expression yield in Expi293 mammalian cells.

[0210] In some embodiments, the expression yield of the antigen-binding construct, minibody, 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, minibody, and / or cys-diabody is determined using chromatographic techniques. In some embodiments, the expression of the antigen-binding construct, minibody, and / or cys-diabody is determined using biolayer interferometry. In some embodiments, the expression yield of an antigen-binding construct, minibody, or cys-diabody is about 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. 120, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500 mg / L, 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 a yield within a range defined by any two of the preceding values. For example, in some embodiments, the expression yield of an antigen-binding construct, minibody, or cys-diabody is about 1-500 mg / L, 1-350 mg / L, 1-200 mg / L, 1-100 mg / L, 1-50 mg / L, 1-25 mg / L, 5-500 mg / L, 5-350 mg / L, 5-200 mg / L, 5-100 mg / L, 5-50 mg / L, 5-25 mg / L, 25-500 mg / L, 25-350 mg / L, 25-20 mg / L, 25-100 mg / L, or 25-50 mg / L. In some embodiments, the expression yield of an antigen-binding construct, minibody, and / or cys-diabody is determined relative to the expression yield of a parent antigen-binding construct, minibody, and / or cys-diabody.For example, in some embodiments, the expression yield of an antigen-binding construct, minibody, or cys-diabody is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold higher than the expression yield of the parent antigen-binding construct, minibody, and / or cys-diabody, or is increased by a range defined by any two of the preceding values. For example, in some embodiments, the expression yield of an antigen-binding construct, minibody, and / or cys-diabody is 1-10-fold, 1-8-fold, 1-5-fold, 1-3-fold, 3-10-fold, 3-8-fold, 3-5-fold, 5-10-fold, or 5-8-fold higher than the expression yield of the parent antigen-binding construct, minibody, and / or cys-diabody.

[0211] FIG. 11 is a bar graph showing some embodiments of the in vivo biodistribution of antigen-binding minibodies in a tumor-bearing mouse model.

[0212] 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, hi some embodiments, the antigen-binding construct is biased in distribution to the subject's blood, liver, kidney, spleen, lung, muscle, bone, heart, stomach, small intestine, large intestine, pancreas, tumor, or any combination thereof.

[0213] In some embodiments, the antigen-binding construct, minibody, or cys-diabody has an activity of 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. ID / g, or 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, or a percentage within the range defined by any two of the preceding values. For example, in some embodiments, the antigen-binding construct, minibody, or cys-diabody is detectable at about 0.1%-35%, 0.1%-30%, 1%-35%, 1%-30%, 5%-35%, or 5%-30% ID / g.

[0214] In some embodiments, the antigen-binding construct, minibody, and / or cys-diabody distributes 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 constructs, minibodies, and / or cys-diabodies exhibit an activity of 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. In some embodiments, the antigen-binding construct, minibody, and / or cys-diabody distributes in the blood, liver, kidney, spleen, lung, muscle, bone, heart, stomach, small intestine, large intestine, pancreas, tumor, or any combination thereof, in ID / g or as a percentage within a range defined by any two of the preceding values. For example, in some embodiments, the antigen-binding construct, minibody, and / or cys-diabody distributes in the blood, liver, kidney, spleen, lung, muscle, bone, heart, stomach, small intestine, large intestine, pancreas, tumor, or any combination thereof, in 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% ID / g.

[0215] FIG. 12 is a bar graph showing some embodiments of the in vivo biodistribution of antigen-binding minibodies.

[0216] In some embodiments, the antigen-binding construct, minibody, and / or cys-diabody distributes 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 constructs, minibodies, and / or cys-diabodies exhibit an activity of 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. In some embodiments, the antigen-binding construct, minibody, and / or cys-diabody distributes in the blood, liver, kidney, spleen, lung, muscle, bone, heart, stomach, small intestine, large intestine, pancreas, tumor, or any combination thereof, in ID / g or as a percentage within a range defined by any two of the preceding values. For example, in some embodiments, the antigen-binding construct, minibody, and / or cys-diabody distributes in the blood, liver, kidney, spleen, lung, muscle, bone, heart, stomach, small intestine, large intestine, pancreas, tumor, or any combination thereof, in 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% ID / g.

[0217] In some embodiments, the antigen-binding construct, minibody, or cys-diabody comprises an LFR2 that is SEQ ID NO: 35 or 37; the antigen-binding construct, minibody, or cys-diabody has improved biodistribution compared to an antigen-binding construct, minibody, or cys-diabody that comprises an LFR2 that is SEQ ID NO: 33.

[0218] FIG. 9 is a graph depicting some embodiments of equilibrium dissociation constant determination of antigen-binding minibodies.

[0219] In some embodiments, the antigen-binding construct, minibody, or cys-diabody is 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 -15 M (e.g., KD for binding to DLL3 protein) or within the range defined by any two of the preceding values. For example, in some embodiments, the antigen-binding construct, minibody, or cys-diabody has a KD of about 10 -5 M~10 -15 M, 10 -5 M~10 -12 M, 10 -5 M~10 -10 M, 10 -7 M~10 -15 M, 10 -7 M~10 -12 M, 10 -7 M~10 -10 M, 10 -10 M~10 -15 M or 10 -10 M~10 -12 The antibody has a KD (e.g., a KD for binding to an antigen) of M. In some embodiments, the KD is determined for the antibody dissolved in a buffer solution. In some embodiments, the buffer solution is a phosphate buffer. If desired, the KD can be determined using any suitable method, such as biolayer interferometry.

[0220] Figure 8 shows the EC of antigen-binding minibodies by ELISA. 50 10 is a graph illustrating some embodiments of the determination.

[0221] Figure 10 shows the EC of antigen-binding minibodies by flow cytometry. 50 10 is a graph illustrating some embodiments of the determination.

[0222] In some embodiments, the antigen-binding construct, minibody, or cys-diabody has a cys-diabody concentration of 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. 0.19, or 0.20 nM, or 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 EC50 of 0.20 nM or 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 50 (e.g., EC 0.05 for binding to DLL3 protein or cells expressing DLL3 protein) 50 ), or the EC within the region defined by any two of the preceding values 50 For example, in some embodiments, the antigen-binding construct, minibody, or cys-diabody has an EC of about 0.001-0.20 nM, 0.001-0.17 nM, 0.001-0.12 nM, 0.01-0.20 nM, 0.01-0.17 nM, 0.01-0.12 nM, 0.05-0.20 nM, 0.05-0.17 nM, 0.05-0.15 nM, 0.05-0.12 nM, 0.08-0.20 nM, 0.08-0.17 nM, or 0.08-0.12 nM. 50 (e.g., EC for binding to an antigen or a cell expressing the antigen) 50 In some embodiments, the antigen-binding construct, minibody, or cys-diabody has an EC 50(e.g., EC 0.05 for binding to DLL3 protein or cells expressing DLL3 protein) 50 ) If desired, EC 50 may be determined using any suitable option, for example using ELISA or flow cytometry.

[0223] Therapeutic Agents and Compositions In some embodiments, the pharmaceutical composition may also include a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier may be a pharmaceutically acceptable material, composition, or vehicle involved in carrying or transporting a compound of interest from one tissue, organ, or part of the body to another tissue, organ, or part of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or some combination thereof. Each component of the carrier is "pharmaceutically acceptable" in that it is compatible with the other components of the formulation. This means that it is suitable for contact with any tissue, organ, or part of the body that it may encounter, and ideally, it will not be associated with a significant risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that unduly outweighs its therapeutic benefit.

[0224] In some embodiments, the therapeutic composition comprises a variable light chain (V) comprising an LCDR1 that is SEQ ID NO: 15; an LCDR2 that is any one of SEQ ID NOs: 19, 21, or 23; and an LCDR3 that is SEQ ID NO: 25. L ) domain; and a variable heavy chain (V) comprising an HCDR1 that is SEQ ID NO:27; an HCDR2 that is SEQ ID NO:29; and an HCDR3 that is SEQ ID NO:31 H ) domain; and a therapeutic agent, a toxic payload, and / or a detectable marker. In some embodiments, the antigen-binding construct comprises a hinge region (e.g., any one of the hinge regions described herein) and an IgG C H 3 sequences (e.g., C HIn some embodiments, the antigen-binding construct is a cys-diabody further comprising an extension sequence at the C-terminus of the monomer (e.g., GGC or GGCPPCPPC (SEQ ID NO: 93)).

[0225] In some embodiments, the therapeutic composition comprises a minibody that binds to DLL3 and comprises a variable heavy chain (V H ) domains linked to the variable light chain (V L a single-chain variable fragment (scFv) that binds to DLL3 and comprises a V L The domain comprises an LCDR1 having SEQ ID NO: 15; an LCDR2 having any one of SEQ ID NOs: 19, 21, or 23; and an LCDR3 having SEQ ID NO: 25, H a single chain variable fragment (scFv) comprising domains: HCDR1 which is SEQ ID NO: 27; HCDR2 which is SEQ ID NO: 29; HCDR3 which is SEQ ID NO: 31; a hinge extension domain comprising an IgG1 hinge region; an IgG C H In some embodiments, the therapeutic composition comprises a minibody that binds to DLL3 and comprises a variable heavy chain (V H ) domains linked to the variable light chain (V L a single-chain variable fragment (scFv) that binds to DLL3 and contains a V L The domain comprises an LCDR1 having SEQ ID NO: 15; an LCDR2 having any one of SEQ ID NOs: 19, 21, or 23; and an LCDR3 having SEQ ID NO: 25, H A single chain variable fragment (scFv) comprising domains: HCDR1, which is SEQ ID NO: 27; HCDR2, which is SEQ ID NO: 29; HCDR3, which is SEQ ID NO: 31; hinge region; IgG C H a minibody comprising the 3 sequences; and a therapeutic agent, a toxic payload, and / or a detectable marker.

[0226] In some embodiments, the therapeutic composition comprises a cys-diabody that binds to DLL3 and comprises a variable heavy chain (V H) domains linked to the variable light chain (V L ) domain, L The domain comprises an LCDR1 of SEQ ID NO: 15; an LCDR2 of SEQ ID NO: 19, 21, or 23; an LCDR3 of SEQ ID NO: 25; H In some embodiments, the therapeutic composition comprises a cys-diabody comprising a polypeptide comprising a single chain variable fragment (scFv), wherein the domains comprise: HCDR1 of HCDR1 of SEQ ID NO: 27; HCDR2 of HCDR2 of SEQ ID NO: 29; and HCDR3 of HCDR3 of SEQ ID NO: 31; and a therapeutic agent, a toxic payload, and / or a detectable marker. In some embodiments, the therapeutic composition comprises a cys-diabody that binds to DLL3, wherein the scFv comprises a variable heavy chain (V H ) domains linked to the variable light chain (V L ) domain, L The domain comprises an LCDR1 of SEQ ID NO: 15; an LCDR2 of SEQ ID NO: 19, 21, or 23; an LCDR3 of SEQ ID NO: 25; H a cys-diabody comprising a polypeptide comprising a single chain variable fragment (scFv) whose domains comprise: HCDR1 of HCDR1 of SEQ ID NO: 27; HCDR2 of HCDR2 of SEQ ID NO: 29; HCDR3 of HCDR3 of SEQ ID NO: 31; an extension sequence; and a therapeutic agent, a toxic payload, and / or a detectable marker.

[0227] In some embodiments, a therapeutic composition comprises one or more of the minibody constructs provided in any one of the figures and / or in a table, such as Table 5.

[0228] In some embodiments, the therapeutic composition comprises one or more of the cys-diabody configurations provided in any one of the figures and / or in a table, such as Table 6.

[0229] In some embodiments, a therapeutic composition comprises an antigen-binding construct, minibody, cys-diabody, VL, VH, CDR, FR, linker, signal peptide, or any combination thereof, that 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 peptide of SEQ ID NO: 1-48. In some embodiments, a therapeutic composition comprises a minibody having any of SEQ ID NO: 1-10. In some embodiments, a therapeutic composition comprises a minibody having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NO: 1-10. In some embodiments, a therapeutic composition comprises a cys-diabody having any of SEQ ID NO: 11-14. In some embodiments, a therapeutic composition comprises a cys-diabody having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a cys-diabody having any of SEQ ID NOs: 11-14.

[0230] In some embodiments, a therapeutic composition comprises an LCDR having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an LCDR having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an LCDR having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an HCDR ... In some embodiments, a 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 set forth in any of SEQ ID NOs: 33-38. In some embodiments, a therapeutic composition comprises a linker set forth in any of SEQ ID NOs: 39-44. In some embodiments, a therapeutic composition comprises a linker having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a linker set forth in any of SEQ ID NOs: 39-44. In some embodiments, a therapeutic composition comprises a signal peptide set forth in any of SEQ ID NOs: 45-46. In some embodiments, a 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 set forth in any of SEQ ID NOs: 45-46. In some embodiments, a therapeutic composition comprises a VH that is SEQ ID NO: 47. In some embodiments, a therapeutic composition comprises 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, a 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 a VL that is SEQ ID NO:48.

[0231] The pharmaceutical or therapeutic compositions described herein may be administered by any suitable route of administration. Route of administration may refer to any route known in the art, including, but not limited to, aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, transdermal (e.g., topical creams or ointments, patches), or vaginal. "Transdermal" administration may be accomplished using a topical cream or ointment or by a transdermal patch. "Parenteral" refers to routes of administration commonly associated with injection, including suborbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intracranial, intraventricular, intrauterine, intravenous, intrathecal, subcapsular, subcutaneous, sublingual, transmucosal, or transtracheal. In some embodiments, the antigen-binding construct may be delivered intraoperatively as a local administration during an intervention or resection.

[0232] In some embodiments, the antigen-binding construct, minibody, or cys-diabody is conjugated to a therapeutic agent. As used herein, a "therapeutic agent" refers to an atom, molecule, or compound that is useful in treating a disorder associated with a target molecule. Examples of therapeutic agents include, but are not limited to, drugs, chemotherapeutic agents, therapeutic antibodies and antibody fragments, toxins, radioisotopes, enzymes (e.g., enzymes that cleave prodrugs into cytotoxic agents at the site of antigen-binding construct binding), nucleases, hormones, immunomodulators, antisense oligonucleotides, chelators, boron compounds, photoactive agents and dyes, elastin-like polypeptides such as PLGA, and nanoparticles. Examples of disorders include those associated with one or more target molecules.

[0233] Chemotherapeutic agents are often cytotoxic or cytostatic in nature and may include alkylating agents, antimetabolites, antitumor antibiotics, topoisomerase inhibitors, antimitotics, hormonal therapies, targeted therapeutic agents, and immunotherapeutic agents. In some embodiments, chemotherapeutic agents that may be used as detectable markers in accordance with embodiments of the present disclosure include 13-cis-retinoic acid, 2-chlorodeoxyadenosine, 5-azacytidine, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine, actinomycin-D, adriamycin, aldesleukin, alemtuzumab, alitretinoin, all-trans retinoic acid, alpha interferon, altretamine, amethopterin, amifostine, anagrelide, anastrozole, and the like. Zol, arabinosylcytosine, arsenic trioxide, amsacrine, aminocamptothecin, aminoglutethimide, asparaginase, azacitidine, bacillus Calmette-Guerin (BCG), bendamustine, bevacizumab, bexarotene, bicalutamide, bortezomib, bleomycin, busulfan, calcium leucovorin, citrovorum factor, capecitabine, canertinib, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, cladribine, cortisone, cyclophosphamide amide, cytarabine, darbepoetin alfa, dasatinib, daunomycin, decitabine, denileukin diftitox, dexamethasone, dexasone, dexrazoxane, dactinomycin, daunorubicin, dacarbazine, docetaxel, doxorubicin, doxifluridine, eniluracil, epirubicin, epoetin alfa, erlotinib, everolimus, exemestane, estramustine, etoposide, filgrastim, fluoxymesterone, fulves Trant, flavopiridol, floxuridine, fludarabine, fluorouracil, flutamide, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, hexamethylmelamine, hydrocortisone hydroxyurea, ibritumomab, interferon alpha, interleukin-2, interleukin-11, isotretinoin, ixabepilone, idarubicin, imatinib mesylate, ifosfamide,Irinotecan, lapatinib, lenalidomide, letrozole, leucovorin, leuprolide, liposomal Ara-C, lomustine, mechlorethamine, megestrol, melphalan, mercaptopurine, mesna, methotrexate, methylprednisolone, mitomycin C, mitotane, mitoxantrone, nelarabine, nilutamide, octreotide, oprelvekin, oxaliplatin, paclitaxel, pamidronate, pemetrexed, panitumumab, PEG-interferon, pegaspargase, pegfilgrastim, PEG-L-asparaginase, pentostatin, plicamycin, prednisolone, prednisone, procarbazine, raloxifene, rituximab Mab, romiplostim, ralitrexed, sapacitabine, sargramostim, satraplatin, sorafenib, sunitinib, semustine, streptozocin, tamoxifen, tegafur, tegafur-uracil, temsirolimus, temozolamide, teniposide, thalidomide, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, trimitrexate, alrubicin, vincristine, vinblastine, vindestine, vinorelbine, vorinostat, or zoledronic acid.

[0234] Toxins that may be used in accordance with embodiments of the present disclosure include, but are not limited to, auristatin E, auristatin F, dolastatin 10, dolastatin 15, combretastatins and their analogs, maytansinoids, calicheamicin, alpha-amanitin, pyrrolobenzodiazepine dimers, epothilones, duocarmycins and their analogs, tubulysin D, bacillistatin, ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.

[0235] In some embodiments, the nanoparticles, when conjugated to an antigen-binding construct, a minibody, or a cys-diabody, are used in therapeutic applications as drug carriers to deliver chemotherapeutic agents, hormonal therapy agents, radiotherapy agents, toxins, or any other cytotoxic or anti-cancer agents known in the art to cancerous cells that overexpress a target on their cell surface.

[0236] In some embodiments, the antigen-binding constructs, minibodies, or cys-diabodies described herein may be further conjugated to one or more immunotherapeutic agents. In some embodiments, the antigen-binding constructs, minibodies, or cys-diabodies described herein may be co-administered with one or more immunotherapeutic agents. For example, in some embodiments, the antigen-binding constructs, minibodies, or cys-diabodies described herein may be further conjugated to or co-administered with multispecific agents, including, but not limited to, anti-PD1 and anti-PD-L1 binding agents, anti-CTLA4 agents, and anti-CTLA-4 / B7-1 / B7-2. Additional immunotherapeutic agents include checkpoint inhibitors such as ipilimumab (Yervoy), pembrolizumab (Keytruda), nivolumab (Opdivo), atezolizumab (Tecentriq), avelumab (Bavencio), and durvalumab (Imfinzi). Immunotherapeutic agents also include tremelimumab and pidilizumab. Small molecule immunotherapeutic agents in development include BMS-1001, BMS-1116, CA-170, CA-327, imiquimod, resiquimod, 852A, VTX-2337, ADU-S100, MK-1454, ibrutinib, 3AC, idelalisib, IPI-549, epacadostat, AT-38, CPI-444, bipadenanthate, preladenanthate, PBF, AZD4635, galunisertib, OTX015 / MK-8628, and CPI-0610 (see Kerr and Chisolm (2019) The Journal of Immunology, 2019, 202: 11-19).

[0237] In some embodiments, the antigen-binding constructs, minibodies, or cys-diabodies described herein may be further conjugated to one or more nephroprotectants and / or radioprotectants. In some embodiments, the antigen-binding constructs, minibodies, or cys-diabodies described herein may be co-administered with one or more nephroprotectants and / or radioprotectants. In some embodiments, the radioprotectants include free lysine, free arginine, probenecid, gelofuscin, 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, fullerenes, fumarate, gallic acid, metformin, and the like. Indirubin, Kaempferol, Sodium Butyrate, Spermidine, Cholecalciferol, Avobenzone, Octinoxate, Titanium Dioxide, Tempol, Gusperimus, Irsogladine, Tetrachlorodecaoxide, Calcium Alginate, Gusperimus Hydrochloride, D-Galacturonic Acid Sodium Salt, Cystamine Hydrobromide, Cystaphos, N-Acetyl-Ser-Asp-Lys-Pro, D08Eai, Prolycopene, Vera Resole, U-74389G Maleate, Trans-2-Hexenyl Salicylate, 3-Benzylidene Camphor, Irsogladine Maleate, Sodium Alginate, Gsh-Mee, Triethylenediamine Diacetate, Cystamine Hydrochloride, Amifostine, Glutathione Reduced Ethyl Ester, Homocysteine Thiolactone, 132316-35-9, Hydroquinone, 13258-59-8, Adeturon, unii-S8Kc 806H2T, nitrogen oxygen, 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-actahydrocyclopenta[A]phenanthren-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, beta-aminoethylisothiourea, zingerone, aeol 10150, artemisinin, 16,16-dimethyl prostaglandin E2, theaflavin, succinic acid, andrographis, balsalazide, N-adenylyl-L-phenylalanine, chlorophyllin, nacu, diequol, equol, diltiazem, edaravone, rhepo, ferulic acid, glutathione, glycyrrhizic acid, hesperidin, indomethacin, misoprostol, morin, myricetin, tiopronin, naringin, resilience sodium, probucol, hydroxyethyl rutoside, cystaphos, sulfasalazine, sucralfate, chugai, silymarin, floxacillin, cyproterone, cysteamine, thymol, mercaptoethanol, desogestrel, cellobiol Su, Kanamycin, Sodium Alginate, Acetylcysteine, Zinc Oxide, Cystaphos, 10-Hydroxy-2-decenoic Acid, Teduglutide, Curcuma, Troxerut, Trichostatin A, Selegiline, Lycopene, Nitrendipine, Glycerol, 21245-02-3, Rosmarinic Acid, Simvastatin, Fisetin, Sirolimus, Oxybenzone, Ascophyllum, Ecamsul, Fsfjezphmoqyqb-Oasotcbpsa-N, Sczygarxjqtndn-Ikfjuqjosa-N, Snzlftjkilzfkf-Uhfffaoysa-M, Alginate, Alginic Acid, Potassium Salt, Potassium Alginate, U-74389G, Calcium Alginate, Hoechst 33342 (trihydrochloride), polydatin, thioctic acid, enalapril, amifostine, ursolic acid, Unii-Sm5Yj88Ltu, carbonyl cyanide m-chlorophenylhydrazone, genistein, resveratrol, (-)-epigallocatechin gallate, ellagic acid, baicalein, valproic acid, pentoxifylline, acacia, melatonin, trehalose, acteoside, kukoamine, atorvastatin, carvacrol, isofraxidin, palifermin, cysteine, vitamin E, heparin, chondroethin sulfate, AET, M40403, EUK-189, EUK-207, vitamin C, vitamin A, lipoic acid, beta-carotene, L-selenomethionine, and / or Q10.

[0238] Any of the antigen-binding constructs, minibodies, or cys-diabodies described herein can be further conjugated with one or more additional therapeutic agents, detectable markers, nanoparticles, carriers, or combinations thereof. For example, the antigen-binding construct can be radiolabeled with iodine-131 and conjugated to a lipid carrier, whereby the anti-target molecule-lipid conjugate forms a micelle. The micelle can incorporate one or more therapeutic agents, isotopes, ions, and / or detectable markers.

[0239] In some embodiments, the antigen-binding construct, minibody, or cys-diabody is conjugated to a therapeutic agent. While these antigen-binding constructs may have a shorter circulating half-life compared to full-length antibodies, in some embodiments, these formats may exhibit improved tumor penetration due to their smaller size and may be therapeutically effective when appropriately equipped with a cytotoxic drug or radioisotope. In some embodiments, a drug-conjugation approach may be employed for the antigen-binding construct, minibody, or cys-diabody. In some embodiments, a therapeutic approach includes radioimmunotherapy by attaching an appropriate radiolabel, such as iodine-131, beta-emitters or alpha-emitters, such as yttrium-90, lutetium-177, copper-67, terbium-149, terbium-161, astatine-211, lead-212, bismuth-212, actinium-225, bismuth-213, and thorium-227, which may cause cell damage and death in target tissues. In some embodiments, treatment with these fragments equipped with cytotoxic drugs or radionuclides results in less nonspecific toxicity because they will be cleared more rapidly from the body. In some embodiments, the radiolabel comprises terbium-149, terbium-161, or lead-212.

[0240] In some embodiments, the label and / or therapeutic agent is 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 Er, 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.

[0241] In some embodiments, the antigen-binding construct, minibody, or cys-diabody can be linked to a therapeutic agent for a disorder associated with expression of the target molecule.

[0242] In some embodiments, the target molecule antigen-binding construct is used as the sole pharmaceutical (eg, the antigen-binding construct in the absence of a therapeutic agent) in the treatment of a disorder associated with expression of the target molecule.

[0243] Methods for detecting the presence or absence of a target molecule The antigen-binding constructs, minibodies, or cys-diabodies can be used to detect the presence or absence of a target molecule in vivo and / or in vitro. Accordingly, some embodiments include methods of detecting the presence or absence of a target.

[0244] 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 phototherapeutic 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 with Auger electron spectroscopy. In some embodiments, the detectable marker comprises a payload. In some embodiments, at least one payload is 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 Er, 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, the marker is a radioactive label. 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 Many radionuclides can be used as imaging labels, including radioisotopes of I and Lu, etc. Those of skill in the art will know of other radionuclides that are particularly well suited for use in the present disclosure.

[0245] The method can kill cancer cells. In some embodiments, the construct recognizes and binds to the DLL3 protein. In additional embodiments, the method further comprises administering a chemotherapy drug, radiation therapy. In some embodiments, the subject is also treated with hormone ablation therapy or hormone antagonist therapy.

[0246] The method may include applying the antigen-binding construct, minibody, or cys-diabody to a subject. In some embodiments, the antigen-binding construct, minibody, or cys-diabody is applied to a sample. The method may include detecting binding or absence of binding of the antigen-binding construct, minibody, or cys-diabody to a target molecule. In some embodiments, any target molecule may be detected by the options provided herein. In some embodiments, the target molecule of interest to be detected is DLL3. In some embodiments, the target molecule is detected using one or more of the minibody configurations provided in the figures and / or in tables, such as Table 5.

[0247] Table 5 provides embodiments that are useful in the compositions and methods described herein.

[0248] [Table 6A]

[0249] [Table 6B]

[0250] [Table 6C]

[0251] In some embodiments, any of the constructs provided herein may be employed without a signal peptide (e.g., the first underlined sequence in Table 5 or Table 6) or may omit the signal peptide. 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, residue 20 (Kabat) of the signal peptide, as numbered in FIG. 3, is immediately adjacent to residue number 1 (Kabat) of the antigen-binding construct, minibody, and / or cys-diabody, as numbered in FIG. 1. In any and all embodiments provided herein, the signal sequence is optional and may be included in or omitted from any of the antigen-binding constructs, minibodies, and / or cys-diabodies disclosed herein. In some embodiments, the target molecule is detected using one or more of the cys-diabody constructs provided in the figures and / or tables, such as Table 6.

[0252] [Table 7]

[0253] Provided herein are methods for detecting the presence or absence of a target molecule. It will be understood that the steps below may be performed in any order and / or optionally repeated and / or eliminated, and that additional steps may optionally be added to the method. In some embodiments, an antigen-binding construct, minibody, or cys-diabody described herein may be applied to a sample. In some embodiments, an optional wash may be performed. Optionally, a second antigen-binding construct may be applied to the sample. Optional washes may be performed. In some embodiments, binding or absence of binding of the antigen-binding construct, minibody, or cys-diabody to the target molecule may be detected.

[0254] In some embodiments, the antigen-binding construct, minibody, or cys-diabody described herein is applied to a sample in vivo. The antigen-binding construct may be administered to a subject. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal, such as a rat, mouse, guinea pig, hamster, rabbit, dog, cat, cow, horse, goat, sheep, donkey, pig, monkey, or ape. In some embodiments, the antigen-binding construct is injected into the subject. In some embodiments, the injection is intravenous. In some embodiments, the injection is intraperitoneal. In some embodiments, the antigen-binding construct, minibody, or cys-diabody is applied to the subject topically or locally (as in interventional or intraoperative applications). In some embodiments, a capsule containing the antigen-binding construct, minibody, or cys-diabody is applied to the subject, for example, orally or intraperitoneally. In some embodiments, the antigen-binding construct, minibody, or cys-diabody is selected to reduce the risk of an immunogenic response by the subject. For example, for human subjects, the antigen-binding construct, minibody, or cys-diabody can be humanized as described herein. In some embodiments, after in vivo application of the antigen-binding construct, minibody, or cys-diabody, a sample or a portion of the sample is removed from the host. In some embodiments, the antigen-binding construct, minibody, or cys-diabody is applied in vivo and incubated in vivo for a period of time described herein, and the sample is removed for in vitro analysis, e.g., in vitro detection of the antigen-binding construct, minibody, or cys-diabody binding to a target molecule described herein, or its absence.

[0255] In some embodiments, the antigen-binding construct, minibody, or cys-diabody is applied to a sample in vitro. In some embodiments, the sample is freshly obtained from a subject, e.g., a biopsy. In some embodiments, the sample is incubated after being obtained from the subject. In some embodiments, the sample is fixed. In some embodiments, the sample comprises a whole organ and / or tissue. In some embodiments, the sample comprises one or more whole cells. In some embodiments, the sample is derived from a cell extract, e.g., a lysate. In some embodiments, the antigen-binding construct in solution is added to a solution in a sample. In some embodiments, the antigen-binding construct, minibody, or cys-diabody in solution is added to a sample that does not contain a solution, e.g., a lyophilized sample, thereby reconstituting the sample. In some embodiments, the lyophilized antigen-binding construct, minibody, or cys-diabody is added to a sample that contains a solution, thereby reconstituting the antigen-binding construct, minibody, or cys-diabody.

[0256] In some embodiments, the antigen-binding construct, minibody, or cys-diabody is optionally incubated with the sample. The antigen-binding construct, minibody, or cys-diabody may be incubated for about 14 days or less, e.g., about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day or less, or for about 23 hours or less, e.g., about 23, 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 hours or less, or within the range defined by any two of the preceding values. In some embodiments, the incubation is within a subject to which the antigen-binding construct, minibody, or cys-diabody has been administered. In some embodiments, the incubation is within an incubator. In some embodiments, the incubator is maintained at a fixed temperature, for example, about 21°C, room temperature, 25°C, 29°C, 34°C, 37°C, or 40°C.

[0257] In some embodiments, antigen-binding constructs, minibodies, or cys-diabodies that are not bound to the target are optionally removed from the sample. In some embodiments, the sample is washed. Washing the sample can include removing a solution containing unbound antigen-binding constructs, minibodies, or cys-diabodies and adding a solution that does not contain antigen-binding constructs, minibodies, or cys-diabodies, such as a buffer solution. In some embodiments, an in vitro sample is washed, for example, by aspirating, pipetting, pumping, or pouring off a solution containing unbound antigen-binding constructs, minibodies, or cys-diabodies, and adding a solution that does not contain antigen-binding constructs, minibodies, or cys-diabodies. In some embodiments, an in vivo sample is washed, for example, by administering a solution that does not contain antigen-binding constructs, minibodies, or cys-diabodies to the subject, or by washing the site of local antigen-binding construct administration. In some embodiments, washing is performed at least twice, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 times. In some embodiments, after one or more washes, at least about 50%, e.g., at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the unbound antibody is removed from the sample.

[0258] In some embodiments, unbound antigen-binding constructs, minibodies, or cys-diabodies are removed from the sample. After application of the antigen-binding constructs, minibodies, or cys-diabodies to the sample, the antigen-binding constructs bound to the target reach equilibrium with the antigen-binding constructs that are not bound to the target, such that at a certain point after application of the antigen-binding constructs, the amount of antigen-binding constructs that are bound to the target does not increase substantially. After this point, at least a portion of the amount of antigen-binding constructs, minibodies, or cys-diabodies that are not bound to the target may be removed. In some embodiments, unbound antigen-binding constructs, minibodies, or cys-diabodies are removed by metabolic or other bodily processes of the subject to which the antigen-binding constructs, minibodies, or cys-diabodies are delivered. In some embodiments, unbound antigen-binding constructs, minibodies, or cys-diabodies are removed by the addition of an agent that destroys or destabilizes the unbound antigen-binding constructs, minibodies, or cys-diabodies, such as a protease or a neutralizing antibody. In some embodiments, one day after application of the antigen-binding construct, minibody, or cys-diabody, at least about 30%, e.g., at least about 30%, 40%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.9% of the applied antigen-binding construct has been cleared. In some embodiments, two days after application of the antigen-binding construct, minibody, or cys-diabody, at least about 40%, e.g., at least about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.9% of the applied antigen-binding construct, minibody, or cys-diabody has been cleared.

[0259] 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. After removal and / or elimination of the antigen-binding construct from the sample, for example 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.

[0260] In some embodiments, the antigen-binding construct comprises a detectable marker as described herein, and thus the presence of the antigen-binding construct can be inferred by detecting the detectable marker.

[0261] In some embodiments, a second antigen-binding construct, minibody, or cys-diabody is used to detect the antigen-binding construct, minibody, or cys-diabody. The second antigen-binding construct, minibody, or cys-diabody can specifically bind to the antigen-binding construct. For example, the second antigen-binding construct, minibody, or cys-diabody can comprise a polyclonal or monoclonal antibody, diabody, minibody, etc., directed against the host type of the antibody or against the antigen-binding construct, minibody, or cys-diabody itself. The second antigen-binding construct, minibody, or cys-diabody can be conjugated to a detectable marker described herein. The second antigen-binding construct, minibody, or cys-diabody can be applied to a sample. In some embodiments, the second antigen-binding construct, minibody, or cys-diabody is applied to a sample in substantially the same manner as the antigen-binding construct, minibody, or cys-diabody. For example, if an antigen-binding construct, minibody, or cys-diabody is injected into a subject, a second antigen-binding construct, minibody, or cys-diabody can also be injected into the subject.

[0262] In some embodiments, binding or absence of binding of the antigen-binding construct, minibody, or cys-diabody is detected via at least one of positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), computed tomography (CT), or detection of fluorescent emissions. PET may include, but is not limited to, small animal PET imaging. In some embodiments, binding or absence of binding of the antigen-binding construct, minibody, or cys-diabody is detected via two or more forms of imaging. In some embodiments, detection may be via near-infrared (NIR) and / or Cerenkov.

[0263] 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. Regarding PET, a positron-emitting drug (e.g., 18F-FDG) can be injected into the human body. When FDG is used, fludeoxyglucose (FDG) has a similar metabolism to glucose, so FDG will accumulate in cells that digest glucose. The positrons released by the decay of 18F and electrons in the tissue will undergo an annihilation reaction to generate two gamma photons with the same energy and in opposite directions. A detector array surrounding the human body can detect the two photons using a coincidence measurement technique and determine the positional information of the positron. Then, a tomographic image of the positrons in the human body can be constructed by processing the positional information using image reconstruction software. In some situations, immuno-PET can be employed, in which a label (e.g., 18F) is attached or linked to an antigen-binding construct. In such embodiments, the distribution of the antigen-binding construct can be monitored, which will depend on the binding and distribution characteristics of the antigen-binding construct. For example, if a CD8-directed minibody is used, PET can be used to monitor the distribution of CD8 molecules through the host's system. PET systems are known in the art, including, for example, U.S. Patent Publication Nos. 20170357015, 20170153337, 20150196266, 20150087974, 20120318988, and 20090159804, each of which is incorporated by reference in its entirety for their description of PET and its uses.

[0264] In some embodiments, imaging is performed by PET scan, PET / CT scan, or SPECT scan. 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 conjugated to at least one metal chelator. In some embodiments, optical imaging is used in assisted surgery. In some embodiments, photodynamic therapy is used. In some embodiments, photodynamic therapy is used in assisted surgery. In some embodiments, infrared fluorescence is used in assisted surgery. In some embodiments, photodynamic therapy is used in theranostics.

[0265] nucleic acid In some embodiments, the polypeptides of the antigen-binding construct, minibody, or cys-diabody can be encoded by a nucleic acid and expressed in vivo or in vitro, or the peptides can be chemically synthesized. Thus, in some embodiments, nucleic acids encoding antigen-binding constructs, minibodies, or cys-diabodies are provided. In some embodiments, the nucleic acid encodes a portion or monomer of the antigen-binding construct, minibody, or cys-diabody. In some embodiments, the nucleic acid encodes two or more monomers, e.g., at least two monomers. A nucleic acid encoding multiple monomers can include a nucleic acid cleavage site between at least two monomers, can encode a transcription or translation initiation 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 the antigen-binding construct, minibody, or cys-diabody VL, VH, CDR, FR, linker, signal peptide, or any combination thereof. In some embodiments, a nucleic acid encodes an antigen-binding construct, minibody, cys-diabody, VL, VH, CDR, FR, linker, signal peptide, or any combination thereof, that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an antigen-binding construct, minibody, cys-diabody, VL, VH, CDR, FR, linker, signal peptide, or any combination thereof, of any of SEQ ID NOs: 1-48. In some embodiments, a nucleic acid encodes a minibody that is any of SEQ ID NOs: 1-10. In some embodiments, a nucleic acid encodes 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 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 having any of SEQ ID NOs: 51-58 or 64-65. In some embodiments, the nucleic acid encodes a cys-diabody having any of SEQ ID NOs: 11-14.In some embodiments, the nucleic acid 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 having any 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 having any of SEQ ID NOs: 59-63. In some embodiments, the nucleic acid encodes an LCDR having any of SEQ ID NOs: 15-26. In some embodiments, the nucleic acid encodes an LCDR having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a LCDR having any of SEQ ID NOs: 15-26. In some embodiments, the nucleic acid encodes an HCDR having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an HCDR having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an HCDR having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an FR ... In some embodiments, the nucleic acid encodes a linker having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a linker having any of SEQ ID NOs: 39-44. In some embodiments, the nucleic acid encodes a signal peptide having any 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 having any 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 SEQ ID NO: 48.

[0266] In some embodiments, the expression vector contains a nucleic acid encoding an antigen-binding construct, minibody, or cys-diabody disclosed herein. In some embodiments, the expression vector comprises the pcDNA3.1™ / myc-His(-) version A vector (Invitrogen, Inc.) or a variant thereof for mammalian expression. The pcDNA3.1 expression vector features a CMV promoter for mammalian expression and both 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 retroviral or adenoviral vector. In embodiments, the vector comprises a cosmid, YAC, or BAC. In some embodiments, the expression vector encodes an antigen-binding construct, minibody, cys-diabody, VL, VH, CDR, FR, linker, signal peptide, or any combination thereof. In some embodiments, an expression vector encodes an antigen-binding construct, minibody, cys-diabody, VL, VH, CDR, FR, linker, signal peptide, or any combination thereof, that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an antigen-binding construct, minibody, cys-diabody, VL, VH, CDR, FR, linker, signal peptide, or any combination thereof, of any of SEQ ID NOs: 1-48. In some embodiments, an expression vector encodes a minibody that is any of SEQ ID NOs: 1-10. In some embodiments, an expression vector encodes 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 of SEQ ID NOs: 1-10. In some embodiments, an expression vector encodes a cys-diabody that is any 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 having any of SEQ ID NOs: 11-14.In some embodiments, the expression vector encodes an LCDR having any one of SEQ ID NOs: 15-26. In some embodiments, the expression vector encodes an LCDR having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an LCDR having any one of SEQ ID NOs: 15-26. In some embodiments, the expression vector encodes an HCDR having any one of SEQ ID NOs: 27-32. In some embodiments, the expression vector encodes an HCDR having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an HCDR having any one of SEQ ID NOs: 27-32. In some embodiments, the expression vector encodes a FR having any one of SEQ ID NOs: 33-38. In some embodiments, the expression vector encodes a FR having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a FR set forth in any of SEQ ID NOs: 33-38. In some embodiments, the expression vector encodes a linker set forth in any of SEQ ID NOs: 39-44. In some embodiments, the expression vector encodes a linker having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a linker set forth in any of SEQ ID NOs: 39-44. In some embodiments, the expression vector encodes a signal peptide set forth in any 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 a signal peptide set forth in any of SEQ ID NOs: 45-46. In some embodiments, the expression vector encodes a VH that is SEQ ID NO: 47. In some embodiments, the nucleic acid encodes 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 expression vector encodes a VL that is 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 a VL that is SEQ ID NO: 48. In any of the embodiments provided herein, the signal peptide may be removed from the amino acid sequence for some embodiments.

[0267] Figure 14 shows some embodiments of nucleic acid sequences for antigen-binding constructs, minibodies, and / or cys-diabodies (SEQ ID NOS: 51-64). In some embodiments, the nucleic acid-binding construct and / or minibody is an antigen-binding construct and / or minibody in Table 6. In some embodiments, the expression vector encodes an antigen-binding construct and / or minibody that is an antigen-binding construct and / or minibody in Table 7.

[0268] [Table 8A]

[0269] [Table 8B]

[0270] [Table 8C]

[0271] [Table 8D]

[0272] [Table 8E]

[0273] In some embodiments, the nucleic acid binding construct and / or minibody is an antigen-binding construct and / or minibody in Table 7. In some embodiments, the expression vector encodes an antigen-binding construct and / or cys-diabody that is an antigen-binding construct and / or cys-diabody in Table 8.

[0274] [Table 9A]

[0275] [Table 9B]

[0276] cell In some embodiments, cell lines expressing at least one of the antigen-binding constructs, minibodies, or cys-diabodies described herein are provided. In some embodiments, mammalian cell lines (e.g., CHO-K1, ExpiCHO, HEK-293, 293f, Expi293 cell lines) are expression systems for producing the antigen-binding constructs, minibodies, cys-diabodies, or other antibodies described herein. In some embodiments, the antigen-binding constructs, minibodies, cys-diabodies, and other antibodies or antibody fragments described herein are non-glycosylated, and therefore do not require mammalian expression systems, and therefore do not require post-translational modifications. Thus, in some embodiments, one or more of a wide variety of mammalian or non-mammalian expression systems are used to produce the antigen-binding constructs, minibodies, and cys-diabodies described herein, including, but not limited to, mammalian expression systems (e.g., CHO-K1 cells), bacterial expression systems (e.g., E. coli, B. subtilis), yeast expression systems (e.g., Pichia, S. cerevisiae), or any other known expression system. Other systems may include insect cells and / or plant cells.

[0277] In some embodiments, the cells express an antigen-binding construct, minibody, cys-diabody, VL, VH, CDR, FR, linker, signal peptide, or any combination thereof. In some embodiments, the cells express an antigen-binding construct, minibody, cys-diabody, VL, VH, CDR, FR, linker, signal peptide, or any combination thereof, that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the antigen-binding construct, minibody, cys-diabody, VL, VH, CDR, FR, linker, signal peptide, or any combination thereof, of any of SEQ ID NOs: 1-48. In some embodiments, the cells express a minibody that is any 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 having any of SEQ ID NOs: 1-10. In some embodiments, the cells express a cys-diabody having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a cys-diabody having any of SEQ ID NOs: 11-14. In some embodiments, the cells express an LCDR having any of SEQ ID NOs: 15-26. In some embodiments, the expression vector encodes an LCDR having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an LCDR set forth in any of SEQ ID NOs: 15-26. In some embodiments, the cell expresses an HCDR set forth in any of SEQ ID NOs: 27-32. In some embodiments, the cell expresses an HCDR set forth in any of SEQ ID NOs: 27-32. In some embodiments, the cell expresses a FR set forth in any 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 having any of SEQ ID NOs: 33-38. In some embodiments, the cells express a linker having any 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 having any of SEQ ID NOs: 39-44. In some embodiments, the cells express a signal peptide having any of SEQ ID NOs: 45-46. In some embodiments, the cells express a signal peptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a signal peptide having any of SEQ ID NOs: 45-46. In some embodiments, the cells express a VH having 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 having SEQ ID NO: 47. In some embodiments, the cells express a VL having SEQ ID NO: 48. In some embodiments, the cells express a VL having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a VL having SEQ ID NO: 48.

[0278] kit In some embodiments, kits are provided. In some embodiments, the kits comprise an antigen-binding construct, a minibody, a cys-diabody, or any combination thereof described herein. In some embodiments, the kits comprise a nucleic acid encoding an antigen-binding construct described herein. In some embodiments, the kits comprise a cell line producing an antigen-binding construct described herein. In some embodiments, the kits comprise a detectable marker described herein. In some embodiments, the kits comprise a therapeutic agent described herein. In some embodiments, the kits comprise a buffer. In some embodiments, the kits comprise a positive control, e.g., target-specific cells or fragments thereof. In some embodiments, the kits comprise a negative control, e.g., a surface or solution substantially free of target. In some embodiments, the kits comprise packaging. In some embodiments, the kits comprise instructions for use. In some embodiments, the kits comprise the antigen-binding construct, minibody, or cys-diabody of any of the preceding embodiments; and a chelator that allows for incorporation of a detectable marker. In some embodiments, the kits comprise the antigen-binding construct, minibody, or cys-diabody of any of the preceding embodiments; and a chelator that allows for incorporation of a therapeutic isotope. In some embodiments, the kit comprises the antigen-binding construct, minibody, or cys-diabody of any of the preceding embodiments; and a linker that allows for the incorporation of a detectable marker. In some embodiments, the kit comprises the antigen-binding construct, minibody, or cys-diabody of any of the preceding embodiments; and a linker that allows for the incorporation of a therapeutic isotope.

[0279] Additional embodiments of the present disclosure are provided in the following numbered configurations: 1. LCDR1, which is SEQ ID NO: 15; LCDR2, which is any one of SEQ ID NOs: 19, 21, or 23; LCDR3, which is SEQ ID NO: 25 The variable light chain (V L ) domain; and HCDR1, which is SEQ ID NO: 27; HCDR2, which is SEQ ID NO: 29; and HCDR3, which is SEQ ID NO: 31 A variable heavy chain (V H )domain 1. An antigen-binding construct comprising: 2. Variable heavy chain (V H ) domains linked to the variable light chain (V L a single-chain variable fragment (scFv) that binds to DLL3 and comprises a V L The domain is LCDR1, which is SEQ ID NO: 15; LCDR2, which is any one of SEQ ID NOs: 19, 21, or 23; LCDR3, which is SEQ ID NO: 25 Including V H The domain is HCDR1, which is SEQ ID NO: 27; HCDR2, which is SEQ ID NO: 29; HCDR3, which is SEQ ID NO: 31 a single chain variable fragment (scFv) comprising: a hinge extension domain comprising a hinge region; and IgG C H 3. Array A minibody that binds to DLL3, including 3. Variable heavy chain (V H ) domains linked to the variable light chain (V L ) domain, L The domain is LCDR1, which is SEQ ID NO: 15; LCDR2, which is any one of SEQ ID NOs: 19, 21, or 23; LCDR3, which is SEQ ID NO: 25 Including V H The domain is HCDR1, which is SEQ ID NO: 27; HCDR2, which is SEQ ID NO: 29; and HCDR3, which is SEQ ID NO: 31 A cys-diabody that binds to DLL3, comprising a polypeptide comprising a single-chain variable fragment (scFv), comprising: 4.V L 4. The antigen-binding construct, minibody or cys-diabody of any one of configurations 1 to 3, wherein the domain further comprises an LFR2 of SEQ ID NO: 35 or 37. 5.V L The domain is V in SEQ ID NO: 48 L 5. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 4, comprising an amino acid sequence having at least 90% identity to the domain, wherein the antigen-binding construct, minibody, or cys-diabody comprises a Y50D and / or Y55N mutation (Kabat). 6.V L The domain is V in SEQ ID NO: 48 L 6. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 5, comprising an amino acid sequence having at least 90% identity to the domain, wherein the antigen-binding construct, minibody, or cys-diabody comprises an A43S and / or Q45R mutation (Kabat). 7.V L The domain is V in SEQ ID NO: 49 L 7. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 6, comprising an amino acid sequence having at least 90% identity to the domain, wherein the antigen-binding construct, minibody, or cys-diabody comprises a Y50D and / or Y55N mutation (Kabat). 8.V L The domain is V in SEQ ID NO: 49 L 8. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 7, comprising an amino acid sequence having at least 90% identity to the domain, 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 of any one of configurations 1 to 8, further comprising a signal peptide which is SEQ ID NO:45. 10. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 9, comprising a linker that is any one of SEQ ID NOs: 39-44. 11.C H 3 domains are IgG C H An antigen-binding construct or minibody as defined in any one of configurations 1 to 10, which is a three domain construct. 12. IgG C H 3 domains, IgG1, IgG2, IgG3, or IgG4 C H An antigen-binding construct or minibody as defined in any one of configurations 1 to 11, which is a three-domain construct. 13. IgG C H The three domains are IgG1 C, which is any one of SEQ ID NOs: 65 to 68. H 12. An antigen-binding construct or minibody as defined in any one of configurations 1 to 12, comprising three domains. 14. IgG C H The three domains are IgG1 C, which is any one of SEQ ID NOs: 69 to 71. H 14. An antigen-binding construct or minibody as defined in any one of configurations 1 to 13, comprising three domains. 15. IgG C H The three domains are IgG1 C, which is any one of SEQ ID NOs: 72 to 82. H 14. An antigen-binding construct or minibody as defined in any one of configurations 1 to 14, comprising three domains. 16. IgG C H The three domains are IgG1 C1 domains having any one of SEQ ID NOs: 83 to 85. H 15. An antigen-binding construct or minibody as defined in any one of configurations 1 to 15, comprising three domains. 17. The antigen-binding construct of configuration 1, which is an antibody. 18. An antigen-binding construct, minibody, or cys-diabody according to any one of configurations 1 to 17, which specifically binds to DLL3. 19. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 18, further comprising a detectable marker. 20. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 19, wherein the detectable marker is a fluorescently detectable marker. 21. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 20, wherein the detectable marker comprises a phototherapy compatible dye. 22. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 21, wherein the detectable marker comprises boron. 23. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 22, 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 one of configurations 1 to 23, wherein the detectable marker is a radioactive label. 25. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 24, wherein the detectable marker is an alpha-emitter radiolabel. 26. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 25, wherein the detectable marker is a beta-emitter radiolabel. 27. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 26, wherein the detectable marker is a positron-emitter radiolabel. 28. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 27, wherein the detectable marker comprises lutetium-177. 29. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 28, wherein the detectable marker is a gamma-emitter radiolabel. 30. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 29, wherein the detectable marker is suitable for use with Auger electron spectroscopy. 31. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 30, wherein the detectable marker comprises an isotope. 32. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 31, wherein the detectable marker comprises a bioluminescent compound. 33. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 32, wherein the detectable marker comprises a chemiluminescent compound. 34. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 33, wherein the detectable marker comprises an enzyme. 35. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 34, wherein the detectable marker comprises a metal chelator. 36. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 35, further comprising a therapeutic agent. 37. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 36, wherein the therapeutic agent comprises a therapeutic isotope or ion. 38. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 37, wherein the therapeutic agent is a radiolabel. 39. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 38, wherein the therapeutic agent is an alpha-emitter radiolabel. 40. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 39, wherein the therapeutic agent is a beta-emitter radiolabel. 41. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 40, wherein the therapeutic agent is a positron emitter radiolabel. 42. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 41, wherein the therapeutic agent is a gamma-emitter radiolabel. 43. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 42, wherein the therapeutic agent comprises lutetium-177. 44. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 43, wherein the therapeutic agent comprises a phototherapy-compatible dye. 45. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 44, wherein the therapeutic agent comprises boron. 46. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 45, 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 one of configurations 1 to 46, wherein the therapeutic agent and / or detectable marker comprises a toxic payload. 48. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 47, which is bispecific. 49. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 48, comprising a monovalent scFv. 50. From the N-terminus to the C-terminus of a polypeptide, V L , V H 50. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 49, wherein: 51. The order of variable domains from the N-terminus to the C-terminus of a polypeptide is V H , V L 51. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 50, wherein: 52. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 51, wherein the minibody is a humanized antigen-binding construct, minibody, or cys-diabody. 53. The humanized antigen-binding construct, minibody, or cys-diabody is LCDR1, which is SEQ ID NO: 15; LCDR2, which is any one of SEQ ID NOs: 19, 21, or 23; LCDR3, which is SEQ ID NO: 25; HCDR1, which is SEQ ID NO: 27; HCDR2, which is SEQ ID NO: 29; and HCDR3, which is SEQ ID NO: 31 53. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 52, comprising: 54. An antigen-binding construct, minibody, or cys-diabody according to any one of configurations 1 to 53, having 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 configurations 1 to 53, having 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 configurations 1 to 53, having 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. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 56, which 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. 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 configuration 57 or 58, which 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 configuration 57 or 58, which is detectable at about 0.1%-35%, 0.1%-30%, 1%-35%, 1%-30%, 5%-35%, or 5%-30% ID / g. 61. An antigen-binding construct, minibody, or cys-diabody according to any one of configurations 1 to 60, comprising an LFR2 of SEQ ID NO: 35 or 37; and having improved biodistribution compared to an antigen-binding construct, minibody, or cys-diabody comprising an LFR2 that is an LFR2 of SEQ ID NO: 33. 62. Approximately 1×10 -10 62. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 61, having a KD of less than M. 63. Approximately 1×10 -12 63. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 62, having a KD of less than M. 64. An EC50 of 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 50 64. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 63, comprising: 65. An EC of approximately 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. 50 65. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 64, comprising: 66. EC of approximately 10 nM 50 66. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 65, comprising: 67. A nucleic acid encoding the antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 66. 68. A cell line producing the antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 66. 69. The nucleic acid of claim 67, which is any one of SEQ ID NOs: 51-65. 70. A cell line producing the antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 66. 71. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 66; and Detectable Markers Kit including: 72. A kit comprising an antigen-binding construct, minibody, or cys-diabody according to any one of configurations 1 to 66; and a chelating agent allowing for the incorporation of a detectable marker. 73. A kit comprising an antigen-binding construct, minibody, or cys-diabody according to any one of configurations 1 to 66; and a chelator allowing incorporation of a therapeutic isotope. 74. A kit comprising an antigen-binding construct, minibody, or cys-diabody according to any one of configurations 1 to 66; and a linker allowing for the incorporation of a detectable marker. 75. A kit comprising an antigen-binding construct, minibody, or cys-diabody according to any one of configurations 1 to 66; and a linker allowing for the incorporation of a therapeutic isotope. 76. A kit comprising the antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 66; and a detectable marker. 77. Applying the antigen-binding construct, minibody, or cys-diabody of any one of configurations 1 to 66 to a sample; and Detecting the presence or absence of the antigen-binding construct, thereby detecting the presence or absence of DLL3. A method for detecting the presence or absence of DLL3, comprising: 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 the subject. 80. The method of any one of aspects 77 to 79, wherein the step of detecting binding 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 configurations 77 to 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 aspects 77 to 81, wherein the antigen-binding construct, minibody, or cys-diabody is incubated with the sample for no more than 20 hours. 83. The method of any one of aspects 77 to 82, wherein the antigen-binding construct, minibody, or cys-diabody is incubated with the sample for no more than 6 hours. 84. The method of any one of configurations 77 to 83, wherein an antigen-binding construct, minibody, or cys-diabody is administered to a host, wherein a first quantity of the antigen-binding construct, minibody, or cys-diabody is not bound to DLL3 and a second quantity of the antigen-binding construct, minibody, or cys-diabody is bound to DLL3, and at least about 80% of the first quantity of the antigen-binding construct, minibody, or cys-diabody is cleared within 12 hours. 85. A method for targeting a therapeutic agent to DLL3, comprising administering to a subject an antigen-binding construct, minibody, or cys-diabody described in any one of configurations 1 to 66, wherein the antigen-binding construct is conjugated to the therapeutic agent. 86. LCDR1 is SEQ ID NO: 15; LCDR2, which is any one of SEQ ID NOs: 19, 21, or 23; LCDR3, which is SEQ ID NO: 25 The variable light chain (V L ) domain; and HCDR1 of SEQ ID NO: 27; HCDR2 of SEQ ID NO: 29; HCDR3 of SEQ ID NO: 31 A variable heavy chain (V H )domain an antigen-binding construct comprising: Therapeutic Agents, Toxic Payloads, and / or Detectable Markers A therapeutic composition that targets DLL3, comprising: 87. A minibody that binds to DLL3, Variable heavy chain (V H ) domains linked to the variable light chain (V L a single-chain variable fragment (scFv) that binds to DLL3 and comprises a V L The domain is LCDR1, which is SEQ ID NO: 15; LCDR2, which is any one of SEQ ID NOs: 19, 21, or 23; LCDR3, which is SEQ ID NO: 25 Including V H The domain is HCDR1, which is SEQ ID NO: 27; HCDR2, which is SEQ ID NO: 29; HCDR3, which is SEQ ID NO: 31 a single chain variable fragment (scFv); a hinge extension domain containing the IgG1 hinge region; IgG C H 3. Array Mini body including; Therapeutic Agents, Toxic Payloads, and / or Detectable Markers A therapeutic composition that targets DLL3, comprising: 88. A cys-diabody that binds to DLL3, Variable heavy chain (V H ) domains linked to the variable light chain (V L ) domain, LThe domain is LCDR1, which is SEQ ID NO: 15; LCDR2, which is SEQ ID NO: 19, 21, or 23; LCDR3, which is SEQ ID NO: 25 Including V H The domain is HCDR1, which is SEQ ID NO: 27; HCDR2, which is SEQ ID NO: 29; HCDR3, which is SEQ ID NO: 31 a cys-diabody comprising a polypeptide comprising a single chain variable fragment (scFv); and Therapeutic Agents, Toxic Payloads, and / or Detectable Markers A therapeutic composition that targets DLL3, comprising: 89. The therapeutic composition of any one of configurations 86 to 88, wherein the detectable marker is a radioactive label. 90. The therapeutic composition of any one of configurations 86 to 89, wherein the detectable marker is an alpha-emitter radiolabel. 91. The therapeutic composition of any one of configurations 86 to 90, wherein the detectable marker is a beta-emitter radiolabel. 92. The therapeutic composition of any one of configurations 86 to 91, wherein the detectable marker is a positron emitter radiolabel. 93. The therapeutic composition of any one of embodiments 86 to 92, wherein the detectable marker comprises lutetium-177. 94. The antigen-binding construct, minibody, or cys-diabody of any one of configurations 1-66, or the composition of any one of configurations 86-93, or the kit of any one of configurations 71-76, or the method of any one of configurations 77-85, wherein the therapeutic agent comprises terbium-149, terbium-161, or lead-212. 95. The antigen-binding construct comprises: A single-chain variable fragment (scFv) that binds to DLL3, comprising a variable heavy chain (V H ) domains linked to the variable light chain (V L ) domain-containing single-chain variable fragment (scFv); a hinge domain; and Fc area 93. The antigen-binding construct of any one of configurations 1 to 66, or the composition of any one of configurations 86 to 93, or the kit of any one of configurations 71 to 76, or the method of any one of configurations 77 to 85, wherein the antigen-binding construct is an scFv-Fc comprising: 96. The variable heavy chain (V) binds to DLL3 and is linked to the Fc region via a hinge domain. H ) domain. 97. Variable heavy chain (V H ) domain, HCDR1, which is SEQ ID NO: 27; HCDR2, which is SEQ ID NO: 29; and HCDR3, which is SEQ ID NO: 31 97. The antigen-binding construct of claim 96, comprising:

[0280] Embodiments of the present disclosure are further defined in the following examples. It should be understood that these examples are given for illustrative purposes only. From the above discussion and these examples, those skilled in the art can ascertain the essential features of the present disclosure and can make various changes and modifications to the embodiments of the present disclosure to adapt it to various uses and conditions without departing from the spirit and scope thereof. Thus, various modifications of the embodiments of the present disclosure, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. The disclosures of each reference specified herein are incorporated herein by reference in their entirety and with respect to the disclosures referenced therein. [Example]

[0281] Example 1 Isolation and characterization of novel antigen-binding constructs Isolation of the series of novel antigen-binding constructs described herein can be carried out using any standard methodology known to those skilled in the art.

[0282] As disclosed herein, a series of antigen-binding constructs specific for DLL3 were designed and isolated (SEQ ID NOS: 1-46). Each antigen-binding construct was either human or murine and was either an antibody, minibody, or cys-diabody. In one example, the variable region from a parent antibody was converted to an scFv and grafted onto a minibody or cys-diabody scaffold. In another example, the variable region from a parent antibody was tested for immunogenic epitopes using in silico methods known in the art, converted to an scFv, and grafted onto a minibody or cys-diabody scaffold. Furthermore, each construct contained heavy and light chains with an HCDR1 of SEQ ID NOS: 27, an HCDR2 of SEQ ID NOS: 29, an HCDR3 of SEQ ID NOS: 31, an LCDR1 of SEQ ID NOS: 15, an LCDR2 of SEQ ID NOS: 19, 21, or 23, and an LCDR3 of SEQ ID NOS: 25. Each antigen-binding construct varied in sequence; for example, some contained a serine at position 43 of the VL, an arginine at position 45 of the VL, an aspartic acid at position 50 of the VL, and / or an asparagine at position 55 of the VL (Kabat). The sequence variations for each antigen-binding construct were selected because they had improved characteristics compared to antigen-binding constructs without the variations. For example, variations in the antigen-binding constructs could improve the dissociation constant (K off This resulted in improved antigen binding kinetics as indicated by a flattening or reduction in the β-glucan-binding domain.

[0283] These antigen-binding constructs were then transfected into the Expi293™ cell line using a liposome transfection protocol (Thermo Exp293 kit). The vector contained a cleavable signal peptide of 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.

[0284] Expression of the constructs was measured relative to the parental minibodies IAB57M1-1 and IAB57M2-2. As shown in Figure 7, the novel antigen-binding constructs disclosed herein had up to four-fold higher expression yields in Expi293™ mammalian cells compared to the parental minibodies.

[0285] The binding kinetics of the novel antigen-binding constructs were also determined by biolayer interferometry (TABLE 9) (Figure 9).

[0286] [Table 10]

[0287] As disclosed herein, the half maximal effective concentration (EC 50 ) was also assessed. 50 was determined (Table 10) (Figure 8). The novel antigen-binding constructs disclosed herein have an EC of 0.01-0.1 nM. 50 and had high affinity for tumor cells in the SHP-77 and NCI-H82 cell lines (Figure 10).

[0288] [Table 11]

[0289] The binding affinity of the novel antigen-binding constructs to purified human, cynomolgus monkey, dog, mouse, rat, or minipig DLL3 antigen was assessed 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 (0.05 M sodium carbonate-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 three times with 300 μl of wash buffer (PBS with 0.05% Tween-20) per well. After the final wash, the plate was blocked by adding 200 μl of blocking buffer (PBS with 1% BSA) warmed to room temperature to each well. The plate was then incubated at room temperature for 1 hour under gentle agitation. After blocking, the plate was washed three times with 300 μl of wash buffer per well. Diluted samples for each minibody were made by making 2.5x dilutions from a starting sample of 100 ng / mL, 250 ng / mL, 1000 ng / mL, or 3000 ng / mL in 100 μL sample buffer (PBS with 1% BSA). The diluted samples were transferred to the assay plate and incubated for 1 hour at room temperature under gentle agitation. The plate was then washed three times with 300 μL of wash buffer per well.

[0290] Detection was performed by adding 100 μl of an appropriate dilution of HRP-labeled secondary antibody and incubating the plate at room temperature for 1 hour under gentle agitation. The plate was then washed three times with 300 μl of wash buffer per well. For color development, 100 μl of TMB substrate reagent pre-warmed to room temperature was added to the plate and incubated at room temperature in the dark for 15 minutes. The reaction was then stopped by adding 100 μl of 650 nm stop solution to the plate and mixing at room temperature for 1 minute under gentle agitation. Within 20 minutes after terminating the reaction, the plate was recorded by reading the OD at 650 nm on a plate reader.

[0291] As disclosed herein, the binding kinetics of novel antigen-binding constructs to purified human / cynomolgus / dog / mouse / rat or minipig DLL3 antigens were assessed using biolayer interferometry (TABLE 9) (FIG. 9). His-tagged DLL3 antigen was diluted to 10 μg / mL in phosphate-buffered saline (PBS) and loaded onto a nickel-NTA chip to saturation. The on-rate (K on ) and off-rate (K off ), and the dissociation constant K D The binding curves were recorded on an Octet BLItz instrument. Diluted samples of the antigen-binding constructs were prepared in PBS at concentrations ranging from 100 nM to 0.1 nM by 2.5x dilution, and binding curves were recorded at each concentration. Off-rate curves were recorded by immersion of the tip in PBS.

[0292] As disclosed herein, binding of the constructs to cellular targets was assessed using flow cytometry. Starting at a concentration of 100 nM, 2.5x serial dilutions of minibodies were made in 180 μl FACS buffer (PBS 2% FBS). 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 minibody dilution series was transferred to the plate containing the cell pellet and incubated at 4°C for 1 hour. The plate was then washed three times with 200 μl / well of FACS buffer, and the supernatant after each wash was discarded by centrifugation at 1000 rpm. 50 μl / well of detection antibody R&D AlexaFluor-647 goat anti-human (H+L) was added at a 1:100 dilution in FACS buffer and incubated at 4°C for 1 hour. The plate was washed three times with 200 μl / well of FACS buffer, and the supernatant after each wash was discarded 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 allophycocyanin fluorescence settings.

[0293] Humanized V in IAB57M1-3 L used the germline sequence of IGVK3-15*01. L 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 positively charged patch may contribute to minibody accumulation in the kidney, leading to excessively rapid clearance of minibodies and potential kidney damage.

[0294] To test this, R45 was substituted with glutamine (Q), as shown in Figure 1. Thus, V of IAB57M1-3 L IAB57M1-13 was generated by changing KPGQAPR (SEQ ID NO: 95) in FR2 to KPGQAPQ (SEQ ID NO: 111). As shown in Figure 1, IAB57M1-14 was generated by further substituting alanine (A) at position 43 with serine (S). Thus, the V of IAB57M1-3 L KPGQAPR (SEQ ID NO: 95) in FR2 was further changed to KPGQSPQ (SEQ ID NO: 110) in IAB57M1-14.

[0295] The in vivo biodistribution of 89Zr-desferoxamine-IAB57M1-3 was assessed in NCr nude mice bearing SHP77 and NCI-H82 tumors (Figure 11). The in vivo biodistribution of 89-Zr-Df-IAB57M1-3, 89-Zr-Df-IAB57M1-13, and 89-Zr-Df-IAB57M1-14 was also assessed (Figure 12). In both cases, the M1-3 antigen-binding construct was relatively highly distributed to the liver and kidney. In contrast, M1-13 and M1-14 exhibited improved biodistribution, as indicated by reduced accumulation of these minibodies in the kidney. The antigen-binding constructs also distributed in vivo to the blood, spleen, lung, muscle, bone, heart, stomach, large intestine, small intestine, pancreas, and tumor.

[0296] Example 2 Use of antigen-binding constructs for the treatment of subjects in need thereof A method of utilizing the novel antigen-binding constructs described herein as a medicine for a subject in need thereof may benefit the subject.

[0297] As disclosed herein, pharmaceutical compositions are prepared comprising novel antigen-binding constructs against DLL3, alone or in combination with a chemotherapy or immuno-oncology agent, and a pharmaceutically acceptable carrier. In another alternative, the dosage is a radioactive dose. In another alternative, the dosage is not fixed. In another alternative, the dosage is provided as a fractionated dose. In another alternative, the dosage is measured in mCi or MBq. In another alternative, the composition may have more than one antigen-binding construct. In another alternative, the antigen-binding construct may be an antibody, a minibody, a cys-diabody, or any combination thereof. In another alternative, the antigen-binding construct may be present in the composition at any pharmaceutically effective concentration, such as from about 0.01 mg / kg to about 25 mg / kg. In another alternative, the immuno-oncology agent may be omitted from the composition. In other alternatives, the immuno-oncology agent may be replaced by one or more small molecules, therapeutic agents, or antigen-binding constructs effective in treating the disease, such as antibodies, chemotherapeutic agents, DNA repair inhibitors, alkylating agents, metabolic inhibitors, radiosensitizers, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, nitrosoureas, corticosteroids, antiangiogenic agents, apoptosis inducers, antimicrotubule agents, vinca alkaloids, taxanes, anthracyclines, antiandrogens, VEGF pathway inhibitors, MAPK / Ras / Raf pathway inhibitors, and EGFR pathway inhibitors.

[0298] The compositions of the present invention may be used in combination with other agents to achieve improved therapeutic outcomes for the subject. Other agents that may be used in combination include therapeutic agents, immunotherapeutic agents, chemotherapeutic agents, radiation protectants, and nephroprotectants. Immunotherapeutic agents useful in combination with the present invention include atezolizumab (Tecentriq), avelumab (Bavencio), dostarlizumab (Gemperli), durvalumab (Imfinzi), ipilimumab (Yervoy), nivolumab (Opdivo), pembrolizumab (Keytruda), and other immunotherapeutic agents in clinical development.

[0299] As disclosed herein, to treat SCLC, a composition is administered to a human subject at a pharmaceutically acceptable dose. In other alternatives, the disease may be other neuroendocrine tumors such as neuroendocrine prostate cancer, glioblastoma, fibrosis, cancer, tumor, solid tumor, autoimmune disease, cardiovascular disease, metabolic disease, bone cancer, bone sarcoma, 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, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, medullary thyroid carcinoma, melanoma, non-small cell lung cancer, osteosarcoma, oral squamous cell carcinoma, oral cancer, ovarian cancer, ovarian cancer, pancreatic adenocarcinoma, pancreatic cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, urothelial cancer, or any other disease associated with altered DLL3 expression. In another alternative, the composition is administered to a subject to inhibit or ameliorate a disease.

[0300] After administration, the disease is monitored through imaging of relevant cells, tissues, or organs in the subject using the methods outlined in the Examples below. As seen through imaging, cancer cells are damaged after administration of the composition to the subject. In another alternative, cancer cells are inhibited, ameliorated, damaged, killed, or have apoptosis induced in response to contact with the composition.

[0301] Example 3 Use of antigen-binding constructs for diagnostic methods For that diagnostic method, methods utilizing the novel antigen-binding constructs described herein may benefit subjects suspected of having the disease.

[0302] As disclosed herein, novel antigen-binding constructs are used to image tissues of human subjects suspected of having cancer. In another alternative, the antigen-binding constructs can be used to image cells, cultured cell lines, tissue fractions, organs, organ sections, multiple tissues, multiple organs, or the entire subject. In another alternative, the subject can be screened for tumors, fibrosis, autoimmune diseases, cardiovascular diseases, or any other disease or abnormality associated with DLL3. In another alternative, the subject can be any mammal, including mice, rats, dogs, minipigs, and non-human primates.

[0303] The antigen-binding construct is administered to the epithelial tissue of a subject and screened for binding to DLL3. The subject is evaluated for the presence of DLL3 by an in vivo diagnostic medical imaging method, 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 the presence of cancer in the tissue. In another alternative, absence of binding of the antigen-binding construct to DLL3 indicates a low likelihood of the presence of cancer in the tissue. In another alternative, binding of the antigen-binding construct to DLL3 is determined by: 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, 124I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154~158 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 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, or 225 The binding of the antigen-binding construct to DLL3 is determined by the payload attached to the antigen-binding construct, such as Ac. In another alternative, the binding of the antigen-binding construct to DLL3 is determined by PET scan.

[0304] Example 4 Use of antigen-binding constructs to reduce renal uptake The methods of modifying antigen-binding constructs described herein can provide the benefit of reduced renal uptake of radiolabeled minibodies in subjects.

[0305] As disclosed herein, minibodies are generated by humanization of mouse hybridomas. In some embodiments, the humanization of antibodies is based on the germline: IGHV1-69*01 / IGHV1-39*01.

[0306] A positively charged patch responsible for accumulating this minibody in the kidney was identified in the germline IGKV1-39*01. As disclosed herein, the patch maps to the sequence KPGKAPK, which contains three charged lysine residues. In some embodiments, mutations on framework 2 of this germline were identified that reduce renal uptake of radiolabeled minibodies.

[0307] As disclosed herein, some human germlines have a pattern of three positively charged residues in 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 to replace framework 2 with a similar sequence from the germline that does not contain this charge pattern. Non-limiting examples of such substitutions are framework 2 from IGKV3-20*01 or IGKV2-28*01.

[0308] As used herein, section headings are for organizational purposes only and should not be construed as limiting the subject matter described in any way. All literature and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, papers, and Internet web pages, including the disclosures specifically referenced herein, are expressly incorporated by reference in their entirety for any purpose. In the event that definitions of terms in incorporated references appear to differ from definitions provided in the present teachings, the definitions provided in the present teachings shall control. It will be understood that there is an implicit "about" before temperatures, concentrations, times, etc. discussed in the present teachings, so that slight and insubstantial deviations are within the scope of the present teachings herein.

[0309] While the present disclosure has been presented in the context of certain specific embodiments and examples, those skilled in the art will appreciate that the present disclosure extends beyond the specifically disclosed embodiments to other alternative embodiments and / or uses of the present disclosure, as well as obvious modifications and equivalents thereof. Additionally, while several variations of the present disclosure have been shown and described in detail, other modifications within the scope of the present disclosure will be readily apparent to those skilled in the art based on the present disclosure. It is also contemplated that various combinations or subcombinations of the specific features and aspects of the embodiments may be made and still fall within the scope of the present disclosure. It should be understood that various features and aspects of the disclosed embodiments may be combined with or substituted for one another to form various modes or embodiments of the present disclosure. Therefore, it is not intended that the scope of the present disclosure presented herein should be limited by the specific disclosed embodiments described above.

[0310] It should be understood, however, that this detailed description, while indicating preferred embodiments of the disclosure, is given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art.

[0311] The terminology used in the description provided herein is not intended to be construed in any limiting or restrictive manner. Rather, the terminology is merely utilized in conjunction with detailed descriptions of embodiments of systems, methods, and associated components. Furthermore, embodiments may include several novel features, no single one of which is solely responsible for its desirable attributes or is considered essential to practicing the present disclosure as described herein.

Claims

1. LCDR1, which is SEQ ID NO: 15; LCDR2, which is any one of SEQ ID NOs: 19, 21, or 23; LCDR3, which is SEQ ID NO: 25 The variable light chain (V L ) domain; and HCDR1, which is SEQ ID NO: 27; HCDR2, which is SEQ ID NO: 29; and HCDR3, which is SEQ ID NO: 31 The variable heavy chain (V H )domain 1. An antigen-binding construct comprising:

2. Variable heavy chain (V H ) domains linked to the variable light chain (V L a single-chain variable fragment (scFv) that binds to DLL3 and comprises a V L The domain is LCDR1, which is SEQ ID NO: 15; LCDR2, which is any one of SEQ ID NOs: 19, 21, or 23; LCDR3, which is SEQ ID NO: 25 Including V H The domain is HCDR1, which is SEQ ID NO: 27; HCDR2, which is SEQ ID NO: 29; HCDR3, which is SEQ ID NO: 31 a single chain variable fragment (scFv) comprising: a hinge extension domain comprising a hinge region; and IgG C H 3. Array A minibody that binds to DLL3, including

3. Variable heavy chain (V H ) domains linked to the variable light chain (V L ) domain, L The domain is LCDR1, which is SEQ ID NO: 15; LCDR2, which is any one of SEQ ID NOs: 19, 21, or 23; LCDR3, which is SEQ ID NO: 25 Including V H The domain is HCDR1, which is SEQ ID NO: 27; HCDR2, which is SEQ ID NO: 29; and HCDR3, which is SEQ ID NO: 31 A cys-diabody that binds to DLL3, comprising a polypeptide comprising a single-chain variable fragment (scFv), comprising:

4. V L 4. The antigen-binding construct, minibody, or cys-diabody of claim 1, wherein the domain further comprises an LFR2 of SEQ ID NO: 35 or 37.

5. V L The domain is V in SEQ ID NO: 48 L 5. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 4, comprising an amino acid sequence having at least 90% identity to the domain, wherein the antigen-binding construct, minibody, or cys-diabody comprises a Y50D and / or Y55N mutation (Kabat).

6. V L The domain is V in SEQ ID NO: 48 L 6. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 5, comprising an amino acid sequence having at least 90% identity to the domain, wherein the antigen-binding construct, minibody, or cys-diabody comprises an A43S and / or Q45R mutation (Kabat).

7. V L The domain is V in SEQ ID NO: 49 L 7. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 6, comprising an amino acid sequence having at least 90% identity to the domain, wherein the antigen-binding construct, minibody, or cys-diabody comprises a Y50D and / or Y55N mutation (Kabat).

8. V L The domain is V in SEQ ID NO: 49 L 8. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 7, comprising an amino acid sequence having at least 90% identity to the domain, wherein the antigen-binding construct, minibody, or cys-diabody comprises an A43S and / or Q45R mutation (Kabat).

9. 9. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 8, further comprising a signal peptide which is SEQ ID NO:

45.

10. 10. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 9, comprising a linker which is any one of SEQ ID NOs: 39 to 44.

11. C H 3 domains are IgG C H 11. The antigen-binding construct or minibody of any one of claims 1 to 10, which is three domains.

12. IgG C H 3 domains, IgG1, IgG2, IgG3, or IgG4 C H 12. The antigen-binding construct or minibody of any one of claims 1 to 11, which is three domains.

13. IgG C H 3 domain is any one of SEQ ID NOs: 65 to 68 H 13. The antigen-binding construct or minibody of any one of claims 1 to 12, comprising three domains.

14. IgG C H 3 domain is any one of SEQ ID NOs: 69-71 H 14. The antigen-binding construct or minibody of any one of claims 1 to 13, comprising three domains.

15. IgG C H 3 domain is any one of SEQ ID NOs: 72 to 82 H 15. The antigen-binding construct or minibody of any one of claims 1 to 14, comprising three domains.

16. IgG C H The IgG1 C3 domain is any one of SEQ ID NOs: 83 to 85. H 16. The antigen-binding construct or minibody of any one of claims 1 to 15, comprising three domains.

17. 2. The antigen-binding construct of claim 1, which is an antibody.

18. 18. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 17, which specifically binds to DLL3.

19. 19. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 18, further comprising a detectable marker.

20. 20. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 19, wherein the detectable marker is a fluorescently detectable marker.

21. 21. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 20, wherein the detectable marker comprises a phototherapy compatible dye.

22. 22. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 21, wherein the detectable marker comprises boron.

23. 23. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 22, wherein the detectable marker is compatible for use with boron neutron capture therapy (BNCT).

24. 24. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 23, wherein the detectable marker is a radioactive label.

25. 25. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 24, wherein the detectable marker is an alpha-emitter radiolabel.

26. 26. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 25, wherein the detectable marker is a beta-emitter radiolabel.

27. 27. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 26, wherein the detectable marker is a positron emitter radiolabel.

28. 28. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 27, wherein the detectable marker comprises lutetium-177.

29. 29. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 28, wherein the detectable marker is a gamma-emitter radiolabel.

30. 30. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 29, wherein the detectable marker is suitable for use with Auger electron spectroscopy.

31. 31. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 30, wherein the detectable marker comprises an isotope.

32. 32. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 31, wherein the detectable marker comprises a bioluminescent compound.

33. 33. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 32, wherein the detectable marker comprises a chemiluminescent compound.

34. 34. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 33, wherein the detectable marker comprises an enzyme.

35. 35. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 34, wherein the detectable marker comprises a metal chelator.

36. 36. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 35, further comprising a therapeutic agent.

37. 37. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 36, wherein the therapeutic agent comprises a therapeutic isotope or ion.

38. 38. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 37, wherein the therapeutic agent is a radiolabel.

39. 39. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 38, wherein the therapeutic agent is an alpha-emitter radiolabel.

40. 40. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 39, wherein the therapeutic agent is a beta-emitter radiolabel.

41. 41. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 40, wherein the therapeutic agent is a positron emitter radiolabel.

42. 42. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 41, wherein the therapeutic agent is a gamma-emitter radiolabel.

43. 43. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 42, wherein the therapeutic agent comprises lutetium-177.

44. 44. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 43, wherein the therapeutic agent comprises a phototherapy compatible dye.

45. 45. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 44, wherein the therapeutic agent comprises boron.

46. 46. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 45, wherein the therapeutic agent is compatible for use with boron neutron capture therapy (BNCT).

47. 47. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 46, wherein the therapeutic agent and / or detectable marker comprises a toxic payload.

48. 48. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 47, which is bispecific.

49. 49. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 48, comprising a monovalent scFv.

50. V from the N-terminus to the C-terminus of the polypeptide L , V H 50. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 49, wherein

51. The order of variable domains from the N-terminus to the C-terminus of a polypeptide is V H , V L 51. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 50, wherein

52. 52. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 51, wherein the minibody is a humanized antigen-binding construct, minibody, or cys-diabody.

53. The humanized antigen-binding construct, minibody, or cys-diabody may be LCDR1, which is SEQ ID NO: 15; LCDR2, which is any one of SEQ ID NOs: 19, 21, or 23; LCDR3, which is SEQ ID NO: 25; HCDR1, which is SEQ ID NO: 27; HCDR2, which is SEQ ID NO: 29; and HCDR3, which is SEQ ID NO: 31 53. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 52, comprising:

54. 54. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 53, having an increased expression yield in mammalian cells compared to an antigen-binding construct, minibody, or cys-diabody comprising SEQ ID NO:

1.

55. 54. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 53, having 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. 54. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 53, having 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. 57. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 56, which 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.

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

59. 59. The antigen-binding construct, minibody, or cys-diabody of claim 57 or 58, which 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. 59. The antigen-binding construct, minibody, or cys-diabody of claim 57 or 58, which 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.

61. 61. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 60, comprising an LFR2 of SEQ ID NO: 35 or 37; and having improved biodistribution compared to an antigen-binding construct, minibody, or cys-diabody comprising an LFR2 that is an LFR2 of SEQ ID NO:

33.

62. Approximately 1×10 -10 62. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 61, having a KD of less than M.

63. Approximately 1×10 -12 63. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 62, having a KD of less than M.

64. an EC50 of 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 50 64. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 63, comprising:

65. an EC50 of about 0.001-0.20 nM, 0.001-0.17 nM, 0.001-0.12 nM, 0.01-0.20 nM, 0.01-0.17 nM, 0.01-0.12 nM, 0.05-0.20 nM, 0.05-0.17 nM, 0.05-0.15 nM, 0.05-0.12 nM, 0.08-0.20 nM, 0.08-0.17 nM, or 0.08-0.12 nM 50 65. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 64, comprising:

66. EC up to approximately 10 nM 50 66. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 65, comprising:

67. 67. A nucleic acid encoding the antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 66.

68. 67. A cell line producing the antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 66.

69. 68. The nucleic acid of claim 67, which is any one of SEQ ID NOs: 51 to 65.

70. 67. A cell line producing the antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 66.

71. 67. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 66; and Detectable Markers Kit including:

72. 67. A kit comprising an antigen-binding construct, minibody, or cys-diabody according to any one of claims 1 to 66; and a chelating agent allowing the incorporation of a detectable marker.

73. 67. A kit comprising an antigen-binding construct, minibody, or cys-diabody according to any one of claims 1 to 66; and a chelating agent allowing for the incorporation of a therapeutic isotope.

74. 67. A kit comprising the antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 66; and a linker allowing the incorporation of a detectable marker.

75. 67. A kit comprising the antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 66; and a linker that allows for the incorporation of a therapeutic isotope.

76. 67. A kit comprising the antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 66; and a detectable marker.

77. applying the antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 66 to a sample; and Detecting the presence or absence of the antigen-binding construct, thereby detecting the presence or absence of DLL3. A method for detecting the presence or absence of DLL3, comprising:

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

79. 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 the subject.

80. 80. The method of any one of claims 77 to 79, wherein detecting binding 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. 81. The method of any one of Claims 77 to 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. 82. The method of any one of claims 77 to 81, wherein the antigen-binding construct, minibody, or cys-diabody is incubated with the sample for no more than 20 hours.

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

84. 84. The method of any one of claims 77-83, wherein an antigen-binding construct, minibody, or cys-diabody is administered to a host, wherein a first quantity of the antigen-binding construct, minibody, or cys-diabody is not bound to DLL3 and a second quantity of the antigen-binding construct, minibody, or cys-diabody is bound to DLL3, and wherein at least about 80% of the first quantity of the antigen-binding construct, minibody, or cys-diabody is cleared within 12 hours.

85. 67. A method of targeting a therapeutic agent to DLL3, comprising administering to a subject the antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 66, wherein the antigen-binding construct is conjugated to the therapeutic agent.

86. LCDR1, which is SEQ ID NO: 15; LCDR2, which is any one of SEQ ID NOs: 19, 21, or 23; LCDR3, which is SEQ ID NO: 25 The variable light chain (V L ) domain; and HCDR1 of SEQ ID NO: 27; HCDR2 of SEQ ID NO: 29; HCDR3 of SEQ ID NO: 31 The variable heavy chain (V H )domain an antigen-binding construct comprising: Therapeutic Agents, Toxic Payloads, and / or Detectable Markers A therapeutic composition that targets DLL3, comprising:

87. A minibody that binds to DLL3, Variable heavy chain (V H ) domains linked to the variable light chain (V L a single-chain variable fragment (scFv) that binds to DLL3 and comprises a V L The domain is LCDR1, which is SEQ ID NO: 15; LCDR2, which is any one of SEQ ID NOs: 19, 21, or 23; LCDR3, which is SEQ ID NO: 25 Including V H The domain is HCDR1, which is SEQ ID NO: 27; HCDR2, which is SEQ ID NO: 29; HCDR3, which is SEQ ID NO: 31 a single chain variable fragment (scFv) comprising: a hinge extension domain containing the IgG1 hinge region; IgG C H 3. Array Mini body including; Therapeutic Agents, Toxic Payloads, and / or Detectable Markers A therapeutic composition that targets DLL3, comprising:

88. A cys-diabody that binds to DLL3, Variable heavy chain (V H ) domains linked to the variable light chain (V L ) domain, L The domain is LCDR1, which is SEQ ID NO: 15; LCDR2, which is SEQ ID NO: 19, 21, or 23; LCDR3, which is SEQ ID NO: 25 Including V H The domain is HCDR1, which is SEQ ID NO: 27; HCDR2, which is SEQ ID NO: 29; HCDR3, which is SEQ ID NO: 31 a cys-diabody comprising a polypeptide comprising a single chain variable fragment (scFv); and Therapeutic Agents, Toxic Payloads, and / or Detectable Markers A therapeutic composition that targets DLL3, comprising:

89. 89. The therapeutic composition of any one of Claims 86 to 88, wherein the detectable marker is a radioactive label.

90. 90. The therapeutic composition of any one of Claims 86 to 89, wherein the detectable marker is an alpha-emitter radiolabel.

91. 91. The therapeutic composition of any one of Claims 86 to 90, wherein the detectable marker is a beta-emitter radiolabel.

92. 92. The therapeutic composition of any one of Claims 86 to 91, wherein the detectable marker is a positron emitter radiolabel.

93. 93. The therapeutic composition of any one of claims 86 to 92, wherein the detectable marker comprises lutetium-177.

94. 85. The antigen-binding construct, minibody, or cys-diabody of any one of claims 1 to 66, or the composition of any one of claims 86 to 93, or the kit of any one of claims 71 to 76, or the method of any one of claims 77 to 85, wherein the therapeutic agent comprises terbium-149, terbium-161, or lead-212.

95. The antigen-binding construct comprises: A single-chain variable fragment (scFv) that binds to DLL3, comprising a variable heavy chain (V H ) domains linked to the variable light chain (V L ) domain-containing single-chain variable fragment (scFv); a hinge domain; and Fc area 94. An antigen-binding construct as defined in any one of claims 1 to 66, or a composition of any one of claims 86 to 93, or a kit of any one of claims 71 to 76, or a method of any one of claims 77 to 85, wherein the construct is an scFv-Fc comprising

96. The variable heavy chain (V) binds to DLL3 and is linked to the Fc region via a hinge domain. H ) domain.

97. Variable heavy chain (V H ) domain, HCDR1, which is SEQ ID NO: 27; HCDR2, which is SEQ ID NO: 29; and HCDR3, which is SEQ ID NO: 31 97. The antigen-binding construct of claim 96, comprising:

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