Antigen binding constructs

Abstract

Provided herein are antigen binding constructs, minibodies, and scFv-Fc fusions. Specifically, in some non-limiting embodiments, antigen binding constructs, minibodies, and scFv-Fc fusions having reduced uptake in healthy organs are provided.

Description

ANTIGEN BINDING CONSTRUCTSREFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 583697, filed September 19, 2023, the disclosure of which is hereby incorporated by reference in their entirety.REFERENCE TO SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled IGNAB 064WO2 SEQLIST.xml, which was created and last modified on September 16, 2024, which is 137,363 bytes in size. The information in the electronic Sequence Listing is hereby incorporated by reference in its entirety.FIELD

[0003] The present disclosure generally relates to antigen binding constructs, minibodies, and scFv-Fc fusions. Specifically, antigen binding constructs, minibodies, and scFv-Fc fusions with modifications to alter biodistribution, including integrin aVp6 specific antigen binding constructs, minibodies, and scFv-Fc fusions.BACKGROUND

[0004] A well-recognized challenge in selecting antibodies and antigen binding fragments as a human clinical agent is finding a product with appropriate biodistribution to meet the clinical need. Monoclonal antibodies typically have long half-lives in blood, such as 2-4 weeks. Smaller agents are often cleared more quickly. But an antigen binding format that accumulates too heavily in an off-target tissue, such as kidney, bone marrow or liver, can do considerable damage to the healthy tissue and may not be suitable for clinical use. Biodistribution of antigen binding fragments is therefore a significant area of research in the early drug discovery phase.SUMMARY

[0005] Some embodiments provided herein are described by way of the following provided embodiments and also provided as possible combinations or overlapping embodiments:

[0006] Provided herein is an antigen binding construct comprising an IgG CH2 having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or a percentage in a range that is defined by any two of the preceding values, identity to an IgG CH2 in SEQ ID NO. 57, wherein at least one mutation in the IgG CH2 reduces uptake of the antigen binding construct by healthy organs as compared to an IgG CH2 sequence in SEQ ID NO. 57, wherein the at least one mutation comprises a substitution of one or more of the following amino acids (EU numbering): E233, D265, E269, D270, E272, D280, E293, and E294, wherein (1) if the at least one mutation comprises a substitution at E233 (EU numbering), then (i) the substitution is with S, V, G, A, Q, N, T, K,R, H, Y, F, W, M, L, or I, optionally the substitution is with S, V, G, A, Q, N, or T, and further optionally the substitution is with S, and / or (ii) the antigen binding construct comprises a second mutation in the IgG CH2 that is different from the substitution at E233 (EU numbering), (2) if the at least one mutation comprises a substitution at D265 (EU numbering), then (i) the substitution is with V, G, Q, N, T, R, P, F, W, M, L, or I, optionally the substitution is with V, G, Q, N, or T, and / or (ii) the antigen binding construct comprises a second mutation in the IgG CH2 that is different from the substitution at D265 (EU numbering), (3) if the at least one mutation comprises a substitution at E269 (EU numbering), then (i) the substitution is with S,V, G, A, T, K, R, H, P, Y, F, W, M, L, or I, optionally the substitution is with S, V, G, A, or T, and further optionally the substitution is with S, and / or (ii) the antigen binding construct comprises a second mutation that is different from the substitution at E269 (EU numbering), (4) if the at least one mutation comprises a substitution at D270 (EU numbering), then (i) the substitution is with S, V, Q, T, K, R, H, P, Y, F, W, L, or I, optionally the substitution is withS, V, Q, or T, and further optionally the substitution is with S, and / or (ii) the antigen binding construct comprises a second mutation that is different from the substitution at D270 (EU numbering), (5) if the at least one mutation comprises a substitution at E272 (EU numbering), then (i) the substitution is with S, V, G, A, Q, T, K, R, H, P, Y, F, W, M, L, or I, optionally the substitution is with S, V, G, A, Q, or T, and further optionally the substitution is with S, and / or (ii) the antigen binding construct comprises a second mutation that is different from the substitution at E272 (EU numbering), (6) if the at least one mutation comprises a substitution at E293 (EU numbering), then (i) the substitution is with V, G, A, Q, N, T, K, R, H, P, Y, F,W, M, L, or I, optionally the substitution is with V, G, A, Q, N, or T, and / or (ii) the antigenbinding construct comprises a second mutation that is different from the substitution at E293 (EU numbering), and / or (7) if the at least one mutation comprises a substitution at E294 (EU numbering), then (i) the substitution is with S, V, G, A, Q, N, T, K, R, H, P, Y, F, W, M, L, or I, optionally the substitution is with S, V, G, A, Q, N, or T, and further optionally the substitution is with S, and / or (ii) the antigen binding construct comprises a second mutation that is different from the substitution at E294 (EU numbering).

[0007] Also provided is a variant antigen binding construct that varies from an original antigen binding construct by having a substitution of at least one surface-exposed, charged amino acid in the original antigen binding construct with an amino acid having a different charge polarity, whereby a localized patch of surface charge comprising the at least one, charged amino acid is disrupted in the variant antigen binding construct, wherein the at least one surface-exposed, charged amino acid is in a heavy chain variable region (VH), a light chain variable region (VL), a hinge region, a heavy chain constant region 2 (CH2) and / or a CH3 of the original antigen binding construct, wherein (1) if the at least one surface-exposed, charged amino acid comprises a positively charged amino acid in a light chain framework region 2 (LFR2) of the VL, then the at least one surface-exposed, charged amino acid comprises: a second charged amino acid outside the LFR2, of the original antigen binding construct, (2) if the at least one surface-exposed, charged amino acid comprises a positively charged amino acid in a hinge region of the original antigen binding construct, then the at least one surface-exposed, charged amino acid comprises: a second charged amino acid outside the hinge region, and / or a negatively charged amino acid in the hinge region, of the original antigen binding construct, (3) if the at least one surface-exposed, charged amino acid comprises E233 (EU numbering) in the CH2 of the original antigen binding construct, then (i) the substitution is with S, V, G, A, Q, N, T, K, R, H, Y, F, W, M, L, or I, optionally the substitution is with S, V, G, A, Q, N, or T, and further optionally the substitution is with S, and / or (ii) the at least one surface-exposed, charged amino acid comprises a second charged amino acid that is different from E233 (EU numbering) in the CH2 of the original antigen binding construct, (4) if the at least one surface-exposed, charged amino acid comprises D265 (EU numbering) in the CH2 of the original antigen binding construct, then (i) the substitution is with V, G, Q, N, T, R, P, F, W, M, L, or I, optionally the substitution is with V, G, Q, N, or T, and / or (ii) the at least one surface-exposed, charged amino acid comprises a second charged amino acid that is differentfrom D265 (EU numbering) in the CH2 of the original antigen binding construct, (5) if the at least one surface-exposed, charged amino acid comprises E269 (EU numbering) in the CH2 of the original antigen binding construct, then (i) the substitution is with S, V, G, A, T, K, R, H, P, Y, F, W, M, L, or I, optionally the substitution is with S, V, G, A, or T, and further optionally the substitution is with S, and / or (ii) the at least one surface-exposed, charged amino acid comprises a second charged amino acid that is different from E269 (EU numbering) in the CH2 of the original antigen binding construct, (6) if the at least one surface-exposed, charged amino acid comprises D270 (EU numbering) in the CH2 of the original antigen binding construct, then (i) the substitution is with S, V, Q, T, K, R, H, P, Y, F, W, L, or I, optionally the substitution is with S, V, Q, or T, and further optionally the substitution is with S, and / or (ii) the at least one surface-exposed, charged amino acid comprises a second charged amino acid that is different from D270 (EU numbering) in the CH2 of the original antigen binding construct, (7) if the at least one surface-exposed, charged amino acid comprises E272 (EU numbering) in the CH2 of the original antigen binding construct, then (i) the substitution is with S, V, G, A, Q, T, K, R, H, P, Y, F, W, M, L, or I, optionally the substitution is with S, V,G, A, Q, or T, and further optionally the substitution is with S, and / or (ii) the at least one surface-exposed, charged amino acid comprises a second charged amino acid that is different from E272 (EU numbering) in the CH2 of the original antigen binding construct, (8) if the at least one surface-exposed, charged amino acid comprises E293 (EU numbering) in the CH2 of the original antigen binding construct, then (i) the substitution is with V, G, A, Q, N, T, K, R,H, P, Y, F, W, M, L, or I, optionally the substitution is with V, G, A, Q, N, or T, and / or (ii) the at least one surface-exposed, charged amino acid comprises a second charged amino acid that is different from E293 (EU numbering) in the CH2 of the original antigen binding construct, and / or (9) if the at least one surface-exposed, charged amino acid comprises E294 (EU numbering) in the CH2 of the original antigen binding construct, then (i) the substitution is with S, V, G, A, Q, N, T, K, R, H, P, Y, F, W, M, L, or I, optionally the substitution is with S, V, G, A, Q, N, or T, and further optionally the substitution is with S, and / or (ii) the at least one surface-exposed, charged amino acid comprises a second charged amino acid that is different from E294 (EU numbering) in the CH2 of the original antigen binding construct.

[0008] Further provided is a variant antigen binding construct that varies from an original antigen binding construct by having a substitution of at least one surface-exposed,charged amino acid in the original antigen binding construct with an amino acid having a different charge polarity, whereby a localized patch of surface charge comprising the at least one, charged amino acid is disrupted in the variant antigen binding construct, wherein the variant antigen binding construct binds to FAP, CD8, CD4, CD3, LFNy, integrin aV06, FOLRa, or PSMA, wherein (1) if the at least one surface-exposed, charged amino acid comprises a positively charged amino acid in a light chain framework region 2 (LFR2) of a VL of the original antigen binding construct, then the at least one surface-exposed, charged amino acid comprises: a second charged amino acid outside the LFR2 of the original antigen binding construct, and / or (2) if the at least one surface-exposed, charged amino acid comprises a positively charged amino acid in a hinge region of the original antigen binding construct, then the at least one surface-exposed, charged amino acid comprises: a second charged amino acid outside the hinge region, and / or a negatively charged amino acid in the hinge region, of the original antigen binding construct.

[0009] Provided herein is an antigen binding construct comprising a CH2 domain comprising (EU numbering): S233, S265, S269, S270, S272S, S280, S293, and / or S294, optionally the CH2 domain comprising S233, S269, and / or S270.

[0010] Also provided is an antigen binding construct comprising: a variable heavy (VH) domain; a variable light (VL) domain; a hinge domain; and a constant heavy domain 3 (CH3), optionally further comprising a CH2 domain, wherein (1) the VH domain comprises (Aho numbering): V13, Q14, A24, D73, S73, S83, N84, S92, and / or T97; (2) the VL domain comprises (Aho numbering): Q29, Q50, Q53K, G110, and / or Si l l; (3) the hinge domain comprises : an upper hinge region comprising the amino acid sequence of SEQ ID NO: 52; or the amino acid sequence of SEQ ID NO: 138; and / or (4) the CH3 comprises: Q355, S356, S376, S380, S382, T382, T414, and S430, optionally (5) the CH2 comprises (EU numbering): S233, S265, S269, S270, S272S, S280, S293, and / or S294, further optionally, the CH2 comprises (EU numbering): S233, S269, and / or S270.

[0011] Provided herein is an antigen binding construct comprising: a means for binding a target antigen; and an IgG CH2 comprising: at least 80% sequence identity to an IgG CH2 in SEQ ID NO. 57; and a substitution at at least one of the following positions (EU numbering): E233, D265, E269, D270, E272, D280, E293, and E294, wherein each substitution is with S, V, G, A, Q, N, T, K, R, H, P, Y, F, W, M, L, or I, optionally wherein theIgG CH2 comprises a substitution at E233, E269, and / or E270, further optionally wherein the IgG CH2 comprises a substitution at E233 / E269 or E233 / D270, further optionally wherein the target antigen is selected from: DLL3, FAP, CD8, CD4, CD3, ZFNy, integrin aVP6, FOLRa, or PSMA.

[0012] Also provided is an antigen binding construct comprising an IgG CH3 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or a percentage in a range that is defined by any two of the preceding values, identity to SEQ ID NO. 77.

[0013] Also provided are pharmaceutical compositions that include an antigen binding construct of the present disclosure; and a therapeutic agent, toxic payload, and / or a detectable marker. Further provided is a method of treating cancer, comprising administering the antigen binding construct or pharmaceutical composition of the present disclosure. Also provided is a method of radiotherapy that includes identifying a subject in need of radiotherapy; and administering to the subject a therapeutically effective amount of the antigen binding construct or pharmaceutical composition of the present disclosure, wherein the antigen binding construct comprises a radionuclide. Provided herein is a method of imaging a subject comprising administering to a subject a composition comprising an effective amount of the antigen binding construct or pharmaceutical composition of the present disclosure.

[0014] Some embodiments herein are directed to an antigen binding construct. In some embodiments, the antigen binding construct disclosed herein comprises: a variable light (VL) domain comprising: a LCDR1 that is SEQ ID NO. 35 or 36; a LCDR2 that is any one of SEQ ID NO. 37; a LCDR3 that is SEQ ID NO. 38 or 39; and a variable heavy (VH) domain comprising: a HCDR1 that is any one of SEQ ID NO. 43-46; a HCDR2 that is any one of SEQ ID NO. 47-48, 119, or 136; and a HCDR3 that is SEQ ID NO. 50 or 51.

[0015] Some embodiments herein are directed to an antigen binding construct. In some embodiments, the antigen binding construct disclosed herein comprises: a variable light (VL) domain comprising: a LCDR1 that is SEQ ID NO. 35; a LCDR2 that is any one of SEQ ID NO. 37; a LCDR3 that is SEQ ID NO. 38; and a variable heavy (VH) domain comprising: a HCDR1 that is SEQ ID NO. 43; a HCDR2 that is SEQ ID NO. 48; and a HCDR3 that is SEQ ID NO. 50

[0016] Some embodiments herein are directed to an antigen binding construct In some embodiments, the antigen binding construct disclosed herein comprises: a variable light (VL) domain comprising: a LCDR1 that is SEQ ID NO. 35; a LCDR2 that is any one of SEQ ID NO. 37; a LCDR3 that is SEQ ID NO. 39; and a variable heavy (VH) domain comprising: a HCDR1 that is SEQ ID NO. 43; a HCDR2 that is SEQ ID NO. 48; and a HCDR3 that is SEQ ID NO. 50

[0017] Some embodiments herein are directed to a single-chain variable fragment- Fc fusion (scFv-Fc) that binds to aVp6. In some embodiments, the scFv-Fc comprises a variable light (VL) domain linked to a variable heavy (Vn) domain, the Vi, domain comprising: a LCDR1 that is SEQ ID NO. 35 or 36; a LCDR2 that is any one of SEQ ID NO. 37; a LCDR3 that is any one of SEQ ID NO. 38-40; and the variable heavy (VH) domain comprising: a HCDR1 that is any one of SEQ ID NO. 43-46; a HCDR2 that is any one of SEQ ID NO. 47- 48,119, or 136; and a HCDR3 that is SEQ ID NO. 49 or 50

[0018] Some embodiments herein are directed to a single-chain variable fragment- Fc fusion (scFv-Fc) that binds to aVp6. In some embodiments, the scFv-Fc comprises a variable light (VL) domain linked to a variable heavy (VH) domain, the V domain comprising: a variable light (VL) domain comprising: a LCDR1 that is SEQ ID NO. 35; a LCDR2 that is any one of SEQ ID NO. 37; a LCDR3 that is SEQ ID NO. 38; and a variable heavy (Vn) domain comprising: a HCDR1 that is SEQ ID NO. 43; a HCDR2 that is SEQ ID NO. 48; and a HCDR3 that is SEQ ID NO. 50.

[0019] Some embodiments herein are directed to a single-chain variable fragment- Fc fusion (scFv-Fc) that binds to aVP6. In some embodiments, the scFv-Fc comprises a variable light (VL) domain linked to a variable heavy (Vn) domain, the VL domain comprising: a variable light (VL) domain comprising: a LCDR1 that is SEQ ID NO. 35; a LCDR2 that is any one of SEQ ID NO. 37; a LCDR3 that is SEQ ID NO. 39; and a variable heavy (VH) domain comprising: a HCDR1 that is SEQ ID NO. 43; a HCDR2 that is SEQ ID NO. 48; and a HCDR3 that is SEQ ID NO. 50.

[0020] Some embodiments herein are directed to a minibody that binds to integrin aVp6. In some embodiments, the minibody comprises: a single-chain variable fragment (scFv) that binds to aVp6, the scFv comprising a variable light (VL) domain linked to a variable heavy (VH) domain, the VL domain comprising: a LCDR1 that is SEQ ID NO. 35 or 36; a LCDR2that is SEQ ID NO. 37; a LCDR3 that is any one of SEQ ID NO. 38-40; and the variable heavy (VH) domain comprising: a HCDR1 that is any one of SEQ ID NO. 43-46; a HCDR2 that is any one of SEQ ID NO. 47-48, 119, or 136; and a HCDR3 that is SEQ ID NO. 49 or 50; a hinge-extension domain comprising a hinge region; and a IgG CH3 sequence.

[0021] Some embodiments herein are directed to a minibody (e.g., a minibody that binds to an antigen target, such as DLL3, FAP, CD8, CD4, CD3, IFNy, FOLRoc, PSMA or integrin aVp6). In some embodiments, the minibody comprises: a single-chain variable fragment (scFv) that binds to aVP6, the scFv comprising a variable light (VL) domain linked to a variable heavy (VH) domain, the VL domain comprising: a LCDR1 that is SEQ ID NO. 35; a LCDR2 that is any one of SEQ ID NO. 37; a LCDR3 that is SEQ ID NO. 38; and the variable heavy (VH) domain comprising: a HCDR1 that is SEQ ID NO. 43; a HCDR2 that is SEQ ID NO. 48; and a HCDR3 that is SEQ ID NO. 50; a hinge-extension domain comprising a hinge region; and a IgG CH3 sequence.

[0022] Some embodiments herein are directed to a minibody that binds to integrin aVp6. In some embodiments, the minibody comprises: a single-chain variable fragment (scFv) that binds to aVp6, the scFv comprising a variable light (VL) domain linked to a variable heavy (VH) domain, the VL domain comprising: a LCDR1 that is SEQ ID NO. 35; a LCDR2 that is any one of SEQ ID NO. 37; a LCDR3 that is SEQ ID NO. 39; and the variable heavy (VH) domain comprising: a HCDR1 that is SEQ ID NO. 43; a HCDR2 that is SEQ ID NO. 48; and a HCDR3 that is SEQ ID NO. 50; a hinge-extension domain comprising a hinge region; and a IgG CH3 sequence.

[0023] Some embodiments herein are directed to a minibody that binds to integrin uVp6. In some embodiments, the minibody comprises: a single-chain variable fragment (scFv) that binds to aVp6, the scFv comprising a variable light (VL) domain linked to a variable heavy (VH) domain, the VL domain comprising: a LCDR1 that is SEQ ID NO. 36; a LCDR2 that is any one of SEQ ID NO. 37; a LCDR3 that is SEQ ID NO. 40; and the variable heavy (VH) domain comprising: a HCDR1 that is SEQ ID NO. 46; a HCDR2 that is SEQ ID NO. 119; and a HCDR3 that is SEQ ID NO. 50; a hinge-extension domain comprising a hinge region; and a IgG CH3 sequence.

[0024] Some embodiments herein are directed to a minibody, scFv or scFv-Fc fusion comprising: a variable light (VL) domain of SEQ ID NO. 1 linked to a variable heavy (VH) domain of SEQ ID NO. 18.

[0025] Some embodiments herein are directed to a minibody, scFv or scFv-Fc fusion comprising: an original antigen binding construct which is a variable light (VL) domain of SEQ ID NO. 1 linked to a variable heavy (VH) domain of SEQ ID NO. 18, wherein the minibody, scFv or scFv-Fc fusion further comprises a disrupted positive patch and / or a disrupted negative patch relative to the original antigen binding construct.

[0026] Also provided are pharmaceutical compositions that include an antigen binding construct of the present disclosure; and a therapeutic agent or toxic payload, for treating cancer, and / or a detectable marker. Also provided is a pharmaceutical composition that includes an antigen binding construct of the present disclosure that further includes a radionuclide, for use in radiotherapy. Also provided is a pharmaceutical composition that includes an antigen binding construct of the present disclosure and a detectable marker for imaging (e.g., non-invasively imaging) a subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 is an illustrative representation of some non-limiting embodiments of an antibody and antigen binding fragments thereof.

[0028] FIG. 2 is an illustrative representation of some non-limiting embodiments of a single-chain variable fragment (scFv).

[0029] FIG. 3 is an illustrative representation of some non-limiting embodiments of a minibody.

[0030] FIG. 4 is an illustrative representation of some non-limiting embodiments of a single-chain variable fragment (scFv) Fc fusion (scFv-Fc).

[0031] FIG. 5 is flow chart illustrating some non-limiting embodiments of a method for reducing tumor size.

[0032] FIG. 6 is flow chart illustrating some non-limiting embodiments of a method of treating cancer.

[0033] FIG. 7 depicts an alignment of some non-limiting embodiments of aVp6 VL domain sequences disclosed herein.

[0034] FIG. 8 depicts an alignment of some non-limiting embodiments of aVp6 VH domain sequences disclosed herein.

[0035] FIG. 9 depicts some non-limiting embodiments of an alignment of IgG CH2 domain sequences disclosed herein.

[0036] FIG. 10 depicts some non-limiting embodiments of an alignment of IgG CH3 domain sequences disclosed herein.

[0037] FIG. 11 depicts an alignment of some non-limiting embodiments of Fc domain sequences disclosed herein.

[0038] FIG. 12 shows some non-limiting embodiments of a polypeptide sequence of LCDR and HCDR sequences disclosed herein.

[0039] FIG. 13 shows some non-limiting embodiments of a polypeptide sequence of aVp6 binding antibody VH and VL domains. (SEQ ID NO. 1 and 18).

[0040] FIG. 14 shows some non-limiting embodiments of a polypeptide sequence of antigen binding minibodies. (SEQ ID NO. 103-108).

[0041] FIG. 15 shows some non-limiting embodiments of a polypeptide sequence of a VH and VL with embodiments of HCDR and LCDR sequences highlighted.

[0042] FIG. 16 shows some non-limiting embodiments of an IAB56M2-14 variable region numbered according to the Aho numbering scheme.

[0043] FIG. 17 shows some non-limiting embodiments of minibody scaffolds and mutations in the scaffold that reduce uptake of the minibody by healthy organs.

[0044] FIG. 18A shows some non-limiting embodiments of charge distribution of the scFv domain of IAB56M2-14 minibodies of the present disclosure.

[0045] FIG. 18B shows some non-limiting embodiments of charge distribution of the scFv domain of IAB56M2-19 minibodies of the present disclosure.

[0046] FIG. 18C shows some non-limiting embodiments of charge distribution of the scFv domain of IAB56M2-27 minibodies of the present disclosure.

[0047] FIG. 19A shows some non-limiting embodiments of differences in charge distribution of the CH3 domain of minibodies of the present disclosure.

[0048] FIG. 19B shows some non-limiting embodiments of a 3D model of scFv IAB56M2-14 indicating the location of exemplary positive patches and negative patches over the exposed surface. Black: positively charged potential; White: negatively charged potential.

[0049] FIG. 19C shows some non-limiting embodiments of a 3D model of scFv IAB56M2-14 indicating the location of exemplary positive patches and negative patches over the exposed surface. Black: positively charged potential; White: negatively charged potential.

[0050] FIG. 19D shows some non-limiting embodiments of a 3D model of scFv IAB56M2-14 indicating the location of exemplary positive patches and negative patches over the exposed surface. Black: positively charged potential; White: negatively charged potential.

[0051] FIG. 20 shows some non-limiting embodiments of a polypeptide sequence of antigen binding minibodies. (SEQ ID NO. 109-118).

[0052] FIG. 21 shows some non-limiting embodiments of a polypeptide sequence of antigen binding scFv-Fc fusions. (SEQ ID NO. 72-81).

[0053] FIG. 22A shows some embodiments of ECso determination of parental murine antibody 12B3 by ELISA.

[0054] FIG. 22B shows some embodiments of parental murine antibody 12B3 binding to A431 cells.

[0055] FIG. 22C shows some embodiments of parental murine antibody 12B3 binding to HT29 cells.

[0056] FIG. 22D shows some embodiments of parental murine antibody 12B3 binding to BxPC3 cells.

[0057] FIG. 22E shows some embodiments of parental murine antibody 12B3 binding to OVMANA cells.

[0058] FIG. 22F shows some embodiments of parental murine antibody 12B3 binding kinetics determined using biolayer interferometry.

[0059] FIG. 23 shows some embodiments of internalization of parental murine antibody 12B3 in BT20, A431, BxPC3, and HT-29 cells.

[0060] FIG. 24 shows some embodiments of parental murine antibody 12B3 nonblocking of aVp6 binding to latency associated protein (LAP).

[0061] FIG. 25 shows some embodiments of parental murine antibody 12B3 nonblocking aVp6 binding to fibronectin.

[0062] FIG. 26 shows some embodiments of parental murine antibody 12B3 crossreactivity with non- aVp6 integrins.

[0063] FIG. 27 is a graph and corresponding table showing some embodiments of ECso determinations of antigen binding minibodies by ELISA.

[0064] FIG. 28 is a graph and corresponding table showing some embodiments of ECso determinations of antigen binding minibodies by ELISA.

[0065] FIG. 29 is a graph and corresponding tables showing some embodiments of equilibrium dissociation constant determinations of antigen binding minibodies.

[0066] FIG. 30A is a graph showing some embodiments of ECso determinations of antigen binding minibodies in a BxPC3 pancreatic adenocarcinoma cell line.

[0067] FIG. 30B is a graph showing some embodiments of ECso determinations of antigen binding minibodies in an A431 epidermoid carcinoma cell line.

[0068] FIG. 30C is a graph showing some embodiments of ECso determinations of antigen binding minibodies in a BT20 breast carcinoma cell line.

[0069] FIG. 30D is a graph showing some embodiments of ECso determinations of antigen binding minibodies in a HT29 colorectal adenocarcinoma cell line.

[0070] FIG. 31 is a graph showing some embodiments of ECso determinations of antigen binding minibodies by ELISA.

[0071] FIG. 32 is a graph and corresponding table showing some embodiments of ECso determinations of antigen binding minibodies by ELISA.

[0072] FIG. 33 is a graph and corresponding tables showing some embodiments of equilibrium dissociation constant determinations of antigen binding minibodies.

[0073] FIG. 34 is a graph and corresponding tables showing some embodiments of equilibrium dissociation constant determinations of antigen binding minibodies.

[0074] FIG. 35 is a graph and corresponding table showing some embodiments of ECso determinations of antigen binding minibodies by flow cytometry.

[0075] FIG. 36A is a graph showing some embodiments of quantification of minibody in-vivo uptake in kidneys.

[0076] FIG. 36B is a graph showing some embodiments of quantification of minibody in-vivo uptake in blood, lungs, muscle, bone, heart, stomach, small intestine, large intestine, and pancreas.

[0077] FIG. 36C is a graph showing some embodiments of quantification of minibody in-vivo uptake.

[0078] FIG. 36D is a graph showing some embodiments of quantification of minibody in-vivo uptake in tumor xenografts in mice.

[0079] FIG. 36E is a graph showing some embodiments of quantification of the ratio between minibody uptake in tumor and kidney organs.

[0080] FIG. 37A is a graph showing some embodiments of differences in tumor size following treatment with (squares) or without (circles) antigen binding minibodies.

[0081] FIG. 37B is a graph showing some embodiments of quantification of differences in tumor size following treatment with or without antigen binding minibodies.

[0082] FIG. 37C is a Kaplan-Meier plot showing differences in survival in animals treated with (squares) or without (circle) antigen binding minibodies.

[0083] FIG. 38 shows some embodiments of the sequence of human IgGl Fc with EU numbering.

[0084] FIG. 39A shows some non-limiting embodiments of modeling of surface electrostatic potential of a IAB56FC2-54 Fc domain.

[0085] FIG. 39B shows some non-limiting embodiments of modeling of surface electrostatic potential of a IAB56FC2-58 Fc domain.

[0086] FIG. 40 is a graph showing quantification of scFv-Fc fusion in-vivo uptake in blood, liver, kidney, spleen, and tumor with or without electrostatic mutations.

[0087] FIG. 41A is a graph and corresponding table showing some non-limiting embodiments of ECso determinations of antigen binding minibodies by ELISA.

[0088] FIG. 41B is a graph and corresponding table showing some non-limiting embodiments of ECso determinations of antigen binding minibodies by flow cytometry.

[0089] FIG. 42 shows some non-limiting embodiments of a polypeptide sequence of antigen binding minibodies. (SEQ ID NO. 133-135).DETAILED DESCRIPTION

[0090] An attractive modality for therapeutic agents towards cell surface expressed targets is to use antibodies, antibody fragments, or antibody constructs (comprising selected domains of antibodies which may be re-ordered or re-organized and mutated at specific sequence locations). The commonality of these biologic agents is that they can be designed to be extremely selective for specific targets, historically termed antigens. A highly specificantigen binding domain can be found in each agent. The agents are sometimes known as antigen binding fragments. ImaginAb, Inc. (Inglewood CA) has been active in development of antigen binding fragments for diagnostics and / or therapeutics as may be seen in WIPO patent publications WO 2011 / 069019, WO 2014 / 164553, WO 2013 / 188693, WO 2017 / 027325, WO 2018 / 147960, WO 2020 / 069433, WO 2019 / 236684, and WO 2023 / 283643.

[0091] Some embodiments herein are directed to an antigen binding construct (e.g., antibody or antigen binding fragment thereof). In some embodiments, the antigen binding construct (e.g., antibody or antigen binding fragment thereof) binds DLL3, FAP, CD8, CD4, CD3, IFNy, FOLRa, or aVp6. In some embodiments, the antigen binding construct (e.g., antibody or antigen binding fragment thereof) is a scFv, minibody, nanobody, or scFv-Fc fusion. In some embodiments, the antigen binding construct comprises: a variable light (VL) domain comprising: a LCDR1 that is SEQ ID NO. 35 or 36; a LCDR2 that is SEQ ID NO. 37; a LCDR3 that is any one of SEQ ID NO. 38-40; and a variable heavy (VH) domain comprising: a HCDR1 that is any one of SEQ ID NO. 43-46; a HCDR2 that is any one of SEQ ID NO. 47-48, 119, or 136; and aHCDR3 that is SEQ ID NO. 49 or 50. In some embodiments, the antigen binding construct further comprises an IgG CH2 sequence. In some embodiments, the antigen binding construct comprises an IgG CH3 sequence. In some embodiments, the antigen binding construct comprises a Fc domain. In some embodiments, the antigen binding construct comprises a hinge-extension domain comprising a hinge region. In some embodiments, the antigen binding construct comprises a linker. In some embodiments, the scFv-Fc comprises a signal peptide.

[0092] Some embodiments herein are directed to a minibody that binds to DLL3, FAP, CD8, CD4, CD3, IFNy, FOLRa, or integrin aVp6. In some embodiments, the minibody comprises a single-chain variable fragment (scFv) that binds to DLL3, FAP, CD8, CD4, CD3, IFNy, FOLRa, or aVp6, the scFv comprising a variable light (VL) domain linked to a variable heavy (VH) domain, the VL domain comprising: a LCDR1 that is SEQ ID NO. 36; a LCDR2 that is any one of SEQ ID NO. 37; a LCDR3 that is SEQ ID NO. 40; and the variable heavy (VH) domain comprising: a HCDR1 that is SEQ ID NO. 46; a HCDR2 that is SEQ ID NO. 119; and a HCDR3 that is SEQ ID NO. 50; a hinge-extension domain comprising a hinge region; and a IgG CH3 sequence. In some embodiments, the antigen binding construct comprises a linker. In some embodiments, the scFv-Fc comprises a signal peptide.

[0093] Some embodiments herein are directed to a minibody, scFv or scFv-Fc fusion. In some embodiments, the minibody, scFv or scFv-Fc fusion comprises: a variable light (VL) domain of SEQ ID NO. 1 linked to a variable heavy (VH) domain of SEQ ID NO. 18.

[0094] Some embodiments herein are directed to a minibody, scFv or scFv-Fc fusion. In some embodiments, the minibody, scFv or scFv-Fc fusion comprises: an original antigen binding construct which is a variable light (VL) domain of SEQ ID NO. 1 linked to a variable heavy (VH) domain of SEQ ID NO. 18, wherein the minibody, scFv or scFv-Fc fusion further comprises a disrupted positive patch and / or a disrupted negative patch relative to the original antigen binding construct. Definitions

[0095] All terms have their ordinary and customary meaning as understood by one of ordinary skill in the art, in view of the present disclosure.

[0096] The term “antigen binding construct” includes all varieties of antibodies, including binding fragments thereof. Further included are constructs that include 1, 2, 3, 4, 5, and / or 6 CDRs. In some embodiments, tandem scFvs can be provided, which can provide two arms with bivalent binding. In some embodiments, these CDRs can be distributed between their appropriate framework regions in a traditional antibody. In some embodiments, the CDRs can be contained within a heavy and / or light chain variable region. In some embodiments, the CDRs can be within a heavy chain and / or a light chain. In some embodiments, the CDRs can be within a single peptide chain. Unless otherwise denoted herein, the antigen binding constructs described herein bind to the noted target molecule. The term “target” or “target molecule” denotes the protein to which the antigen binding construct binds. Examples of target proteins are known in the art.

[0097] The term “antibody” includes, but is not limited to, genetically engineered or otherwise modified forms of immunoglobulins, such as intrabodies, chimeric antibodies, fully human antibodies, humanized antibodies, antibody fragments, single-chain variable fragment (scFv), and heteroconjugate antibodies (for example, bispecific antibodies, diabodies, triabodies, tetrabodies, and nanobodies, etc.). The term “antibody” includes minibodies and diabodies. The term “antibody” includes a polypeptide of the immunoglobulin family or a polypeptide comprising fragments of an immunoglobulin that is capable of noncovalently, reversibly, and in a specific manner binding a corresponding antigen. Anexemplary antibody structural unit comprises a tetramer. In some embodiments, a full-length antibody can be composed of two identical pairs of polypeptide chains, each pair having one “light” and one “heavy” chain (connected through a disulfide bond). The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, hinge, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. For full length chains, the light chains are classified as either kappa or lambda. For full length chains, the 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. The terms variable light chain (VL) and variable heavy chain (VH) refer to these regions of light and heavy chains respectively. As used in this application, an “antibody” encompasses all variations of antibody and fragments thereof. Also, the term “antibody” includes camelid derived immunoglobulins like single heavy-chain antibodies and nanobodies®. Thus, within the scope of this concept are full length antibodies, chimeric antibodies, humanized antibodies, single chain antibodies (scFv), Fab, Fab', and multimeric versions of these fragments (for example, F(ab')2) with the same binding specificity, scFv-Fc, single domain fragments (e.g., nanobodies®), peptibodies, nanobodies®, nanobody®-Fc, minibodies, and diabodies. In some embodiments, the antibody binds specifically to a desired target.

[0098] The term "complementarity-determining domains" or "complementaritydetermining regions ("CDRs") interchangeably refer to the hypervariable regions of VL and VH. The CDRs are the target molecule-binding site of the antibody chains that harbors specificity for such target molecule. In some embodiments, there are three CDRs (CDR1-3, numbered sequentially from the N-terminus) in each VL and / or VH, constituting about 15-20% of the variable domains. The CDRs are structurally complementary to the epitope of the target molecule and are thus directly responsible for the binding specificity. The remaining stretches of the VL or VH, the so-called FRs, exhibit less variation in amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4. W.H. Freeman & Co., New York, 2000).

[0099] The positions of the CDRs and framework regions can be determined using various well known definitions in the art, for example, Kabat (Wu, T. T. et al., “An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains andtheir 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); Chotia (Chothia C. et al., “Canonical structures for the hypervariable regions of immunoglobulins,” I. Mol. Biol., Vol. 196, No. 4, pp. 901-917, 1987; Chothia C. et al., “Conformations of immunoglobulin hypervariable regions,” Nature, Vol. 342, No. 6252, pp. 877-883, 1989; Chothia C. et al., "Structural repertoire of the human VH segments," I. Mol. Biol., Vol. 227, No. 3, pp. 799-817, 1992; Al- Lazikani B. et al., “Standard conformations for the canonical structures of immunoglobulins,” J. Mol. Biol., Vol. 273, No. 4, pp. 927-748, 1997), ImMunoGeneTics database (IMGT) (on the worldwide 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, M. P. et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev. Comp. Immunol., Vol. 27, No. 1, pp. 55-77, 2003; Brochet, X. et al., “IMGT / V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis,” Nucleic Acids Res., Vol. 36 (Web Server Issue), pp. W503-508, 2008); AbM (Martin, A. C. et al., “Modeling antibody hypervariable loops: a combined algorithm.” Proc. Natl. Acad. Sci. U.S.A., Vol. 86, No. 23, pp. 9268-9272, 1989); Aho (Honegger A, 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 RLI. A new clustering of antibody CDR loop conformations. J Mol Biol. (2011) 406:228-56); the contact definition (MacCallum, R. M. et al., “Antibody-antigen interactions: contact analysis and binding site topography,” J. Mol. Biol., Vol. 262, No. 5, pp. 732-745, 1996), and / or the automatic modeling and analysis tool (Honegger, A. et al., Accessible on the world wide web at bioc.uzh.ch / plueckthun / antibody / Numbering / ). In some embodiments, the polypeptide is numbered from the beginning of the polypeptide signal sequence. In some embodiments, the polypeptide is numbered according from the beginning of the polypeptide and not includingthe signal sequence. It is understood that K47, Q50 and K53 in Aho numbering correspond to K45, Q48 and K51 in IMGT numbering, respectively.

[0100] An "antibody variable light chain" or an "antibody variable heavy chain" as used herein refers to a polypeptide comprising the VL or VH, respectively. The endogenous VL is encoded by the gene segments V (variable) and J (junctional), and the endogenous VH by V, D (diversity), and J. Each of VL or VH includes the CDRs as well as the framework regions. In this application, antibody variable light chains and / or antibody variable heavy chains may, from time to time, be collectively referred to as "antibody chains." These terms encompass antibody chains containing mutations that do not disrupt the basic structure of Vi. or VH, as one skilled in the art will readily recognize. In some embodiments, full length heavy and / or light chains are contemplated. In some embodiments, only the variable region of the heavy and / or light chains are contemplated as being present.

[0101] The term “hinge” denotes at least a part of a hinge region for an antigen binding construct, such as an antibody, a minibody, a scFv-Fc, or a nanobody®-Fc. A hinge region can include a combination of the 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. Native IgGl, IgG2, and IgG4 antibodies have hinge regions having of 12-15 amino acids. IgG3 has a hinge region, having 62 amino acids, including 21 prolines and 11 cysteines. The functional hinge region of naturally occurring antibodies, deduced from crystallographic studies, extends from amino acid residues 216-237 of the IgGl H chain (EU numbering; ref. 12) and includes a small segment of the N terminus of the CH2 domain in the lower hinge, with the lower hinge being the N terminus of CH2 domain. The hinge can be divided into three regions; the "upper hinge," the "core," and the "lower hinge".

[0102] The term “artificial” or “non-natural” when modifying a hinge (or a subpart thereof) denotes that the sequence in question is not present, in the noted state, in nature. In the present context the hinges have been altered from their native state, so that their sequences are no longer those found in wild-type antibodies. As will be appreciated by those of skill in the art, minibodies do not naturally occur in nature, and thus, any construct which is a minibody construct is also not found in nature. In some embodiments, any of the hinge subparts or full hinge sequences can be artificial hinge sequences, as long as the sequence (or resulting combination for the hinge) does not occur in nature.

[0103] The term “full hinge region” or “entire hinge region” denotes the presence of the entire upper, core, and lower hinge regions as a single construct. The upper, core, and lower regions can be positioned immediately adjacent to one another, or additional residues can be added between, or N- or C-terminal to the regions. In some embodiments, the native lower hinge can be replaced with an extension sequence. In some embodiments, one can combine a native lower hinge with the extension sequence. In some embodiments, an extension or other set of sequences can be added after the upper and / or core sequences.

[0104] The phrase “effective hinge region” denotes that an adequate amount of part of at least one of the upper, core and lower hinge regions is present to allow the hinge region to be effective for its intended purpose. Thus, the phrase encompasses variants of hinge regions and fragments of the various hinge regions. In some embodiments, the function of the hinge region is one or more of the following: to link the scFv with the Cu3 domain, provide flexibility and spacing for the two scFvs to bind to the target properly, to link two half molecules together, to provide overall stability to the molecule, and / or to provide a site for site-specific conjugation due to its solvent exposure. In some embodiments, the hinge should be close to natural as to reduce potential immunogenicity. In some embodiments, the upper hinge provides flexibility to scFv (starts at residue 216 in native IgGs), the middle hinge provides stability, and the lower hinge mediates flexibility to Cn3 (starts at residue 231 in native IgGs).

[0105] The term “upper hinge” denotes the first part of the hinge that starts at the end of the scFv. The upper hinge includes the amino acids from the end of the scFv up to, but not including the first cysteine residue in the core hinge. As above, the term “effective upper hinge” denotes that enough of the sequence is present to allow the section to function as an upper hinge; the term encompasses functional variants and fragments of the designated hinge section.

[0106] The term “core hinge” denotes the second part of the hinge region that is C- terminal to the upper hinge. The core hinge contains the inter-chain disulfide bridges and a high content of prolines. As above, the term “effective core hinge” denotes that enough of the sequence is present to allow the section to function as a core hinge; the term encompasses functional variants and fragments of the designated hinge section.

[0107] The term “lower hinge” denotes the third part of the hinge region that is C terminal to the core hinge. In the context of a minibody or antibody fragment, the lower hingeconnects to the CH3 domain. As above, the term “effective lower hinge” denotes that enough of the sequence is present to allow the section to function as a lower hinge; the term encompasses functional variants and fragments of the designated hinge section. The term “lower hinge” as used herein can encompass various amino acid sequences including naturally occurring IgG lower hinge sequences and artificial extension sequences in place of one another or a combination thereof provided herein. In some embodiments, the various extensions can be considered to be a lower hinge region in its entirety or a replacement.

[0108] Antibodies can exist as intact immunoglobulins or as a number of fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab' which itself is a light chain (VL-CL) joined to VH-CH1 by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into an Fab' monomer. The Fab' monomer is a Fab with part of the hinge region. (Paul, W. E., “Fundamental Immunology,” 3d Ed., New York: Raven Press, 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term "antibody," as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (for example, single chain Fv) or those identified using phage display libraries (see, for example, McCafferty, J. et al., “Phage antibodies: filamentous phage displaying antibody variable domains,” Nature, Vol. 348, No. 66301, pp. 552-554, 1990).For preparation of monoclonal or polyclonal antibodies, any technique known in the art can be used (see, for example, 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. l, 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, J. S., “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 to polypeptides of the present disclosure. Also, transgenic mice, or other organisms such as other mammals, may be used to express fully human monoclonal antibodies. Furthermore, E. Coli or yeast may be used to express and manufacture recombinant antibodies and antibody fragments (Simmons L. C., Reilly D., Klimowski L., Shantha Raju T., Meng G., Sims P., Hong K., Shields R. L., Damico L. A., Rancatore P., Yansura D. G.; Expression of full-length immunoglobulins in Escherichia coli: rapid and efficient production of aglycosylated antibodies, J Immunol Methods. 2002 May l;263(l-2): 133-47; Kulagina N, Besseau S, Godon C, Goldman G. H., Papon N., Courdavault V., Yeasts as biopharmaceutical production platforms; Front. Fungal Biol., 22 September 2021). Alternatively, phage display and yeast display technologies can be used to identify high affinity binders to selected antigens (see, for example, McCafferty et al., supra; Marks, J. D. et al., “By-passing immunization: building high affinity human antibodies by chain shuffling,” Biotechnology (N. Y.), Vol. 10, No. 7, pp. 779-783, 1992; Feldhaus M. J., Siegel R. W ., Yeast display of antibody fragments: a discovery and characterization platform, J Immunol Methods, 2004 Iul;290(l-2):69-80). Alternatively, antibodies can be produced through B-cell screening technologies from human hosts (Pedrioli A., Oxenius A., Single B cell technologies for monoclonal antibody discovery, Trends in Immunology, (2021) volume 42, issue 12, pl 143- 1158). Furthermore, antibodies can be derived from immunization of camelid animals or screening of camelid phage libraries (Harmsen M. M., De Haard H. I., Properties, production, and applications of camelid single-domain antibody fragments, Appl Microbiol Biotechnol. 2007; 77(1): 13-22). Alternatively, antibodies can be derived from in-silico screening simulations through 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).

[0109] 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 which 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 can be employed. Humanization can be essentially performed following the method of Winter and co-workers(see, for example, 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), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. 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.

[0110] The term "Fc region" or “Fc domain” or “Fc” denotes a 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 reduces an effector function, e.g., FcyR binding, and / or binding to the Fc neonatal receptor (FcRn), etc ). The Fc region of an immunoglobulin (e.g., IgG) generally comprises two constant domains, CH2 and CH3. The Fc region can include a hinge region or sequence, as described herein. An Fc region can be present in dimer or monomeric form. In some embodiments, the Fc region is a human Fc region, or a variant thereof.[0U1] A "chimeric antibody" is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and / or species, or an entirely different molecule which confers new properties to the chimeric antibody, for example, an enzyme, toxin, hormone, growth factor, and drug; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.

[0112] The term “antibody fragment” includes but is not limited to one or more antigen binding fragments of antibodies alone or in combination with other molecules, including, but not limited to Fab', F(ab')2, Fab, Fv, rlgG (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 chain and of the light chain of a traditional two chain antibody have been joined to form one chain.

[0113] The term “artificial” or “non-natural” when modifying a CDR or FR (or a subpart thereof) denotes that the sequence in question is not present, in the noted state, in nature. In the present context the CDRs or FRs have been altered from their native state, so that their sequences are no longer those found in wild-type antibodies. As will be appreciated by those of skill in the art, minibodies and cys-diabodies do not naturally occur in nature, and thus, any construct which is a minibody or a cys-diabody construct is also not found in nature. This also applies to at least some of the constructs found in and / or incorporating the sequences of any of the CDR or FR sequence tables provided herein. In some embodiments, any of the CDR or FR sequences in the figures or tables can be artificial CDR or FR sequences, as long as the sequence (or resulting combination for the CDR or FR) does not occur in nature.

[0114] A “minibody” is an antibody format that has a smaller molecular weight than the full-length antibody while maintaining the bivalent binding property against an antigen. Because of its smaller size, absence of CH2 domain that binds Fc-gamma and FcRn receptors, absence of glycosylation, the minibody has a faster clearance from the system and potentially enhanced penetration when targeting tumor tissue. With the ability for strong targeting combined with rapid clearance, the minibody is advantageous for diagnostic imaging and delivery of radioactive payloads for which prolonged circulation times may result in adverse patient dosing or dosimetry. In some embodiments, it can also be advantageous for delivery of a cytotoxic payload due to the above-mentioned features such as tumor penetration and faster clearance. A “minibody” as described herein, encompasses a homodimer, wherein each monomer is a single-chain variable fragment (scFv) linked to a human IgG CH3 domain by a hinge sequence. In some embodiments, a minibody is a bivalent or bispecific, covalently bound homodimer of ~80 kDa. In some embodiments, each monomer (half-molecule) is comprised of a variable heavy (VH) domain linked to the corresponding variable light (VL) domain by an approximate 15-18 amino acid Gly-Ser-rich linker sequence. As used herein, “minibody” and “antigen binding minibody” are used interchangeably.

[0115] The term “extension sequence” (e.g., in a diabody context) denotes a region that connects a first VH domain to a second VH domain, or a first VL to a second VL domain, in for example, a diabody. Extension sequences can connect the domains through the C- terminus of each domain. In some embodiments, extension sequences connect the domains through covalent bonds. In some embodiments, the extension sequence will include one or more cysteine, allowing for one or more disulfide bonds to be formed between two such extension sequences. A non-limiting example of an extension sequence includes -(Gly)2-(Cys). In some embodiments, the extension sequence includes 1, 2, 3, or more cysteines per monomer chain. While the extension sequence will be towards the C-terminus of the constructs, it need not be the absolute last amino acid in the variable domain. That is, the extension sequence can be positioned slightly N-terminal to the C-terminus. For example, the extension sequence can be placed within the 10 amino acids of the C-terminus of the monomer. Similarly, additional sequence can be placed between the native C-terminus and where the extension sequence starts. The extension sequence can connect VH to VH or VL to VL through a disulfide bond.

[0116] As used herein, “pharmaceutically acceptable” has its plain and ordinary meaning as understood in light of the specification and refers to carriers, excipients, and / or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed or that have an acceptable level of toxicity. A “pharmaceutically acceptable” “diluent,” “excipient,” and / or “carrier” as used herein have their plain and ordinary meaning as understood in light of the specification and are intended to include any and all solvents, dispersion media, coatings, antibacterial or antifungal agents, isotonic or absorption delaying agents, compatible with administration to humans, primates, cats, dogs, or other vertebrate hosts. Typically, a pharmaceutically acceptable diluent, excipient, and / or carrier is a diluent, excipient, and / or carrier approved by a regulatory agency of a Federal, a state government, or other regulatory agency, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans as well as non-human mammals, such as cats and dogs. The term diluent, excipient, and / or “carrier” can refer to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Such pharmaceutical diluent, excipient, and / or carriers, which can be incorporated in 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 solutions or aqueous dextrose and glycerol solutions can be employed as liquid diluents, excipients, and / or carriers. Suitable pharmaceutical diluents and / or excipients, which can be incorporated in 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. The physiologically acceptable carrier may also comprise 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 dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming counterions such as sodium, and nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), PLURONIC® or preservatives such as an essential oil, methyl paraben, propyl paraben, or sodium salt of parabens. In some embodiments, the preservative is bronidiol. The composition, if desired, can also contain minor amounts of wetting, bulking, emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, sustained release formulations and the like. The formulation should suit the mode of administration.

[0117] Additional excipients with desirable properties include but are not limited to preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizing agents, 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 any combination thereof. In some embodiments, the formulation includes an at least one agent that acts to reduce radiolysis (also known as “radioprotectors”) or is a kidney protecting agent. Non-limiting examples of radiolysis reducing agents include Gentisic acid, Acetylcholine, AET, ACE inhibitors, acteoside, alpha-tocopherol acetate, amifostine, ascorbic acid, aspirin, atorvastatin, beta-carotene, Bowman-Birk proteinase inhibitor, Caffeic acid, Captopril,carbaminoyl choline, Carvacrol, Celecoxib, coenzyme Q10, COX2 inhibitors / NSAIDs, curcumin, cysteine, cysteamine, cystamine, dendrodine analog, Dithiolthione, Dopamine, enalapril, epigallocatechin-3 -gallate, Epinephrine, 17-P-estradiol, GANRA-5, Genistein, green tea abstract, growth factors, guanine nucleotides, Halofuginone, Hmg-CoA reductase inhibitors (statins), heroin, histamine, ibuprofen, inapoyl-E-glucoside, Isoflavone, isofraxidin, kukoamine A, lactoferrin amifostine, lipoic acid, lovastatin, luteolin-7-O-(2-apiosyl)- glucoside, 2-mercaptoethylguanidine, melatonin, methacholine, morphine, N-acetyl cysteine, Oltipraz, palifermin, phenethyl ester, 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, tocopherols, vitamin C, vitamin E, watermelon juice, black grape juice, and thiols such as glutathione. Kidney protecting agents include free lysine, arginine, probenecid, gelofusin, and other compositions. Some excipients may be in residual amounts or contaminants from the process of manufacturing, including but not limited to serum, albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde, glutaraldehyde, P -propiolactone, gelatin, cell debris, nucleic acids, peptides, amino acids, or growth medium components or any combination thereof. The amount of the excipient may be found in the composition at a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w / w or any percentage by weight in a range defined by any two of the aforementioned numbers.

[0118] As used herein, a “carrier” has its plain and ordinary meaning as understood in light of the specification and refers to a compound, particle, solid, semi-solid, liquid, or diluent that facilitates the passage, delivery and / or incorporation of a compound to cells, tissues and / or bodily organs.

[0119] As used herein, a “diluent” has its plain and ordinary meaning as understood in light of the 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 bulk of a potent drug whose mass is too small for manufacture and / or administration. It may also be a liquid for the dissolution of a drug to be administered by injection, ingestion or inhalation. A common form of diluent in the art is abuffered aqueous solution such as, without limitation, phosphate buffered saline that mimics the composition of human blood.

[0120] The term “target molecule dependent disorder” “or “target molecule associated disorder” includes any disorder in which the target molecule plays a role in the disorder itself. In some embodiments, this denotes over-expression of the target molecule. In some embodiments, the disorders can include any of the disorders discussed herein. In some embodiments, the disorder can be any for which there is a target molecule that can be targeted by binding, whose binding will result in the detection and / or treatment of the disorder.

[0121] The term “treating” or “treatment” of a condition can refer to preventing the condition, slowing the onset and / or rate of development of the condition, reducing the risk of developing the condition, preventing and / or delaying the development of symptoms associated with the condition, reducing or ending symptoms associated with the condition, generating a complete or partial regression of the condition, or some combination thereof. The term “prevent” does not require the absolute prohibition of the disorder or disease. Examples of disorders or diseases include fibrosis, cancer, tumor and neoplasms, autoimmune disease, cardiovascular, neurodegenerative, metabolic and endocrine disorders, inflammatory, immunity, genetic disorders, infectious disorders, hematological, congenital disorders, musculoskeletal, oral and gastrointestinal, renal and urogenital disorders, reproductive disorders, respiratory disorders, skin and / or epithelial, as well as disorders with disputed or unknown etiology.

[0122] “Tumor,” as used herein, 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” are not mutually exclusive as referred to herein. The term “neoplasia” encompasses the term tumor.

[0123] 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 particular examples of such cancers include lung cancer including small-cell lung cancer, non-small cell lung cancer and lung adenocarcinomas with neuroendocrine features; neuroendocrine prostate cancer, melanoma, gliomas, low-gradegliomas and glioblastoma, medullary thyroid cancer, carcinoid tumors, neuroendocrine tumors in the pancreas, bladder cancer, testicular cancer squamous cell cancer (e.g. epithelial squamous cell cancer), neuroendocrine neoplasms, such as neuroendocrine tumors of unknown primary, neuroendocrine neoplasms of the small bowel, carotid body, adrenal gland, colorectal gynecological organ, abdomen, esophagus, GI tract, bile duct, nervous system, appendix, liver, anal, thymus, ileocecal junction, head and neck, breast, peritoneum and retroperitoneum, kidney, thyroid, stomach, bone,; adenocarcinomas, such as adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, bone cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, multiple myeloma and B-cell lymphoma, brain, as well as 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 highly fibrotic tumor or cancer. In some embodiments, the cancer is a desmoplasia.

[0124] A “therapeutically effective amount” or a “therapeutically effective dose” is an amount that produces a desired therapeutic effect in a subject, such as preventing, treating a target condition, delaying the onset of the disorder and / or symptoms, and / or alleviating symptoms associated with the condition. This amount will vary depending upon 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 or carriers in the formulation, and / or the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for example by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly, given the present disclosure. For additional guidance, see Remington: The Science and Practiceof Pharmacy 21stEdition, Univ, of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005.

[0125] Label”, “detectable label” or “detectable marker” are used interchangeably herein and refer to a detectable compound or composition which is conjugated directly or indirectly associated with the antibody so as to generate a “labeled” antibody. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.

[0126] The term “payload” denotes an atom or molecule or other entity that is associated (covalently or otherwise) to an antigen binding construct. It includes labels or markers for aspects for diagnostics for example, as well as toxins, cytotoxic agents, chemotherapeutic agents for various therapies. In some embodiments, the payload involves as chelator so as to attach the antigen binding construct to the molecule or atom to be delivered via or colocalized via the antigen binding construct.

[0127] The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents a cellular function and / or causes cell death or destruction. The term is intended to include non-radioactive isotopes (ADC), radioactive isotopes (e.g.,177Lu,225Ac,67Cu,227Th,211At,131I,125I,90Y,186Re,188Re,153Sm,212Bi,213Bi,32P,149Tb,161Tb,212Pb, and radioactive isotopes of Lu), chemotherapeutic agents (as defined elsewhere herein). Other cytotoxic agents are described below. A tumoricidal agent causes destruction of tumor cells.

[0128] A “toxin” is any substance capable of having a detrimental effect on the growth or proliferation of a cell. Non-radioactive payloads include those commonly used for antibody drug conjugates (ADC) and fragment drug conjugates (FDC), such as toxins belonging to the families of auristatins, maytansines, maytansinoids, calicheamicins, duocarymycins, pyrrolobenzodiazepines dimers and amatoxins.

[0129] A “therapeutic ion” refers to an electrically charged particle that that is useful in the treatment of a disorder related to a target molecule. Examples of therapeutic ions include18F,18F-FDG,32P,33P,45Ti,47Sc,52Fe,59Fe,62Cu,64Cu,67Cu,67Ga,68Ga,75Sc,77As,86Y,90Y,89Sr,89Zr,94Tc,94Tc,99mTc, "Mo,105Pd,105Rh,mAg,mIn,123I,124I,125I,131I,142Pr,143Pr,149Pm,149Tb,153Sm,154’158Gd,161Tb,166Dy,166Ho,169Er,175Lu,177Lu,186Re,188Re,189Re,194Ir,198Au,199Au,211At,211Pb,212Bi,212Pb,213Bi,223Ra,227Th, and223Ac. These are also options of therapeutic agents.

[0130] A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXANTM 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 (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOLTM); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTINTM), CPT-11 (irinotecan, CAMPTOSARTM), acetyl camptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem Inti. Ed. Engl., 33 : 183-186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, ADRIAMYCINTM doxorubicin (including morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin,zorubicin; metabolic inhibitor such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINETM, FILDESINTM); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); thiotepa; taxoids, e.g., TAXOL.RTM. paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANETM Cremophor- free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERETM docetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (GEMZARTM); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBANTM); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVINTM); oxaliplatin; leucovovin; vinorelbine (NAVELBINETM); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODATM); pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATINTM) combined with 5-FU and leucovovin. These are also options of therapeutic agents.

[0131] “Radiotherapy” also sometimes called “radiopharmaceutical therapy” or “RPT” means treatment using a radioisotope with a therapeutic purpose. Embodiments of the invention are radiopharmaceutical agents and useful in RPT. It includes radiation therapy intended to have abscopal effect as described in Yang Liu, Yinping Dong, Li Kong, Fang Shi, Hui Zhu & Jinming Yu; “Abscopal effect of radiotherapy combined with immune checkpoint inhibitors”; Journal of Hematology & Oncology volume 11, Article number: 104 (2018); and in Melek Tugce Yilmaz, Aysenur Elmali, and Gozde Yazici; “Abscopal Effect, From Myth to Reality: From Radiation Oncologists' Perspective”; Cureus. 2019 Jan; 11(1).

[0132] As used herein, the term “pretargeting” has its plain and ordinary meaning and may refer to target site localization of a targeting moiety conjugated with one member of a ligand / anti-ligand pair; after a time period sufficient for optimal target-to-non-target accumulation of this targeting moiety conjugate, an active agent conjugated to the opposite member of the ligand / anti-ligand pair is administered and is bound (directly or indirectly) to the targeting moiety conjugate at the target site.

[0133] As used herein, the term “chelation” has it’s plain and ordinary meaning and also may refer to the formation of multiple coordination bonds between organic molecules and a transition metal ion leading to sequestration of the metal. In some embodiments, the chelating ligand comprises ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTP A), dodecane tetraacetic acid (DOTA), 1,4,7,10- tetraazacyclododececane, 1 -(glutaric acid)-4,7,10-triacetic acid (DOTAGA), 1,4,7- triazacyclononane-l,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane, 1-glutaric acid-5,7 acetic acid (NODAGA), 4-[2-(bis-carboxymethyl-amino)-ethyl]-7-carboxymethyl- [l,4,7]triazonan-l-yl-acetic acid (NETA), deferoxamine (Df, which may also be referred to as DFO), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and like groups.

[0134] The terms "subject," "patient," and "individual" interchangeably refer to an entity that is being examined and / or treated. The term “mammal” is used in its usual biological sense. Thus, it specifically includes, but is not limited to, primates, including simians (chimpanzees, apes, monkeys), humans, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rodents, rats, mice, or guinea pigs.

[0135] The term "co-administer" refers to the administration of two active agents in the blood of an individual or in a sample to be tested. Active agents that are co-administered in combination or sequentially delivered. “In combination” means that two (or more) different compositions are delivered to the subject during the course of the subject's affliction with the disorder, e.g., the two or more compositions are delivered after the subject has been diagnosed or selected as one having the disorder and before the disorder has been cured or eliminated. In some embodiments the subject is selected to receive any one or more of the compositions described herein by diagnostic analysis or clinical evaluation or both. In some embodiments, the delivery of one therapy is still occurring when the delivery of the second begins, so that there is overlap. This is sometimes referred to herein as "simultaneous" or "concomitant" or "concurrent delivery". In other embodiments, the delivery of one therapy ends before the delivery of the other therapy begins. This is sometimes referred to herein as "successive" or "sequential delivery." In embodiments of either case, the therapy is more effective because of combined administration. For example, the second therapy is a more effective, e.g., an equivalent effect is seen with less of the second therapy, or the second therapy reduces symptoms to a greater extent, than would be seen if the second therapy were administered in the absence of the first therapy, or the analogous situation is seen with the first therapy. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one therapy delivered in the absence of the other. The effect of the two therapies can be partially additive, wholly additive, or greater than additive (e.g., synergistic). The delivery can be such that an effect of the first therapy delivered is still detectable when the second is delivered.

[0136] As used herein, the term “palliative care” has its plain and ordinary meaning and may refer to specialized medical care focused on providing relief from pain and other symptoms of a serious illness and / or side effects from medical treatments. The availability of palliative care does not depend on whether the serious illness can be cured. Palliative care may be offered alongside one or more other treatments.

[0137] The phrase "specifically (or selectively) bind," when used in the context of describing the interaction between an antigen, for example, a protein, to an antibody or antibody-derived binding agent, refers to a binding reaction that is determinative of the presence of the antigen in a heterogeneous population of proteins and other biologies, forexample, in a biological sample, for example, a blood, serum, plasma or tissue sample. Thus, under designated immunoassay conditions, in some embodiments, the antibodies or binding agents with a particular binding specificity bind to a particular antigen at least two times the background and do not substantially bind in a significant amount to other antigens present in the sample. Specific binding to an antibody or binding agent under such conditions may require the antibody or agent to have been selected for its specificity for a particular protein. A variety of immunoassay formats may 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, for example, 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 at least twice over the background signal and more typically at least than 10 to 100 times over the background.

[0138] The term "equilibrium dissociation constant (KD, M)" refers to the dissociation rate constant (kd, time'1) divided by the association rate constant (ka, time'1M'1). Equilibrium dissociation constants can be measured using any known method in the art. The antibodies of the present disclosure generally will have an equilibrium dissociation constant of less (that is superior binding) than about 10'7or 10'8M, for example, less than about 10'9M or IO'10M, in some embodiments, less than about 10'11M, 10'12M, or 10'13M.

[0139] 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 residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer.

[0140] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or doublestranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues 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 encompassesconservatively modified variants thereof (for example, degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer, M. A. 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, G. M. 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).

[0141] 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 the 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, for example, hydroxyproline, gamma-carboxy glutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, for example, an alpha-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, for example, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (for example, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0142] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where analanine 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 which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0143] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results 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.

[0144] 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, for example, Creighton, T. E., “Proteins - Structures and Molecular Properties,” W. H. Freeman & Co. Ltd., 1984).

[0145] The term "percentage of sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (for example, a polypeptide of the present disclosure), which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0146] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same sequences. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (for example, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over a specified region, or, when not specified, over the entire sequence of a 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, respectively, exemplified herein. Optionally, the 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 to 500 or 1000 or more nucleotides in length, or over the full length of the reference sequence. With respect to amino acid sequences, identity or substantial identity can exist over a region that is at least 5, 10, 15 or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75 or 100 amino acids in length, optionally at least about 150, 200 or 250 amino acids in length, or over the full length of the reference sequence. With respect to shorter amino acid sequences, for example, 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 the conservative substitutions defined herein.

[0147] In some embodiments, the percent identity is over the CDR and / or FR regions noted herein. In such situations, the percent identity of the CDR or FR can be identified separately from the rest 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 (for example, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over a specified region, or, when not specified, over the entire sequence of a reference sequence), while allowing for the remainder of the protein to either stay 100% identical to the comparison protein, our while also allowing the remainder of the protein to also have variation by a specified percent identity.

[0148] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0149] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in 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 of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman, S. B. 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 search for similarity method of Pearson, W. R. et al., “Improved tools for biological sequence comparison,” Proc. Natl. Acad. Sci. U.S.A., Vol. 85, No. 8, pp. 2444-2448, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Supplement, 1995).

[0150] Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described 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, respectively. 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 inthe query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul, S. F. 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. Cumulative scores are calculated using, for nucleotide sequences, 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 are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negativescoring residue alignments; or 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) or 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, and 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. U.S.A., Vol. 89, No. 22, pp. 10915-10919, 1992) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0151] 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. U.S.A., 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 by which 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 a comparison of the test nucleic acid to 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.

[0152] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid isimmunologically cross reactive with the antibodies 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 sequence.Antigen Binding Constructs and Methods of Use Thereof:

[0153] Provided herein are antigen binding constructs (e.g., antibodies, minibodies, scFv-Fc fusions, nanobody®-Fc) having altered biodistribution compared to an original antigen binding construct from which the antigen binding constructs are derived. Without being bound by theory, it is thought that localized patches of surface charges as described by an iso-potential surface over the antibody may contribute to accumulation of the antibody in radiosensitive or drug sensitive tissues, such as the kidneys, when administered to a subject.

[0154] Provided herein is a antigen binding construct comprising an IgG CH2 having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or a percentage in a range that is defined by any two of the preceding values, identity to an IgG CH2 in SEQ ID NO. 57, wherein at least one mutation in the IgG CH2 reduces uptake of the antigen binding construct by healthy organs as compared to an IgG CH2 sequence in SEQ ID NO. 57.

[0155] FIG. 1 is an illustrative representation of some non-limiting embodiments of an antibody and antigen binding fragments thereof. Some embodiments herein are directed to an antibody or antigen binding fragment thereof 100. In some embodiments, the antibody or antigen binding fragment thereof is aDLL3, FAP, CD8, CD4, CD3, ZFNy, FOLROC, or aVp6 antibody or antigen binding fragment thereof. In some embodiments, the antibody of antigen binding fragment thereof 101 comprises: a variable light (VL) domain 102 and a variable heavy (VH) domain 103. The VL and VH domains can comprise one or more CDRs 104. The VL domain can comprise an LCDR1 105, LCDR2 106, and LCDR3 107. The VH domain can comprise an HCDR1 108, HCDR2 109, and HCDR3 110. In some embodiments, the antibody or antigen binding fragment thereof comprises a hinge domain 111 comprising a hinge region.In some embodiments, the hinge region comprises an upper hinge, a core hinge, and a lower hinge region. In some embodiments, the upper hinge comprises a sequence that is SEQ ID NO. 51 or 52. In some embodiments, the core hinge comprises a sequence that is SEQ ID NO.53. In some embodiments, the lower hinge region comprises a sequence that is SEQ ID NO.54. In some embodiments, the antibody or binding fragment thereof comprises a hingeextension domain comprising a hinge region. In some embodiments, the antibody or antigen binding fragment thereof comprises a linker. In some embodiments, the linker comprises a sequence that is SEQ ID NO. 55. In some embodiments, the antibody or antigen binding fragment thereof comprises a signal peptide. In some embodiments, the signal peptide comprises a sequence that is SEQ ID NO. 56. In some embodiments, the antibody or antigen binding fragment thereof comprises an IgG CH2 domain 112. In some embodiments, the CH2 domain comprises a sequence that is any one of SEQ ID NO. 47-76. In some embodiments, the antibody or antigen binding fragment thereof comprises an IgG CH3 domain 113. In some embodiments, the CH3 domain comprises any one of SEQ ID NO. 77-80. In some embodiments, the CH2 and / or CH3 domain comprises one or more substitutions. In some embodiments, the one or more substitutions in the CH2 and / or CH3 domain alters biodistribution of the antibody or antigen binding fragment thereof. In some embodiments, the one or more substitutions in the CH2 and / or CH3 domain alter distribution of the antibody or antigen binding fragment thereof to a subject’s blood, lungs, muscle, bone (bone marrow), liver, kidneys, heart, stomach, small intestine, large intestine, pancreas, tumor, testis, ovaries, and / or any combination thereof. In some embodiments, the antibody or antigen binding fragment thereof comprises a Fc domain 114. In some embodiments, the antibody or antigen binding fragment thereof comprises a Fc domain comprises a sequence that is any one of SEQ ID NO. 83-102. In some embodiments, the Fc domain comprises one or more substitutions. In some embodiments, the one or more substitutions in the Fc domain alters biodistribution of the antibody or antigen binding fragment thereof. In some embodiments, the one or more substitutions in the Fc domain alters distribution of the antibody or antigen binding fragment thereof to a subject’s blood, lungs, muscle, bone (bone marrow), liver, kidneys, heart, stomach, small intestine, large intestine, pancreas, tumor, testis, ovaries, and / or any combination thereof. In some embodiments, the antibody or antigen binding fragment thereof further comprise a payload. In some embodiments, the payload comprises a therapeutic agent, toxin, and / or adetectable marker. In some embodiments, the detectable marker is a radio label. In some embodiments, the antibody or antigen binding fragment thereof is administered to a subject having one or more diseases. In some embodiments, the disease is a disease of the blood, lungs, muscle, bone (bone marrow), liver, kidneys, heart, stomach, small intestine, large intestine, pancreas, tumor, testis, ovaries, and / or any combination thereof. In some embodiments, administration of the antibody or antigen binding fragment thereof reduces or maintains the severity of one or more symptoms of the disease. In some embodiments, administration of the antibody or antigen binding fragment thereof eliminates on or more symptoms of the disease. In some embodiments, the antibody or antigen binding fragment thereof is administered as palliative care. In some embodiments, the antigen binding fragment does not comprise a disrupted positive patch. In some embodiments, the antigen binding fragment does not comprise a disrupted positive patch. In some embodiments, the antigen binding fragment does not comprise a disrupted positive patch but does comprise a disrupted negative patch. In some embodiments, the antigen binding fragment comprises a disrupted positive patch but does not comprise a disrupted negative patch. In some embodiments, the VL domain comprises a sequence that is any one of SEQ ID NO. 2-17. In some embodiments, the VH domain comprises a sequence that is any one of SEQ ID NO. 19-34, 139 or 140.

[0156] In some embodiments, the LCDR1 has a sequence that is SEQ ID NO. 35 or 36. In some embodiments, the LCDR2 has a sequence that is SEQ ID NO. 37. In some embodiments, the LCDR3 has a sequence that is any one of SEQ ID NO. 38-40. In some embodiments, the VL domain comprises a framework region (LFR). In some embodiments, the VL domain comprises a LFR 1, 2, 3, and 4. In some embodiments, the LFR2 domain comprises a sequence that is SEQ ID NO. 41 or 42. In some embodiments, the LFR2 domain comprises a sequence that is X1X2X3X4X5X6X7 (SEQ ID NO. 81), wherein X2, X3, X5, and Xe are each independently a negatively charged or a non-charged amino acid, and Xi, X4, and X7 are each a lysine (K) (SEQ ID NO. 82). In some embodiments, Xi, X4, and / or X7, are substituted (e.g., is not K). In some embodiments, substitution of Xi, X4, and / or X7, in the LFR2 domain alter distribution of the antibody or binding fragment thereof to a subject’s blood, lungs, muscle, bone (bone marrow), liver, kidneys, heart, stomach, small intestine, large intestine, pancreas, tumor, testis, ovaries, and / or any combination thereof.

[0157] In some embodiments, the HCDR1 has a sequence that is any one of SEQ ID NO. 43-46. In some embodiments, the HCDR2 has a sequence that is any one of SEQ ID NO. 47-48, 119, 136. In some embodiments, the HCDR3 has a sequence that is SEQ ID NO. 49 or 50.

[0158] Some embodiments herein are directed to an antibody of antigen binding fragment thereof having altered biodistribution. In some embodiments, the antibody or antigen binding fragment thereof having altered biodistribution is a DLL3, FAP, CD8, CD4, CD3, IFNy, FOLRa, or aVp6 antibody or antigen binding fragment thereof.

[0159] FIG. 2 is an illustrative representation of some embodiments of a singlechain variable fragment (scFv). Some embodiments herein are directed to a single-chain variable fragment (scFv) 200. In some embodiments, the scFv is a DLL3, FAP, CD8, CD4, CD3, IFNy, FOLRa, or aVp6 binding scFv. The scFv can comprise: a variable light (VL) domain 201 and a variable heavy (VH) domain 205. In some embodiments, the VL domain is N-terminal to the VH domain in the scFv. In some embodiments, the VH domain is N-terminal to the VL domain in the scFv. In some embodiments, the scFv comprises a hinge-extension domain comprising a hinge region. In some embodiments, the hinge region comprises an upper hinge, a core hinge, and a lower hinge region. In some embodiments, the upper hinge comprises a sequence that is SEQ ID NO. 51 or 52. In some embodiments, the core hinge comprises a sequence that is SEQ ID NO. 53. In some embodiments, the lower hinge region comprises a sequence that is SEQ ID NO. 54. In some embodiments, the scFv comprises a linker 209. In some embodiments, the linker comprises a sequence that is SEQ ID NO. 55. In some embodiments, the scFv comprises a signal peptide. In some embodiments, the signal peptide comprises a sequence that is SEQ ID NO. 56. In some embodiments, the scFv further comprise a payload. In some embodiments, the payload comprises a therapeutic agent, toxin, and / or a detectable marker. In some embodiments, the detectable marker is a radio label. In some embodiments, the scFv is administered to a subject having one or more diseases. In some embodiments, the disease is a disease of the blood, lungs, muscle, bone (bone marrow), liver, kidneys, heart, stomach, small intestine, large intestine, pancreas, testis, ovaries, and / or any combination thereof. In some embodiments, administration of the scFv reduces or maintains the severity of one or more symptoms of the disease. In some embodiments, administration ofthe scFv eliminates on or more symptoms of the disease. In some embodiments, the scFv is administered as palliative care.

[0160] Some embodiments herein are directed to a single-chain variable fragment (scFv) having altered biodistribution. In some embodiments, the scFv having altered biodistribution is a DLL3, FAP, CD8, CD4, CD3, IFNy, FOLRoc, or aVp6 binding scFv. In some embodiments, the scFv comprises a linker. In some embodiments, the linker comprises a sequence that is SEQ ID NO. 55. In some embodiments, the scFv comprises a signal peptide. In some embodiments, the signal peptide comprises a sequence that is SEQ ID NO. 56. In some embodiments, the scFv is administered to a subject having one or more diseases. In some embodiments, the disease is a disease of the blood, lungs, muscle, bone (bone marrow), liver, kidneys, heart, stomach, small intestine, large intestine, pancreas, testis, ovaries, and / or any combination thereof. In some embodiments, the scFv further comprise a payload. In some embodiments, the payload comprises a therapeutic agent, toxin, and / or a detectable marker. In some embodiments, the detectable marker is a radio label. In some embodiments, administration of the scFv reduces or maintains the severity of one or more symptoms of the disease. In some embodiments, administration of the scFv eliminates on or more symptoms of the disease. In some embodiments, the scFv is administered as palliative care.

[0161] FIG. 3 is an illustrative representation of some embodiments of a minibody. Some embodiments herein are directed to a minibody 300. In some embodiments, the minibody is a DLL3, FAP, CD8, CD4, CD3, IFNy, FOLRa, or aVp6 binding minibody. The minibody 300 can comprise: a variable light (VL) domain 301 and a variable heavy (VH) domain 302. The minibody 300 can comprise a scFv comprising the VL and VH domains. In some embodiments, the VL domain is N-terminal to the VH domain in the minibody or the scFv portion thereof. In some embodiments, the VH domain is N-terminal to the VL domain in the minibody or the scFv portion thereof. In some embodiments, the antibody or binding fragment thereof comprises a hinge-extension domain 310 comprising a hinge region. In some embodiments, the hinge region comprises an upper hinge, a core hinge, and a lower hinge region. In some embodiments, the upper hinge comprises a sequence that is SEQ ID NO. 51 or 52. In some embodiments, the core hinge comprises a sequence that is SEQ ID NO. 53. In some embodiments, the lower hinge region comprises a sequence that is SEQ ID NO. 54. In some embodiments, the minibody comprises a linker. In some embodiments, the linkercomprises a sequence that is SEQ ID NO. 55. In some embodiments, the minibody comprises a signal peptide. In some embodiments, the signal peptide comprises a sequence that is SEQ ID NO. 56. In some embodiments, the minibody comprises an IgG CH3 domain 311. In some embodiments, the CH3 domain comprises any one of SEQ ID NO. 77-80. In some embodiments, the CH3 domain comprises one or more substitutions. In some embodiments, the one or more substitutions in the CH3 domain alters biodistribution of the minibody. In some embodiments, the one or more substitutions in the CH3 domain alters distribution of the minibody to a subject’s blood, lungs, muscle, bone (bone marrow), liver, kidneys, heart, stomach, small intestine, large intestine, pancreas, tumor, testis, ovaries, and / or any combination thereof. In some embodiments, the minibody further comprise a payload. In some embodiments, the payload comprises a therapeutic agent, toxin, and / or a detectable marker. In some embodiments, the detectable marker is a radio label. In some embodiments, the minibody is administered to a subject having one or more diseases. In some embodiments, the disease is a disease of the blood, lungs, muscle, bone (bone marrow), liver, kidneys, heart, stomach, small intestine, large intestine, pancreas, testis, ovaries, and / or any combination thereof. In some embodiments, administration of the minibody reduces or maintains the severity of one or more symptoms of the disease. In some embodiments, administration of the minibody eliminates on or more symptoms of the disease. In some embodiments, the minibody is administered as palliative care. In some embodiments, the minibody does not comprise a disrupted positive patch. In some embodiments, the minibody does not comprise a disrupted positive patch but does comprise a disrupted negative patch. In some embodiments, the minibody comprises a disrupted positive patch but does not comprise a disrupted negative patch.

[0162] Some embodiments herein are directed to a minibody having altered biodistribution. In some embodiments, the minibody having altered biodistribution is a DLL3, FAP, CD8, CD4, CD3, IFNy, FOLRa, or aV 6 binding minibody. In some embodiments, the minibody comprises a hinge-extension domain comprising a hinge region. In some embodiments, the hinge region comprises an upper hinge, a core hinge, and a lower hinge region. In some embodiments, the upper hinge comprises a sequence that is SEQ ID NO. 51 or 52. In some embodiments, the core hinge comprises a sequence that is SEQ ID NO. 53. In some embodiments, the lower hinge region comprises a sequence that is SEQ ID NO. 54. In some embodiments, the minibody comprises a linker. In some embodiments, the linkercomprises a sequence that is SEQ ID NO. 55. In some embodiments, the minibody comprises a signal peptide. In some embodiments, the signal peptide comprises a sequence that is SEQ ID NO. 56. In some embodiments, the minibody comprises an IgG CH3 domain. In some embodiments, the CH3 domain comprises any one of SEQ ID NO. 77-80. In some embodiments, the CH3 domain comprises one or more substitutions. In some embodiments, the one or more substitutions in the CH3 domain alters biodistribution of the minibody. In some embodiments, the one or more substitutions in the CH3 domain alter distribution of the minibody to a subject’s blood, lungs, muscle, bone (bone marrow), liver, kidneys, heart, stomach, small intestine, large intestine, pancreas, tumor, testis, ovaries, and / or any combination thereof. In some embodiments, the minibody further comprise a payload. In some embodiments, the payload comprises a therapeutic agent, toxin, and / or a detectable marker. In some embodiments, the detectable marker is a radio label. In some embodiments, the minibody is administered to a subject having one or more diseases. In some embodiments, the disease is a disease of the blood, lungs, muscle, bone (bone marrow), liver, kidneys, heart, stomach, small intestine, large intestine, pancreas, testis, ovaries, and / or any combination thereof. In some embodiments, administration of the minibody reduces or maintains the severity of one or more symptoms of the disease. In some embodiments, administration of the minibody eliminates on or more symptoms of the disease. In some embodiments, the minibody does not comprise a disrupted positive patch. In some embodiments, the minibody does not comprise a disrupted positive patch. In some embodiments, the minibody does not comprise a disrupted positive patch but does comprise a disrupted negative patch. In some embodiments, the minibody comprises a disrupted positive patch but does not comprise a disrupted negative patch.

[0163] FIG. 4 is an illustrative representation of some embodiments of a singlechain variable fragment (scFv) Fc fusion (scFv-Fc). Some embodiments herein are directed to a single-chain variable fragment Fc fusion (scFv-Fc) 400. In some embodiments, the scFv-Fc fusion is a DLL3, FAP, CD8, CD4, CD3, IFNy, FOLRa, or aVp6 binding scFv-Fc fusion. The scFv-Fc fusion can comprise a scFv 401 that comprises a variable light (VL) domain 402 and a variable heavy (VH) domain 403. In some embodiments, the VL domain is N-terminal to the VH domain in the scFv. In some embodiments, the VH domain is N-terminal to the VL domain in the scFv. In some embodiments, the scFv-Fc fusion comprises a hinge-extension domain 411 comprising a hinge region. In some embodiments, the hinge region comprises an upperhinge, a core hinge, and a lower hinge region. In some embodiments, the upper hinge comprises a sequence that is SEQ ID NO. 51 or 52. In some embodiments, the core hinge comprises a sequence that is SEQ ID NO. 53. In some embodiments, the lower hinge region comprises a sequence that is SEQ ID NO. 54. In some embodiments, the scFv-Fc fusion comprises a linker 412. In some embodiments, the linker comprises a sequence that is SEQ ID NO. 55. In some embodiments, the scFv-Fc fusion comprises a signal peptide. In some embodiments, the signal peptide comprises a sequence that is SEQ ID NO. 56. In some embodiments, the scFv-Fc fusion comprises a Fc domain 413. In some embodiments, the scFv-Fc fusion comprises a Fc domain comprises a sequence that is any one of SEQ ID NO. 83-102. In some embodiments, the Fc domain comprises one or more substitutions. In some embodiments, the one or more substitutions in the Fc domain alters biodistribution of the scFv-Fc fusion. In some embodiments, the one or more substitutions in the Fc domain alters distribution of the scFv-Fc fusion to a subject’s blood, lungs, muscle, bone (bone marrow), liver, kidneys, heart, stomach, small intestine, large intestine, pancreas, tumor, testis, ovaries, and / or any combination thereof. In some embodiments, the scFv-Fc fusion comprises an IgG CH2 domain 414. In some embodiments, the CH2 domain comprises a sequence that is any one of SEQ ID NO. 47-76. In some embodiments, the scFv-Fc fusion comprises an IgG CH3 domain 415. In some embodiments, the CH3 domain comprises any one of SEQ ID NO. 77-80. In some embodiments, the CH2 and / or CH3 domain comprises one or more substitutions. In some embodiments, the one or more substitutions in the CH2 and / or CH3 domain alters biodistribution of the antibody or binding fragment thereof. In some embodiments, the one or more substitutions in the CH2 and / or CH3 domain alter distribution of the scFv-Fc fusion to a subject’s blood, lungs, muscle, bone (bone marrow), liver, kidneys, heart, stomach, small intestine, large intestine, pancreas, tumor, testis, ovaries, and / or any combination thereof. Some embodiments herein are directed to a single-chain variable fragment Fc fusion (scFv-Fc fusion) having altered biodistribution. In some embodiments, the scFv-Fc fusion having altered biodistribution is a DLL3, FAP, CD8, CD4, CD3, IFNy, FOLRa, or aVp6 binding scFv-Fc fusion.

[0164] FIG. 5 is flow chart illustrating some embodiments of a method for reducing tumor size. Some embodiments herein are directed to a method of reducing tumor size in a subject 500. In some embodiments, the method comprises administering an antibodyor binding fragment thereof to a subject having a tumor 501. In some embodiments, the antibody or binding fragment thereof is a DLL3, FAP, CD8, CD4, CD3, IFNy, FOLRa, or aVp6 antibody antigen binding fragment thereof. In some embodiments, the antibody or binding fragment thereof comprises a hinge-extension domain comprising a hinge region. In some embodiments, the hinge region comprises an upper hinge, a core hinge, and a lower hinge region. In some embodiments, the upper hinge comprises a sequence that is SEQ ID NO. 51 or 52. In some embodiments, the core hinge comprises a sequence that is SEQ ID NO. 53. In some embodiments, the lower hinge region comprises a sequence that is SEQ ID NO. 54. In some embodiments, the antibody or binding fragment thereof comprises a linker. In some embodiments, the linker comprises a sequence that is SEQ ID NO. 55. In some embodiments, the antibody or binding fragment thereof comprises a signal peptide. In some embodiments, the signal peptide comprises a sequence that is SEQ ID NO. 56. In some embodiments, the antibody or binding fragment thereof comprises an IgG CH2 domain. In some embodiments, the CH2 domain comprises a sequence that is any one of SEQ ID NO. 47-76. In some embodiments, the antibody or binding fragment thereof comprises an IgG CH3 domain. In some embodiments, the CH3 domain comprises any one of SEQ ID NO. 77-80. In some embodiments, the CH2 and / or CH3 domain comprises one or more substitutions. In some embodiments, the one or more substitutions in the CH2 and / or CH3 domain alters biodistribution of the antibody or binding fragment thereof. In some embodiments, the one or more substitutions in the CH2 and / or CH3 domain alter distribution of the antibody or binding fragment thereof to a subject’s blood, lungs, muscle, bone (bone marrow), liver, kidneys, heart, stomach, small intestine, large intestine, pancreas, tumor, testis, ovaries, and / or any combination thereof. In some embodiments, the antibody or binding fragment thereof comprises a Fc domain. In some embodiments, the antibody or binding fragment thereof comprises a Fc domain comprises a sequence that is any one of SEQ ID NO. 83-102. In some embodiments, the Fc domain comprises one or more substitutions. In some embodiments, the one or more substitutions in the Fc domain alters biodistribution of the antibody or binding fragment thereof. In some embodiments, the one or more substitutions in the Fc domain alters distribution of the antibody or binding fragment thereof to a subject’s blood, lungs, muscle, bone (bone marrow), liver, kidneys, heart, stomach, small intestine, large intestine, pancreas, tumor, testis, ovaries, and / or any combination thereof. In some embodiments, the one or moresubstitutions in the Fc domain alters the ratio of biodistribution of the antibody or binding fragment thereof in a subj ect’ s tumor as compared to biodistribution of the antibody or binding fragment thereof to the subject’s blood, lungs, muscle, bone (bone marrow), liver, kidneys, heart, stomach, small intestine, large intestine, pancreas, testis, ovaries, and / or any combination thereof. In some embodiments, administration of the antibody or binding fragment thereof reduces tumor size as compared to tumor size in the subject prior to administration of the antibody or binding fragment thereof. In some embodiments, reduction in tumor size reduces or maintains the severity of one or more symptoms of the disease. In some embodiments, reduction of tumor size eliminates on or more symptoms of the disease. In some embodiments, the antibody or binding fragment thereof does not comprise a disrupted positive patch. In some embodiments, the antibody or antigen binding fragment thereof does not comprise a disrupted positive patch but does comprise a disrupted negative patch. In some embodiments, the antibody or binding fragment thereof comprises a disrupted positive patch but does not comprise a disrupted negative patch.

[0165] FIG. 6 is flow chart illustrating some embodiments of a method of treating cancer. Some embodiments herein are directed to a method of treating cancer 600. In some embodiments, the method comprises administering an antigen binding construct (e.g., antibody, minibody, scFv-Fc fusion) or antigen binding fragment thereof to a subject having a tumor 601. In some embodiments, the antigen binding construct (e.g., antibody, minibody, scFv-Fc fusion) or antigen binding fragment thereof is a DLL3, FAP, CD8, CD4, CD3, IFNy, FOLRa, or aVp6 antigen binding construct (e.g., antibody, minibody, scFv-Fc fusion) or antigen binding fragment thereof.

[0166] In some embodiments, the antigen binding construct is a aVp6 antibody or antigen binding fragment thereof comprising a VL domain having a sequence that is any one of SEQ ID NO. 1-17 In some embodiments, the VL domain comprises a sequence having, having about, or having at least 70%, 71%, 72%, 73%, 74%, 75%, 75%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to any one of SEQ ID NO. 1-17, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the VL domain comprises a sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 70%-75%, 57%-100%, 75%-95%, 75%-90%, 75%-80%, 80%-100%, 80%-95%,80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to any one of SEQ ID NO. 1-17. In some embodiments, the aV 6 antibody or antigen binding fragment thereof comprises a LFR2 having SEQ ID NO. 41 or 42. In some embodiments, the aVP6 antibody or antigen binding fragment thereof comprises a LFR2 in Table 2. In some embodiments, the VL is based on a human germline sequence of IGKV1-39*O1. In some embodiments, the VL is based on a human germline sequence of IGKJ4*01.

[0167] Table 1 depicts some non-limiting embodiments of VL domain sequences disclosed herein.Table 1.

[0168] Table 2 depicts some non-limiting embodiments of LFR2 sequences disclosed herein.Table 2

[0169] FIG. 7 depicts an alignment of some non-limiting embodiments of aVp6 VL domain sequences disclosed herein. In some embodiments, the aVp6 antibody or antigen binding fragment thereof comprises a VL domain having a VL domain sequence in FIG. 7. In some embodiments, the aVP6 antibody or antigen binding fragment thereof comprises a VL domain having, having about, or having at least 70%, 71%, 72%, 73%, 74%, 75%, 75%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to a VL domain sequence in FIG. 7, or has a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the aVp6 antibody or antigen binding fragment thereof comprises a VL domain having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 70%-75%, 57%- 100%, 75%-95%, 75%-90%, 75%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%- 95%, or 95%-100% identity to a VL domain sequence in FIG. 7. In some embodiments, the VL is based on a human germline sequence of IGKV1 -39*01. In some embodiments, the VL is based on a human germline sequence of IGKJ4*01.

[0170] In some embodiments, the VL domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VL domain in SEQ ID NO. 9, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the VL domain comprises an amino acid sequence having between about 70%-100%, 70%- 95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-l 00%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%- 95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VL domain in SEQ ID NO. 9 In some embodiments, the antibody or antigen binding fragment thereof comprises a LFR2 having SEQ ID NO. 41 or 42

[0171] In some embodiments, the antibody or antigen binding fragment thereof comprises a VL domain comprising the polypeptide sequence X1X2X3X4X5X6X7 (SEQ ID NO. 81), wherein Xi is a positively charged amino acid, wherein X2, X3, X5, and Xc> are each independently a negatively charged or a non-charged amino acid, and wherein X4 and X7 are each independently any amino acid with the proviso that at least one is a positively charged amino acid. In some embodiments, Xi is lysine (K), arginine (R), or histidine (H). In some embodiments, X2, X3, X5, and Xe are each independently a glycine (G), alanine (A), valine (V), cysteine (C), proline (P), leucine (L), isoleucine (I), methionine (M), tryptophan (W), phenylalanine (F), serine (S), threonine (T), tyrosine (Y), asparagine (N), glutamine (Q), aspartic acid (D), or glutamic acid (E). In some embodiments, the sequence is within a VL framework region (FR) of the antibody. In some embodiments, X4 and X7 are each independently a lysine (K), arginine (R), histidine (H), glycine (G), alanine (A), valine (V), cysteine (C), proline (P), leucine (L), isoleucine (I), methionine (M), tryptophan (W), phenylalanine (F), serine (S), threonine (T), tyrosine (Y), asparagine (N), glutamine (Q), aspartic acid (D), or glutamic acid (E) with the proviso that at least one is lysine (K), arginine (R), or histidine (H). In some embodiments, Xi, X4. and X7, are lysine (K). In some embodiments, the sequence is within a LFR2 of the antibody or binding fragment thereof. In some embodiments, the sequence is within a LFR2 of a DLL3, FAP, CD8, CD4, CD3, IFNy, FOLRot, or aVp6 antibody or binding fragment thereof. In some embodiments, one or more mutations in the LFR2 alter biodistribution of the antibody or antigen binding fragment thereof in a subject’s blood, lungs, muscle, bone (bone marrow), liver, kidneys, heart, stomach, small intestine, large intestine, pancreas, tumor, testis, ovaries, and / or any combination thereof. In some embodiments, the sequence is in a LFR2 having SEQ ID NO. 41 or 42. In some embodiments, the sequence is in an antibody or antigen binding fragment thereof comprising a LFR2 in Table 2. In some embodiments, the sequence is in a VL is based on a human germline sequence of IGKV1-39*O1. In some embodiments, the sequence is in a VL is based on a human germline sequence of IGKJ4*01.

[0172] In some embodiments, the antibody or antigen binding fragment thereof comprises a VL domain comprising the polypeptide sequence X1X2X3X4X5X6X7 (SEQ ID NO. 81), wherein Xi, X4, and X7 are each a lysine (K) (SEQ ID NO. 82). In some embodiments, one or more of Xi, X4, and / or X7, are mutated. In some embodiments, one or more of Xi, X4, and / or X7, are each independently mutated to (or substituted with) a glycine (G), alanine (A), valine (V), cysteine (C), proline (P), leucine (L), isoleucine (I), methionine (M), tryptophan (W), phenylalanine (F), serine (S), threonine (T), tyrosine (Y), asparagine (N), glutamine (Q), aspartic acid (D), or glutamic acid (E). In some embodiments, one or more of Xi, X4, and / or X7, are each independently mutated to (or substituted with) a negative or noncharged amino acid. In some embodiments, X2, X3, X5, and Xc> are each independently a negatively charged or a non-charged amino acid. In some embodiments, X2, X3, X5, and Xe are each independently a glycine (G), alanine (A), valine (V), cysteine (C), proline (P), leucine (L), isoleucine (I), methionine (M), tryptophan (W), phenylalanine (F), serine (S), threonine (T), tyrosine (Y), asparagine (N), glutamine (Q), aspartic acid (D), or glutamic acid (E). In some embodiments, the sequence is within a LFR2 of the antibody or binding fragment thereof. In some embodiments, the sequence is within a LFR2 of a DLL3, FAP, CD8, CD4, CD3, IFNy, FOLRa, or aVp6 antibody or antigen binding fragment thereof.

[0173] In some embodiments, the VL domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VL domain in SEQ ID NO. 1, wherein the VL domain comprises a K47X1 and / or K 3X2 mutation (Aho), wherein Xi and X2 are any non-lysine (and non-arginine) residue. For example, in some embodiments, the VL domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%- 100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%- 97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VL domain in SEQ ID NO. 1, wherein the VL domain comprises a K47Xi and / or K53X2 mutation (Aho), wherein X is any non-lysine (and non-arginine) residue. In some embodiments, Xi and X2 are a histidine (H), glycine (G), alanine (A), valine (V), cysteine (C), proline (P), leucine (L), isoleucine (I), methionine (M), tryptophan (W), phenylalanine (F), serine (S), threonine (T), tyrosine (Y), asparagine (N), glutamine (Q),aspartic acid (D), or glutamic acid (E). In some embodiments, positions 47, 50, and 53 of the VL domain according to Aho numbering corresponds to positions 45, 48, and 51, respectively, according to IMGT numbering.

[0174] In some embodiments, the VL domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VL domain in SEQ ID NO. 1, wherein the VL domain comprises a K53Q mutation (Aho).

[0175] In some embodiments, the VL domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VL domain in SEQ ID NO. 9, wherein the VL domain comprises a Q to K substitution at a position corresponding to residue number 46 and / or residue number 49 of SEQ ID NO. 9 For example, in some embodiments, the VL domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VL domain in SEQ ID NO. 9, wherein the VL domain comprises a Q to K substitution at a position corresponding to residue number 46 and / or residue number 49 of SEQ ID NO. 9

[0176] In some embodiments, the VL domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VL domain in SEQ ID NO. 1 wherein the VL domain comprises a E29Q mutation (Aho). For example, in some embodiments, the VL domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VL domain in SEQ ID NO. 1, wherein the VL domain comprises a E29Q mutation (Aho).

[0177] In some embodiments, the VL domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VL domain in SEQ ID NO. 9, wherein the VL domain comprises an E to Q substitution at a position corresponding to residue number 27 of SEQ ID NO. 9. For example, in some embodiments, the VL domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VL domain in SEQ ID NO. 9, wherein the VL domain comprises an E to Q substitution at a position corresponding to residue number 46 and / or residue number 27 of SEQ ID NO. 9.

[0178] In some embodiments, the VL domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VL domain in SEQ ID NO. 9, wherein the VL domain comprises a S to A substitution at a position corresponding to residue number 47 of SEQ ID NO. 9. For example, in some embodiments, the VL domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VL domain in SEQ ID NO. 9, wherein the VL domain comprises a S to A substitution at a position corresponding to residue number 47 of SEQ ID NO. 9.

[0179] In some embodiments, the VL domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VL domain in SEQ ID NO. 9, wherein the VL domain comprises a G to K substitution at a position corresponding to residue number 96 of SEQ ID NO. 9. For example, in some embodiments, the VL domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identityto a VL domain in SEQ ID NO. 9, wherein the VL domain comprises a G to K substitution at a position corresponding to residue number 96 of SEQ ID NO. 9.

[0180] In some embodiments, the VL domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VL domain in SEQ ID NO. 1, wherein the VL domain comprises a El l IS mutation (Aho). For example, in some embodiments, the VL domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VL domain in SEQ ID NO. 1, wherein the VL domain comprises a El 11 S mutation (Aho).

[0181] In some embodiments, the VL domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VL domain in SEQ ID NO. 9, wherein the VL domain comprises an E to S substitution at a position corresponding to residue number 97 of SEQ ID NO. 9. For example, in some embodiments, the VL domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VL domain in SEQ ID NO. 9, wherein the VL domain comprises an E to S substitution at a position corresponding to residue number 46 and / or residue number 97 of SEQ ID NO. 9.

[0182] In some embodiments, the VL domain comprises any one or more of the VL domain mutations (e.g., any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or all of the non-overlapping VL domain mutations) provided herein.

[0183] Table 3 depicts some embodiments of LCDR sequences disclosed herein. In some embodiments, the antibody or antigen binding fragment thereof comprises a VL domain with an LCDR1 sequence that is an LCDR1 sequence in Table 3. In some embodiments, the antibody or antigen binding fragment thereof comprises a VL domain with an LCDR2 sequence that is an LCDR2 sequence in Table 3. In some embodiments, theantibody or antigen binding fragment thereof comprises a VL domain with an LCDR3 sequence that is an LCDR3 sequence in Table 3.Table 3

[0184] FIG. 8 depicts an alignment of some non-limiting embodiments of aVp6 VH domain sequences disclosed herein. In some embodiments, the aV 6 antibody or antigen binding fragment thereof comprises a VH domain having a sequence in FIG. 8. In some embodiments, the VH domain comprises a sequence having, having about, or having at least 70%, 71%, 72%, 73%, 74%, 75%, 75%, 1 / 0, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to a VH domain having a sequence in FIG. 8, or a percentage identity that is in a range defined by any two of the preceding values. For example, in some embodiments, the VH domain comprises a sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 70%-75%, 57%-100%, 75%-95%, 75%-90%, 75%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to a VH domain having a sequence in FIG. 8. In some embodiments, the VH is based on a human germline sequence of IGHV1 -69*01. In some embodiments, the VH is based on a human germline sequence of IGHJ4*01.

[0185] Table 4 depicts some embodiments of VL domain sequences disclosed herein. In some embodiments, the antibody or antigen binding fragment thereof comprises a VH domain having a sequence in Table 4. In some embodiments, the VH domain comprises a sequence having, having about, or having at least 70%, 71%, 72%, 73%, 74%, 75%, 75%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to a VH domain sequence in Table 4, or a percentage identity that is in a range defined by any two of the preceding values. Forexample, in some embodiments, the VH domain comprises a sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 70%-75%, 57%-100%, 75%-95%, 75%-90%, 75%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to a VH domain sequence in Table 4.Table 4

[0186] Also provided herein is an antigen binding construct (e.g., an antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) that includes a variable light (VL) domain that includes an amino acid sequence of a VL in Table 1; and a variable heavy (VH) domain that includes an amino acid sequence of a VH in Table 4. In some embodiments, the VH sequence from Table 4 and the VL sequence from Table 1 are paired as they are combined in any one of the antigen binding constructs set forth in FIGs. 13, 14, 15, 20, or 42.

[0187] In some embodiments, the antigen binding construct (e.g., an antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) includes a VL that is, is about, or is at least 70%, 71%, 72%, 73%, 74%, 75%, 75%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to any one of SEQ ID NOs: l-17, optionally the amino acid sequence has percent sequence identity to the any one of SEQ ID NOs: l-17 in a range defined by any two of the preceding values (e.g., 70%-100%, 75%-95%, 80%-100%, 80%-95%, 90%-100%, 95%- 100%, etc.); and a VH that is, is about, or is at least 70%, 71%, 72%, 73%, 74%, 75%, 75%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to any one of SEQ ID NOs: 18-34, 139, or 140, optionally the amino acid sequence has percent sequence identity to the any one of SEQ ID NOs: 18-34, 139, or 140 in a range defined by any two of the preceding values (e.g., 70%-100%, 75%-95%, 80%-100%, 80%-95%, 90%-100%, 95%-100%, etc.). In some embodiments, the VH sequence from Table 4 and the VL sequence from Table 1 are paired as they are combined in any one of the antigen binding constructs set forth in FIGs. 13, 14, 15, 20, or 42.

[0188] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K73D mutation (Aho). For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 70%-75%, 57%-100%, 75%-95%, 75%-90%, 75%-80%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-95%, or 95%-100% identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K73D mutation (Aho).

[0189] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K73S mutation (Aho). For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K73S mutation (Aho).

[0190] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K13V mutation (Aho). For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K13V mutation (Aho).

[0191] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K14Q mutation (Aho). For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K14Q mutation (Aho).

[0192] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K13V and K14Q mutation (Aho). For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%- 90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%- 90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%- 100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K13V and K14Q mutation (Aho).

[0193] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a D83S mutation (Aho). For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a D83S mutation (Aho).

[0194] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a E84N mutation (Aho). For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a E84N mutation (Aho).

[0195] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a D83S and E84N mutation (Aho). For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%- 90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%- 90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%- 100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a D83S and E84N mutation (Aho).

[0196] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a E92S mutation (Aho). For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a E92S mutation (Aho).

[0197] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K24A mutation (Aho). For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K24A mutation (Aho).

[0198] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a R97T A mutation (Aho). For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18 wherein the VH domain comprises a R97T mutation (Aho).

[0199] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises an E to Q mutation at a position corresponding to residue 1 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a E to Q mutation at a position corresponding to residue 1 of SEQ ID NO. 18.

[0200] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a Q to V mutation at a position corresponding to residue 5 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to aVH domain in SEQ ID NO. 18, wherein the VH domain comprises a Q to V mutation at a position corresponding to residue 5 of SEQ ID NO. 18.

[0201] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a P to A mutation at a position corresponding to residue 9 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a P to A mutation at a position corresponding to residue 9 of SEQ ID NO. 18.

[0202] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a L to V mutation at a position corresponding to residue 11 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a L to V mutation at a position corresponding to residue 11 of SEQ ID NO. 18.

[0203] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a V to K mutation at a position corresponding to residue 12 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-l 00%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a V to K mutation at a position corresponding to residue 12 of SEQ ID NO. 18.

[0204] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K to Q mutation at a position corresponding to residue 13 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K to Q mutation at a position corresponding to residue 13 of SEQ ID NO. 18.

[0205] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises an A to S mutation at a position corresponding to residue 16 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises an A to S mutation at a position corresponding to residue 16 of SEQ ID NO. 18.

[0206] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises an I to V mutation at a position corresponding to residue 20 of SEQ ID NO. 18. Forexample, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises an I to V mutation at a position corresponding to residue 20 of SEQ ID NO. 18.

[0207] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K to A mutation at a position corresponding to residue 23 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K to A mutation at a position corresponding to residue 23 of SEQ ID NO. 18.

[0208] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises an A to G mutation at a position corresponding to residue 26 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises an A to G mutation at a position corresponding to residue 26 of SEQ ID NO. 18.

[0209] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two ofthe preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises an A to S mutation at a position corresponding to residue 31 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises an A to S mutation at a position corresponding to residue 31 of SEQ ID NO. 18.

[0210] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K to R mutation at a position corresponding to residue 38 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K to R mutation at a position corresponding to residue 38 of SEQ ID NO. 18.

[0211] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a S to A mutation at a position corresponding to residue 40 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a S to A mutation at a position corresponding to residue 40 of SEQ ID NO. 18.

[0212] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a H to P mutation at a position corresponding to residue 41 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a H to P mutation at a position corresponding to residue 41 of SEQ ID NO. 18.

[0213] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K to Q mutation at a position corresponding to residue 43 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K to Q mutation at a position corresponding to residue 43 of SEQ ID NO. 18.

[0214] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a S to G mutation at a position corresponding to residue 44 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to aVH domain in SEQ ID NO. 18, wherein the VH domain comprises a S to G mutation at a position corresponding to residue 44 of SEQ ID NO. 18.

[0215] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises an I to M mutation at a position corresponding to residue 48 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises an I to M mutation at a position corresponding to residue 48 of SEQ ID NO. 18.

[0216] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a N to A mutation at a position corresponding to residue 61 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a N to A mutation at a position corresponding to residue 61 of SEQ ID NO. 18.

[0217] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K to D mutation at a position corresponding to residue 63 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-l 00%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K to D mutation at a position corresponding to residue 63 of SEQ ID NO. 18.

[0218] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K to S mutation at a position corresponding to residue 63 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K to S mutation at a position corresponding to residue 63 of SEQ ID NO. 18.

[0219] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K to Q mutation at a position corresponding to residue 65 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K to Q mutation at a position corresponding to residue 65 of SEQ ID NO. 18.

[0220] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K to R mutation at a position corresponding to residue 67 of SEQ ID NO. 18. Forexample, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K to R mutation at a position corresponding to residue 67 of SEQ ID NO. 18.

[0221] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises an A to V mutation at a position corresponding to residue 68 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises an A to V mutation at a position corresponding to residue 68 of SEQ ID NO. 18.

[0222] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a L to I mutation at a position corresponding to residue 70 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a L to I mutation at a position corresponding to residue 70 of SEQ ID NO. 18.

[0223] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two ofthe preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a V to A mutation at a position corresponding to residue 72 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a V to A mutation at a position corresponding to residue 72 of SEQ ID NO. 18.

[0224] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K to E mutation at a position corresponding to residue 74 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K to E mutation at a position corresponding to residue 74 of SEQ ID NO. 18.

[0225] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K to N mutation at a position corresponding to residue 74 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a K to N mutation at a position corresponding to residue 74 of SEQ ID NO. 18.

[0226] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a S to T mutation at a position corresponding to residue 76 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a S to T mutation at a position corresponding to residue 76 of SEQ ID NO. 18.

[0227] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a H to Y mutation at a position corresponding to residue 80 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a H to Y mutation at a position corresponding to residue 80 of SEQ ID NO. 18.

[0228] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a D to E mutation at a position corresponding to residue 82 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to aVH domain in SEQ ID NO. 18, wherein the VH domain comprises a D to E mutation at a position corresponding to residue 82 of SEQ ID NO. 18.

[0229] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a D to S mutation at a position corresponding to residue 82 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a D to S mutation at a position corresponding to residue 82 of SEQ ID NO. 18.

[0230] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a L to S mutation at a position corresponding to residue 84 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a L to S mutation at a position corresponding to residue 84 of SEQ ID NO. 18.

[0231] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a T to R mutation at a position corresponding to residue 87 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-l 00%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a T to R mutation at a position corresponding to residue 87 of SEQ ID NO. 18.

[0232] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a S to T mutation at a position corresponding to residue 91 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a S to T mutation at a position corresponding to residue 91 of SEQ ID NO. 18.

[0233] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a G to A mutation at a position corresponding to residue 97 of SEQ ID NO. 18. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a G to A mutation at a position corresponding to residue 97 of SEQ ID NO. 18.

[0234] In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a S to L mutation at a position corresponding to residue 117 of SEQ ID NO. 18.For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%- 90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%- 93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises a S to L mutation at a position corresponding to residue 117 of SEQ ID NO. 18.

[0235] In some embodiments, the VH domain comprises any one or more of the VH domain mutations (e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 or all of the non-overlapping VH domain mutations) provided herein.

[0236] Table 5 depicts some embodiments of HCDR sequences disclosed herein. In some embodiments, the antibody or antigen binding fragment thereof comprises a VH domain with a HCDR1 sequence that is a HCDR1 sequence in Table 5. In some embodiments, the antibody or antigen binding fragment thereof comprises a VH domain with a HCDR2 sequence that is a HCDR2 sequence in Table 5. In some embodiments, the antibody or antigen binding fragment thereof comprises a VH domain with an HCDR3 sequence that is a HCDR3 sequence in Table 5. In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain comprising a HCDR1, HCDR2, and / or HCDR3 in Table 5. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%- 100%, identity to a VH domain comprising a HCDR1, HCDR2, and / or HCDR3 in Table 5.Table 5.

[0237] Provided herein is an antigen binding construct (e.g., an antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) that includes a variable heavy (VH) domain that includes a HCDR1 that is an HCDR1 in any one of SEQ ID NOs: 18-34, 139 or 140, a HCDR2 that is an HCDR2 in any one of SEQ ID NOs: 18-34, 139 or 140, and a HCDR3 that is an HCDR3 in any one of SEQ ID NOs: 18-34, 139 or 140. In some embodiments, the HCDR1 , HCDR2 and LCR3 are combined as they are arranged in each one of SEQ ID NOs: 18- 34, 139 or 140.

[0238] Also provided herein is an antigen binding construct (e.g., an antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) that includes a variable light (VL) domain that includes a LCDR1 that is an LCDR1 in Table 6, a LCDR2 that is an LCDR2 in Table 6, and a LCDR3 that is an LCDR3 in Table 6. In some embodiments, the LCDR1, LCDR2 and LCR3 are combined as they are arranged in each construct in Table 6. In some embodiments, the VL domain includes a LCDR1 of SEQ ID NO:35, a LCDR2 of SEQ ID NO:37, aLCDR3 of SEQ ID NO:38. In some embodiments, the VL domain includes a LCDR1 of SEQ ID NO:35, a LCDR2 of SEQ ID NO:37, a LCDR3 of SEQ ID NO:39. In some embodiments, the VL domain includes a LCDR1 of SEQ ID NO:36, a LCDR2 of SEQ ID NO:37, aLCDR3 of SEQ ID NO:39. In some embodiments, the VL domain includes a LCDR1 of SEQ ID NO:36, a LCDR2 of SEQ ID NO:37, a LCDR3 of SEQ ID NO:40.Table 6

[0239] Also provided herein is an antigen binding construct (e.g., an antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) that includes a variable heavy (VH) domain that includes a HCDR1 that is an HCDR1 in Table 6, a HCDR2 that is an HCDR2 in Table 6, and a HCDR3 that is an HCDR3 in Table 6. In some embodiments, the HCDR1, HCDR2 and LCR3 are combined as they are arranged in each construct in Table 6. Also provided herein is an antigen binding construct (e.g., an antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) that includes a variable light (VL) domain that includes a LCDR1 that is an LCDR1 in Table 6, a LCDR2 that is an LCDR2 in Table 6, and a LCDR3 that is an LCDR3 in Table 6; and a variable heavy (VH) domain that includes a HCDR1 that is an HCDR1 in Table 6, a HCDR2 that is an HCDR2 in Table 6, and a HCDR3 that is an HCDR3 in Table 6. In some embodiments, the LCDR1, LCDR2 and LCDR3 are combined as they are arranged in each construct in Table 6, and the HCDR1, HCDR2 and LCR3 are combined as they are arranged in a corresponding construct in Table 6.

[0240] Provided herein is a minibody of the present disclosure (e g., that binds to aV 6 integrin) includes an amino acid sequence that is, is about, or is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the sequences set forth in SEQ ID NOs: 103-118 or 133-135, optionally the amino acid sequence has a sequenceidentity to any one of the sequences set forth in SEQ ID NOs: 103-118 or 133-135 in a range defined by any two of the preceding values (e.g., 70-100%, 80-100%, 90-100%, 85-98%, 80- 95%, etc.). In some embodiments, the minibody includes any one of the sequences set forth in SEQ ID NOs: 103-118 or 133-135.

[0241] Table 7 lists some embodiments of substitutions in the VL, VH, CH2, CH3, and / or Fc domains of an antibody or antigen binding fragment thereof that, either alone, or in combination, may reduce healthy organ uptake of the antibody or antigen binding fragment thereof. In some embodiments, the VH domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises one or more mutations (e g. any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, or all of the non-overlapping mutations) in Table 7. For example, in some embodiments, the VH domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VH domain comprises one or more mutations in Table 7. In some embodiments, the VL domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VL domain in SEQ ID NO. 1, wherein the VL domain comprises one or more mutations in Table 7. For example, in some embodiments, the VL domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%- 100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%- 97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VL domain in SEQ ID NO. 1, wherein the VL domain comprises one or more mutations in Table 7. In some embodiments, the VL domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to a VL domain in SEQ ID NO. 1, and the VH domaincomprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VL and / or the VH domain comprises one or more mutations in Table 7. For example, in some embodiments, the VL domain comprises an amino acid sequence having between about 70%- 100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%- 97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VL domain in SEQ ID NO. 1, and the VH domain comprises an amino acid sequence having between about 70%- 100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%- 100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%- 97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 18, wherein the VL and / or the VH domain comprises one or more mutations in Table 7.Table 7.* X can be any amino acid other than K or R.

[0242] Also provided herein is an antigen binding construct (e.g., antibody or binding fragment thereof) that includes any one or more of the mutations or a combination thereof (e.g. any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or all of the non-overlapping mutations) in the VL, VH, CH2, CH3, and / or Fc domains set forth in Table 7. In some embodiments, the antigen binding construct (e.g., antibody or binding fragment thereof) binds specifically to any suitable antigen target. In some embodiments, the antigen binding construct (e.g., antibody or binding fragment thereof) specifically binds to DLL3, FAP, CD8, CD4, CD3, IFNy, Integrin ocVp6, FOLRoc, or PSMA. In some embodiments, the antigen binding construct (e.g., antibody or binding fragment thereof) includes any suitable combination of heavy chain CDR sequences and light chain CDR sequences. Non-limiting examples of combinations of suitable heavy chain and light chain CDR sequences are disclosed in, e.g., International Pub. Nos. WO2011 / 069019, WO2014 / 164553, WO2013 / 188693, WO2019 / 236684, and WO2023 / 283643, and International App. No. PCT / US2023 / 069710, filed July 6, 2023, each of which is incorporated herein by reference in its entirety. In some embodiments, the antigen binding construct is a minibody, diabody (e.g., cys-diabody), an scFv-Fc fusion, or a nanobody®-Fc.

[0243] Provided herein is a variant antigen binding construct (e.g., antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) that includes at least one disruptedcluster of surface-exposed, positively charged amino acids and / or at least one disrupted cluster of surface-exposed, negatively charged amino acids, where the variant antigen binding construct varies from an original antigen binding construct that includes an original cluster having at least two (e.g., 2, 3, 4, 5, 6 or more) surface-exposed, positively charged amino acids that are within about 30 angstroms of each other, and / or at least two (e.g., 2, 3, 4, 5, 6 or more) surface-exposed, negatively charged amino acids that are within about 30 angstroms of each other. The variant antigen binding construct can vary from the original antigen binding construct by having a substitution of at least one of the surface-exposed, positively charged and / or negatively charged amino acids of the original cluster, e.g., substitution of surface- exposed, positively charged amino acids, such as lysine and / or arginine, with negatively charged or non-charged amino acids, and / or substitution of surface-exposed, negatively charged amino acids, such as aspartate and / or glutamate, with non-charged amino acids. The substitution of the surface-exposed, positively charged and / or negatively charged amino acid(s) of the cluster can disrupt the original cluster (e.g., by having less surface-exposed positive or negative charges at the corresponding location on the variant antigen binding construct compared to the original antigen binding construct). In some embodiments, the original antigen binding construct can include a heavy chain and / or light chain, and the original cluster can include at least one surface-exposed positively charged and / or negatively charged amino acid from a non-FR2 domain of the light chain (e.g., a light chain CDR domain, and / or a FR1, FR3, and / or FR4 domain, and / or a CL domain), and / or from the heavy chain. In some embodiments, the original or variant antigen binding construct is any antibody construct. In some embodiments, this disruption is useful for constructs equal in size to or smaller than a minibody. In some embodiments, the antigen binding construct is a cys-diabody. In some embodiments, the antigen binding construct is a scFv-Fc (e g., a scFv fused to a Fc) or a nanobody®-Fc (e.g., a camelid nanobody® or single domain fragment fused to a Fc). In some embodiments, the antibody or binding fragment thereof does not comprise a disrupted positive patch. In some embodiments, the antibody or binding fragment thereof does not comprise a disrupted positive patch but does comprise a disrupted negative patch. In some embodiments, the antibody or binding fragment thereof comprises a disrupted positive patch but does not comprise a disrupted negative patch.

[0244] The distance between the surface-exposed positively charged and / or negatively charged amino acids in the cluster can be the distance between the center of mass of each amino acid residue in a suitable three-dimensional model of the antigen binding construct or a portion thereof. The positively charged and / or negatively charged amino acids of the cluster are surface exposed as determined by the Van der Waals radii, the Solvent Accessible Area, an isopotential surface representation of the electrostatic field as calculated by the Adaptive Poisson-Boltzmann Solver, or a combination thereof.

[0245] In general, the positively charged amino acids are amino acids that are positively charged under physiological conditions (e.g., physiological pH, or pH 5.6-7.5, pH 6.0-7.5, or pH 7.3-7.4). In some embodiments, the positively charged amino acids are selected from: arginine, lysine, and histidine. In some embodiments, the positively charged amino acids are selected from: arginine, and lysine. The amino group at the N-terminus of a polypeptide or protein can also provide a positive charge. The negatively charged or non-charged amino acids include amino acids that are not the positively charged amino acids under physiological conditions (e.g., physiological pH, or pH 5.6-7.5, pH 6.0-7.5, or pH 7.3-7.4). In some embodiments, the negatively charged amino acid is selected from: aspartate and glutamate. The carboxyl group at the C-terminus of a polypeptide or protein can also provide a negative charge. In some embodiments, the non-charged amino acid is selected from: alanine, glycine, asparagine, glutamine, isoleucine, leucine, methionine, valine, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, and methionine. In some embodiments, the negatively charged or non-charged amino acid is selected from: alanine, glycine, asparagine, glutamine, valine, serine, and threonine.

[0246] The surface-exposed, positively charged or negatively charged amino acids within a cluster can form a continuous patch of positive or negative charge, respectively, on the surface (e.g., isopotential electrostatic surface) of the antibody (e.g., original antibody having the cluster). In some embodiments, the cluster has at least two (e.g., 2, 3, 4, 5, 6 or more) surface-exposed, positively charged amino acids within about 30, 25, 20, 15, 14, 13, 12, 11, 10, or 5 angstroms or less of each other, or within a distance of each other in a range defined by any two of the preceding values (e.g., 30-5 angstroms, 30-10 angstroms, 20-5 angstroms, 15-10 angstroms, 12-10 angstroms of each other). In some embodiments, the at least two surface-exposed, positively charged amino acids of the cluster are within about 15 angstromsof each other. In some embodiments, where a cluster includes 3 or more surface-exposed, positively charged amino acids, the distance between the two residues that are the furthest apart from each other is within about 30 angstroms, e.g., 25, 20, 15, 14, 13, 12, 11, 10, or about 5 angstroms or less, or within a distance in a range defined by any two of the preceding values (e g., 30-5 angstroms, 30-10 angstroms, 20-5 angstroms, 15-10 angstroms, 12-10 angstroms). In some embodiments, there are no intervening surface-exposed, negatively charged amino acids between at least two (e.g., 2, 3, 4, 5, 6 or more) of the surface-exposed, positively charged amino acids of the cluster. In some embodiments, there are no surface-exposed, negatively charged amino acids between any two of the surface-exposed, positively charged amino acids of the cluster.

[0247] In some embodiments, the cluster has at least two (e.g., 2, 3, 4, 5, 6 or more) surface-exposed, negatively charged amino acids within about 30, 25, 20, 15, 14, 13, 12, 11, 10, or 5 angstroms or less of each other, or within a distance of each other in a range defined by any two of the preceding values (e.g., 30-5 angstroms, 30-10 angstroms, 20-5 angstroms, 15-10 angstroms, 12-10 angstroms of each other). In some embodiments, the at least two surface-exposed, negatively charged amino acids of the cluster are within about 15 angstroms of each other. In some embodiments, where a cluster includes 3 or more surface-exposed, negatively charged amino acids, the distance between the two residues that are the furthest apart from each other is within about 30 angstroms, e.g., 25, 20, 15, 14, 13, 12, 11, 10, or about 5 angstroms or less, or within a distance in a range defined by any two of the preceding values (e.g., 30-5 angstroms, 30-10 angstroms, 20-5 angstroms, 15-10 angstroms, 12-10 angstroms). In some embodiments, there are no intervening surface-exposed, positively charged amino acids between at least two (e.g., 2, 3, 4, 5, 6 or more) of the surface-exposed, negatively charged amino acids of the cluster. In some embodiments, there are no surface-exposed, positively charged amino acids between any two of the surface-exposed, negatively charged amino acids of the cluster.

[0248] In some embodiments, the original cluster is disrupted where the number of surface-exposed, positively charged and / or negatively charged amino acids is reduced by at least one (e.g., by one, two, three or more) in the variant antibody compared to the original antibody. In some embodiments, the variant antibody varies from the original antibody by up to 3 (e.g., 1, 2, or 3) amino acid substitutions (e.g., substitution of a positively charged with anegatively charged or non-charged amino acid, or substitution of a negatively charged with a non-charged amino acid) that disrupt the cluster. In some embodiments, the total positive charge attributed to the cluster is determined by use of algorithms suitable for solving the Poisson-Boltzmann equation such as the Adaptive Poisson-Boltzmann Solver (APBS).

[0249] In some embodiments, the cluster (e.g., cluster of two or more surface- exposed, positively charged amino acids) is disrupted where the number of surface-exposed, positively charged amino acids is reduced by, by about, or by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or by about 100%, or by a percentage in a range defined by any two of the preceding values (e.g., by 30-90%, 30-70%, 50-100%, 30-60%, etc.) in the variant antibody compared to the original antibody. In some embodiments, the original cluster is disrupted where the number of surface-exposed, positively charged amino acids is reduced by about 33% or more in the variant antibody compared to the original antibody. In some embodiments, the original cluster is disrupted where the number of surface-exposed, positively charged amino acids is reduced by about 66% or more in the variant antibody compared to the original antibody. In some embodiments, the variant antibody includes a disrupted cluster that has or has about 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 0%, or a percentage in a range defined by any two of the preceding values (e.g., 0-70%, 30-70%, 0-40%, etc.) of the total positive charge attributed to the original cluster in the original antibody due to disruption of the cluster. For example, and without limitation, the original antibody can have a cluster of 3 positively charged amino acids (e.g., lysines and / or arginines), and one of the positively charged amino acids can be substituted by a non-charged or negatively charged amino acid in the variant antibody, whereby the original cluster is disrupted. In some embodiments, the variant antibody includes a disrupted cluster that has about 66% or less total positive charge attributed to the cluster in the original antibody due to disruption of the cluster. In some embodiments, the variant antibody includes a disrupted cluster that has about 33% or less total positive charge attributed to the cluster in the original antibody due to disruption of the cluster.

[0250] In some embodiments, the cluster (e.g., cluster of two or more surface- exposed, negatively charged amino acids) is disrupted where the number of surface-exposed, negatively charged amino acids is reduced by, by about, or by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or by about 100%, or by a percentage in a range defined by any two of the preceding values (e.g., by 30-90%, 30-70%, 50-100%, 30-60%, etc.) in the variant antibodycompared to the original antibody. In some embodiments, the original cluster is disrupted where the number of surface-exposed, negatively charged amino acids is reduced by about 33% or more in the variant antibody compared to the original antibody. In some embodiments, the original cluster is disrupted where the number of surface-exposed, negatively charged amino acids is reduced by about 66% or more in the variant antibody compared to the original antibody. In some embodiments, the variant antibody includes a disrupted cluster that has or has about 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 0%, or a percentage in a range defined by any two of the preceding values (e.g., 0-70%, 30-70%, 0-40%, etc.) of the total negative charge attributed to the original cluster in the original antibody due to disruption of the cluster. For example and without limitation, the original antibody can have a cluster of 3 negatively charged amino acids (e.g., glutamates and / or aspartates), and one of the negatively charged amino acids can be substituted by a non-charged amino acid in the variant antibody, whereby the original cluster is disrupted. In some embodiments, the variant antibody includes a disrupted cluster that has about 66% or less total negative charge attributed to the cluster in the original antibody due to disruption of the cluster. In some embodiments, the variant antibody includes a disrupted cluster that has about 33% or less total negative charge attributed to the cluster in the original antibody due to disruption of the cluster. In some embodiments, the antibody or binding fragment thereof does not comprise a disrupted positive patch. In some embodiments, the antibody or binding fragment thereof does not comprise a disrupted positive patch but does comprise a disrupted negative patch. In some embodiments, the antibody or binding fragment thereof comprises a disrupted positive patch but does not comprise a disrupted negative patch.

[0251] The relevant features of amino acid residues (e.g., surface exposure, distance between amino acids) of the antibody can be determined using any suitable option. Suitable options include, without limitation, a resolved crystal structure, homology modeling, molecular dynamics simulation, Adaptive Poisson-Boltzmann Solver, Van der Waals radii, Solvent Accessible Area, or a combination thereof.

[0252] In some embodiments, at least two of the surface-exposed, positively charged amino acids of the cluster in the original antibody are within a contiguous stretch of 15 or fewer (e g., 15, 12, 10, 8, 7, 6, 5, 4, 3, or 2) residues in a polypeptide of the original antibody. In some embodiments, at least two of the surface-exposed, positively charged aminoacids of the cluster in the original antibody are at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, least 75, at least 100, at least 150, at least 200, or more residues apart, or are apart by a number of residues in a range defined by any two of the preceding values (e.g., 20-200, 20-50, 40-75, 30-100, 50-150, 100-200, etc.), in a polypeptide of the original antibody.

[0253] In some embodiments, at least two of the surface-exposed, negatively charged amino acids of the cluster in the original antibody are within a contiguous stretch of 15 or fewer (e.g., 15, 12, 10, 8, 7, 6, 5, 4, 3, or 2) residues in a polypeptide of the original antibody. In some embodiments, at least two of the surface-exposed, negatively charged amino acids of the cluster in the original antibody are at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, least 75, at least 100, at least 150, at least 200, or more residues apart, or are apart by a number of residues in a range defined by any two of the preceding values (e.g., 20-200, 20-50, 40-75, 30-100, 50-150, 100-200, etc.), in a polypeptide of the original antibody.

[0254] In some embodiments, the original antigen binding construct can include a heavy chain and / or light chain, and the cluster can include at least one surface-exposed, positively charged and / or negatively charged amino acid from a non-FR2 domain of the light chain (e.g., a light chain CDR domain, and / or a FR1, FR3, and / or FR4 domain, and / or a CL domain), and / or from the heavy chain, that disrupts the cluster when mutated. Any suitable original antigen binding construct can be used, as described herein. In some embodiments, the antibody or binding fragment thereof does not comprise a disrupted positive patch. In some embodiments, the antibody or binding fragment thereof does not comprise a disrupted positive patch but does comprise a disrupted negative patch. In some embodiments, the antibody or binding fragment thereof comprises a disrupted positive patch but does not comprise a disrupted negative patch.

[0255] A surface-exposed positively charged cluster is sometimes referred to herein as a “positive patch”. A surface-exposed negatively charged cluster is sometimes referred to herein as a “negative patch”. FIG. 19B-D provides illustrations of an original antigen binding construct, in this case an scFv, in 3-dimensional orientation highlighting exemplary positive patches and negative patches. Some embodiments herein provide substitution mutations for these patches which change and / or enhance biodistribution of the antigen binding construct, relative to the original antigen binding construct, for use in diagnostic, therapeutic and radiopharmaceutical therapy (RPT). The identity of the identifiedpositive patches and negative patches is provided in Table 12 and Table 13, respectively, infra. In some embodiments, the antibody or binding fragment thereof does not comprise a disrupted positive patch. In some embodiments, the antibody or binding fragment thereof does not comprise a disrupted positive patch but does comprise a disrupted negative patch. In some embodiments, the antibody or binding fragment thereof comprises a disrupted positive patch but does not comprise a disrupted negative patch.

[0256] In some embodiments, the variant antigen binding construct includes a substitution of one or more residues in Table 12 and / or Table 13. In some embodiments, the original antigen binding construct (e.g., antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) includes a heavy chain comprising one or more (e.g., any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all 11) of the following amino acid residues (Aho): K13, K14, K24, R45, K73, R77, R97, K47, K50, K53, and / or KI 10. In some embodiments, the variant antigen binding construct includes a substitution of one or more of K13, K14, K24, R45, K73, R77, R97, K47, K50, K53, and / or KI 10, with a negatively charged (Asp or Glu) or a non-charged amino acid (Gly, Ala, Vai, Cys, Pro, Leu, He, Met, Trp, Phe, Ser, Thr, Tyr, Asn, Gin). In some embodiments, the original antigen binding construct (e.g., antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) includes a light chain comprising one or more (e.g., any 1, 2, 3, or all 4) of the following amino acid residues (Aho): K47, K50, K53, and / or KI 10. In some embodiments, the variant antigen binding construct includes a substitution of one or more of K47, K50, K53, and / or KI 10, with a negatively charged (Asp or Glu) or a non-charged amino acid (Gly, Ala, Vai, Cys, Pro, Leu, He, Met, Trp, Phe, Ser, Thr, Tyr, Asn, Gin). In some embodiments, the original antigen binding construct (e.g., antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) includes a heavy chain comprising one or more (e.g., any 1,2, 3, or all 4) of the following amino acid residues (Aho): D67, D83, E84, and / or E92. In some embodiments, the variant antigen binding construct includes a substitution of one or more of D67, D83, E84, and / or E92, with a positively charged (Lys, Arg, His) or a non-charged amino acid (Gly, Ala, Vai, Cys, Pro, Leu, He, Met, Trp, Phe, Ser, Thr, Tyr, Asn, Gin). In some embodiments, the original antigen binding construct (e.g., antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) includes a light chain comprising one or more (e.g., any 1, 2,3, or all 4) of the following amino acid residues (Aho): E29, El 11, D17, and / or E147. In some embodiments, the variant antigen binding construct includes a substitution of one or more ofE29, Bi l l , D17, and / or E147, with a positively charged (Lys, Arg, His) or a non-charged amino acid (Gly, Ala, Vai, Cys, Pro, Leu, He, Met, Trp, Phe, Ser, Thr, Tyr, Asn, Gin). In some embodiments, the antibody or binding fragment thereof does not comprise a disrupted positive patch. In some embodiments, the antibody or binding fragment thereof does not comprise a disrupted positive patch but does comprise a disrupted negative patch. In some embodiments, the antibody or binding fragment thereof comprises a disrupted positive patch but does not comprise a disrupted negative patch.

[0257] In some embodiments, the original antigen binding construct (e.g., antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) includes a heavy chain comprising one or more (e.g., any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all 15) of the following amino acid residues (Aho): K13, K14, K24, R45, D67, K73, R77, D83, E84, E92, R97, K47, K50, K53, KI 10. In some embodiments, the variant antigen binding construct includes a substitution of one or more of K13, K14, K24, R45, K73, R77, R97, K47, K50, K53, KI 10 with a negatively charged (Asp or Glu) or a non-charged amino acid (Gly, Ala, Vai, Cys, Pro, Leu, He, Met, Trp, Phe, Ser, Thr, Tyr, Asn, Gin) and / or a substitution of one or more of D67, D83, E84, and / or E92, with a positively charged (Lys, Arg, His) or a non-charged amino acid (Gly, Ala, Vai, Cys, Pro, Leu, He, Met, Trp, Phe, Ser, Thr, Tyr, Asn, Gin). In some embodiments, the original antigen binding construct (e.g., antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) includes a light chain comprising one or more (e.g., any 1, 2, 3, 4, 5, 6, 7, or all 8) of the following amino acid residues (Aho): D17, E29, K47, K50, K53, KI 10, El 11, and / or E147. In some embodiments, the variant antigen binding construct includes a substitution of one or more of K47, K50, K53, and / or KI 10, with a negatively charged (Asp or Glu) or a non-charged amino acid (Gly, Ala, Vai, Cys, Pro, Leu, He, Met, Trp, Phe, Ser, Thr, Tyr, Asn, Gin) and / or a substitution of one or more of E29, El 11, D17, and / or E147, with a positively charged (Lys, Arg, His) or a non-charged amino acid (Gly, Ala, Vai, Cys, Pro, Leu, He, Met, Trp, Phe, Ser, Thr, Tyr, Asn, Gin). In some embodiments, the antibody or binding fragment thereof does not comprise a disrupted positive patch. In some embodiments, the antibody or binding fragment thereof does not comprise a disrupted positive patch but does comprise a disrupted negative patch. In some embodiments, the antibody or binding fragment thereof comprises a disrupted positive patch but does not comprise a disrupted negative patch.

[0258] In some embodiments, the antigen binding construct comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to an antigen binding construct in Table 12. For example, in some embodiments, the antigen binding construct comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%- 100%, identity to the amino acid sequence of an antigen binding construct in Table 12. In some embodiments, the antibody or binding fragment thereof does not comprise a disrupted positive patch. In some embodiments, the antibody or binding fragment thereof does not comprise a disrupted positive patch but does comprise a disrupted negative patch. In some embodiments, the antibody or binding fragment thereof comprises a disrupted positive patch but does not comprise a disrupted negative patch.

[0259] In some embodiments, the antigen binding construct comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to an antigen binding construct in Table 13. For example, in some embodiments, the antigen binding construct comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%- 100%, identity to the amino acid sequence of an antigen binding construct in Table 13. In some embodiments, the antibody or binding fragment thereof does not comprise a disrupted positive patch. In some embodiments, the antibody or binding fragment thereof does not comprise a disrupted positive patch but does comprise a disrupted negative patch. In some embodiments, the antibody or binding fragment thereof comprises a disrupted positive patch but does not comprise a disrupted negative patch.

[0260] On the basis of this description, those skilled in the art may identify additional positive and / or negative patches on antigen binding constructs which can besubstituted to change and / or enhance biodistribution relative to an original antigen binding construct.

[0261] In some embodiments, the original antigen binding construct (e.g., antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) includes a light chain variable region (VL), where the VL includes one or more (e.g., any 1, 2, or all 3) of the following amino acid residues that is mutated in the variant antigen binding construct (Aho): E29, KI 10, and El 11. In some embodiments, a glutamate (e.g., E29, El l 1) in the VL of the original antigen binding construct is substituted with a non-charged amino acid (e.g., glutamine or serine). In some embodiments, a lysine (e.g., KI 10) in the VL of the original antigen binding construct is substituted with a negatively charged or a non-charged amino acid (e.g., to a noncharged amino acid, for example, glycine). In some embodiments, the variant antigen binding construct includes one or more (e.g., any 1, 2, or all 3) of the following mutations in the VL relative to the original antigen binding construct (Aho): E29Q, K110G, El l IS. In some embodiments, the VL of the original antigen binding construct is further mutated at one or both of the following lysine residues to a negatively charged or a non-charged amino acid (e.g., to a non-charged amino acid, for example, glutamine) (Aho): K50, K53. In some embodiments, the variant antigen binding construct further includes one or more of the following mutations in the VL relative to the original antigen binding construct (Aho): K50Q, K53Q. In some embodiments, the variant antigen binding construct includes a substitution in the following residues in the VL from the original antigen binding construct to a negatively charged or a noncharged amino acid (e.g., to a non-charged amino acid, for example, glutamine or glycine) (Aho): K50, K53, KI 10. In some embodiments, the variant antigen binding construct includes the following mutations in the VL relative to the original antigen binding construct (Aho): K50Q, K53Q, K110G.

[0262] In some embodiments, the variant antigen binding construct includes a VL having one or more (e.g., any 1, 2, or all 3) of the following amino acid residues (Aho): Q29, G110, Si l l. In some embodiments, the variant antigen binding construct includes a VL having the following amino acid residues (Aho): Q50, Q53, Gi l l .

[0263] In some embodiments, the original antigen binding construct (e.g., antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) includes a polypeptide (e.g., a light chain polypeptide) having a cluster of positively charged amino acids that is withina VL FR2. In some embodiments, the VL FR2 includes the amino acid sequence of XiX2X3X4X5X6X7 (SEQ ID NO: 81), wherein Xi, X2, X3, X4, X5, X6, and X7are each independently any amino acid, wherein the at least two (e.g., 2, 3, 4, or more) surface-exposed, positively charged amino acids comprise at least two of Xi, X2, X3, X4, Xs, Xe, and X7. In some embodiments, X4is position 50 and X7is position 53, as defined by Aho numbering. In some embodiments, the at least two surface-exposed, positively charged amino acids comprise at least Xi. In some embodiments, the at least two surface-exposed, positively charged amino acids comprise at least X4and X7. In some embodiments, X2, X3, Xs, and Xe, are each independently any negatively charged or non-charged amino acid residue in the original polypeptide. In some embodiments, X2, X3, Xs, and Xe are each independently any noncharged amino acid. In some embodiments, at least one of X4and X7are substituted with a negatively charged or non-charged amino acid in the variant antigen binding construct. In some embodiments, each of X4and X7is independently substituted with a non-charged amino acid in the variant antigen binding construct. In some embodiments, the at least two surface- exposed, positively charged amino acids comprise arginine, lysine and / or histidine.

[0264] In some embodiments, the original antigen binding construct (e.g., antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) includes at least one of the following sequences as at least a portion of FR2: KX2X3KX5Xr,K (SEQ ID NO: 82), where X2, X3, Xs, and Xe are each independently a negatively charged or a non-charged amino acid (e.g., where the second K is position 50 and the third K is position 53 as defined by Aho numbering); KX2X3X4X5X6R (SEQ ID NO: 123), where X2, X3, X4, X5, and X6are each independently a negatively charged or a non-charged amino acid; or KX2X3X4XsXeK (SEQ ID NO: 124), where X2, X3, X4, Xs, and Xe are each independently a negatively charged or a non-charged amino acid. In some embodiments, the original antigen binding construct includes at least one of the following sequences as at least a portion of FR2: KPGKAPK (SEQ ID NO: 125), KPGQAPR (SEQ ID NO: 126), KPEKAPK (SEQ ID NO: 127), KPGKVPK (SEQ ID NO: 128), KPGQPPR (SEQ ID NO: 129), KPGQSPR (SEQ ID NO: 130), KPGLAPR (SEQ ID NO: 131), or KPGQPPK (SEQ ID NO: 132), corresponding to XiX2X3X4X5X6X7(SEQ ID NO: 81) (e g , where X4is position 50 and X7is position 53, as defined by Aho numbering). In some embodiments, the original antigen binding construct includes at least KPGKAPK (SEQ ID NO: 125) or KPGQAPR (SEQ ID NO: 126), corresponding toX1X2X3X4X5X6X7 (SEQ ID NO: 81), as at least a portion of FR2 (e g., where X4 is position 50 and X7 is position 53, as defined by Aho numbering).

[0265] In some embodiments, the variant antigen binding construct includes a VL having the following in at least a portion of FR2: KPGQSPQ (SEQ ID NO: 127) or KPGQAPQ (SEQ ID NO: 128) (e.g., where the first Q is position 50 and the second is position 53, as defined by Aho numbering).

[0266] In some embodiments, the original antigen binding construct (e.g., antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) includes a heavy chain variable region (VH), where the VH includes one or more (e.g., any 1, 2, 3, 4, 5, 6, 7, or all 8) of the following amino acid residues (Aho): K13, K14, K24, K73, D83, E84, E92, R97. In some embodiments, a glutamate or aspartate (e.g., D83, E84, E92) in the VH of the original antigen binding construct is substituted with a non-charged amino acid (e.g., serine or asparagine). In some embodiments, a lysine or arginine (e.g., K13, K14, K24, K73, R97) in the VH of the original antigen binding construct is substituted with a negatively charged or a non-charged amino acid (e.g., valine, glutamine, alanine, aspartate, serine, threonine). In some embodiments, the variant antigen binding construct includes one or more (e g., any 1, or any non-overlapping 2, 3, 4, 5, 6, 7, 8) of the following mutations in the VH relative to the original antigen binding construct (Aho): K13V, K14Q, K24A, K73D, K73S, D83S, E84N, E92S, R97T. In some embodiments, the variant antigen binding construct includes a substitution in the following residue in the VH from the original antigen binding construct to a negatively charged or a non-charged amino acid (e.g., aspartate or serine) (Aho): K73. In some embodiments, the variant antigen binding construct includes the following mutation in the VH relative to the original antigen binding construct (Aho): K73D. In some embodiments, the variant antigen binding construct includes the following mutation in the VH relative to the original antigen binding construct (Aho): K73S. In some embodiments, the variant antigen binding construct includes a substitution in the following residues in the VH from the original antigen binding construct to a negatively charged or a non-charged amino acid (e.g., to a noncharged amino acid, for example, valine, glutamine or serine) (Aho): K13, K14, K73. In some embodiments, the variant antigen binding construct includes the following mutations in the VH relative to the original antigen binding construct (Aho): K13V, K14Q, K73S. In some embodiments, the variant antigen binding construct includes a substitution in the followingresidues in the VH from the original antigen binding construct (e.g., substitution of a lysine with a negatively charged or a non-charged amino acid, and substitution of an aspartate or glutamate with a non-charged amino acid) (Aho): K13, K14, K73, D83, E84, E92. In some embodiments, the variant antigen binding construct includes the following mutations in the VH relative to the original antigen binding construct (Aho): K13V, K14Q, K73S, D83S, E84N, E92S. In some embodiments, the variant antigen binding construct includes a substitution in the following residues in the VH from the original antigen binding construct (e.g., substitution of a lysine with a negatively charged or a non-charged amino acid, and substitution of an aspartate or glutamate with a non-charged amino acid) (Aho): K13, K14, K24, K73, D83, E84, E92. In some embodiments, the variant antigen binding construct includes the following mutations in the VH relative to the original antigen binding construct (Aho): K13V, K14Q, K24A, K73S, D83S, E84N, E92S.

[0267] In some embodiments, the variant antigen binding construct (e.g., antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) includes a VH having one or more (e.g., any 1, 2, 3, 4, 5, 6, 7, 8, or all 9) of the following amino acid residues (Aho): V13, Q14, A24, D73, S73, S83, N84, S92, T97. In some embodiments, the variant antigen binding construct includes a VH having the following amino acid residue (Aho): S73. In some embodiments, the variant antigen binding construct includes a VH having the following amino acid residue (Aho): D73. In some embodiments, the variant antigen binding construct includes a VH having the following amino acid residues (Aho): V13, Q14, S73. In some embodiments, the variant antigen binding construct includes a VH having the following amino acid residues (Aho): V13, Q14, S73, S83, N84, S92. In some embodiments, the variant antigen binding construct includes a VH having the following amino acid residues (Aho): V13, Q14, A24, S73, S83, N84, S92.

[0268] In some embodiments, the variant antigen binding construct (e.g., antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) includes a substitution in the following residues in the VL from the original antigen binding construct to a negatively charged or a non-charged amino acid (e.g., to a non-charged amino acid, for example, glutamine or glycine) (Aho): K50, K53, KI 10; and a substitution in the following residue in the VH from the original antigen binding construct to a negatively charged or a non-charged amino acid (e.g., aspartate or serine) (Aho): K73. In some embodiments, the variant antigenbinding construct includes the following mutations in the VL relative to the original antigen binding construct (Aho): K50Q, K53Q, KI 10G; and the following mutation in the VH relative to the original antigen binding construct (Aho): K73D. In some embodiments, the variant antigen binding construct includes the following mutations in the VL relative to the original antigen binding construct (Aho): K50Q, K53Q, KI 10G; and the following mutation in the VH relative to the original antigen binding construct (Aho): K73S. In some embodiments, the variant antigen binding construct includes a substitution in the following residues in the VL relative to the original antigen binding construct to a negatively charged or a non-charged amino acid (e.g., substitution of a lysine with a negatively charged or a non-charged amino acid, and substitution of a glutamate with a non-charged amino acid) (Aho): E29, K50, K53, KI 10, El 11; and a substitution in the following residue in the VH from the original antigen binding construct to a negatively charged or a non-charged amino acid (e.g., valine, glutamine, serine) (Aho): KI 3, KI 4, K73. In some embodiments, the variant antigen binding construct includes the following mutations in the VL relative to the original antigen binding construct (Aho): E29Q, K50Q, K53Q, KI 10G, El 11 S; and the following mutation in the VH relative to the original antigen binding construct (Aho): K13V, K14Q, K73S. In some embodiments, the variant antigen binding construct includes a substitution in the following residues in the VL from the original antigen binding construct to a negatively charged or a non-charged amino acid (e.g., substitution of a lysine with a negatively charged or a non-charged amino acid, and substitution of a glutamate with a non-charged amino acid) (Aho): E29, K50, K53, KI 10, El 11; and a substitution in the following residues in the VH from the original antigen binding construct (e.g., substitution of a lysine with a negatively charged or a non-charged amino acid, and substitution of an aspartate or glutamate with a non-charged amino acid) (Aho): K13, K14, K24, K73, D83, E84, E92. In some embodiments, the variant antigen binding construct includes the following mutations in the VL relative to the original antigen binding construct (Aho): E29Q, K50Q, K53Q, KI 10G, El 11 S; and the following mutation in the VH relative to the original antigen binding construct (Aho): K13V, K14Q, K24A, K73S, D83S, E84N, E92S. In some embodiments, the variant antigen binding construct includes a substitution in the following residues in the VL from the original antigen binding construct to a negatively charged or a non-charged amino acid (e g., substitution of a lysine with a negatively charged or a non-charged amino acid, and substitution of a glutamate with a non-charged amino acid)(Aho): E29, KAO, K53, KI 10, El 11 ; and a substitution in the following residues in the VH from the original antigen binding construct (e.g., substitution of a lysine or arginine with a negatively charged or a non-charged amino acid, and substitution of an aspartate or glutamate with a non-charged amino acid) (Aho): K13, K14, K24, K73, D83, E84, E92, R97. In some embodiments, the variant antigen binding construct includes the following mutations in the VL relative to the original antigen binding construct (Aho): E29Q, K50Q, K53Q, KI 10G, El 1 IS; and the following mutation in the VH relative to the original antigen binding construct (Aho): K13V, K14Q, K24A, K73S, D83S, E84N, E92S, R97T.

[0269] In some embodiments, the variant antigen binding construct (e.g., antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) includes a VL having the following amino acid residues (Aho): Q50, Q53, Gi l l; and a VH having the following amino acid residue (Aho): D73. In some embodiments, the variant antigen binding construct includes a VL having the following amino acid residues (Aho): Q50, Q53, Gi l l; and a VH having the following amino acid residue (Aho): S73. In some embodiments, the variant antigen binding construct includes a VL having the following amino acid residues (Aho): Q29, Q50, Q53, G110, Si l l; and a VH having the following amino acid residues (Aho): V13, Q14, S73. In some embodiments, the variant antigen binding construct includes a VL having the following amino acid residues (Aho): Q29, Q50, Q53, G110, Si l l; and a VH having the following amino acid residues (Aho): V13, Q14, A24, S73, S83, N84, S92. In some embodiments, the variant antigen binding construct includes a VL having the following amino acid residues (Aho): Q29, Q50, Q53, G110, Si l l; and a VH having the following amino acid residues (Aho): V13, Q14, A24, S73, S83, N84, S92, T97.

[0270] In some embodiments, the original antigen binding construct (e.g., antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) includes a variable region (e.g., VL and / or VH) that is based on a human germline sequence. In some embodiments, the original antigen binding construct includes a VL that is based on a human germline sequence IGKV1 family. In some embodiments, the FR of the VL is the corresponding FR of a human germline sequence belonging to the IGKV1 family, with no substitution, or up to 1, 2, 3, 4, or 5 substitutions thereto. In some embodiments, the VL (e.g., the VL FR sequences) is based on a human germline sequence IGKV1-39*O1. In some embodiments, the FR of the VL is the corresponding FR of a human germline sequence IGKV1-39*O1, with no substitution, or up to1, 2, 3, 4, or 5 or more substitutions thereto. In some embodiments, the FR of the VL is the corresponding FR of a human germline sequence IGKJ4*01, with no substitution, or up to 1,2, 3, 4, or 5 or more substitutions thereto. In some embodiments, the FR of the VH is the corresponding FR of a human germline sequence belonging to the IGHV1-69*O1 family, with no substitution, or up to 1, 2, 3, 4, or 5 substitutions thereto. In some embodiments, the VH (e.g., the VH FR sequences) is based on a human germline sequence IGHJ4*01. In some embodiments, the FR of the VH is the corresponding FR of a human germline sequence IGHV1-69*O1, with no substitution, or up to 1, 2, 3, 4, or 5 or more substitutions thereto. In some embodiments, the FR of the VH is the corresponding FR of a human germline sequence IGHJ4*01, with no substitution, or up to 1, 2, 3, 4, or 5 or more substitutions thereto.

[0271] In some embodiments, the original antigen binding construct (e.g., a full- length antibody, minibody, a scFv-Fc fusion, a nanobody®-Fc) includes a heavy chain having a CH3 domain, where the CH3 domain includes one or more (e.g., any 1, 2, 3, 4, 5, 6, or all 7) of the following amino acid residues (EU numbering): R355, E356, D376, E380, E382, K414, and E430. In some embodiments, a glutamate or aspartate (e.g., E356, D376, E380, E382, E430) in the CH3 domain of the original antigen binding construct is substituted with a noncharged amino acid (e.g., serine, threonine). In some embodiments, a lysine or arginine (e.g., R355, K414) in the CH3 domain of the original antigen binding construct is substituted with a negatively charged or a non-charged amino acid (e.g., to a non-charged amino acid, for example, glutamine, threonine). In some embodiments, the variant antigen binding construct includes one or more (e.g., any 1, 2, 3, 4, 5, 6, or all 7) of the following mutations in the CH3 domain relative to the original antigen binding construct (EU numbering): R355Q, E356S, D376S, E380S, E382T, K414T, and E430S. In some embodiments, the variant antigen binding construct includes a substitution in the following residues in the CH3 domain of the original antigen binding construct (e.g., substitution of a lysine or arginine with a negatively charged or a non-charged amino acid, and substitution of an aspartate or glutamate with a non-charged amino acid) (EU numbering): R355, D376, E380, E382, K414, and E430. In some embodiments, the variant antigen binding construct includes the following mutations in the CH3 domain relative to the original antigen binding construct (Aho): R355Q, D376S, E380S, E382T, K414T, and E430S.

[0272] In some embodiments, the variant antigen binding construct (e.g., antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) includes a CH3 domain having any one (e.g., any 1, 2, 3, 4, 5, 6, or all 7) of the following amino acid residues (EU numbering): Q355, S356, S376, S380, T382, T414, and S430. In some embodiments, the variant antigen binding construct includes a CH3 domain having the following amino acid residues (EU numbering): Q355, S356, S376, S380, T382, T414, and S430.

[0273] In some embodiments, the original antigen binding construct (e.g., antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) includes a CH3 domain of a human IgGl . In some embodiments, the original antigen binding construct includes a CH3 domain having an amino acid sequence of a CH3 domain in SEQ ID NO: 120. In some embodiments, the original antigen binding construct includes a CH3 domain having an amino acid sequence that is, that is about, or that is at least 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or that is 100%, or a percentage in a range defined by any two of the preceding values (e.g., 75-100%, 80-99%, 85-95%, 80-98%, etc.) identical to a CH3 domain in SEQ ID NO: 120. In some embodiments, the variant antigen binding construct includes a CH3 domain having an amino acid sequence of a CH3 domain in SEQ ID NO: 121. In some embodiments, the variant antigen binding construct includes a CH3 domain having an amino acid sequence that is, that is about, or that is at least 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or that is 100%, or a percentage in a range defined by any two of the preceding values (e.g., 75-100%, 80-99%, 85-95%, 80-98%, etc.) identical to a CH3 domain in SEQ ID NO: 120.

[0274] In some embodiments, the original antigen binding construct (e.g., antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) includes a hinge region comprising an upper hinge that includes the sequence EPKSSDKTHT (SEQ ID NO: 51), and the variant antigen binding construct varies by having a mutation in the upper hinge (e.g., to substitute a lysine with a negatively charged or a non-charged amino acid, for example, a serine). In some embodiments, the variant antigen binding construct varies by having a substitution of the upper hinge sequence of the original antigen binding construct of EPKSSDKTHT (SEQ ID NO: 51) with a Gly-Ser-rich linker sequence. In some embodiments, the variant antigen binding construct varies by having a substitution of the upper hinge sequence of the original antigen binding construct of EPKSSDKTHT (SEQ ID NO:51) with GGSGGGSTHT (SEQ ID NO: 52). In some embodiments, the original antigen binding construct includes a hinge region comprising EPKSSDKTHTCPPCPPCGGGSSGGGSG (SEQ ID NO: 137), and the variant antigen binding construct varies by having a mutation in the hinge region (e.g., to substitute a lysine with a negatively charged or a non-charged amino acid, for example, a serine). In some embodiments, the variant antigen binding construct varies by having a substitution of the upper hinge sequence of the original antigen binding construct of EPKSSDKTHTCPPCPPCGGGSSGGGSG (SEQ ID NO: 137) with GGSGGGSTHTCPPCPPCGGGSSGGGSGG (SEQ ID NO: 138) In some embodiments, the variant antigen binding construct includes a hinge region comprising at least one of EPKSSDKTHT (SEQ ID NO: 51) and EPKSSDKTHTCPPCPPCGGGSSGGGSG (SEQ ID NO: 137)

[0275] In some embodiments, the variant antigen binding construct (e.g., antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) includes a substitution in the following residues in the VL from the original antigen binding construct to a negatively charged or a non-charged amino acid (e g., substitution of a lysine with a negatively charged or a non-charged amino acid, and substitution of a glutamate with a non-charged amino acid) (Aho): E29, K50, K53, KI 10, El 11; a substitution in the following residues in the VH from the original antigen binding construct (e.g., substitution of a lysine with a negatively charged or a non-charged amino acid, and substitution of an aspartate or glutamate with a non-charged amino acid) (Aho): K13, K14, K24, K73, D83, E84, E92; and a substitution in the following residues in the CH3 domain from the original antigen binding construct (e.g., substitution of a lysine or arginine with a negatively charged or a non-charged amino acid, and substitution of an aspartate or glutamate with a non-charged amino acid) (EU numbering): R355, D376, E380, E382, K414, and E430. In some embodiments, the variant antigen binding construct includes the following mutations in the VL relative to the original antigen binding construct (Aho): E29Q, K50Q, K53Q, KI 10G, El 11 S; the following mutation in the VH relative to the original antigen binding construct (Aho): K13V, K14Q, K24A, K73S, D83S, E84N, E92S; and the following mutations in the CH3 domain relative to the original antigen binding construct (Aho): R355Q, D376S, E380S, E382T, K414T, and E430S.

[0276] In some embodiments, the variant antigen binding construct (e.g., antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) includes a substitution in the following residues in the VL from the original antigen binding construct to a negatively charged or a non-charged amino acid (e.g., substitution of a lysine with a negatively charged or a non-charged amino acid, and substitution of a glutamate with a non-charged amino acid) (Aho): E29, K50, K53, KI 10, El 11; a substitution in the following residues in the VH from the original antigen binding construct (e.g., substitution of a lysine with a negatively charged or a non-charged amino acid, and substitution of an aspartate or glutamate with a non-charged amino acid) (Aho): K13, K14, K24, K73, D83, E84, E92; a substitution in the following residues in the CH3 domain from the original antigen binding construct (e.g., substitution of a lysine or arginine with a negatively charged or a non-charged amino acid, and substitution of an aspartate or glutamate with a non-charged amino acid) (EU numbering): R355, D376, E380, E382, K414, and E430; and a substitution of the upper hinge sequence of EPKSSDKTHT (SEQ ID NO: 51) with a Gly-Ser-rich linker sequence (e.g., GGSGGGSTHT (SEQ ID NO: 52)). In some embodiments, the variant antigen binding construct includes the following mutations in the VL relative to the original antigen binding construct (Aho): E29Q, K50Q, K53Q, K110G, El l IS; the following mutation in the VH relative to the original antigen binding construct (Aho): K13V, K14Q, K24A, K73S, D83S, E84N, E92S; the following mutations in the CH3 domain relative to the original antigen binding construct (Aho): R355Q, D376S, E380S, E382T, K414T, and E430S; and a substitution of the hinge region having a sequence EPKSSDKTHTCPPCPPCGGGSSGGGSG (SEQ ID NO: 137) with GGSGGGSTHTCPPCPPCGGGSSGGGSG (SEQ ID NO: 138)

[0277] In some embodiments, the variant antigen binding construct (e.g., antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) includes a substitution in the following residues in the VL from the original antigen binding construct to a negatively charged or a non-charged amino acid (e.g., substitution of a lysine with a negatively charged or a non-charged amino acid, and substitution of a glutamate with a non-charged amino acid) (Aho): E29, K50, K53, KI 10, El 11; a substitution in the following residues in the VH from the original antigen binding construct (e.g., substitution of a lysine with a negatively charged or a non-charged amino acid, and substitution of an aspartate or glutamate with a non-charged amino acid) (Aho): K13, K14, K24, K73, D83, E84, E92; a substitution in the followingresidues in the CH3 domain from the original antigen binding construct (e.g., substitution of a lysine or arginine with a negatively charged or a non-charged amino acid, and substitution of an aspartate or glutamate with a non-charged amino acid) (EU numbering): R355, E356, D376, E380, E382, K414, and E430; and a substitution of the upper hinge sequence of EPKSSDKTHT (SEQ ID NO: 51) with a Gly-Ser-rich linker sequence (e.g., GGSGGGSTHT (SEQ ID NO: 52)). In some embodiments, the variant antigen binding construct includes the following mutations in the VL relative to the original antigen binding construct (Aho): E29Q, K50Q, K53Q, K110G, El l IS; the following mutation in the VH relative to the original antigen binding construct (Aho): K13V, K14Q, K24A, K73S, D83S, E84N, E92S; the following mutations in the CH3 domain relative to the original antigen binding construct (Aho): R355Q, E356S, D376S, E380S, E382T, K414T, and E430S; and a substitution of the hinge region having a sequence EPKSSDKTHTCPPCPPCGGGSSGGGSG (SEQ ID NO: 137) with GGSGGGSTHTCPPCPPCGGGSSGGGSG (SEQ ID NO: 138)

[0278] In some embodiments, the variant antigen binding construct (e.g., antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) includes a VL having the following amino acid residues (Aho): Q29, Q50, Q53, G110, Si l l; a VH having the following amino acid residues (Aho): V13, Q14, A24, S73, S83, N84, S92; and a CH3 domain having the following amino acid residues (EU numbering): Q355, S356, S376, S380, T382, T414, and S430. In some embodiments, the variant antigen binding construct includes a hinge region comprising at least one of EPKSSDKTHT (SEQ ID NO: 51), GGSGGGSTHT (SEQ ID NO: 52), EPKSSDKTHTCPPCPPCGGGSSGGGSG (SEQ ID NO: 137), and GGSGGGSTHTCPPCPPCGGGSSGGGSG (SEQ ID NO: 138) In some embodiments, the variant antigen binding construct includes a VL having the following amino acid residues (Aho): Q29, Q50, Q53, G110, Si l l; a VH having the following amino acid residues (Aho): V13, Q14, A24, S73, S83, N84, S92; a CH3 domain having the following amino acid residues (EU numbering): Q355, S376, S380, T382, T414, and S430; and an upper hinge region having the sequence GGSGGGSTHT (SEQ ID NO: 52). In some embodiments, the variant antigen binding construct includes a VL having the following amino acid residues (Aho): Q29, Q50, Q53, G110, Si l l; a VH having the following amino acid residues (Aho): V13, Q14, A24, S73, S83, N84, S92; a CH3 domain having the following amino acid residues (EU numbering):Q355, S356, S376, S380, T382, T414, and S430; and an upper hinge region having the sequence GGSGGGSTHT (SEQ ID NO: 52). In some embodiments, the variant antigen binding construct includes a VL having the following amino acid residues (Aho): Q29, Q50, Q53, G110, Si l l; a VH having the following amino acid residues (Aho): V13, Q14, A24, S73, S83, N84, S92; a CH3 domain having the following amino acid residues (EU numbering): Q355, S376, S380, T382, T414, and S430; and a hinge region having the sequence GGSGGGSTHTCPPCPPCGGGSSGGGSG (SEQ ID NO: 138) In some embodiments, the variant antigen binding construct includes a VL having the following amino acid residues (Aho): Q29, Q50, Q53, G110, Si l l; a VH having the following amino acid residues (Aho): V13, Q14, A24, S73, S83, N84, S92; a CH3 domain having the following amino acid residues (EU numbering): Q355, S356, S376, S380, T382, T414, and S430; and a hinge region having the sequence GGSGGGSTHTCPPCPPCGGGSSGGGSG (SEQ ID NO: 138)

[0279] In some embodiments, the original antigen binding construct (e.g., a full- length antibody, a scFv-Fc fusion, a nanobody®-Fc) includes a Fc domain, where the Fc domain includes one or more (e.g., any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or all 14) of the following amino acid residues (EU numbering): E233, D265, E269, D270, E272, D280, E294, R355, E356, D376, E380, E382, K414, and E430. In some embodiments, a glutamate or aspartate (e.g., E233, D265, E269, D270, E272, D280, E294, E356, D376, E380, E382, E430) in the Fc domain of the original antigen binding construct is substituted with a non-charged amino acid (e.g., serine or threonine). In some embodiments, a lysine or arginine (e.g., R355, K414) in the Fc domain of the original antigen binding construct is substituted with a negatively charged or a non-charged amino acid (e.g., to a non-charged amino acid, for example, glutamine, threonine). In some embodiments, the variant antigen binding construct includes one or more (e.g., any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or all 14) of the following mutations in the Fc domain relative to the original antigen binding construct (EU numbering): E233S, D265S, E269S, D270S, E272S, D280S, E294S, R355Q, E356S, D376S, E380S, E382T, K414T, and E430S. In some embodiments, the variant antigen binding construct includes a substitution in the following residue in the Fc domain from the original antigen binding construct (e.g., to a non-charged amino acid, for example, serine) (EU numbering): E269. In some embodiments, the variant antigen binding construct includes the following mutation in the Fc domain relative to the original antigen binding construct (EU numbering):E269S. In some embodiments, the variant antigen binding construct includes a substitution in the following residues in the Fc domain from the original antigen binding construct (e.g., to a non-charged amino acid, for example, serine) (EU numbering): E233, E269. In some embodiments, the variant antigen binding construct includes the following mutations in the Fc domain relative to the original antigen binding construct (EU numbering): E233S, E269S. In some embodiments, the variant antigen binding construct includes a substitution in the following residues in the Fc domain from the original antigen binding construct (e.g., to a noncharged amino acid, for example, serine) (EU numbering): E233, D265, E269. In some embodiments, the variant antigen binding construct includes the following mutations in the Fc domain relative to the original antigen binding construct (EU numbering): E233S, D265S, E269S. In some embodiments, the variant antigen binding construct includes a substitution in the following residues in the Fc domain from the original antigen binding construct (e.g., to a non-charged amino acid, for example, serine) (EU numbering): E233, D265, E269, D270. In some embodiments, the variant antigen binding construct includes the following mutations in the Fc domain relative to the original antigen binding construct (EU numbering): E233S, D265S, E269S, D270S.

[0280] In some embodiments, the variant antigen binding construct (e.g., a full- length antibody, a scFv-Fc fusion, a nanobody®-Fc) includes a Fc domain, where the Fc domain includes one or more (e.g., any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or all 14) of the following amino acid residues (EU numbering): S233, S265, S269, S270, S272, S280, S294, Q355, S356, S376S, S380, T382, T414, and S430. In some embodiments, the variant antigen binding construct includes a Fc domain that includes the following amino acid residue (EU numbering): S269. In some embodiments, the variant antigen binding construct includes a Fc domain that includes the following amino acid residues (EU numbering): S233, S269. In some embodiments, the variant antigen binding construct includes a Fc domain that includes the following amino acid residues (EU numbering): S233, S265, S269. In some embodiments, the variant antigen binding construct includes a Fc domain that includes the following amino acid residues (EU numbering): S233, S265, S269, S270.

[0281] In some embodiments, the variant antigen binding construct (e.g., antibody, minibody, cys-diabody, scFv-Fc fusion, nanobody®-Fc) includes any one or any combination of two or more of the mutations in the VL, VH, CH3 and / or Fc domains in the original antigenbinding construct, as described herein. In some embodiments, the Fc region (e.g., of the original or the variant antigen binding construct) includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) mutations (e.g., substitutions) that changes (e.g., increase or reduce) an effector function (e.g., FcyR binding) and / or binding to the Fc neonatal receptor (FcRn). In some embodiments, the one or more mutations that changes an effector function (e.g., FcyR binding) and / or binding to the Fc neonatal receptor (FcRn) is a mutation in one or more of the following (EU numbering): 1253, N297, H310, and H435. In some embodiments, the Fc region (e.g., of the original or the variant antigen binding construct) includes one or more of the following mutations following (EU numbering): I253A, N297Q, H310A, and H435A.

[0282] In some embodiments, the original antigen binding construct (e.g., antibody, scFv-Fc fusion, nanobody®-Fc) includes a Fc domain of a human IgGl . In some embodiments, the original antigen binding construct includes a Fc domain having an amino acid sequence of a Fc domain in SEQ ID NO:83. In some embodiments, the original antigen binding construct includes a Fc domain having an amino acid sequence that is, that is about, or that is at least 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or that is 100%, or a percentage in a range defined by any two of the preceding values (e.g., 75-100%, 80-99%, 85-95%, 80-98%, etc.) identical to a Fc domain in SEQ ID NO:83.

[0283] In some embodiments, the antibody or binding fragment thereof further comprises an IgG CH2 sequence. In some embodiments, the CH2 sequence comprises a sequence that is any one of SEQ ID NO. 57-76. In some embodiments, the antibody or binding fragment thereof further comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to an IgG CH2 that is any one of SEQ ID NO. 57-76. For example, in some embodiments, the antibody or binding fragment thereof further comprises an IgG CH2 sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to an IgG CH2 that is any one of SEQ ID NO. 57-76.

[0284] FIG. 9 depicts some embodiments of an alignment of IgG CH2 domain sequences disclosed herein. In some embodiments, the CH2 sequence comprises a sequenceFIG. 9. In some embodiments, the antibody or binding fragment thereof further comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to an IgG CH2 in FIG. 9. For example, in some embodiments, the antibody or binding fragment thereof further comprises an IgG CH2 sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%- 100%, identity to an IgG CH2 in FIG. 9.

[0285] Table 8 lists some embodiments of IgG CH2 sequences disclosed herein. In some embodiments, the CH2 sequence comprises a sequence that is any one of SEQ ID NO. 57-76. In some embodiments, the antibody or binding fragment thereof further comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to an IgG CH2 in Table 8. For example, in some embodiments, the antibody or binding fragment thereof further comprises an IgG CH2 sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%- 100%, identity to an IgG CH2 in Table 8.Table 8

[0286] In some embodiments, the antibody or antigen binding fragment thereof comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity to an IgG CH2 having a sequence that is SEQ ID NO. 57 or a percent identity to an IgG CH2 having a sequence that is SEQ ID NO. 57 that is in a range defined by any two of the preceding values. For example, in some embodiments, the antibody or antigen binding fragment thereof comprises an IgG CH2 sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to an IgG CH2 having a sequence that is SEQ ID NO. 57. In some embodiments, if the at least one mutation comprises a substitution at E233 (EU numbering), then (i) the substitution is with S, V, G, A, Q, N, T, K, R, H, Y, F, W, M, L, or I, optionally the substitution is with S, V, G, A, Q, N, or T, and further optionally the substitutionis with S, and / or (ii) the antigen binding construct comprises a second mutation in the IgG CH2 that is different from the substitution at E233 (EU numbering). In some embodiments, if the at least one mutation comprises a substitution at D265 (EU numbering), then (i) the substitution is with V, G, Q, N, T, R, P, F, W, M, L, or I, optionally the substitution is with V, G, Q, N, or T, and / or (ii) the antigen binding construct comprises a second mutation in the IgG CH2 that is different from the substitution at D265 (EU numbering). In some embodiments, if the at least one mutation comprises a substitution at E269 (EU numbering), then (i) the substitution is withS, V, G, A, T, K, R, H, P, Y, F, W, M, L, or I, optionally the substitution is with S, V, G, A, orT, and further optionally the substitution is with S, and / or (ii) the antigen binding construct comprises a second mutation that is different from the substitution at E269 (EU numbering). In some embodiments, if the at least one mutation comprises a substitution at D270 (EU numbering), then (i) the substitution is with S, V, Q, T, K, R, H, P, Y, F, W, L, or I, optionally the substitution is with S, V, Q, or T, and further optionally the substitution is with S, and / or (ii) the antigen binding construct comprises a second mutation that is different from the substitution at D270 (EU numbering). In some embodiments, if the at least one mutation comprises a substitution at E272 (EU numbering), then (i) the substitution is with S, V, G, A, Q, T, K, R, H, P, Y, F, W, M, L, or I, optionally the substitution is with S, V, G, A, Q, or T, and further optionally the substitution is with S, and / or (ii) the antigen binding construct comprises a second mutation that is different from the substitution at E272 (EU numbering). In some embodiments, if the at least one mutation comprises a substitution at E293 (EU numbering), then (i) the substitution is with V, G, A, Q, N, T, K, R, H, P, Y, F, W, M, L, or I, optionally the substitution is with V, G, A, Q, N, or T, and / or (ii) the antigen binding construct comprises a second mutation that is different from the substitution at E293 (EU numbering). In some embodiments, if the at least one mutation comprises a substitution at E294 (EU numbering), then (i) the substitution is with S, V, G, A, Q, N, T, K, R, H, P, Y, F, W, M, L, or I, optionally the substitution is with S, V, G, A, Q, N, or T, and further optionally the substitution is with S, and / or (ii) the antigen binding construct comprises a second mutation that is different from the substitution at E294 (EU numbering).

[0287] In some embodiments, the antibody or antigen binding fragment thereof comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity to an IgGCH2 having a sequence that is SEQ ID NO. 57, or a percent identity to an IgG CH2 having a sequence that is SEQ ID NO. 57 that is in a range defined by any two of the preceding values, wherein the IgG CH2 sequence comprises one or more mutations selected from E233S, E269S, N297Q, D265S, D270S, E380S, E382S, E272S, E294S, and D280S (EU Numbering). For example, in some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%- 100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%- 97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to an IgG CH2 in SEQ ID NO. 57, wherein the IgG CH2 sequence comprises one or more mutations selected from E233S, E269S, N297Q, D265S, D270S, E272S, E294S, and D280S (EU Numbering).

[0288] Table 7 lists some embodiments of substitutions in the VL, VH, CH2, CH3, and Fc domains of an antibody or antigen binding fragment thereof that, either alone, or in combination, may reduce healthy organ uptake of the antibody or antigen binding fragment thereof. In some embodiments, the CH2 domain comprises an amino acid sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or a percentage in a range that is defined by any two of the preceding values, identity to a CH2 domain in SEQ ID NO. 57, wherein the CH2 domain comprises one or more mutations in Table 7. For example, in some embodiments, the CH2 domain comprises an amino acid sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to a VH domain in SEQ ID NO. 57, wherein the CH2 domain comprises one or more mutations in Table 7.

[0289] In some embodiments, the antibody or antigen binding fragment thereof comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity to an IgG CH2 in SEQ ID NO. 57, or a percent identity to an IgG CH2 in SEQ ID NO. 57 that is in a range defined by any two of the preceding values, wherein the IgG CH2 sequence comprises an E233S mutation (EU Numbering). For example, in some embodiments, the antibody or antigen binding fragment thereof comprises an IgG CH2 sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%- 100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%- 97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to an IgG CH2 in SEQ ID NO. 57, wherein the IgG CH2 sequence comprises an E233S mutation (EU Numbering).

[0290] In some embodiments, the antibody or antigen binding fragment thereof comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity to an IgG CH2 in SEQ ID NO. 57, or a percent identity to an IgG CH2 in SEQ ID NO. 57 that is in a range defined by any two of the preceding values, wherein the IgG CH2 sequence comprises an E233X mutation (EU Numbering), wherein X is any amino acid. For example, in some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to an IgG CH2 in SEQ ID NO. 57, wherein the IgG CH2 sequence comprises an E233X mutation (EU Numbering), wherein X is any amino acid. In some embodiments, X is an arginine (R), histidine (H), glycine (G), alanine (A), valine (V), cysteine (C), proline (P), leucine (L), isoleucine (I), methionine (M), tryptophan (W), phenylalanine (F), serine (S), threonine (T), tyrosine (Y), asparagine (N), glutamine (Q), aspartic acid (D), glutamic acid (E), or lysine (K). In some embodiments, X is a S, V, G, A, Q, N, T, K, R, H, Y, F, W, M, L, or I. In some embodiments, X is a S, V, G, A, Q, N, or T.

[0291] In some embodiments, the antibody or antigen binding fragment thereof comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity to an IgG CH2 in SEQ ID NO. 57, or a percent identity to an IgG CH2 in SEQ ID NO. 57 that is in a range defined by any two of the preceding values, wherein the IgG CH2 sequence comprises an D265X mutation (EU Numbering), wherein X is any amino acid. For example, in some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having between about 70%-100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identityte an IgG CH2 in SEQ ID NO. 57, wherein the IgG CH2 sequence comprises an D265X mutation (EU Numbering), wherein X is any amino acid. In some embodiments, X is an arginine (R), histidine (H), glycine (G), alanine (A), valine (V), cysteine (C), proline (P), leucine (L), isoleucine (I), methionine (M), tryptophan (W), phenylalanine (F), serine (S), threonine (T), tyrosine (Y), asparagine (N), glutamine (Q), aspartic acid (D), glutamic acid (E), or lysine (K). In some embodiments, X is a V, G, Q, N, T, R, P, F, W, M, L, or I. In some embodiments, X is a V, G, Q, N, or T.

[0292] In some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity to an IgG CH2 in SEQ ID NO. 57, or a percent identity to an IgG CH2 in SEQ ID NO. 57 that is in a range defined by any two of the preceding values, wherein the IgG CH2 sequence comprises a D265S mutation (EU Numbering). For example, in some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having between about 70%- 100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%- 100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%- 97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to an IgG CH2 in SEQ ID NO. 57, wherein the IgG CH2 sequence comprises a D265S mutation (EU Numbering).

[0293] In some embodiments, the antibody or binding fragment thereof, comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity to an IgG CH2 in SEQ ID NO. 57, or a percent identity to an IgG CH2 in SEQ ID NO. 57 that is in a range defined by any two of the preceding values, wherein the IgG CH2 sequence comprises an E269S mutation (EU Numbering). For example, in some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having between about 70%-100%, 70%- 95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%- 95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%- 95%, 95%-100%, 95%-97%, or 97%-100%, identity to an IgG CH2 in SEQ ID NO. 57, wherein the IgG CH2 sequence comprises an E269S mutation (EU Numbering).

[0294] In some embodiments, the antibody or binding fragment thereof comprises an IgG CH2 sequence having at least 90% identity to an IgG CH2 in SEQ ID NO. 57. In some-noembodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity to an IgG CH2 in SEQ ID NO. 57, or a percent identity to an IgG CH2 in SEQ ID NO. 57 that is in a range defined by any two of the preceding values, wherein the IgG CH2 sequence comprises an E269X mutation (EU Numbering), wherein X is any amino acid. For example, in some embodiments, the antibody or binding fragment thereof comprises an IgG CH2 sequence having between about 70%-100%, 70%- 95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%- 95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%- 95%, 95%-100%, 95%-97%, or 97%-100%, identity to an IgG CH2 in SEQ ID NO. 57, wherein the IgG CH2 sequence comprises an E269X mutation (EU Numbering), wherein X is any amino acid. In some embodiments, X is an arginine (R), histidine (H), glycine (G), alanine (A), valine (V), cysteine (C), proline (P), leucine (L), isoleucine (I), methionine (M), tryptophan (W), phenylalanine (F), serine (S), threonine (T), tyrosine (Y), asparagine (N), glutamine (Q), aspartic acid (D), glutamic acid (E), or lysine (K). In some embodiments, X is a S, V, G, A, T, K, R, H, P, Y, F, W, M, L, or I. In some embodiments, X is a S, V, G, A, or T.

[0295] In some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity to an IgG CH2 in SEQ ID NO. 57, or a percent identity to an IgG CH2 in SEQ ID NO. 57 that is in a range defined by any two of the preceding values, wherein the IgG CH2 sequence comprises a D270S mutation (EU Numbering). For example, in some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having between about 70%- 100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%- 100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%- 97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to an IgG CH2 in SEQ ID NO. 57, wherein the IgG CH2 sequence comprises a D270S mutation (EU Numbering).

[0296] In some embodiments, the antibody or binding fragment thereof comprises an IgG CH2 sequence having at least 90% identity to an IgG CH2 in SEQ ID NO. 57. In some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence-I l l-having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity to an IgG CH2 in SEQ ID NO. 57, or a percent identity to an IgG CH2 in SEQ ID NO. 57 that is in a range defined by any two of the preceding values, wherein the IgG CH2 sequence comprises an D270X mutation (EU Numbering), wherein X is any amino acid. For example, in some embodiments, the antibody or binding fragment thereof comprises an IgG CH2 sequence having between about 70%-100%, 70%- 95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%- 95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%- 95%, 95%-100%, 95%-97%, or 97%-100%, identity to an IgG CH2 in SEQ ID NO. 57, wherein the IgG CH2 sequence comprises an D270X mutation (EU Numbering), wherein X is any amino acid. In some embodiments, X is an arginine (R), histidine (H), glycine (G), alanine (A), valine (V), cysteine (C), proline (P), leucine (L), isoleucine (I), methionine (M), tryptophan (W), phenylalanine (F), serine (S), threonine (T), tyrosine (Y), asparagine (N), glutamine (Q), aspartic acid (D), glutamic acid (E), or lysine (K). In some embodiments, X is a S, V, Q, T, K, R, H, P, Y, F, W, L, or I. In some embodiments, X is a S, V, Q, or T.

[0297] In some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having at least 90% identity to an IgG CH2 in SEQ ID NO. 57. In some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity to an IgG CH2 in SEQ ID NO. 57, or a percent identity to an IgG CH2 in SEQ ID NO. 57 that is in a range defined by any two of the preceding values, wherein the IgG CH2 sequence comprises an E272S mutation (EU Numbering). For example, in some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having between about 70%-100%, 70%-95%, 70%- 90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%- 90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%- 100%, 95%-97%, or 97%-100%, identity to an IgG CH2 in SEQ ID NO. 57, wherein the IgG CH2 sequence comprises an E272S mutation (EU Numbering).

[0298] In some embodiments, the antibody or binding fragment thereof comprises an IgG CH2 sequence having at least 90% identity to an IgG CH2 in SEQ ID NO. 57. In some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequencehaving, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity to an IgG CH2 in SEQ ID NO. 57, or a percent identity to an IgG CH2 in SEQ ID NO. 57 that is in a range defined by any two of the preceding values, wherein the IgG CH2 sequence comprises an E272X mutation (EU Numbering), wherein X is any amino acid. For example, in some embodiments, the antibody or binding fragment thereof comprises an IgG CH2 sequence having between about 70%-100%, 70%- 95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%- 95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%- 95%, 95%-100%, 95%-97%, or 97%-100%, identity to an IgG CH2 in SEQ ID NO. 57, wherein the IgG CH2 sequence comprises an E272X mutation (EU Numbering), wherein X is any amino acid. In some embodiments, X is an arginine (R), histidine (H), glycine (G), alanine (A), valine (V), cysteine (C), proline (P), leucine (L), isoleucine (I), methionine (M), tryptophan (W), phenylalanine (F), serine (S), threonine (T), tyrosine (Y), asparagine (N), glutamine (Q), aspartic acid (D), glutamic acid (E), or lysine (K). In some embodiments, X is a S, V, G, A, Q, T, K, R, H, P, Y, F, W, M, L, or I. In some embodiments, X is a S, V, G, A, Q, or T.

[0299] In some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having at least 90% identity to an IgG CH2 in SEQ ID NO. 57. In some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity to an IgG CH2 in SEQ ID NO. 57, or a percent identity to an IgG CH2 in SEQ ID NO. 57 that is in a range defined by any two of the preceding values, wherein the IgG CH2 sequence comprises a D280S mutation (EU Numbering). For example, in some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having between about 70%-100%, 70%-95%, 70%- 90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%- 90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%- 100%, 95%-97%, or 97%-100%, identity to an IgG CH2 in SEQ ID NO. 57, wherein the IgG CH2 sequence comprises a D280S mutation (EU Numbering).

[0300] In some embodiments, the antibody or binding fragment thereof comprises an IgG CH2 sequence having at least 90% identity to an IgG CH2 in SEQ ID NO. 57. In someembodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity to an IgG CH2 in SEQ ID NO. 57, or a percent identity to an IgG CH2 in SEQ ID NO. 57 that is in a range defined by any two of the preceding values, wherein the IgG CH2 sequence comprises an E293X mutation (EU Numbering), wherein X is any amino acid. For example, in some embodiments, the antibody or binding fragment thereof comprises an IgG CH2 sequence having between about 70%-100%, 70%- 95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%- 95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%- 95%, 95%-100%, 95%-97%, or 97%-100%, identity to an IgG CH2 in SEQ ID NO. 57, wherein the IgG CH2 sequence comprises an E293X mutation (EU Numbering), wherein X is any amino acid. In some embodiments, X is an arginine (R), histidine (H), glycine (G), alanine (A), valine (V), cysteine (C), proline (P), leucine (L), isoleucine (I), methionine (M), tryptophan (W), phenylalanine (F), serine (S), threonine (T), tyrosine (Y), asparagine (N), glutamine (Q), aspartic acid (D), glutamic acid (E), or lysine (K). In some embodiments, X is a S, V, G, A, Q, N, T, K, R, H, P, Y, F, W, M, L, or I. In some embodiments, X is a V, G, A, Q, N, or T. In some embodiments, X is not S.

[0301] In some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having at least 90% identity to an IgG CH2 in SEQ ID NO. 57. In some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity to an IgG CH2 in SEQ ID NO. 57, or a percent identity to an IgG CH2 in SEQ ID NO. 57 that is in a range defined by any two of the preceding values, wherein the IgG CH2 sequence comprises an E294S mutation (EU Numbering). For example, in some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having between about 70%-100%, 70%-95%, 70%- 90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%- 90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%- 100%, 95%-97%, or 97%-100%, identity to an IgG CH2 in SEQ ID NO. 57, wherein the IgG CH2 sequence comprises an E294S mutation (EU Numbering).

[0302] In some embodiments, the antibody or binding fragment thereof comprises an IgG CH2 sequence having at least 90% identity to an IgG CH2 in SEQ ID NO. 57. In some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity to an IgG CH2 in SEQ ID NO. 57, or a percent identity to an IgG CH2 in SEQ ID NO. 57 that is in a range defined by any two of the preceding values, wherein the IgG CH2 sequence comprises an E294X mutation (EU Numbering), wherein X is any amino acid. For example, in some embodiments, the antibody or binding fragment thereof comprises an IgG CH2 sequence having between about 70%-100%, 70%- 95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%- 95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%- 95%, 95%-100%, 95%-97%, or 97%-100%, identity to an IgG CH2 in SEQ ID NO. 57, wherein the IgG CH2 sequence comprises an E294X mutation (EU Numbering), wherein X is any amino acid. In some embodiments, X is an arginine (R), histidine (H), glycine (G), alanine (A), valine (V), cysteine (C), proline (P), leucine (L), isoleucine (I), methionine (M), tryptophan (W), phenylalanine (F), serine (S), threonine (T), tyrosine (Y), asparagine (N), glutamine (Q), aspartic acid (D), glutamic acid (E), or lysine (K). In some embodiments, X is a S, V, G, A, Q, N, T, K, R, H, P, Y, F, W, M, L, or I. In some embodiments, X is a S, V, G, A, Q, N, or T.

[0303] In some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having at least 90% identity to an IgG CH2 in SEQ ID NO. 57. In some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity to an IgG CH2 in SEQ ID NO. 57, or a percent identity to an IgG CH2 in SEQ ID NO. 57 that is in a range defined by any two of the preceding values, wherein the IgG CH2 sequence comprises a N297Q mutation (EU Numbering). For example, in some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having between about 70%-100%, 70%-95%, 70%- 90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%- 90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to an IgG CH2 in SEQ ID NO. 57, wherein the IgG CH2 sequence comprises a N297Q mutation (EU Numbering).

[0304] In some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having at least 90% identity to an IgG CH2 in SEQ ID NO. 57. In some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity to an IgG CH2 in SEQ ID NO. 57, or a percent identity to an IgG CH2 in SEQ ID NO. 57 that is in a range defined by any two of the preceding values, wherein the IgG CH2 sequence comprises a I253A mutation (EU Numbering). For example, in some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having between about 70%-100%, 70%-95%, 70%- 90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%- 90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%- 100%, 95%-97%, or 97%-100%, identity to an IgG CH2 in SEQ ID NO. 57, wherein the IgG CH2 sequence comprises a I253A mutation (EU Numbering).

[0305] In some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having at least 90% identity to an IgG CH2 in SEQ ID NO. 57. In some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity to an IgG CH2 in SEQ ID NO. 57, or a percent identity to an IgG CH2 in SEQ ID NO. 57 that is in a range defined by any two of the preceding values, wherein the IgG CH2 sequence comprises an H310A mutation (EU Numbering). For example, in some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having between about 70%-100%, 70%-95%, 70%- 90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%- 90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%- 100%, 95%-97%, or 97%-100%, identity to an IgG CH2 in SEQ ID NO. 57, wherein the IgG CH2 sequence comprises an H310A mutation (EU Numbering).

[0306] In some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity to an IgGCH2 in SEQ ID NO. 57, wherein the IgG CH2 sequence comprises one or more mutations resulting in decreased uptake by one or more organs as compared to an antigen binding construct or scFv-Fc fusion comprising an IgG CH2 sequence that is SEQ ID NO. 57. In some embodiments, uptake of the antigen binding construct or scFv-Fc fusion by one or more organs is decreased by 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%, or by a percentage that is in a range defined by any two of the preceding values, as compared to an antigen binding construct or scFv-Fc fusion comprising an IgG CH2 sequence that is SEQ ID NO. 57. For example, in some embodiments, uptake of the antigen binding construct or scFv-Fc fusion by one or more organs is decreased by between about l%-100%, l%-95%, l%-90%, l%-75%, l%-50%, 1%- 25%, l%-10%, l%-5%, 5%-100%, 5%-95%, 5%-90%, 5%-75%, 5%-50%, 5%-25%, 5%- 10%, 10%-100%, 10%-95%, 10%-90%, 10%-75%, 10%-50%, 10%-25%, 25%-100%, 25%- 95%, 25%-90%, 25%-75%, 25%-50%, 50%-100%, 50%-95%, 50%-90%, 50%-75%, 75%- 100%, 75%-95%, 75%-90%, 90%-100%, 90%-95%, or 95%-100%, as compared to an antigen binding construct or scFv-Fc fusion comprising an IgG CH2 sequence that is SEQ ID NO. 57.

[0307] In some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity to an IgG CH2 in SEQ ID NO. 57, wherein the IgG CH2 sequence comprises one or more mutations has decreased uptake by blood, lungs, muscle, bone (bone marrow), liver, kidneys, heart, stomach, small intestine, large intestine, pancreas, testis, ovaries, and / or any combination therein, as compared to an antigen binding construct or scFv-Fc fusion comprising an IgG CH2 sequence that is SEQ ID NO. 57. In some embodiments, uptake of the antigen binding construct or scFv- Fc fusion by one or more organs is decreased by 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%, or by a percentage that is in a range defined by any two of the preceding values, as compared to an antigen binding construct or scFv-Fc fusion comprising an IgG CH2 sequence that is SEQ ID NO. 57. For example, in some embodiments, uptake of the antigen binding construct or scFv-Fc fusion by blood, lungs, muscle, bone (bone marrow), liver, kidneys, heart, stomach, small intestine, large intestine, pancreas, testis, ovaries, and / or any combination therein, is decreased by between about l%-100%, l%-95%, l%-90%, l%-75%, l%-50%, l%-25%, 1%-10%, l%-5%, 5%-100%, 5%-95%, 5%-90%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%- 100%, 10%-95%, 10%-90%, 10%-75%, 10%-50%, 10%-25%, 25%-100%, 25%-95%, 25%- 90%, 25%-75%, 25%-50%, 50%-100%, 50%-95%, 50%-90%, 50%-75%, 75%-100%, 75%- 95%, 75%-90%, 90%-100%, 90%-95%, or 95%-100%, as compared to an antigen binding construct or scFv-Fc fusion comprising an IgG CH2 sequence that is SEQ ID NO. 57.

[0308] In some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity to an IgG CH2 in SEQ ID NO. 57, wherein the IgG CH2 sequence comprises one or more mutations, has decreased uptake by the liver as compared to an antigen binding construct or scFv-Fc fusion comprising an IgG CH2 sequence that is SEQ ID NO. 57. In some embodiments, uptake of the antigen binding construct or scFv-Fc fusion by one or more organs is decreased by 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%, or by a percentage that is in a range defined by any two of the preceding values, as compared to an antigen binding construct or scFv-Fc fusion comprising an IgG CH2 sequence that is SEQ ID NO. 57. For example, in some embodiments, uptake of the antigen binding construct or scFv-Fc fusion by the liver is decreased by between about 1%- 100%, l%-95%, l%-90%, l%-75%, l%-50%, l%-25%, l%-10%, l%-5%, 5%-100%, 5%- 95%, 5%-90%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-100%, 10%-95%, 10%-90%, 10%-75%, 10%-50%, 10%-25%, 25%-100%, 25%-95%, 25%-90%, 25%-75%, 25%-50%, 50%-100%, 50%-95%, 50%-90%, 50%-75%, 75%-100%, 75%-95%, 75%-90%, 90%-100%, 90%-95%, or 95%-100%, as compared to an antigen binding construct or scFv-Fc fusion comprising an IgG CH2 sequence that is SEQ ID NO. 57.

[0309] In some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity to an IgG CH2 in SEQ ID NO. 57, or a percent identity to an IgG CH2 in SEQ ID NO. 57 that is in a range defined by any two of the preceding values, wherein the IgG CH2 sequence comprises an E233X1 and E269X2 mutation (EU Numbering), wherein Xi and X2 are each any amino acid. For example, in some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having between about 70%-100%, 70%-95%, 70%-90%,70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%-100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%-97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to an IgG CH2 in SEQ ID NO. 57, wherein the IgG CH2 sequence comprises an E233X1 and E269X2 mutation (EU Numbering), wherein Xi and X2 are each any amino acid. In some embodiments, X is an arginine (R), histidine (H), glycine (G), alanine (A), valine (V), cysteine (C), proline (P), leucine (L), isoleucine (I), methionine (M), tryptophan (W), phenylalanine (F), serine (S), threonine (T), tyrosine (Y), asparagine (N), glutamine (Q), aspartic acid (D), glutamic acid (E), or lysine (K). In some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity to an IgG CH2 in SEQ ID NO. 57, or a percent identity to an IgG CH2 in SEQ ID NO. 57 that is in a range defined by any two of the preceding values, wherein the IgG CH2 sequence comprises an E233X1 and E269X2 mutation (EU Numbering), wherein Xi and X2 are each an arginine (R), histidine (H), glycine (G), alanine (A), valine (V), cysteine (C), proline (P), leucine (L), isoleucine (I), methionine (M), tryptophan (W), phenylalanine (F), serine (S), threonine (T), tyrosine (Y), asparagine (N), glutamine (Q), aspartic acid (D), glutamic acid (E), or lysine (K). For example, in some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having between about 70%- 100%, 70%-95%, 70%-90%, 70%-80%, 75%-100%, 75%-95%, 75%-90%, 75%-85%, 80%- 100%, 80%-95%, 80%-90%, 90%-100%, 90%-97%, 90%-95%, 90%-93%, 93%-100%, 93%- 97%, 93%-95%, 95%-100%, 95%-97%, or 97%-100%, identity to an IgG CH2 in SEQ ID NO.57, wherein the IgG CH2 sequence comprises an E233X1 and E269X2 mutation (EU Numbering), wherein Xi and X2 are each an arginine (R), histidine (H), glycine (G), alanine (A), valine (V), cysteine (C), proline (P), leucine (L), isoleucine (I), methionine (M), tryptophan (W), phenylalanine (F), serine (S), threonine (T), tyrosine (Y), asparagine (N), glutamine (Q), aspartic acid (D), glutamic acid (E), or lysine (K). In some embodiments, Xi and X2 are each S, V, G, A, Q, N, T, K, R, H, P, Y, F, W, M, L, or I. In some embodiments, Xi and X2 are each S, V, G, A, Q, N, or T. In some embodiments, uptake of the antigen binding construct or scFv-Fc fusion by one or more organs (e.g., blood, lungs, muscle, bone (bone marrow), liver, kidneys, heart, stomach, small intestine, large intestine, pancreas, testis, ovaries, and / or any combination therein) is decreased by, by about, or by at least 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 100%, or by a percentage that is in a range defined by any two of the preceding values, as compared to an antigen binding construct or scFv-Fc fusion comprising an IgG CH2 sequence that is SEQ ID NO. 57. For example, in some embodiments, uptake of the antigen binding construct or scFv-Fc fusion by one or more organs (e.g., blood, lungs, muscle, bone (bone marrow), liver, kidneys, heart, stomach, small intestine, large intestine, pancreas, testis, ovaries, and / or any combination therein) is decreased by between about 1%-100%, l%-95%, l%-90%, l%-75%, l%-50%, l%-25%, l%-10%, l%-5%, 5%-100%, 5%-95%, 5%-90%, 5%- 75%, 5%-50%, 5%-25%, 5%-10%, 10%-100%, 10%-95%, 10%-90%, 10%-75%, 10%-50%, 10%-25%, 25%-100%, 25%-95%, 25%-90%, 25%-75%, 25%-50%, 50%-100%, 50%-95%, 50%-90%, 50%-75%, 75%-100%, 75%-95%, 75%-90%, 90%-100%, 90%-95%, or 95%- 100%, as compared to an antigen binding construct or scFv-Fc fusion comprising an IgG CH2 sequence that is SEQ ID NO. 57. In some embodiments, uptake by the liver of the antigen binding construct or scFv-Fc fusion is reduced by, by about, or by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 100%, or by a percentage that is in a range defined by any two of the preceding values, as compared to an antigen binding construct or scFv-Fc fusion comprising an IgG CH2 sequence that is SEQ ID NO. 57. For example, in some embodiments, uptake of the antigen binding construct or scFv-Fc fusion by the liver is decreased by between about 1 %-l 00%, 1%- 95%, l%-90%, l%-75%, l%-50%, l%-25%, l%-10%, l%-5%, 5%-100%, 5%-95%, 5%- 90%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-100%, 10%-95%, 10%-90%, 10%-75%, 10%-50%, 10%-25%, 25%-100%, 25%-95%, 25%-90%, 25%-75%, 25%-50%, 50%-100%, 50%-95%, 50%-90%, 50%-75%, 75%-100%, 75%-95%, 75%-90%, 90%-100%, 90%-95%, or 95%-100%, as compared to an antigen binding construct or scFv-Fc fusion comprising an IgG CH2 sequence that is SEQ ID NO. 57. In some embodiments, uptake of the antigen binding construct or scFv-Fc fusion by tumor cells is increased by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to an antigen binding construct or scFv-Fc fusion comprising an IgG CH2 sequence that is SEQ ID NO. 57. For example, in some embodiments, uptake of the antigen binding construct or scFv-Fc fusion by tumor cells is increased by between about 1 %- 1000%, l%-750%, l%-500%, 1%- 250%, l%-100%, l%-75%, l%-50%, l%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%- 250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 50%-75%, 75%-1000%, 75%-750%, 75%-500%, 75%-250%, 75%-100%, 100%- 1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, as compared to an antigen binding construct or scFv-Fc fusion comprising an IgG CH2 sequence that is SEQ ID NO. 57. In some embodiments, the ratio between the amount of antigen binding construct or scFv-Fc fusion comprising an E233X, and E269X2 mutation (EU Numbering) taken up by tumor cells as compared to the amount of antigen binding construct or scFv-Fc fusion comprising an IgG CH2 sequence that is SEQ ID NO. 57 taken up by organs is greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or greater than a ratio that is in a range defined by any two of the preceding values. For example, in some embodiments, the ratio between the amount of antigen binding construct or scFv-Fc fusion comprising an E233X1 and E269X2 mutation (EU Numbering) taken up by tumor cells as compared to the amount of antigen binding construct or scFv-Fc fusion comprising an IgG CH2 sequence that is SEQ ID NO. 57 taken up by one or more organs is between about 1-10, 1-7, 1-5, 1-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7-10.

[0310] In some embodiments, the antigen binding construct or scFv-Fc fusion comprises an IgG CH2 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identity to an IgG CH2 in SEQ ID NO. 57, wherein the IgG CH2 sequence comprises an E233S and E269S mutation (EU Numbering). In some embodiments, the E233S and E269S mutation (EU Numbering) results in decreased uptake of the antigen binding construct or scFv-Fc fusion by one or more organs by 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%, or by a percentage that is in a range defined by any two of the preceding values, as compared to an antigen binding construct or scFv-Fc fusion comprising an IgG CH2 sequence that is SEQ ID NO. 57. For example, in some embodiments, uptake of the antigen binding construct or scFv-Fc fusion comprising an E233S and E269S mutation (EU Numbering), by one or more organs is decreased by between about l%-100%, l%-95%, l%-90%, l%-75%, l%-50%, l%-25%, l%-10%, l%-5%, 5%- 100%, 5%-95%, 5%-90%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-100%, 10%-95%,10%-90%, 10%-75%, 10%-50%, 10%-25%, 25%-100%, 25%-95%, 25%-90%, 25%-75%, 25%-50%, 50%-100%, 50%-95%, 50%-90%, 50%-75%, 75%-100%, 75%-95%, 75%-90%, 90%-100%, 90%-95%, or 95%-100%, as compared to an antigen binding construct or scFv-Fc fusion comprising an IgG CH2 sequence that is SEQ ID NO. 57. In some embodiments, uptake of the antigen binding construct or scFv-Fc fusion by blood, lungs, muscle, bone (bone marrow), liver, kidneys, heart, stomach, small intestine, large intestine, pancreas, testis, ovaries, and / or any combination therein, is decreased by, by about, or by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 100%, or by a percentage that is in a range defined by any two of the preceding values, as compared to an antigen binding construct or scFv-Fc fusion comprising an IgG CH2 sequence that is SEQ ID NO. 57. In some embodiments, the antigen binding construct or scFv- Fc fusion comprises an IgG CH2 sequence having at least 90% identity to an IgG CH2 in SEQ ID NO. 57, wherein the IgG CH2 sequence comprises an E233S and E269S mutation (EU Numbering) in the IgG CH2 sequence results in decreased uptake of the antigen binding construct or scFv-Fc fusion by blood, lungs, muscle, bone (bone marrow), liver, kidneys, heart, stomach, small intestine, large intestine, pancreas, testis, ovaries, and / or any combination therein, by between about l%-100%, l%-95%, l%-90%, l%-75%, l%-50%, l%-25%, 1%- 10%, l%-5%, 5%-100%, 5%-95%, 5%-90%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%- 100%, 10%-95%, 10%-90%, 10%...

Claims

WHAT IS CLAIMED IS:

1. An antigen binding construct comprising an IgG CH2 having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or a percentage in a range that is defined by any two of the preceding values, identity to an IgG CH2 in SEQ ID NO. 57, wherein at least one mutation in the IgG CH2 reduces uptake of the antigen binding construct by healthy organs as compared to an IgG CH2 sequence in SEQ ID NO. 57, wherein the at least one mutation comprises a substitution of one or more of the following amino acids (EU numbering): E233, D265, E269, D270, E272, D280, E293, and E294, wherein(1) if the at least one mutation comprises a substitution at E233 (EU numbering), then (i) the substitution is with S, V, G, A, Q, N, T, K, R, H, Y, F, W, M, L, or I, optionally the substitution is with S, V, G, A, Q, N, or T, and further optionally the substitution is with S, and / or (ii) the antigen binding construct comprises a second mutation in the IgG CH2 that is different from the substitution at E233 (EU numbering),(2) if the at least one mutation comprises a substitution at D265 (EU numbering), then (i) the substitution is with V, G, Q, N, T, R, P, F, W, M, L, or I, optionally the substitution is with V, G, Q, N, or T, and / or (ii) the antigen binding construct comprises a second mutation in the IgG CH2 that is different from the substitution at D265 (EU numbering),(3) if the at least one mutation comprises a substitution at E269 (EU numbering), then (i) the substitution is with S, V, G, A, T, K, R, H, P, Y, F, W, M, L, or I, optionally the substitution is with S, V, G, A, or T, and further optionally the substitution is with S, and / or (ii) the antigen binding construct comprises a second mutation that is different from the substitution at E269 (EU numbering),(4) if the at least one mutation comprises a substitution at D270 (EU numbering), then (i) the substitution is with S, V, Q, T, K, R, H, P, Y, F, W, L, or I, optionally the substitution is with S, V, Q, or T, and further optionally the substitution is with S, and / or (ii) the antigen binding construct comprises a second mutation that is different from the substitution at D270 (EU numbering),(5) if the at least one mutation comprises a substitution at E272 (EU numbering), then (i) the substitution is with S, V, G, A, Q, T, K, R, H, P, Y, F, W, M, L, or I, optionally the substitution is with S, V, G, A, Q, or T, and further optionally the substitution is with S, and / or (ii) the antigen binding construct comprises a second mutation that is different from the substitution at E272 (EU numbering),(6) if the at least one mutation comprises a substitution at E293 (EU numbering), then (i) the substitution is with V, G, A, Q, N, T, K, R, H, P, Y, F, W, M,L, or I, optionally the substitution is with V, G, A, Q, N, or T, and / or (ii) the antigen binding construct comprises a second mutation that is different from the substitution at E293 (EU numbering), and / or(7) if the at least one mutation comprises a substitution at E294 (EU numbering), then (i) the substitution is with S, V, G, A, Q, N, T, K, R, H, P, Y, F, W,M, L, or I, optionally the substitution is with S, V, G, A, Q, N, or T, and further optionally the substitution is with S, and / or (ii) the antigen binding construct comprises a second mutation that is different from the substitution at E294 (EU numbering).

2. The antigen binding construct of claim 1, wherein the IgG CH2 comprises an E233X mutation (EU Numbering), wherein X is S, V, G, A, Q, N, T, K, R, H, Y, F, W, M, L, or I, optionally wherein X is S, V, G, A, Q, N, or T, further optionally wherein the IgG CII2 comprises an E233S mutation (EU Numbering).

3. The antigen binding construct of claim 1 or 2, wherein the IgG CH2 comprises a D265X mutation (EU Numbering), wherein X is V, G, Q, N, T, R, P, F, W, M, L, or I, optionally wherein X is V, G, Q, N, or T, further optionally wherein the IgG CH2 comprises a D265S mutation (EU Numbering).

4. The antigen binding construct of any one of the preceding claims, wherein the IgG CH2 comprises an E269X mutation (EU Numbering), wherein X is S, V, G, A, T, K, R, H, P, Y, F, W, M, L, or I, optionally wherein X is S, V, G, A, or T , further optionally wherein the IgG CH2 comprises an E269S mutation (EU Numbering).

5. The antigen binding construct of any one of the preceding claims, wherein the IgG CH2 comprises an D270X mutation (EU Numbering), wherein X is S, V, Q, T, K, R, H, P, Y, F, W, L, or I, optionally wherein X is S, V, Q, or T, further optionally wherein the IgG CH2 comprises an D270S mutation (EU Numbering).

6. The antigen binding construct of any one of the preceding claims, wherein the IgG CH2 comprises an E272X mutation (EU Numbering), wherein X is S, V, G, A, Q, T, K, R, H, P, Y, F, W, M, L, or I, optionally wherein X is S, V, G, A, Q, or T, further optionally wherein the IgG Cu2 comprises an E272S mutation (EU Numbering).

7. The antigen binding construct of any one of the preceding claims, wherein the IgG CH2 comprises an D293X mutation (EU Numbering), wherein X is S, V, G, A, Q, N, T, K, R, H, P, Y, F, W, M, L, or I, optionally wherein X is S, V, G, A, Q, N, or T, further optionally wherein the IgG CH2 comprises an D280S mutation (EU Numbering).

8. The antigen binding construct of any one of the preceding claims, wherein the IgG CH2 comprises an E293X mutation (EU Numbering), wherein X is S, V, G, A, Q, N, T, K, R, H, P, Y, F, W, M, L, or I, optionally wherein X is V, G, A, Q, N, or T.

9. The antigen binding construct of any one of the preceding claims, wherein the IgG CH2 comprises an E294X mutation (EU Numbering), wherein X is S, V, G, A, Q, N, T, K, R, H, P, Y, F, W, M, L, or I, optionally wherein X is S, V, G, A, Q, N, or T, further optionally wherein the IgG Cu2 comprises an E294S mutation (EU Numbering).

10. The antigen binding construct of any one of the preceding claims, wherein the IgG CH2 comprises an N297Q, N297G, or N297A mutation (EU Numbering).

11. The antigen binding construct of any one of the preceding claims, wherein the IgG CH2 comprises an H310A mutation (EU Numbering).

12. The antigen binding construct of any one of the preceding claims, wherein the IgG CH2 comprises one or more mutations selected from E233S, E269S, N297Q, D265S, D270S, E272S, E294S, and D280S (EU Numbering).

13. The antigen binding construct of any one of the preceding claims, wherein the IgG CH2 comprises an E233X1 and E269X2 mutations (EU Numbering), wherein Xi and X2 are each independently S, V, G, A, Q, N, or T, optionally wherein Xi and X2 are both S.

14. The antigen binding construct of any one of the preceding claims, wherein the IgG CH2 comprises an E233X1 and D270X2 mutation (EU Numbering), wherein Xi and X2 are each independently S, V, G, A, Q, N, or T, optionally wherein Xi and X2 are both S.

15. The antigen binding construct of any one of the preceding claims, comprising an IgG CH2 sequence comprising an I253A mutation (EU Numbering).

16. The antigen binding construct of any one of the preceding claims, wherein the antigen binding construct comprises an IgG CH2 sequence that is an IgG CH2 sequence in any one of SEQ ID NO: 58-7617. The antigen binding construct of any one of the preceding claims, wherein the antigen binding construct comprises a Fc domain having an amino acid sequence that is, is about, or is at least 80% identical to the Fc domain sequence set forth in SEQ ID NO: 87.

18. The antigen binding construct of any one of the preceding claims, wherein the antigen binding construct comprises a Fc domain that is a Fc domain in any one of claims SEQ ID NOs: 84-10219. The antigen binding construct of any one of the preceding claims, wherein the antigen binding construct comprising one or more mutations in a Fc domain that blocks FcRn recycling, blocks binding to receptors of the FcyR family, and / or silences FcyR effector function.

20. The antigen binding construct of any one of the preceding claims, comprising a Fc domain sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or a percentage in a range that is defined by any two of the preceding values, identity to a Fc domain sequence in SEQ ID NO. 83, wherein the Fc domain sequence comprises one or more mutations selected from H310A (EU Numbering), H435A (EU Numbering), H310A / H435A (EU Numbering), I253A (EU Numbering), I253A / H310A / H435A (EU Numbering), N297Q (EU Numbering), E233S (EU Numbering), E269S (EU Numbering), D265S (EU Numbering), D270S (EU Numbering), R355Q (EU Numbering), E356S (EU Numbering), E376S (EU Numbering), E380S (EU Numbering), E382S (EU Numbering), E272S (EU Numbering), E294S (EU Numbering), D280S (EU Numbering), K414T (EU Numbering), E430S (EU Numbering).

21. The antigen binding construct of claim 20, wherein the Fc domain sequence comprises (EU Numbering):(a) a H435A mutation;(b) a H310A mutation;(c) a H435A and H310A mutation;(d) an I253A mutation;(e) an I253A, H435A, and H310A triple mutation;(f) a N297Q mutation;(g) a E380S mutation;(h) a E382S mutation;(i) a R355Q mutation;(j) a E356S mutation;(k) a E376S mutation;(l) a K414T mutation;(m) a E430S mutation;(n) a H310A, H435A, and N297Q mutation;(o) a I253A, H310A, H435A, and N297Q mutation;(p) an I253A, H310A, H435A, N297Q, and E233S mutation;(q) an I253A, H310A, H435A, N297Q, and E269S mutation;(r) an I253A, H310A, H435A, N297Q, E233S, and E269S mutation;(s) an H435A and N297Q mutation;(t) an H435A, E233S, and E269S, mutation;(u) an H435A, N297Q, E233S, and E269S, mutation;(v) an I253A, H310A, H435A, N297Q, E233S, E269S, and D265S, mutation;(w) an I253A, H310A, H435A, N297Q, E233S, E269S, D265S, and D270S, mutation;(x) a H435A, N297Q, E233S, E269S, and D265S, mutation; and / or(y) a H435A, N297Q, E233S, E269S, D265S, and D270S mutation.

22. The antigen binding construct of any one of the preceding claims, further comprising a linker, optionally wherein the linker comprises GSTSGGGSGGGSGGGGSS (SEQ ID NO. 55)23. The antigen binding construct of any one of the preceding claims, wherein the VL is based on a human germline sequence of IGKV1-39*O1 or IGKJ4*01.

24. The antigen binding construct of any one of the preceding claims, wherein the VL domain comprises a framework region 2 (LFR2) comprising a sequence of X1X2X3X4X5X6X7 (SEQ ID NO. 81), wherein Xi is a positively charged amino acid, wherein X2, X3, X5, and Xe are each independently a negatively charged or a non-charged amino acid, and wherein X4 and X7 are each independently any amino acid with the proviso that at least one is a positively charged amino acid.

25. The antigen binding construct of claim 24, wherein the VL domain comprises aLFR2 of SEQ ID NO. 41 or 4226. The antigen binding construct of any one of claims 1-23, wherein the VLdomain comprises a framework region 2 (FR2) comprising a sequence of X1X2X3X4X5X6X7 (SEQ ID NO. 81), where Xi, X4, and X7 are each a lysine (K), wherein one or more of Ki, K.4, and / or K.7, are mutated; wherein X2, X3, X5, and Xe are each independently a negatively charged or a non-charged amino acid.

27. The antigen binding construct of any one of the preceding claims, wherein the Vi. domain comprises:(a) a K47Xi and / or K53X2 mutation (Aho), wherein Xi and X2 are each a non-lysine and non-arginine residue;(b) a K50Q, and / or K53Q mutation (Aho)(c) a E29Q mutation (Aho);(d) a K50X3 mutation (Aho), wherein X3 is a non-lysine and non-arginine residue;(e) a K50X3, and / or K53X2 mutation (Aho), wherein X2 and X3 are a non-lysine and non-arginine residue;(f) a KI 10G mutation (Aho); and / or(g) a El 11 S mutation (Aho).

28. The antigen binding construct of any one of the preceding claims, wherein the VH domain comprises:(a) a K73D mutation (Aho);(b) a K73S mutation (Aho);(c) a K13V mutation (Aho);(d) a K14Q mutation (Aho);(e) a K13V and K14Q mutation (Aho);(f) a D83S mutation (Aho);(g) a E84N mutation (Aho);(h) a D83S and E84N mutation (Aho);(i) a E92S mutation (Aho);(j) a K24A mutation (Aho); and / or(k) a R97T mutation (Aho).

29. The antigen binding construct, minibody, or scFv-Fc fusion, of any one of the preceding claims, wherein the hinge region comprises an upper hinge region comprising SEQ ID NO. 51 or 5230. The antigen binding construct of any one of the preceding claims, comprising an IgG CH3 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or a percentage in a range that is defined by any two of the preceding values, identity to SEQ ID NO. 77, wherein the Cu3 sequence comprises:(a) an R355Q mutation (EU Numbering);(b) an E356S mutation (EU Numbering);(c) an D376S mutation (EU Numbering);(d) an E380S mutation (EU Numbering);(e) an E382T mutation (EU Numbering);(f) an K414T mutation (EU Numbering); and / or(g) one or more mutations selected from R355Q (EU Numbering), E356S (EU Numbering), D376S (EU Numbering), E380S (EU Numbering), E382T (EU Numbering), K414T (EU Numbering), and E430S (EU Numbering).

31. The antigen binding construct, of any one of the preceding claims, comprising an IgG CH3 sequence that is any one of SEQ ID NO. 77-80.

32. The antigen binding construct of any one of the preceding claims, comprising a VL domain comprising framework region (LFR) sequences of LFR sequences in a VL domain in any one of SEQ ID NO. 1-17.

33. The antigen binding construct, minibody, or scFv-Fc fusion, of any one of the preceding claims, comprising a VH domain comprising framework region (HFR) sequences of HFR sequences in a VH domain in any one of SEQ ID NO. 18-34, or a sequence that has, has about, or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or a percentage in a range that is defined by any two of the preceding values, identity to the HFR sequences in a VH domain in any one of SEQ ID NO. 18-34.

34. An antigen binding construct comprising an IgG CH3 sequence having, having about, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more, or a percentage in a range that is defined by any two of the preceding values, identity to SEQ ID NO. 77.

35. The antigen binding construct of any one of the preceding claims, wherein the VL domain comprises a mutation, relative to wild-type, at one or more negative patch amino acids D17Xi, E29X2, EI I IX3 and / or E147X4 (Aho), and / or the VH domain comprises a mutation relative to wild-type at one or more negative patch amino acids D67Xs, D83Xe, E84X7, and / or E92Xs (Aho).

36. A variant antigen binding construct that varies from an original antigen binding construct by having a substitution of at least one surface-exposed, charged amino acid in the original antigen binding construct with an amino acid having a different charge polarity, whereby a localized patch of surface charge comprising the at least one, charged amino acid is disrupted in the variant antigen binding construct, wherein the at least one surface-exposed, charged amino acid is in a heavy chain variable region (VH), a light chain variable region (VL), a hinge region, a heavy chain constant region 2 (CH2) and / or a CH3 of the original antigen binding construct, wherein(1) if the at least one surface-exposed, charged amino acid comprises a positively charged amino acid in a light chain framework region 2 (LFR2) of the VL, then the at least one surface-exposed, charged amino acid comprises: a second charged amino acid outside the LFR2, of the original antigen binding construct,(2) if the at least one surface-exposed, charged amino acid comprises a positively charged amino acid in a hinge region of the original antigen binding construct, then the at least one surface-exposed, charged amino acid comprises: a second charged amino acid outside the hinge region, and / or a negatively charged amino acid in the hinge region, of the original antigen binding construct,(3) if the at least one surface-exposed, charged amino acid comprises E233 (EU numbering) in the CH2 of the original antigen binding construct, then (i) the substitution is with S, V, G, A, Q, N, T, K, R, H, Y, F, W, M, L, or I, optionally the substitution is with S, V, G, A, Q, N, or T, and further optionally the substitution is with S, and / or (ii) the at least one surface-exposed, charged amino acid comprises a second charged amino acid that is different from E233 (EU numbering) in the CH2 of the original antigen binding construct,(4) if the at least one surface-exposed, charged amino acid comprises D265 (EU numbering) in the CH2 of the original antigen binding construct, then (i) the substitution is with V, G, Q, N, T, R, P, F, W, M, L, or I, optionally the substitution is with V, G, Q, N, or T, and / or (ii) the at least one surface-exposed, charged amino acid comprises a second charged amino acid that is different from D265 (EU numbering) in the CH2 of the original antigen binding construct,(5) if the at least one surface-exposed, charged amino acid comprises E269 (EU numbering) in the CH2 of the original antigen binding construct, then (i) the substitution is with S, V, G, A, T, K, R, H, P, Y, F, W, M, L, or I, optionally the substitution is with S, V, G, A, or T, and further optionally the substitution is with S, and / or (ii) the at least one surface-exposed, charged amino acid comprises a second charged amino acid that is different from E269 (EU numbering) in the CH2 of the original antigen binding construct,(6) if the at least one surface-exposed, charged amino acid comprises D270 (EU numbering) in the CH2 of the original antigen binding construct, then (i) the substitution is with S, V, Q, T, K, R, H, P, Y, F, W, L, or I, optionally the substitution is with S, V, Q, or T, and further optionally the substitution is with S, and / or (ii) the at least one surface-exposed, charged amino acid comprises a second charged amino acid that is different from D270 (EU numbering) in the CH2 of the original antigen binding construct,(7) if the at least one surface-exposed, charged amino acid comprises E272 (EU numbering) in the CH2 of the original antigen binding construct, then (i) the substitution is with S, V, G, A, Q, T, K, R, H, P, Y, F, W, M, L, or I, optionally the substitution is with S, V, G, A, Q, or T, and further optionally the substitution is with S, and / or (ii) the at least one surface-exposed, charged amino acid comprises a second charged amino acid that is different from E272 (EU numbering) in the CH2 of the original antigen binding construct,(8) if the at least one surface-exposed, charged amino acid comprises E293 (EU numbering) in the CH2 of the original antigen binding construct, then (i) the substitution is with V, G, A, Q, N, T, K, R, H, P, Y, F, W, M, L, or I, optionally the substitution is with V, G, A, Q, N, or T, and / or (ii) the at least one surface-exposed,charged amino acid comprises a second charged amino acid that is different from E293 (EU numbering) in the CH2 of the original antigen binding construct, and / or(9) if the at least one surface-exposed, charged amino acid comprises E294 (EU numbering) in the CH2 of the original antigen binding construct, then (i) the substitution is with S, V, G, A, Q, N, T, K, R, H, P, Y, F, W, M, L, or I, optionally the substitution is with S, V, G, A, Q, N, or T, and further optionally the substitution is with S, and / or (ii) the at least one surface-exposed, charged amino acid comprises a second charged amino acid that is different from E294 (EU numbering) in the CH2 of the original antigen binding construct.

37. A variant antigen binding construct that varies from an original antigen binding construct by having a substitution of at least one surface-exposed, charged amino acid in the original antigen binding construct with an amino acid having a different charge polarity, whereby a localized patch of surface charge comprising the at least one, charged amino acid is disrupted in the variant antigen binding construct, wherein the variant antigen binding construct binds to FAP, CD8, CD4, CD3, IFNy, integrin aVp6, FOLRa, or PSMA, wherein(1) if the at least one surface-exposed, charged amino acid comprises a positively charged amino acid in a light chain framework region 2 (LFR2) of a VL of the original antigen binding construct, then the at least one surface-exposed, charged amino acid comprises: a second charged amino acid outside the LFR2 of the original antigen binding construct, and / or(2) if the at least one surface-exposed, charged amino acid comprises a positively charged amino acid in a hinge region of the original antigen binding construct, then the at least one surface-exposed, charged amino acid comprises: a second charged amino acid outside the hinge region, and / or a negatively charged amino acid in the hinge region, of the original antigen binding construct.

38. The variant antigen binding construct of claim 36 or 37, wherein substitution of the at least one surface-exposed, charged amino acid of the localized patch with the amino acid having a different charge polarity comprises a substitution of: K with S, V, G, A, Q, N, D, or T; R with S, V, G, A, Q, N, or T; D with S, V, G, A, Q, N, or T; and / or E with S, V, G, A, Q, N, or T.

39. The variant antigen binding construct any one of claims, wherein the at least one surface-exposed, charged amino acid comprises: (a) at least one amino acid in the VH selected from (Aho numbering) K13, K14, K24, R45, D67, K73, R77, D83, E84, E92, and R97; (b) at least one amino acid in the VL selected from (Aho numbering) DI 7, E29, K47, K50, K53, KI 10, El 11, and E147; (c) at least one amino acid in the CH2 selected from (EU numbering) E233, D265, E269, D270, E272, D280, E293, and E294; (d) at least one amino acid in the CH3 selected from (EU numbering) R355, E356, D376, E380, E382, K414, and E430; and / or (e) at least one amino acid in the hinge region.

40. The variant antigen binding construct of any one of claims 36-39, wherein the at least one surface-exposed, charged amino acid comprises at least one amino acid in the CH2 of the original antigen binding construct.

41. The variant antigen binding construct of any one of claims 36-40, wherein the at least one surface-exposed, charged amino acid comprises at least one amino acid in the CH3 of the original antigen binding construct.

42. The variant antigen binding construct of any one of claims 36-41, wherein the at least one surface-exposed, charged amino acid comprises at least one amino acid in the VH of the original antigen binding construct.

43. The variant antigen binding construct of any one of claims 36-42, wherein the at least one surface-exposed, charged amino acid comprises at least one amino acid in the VL of the original antigen binding construct.

44. The variant antigen binding construct of any one of claims 36-43, wherein the at least one surface-exposed, charged amino acid comprises at least one amino acid in the hinge region of the original antigen binding construct.

45. The variant antigen binding construct of any one of claims 36-44, wherein the at least one surface-exposed, charged amino acid comprises (EU numbering): E233, D265, E269, D270, E272, E293, and / or E294, optionally the at least one surface-exposed, charged amino acid comprises (EU numbering): E233, E269, D270, E272, and / or E294, further optionally the at least one surface-exposed, charged amino acid comprises (EU numbering): E233, E269, and / or D270.

46. The variant antigen binding construct of any one of claims 36-45, wherein the at least one surface-exposed, charged amino acid comprises at least one of the following pairsof amino acids (EU numbering): E233 / E269 or D265 / D270, optionally the at least one surface- exposed, charged amino acid comprises at least one of the following trios of amino acids (EU numbering): E233 / D265 / E269 or E233 / E269 / D270, further optionally the at least one surface- exposed, charged amino acid comprises at least the following amino acids E233 / D265 / E269 / D270 (EU numbering).

47. The variant antigen binding construct of any one of claims 36-46, wherein the substitution of the at least one surface-exposed, charged amino acid of the localized patch with the amino acid having a different charge polarity comprises at least one substitution in the CH2 selected from (EU numbering) E233S, D265S, E269S, D270S, E272S, D280S, E293S, and E294S, optionally at least one substitution in the CH2 selected from (EU numbering) E233S, E269S, D270S, E272S, and E294S, further optionally at least one substitution in the CH2 selected from (EU numbering) E233S, E269S, and D270S.

48. The variant antigen binding construct of any one of claims 36-47, wherein the substitution of the at least one surface-exposed, charged amino acid of the localized patch with the amino acid having a different charge polarity comprises at least one of the following pairs of substitutions (EU numbering): E233S / E269S or D265S / D270S, optionally at least one of the following trios of substitutions (EU numbering): E233S / D265S / E269S or E233S / E269S / D270S, further optionally at least the following substitutions E233S / D265S / E269S / D270S (EU numbering).

49. The variant antigen binding construct of any one of claim 36-46, wherein the substitution of the at least one surface-exposed, charged amino acid of the localized patch with the amino acid having a different charge polarity comprises at least one substitution in the CH2 selected from E233S, D265X, E269S, D270S, E272S, D280S, E293S, and E294S (EU numbering), wherein X is V, G, Q, N, or T.

50. The variant antigen binding construct of any one of claims 36-46, wherein the substitution of the at least one surface-exposed, charged amino acid of the localized patch with the amino acid having a different charge polarity comprises at least one of the following pairs of substitutions: E233S / E269S or D265XZD270S (EU numbering), wherein X is V, G, Q, N, or T, optionally at least one of the following trios of substitutions: E233S / D265X / E269S or E233S / E269S / D270S (EU numbering), wherein X is V, G, Q, N, or T, further optionally atleast the following substitutions E233S / D265X / E269S / D270S (EU numbering), wherein X is V, G, Q, N, or T.

51. The variant antigen binding construct of any one of claims 36-50, wherein the at least one surface-exposed, charged amino acid comprises (EU numbering): R355, E356, D376, E380, E382, K414, and / or E430.

52. The variant antigen binding construct of any one of claims 36-51, wherein the substitution of the at least one surface-exposed, charged amino acid of the localized patch with the amino acid having a different charge polarity comprises at least one substitution in the CH3 selected from (EU numbering) R355Q, E356S, D376S, E380S, E382S, E382T, K414T, and E430S.

53. The variant antigen binding construct of any one of claims 36-52, wherein the at least one surface-exposed, charged amino acid comprises at least one amino acid in the VH selected from (Aho numbering) K13, K14, K24, K73, D83, E84, E92, and R97.

54. The variant antigen binding construct of any one of claims 36-53, wherein the substitution of the at least one surface-exposed, charged amino acid of the localized patch with the amino acid having a different charge polarity comprises at least one substitution in the VH selected from (Aho numbering) K13V, K14Q, K24A, K73D, K73S, D83S, E84N, E92S, and R97T.

55. The variant antigen binding construct of any one of claims 36-54, wherein the at least one surface-exposed, charged amino acid comprises at least one amino acid in the VL selected from (Aho numbering) E29, K50, K53, KI 10, and El 11.

56. The variant antigen binding construct of any one of claims 36-55, wherein the substitution of the at least one surface-exposed, charged amino acid of the localized patch with the amino acid having a different charge polarity comprises at least one substitution in the VL selected from (Aho numbering) E29Q, K50Q, K53Q, KI 10G, and El 1 IS57. The variant antigen binding construct of any one of claims 36-56, wherein the substitution of the at least one surface-exposed, charged amino acid of the localized patch with the amino acid having a different charge polarity comprises a substitution of an upper hinge sequence of the hinge region of the original antigen binding construct with GGSGGGSTHT (SEQ ID NO:52), optionally wherein the substitution of the at least one surface-exposed, charged amino acid of the localized patch with the amino acid having a different chargepolarity comprises a substitution of the hinge region of the original antigen binding construct with GGSGGGSTHTCPPCPPCGGGSSGGGSGG (SEQ ID NO: 138).

58. The variant antigen binding construct of any one of claims 36-57, wherein the CH2 of the original antigen binding construct comprises (EU numbering): E233, D265, E269, D270, E272, D280, E293, and E294; and an amino acid sequence that is, is at least, or is about 80% identical to the sequence set forth in SEQ ID NO:57.

59. The variant antigen binding construct of any one of claims 36-58, wherein the CH3 of the original antigen binding construct comprises (EU numbering): R355, E356, D376, E380, E382, K414, and E430; and an amino acid sequence that is, is at least, or is about 80% identical to the sequence set forth in SEQ ID NO:77.

60. The variant antigen binding construct of any one of claims 36-59, wherein the VH of the original antigen binding construct comprises (Aho numbering): KI 3, K14, K24, R45, D67, K73, R77, D83, E84, E92, and R97; and an amino acid sequence that is, is at least, or is about 80% identical to sequences outside a HCDR in SEQ ID NO: 19.

61. The variant antigen binding construct of any one of claims 36-60, wherein the VL of the original antigen binding construct comprises (Aho numbering): D17, E29, K47, K50, K53, KI 10, El 11, and E147; and an amino acid sequence that is, is at least, or is about 80% identical to sequences outside a LCDR in SEQ ID NO:2.

62. The variant antigen binding construct of any one of claims 36-61, comprising at least one mutation in a Fc region that changes an effector function and / or binding to the Fc neonatal receptor, optionally wherein the at least one mutation is at (EU numbering) 1253, N297, H310, and / or H435 of the original antigen binding construct, further optionally wherein the at least one mutation is selected from I253A, N297Q, H310A, and H435A (EU numbering).

63. The variant antigen binding construct of any one of claims 36-62, wherein the hinge region of the original antigen binding construct comprises an upper hinge comprising an amino acid sequence of SEQ ID NO: 51.

64. The variant antigen binding construct of any one of claims 36-63 that is an antibody, minibody, cys-diabody, scFv-Fc fusion, or a nanobody®-Fc, optionally wherein the variant antigen binding construct is a minibody or scFv-Fc fusion.

65. The variant antigen binding construct of any one of claims 37-64, wherein the localized patch of surface charge is mapped using molecular modeling of iso-potential surfaces with a cut-off at + / -1.0 kT / e.

66. The variant antigen binding construct of any one of claims 37-65, wherein the localized patch of surface charge comprises at least two surface-exposed, charged amino acids within about 30 angstroms of each other, optionally the at least two surface-exposed, charged amino acids are within about 15 angstroms of each other.

67. An antigen binding construct comprising a CH2 domain comprising (EU numbering): S233, S265, S269, S270, S272S, S280, S293, and / or S294, optionally the CH2 domain comprising S233, S269, and / or S270.

68. The antigen binding construct of claim 67, wherein the antigen binding construct comprises a variable heavy (VH) domain; a variable light (VL) domain; a hinge domain; and a constant heavy domain 3 (CH3), wherein(1) the VH domain comprises (Aho numbering): V13, Q14, A24, D73, S73, S83, N84, S92, and / or T97;(2) the VL domain comprises (Aho numbering): Q29, Q50, Q53K, G110, and / or Si l l;(3) the hinge domain comprises: an upper hinge region comprising the amino acid sequence of SEQ ID NO: 52; or the amino acid sequence of SEQ ID NO: 138; and / or(4) the CH3 comprises: Q355, S356, S376, S380, S382, T382, T414, and S430.

69. An antigen binding construct comprising: a variable heavy (VH) domain; a variable light (VL) domain; a hinge domain; and a constant heavy domain 3 (CH3), optionally further comprising a CH2 domain, wherein(1) the VH domain comprises (Aho numbering): VI 3, QI 4, A24, D73, S73, S83, N84, S92, and / or T97;(2) the VL domain comprises (Aho numbering): Q29, Q50, Q53K, G110, and / or Si l l;(3) the hinge domain comprises : an upper hinge region comprising the amino acid sequence of SEQ ID NO: 52; or the amino acid sequence of SEQ ID NO: 138; and / or(4) the CH3 comprises: Q355, S356, S376, S380, S382, T382, T414, and S430,optionally (5) the CH2 comprises (EU numbering): S233, S265, S269, S270, S272S, S280, S293, and / or S294, further optionally, the CH2 comprises (EU numbering): S233, S269, and / or S270.

70. The antigen binding construct of any one of claims 68 or 69, wherein (1) the VH domain comprises an amino acid sequence that is, is at least, or is about 80% identical to sequences outside a HCDR in SEQ ID NO: 19; (2) the VL domain comprises an amino acid sequence that is, is at least, or is about 80% identical to sequences outside a LCDR in SEQ ID NO:2; (3) the CH3 comprises an amino acid sequence that is, is at least, or is about 80% identical to the sequence set forth in SEQ ID NO:77.

71. The antigen binding construct of any one of claims 67-70, wherein the CH2 comprises an amino acid sequence that is, is at least, or is about 80% identical to the sequence set forth in SEQ ID NO: 57.

72. The antigen binding construct of any one of the preceding claims that is an antibody, minibody, cys-diabody, scFv-Fc fusion, or a nanobody®-Fc, optionally wherein the antigen binding construct is a minibody or scFv-Fc fusion.

73. The antigen binding construct of any one of the preceding claims, wherein the antigen binding construct binds to DLL3, FAP, CD8, CD4, CD3, IFNy, integrin otVP6, FOLRa, or PSMA.

74. An antigen binding construct comprising: a means for binding a target antigen; and an IgG CH2 comprising: at least 80% sequence identity to an IgG CH2 in SEQ ID NO. 57; and a substitution at at least one of the following positions (EU numbering): E233, D265, E269, D270, E272, D280, E293, and E294, wherein each substitution is with S, V, G, A, Q, N, T, K, R, H, P, Y, F, W, M, L, or I, optionally wherein the IgG CH2 comprises a substitution at E233, E269, and / or E270, further optionally wherein the IgG CH2 comprises a substitution at E233 / E269 or E233 / D270.

75. A pharmaceutical composition comprising the antigen binding construct of any one of the preceding claims; and a therapeutic agent, toxic payload, and / or a detectable marker.

76. The pharmaceutical composition of claim 75, wherein the therapeutic agent, toxic payload, and / or a detectable marker is selected from a cytotoxic agent, a radioisotope, a chemotherapeutic agent, an immunotherapeutic agent, a DNA damage or DNA repair specificagent, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator or an enzyme.

77. A method of treating cancer, comprising administering the antigen binding construct of any one of claims 1-74 or the pharmaceutical composition of claim 75 or 76 to a subject in need of treatment for cancer.

78. A method of radiotherapy, comprising: identifying a subject in need of radiotherapy; and administering to the subject a therapeutically effective amount of the antigen binding construct of any one of claims 1-74 or the pharmaceutical composition of claim 75 or 76, wherein the antigen binding construct comprises a radionuclide.

79. A method of imaging a subject, comprising: administering to a subject a composition comprising an effective amount of the antigen binding construct of any one of claims 1-74 or the pharmaceutical composition of claim 75 or 76, wherein the antigen binding construct is detectably labeled; and imaging the subject to detect the labeled antigen binding construct in the subject.

80. The antigen binding construct of any one of claims 1-74, for treatment of cancer in a subject in need thereof.

81. The pharmaceutical composition of claim 75 or 76, for treatment of cancer in a subject in need thereof.

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