Multispecific binding protein degradation platform and methods of use

Abstract

The present disclosure relates to a multispecific antibody platform for targeted degradation of cell surface proteins. The present disclosure relates to multispecific (e.g., bispecific or trispecific) binding molecules, such as multispecific antibodies, that target at least one transmembrane E3 ubiquitin ligase protein and at least one cell surface protein, e.g., cell surface proteins for degradation, and methods of use thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 145,336, filed February 3, 2021; U.S. Provisional Application No. 63 / 217,470, filed July 1, 2021; and U.S. Provisional Application No. 63 / 274,288, filed November 1, 2021. The disclosures of each of these provisional applications are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a computer-readable sequence listing entitled "01164-0011-00 PCT_ST 25", created on February 1, 2022, having a size of 501 KB, which is incorporated herein by reference.

[0003] Field The present disclosure relates to a multispecific antibody platform for targeted degradation of cell surface proteins. The present disclosure relates to multispecific (e.g., bispecific or trispecific) binding molecules, such as multispecific antibodies, that target at least one transmembrane E3 ubiquitin ligase protein and at least one cell surface protein, e.g., cell surface proteins for degradation, and methods of use thereof. [Background technology]

[0004] background Various platforms have been constructed with the aim of targeting the degradation of proteins of interest. For example, PROTACs (Proteolysis Targeting Chimeras) are small molecule constructs intended to target cytoplasmic proteins to the 26S proteosome for degradation. They may contain a domain that binds to ubiquitin E3 ligase, a domain that binds to the protein to be targeted for degradation (i.e., the protein of interest), and a linker molecule that connects these two binding domains. However, PROTACs target cytoplasmic protein domains, which means that this approach cannot be used with certain membrane-bound or cell surface protein targets. (See, for example, Ahn et al., ChemRxiv, doi.org / 10.26434 / chemrxiv.12736778.v 1, submitted July 30, 2020). LYTAC (lysosome-targeting chimera) constructs are, for example, multivalent constructs that bind to membrane proteins of interest and also contain a ligand, such as mannose-6-phosphate, which binds to the cation-independent mannose-6-phosphate receptor (CI-M6PR). Linking the targeted membrane protein to CI-M6PR directs the protein to the lysosome for degradation. See ibid. However, unlike PROTACs, LYTACs require, for example, chemical conjugation to mannose-6-phosphate, which may need to be controlled during production, such as to avoid product heterogeneity. Furthermore, CI-M6PR is expressed in almost all tissues, so this approach may not be useful for selectively degrading proteins in specific tissues. A bispecific antibody that binds the cell surface E3 ubiquitin ligase RNF43 and the cell surface immune checkpoint protein PD-L1 was described by Cotton et al. (J. Am. Chem. Soc. 2021, available at https: / / dx.doi.org / 10.1021 / jacs.0c10008). Lysosomal degradation of PD-L1 was demonstrated in tumor cell lines exposed to the bispecific antibody in vitro.WO2021 / 176034 describes a bispecific single chain (VHH) "anti-tag" antibody capable of degrading tagged cell surface proteins that are also transiently expressed in HEK293T cells that transiently express a tagged E3 ligase. However, the general applicability of this approach, including the efficiency of degradation of endogenous cell surface proteins in cells expressing endogenous E3 ligases and efficacy in vivo, remains unknown, as do the optimal attributes of a platform that utilizes antibodies against cell surface ligases. Summary of the Invention

[0005] overview The present disclosure relates to improved approaches to targeted degradation of membrane-bound and cell surface proteins using at least one transmembrane E3 ubiquitin ligase (e.g., RNF43 or ZNRF3, or RNF128, RNF130, RNF133, RNF148, RNF149, RNF150, RNF167 or ZNRF4) or other cell surface ligase and a multispecific binding protein, such as a multispecific (e.g., bispecific or trispecific) antibody that binds to the cell surface protein targeted for degradation. The multispecific binding protein uses the transmembrane E3 ubiquitin ligase to target the cell surface protein of interest, for example, to the lysosome for degradation. Zinc and RING finger 3 (ZNRF3) and its homolog RING finger 43 (RNF43) are exemplary transmembrane E3 ubiquitin ligases that normally promote the degradation and turnover of Frizzled (FZD) and LRP6 receptors on the cell surface (Hao et al., Nature 485:195-200 (2012); Koo et al., Nature 488:665-669 (2012)). RNF43 and ZNRF3 are also members of the Wnt signaling pathway, and can be activated, for example, in certain types of cancer. (See, for example, FIG. 1C). Thus, tumor cells from various cancers can express relatively high levels of RNF43 and ZNRF3 compared to other cells, which in some embodiments can allow selective degradation of target proteins in tumor cells. Various other transmembrane E3 ubiquitin ligases can also be used as targets of the multispecific binding proteins herein. Optimal attributes of multispecific antibodies, including, for example, optimized binding affinity and multispecific formats, are also provided herein. In some embodiments, the multispecific binding proteins herein are referred to as PROTABs ("Proteolysis Targeting Antibodies").

[0006] In addition to providing a general platform for multispecific binding proteins that target transmembrane E3 ubiquitin ligases and cell surface proteins for destruction, the present disclosure also provides various antibody heavy and light chain variable regions that target RNF43 or ZNRF3 that may be useful in constructing such multispecific antibodies and binding proteins. The binding proteins herein may be used therapeutically in some embodiments, for example, but not limited to, in cells in which the Wnt pathway is activated and expresses transmembrane E3 ubiquitin ligases, for example, to degrade proteins that contribute to disease progression. For example, certain cancers, such as colorectal cancer (CRC), are characterized by overexpression of RNF43 and ZNRF3. Thus, the multispecific binding proteins herein that target one or both of these ligases may be selectively targeted to cancer cells due to those cells overexpressing these proteins. The binding proteins herein may also be used in vitro, for example, to degrade specific membrane proteins in cell culture or tissue assays, thereby reducing their levels and knocking down their associated signaling. For example, certain E3 ubiquitin ligases (e.g., RNF130, RNF149, and RNF167) are expressed in hematopoietic cells, and in some embodiments, multispecific binding proteins targeting those ligases can be used to target hematopoietic cells in vivo or in vitro. Similarly, E3 ubiquitin ligases such as RNF133 and RNF148 are expressed in testicular cells, and multispecific binding proteins targeting those ligases can be used in some embodiments to target testicular cells in vivo or in vitro.

[0007] The present invention provides, inter alia, a multispecific binding protein that binds at least a first cell surface target protein and a second cell surface protein, wherein the first cell surface target protein is a transmembrane E3 ubiquitin ligase. In some embodiments, the multispecific binding protein reduces the level of the second cell surface protein on the surface of a cell compared to the level observed in the absence of the multispecific binding protein. In certain embodiments, the multispecific binding protein reduces the level of the second cell surface protein on the surface of a cell in vitro compared to the level observed in the absence of the multispecific binding protein. In some such cases, the multispecific binding protein reduces the level of the second cell surface protein on the surface of a cell in vitro as measured by flow cytometry or luminescence assay. In some cases, the multispecific binding protein reduces the level of the second cell surface protein on the surface of a cell in vivo compared to the level observed in the absence of the multispecific binding protein or both in vitro and in vivo.

[0008] In some embodiments, the multispecific binding protein herein is a multispecific antibody. For example, in some embodiments, the protein is a bispecific or trispecific antibody, such as a 1+1 FabIgG, a 1+1 FvIgG, a 2+1 FvIgG, a 2+1 FabIgG, a 1-arm FvIgG, or a 1-arm FabIgG. In some such cases, the protein comprises an IgG Fc region that comprises at least one knob-into-hole modification.

[0009] In some embodiments, the transmembrane E3 ubiquitin ligase does not have catalytic activity and the lack of catalytic activity is determined in a cell surface degradation assay. In other embodiments, the transmembrane E3 ubiquitin ligase has catalytic activity and the presence of catalytic activity is determined in a cell surface degradation assay.

[0010] In some embodiments, the multispecific binding protein herein binds to a transmembrane E3 ubiquitin ligase selected from RNF43, ZNRF3, RNF13, RNF128, RNF130, RNF133, RNF148, RNF149, RNF150, RNF167, ZNRF4, RSPRY1, SYVN1, LNX1 isoform 2 and TRIM7 isoform 3. In some embodiments, the multispecific binding protein herein binds to a transmembrane E3 ubiquitin ligase selected from RNF43, RNF128, RNF130, RNF133, RNF149, RNF150 or ZNRF3. In some embodiments, the transmembrane E3 ubiquitin ligase is RNF130, RNF133, RNF149 or RNF150. In some embodiments, the transmembrane E3 ubiquitin ligase is RNF130, RNF149, or RNF167. In some embodiments, the transmembrane E3 ubiquitin ligase is RNF133 or RNF148. In some embodiments, the transmembrane E3 ubiquitin ligase is RNF43 or ZNFR3, or both, such that the protein binds to RNF43, ZNRF3, or both RNF43 and ZNRF3, and optionally, the protein does not block the binding between RNF43 and / or ZNRF3 and FZD and / or LRP6.

[0011] In some embodiments, the multispecific binding protein is a multispecific antibody that binds to RNF43 or ZNRF3; or a multispecific antibody that binds to RNF43 or ZNRF3 and comprises an antibody heavy chain variable region (VH) comprising (a) CDR-H1, (b) CDR-H2 and (c) CDR-H3, and a light chain variable domain (VL) comprising (d) CDR-L1, (e) CDR-L2 and (f) CDR-L3; or a multispecific antibody that binds to RNF43 or ZNRF3 and comprises a VH and a VL.

[0012] <h2 style=";text-align:left;direction:ltr">RNF 43-104、RNF43-106、RNF43-107、RNF4 3-108、RNF43-116、RNF43-117、RNF43-123、RNF43-126、RNF43-128、RNF43-1 29、RNF43-130、RNF43-136、RNF43-145、RNF43-152、RNF43-156、RNF43-168 、RNF43-170、RNF43-176、RNF43-177、RNF43-179、RNF43-180、RNF43-181、RN F43-186, RNF43-187, RNF43-196, RNF43-200, RNF43-201, RNF43-206, RNF43-210, RNF43-213, RNF43-217, RNF43-221, RNF43-224, RNF43-25, RNF43-31 RNF43-33, RNF43-35, RNF43-38, RNF43-41, RNF43-53, RNF43-56, RNF43-61, RNF43-67, RNF43-69, RNF43-71, RNF43-74, RNF43-75, RNF43-76, RNF43-8 0、RNF43-86、RNF43-90、ZNRF3-101、ZNRF3-117、ZNRF3-128、ZNRF3-131、ZN RF3-163、ZNRF3-17、ZNRF3-170、ZNRF3-171、ZNRF3-172、ZNRF3-179、ZNRF3- 182、ZNRF3-195、ZNRF3-219、ZNRF3-222、ZNRF3-223、ZNRF3-231、ZNRF3-23 7、ZNRF3-244、ZNRF3-247、ZNRF3-253、ZNRF3-254、ZNRF3-255、ZNRF3-265、Z NRF3-269、ZNRF3-270、ZNRF3-275、ZNRF3-279、ZNRF3-287、ZNRF3-296、ZNR F3-30、ZNRF3-300、ZNRF3-301、ZNRF3-305、ZNRF3-312、ZNRF3-314、ZNRF3-3 22, ZNRF3-329, ZNRF3-35, ZNRF3-55, ZNRF3-6, ZNRF3-90, RNF43-1, RNF43-24, RNF43-8, RNF43-12, RNF43-20, RNF43-11, RNF43-23, RNF43-6, RNF43-5,The heavy chain variable domain (VH) includes the heavy chain complementarity determining region 1 (CDR-H1), CDR-H2 and / or CDR-H3 of any one of RNF43-15, RNF43-2, RNF43-16, RNF43-14, RNF43-17, RNF43-22, RNF43-9, RNF43-21, RNF43-13, RNF43-19, ZNRF3-331, ZNRF3-333 or ZNRF3-332.

[0013] <h2 style=";text-align:left;direction:ltr">RNF 43-104、RNF43-106、RNF43-107、RNF4 3-108、RNF43-116、RNF43-117、RNF43-123、RNF43-126、RNF43-128、RNF43-1 29、RNF43-130、RNF43-136、RNF43-145、RNF43-152、RNF43-156、RNF43-168 、RNF43-170、RNF43-176、RNF43-177、RNF43-179、RNF43-180、RNF43-181、RN F43-186, RNF43-187, RNF43-196, RNF43-200, RNF43-201, RNF43-206, RNF43-210, RNF43-213, RNF43-217, RNF43-221, RNF43-224, RNF43-25, RNF43-31 RNF43-33, RNF43-35, RNF43-38, RNF43-41, RNF43-53, RNF43-56, RNF43-61, RNF43-67, RNF43-69, RNF43-71, RNF43-74, RNF43-75, RNF43-76, RNF43-8 0、RNF43-86、RNF43-90、ZNRF3-101、ZNRF3-117、ZNRF3-128、ZNRF3-131、ZN RF3-163、ZNRF3-17、ZNRF3-170、ZNRF3-171、ZNRF3-172、ZNRF3-179、ZNRF3- 182、ZNRF3-195、ZNRF3-219、ZNRF3-222、ZNRF3-223、ZNRF3-231、ZNRF3-23 7、ZNRF3-244、ZNRF3-247、ZNRF3-253、ZNRF3-254、ZNRF3-255、ZNRF3-265、Z NRF3-269、ZNRF3-270、ZNRF3-275、ZNRF3-279、ZNRF3-287、ZNRF3-296、ZNR F3-30、ZNRF3-300、ZNRF3-301、ZNRF3-305、ZNRF3-312、ZNRF3-314、ZNRF3-3 22, ZNRF3-329, ZNRF3-35, ZNRF3-55, ZNRF3-6, ZNRF3-90, RNF43-1, RNF43-24, RNF43-8, RNF43-12, RNF43-20, RNF43-11, RNF43-23, RNF43-6, RNF43-5,The heavy chain variable region (VH) includes CDR-H1, CDR-H2 and CDR-H3 of any one of RNF43-15, RNF43-2, RNF43-16, RNF43-14, RNF43-17, RNF43-22, RNF43-9, RNF43-21, RNF43-13, RNF43-19, ZNRF3-331, ZNRF3-333 or ZNRF3-332.

[0014] <h2 style=";text-align:left;direction:ltr">RNF 43-104、RNF43-106、RNF43-107、RNF4 3-108、RNF43-116、RNF43-117、RNF43-123、RNF43-126、RNF43-128、RNF43-1 29、RNF43-130、RNF43-136、RNF43-145、RNF43-152、RNF43-156、RNF43-168 、RNF43-170、RNF43-176、RNF43-177、RNF43-179、RNF43-180、RNF43-181、RN F43-186, RNF43-187, RNF43-196, RNF43-200, RNF43-201, RNF43-206, RNF43-210, RNF43-213, RNF43-217, RNF43-221, RNF43-224, RNF43-25, RNF43-31 RNF43-33, RNF43-35, RNF43-38, RNF43-41, RNF43-53, RNF43-56, RNF43-61, RNF43-67, RNF43-69, RNF43-71, RNF43-74, RNF43-75, RNF43-76, RNF43-8 0、RNF43-86、RNF43-90、ZNRF3-101、ZNRF3-117、ZNRF3-128、ZNRF3-131、ZN RF3-163、ZNRF3-17、ZNRF3-170、ZNRF3-171、ZNRF3-172、ZNRF3-179、ZNRF3- 182、ZNRF3-195、ZNRF3-219、ZNRF3-222、ZNRF3-223、ZNRF3-231、ZNRF3-23 7、ZNRF3-244、ZNRF3-247、ZNRF3-253、ZNRF3-254、ZNRF3-255、ZNRF3-265、Z NRF3-269、ZNRF3-270、ZNRF3-275、ZNRF3-279、ZNRF3-287、ZNRF3-296、ZNR F3-30、ZNRF3-300、ZNRF3-301、ZNRF3-305、ZNRF3-312、ZNRF3-314、ZNRF3-3 22, ZNRF3-329, ZNRF3-35, ZNRF3-55, ZNRF3-6, ZNRF3-90, RNF43-1, RNF43-24, RNF43-8, RNF43-12, RNF43-20, RNF43-11, RNF43-23, RNF43-6, RNF43-5,The light chain variable domain (VL) comprises a light chain complementarity determining region 1 (CDR-L1), CDR-L2 and / or CDR-L3 of any one of RNF43-15, RNF43-2, RNF43-16, RNF43-14, RNF43-17, RNF43-22, RNF43-9, RNF43-21, RNF43-13, RNF43-19, ZNRF3-331, ZNRF3-333 or ZNRF3-332.

[0015] <h2 style=";text-align:left;direction:ltr">RNF 43-104、RNF43-106、RNF43-107、RNF4 3-108、RNF43-116、RNF43-117、RNF43-123、RNF43-126、RNF43-128、RNF43-1 29、RNF43-130、RNF43-136、RNF43-145、RNF43-152、RNF43-156、RNF43-168 、RNF43-170、RNF43-176、RNF43-177、RNF43-179、RNF43-180、RNF43-181、RN F43-186, RNF43-187, RNF43-196, RNF43-200, RNF43-201, RNF43-206, RNF43-210, RNF43-213, RNF43-217, RNF43-221, RNF43-224, RNF43-25, RNF43-31 RNF43-33, RNF43-35, RNF43-38, RNF43-41, RNF43-53, RNF43-56, RNF43-61, RNF43-67, RNF43-69, RNF43-71, RNF43-74, RNF43-75, RNF43-76, RNF43-8 0、RNF43-86、RNF43-90、ZNRF3-101、ZNRF3-117、ZNRF3-128、ZNRF3-131、ZN RF3-163、ZNRF3-17、ZNRF3-170、ZNRF3-171、ZNRF3-172、ZNRF3-179、ZNRF3- 182、ZNRF3-195、ZNRF3-219、ZNRF3-222、ZNRF3-223、ZNRF3-231、ZNRF3-23 7、ZNRF3-244、ZNRF3-247、ZNRF3-253、ZNRF3-254、ZNRF3-255、ZNRF3-265、Z NRF3-269、ZNRF3-270、ZNRF3-275、ZNRF3-279、ZNRF3-287、ZNRF3-296、ZNR F3-30、ZNRF3-300、ZNRF3-301、ZNRF3-305、ZNRF3-312、ZNRF3-314、ZNRF3-3 22, ZNRF3-329, ZNRF3-35, ZNRF3-55, ZNRF3-6, ZNRF3-90, RNF43-1, RNF43-24, RNF43-8, RNF43-12, RNF43-20, RNF43-11, RNF43-23, RNF43-6, RNF43-5,The light chain variable region (VL) comprises CDR-L1, CDR-L2 and / or CDR-L3 of any one of RNF43-15, RNF43-2, RNF43-16, RNF43-14, RNF43-17, RNF43-22, RNF43-9, RNF43-21, RNF43-13, RNF43-19, ZNRF3-331, ZNRF3-333 or ZNRF3-332.

[0016] <h2 style=";text-align:left;direction:ltr">RNF 43-104、RNF43-106、RNF43-107、RNF4 3-108、RNF43-116、RNF43-117、RNF43-123、RNF43-126、RNF43-128、RNF43-1 29、RNF43-130、RNF43-136、RNF43-145、RNF43-152、RNF43-156、RNF43-168 、RNF43-170、RNF43-176、RNF43-177、RNF43-179、RNF43-180、RNF43-181、RN F43-186, RNF43-187, RNF43-196, RNF43-200, RNF43-201, RNF43-206, RNF43-210, RNF43-213, RNF43-217, RNF43-221, RNF43-224, RNF43-25, RNF43-31 RNF43-33, RNF43-35, RNF43-38, RNF43-41, RNF43-53, RNF43-56, RNF43-61, RNF43-67, RNF43-69, RNF43-71, RNF43-74, RNF43-75, RNF43-76, RNF43-8 0、RNF43-86、RNF43-90、ZNRF3-101、ZNRF3-117、ZNRF3-128、ZNRF3-131、ZN RF3-163、ZNRF3-17、ZNRF3-170、ZNRF3-171、ZNRF3-172、ZNRF3-179、ZNRF3- 182、ZNRF3-195、ZNRF3-219、ZNRF3-222、ZNRF3-223、ZNRF3-231、ZNRF3-23 7、ZNRF3-244、ZNRF3-247、ZNRF3-253、ZNRF3-254、ZNRF3-255、ZNRF3-265、Z NRF3-269、ZNRF3-270、ZNRF3-275、ZNRF3-279、ZNRF3-287、ZNRF3-296、ZNR F3-30、ZNRF3-300、ZNRF3-301、ZNRF3-305、ZNRF3-312、ZNRF3-314、ZNRF3-3 22, ZNRF3-329, ZNRF3-35, ZNRF3-55, ZNRF3-6, ZNRF3-90, RNF43-1, RNF43-24, RNF43-8, RNF43-12, RNF43-20, RNF43-11, RNF43-23, RNF43-6, RNF43-5,(a) a heavy chain variable domain (VH) comprising heavy chain complementarity determining region 1 (CDR-H1), CDR-H2 and CDR-H3, and (b) a light chain variable domain (VL) comprising light chain complementarity determining region 1 (CDR-L1), CDR-L2 and CDR-L3, of any one of RNF43-15, RNF43-2, RNF43-16, RNF43-14, RNF43-17, RNF43-22, RNF43-9, RNF43-21, RNF43-13, RNF43-19, ZNRF3-331, ZNRF3-333 or ZNRF3-332.

[0017] In any of the above cases, the heavy and / or light chain CDRs may be Kabat, Chothia or McCallum CDRs. The amino acid and DNA sequences of the relevant VH and VL of the antibodies listed above are provided in the sequence listing below with SEQ ID NOs: 33-444.

[0018] <h2 style=";text-align:left;direction:ltr">RNF 43-104、RNF43-106、RNF43-107、RNF4 3-108、RNF43-116、RNF43-117、RNF43-123、RNF43-126、RNF43-128、RNF43-1 29、RNF43-130、RNF43-136、RNF43-145、RNF43-152、RNF43-156、RNF43-168 、RNF43-170、RNF43-176、RNF43-177、RNF43-179、RNF43-180、RNF43-181、RN F43-186, RNF43-187, RNF43-196, RNF43-200, RNF43-201, RNF43-206, RNF43-210, RNF43-213, RNF43-217, RNF43-221, RNF43-224, RNF43-25, RNF43-31 RNF43-33, RNF43-35, RNF43-38, RNF43-41, RNF43-53, RNF43-56, RNF43-61, RNF43-67, RNF43-69, RNF43-71, RNF43-74, RNF43-75, RNF43-76, RNF43-8 0、RNF43-86、RNF43-90、ZNRF3-101、ZNRF3-117、ZNRF3-128、ZNRF3-131、ZN RF3-163、ZNRF3-17、ZNRF3-170、ZNRF3-171、ZNRF3-172、ZNRF3-179、ZNRF3- 182、ZNRF3-195、ZNRF3-219、ZNRF3-222、ZNRF3-223、ZNRF3-231、ZNRF3-23 7、ZNRF3-244、ZNRF3-247、ZNRF3-253、ZNRF3-254、ZNRF3-255、ZNRF3-265、Z NRF3-269、ZNRF3-270、ZNRF3-275、ZNRF3-279、ZNRF3-287、ZNRF3-296、ZNR F3-30、ZNRF3-300、ZNRF3-301、ZNRF3-305、ZNRF3-312、ZNRF3-314、ZNRF3-3 22, ZNRF3-329, ZNRF3-35, ZNRF3-55, ZNRF3-6, ZNRF3-90, RNF43-1, RNF43-24, RNF43-8, RNF43-12, RNF43-20, RNF43-11, RNF43-23, RNF43-6, RNF43-5,and a heavy chain variable region (VH) comprising an amino acid sequence at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the VH of any one of RNF43-15, RNF43-2, RNF43-16, RNF43-14, RNF43-17, RNF43-22, RNF43-9, RNF43-21, RNF43-13, RNF43-19, ZNRF3-331, ZNRF3-333 or ZNRF3-332, and optionally the amino acid sequence of the CDR-H1, CDR-H2 and / or CDR-H3 of the VH is 100% identical to the amino acid sequence of the selected antibody.

[0019] <h2 style=";text-align:left;direction:ltr">RNF 43-104、RNF43-106、RNF43-107、RNF4 3-108、RNF43-116、RNF43-117、RNF43-123、RNF43-126、RNF43-128、RNF43-1 29、RNF43-130、RNF43-136、RNF43-145、RNF43-152、RNF43-156、RNF43-168 、RNF43-170、RNF43-176、RNF43-177、RNF43-179、RNF43-180、RNF43-181、RN F43-186, RNF43-187, RNF43-196, RNF43-200, RNF43-201, RNF43-206, RNF43-210, RNF43-213, RNF43-217, RNF43-221, RNF43-224, RNF43-25, RNF43-31 RNF43-33, RNF43-35, RNF43-38, RNF43-41, RNF43-53, RNF43-56, RNF43-61, RNF43-67, RNF43-69, RNF43-71, RNF43-74, RNF43-75, RNF43-76, RNF43-8 0、RNF43-86、RNF43-90、ZNRF3-101、ZNRF3-117、ZNRF3-128、ZNRF3-131、ZN RF3-163、ZNRF3-17、ZNRF3-170、ZNRF3-171、ZNRF3-172、ZNRF3-179、ZNRF3- 182、ZNRF3-195、ZNRF3-219、ZNRF3-222、ZNRF3-223、ZNRF3-231、ZNRF3-23 7、ZNRF3-244、ZNRF3-247、ZNRF3-253、ZNRF3-254、ZNRF3-255、ZNRF3-265、Z NRF3-269、ZNRF3-270、ZNRF3-275、ZNRF3-279、ZNRF3-287、ZNRF3-296、ZNR F3-30、ZNRF3-300、ZNRF3-301、ZNRF3-305、ZNRF3-312、ZNRF3-314、ZNRF3-3 22, ZNRF3-329, ZNRF3-35, ZNRF3-55, ZNRF3-6, ZNRF3-90, RNF43-1, RNF43-24, RNF43-8, RNF43-12, RNF43-20, RNF43-11, RNF43-23, RNF43-6, RNF43-5,and a light chain variable region (VL) comprising an amino acid sequence at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the VL of any one of RNF43-15, RNF43-2, RNF43-16, RNF43-14, RNF43-17, RNF43-22, RNF43-9, RNF43-21, RNF43-13, RNF43-19, ZNRF3-331, ZNRF3-333 or ZNRF3-332, and optionally the amino acid sequence of the CDR-H1, CDR-H2 and / or CDR-H3 of the VH is 100% identical to the amino acid sequence of the selected antibody.

[0020] <h2 style=";text-align:left;direction:ltr">RNF 43-104、RNF43-106、RNF43-107、RNF4 3-108、RNF43-116、RNF43-117、RNF43-123、RNF43-126、RNF43-128、RNF43-1 29、RNF43-130、RNF43-136、RNF43-145、RNF43-152、RNF43-156、RNF43-168 、RNF43-170、RNF43-176、RNF43-177、RNF43-179、RNF43-180、RNF43-181、RN F43-186, RNF43-187, RNF43-196, RNF43-200, RNF43-201, RNF43-206, RNF43-210, RNF43-213, RNF43-217, RNF43-221, RNF43-224, RNF43-25, RNF43-31 RNF43-33, RNF43-35, RNF43-38, RNF43-41, RNF43-53, RNF43-56, RNF43-61, RNF43-67, RNF43-69, RNF43-71, RNF43-74, RNF43-75, RNF43-76, RNF43-8 0、RNF43-86、RNF43-90、ZNRF3-101、ZNRF3-117、ZNRF3-128、ZNRF3-131、ZN RF3-163、ZNRF3-17、ZNRF3-170、ZNRF3-171、ZNRF3-172、ZNRF3-179、ZNRF3- 182、ZNRF3-195、ZNRF3-219、ZNRF3-222、ZNRF3-223、ZNRF3-231、ZNRF3-23 7、ZNRF3-244、ZNRF3-247、ZNRF3-253、ZNRF3-254、ZNRF3-255、ZNRF3-265、Z NRF3-269、ZNRF3-270、ZNRF3-275、ZNRF3-279、ZNRF3-287、ZNRF3-296、ZNR F3-30、ZNRF3-300、ZNRF3-301、ZNRF3-305、ZNRF3-312、ZNRF3-314、ZNRF3-3 22, ZNRF3-329, ZNRF3-35, ZNRF3-55, ZNRF3-6, ZNRF3-90, RNF43-1, RNF43-24, RNF43-8, RNF43-12, RNF43-20, RNF43-11, RNF43-23, RNF43-6, RNF43-5,The heavy chain variable region (VH) of any one of RNF43-15, RNF43-2, RNF43-16, RNF43-14, RNF43-17, RNF43-22, RNF43-9, RNF43-21, RNF43-13, RNF43-19, ZNRF3-331, ZNRF3-333, or ZNRF3-332.

[0021] <h2 style=";text-align:left;direction:ltr">RNF 43-104、RNF43-106、RNF43-107、RNF4 3-108、RNF43-116、RNF43-117、RNF43-123、RNF43-126、RNF43-128、RNF43-1 29、RNF43-130、RNF43-136、RNF43-145、RNF43-152、RNF43-156、RNF43-168 、RNF43-170、RNF43-176、RNF43-177、RNF43-179、RNF43-180、RNF43-181、RN F43-186, RNF43-187, RNF43-196, RNF43-200, RNF43-201, RNF43-206, RNF43-210, RNF43-213, RNF43-217, RNF43-221, RNF43-224, RNF43-25, RNF43-31 RNF43-33, RNF43-35, RNF43-38, RNF43-41, RNF43-53, RNF43-56, RNF43-61, RNF43-67, RNF43-69, RNF43-71, RNF43-74, RNF43-75, RNF43-76, RNF43-8 0、RNF43-86、RNF43-90、ZNRF3-101、ZNRF3-117、ZNRF3-128、ZNRF3-131、ZN RF3-163、ZNRF3-17、ZNRF3-170、ZNRF3-171、ZNRF3-172、ZNRF3-179、ZNRF3- 182、ZNRF3-195、ZNRF3-219、ZNRF3-222、ZNRF3-223、ZNRF3-231、ZNRF3-23 7、ZNRF3-244、ZNRF3-247、ZNRF3-253、ZNRF3-254、ZNRF3-255、ZNRF3-265、Z NRF3-269、ZNRF3-270、ZNRF3-275、ZNRF3-279、ZNRF3-287、ZNRF3-296、ZNR F3-30、ZNRF3-300、ZNRF3-301、ZNRF3-305、ZNRF3-312、ZNRF3-314、ZNRF3-3 22, ZNRF3-329, ZNRF3-35, ZNRF3-55, ZNRF3-6, ZNRF3-90, RNF43-1, RNF43-24, RNF43-8, RNF43-12, RNF43-20, RNF43-11, RNF43-23, RNF43-6, RNF43-5,The VL includes an amino acid sequence of the light chain variable region (VL) of any one of RNF43-15, RNF43-2, RNF43-16, RNF43-14, RNF43-17, RNF43-22, RNF43-9, RNF43-21, RNF43-13, RNF43-19, ZNRF3-331, ZNRF3-333, or ZNRF3-332. As mentioned above, the amino acid sequences and DNA sequences of the VH and VL of the antibodies listed above are those in SEQ ID NOs: 33 to 444 in the sequence listing below.

[0022] In some embodiments, the multispecific binding protein binds ZNRF3 and / or RNF43 and has a binding affinity for ZNRF3 or RNF43 of less than 50 nM, or less than 10 nM, or less than 1 nM, or less than 0.5 nM, or less than 0.05 nM, or between 50 nM and 10 nM, or between 10 nM and 1 nM, or between 1 nM and 0.5 nM, or between 0.5 nM and 0.05 nM, or between 0.05 nM and 0.01 nM. In some embodiments, the protein is a multispecific antibody comprising the heavy chain variable region and / or the light chain variable region of a rat anti-human RNF43 B cell antibody, a rat anti-human ZNRF3 B cell antibody, a rabbit anti-human RNF43 B cell antibody, or a rabbit anti-human ZNRF3 B cell antibody. In some embodiments, the protein is a trispecific antibody comprising a 2+1 FvIgG or 2+1 FabIgG format, which binds to both ZNRF3 and RNF43 and at least one second cell surface protein, and optionally does not block binding of ZNRF3 or RNF43 to FZD and LRP6.

[0023] In some embodiments, the multispecific binding protein herein may be a multispecific antibody that includes a heavy or light chain variable region that is chimeric, or may be a multispecific antibody that includes a humanized variable region. In some cases, the protein includes a wild-type human Fc region. In some cases, the Fc region is an IgG1, IgG2, IgG3, or IgG4 Fc region, and is optionally either a wild-type human Fc region or a human Fc region with one or more engineered mutations. In some cases, the protein includes an Fc region that has effector function. In some cases, the protein includes a human IgG1 Fc region that includes a LALAPG mutation or a substitution at position N297, such as N297G or N297Q, and / or the Fc region lacks effector function.

[0024] In some embodiments, the second cell surface protein to which the multispecific binding protein binds is a receptor tyrosine kinase, a growth factor receptor, a cytokine, a mucin, a Siglec receptor, or an immune checkpoint regulator, or is HER2, HER3, IGF1R, EGFR, FGFR, VEGFR, PDGFR, EpCAM, FZD, PD-L1, CTLA4, PD-1, TIM3, LAG3, TIGIT, CEACAM1, CD25, ILT-2, ILT-3, ILT-4, ILT-5, LAIR-1, PECAM-1 (CD31), PILR-alpha, SIRL-1, or SIRP-alpha. In some cases, the second cell surface protein is HER2, EGFR, or IGF1R. In some cases, the multispecific binding protein comprises the heavy and light chain variable regions of an anti-HER2 antibody, e.g., 4D5, 7C2, or 2C4, or an anti-IGF1R antibody, e.g., cizutumumab, ganitumab, dalotuzumab, figitumumab, lobatumumab, teprotumumab, or istiratumumab.

[0025] The present disclosure also relates to an isolated nucleic acid or set of nucleic acids (e.g., comprising two or more nucleic acids each encoding a portion of a multimeric protein, such as a light or heavy chain or a half antibody) encoding a multispecific binding protein as described herein. The present disclosure also relates to a host cell comprising such a nucleic acid or set of nucleic acids. The present disclosure also encompasses a method of making a multispecific binding protein herein, comprising culturing a host cell comprising a nucleic acid or set of nucleic acids encoding the protein under conditions suitable for expression of the protein. Such a method may further comprise recovering the protein from the host cell. The present disclosure further encompasses a multispecific binding protein produced by the methods herein.

[0026] Pharmaceutical compositions comprising a multispecific binding protein as described herein and a pharma- ceutically acceptable carrier can also be prepared.

[0027] The disclosure also encompasses multispecific binding proteins and related pharmaceutical compositions for use as drugs for use in treating, for example, a subject's cancer, an autoimmune condition, an inflammatory condition, a neurodegenerative condition, or an infectious disease, and / or for use in reducing the level of a cell surface protein in a subject in need of such a reduction (in some cases, the subject has cancer, an autoimmune condition, an inflammatory condition, a neurodegenerative condition, or an infectious disease), and / or for use in increasing an immune response in a subject, such as a subject having cancer, an autoimmune condition, an inflammatory condition, a neurodegenerative condition, or an infectious disease. In some embodiments, (a) the subject has a mutation in RNF43 and the multispecific binding protein does not bind to or activate RNF43, or (b) the subject has a mutation in ZNRF3 and the multispecific binding protein does not bind to or activate ZNRF3. In some such cases, if the subject has an RNF43 or ZNRF3 mutation, the subject has a cancer that includes the mutation.

[0028] The present disclosure also relates to the use of the multispecific binding protein or pharmaceutical composition herein in the manufacture of a medicament for treating a cancer, an autoimmune condition, an inflammatory condition, a neurodegenerative condition or an infectious disease in a subject; and / or a medicament for reducing the level of a cell surface protein in a subject in need of such a reduction (optionally the subject has cancer, an autoimmune condition, an inflammatory condition, a neurodegenerative condition or an infectious disease); and / or a medicament for increasing an immune response in a subject, such as a subject having cancer, an autoimmune condition, an inflammatory condition, a neurodegenerative condition or an infectious disease. In some embodiments, (a) the subject has a mutation in RNF43 and the multispecific binding protein does not bind to or activate RNF43, or (b) the subject has a mutation in ZNRF3 and the multispecific binding protein does not bind to or activate ZNRF3. In some such cases, if the subject has an RNF43 or ZNRF3 mutation, the subject has a cancer in which the cancer comprises the mutation.

[0029] The present disclosure also relates to a method of treating cancer, an autoimmune condition, an inflammatory condition, a neurodegenerative condition, or an infectious disease in a subject in need of such treatment, comprising administering to the subject an effective amount of a multispecific binding protein or pharmaceutical composition herein. The present disclosure further relates to a method of decreasing the level of a cell surface protein in a subject in need of such treatment, comprising administering to the subject an effective amount of a multispecific binding protein or pharmaceutical composition herein, optionally wherein the subject has cancer, an autoimmune condition, an inflammatory condition, a neurodegenerative condition, or an infectious disease. The present disclosure also relates to a method of increasing an immune response in a subject in need of such treatment, comprising administering to the subject an effective amount of a multispecific binding protein or pharmaceutical composition herein, optionally wherein the subject has cancer, an autoimmune condition, an inflammatory condition, a neurodegenerative condition, or an infectious disease. In some embodiments, (a) the subject has a mutation in RNF43, and the multispecific binding protein does not bind to or activate RNF43, or (b) the subject has a mutation in ZNRF3, and the multispecific binding protein does not bind to or activate ZNRF3. In some such cases, if the subject has an RNF43 or ZNRF3 mutation, the subject has a cancer in which the cancer comprises a mutation. In some cases, the method further comprises determining whether the subject has a mutation in RNF43 or ZNRF3 before administering the multispecific binding protein, and (a) if the subject has a mutation in RNF43, the multispecific binding protein does not bind to or activate RNF43, and (b) if the subject has a mutation in ZNRF3, the multispecific binding protein does not bind to or activate ZNRF3.

[0030] In some embodiments, the multispecific binding proteins herein can be used to deplete the levels of cell surface proteins on specific cell types by targeting E3 ubiquitin ligases that are primarily expressed on those cell types. For example, the present disclosure also includes a method of inducing the degradation of cell surface proteins on the surface of hematopoietic cells using the multispecific binding proteins herein, in which the transmembrane E3 ubiquitin ligase used in the multispecific protein is RNF130, RNF149 or RNF167. Furthermore, the present disclosure also includes a method of inducing the degradation of cell surface proteins on the surface of testicular cells using the multispecific binding proteins herein, in which the transmembrane E3 ubiquitin ligase used in the multispecific protein is RNF133 or RNF148. In certain embodiments, the multispecific binding proteins reduce the levels of cell surface proteins on the surface of specific cell types, such as hematopoietic or testicular cells, in vitro, compared to the levels observed in the absence of the multispecific binding proteins. In some such cases, the multispecific binding protein reduces the level of a cell surface protein on the surface of a particular cell type, such as hematopoietic cells or testicular cells, in vitro as measured by flow cytometry or luminescence assays. In some cases, the multispecific binding protein reduces the level of a cell surface protein on the surface of a particular cell type, such as these cells, in vivo, compared to the level observed in the absence of the multispecific binding protein or both in vitro and in vivo. Thus, the present disclosure also includes a method of reducing the level of a cell surface protein on the surface of hematopoietic cells in a cell or tissue sample in vitro or in vivo in a subject, the method comprising administering to the cell or tissue sample or subject a multispecific binding protein that targets one or more of RNF130, RNF149 and RNF167.The disclosure also includes a method of reducing the level of a cell surface protein on the surface of testicular cells in a cell or tissue sample in vitro or in vivo in a subject, the method comprising administering to the cell or tissue sample or to a subject a multispecific binding protein that targets one or both of RNF133 and RNF148.

[0031] The disclosure also encompasses kits comprising a multispecific binding protein, nucleic acid, set of nucleic acids, or host cells described herein, which further comprise one or more reagents for expressing or purifying the multispecific binding protein and / or one or more reagents for incubating the protein with a cell or tissue sample in vitro to reduce the level of a cell surface protein in the sample. The disclosure also encompasses a method of reducing the level of a cell surface protein in a cell or tissue sample in vitro comprising incubating the sample with a multispecific binding protein described herein. [Brief description of the drawings]

[0032] [Fig. 1A-1F]Figure 1A-F show that cell surface ligases driven by oncogenic Wnts can be repurposed for targeted degradation. Figure 1A) Schematic showing the role of RNF43 in controlling cell surface abundance of frizzled (FZD) receptors via ubiquitination. Figure 1B) Expression of RNF43 (left panel) and ZNRF3 (right panel) in human adenoma primary tissue (see Galamb O., et al., Cancer Epidemiol Biomarkers Prev 17(10):2835-2845, 2008). Figure 1C) pan-TCGA (The Cancer Genome Atlas) data showing expression of RNF43 in various cancer subtypes. Note high expression in colorectal cancer and other tumor subtypes showing elevated Wnt activity (red dots indicate tumor tissue; black dots indicate normal tissue). Figure 1D) Doxycycline-inducible expression of wild-type (WT) RNF43, but not delta-RING RNF43, a mutant deficient in ligase activity, leads to a reduction in cell surface expression of FZD. Figure 1E) Schematic of the strategy used to chemically dimerize RNF43 and / or ZNRF3 to non-native cell surfaces using A / C heterodimerizers with receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). Figure 1F) Immunoblot of tagged HER2 following immunoprecipitation of tagged RNF43 in transiently transfected HEK293T cells after treatment with A / C heterodimerizers. [Figures 2A-2E]Figures 2A-E show antibody-mediated dimerization of gD-tagged cell surface RNF43 or ZNRF3 ligase to HER2. Figure 2A) Schematic showing dimerization of N-terminal gD-tagged RNF43 or ZNRF3 ligase to a protein of interest using an anti-gD / anti-protein of interest (POI) bispecific antibody. Figure 2B) Bispecific antibodies against gD and three different HER2 epitopes. Figure 2C) Fluorescence assisted cell sorting (FACS) plot showing cell surface gD expression in HEK293T cells transiently transfected with either gD-tagged RNF43 or ZNRF3. Figure 2D) Western blot of HER2 in HEK293T transiently transfected with gD-tagged RNF43, gD-tagged ZNRF3 or control ("mock") after treatment with a bispecific antibody targeting gD and HER2 ("gD / HER2"). Actin is used as a loading control. Figure 2E) Western blot of HER2 in MCF7, KPL4 and SKBr3 cells stably transfected with gD-RNF43 and gD-ZNRF3 after treatment with the indicated bispecific antibodies. Downstream regulation of the HER2 pathway with p-Erk is shown in SKBr3. Actin is used as a loading control. [Figure 3A-3E]Figures 3A-E show the mechanisms of cell surface clearance and receptor degradation upon RNF43 / ZNRF3 dimerization. Figure 3A) Mean fluorescence intensity (MFI) of cell surface HER2 in HT29 cells transfected with gD-ZNRF3 after treatment with various antibodies against gD / HER2 or CD3 / HER2. Figure 3B) Western blot of HER2 in HEK293T cells transiently transfected with gD-tagged ZNRF3 or transfected with guide RNA driving genetic deletion of HER2 after treatment with the indicated bispecific antibodies against gD / HER2 or CD3 / HER2. Tubulin is used as a loading control. Figure 3C) Immunoblot of ubiquitin in HEK293T transiently transfected with gD-tagged RNF43, ZNRF3 or control ("mock") after immunoprecipitation of HER2 followed by treatment with three different bispecific antibodies against gD and HER2 as indicated: T (Trastuzumab, 4D5), P (Pertuzumab, 2C4) and 7 (7C2). Figure 3D) Western blot of HER2 in HEK293T transiently transfected with gD-tagged RNF43, ZNRF3 or control with good (WT RING) or poor (delta-RING) ligase activity after treatment with bispecific antibodies against gD / HER2 as indicated. Actin is used as a loading control. FIG. 3E) Western blot of HER2 in HEK293T transiently transfected with gD-tagged RNF43, ZNRF3 or control after treatment with the indicated bispecific antibody against gD / HER2 and the indicated inhibitors (Bortezomib=proteasome inhibitor; E1-activating enzyme inhibitor, BafA=lysosomal inhibitor). Actin is used as a loading control. (T=trastuzumab (4D5), P=pertuzumab (2C4), 7=7C2) [Figure 4A-4F]Figures 4A-F show the phenotypic impact of cell surface ligase-mediated receptor degradation. Figure 4A) Immunoblot of tagged IGF1R after immunoprecipitation of tagged RNF43 in transiently transfected HEK293T cells after treatment with AC heterodimerizer. Tubulin is used as a loading control. Figure 4B) Western blot of IGF1R in HT29 cells transfected with dox-inducible gD-tagged ZNRF3 after treatment with doxycycline ("dox") and the indicated bispecific antibodies against gD and various IGF1R epitopes. Tubulin is used as a loading control. Figure 4C) Clonal growth assay of HT55 cells transfected with dox-inducible CRISPR-Cas9 and IGF1R-targeting sgRNA or non-targeting control (NTC) sgRNA. (Figure 4D) In ​​vivo tumor growth of HT55 cells transfected with dox-inducible CRISPR-Cas9 and IGF1R-targeting sgRNA or non-targeting control (NTC) sgRNA. (Figure 4E) Clonal growth assay of HT55 cells transfected with dox-inducible gD-ZNRF3 after treatment with anti-Citxu / gD or anti-Citxu / NIST bispecific antibodies. Licor-based quantification of (Figure 4E) is shown in (Figure 4F). [Figure 5A-5F]Figures 5A-5F provide characterization of RNF43 and ZNRF3 targeting antibodies and related bispecific antibodies. Figure 5A) SPR binding of antibodies discovered from rat or rabbit immunization against RNF43 or ZNRF3. Figures 5B and 5C) Cross-blocking analysis of a subset of ZNRF3 (Figure 5B) and RNF43 (Figure 5C) binding antibodies. Figure 5D) Time course of cizutumumab / hSC 37.39 bispecific antibody driving cell surface clearance of IGF-1R at concentrations between 0 and 10 μg / mL as measured by following IGF1R MFI after 1-24 hours. Figure 5E and 5F) Clearance of IGF1R using a panel of ligase / cizutumumab bispecific antibodies that bind to human ZNRF3 ("hu ZNRF3"; Figure 5E) or human RNF43 ("hu RNF43"; Figure 5F). Percent clearance is plotted against monovalent affinity for each respective ligase. [Figure 6A-6C] Figures 6A-C show characterization of the cleavage capacity of novel cell surface ligase antibodies. Figures 6A and 6B) Mean fluorescence intensity (MFI) of cell surface IGF1R in SW1417 or HT29 cells transfected with gD-ZNRF3 after treatment with various antibodies against IGF1R / ZNRF3 (Figure 6A) or IGF1R / NIST (Figure 6B). Figure 6C) Western blot of IGF1R in parental SW1417 cells after treatment with the indicated bispecific antibodies. Actin is used as a loading control. [Figure 7A-7F]Figures 7A-7F show the characterization of the degradation capacity of a novel format of cell surface ligase antibodies. Figures 7A and 7B show schematics of the antibodies tested. Figures 7C)-7F) show flow cytometry analysis of IGF1R to assess antibody activity in HT29 cells overexpressing the relevant ligase. FIG. 7C shows results for cizutumumab / anti-RNF43 constructs with one or two cizutumumab (anti-IGFR; "cixu") binding regions, FIG. 7D shows results for cizutumumab / anti-RNF43 constructs with one or two RNF43 binding regions, FIG. 7E shows results for istiratuzumab / anti-RNF43 constructs with one or two istiratuzumab (anti-IGFR; "istira") binding regions, and FIG. 7F shows results for istiratuzumab / anti-RNF43 constructs with one or two RNF43 binding regions. [Figure 8A-8D] Figures 8A-D show the characterization of the degradation capacity of additional novel formats of cell surface ligase antibodies. Figure 8A) Schematic diagram of tested FvIgG or FabIbG format antibodies is shown. Figure 8B) Flow cytometric analysis of IGF1R is used to evaluate the activity of FvIgG format in HT29 cells overexpressing the relevant ligase. Figures 8C and 8D) Flow cytometric analysis of IGF1R is used to evaluate the activity of FabIgG format in HT29 cells overexpressing the relevant ligase (Figure 8C = cizutumumab / anti-RNF43; Figure 8D = istiratuzumab / anti-RNF43). [Figure 9] Figure 9 shows characterization of the degradative potential of EGFR-targeting multispecific antibodies. Flow cytometric analysis of EGFR is used to assess the activity of multispecific antibodies in HT29 cells overexpressing the relevant ligase. [Figure 10A-10F]Figures 10A-F show characterization of the cleavage ability of novel cell surface ligase antibodies. Figure 10A) Schematic showing the structural domains of both RNF43 and ZNRF3, including the signal peptide (SP), transmembrane domain (TM) and ligase domain (RING). Figure 10B) Identification and gD tagging of additional E3 ligases with similar structural features to RNF43 and ZNRF3, including SP and TM. Figure 10C) FACS plot showing transfection of gD-ZNRF3 in HEK293T cells (FITC positive) and gD cell surface detection with anti-gD antibody (conjugated to APC). Figure 10D) Cell surface MFI of gD signal from the indicated gD tagged ligases transiently transfected into HEK293T cells. gD is detected using an anti-gD APC conjugated antibody. FIG. 10E and FIG. 10F) Western blots of HER2 in HEK293T cells transiently transfected with the indicated dox-inducible gD-tagged ligases after treatment with anti-HER2 / gD bispecific antibody. Western blots for gD and tubulin are used for transfection assessment and loading control, respectively. FIG. 10E shows data for ligases RNF13, RNF43, RNF128, RNF130, RNF133, RNF148 and RNF149. FIG. 10F shows data for ligases RNF150, RNF167, ZNRF3, LNX1, RSPRY1, SYVN1 and TRIM7. [Figure 11A-11B]Figure 11A and Figure 11B show activation of the Wnt / β-catenin signaling pathway via the TCF reporter by ZNRF3 antibodies. Figure 11A) Rat clone ZNRF3 bivalent antibody induced slight activation of Wnt / β-catenin in the presence of 100ng / mL recombinant Wnt3a in HEK293 cells. Untreated cells, cells treated with DMSO and cells treated with 100ng / mL Wnt3a served as negative controls. Cells treated with 2μM GSK3beta or various concentrations of RSPO3 (500ng / μL, 10ng / μL, 0.2ng / μL) in combination with 100ng / mL Wnt3a served as positive controls. Figure 11B) Rabbit clone ZNRF3 bivalent antibody induced slight activation of Wnt / β-catenin. Positive and negative controls used are described above. Data shown are from two independent experiments (n=2). [Fig. 12A-12F] Figures 12A-12F show the kinetics of bispecific antibody-mediated surface IGF1R-HibiT clearance. Figures 12A-12C) The percentage of cell surface IGF1R-HibiT remaining was assessed after 0, 4, 8 and 24 hours using bispecific antibodies Cixu / ZNRF3-6 (Figure 12A), Cixu / ZNRF3-55 (Figure 12B) and Cixu / RNF43-67 (Figure 12C) at concentrations of 10 μg / mL and 1 μg / mL. A time-dependent clearance was observed. Cixu / RNF43 is a positive control. (Figures 12D-12F) When using the novel formats Cixu-RNF43 Fv-IgG (Figure 12D) and Istira-RNF43 Fv-IgG (Figure 12E), time-dependent clearance was observed, with 10 μg / mL Istira-RNF43 Fv-IgG (Figure 12F) exhibiting a "hook effect." [Figures 13A-13C]Figures 13A-C show cytotoxicity and cell surface clearance assessed by multiplexing the HiBiT extracellular detection assay with LDH or CellTiter-Glo® assays. Figure 13A) IGF1R-HibiT remained on the cell surface after 48 hours of treatment with 1 μg / mL of cixu / ZNRF3 bispecific antibody. Figure 13B) Lactate dehydrogenase released by HT29 cells treated with 1 μg / mL of cixu / ZNRF3 bispecific antibody for 48 hours was measured using the LDH cytotoxicity assay. No toxicity was observed in these treatment conditions. Figure 13C) CellTiter-Glo® assay measured luminescence signal (RLU) after detection of IGF1R-HiBiT on the cell surface using the HibiT extracellular detection system. Similar to the LDH assay, no toxicity was observed in these treatment conditions. [Figure 14A-14B] Figures 14A and 14B show lytic detection of total levels of IGF1R-HibiT. Figure 14A) HiBiT extracellular system is used to detect the remaining amount of IGF1R-HibiT on the surface of HT29 cells. Lytic detection shows the total level of IGF1R-HiBiT (extracellular and intracellular). Both systems measure luminescence signal. Cixu-RNF43 Fv-IgG format left 20% of IGF1R-HibiT on the cell surface, resulting in 80% intact IGF1R-HiBiT. Cixu / RNF43.hSC 37.39 was the positive control. Cixu / NIST, Cixu / Cixu and UT were the negative controls. Figure 14B) Western blot showing the total amount of IGF1R_HibiT or IGF1R in WT HT29 cells upon 24 hours of treatment with the new format and bispecific antibodies. ProIGF1R-HiBiT / IGF1R is the precursor of IGF1R-HibiT / IGF1R. B-actin is an internal control. [Figures 15A-15G]Figures 15A-15G show various exemplary formats for constructing multispecific antibodies compatible with embodiments herein: "2+1 FabIgG" format (Figure 15A), "1-arm FvIgG" or "OA FvIgG" format (Figure 15B) and "1-arm FabIgG" or "OA FabIgG" format (Figure 15C). Figures 15D-15E show two different optional versions of a trispecific anti-EGFR-RNF43-ZNRF3 antibody, and Figures 15F-15G show two different optional versions of a trispecific anti-IGF1R-RNF43-ZNRF3 antibody. [Figure 16] FIG. 16 shows that the combination of bispecific antibodies targeting both RNF43 / IGF1R and ZNRF3 / IGF1R is more effective at removing IGF1R from the cell surface than either bispecific antibody alone. [Figure 17] FIG. 17 shows that RNF43 is uniformly expressed throughout the tumor mass in an implanted mouse APC mutant colorectal cancer model. [Figure 18A-18B] Figures 18A-B show Western blots showing the total amount of IGF1R in WT LS180 cells upon 24 hour treatment with various bispecific antibodies (Figure 18A). Pro-IGF1R is the precursor of IGF1R. Tubulin is used as a loading control. The degradation percentage is summarized across the various cell lines (Figure 18B). [Figure 19A-19B]Figures 19A-B show Western blot analysis of lysates from HT29 cells subjected to the indicated treatments: IGF1 stimulation (+IGF1; 50 ng / ml; 5 min), no treatment (-), bivalent antibody treatment (Cixu; 1 μg / ml; 135 min), control bispecific antibody (NIST; 1 μg / ml; 135 min), RNF43-based bispecific antibody (RNF43-35; 1 μg / ml; 135 min) or ZNRF3-based bispecific antibody (ZNRF3-55; 1 μg / ml; 135 min) after IgG or IGF1Rβ immunoprecipitation (IP). IGF1R ubiquitination was assessed by immunoblotting IP samples using an antibody against ubiquitin. Immunoprecipitation and total IGF1Rβ levels were assessed using an antibody against IGF1Rβ, and α-tubulin was used as a loading control (Figure 19A). Western blot analysis of lysates from parental HEK293T cells or HEK293T cells expressing ZNRF3 wild type (WT) or delta RING mutant (ΔRING) tagged with N-terminus gD and C-terminus FLAG after incubation with HA epitope tag antibody agarose conjugate or agarose-TUBE2. Total and co-precipitated IGF1R levels were assessed by immunoblotting with an antibody against IGF1Rβ. ZNRF3 ubiquitination and expression levels were assessed with an antibody against the C-terminal FLAG tag, and α-tubulin was used as a loading control (Figure 19B). [Figure 20] Figure 20 shows a Western blot of IGF1R in HEK293T transiently transfected with gD-tagged ZNRF3, either with (WT RING) or lacking (delta-RING) ligase activity, or in controls after treatment with the indicated bispecific antibodies against gD / cizutumumab. Tubulin is used as a loading control. [Figures 21A-21C]Figures 21A-C show the distribution of RNF43 expression plotted against the presence of RNF43 mutations. Special emphasis is placed on SW48 CRC lines that show elevated expression of RNF43 and a frameshift variant in the RING domain (Figure 21A). Cell surface expression of RNF43 is shown for the non-expressing line RKO compared to the RING domain mutant SW48 (Figure 21B). Western blot of IGF1R in SW48 after treatment with the indicated bispecific antibodies against RNF43 / IGF1R or ZNRF3 / IGF1R (Figure 21C). [Figures 22A-22C] Figures 22A-C show schematics of indel generation in RNF43 (Figure 22A) and ZNRF3 (Figure 22B) using the CRISPR CAS 9 system. FACS plots show cell surface ligase expression of RNF43 or ZNRF3 in the presence of n-term truncation or RING indels. Western blots of IGF1R in HT29 cells with N-terminus knockout or RING deletion, transiently transfected with gD-tagged ZNRF3, showing good ligase potency, after treatment with the bispecific antibodies shown (Figure 22C). [Figure 23] Figure 23 shows a Western blot of IGF1R in DLD1 after treatment with the indicated bispecific antibody against gD / IGF1R and the indicated inhibitors (Bortezomib = proteasome inhibitor; E1 activating enzyme inhibitor, BafA = lysosomal inhibitor). Tubulin was used as a loading control and ubiquitin is used to verify inhibition of the proteasome and E1 activating enzyme. LC3B is used to confirm inhibition of the lysosomal. [Figure 24] FIG. 24 shows a Western blot of IGF1R in DLD1 upon 24 hour treatment of the new formats: bispecific antibodies showing three different exemplary formats for constructing bispecific antibodies compatible with embodiments herein: "2+1 FabIgG" format (FIG. 15A), "1-arm FvIgG" or "OA FvIgG" format. [Figures 25A-25C] Figures 25A-C show Western blots of IGF1R, pIGF1R, and downstream components of the IGF1R signaling axis (including pAKT and pS6) in SW48 cells treated with various indicated antibodies (Figure 25A). Note the near complete inhibition of pAKT signaling upon ligase-mediated degradation of IGF1R. Clonal expansion of SW48 cells after 14 days of treatment with indicated antibodies (Figure 25B) and licor-based quantification of (Figure 25B) is shown in (Figure 25C). [Figures 26A-26D] Figures 26A-D show clonal proliferation assays of HT29 cells transfected with dox-inducible gD-ZNRF3 after treatment with various anti-Citxu / ZNRF3 or control bispecific antibodies. Licor-based quantification of Figure 26A (results for gD-ZNRF3 WT-FLAG) and Figure 26C (results for gD-ZNRF3 delta-RING-FLAG mutant) is shown in Figure 26B and Figure 26D, respectively. [Figure 27] Figure 27 shows a western blot of PD-L1 in SW48 cells treated with various indicated antibodies. Note the extensive degradation seen when using the ZNRF3 / PD-L1 antibody. [Fig. 28A-28B] Figures 28A-B show characterization of the cleavage capacity of novel cell surface ligase antibodies. FACS plots show cell surface expression of gD-RNF43, RNF13 and RNF128 stable HT29 cells (Figure 28A). gD cell surface detection using anti-gD antibody (conjugated to APC). Figure 28B: Cell surface MFI of gD signal from the indicated gD tagged ligase stably integrated into HT29 cells. gD is detected using anti-gD APC conjugated antibody. [Figure 29] Figure 29 shows a Western blot of IGF1R in HT29 cells with dox-inducible gD tagged ligase as indicated after treatment with anti-IGF1R / gD bispecific antibody. Western blots against gD and tubulin are used for transfection assessment and loading control, respectively. [Diagram 30] FIG. 30 shows that bispecific antibodies designed to tether endogenous RNF43 or ZNRF3 to IGF1R induce IGF1R targeted degradation in the SW48 in vivo xenograft model. [Diagram 31] Figure 31 shows a heat map depicting expression of the indicated cell surface ligases across normal tissues (source GTEX) as described in Example 18. Data are z-score normalized. [Diagram 32] Figure 32 shows Western blot analysis of lysates from doxycycline-treated HT29 cells carrying the indicated doxycycline-inducible gD-ligase-FLAG expression constructs after 24 hours of incubation with gD*IGF1R (Cixu) bispecific PROTAB. Endogenous IGF1Rβ and exogenous gD-ligase-FLAG protein levels were detected. Data are representative of three independent experiments. [Diagram 33] Figure 33 shows further Western blot analysis of lysates from doxycycline-treated HT29 cells carrying the indicated doxycycline-inducible gD-ligase-FLAG expression constructs after 24 hours of incubation with gD*IGF1R (Cixu) bispecific PROTAB. Endogenous IGF1Rβ and exogenous gD-ligase-FLAG protein levels were detected. Data are representative of three independent experiments. [Diagram 34] Figure 34 shows Western blot analysis of lysates from doxycycline-treated HT29 cells carrying the indicated doxycycline-inducible gD-ligase-FLAG expression constructs after 24 hours of incubation with gD*HER2 (4D5) bispecific PROTAB. Endogenous HER2 and exogenous gD-ligase-FLAG protein levels were detected. Data are representative of two independent experiments. [Diagram 35]Figure 35 shows Western blot analysis of lysates from doxycycline-treated HT29 cells harboring the indicated doxycycline-inducible gD-ligase-FLAG expression constructs after 24 hours of incubation with gD*PD-L1 (Atezo) bispecific PROTAB. Endogenous PD-L1 and exogenous gD-ligase-FLAG protein levels were detected. Data are representative of two independent experiments. [Diagram 36] Figure 36 shows further Western blot analysis of lysates from doxycycline-treated HT29 cells carrying the indicated doxycycline-inducible gD-ligase-FLAG expression construct after 24 hours of incubation with gD*HER2 (4D5) bispecific PROTAB. Endogenous HER2 and exogenous gD-ligase-FLAG protein levels were detected. Data are representative of two independent experiments. [Figure 37] Figure 37 shows further Western blot analysis of lysates from doxycycline-treated HT29 cells carrying the indicated doxycycline-inducible gD-ligase-FLAG expression constructs after 24 hours of incubation with gD*PD-L1 (Atezo) bispecific PROTAB. Endogenous PD-L1 and exogenous gD-ligase-FLAG protein levels were detected. Data are representative of two independent experiments. [Fig. 38A-38L]Figures 38A-L show cell surface clearance of EpCAM (Figures 38A-38F) and degradation of EpCAM in doxycycline-treated HT29 cells bearing the indicated doxycycline-inducible gD-ligase expression constructs (Figures 38G-38J) after 24 hours of incubation with 1 μg / ml and 10 μg / ml of gD-EpCAM or control gD-NIST bispecific PROTAB. Figures 38A-F show % EpCAM clearance assessed by FACS and Figures 38G-L show degradation of EpCAM assessed by MFI. Figures 38A and 38G: HT29 cells; Figures 38B and 38H: HT29-gD-RNF43; Figures 38C and 38I: HT29-gD-RNF133; Figures 38D and 38J HT29-gD-RNF149; Figures 38E and 38K: HT29-gD-RNF150; and Figures 38F and 38L: HT29-gD-ZNRF3. Figures 38C and 38D show that HT29-gD-RNF133 and HT29-gD-RNF149 had the highest EpCAM clearance (20-30%) compared to other HT-29-gD-ligases, and Figures 38I and 38J show that HT29-gD-RNF133 and HT29-gD-RNF149 had the highest EpCAM degradation compared to other HT29-gD-ligases. Similar results were observed in HT29-gD-RNF133 and HT29-gD-RNF149 showing the highest clearance and degradation of HER2, EGFR and IGF1R by gD-HER2, gD-EGFR or gD-IGF1R PROTABs, respectively, compared to other HT-29-gD-ligases (data not shown). [Figure 39A-39B]Figure 39A and Figure 39B show HER2 degradation in SW48 after bivalent antibody treatment or PROTAB antibody treatment. Figure 39A shows the level of HER2 degradation by Western blot analysis in SW48 cells left untreated (-) or subjected to HER2 bivalent antibody (7C2), NIST*HER2 (NIST) control bispecific antibody or HER2 bispecific PROTAB (ZNRF3-6 and RNF43-37.39) for 48 hours. Figure 39B shows the level of HER2 degradation by Western blot analysis in SW48 cells left untreated (-) or subjected to HER2 bivalent antibody (4D5), NIST*HER2 bispecific antibody (NIST), ZNRF3*HER2 PROTAB antibody (ZNRF3-6) or ZNRF3*NIST control bispecific antibody for 48 hours. Alpha-tubulin levels were used as a loading control. Data are representative of two independent experiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] I. Definition As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0034] "Transmembrane E3 ubiquitin ligase" refers to a class of proteins that contain at least one cell membrane spanning domain and have E3 ubiquitin ligase activity. A transmembrane E3 ubiquitin ligase has "catalytic activity" herein if it can facilitate the final transfer of ubiquitin from a ubiquitin conjugating enzyme (E2) to a substrate to change the substrate's function. In some embodiments, catalytic activity can be measured in a cell surface assay (see, for example, Example 18 below).

[0035] "Protein of interest" or "protein targeted for degradation" or "protein targeted for degradation" and the like refer to a protein intended to be directed to lysosomes for degradation by the molecules described herein. In some embodiments herein, such proteins intended for degradation are "cell surface proteins". As used herein, "cell surface protein" refers broadly to a protein located on the cell surface, either as a transmembrane protein with an extracellular domain, or as a protein that is otherwise localized to the cell surface (e.g., a protein bound to a transmembrane protein).

[0036] Proteins such as transmembrane E3 ubiquitin ligases and degradation targets herein may be from any vertebrate origin, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats) or domesticated mammals (e.g., dogs, cats, horses, livestock such as cows, pigs, sheep, goats, etc.), birds (e.g., poultry, chickens, turkeys) or fish, unless otherwise indicated.

[0037] As used herein, "multispecific binding protein" refers to a protein molecule that can be used to bind to a transmembrane E3 ubiquitin ligase and a degradation target protein. The protein is "multispecific" because it binds to at least two target proteins (i.e., a transmembrane E3 ubiquitin ligase and a degradation target). In some embodiments, the binding protein is an antibody, such as a bispecific or multispecific antibody, or a bispecific or multispecific protein that includes an antibody or antibody fragment and, optionally, another specific protein binding domain.

[0038] The term "antibody" as used herein refers to a molecule that comprises at least the complementarity determining region (CDR)1, CDR2 and CDR3 of a heavy chain and at least the CDR1, CDR2 and CDR3 of a light chain, which molecule can bind to an antigen. This term is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, diabodies, etc.), full-length antibodies, single-chain antibodies, antibody conjugates and antibody fragments, so long as they exhibit the desired binding activity.

[0039]

[0001] An "isolated" antibody is one that is separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as measured by electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0040] "Antigen" refers to the target of an antibody, i.e., the molecule to which the antibody specifically binds. The term "epitope" refers to the site on an antigen, either proteinaceous or non-proteinaceous, to which an antibody binds. Epitopes on proteins can be both formed from a continuous stretch of amino acids (linear epitopes) or can include non-contiguous amino acids that are in spatial proximity, for example, due to antigen folding (i.e., by tertiary folding of a proteinaceous antigen) (conformational epitopes). Linear epitopes are usually still bound by antibodies after exposure of the proteinaceous antigen to a denaturing agent, whereas conformational epitopes are usually destroyed by treatment with a denaturing agent.

[0041] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). In general, the affinity of a molecule X for its partner Y is determined by the dissociation constant (K D Affinity can be measured by methods known in the art, including those described herein.

[0042] In this disclosure, "binds" or "binding" or "specific binding" and similar terms, for example when referring to a protein and its ligand or an antibody and its antigen target, means that the binding affinity is strong enough that the interaction between the members of the binding pair cannot be due to random molecular association (i.e., "non-specific binding"). Such binding typically has a dissociation constant (K D ) and a K of 100 nM or less D is often necessary.

[0043] "Anti-RNF43 antibody" or "RNF43 antibody" or "antibody that specifically binds to RNF43" or "antibody that binds to RNF43" and similar phrases (e.g., an anti-gD antibody or an anti-ZNRF3 antibody or an anti-HER2 antibody, etc.) refer to an antibody that specifically binds to the described protein.

[0044] The term "heavy chain" refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some embodiments, a heavy chain includes at least a portion of a heavy chain constant region. The term "full-length heavy chain" refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

[0045] The term "light chain" refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, a light chain also comprises at least a portion of a light chain constant region. The term "full-length light chain" refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

[0046] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable region that is hypervariable in sequence and determines antigen binding specificity, e.g., "complementarity determining regions" (CDRs). Generally, antibodies contain six CDRs; three in the VH (CDR-H1 or heavy chain CDR1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include: (a) "Chothia CDRs": hypervariable loops located at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) "Kabat CDRs": CDRs located at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) "McCallum CDR": antigenic contacts present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)).

[0047] Unless otherwise indicated, CDRs are determined according to Kabat et al., supra. One of skill in the art will understand that the designations of CDRs may also be determined according to McCallum or any other scientifically accepted nomenclature system.

[0048] "Framework" or "FR" refers to the residues of variable region residues that are not part of the complementarity determining region (CDR). The FR of a variable region generally consists of four FRs: FR1, FR2, FR3 and FR4. Thus, the CDR and FR sequences generally occur in the VH (or VL) in the following sequence: FR1-CDR-H1 (CDR-L1)-FR2-CDR-H2 (CDR-L2)-FR3-CDR-H3 (CDR-L3)-FR4. An "acceptor human framework" for the purposes herein is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or may comprise changes in the amino acid sequence. In some embodiments, the number of amino acid changes is no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3 or no more than 2. In some embodiments, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

[0049] The terms "variable region" or "variable domain" refer interchangeably to the domains of the heavy or light chain of an antibody involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three complementarity determining regions (CDR). See, for example, Kindt et al. Kuby Immunology, 6 thed., W. H. Freeman and Co., page 91 (2007). The variable domain may comprise heavy chain (HC) CDR1-FR2-CDR2-FR3-CDR3, with or without all or a portion of FR1 and / or FR4; and light chain (LC) CDR1-FR2-CDR2-FR3-CDR3, with or without all or a portion of FR1 and / or FR4. That is, the variable domain may lack a portion of FR1 and / or FR4, so long as it retains antigen-binding activity. A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, a library of complementary VL or VH domains may be screened by isolating antibodies that bind to a particular antigen using the VH or VL domain of the antibody that binds the antigen, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0050] The "constant region" of the light and heavy chains of an antibody refers to the FR and CDR and additional sequence parts outside the variable region. Certain antibody fragments may lack all or part of the constant region. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called the variable heavy domain or heavy chain variable region, followed by three constant heavy chain domains (CH1, CH2 and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called the variable light domain or light chain variable region, followed by a constant light chain (CL) domain.

[0051] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the antibody produced by the host cell may undergo post-translational truncation of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, the antibody produced by the host cell by expression of a particular nucleic acid molecule encoding a full-length heavy chain may comprise a full-length heavy chain or a truncated variant of the full-length heavy chain. This may be the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the EU index). Thus, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. Thus, for example, "full-length IgG1" includes IgG1 with Gly446 and Lys447, or without Lys447, or without both Gly446 and Lys447. The amino acid sequence of the heavy chain comprising the Fc region is shown herein without the C-terminal glycine-lysine dipeptide, unless otherwise indicated. In one embodiment, the heavy chain comprising the Fc region as specified herein, included in the antibody of the present invention, may comprise Gly446 and Lys447 (numbering according to the EU index). In one embodiment, the heavy chain comprising the Fc region as specified herein, included in the antibody of the present invention, may comprise Gly446 (numbering according to the EU index). Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0052] "Effector function" refers to the biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0053] The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, an antibody is of the IgG1, IgG2, IgG3, or IgG4 isotype. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0054] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen (i.e., RNF43, ZNRF3, gD, or degradation target protein) to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0055] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody or, in the case of IgG antibodies, an antibody having a heavy chain including an Fc region as defined herein.

[0056] The term "multispecific" herein refers to a molecule that can bind to more than one different target or antigen, for example, two or more different targets or antigens. The term "bispecific" herein refers to a molecule, such as a binding protein or antibody, that can specifically bind to two different targets or antigens. A "multispecific" or "bispecific" antibody herein may comprise a suitable full-length heavy chain and a suitable full-length light chain for binding to two different antigens, or may comprise a suitable antibody fragment for binding to two different antigens. There are a variety of different platforms for making multispecific binding proteins that are compatible with the present disclosure.

[0057] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies that make up the population are identical and / or bind to the same epitope, except for possible variant antibodies (usually present in small amounts), including, for example, naturally occurring mutations or arising during the generation of the monoclonal antibody preparation. Each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen, in contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes). Thus, the modifier "monoclonal" refers to the character of the antibody obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies of the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin locus, and such methods, as well as other exemplary methods for producing monoclonal antibodies, are described herein.

[0058] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular origin or species, and the remainder of the heavy and / or light chain is derived from a different origin or species.

[0059] A "humanized" antibody refers to a chimeric antibody that contains amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody contains substantially all of at least one, usually two, variable domains, with all or substantially all of the CDRs corresponding to the variable domains of a non-human antibody and all or substantially all of the FRs corresponding to the variable domains of a human antibody. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0060] A "human" antibody is one having an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes sequences encoding human antibodies or other human antibodies. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0061] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including, but not limited to, a cytotoxic agent. A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. The naked antibody may be present in a pharmaceutical composition.

[0062] The term "nucleic acid molecule" or "polynucleotide" includes any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are often described by the sequence of bases, whereby the bases represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is usually represented from 5' to 3'. As used herein, the term nucleic acid molecule encompasses, for example, deoxyribonucleic acid (DNA), including complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms.Furthermore, the nucleic acid molecules described herein may include naturally occurring or non-naturally occurring nucleotides.Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages or chemically modified residues.Nucleic acid molecules also include DNA and RNA molecules suitable as vectors for direct expression of the antibody of the present invention in vitro and / or in vivo, for example in a host or patient.Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be modified or unmodified. For example, the mRNA can be chemically modified to increase the stability of the RNA vector and / or the expression of the encoded molecule, such that the mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler ert al, Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or EP2101823B1).

[0063] An "isolated" nucleic acid refers to a nucleic acid molecule that is separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is contained within a cell that normally contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0064] "Isolated nucleic acid encoding an antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof) herein, including nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present in one or more locations within a host cell.

[0065] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as autonomously replicating nucleic acid structures as well as vectors integrated into the genome of a host cell into which it is introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0066] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably to refer to such cells, including the progeny of cells into which exogenous nucleic acid has been introduced. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and its progeny, regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0067] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity, without considering any conservative substitutions as part of sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways that are within the skill of the art, for example, using publicly available computer software (e.g., BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or FASTA program package). Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared. Alternatively, percent identity values ​​can be generated using sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc. and the source code has been filed in the user documentation of the U.S. Copyright Office, Washington DC, 20559, registered under U.S. Copyright Registration No. TXU510087, and described in WO2001 / 007611.

[0068] Unless otherwise indicated, for purposes herein, percent amino acid sequence identity values ​​are generated using the ggsearch program of the FASTA package version 36.3.8c or subsequent BLOSUM50 comparison matrix. The FASTA program package was developed by WR Pearson and DJ Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448; WR Pearson (1996) "Effective protein sequence comparison" Meth. Enzymol. 266:227-258; and Pearson et.al. (1997) Genomics 46:24-36, and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, global rather than local alignments are performed by comparing sequences using the public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi, using the ggsearch (global protein:protein) program and default options (BLOSUM50; open:-10; extend:-2; Ktup=2). Percent amino acid identity is given in the output alignment header.

[0069] By "reduce" is meant the ability to result in an overall decrease of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more. In some embodiments, reduce or inhibit can refer to a relative decrease compared to a reference (e.g., a reference level of biological activity (e.g., wnt signaling) or binding). In some embodiments, reduce can refer to, for example, a decrease in the "level" (i.e., amount or concentration) of a cell surface protein on a cell. For example, degradation of a protein can result in a decrease in the level of that protein observed in a cell or tissue sample, such as by flow cytometry or qualitative analysis of fluorescent staining.

[0070] A multispecific binding protein that "blocks the binding" of transmembrane E3 ubiquitin ligase to a ligand refers to its ability to inhibit the interaction between the ligase and one of its ligands. For example, ligases such as RNF43 and ZNRF3 bind to natural ligands such as frizzled (FZD) and LRP6. In some embodiments, the multispecific binding protein does not block such binding. Such inhibition can occur by any mechanism, including direct interference with ligand binding, for example, because of overlapping binding sites on the ligase for an antibody and one or more ligands, and / or indirect interference with ligand binding, such as allosteric interference with binding, for example, by causing conformational changes in the ligase that change ligand affinity.

[0071] As used herein, the term "about" refers to numerical values, including, for example, integers, fractions, and percentages, whether or not explicitly stated. The term "about" generally refers to a range of numerical values ​​(e.g., + / - 5 to 10% of the stated range) that one of ordinary skill in the art would consider equivalent to the stated value (e.g., having the same function or result). When terms such as "at least" and "about" precede a list of numerical values ​​or ranges, those terms modify all of the values ​​or ranges provided in that list. In some cases, the term about may include numerical values ​​that are rounded to the nearest significant figure.

[0072] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include plurals and plural terms shall include the singular.

[0073] In this application, the use of "or" means "and / or" unless stated otherwise. In the context of multiple dependent claims, the use of "or" refers only in the alternative to more than one preceding independent or dependent claim. Also, terms such as "element" or "component" encompass both elements and components that contain one unit and element, and components that contain more than one subunit, unless specifically stated otherwise.

[0074] Exemplary techniques used in connection with recombinant DNA, oligonucleotide synthesis, tissue culture and transformation (e.g., electroporation, lipofection), enzymatic reactions and purification methods are described, for example, in Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1989)), among others.

[0075] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in a form such that the biological activity of the active ingredient contained therein is effective, and that does not contain additional components that are unacceptably toxic to a subject to which the pharmaceutical composition may be administered.

[0076] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition or formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0077] An "individual" or "subject" is a human unless otherwise specified. In some cases, when specified, an "individual" or "subject" is or includes a non-human mammal (e.g., a "mammalian subject" or a "non-human mammalian subject"). Mammals include, but are not limited to, livestock animals (e.g., cows, sheep, cats, dogs and horses), primates (e.g., humans and non-human primates, e.g., monkeys), rabbits and rodents (e.g., mice and rats). In some cases, non-mammals whose cells express transmembrane E3 ubiquitin ligase can also be specified (e.g., birds or fish).

[0078] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and may be performed for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, diminishing direct or indirect pathological consequences of a disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating the disease state, and remission or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or to slow the progression of a disease.

[0079] An "effective amount" of an agent, eg, a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0080] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is usually characterized by uncontrolled cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of such cancer include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

[0081] II. Multispecific Binding Proteins In one aspect, the disclosure herein relates to a multispecific binding protein that can specifically bind to both at least one first cell surface protein, including a transmembrane E3 ubiquitin ligase, and at least one second cell surface protein. In some embodiments, the second cell surface protein can be intended for degradation (i.e., a degradation target protein). The multispecific binding protein molecule can have a variety of general formats. For example, the multispecific binding protein can be bispecific (i.e., binds to two targets) in some embodiments, or can bind to more than two targets or more than one site on one target in some embodiments. In some embodiments, the multispecific antibodies can be trispecific, i.e., bind to three targets or sites. In other embodiments, they can bind to more than three targets or sites. In some embodiments, the multispecific binding protein is a multispecific antibody, including multispecific antibody fragments as well as multispecific full-length antibodies, such as full-length IgG antibodies. As described below, there are a variety of bispecific and trispecific and other multispecific antibody formats that are compatible with the molecules herein. In other embodiments, the multispecific binding proteins herein may include other types of specific ligands for the target molecule, or a combination of an antibody variable region specific for one target with a different type of protein ligand specific for another target. In some embodiments, the multispecific binding proteins herein are known by the acronym PROTAB.

[0082] In some embodiments, the transmembrane E3 ubiquitin ligase is any one of RNF43, ZNRF3, RNF13, RNF128, RNF130, RNF133, RNF148, RNF149, RNF150, RNF167, ZNRF4, RSPRY1, SYVN1, LNX1 isoform 2, or TRIM7 isoform 3. In some embodiments, the transmembrane E3 ubiquitin ligase is RNF13, RNF43, SYVN1, RNF130, RNF148, RNF149, LNX1_isoform 2, RNF128, RNF133, ZNRF4, RSPRY1, TRIM7_isoform 3, RNF167, RNF150, or ZNRF3, each of which comprises an amino acid sequence selected from any one of SEQ ID NOs: 445-459. (See sequence listing below.) In some embodiments, the transmembrane E3 ubiquitin ligase is RNF43, RNF128, RNF130, RNF133, RNF149, RNF150, or ZNRF3. In some embodiments, the transmembrane E3 ubiquitin ligase is RNF130, RNF133, RNF149, or RNF150. In some embodiments, the transmembrane E3 ubiquitin ligase is RNF130, RNF149, or RNF167. In some embodiments, the transmembrane E3 ubiquitin ligase is RNF133 or RNF148. In some embodiments, the transmembrane E3 ubiquitin ligase is RNF43 or ZNRF3. In some embodiments, the multispecific binding protein binds to one type of transmembrane E3 ubiquitin ligase, e.g., ZNRF3 or RNF43. In other embodiments, the multispecific binding protein binds two transmembrane E3 ubiquitin ligases, such as both RNF43 and ZNRF3. For example, the multispecific binding protein can comprise two different antibody fragments, one that recognizes RNF43 and the other that recognizes ZNRF3, thereby allowing binding to both ligases.

[0083] As shown in FIG. 1C, for example, RNF43 is expressed in some cancer cell lines. Also, both RNF43 and ZNRF3 are members of the Wnt signaling pathway that are activated in some tumor cell lines. Since these proteins are expected to be expressed on numerous types of tumors, the constructs herein may be particularly useful in reducing the levels of one or more target cell surface proteins in a subject's tumor. For example, the constructs herein may be useful in reducing the levels of target cell surface proteins, where high levels of such proteins are known to promote cancer growth. Additionally, certain target cells, such as cancer cells, may express high levels of a transmembrane E3 ubiquitin ligase, such as RNF43 or ZNRF3, which may allow the multispecific binding protein to preferentially bind to those target cells compared to other normal or non-diseased cells that express lower levels of that ligase. Thus, the constructs herein may be useful in the treatment of cancer. Also, depending on the cell surface protein selected for degradation, the constructs herein may be useful in increasing the immune response in a subject, such as a cancer subject.

[0084] The disclosure herein also relates to exemplary anti-RNF43 and anti-ZNRF3 antibodies that may be useful in constructing the multispecific binding proteins (e.g., multispecific or bispecific antibodies) herein, which are described in detail below.

[0085] In some embodiments, the multispecific binding protein can also block the ability of the normal ligand that the transmembrane E3 ubiquitin ligase binds to. Thus, for example, in some embodiments, the proteins can block the ability of FZD or LRP6 to bind to RNF43 or ZNRF3. In other embodiments, the multispecific binding protein does not block the binding of the transmembrane E3 ubiquitin ligase to its endogenous ligand.

[0086] In some embodiments, the multispecific binding protein reduces the level of a cell surface protein on the surface of a cell compared to the level observed in the absence of the multispecific binding protein. In some cases, the multispecific binding protein reduces the level of a cell surface protein on the surface of a cell in vitro, such as observed by flow cytometry, compared to the level observed in the absence of the multispecific binding protein. In some cases, the multispecific binding protein reduces the level of a cell surface protein on the surface of a cell in vivo, compared to the level observed in the absence of the multispecific binding protein.

[0087] The multispecific binding protein of the present disclosure can recognize a second cell surface protein, such as a protein that can be targeted for degradation by being in close proximity to a transmembrane E3 ubiquitin ligase, in addition to a transmembrane E3 ubiquitin ligase. There are a wide variety of cell surface proteins that can be targeted for degradation by the multispecific binding protein of the present invention. Examples include, for example, receptor tyrosine kinases, growth factor receptors, cytokines, including cytokine receptors, mucins, Siglec receptors, and immune checkpoint regulators. Examples of growth factor receptors include, for example, fibroblast growth factor receptor FGFR, vascular endothelial growth factor receptor VEGFR, epidermal growth factor receptor EGFR, and platelet-derived growth factor receptor PDGFR. Various growth factor receptors can be overexpressed, for example, in certain cancers. Examples of mucins include, for example, MUC1, MUC2, MUC3A, MUC3B, MUC4, MUC5B, MUC6, MUC7, MUC8, MUC12, MUC13, MUC15, MUC16, MUC17, MUC19 and MUC20, and MUC21 and MUC22. Mucins such as MUC1 can be overexpressed in certain cancers. Examples of Siglec (sialic acid-binding immunoglobulin-type lectin) receptors include, for example, CD22, CD33, MAG / Siglec-4, Siglec-5, Siglec-7 and Siglec-9. Exemplary cytokine receptors include, for example, members of the TNFR superfamily, such as CD40, CD27, OX40, 4-1BB. Examples of particular cell surface proteins that can be targeted by the multispecific binding proteins herein include, for example, HER2, HER3, IGF1R, EGFR, FGFR, VEGFR, PDGFR, EpCAM, FZD, PD-L1, CTLA4, PD-1, TIM3, LAG3, TIGIT, CEACAM1, CD25, ILT-2, ILT-3, ILT-4, ILT-5, LAIR-1, PECAM-1 (CD31), PILR-alpha, SIRL-1, or SIRP-alpha.Specific examples of cell surface proteins that can be targeted by the multispecific binding proteins herein include, for example, HER2, IGF1R, EGFR, FZLD5, EpCAM, and PD-L1. Depending on the particular target cell surface protein selected, the multispecific binding proteins herein include, for example, antibodies or antibody fragments derived from anti-HER2 antibodies, such as 4D5, 7C2, or 2C4, or anti-IGF1R antibodies, such as cixutumumab, ganitumab, dalotuzumab, figitumumab, robatumumab, teprotumumab, or istiratumab.

[0088] Reducing the cell surface levels of these or other proteins can be useful, for example, in treatments such as cancer, but the present multispecific binding proteins are useful in a wide variety of situations, indeed whenever it is necessary to reduce the levels of a particular protein at the cell surface by inducing its degradation. For example, selectively reducing the levels of a particular cell surface protein in an in vitro cell culture or tissue sample can be useful, for example, in a variety of experimental settings to study the effect of that protein on a particular mechanism. Thus, the cell surface proteins targeted for destruction can include a wide variety of cell surface proteins.

[0089] A. Exemplary Multispecific Binding Protein Formats In certain embodiments, the multispecific binding proteins provided herein are multispecific antibodies, such as bispecific or trispecific antibodies. In other embodiments, the multispecific binding proteins may contain one or more antigen-binding fragments from an antibody that bind to other protein domains, such as ligand-binding domains from non-antibody proteins. A "multispecific antibody" is generally a monoclonal antibody that has binding specificities for at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain embodiments, a multispecific antibody has three or more binding specificities. A multispecific antibody may be prepared as a full-length antibody or antibody fragment. A multispecific molecule that binds to two sites or targets is "bispecific" and a multispecific molecule that binds to three sites or targets is "trispecific". Thus, a bispecific binding protein herein, such as a bispecific antibody, binds to both a transmembrane E3 ubiquitin ligase and a cell surface protein. Trispecific binding proteins, such as trispecific antibodies, can be constructed to bind, for example, a cell surface protein and more than one transmembrane E3 ubiquitin ligase, more than one cell surface protein and one transmembrane E3 ubiquitin ligase, or one cell surface protein and one transmembrane E3 ubiquitin ligase, but to two sites on either the cell surface protein or the ligase.

[0090] For example, in some embodiments, the multispecific binding protein herein can be engineered to bind to RNF43 and one or more second cell surface proteins. In other embodiments, the protein can be engineered to bind to ZNRF3 and one or more second cell surface proteins. Or, the protein can be engineered to bind to both RNF43 and ZNRF3 and one or more second cell surface proteins. In yet other embodiments, the multispecific binding protein herein can be engineered to bind to RNF13, RNF128, RNF130, RNF133, RNF148, RNF149, RNF150, RNF167, ZNRF4, RSPRY1, SYVN1, LNX1 isoform 2 or TRIM7 isoform 3 and one or more second cell surface proteins.

[0091] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)) and "knobs-in-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168, and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be produced by manipulating electrostatic steering effects to create antibody Fc-heterodimeric molecules (see, e.g., WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); producing bispecific antibodies using leucine zippers (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605); using general light chain technology to avoid light chain mispairing problems (see, e.g., WO 98 / 50431); using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605). al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), and the use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and by preparation of trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991).

[0092] Engineered antibodies with three or more antigen binding sites, including, for example, "Octopus antibodies," or DVD-Igs, are also included herein (see, for example, WO2001 / 77342 and WO2008 / 024715). Other examples of multispecific antibodies with three or more antigen binding sites can be found in WO2010 / 115589, WO2010 / 112193, WO2010 / 136172, WO2010 / 145792, and WO2013 / 026831. Bispecific antibodies or antigen-binding fragments thereof also include "dual acting FAbs" or "DAFs" that contain an antigen binding site that binds to a cell surface protein and another antigen binding site that binds to a transmembrane E3 ubiquitin ligase (see, for example, US2008 / 0069820 and WO2015 / 095539).

[0093] Multispecific antibodies can also be provided in an asymmetric form with domain crossovers in one or more binding arms of the same antigen specificity, i.e., by exchanging VH / VL domains (see, e.g., WO2009 / 080252 and WO2015 / 150447), CH1 / CL domains (see WO2009 / 080253) or complete Fab arms (see WO2009 / 080251, WO2016 / 016299; see also Schaefer et al, PNAS, 108 (2011) 1187-1191 and Klein at al., MAbs 8 (2016) 1010-20). In one embodiment, the multispecific antibody comprises a cross-Fab fragment. The term "cross-Fab fragment" or "xFab fragment" or "crossover Fab fragment" refers to a Fab fragment in which either the variable or constant regions of the heavy and light chains are exchanged. A cross-Fab fragment contains a polypeptide chain composed of a light chain variable region (VL) and a heavy chain constant region 1 (CH1) and a polypeptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL). Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations at the domain interface to direct correct Fab pairing. See, for example, WO2016 / 172485.

[0094] A variety of additional molecular formats for multispecific antibodies are known in the art and are included herein (see, e.g., Spiess et al., Mol Immunol 67 (2015) 95-106).

[0095] Also included herein is a specific type of multispecific antibody that is designed to simultaneously bind to a surface antigen on a target cell, e.g., a tumor cell, and to another cell surface protein that can target the surface antigen for lysosomal degradation. Thus, in certain embodiments, the antibodies provided herein are multispecific antibodies, in particular bispecific antibodies, one of whose binding specificities is for a cell surface protein to be degraded and the other binding specificity is for a transmembrane E3 ubiquitin ligase.

[0096] Examples of bispecific antibody formats that may be useful for this purpose include molecules in which two scFv molecules are fused by a flexible linker (see, e.g., WO2004 / 106381, WO2005 / 061547, WO2007 / 042261 and WO2008 / 119567; Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011)); diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and their derivatives, such as tandem diabodies ("TandAbs"; Kipriyanov et al., J Mol Biol 293, 41-56 (1999)); "DART" (dual affinity retargeting) molecules, which are based on the diabody format but feature a C-terminal disulfide bridge for additional stabilization (Johnson et al., J Mol Biol 399,436-449(2010)) and the so-called triomabs, which are whole molecules of hybrid mouse / rat IgG molecules (reviewed in Seimetz et al., Cancer Treat Rev 36,458-467(2010)). Certain bispecific antibody formats included herein are described in WO2013 / 026833, WO2013 / 026839, WO2016 / 020309; Bacac et al., Oncoimmunology 5(8)(2016)e1203498. The bispecific antibody formats described in the above documents that bind to T cells (e.g., by binding to CD3, an invariant component of the T cell receptor complex) can be adapted to bind to targets as described herein instead of T cells, for example, by replacing the antibody fragment that binds to CD3 with an antibody fragment that binds to a cell surface ligase.

[0097] In some embodiments, the multispecific antibodies herein may have a symmetrical "1+1 FabIgG" or "1+1 FvIgG" bispecific format, comprising two Fv domains that recognize different targets linked to an interactive Fc region, or two Fab regions that recognize different targets linked to an interactive Fc region. Some further examples of bispecific antibody formats herein include those provided in Figures 15A-15C: 2+1 FabIgG, 1-arm FvIgG, and 1-arm FabIgG, respectively. These formats are, for example, asymmetric. Illustrative examples of trispecific antibodies that bind cell surface protein targets as well as both RNF43 and ZNFR3 are shown in Figures 15D-15G. The 2+1 FabIgG format shown in Figure 15A is described, for example, in WO2015 / 095539. In some cases, antibodies such as those described above comprise an interactive Fc region that includes at least one "knob-into-hole" modification. For example, knob-into-hole modifications can help prevent mispairing of Fc regions. Exemplary knob-into-hole modifications include, for example, the substitution of one or more amino acids on the CH3 or CH2 regions of one Fc domain with an amino acid larger than that naturally found at that position, and the substitution of one or more adjacent amino acids on the partner Fc domain with an amino acid smaller than that normally found. (See, e.g., U.S. Pat. No. 5,731,168 and Atwell et al., J. Mol. Biol. 270:26 (1997), and, e.g., WO2009 / 089004, WO2012 / 106587, and U.S. Patent Publication No. 2009 / 0182127). In some embodiments, knob-into-hole modifications can be, for example, substitutions at amino acid positions 366, 368, and 407 of human IgG1 Fc.For example, in some embodiments, a "knob mutation" may involve the substitution of T366 in human IgG1 Fc with a larger amino acid such as W (and optionally one of S354C or Y349C), and a concomitant "hole mutation" in the partner Fc may involve the substitution of T366, L368, and Y407 with smaller amino acids (e.g., T366S, L368A, and Y407V) (and optionally Y349C or S354C). (See, e.g., Carter, P. et al., Immunotechnol. 2 (1996) 73). Numbering is according to EU index numbering.

[0098] In some embodiments, the multispecific binding proteins herein can have one of the following formats or another format tested in the Examples section or shown in the figures herein. a) 2+1 FabIgG, which contains an arm with two Fab regions that bind to a transmembrane E3 ubiquitin ligase and an arm with one Fab region that binds to a cell surface protein; (see, e.g., Figures 7B and 15A) b) 2+1 FabIgG, which comprises an arm with two Fab regions that bind to a cell surface protein and an arm with one Fab region that binds to a transmembrane E3 ubiquitin ligase; (see, e.g., Figures 7A and 15A); c) 1-arm FvIgG, which comprises an Fv region that binds to a cell surface protein and a Fab region that binds to a transmembrane E3 ubiquitin ligase; (see, e.g., Figure 8A, far left, Figure 15B); d) one-arm FvIgG comprising an Fv that binds to a transmembrane E3 ubiquitin ligase and a Fab that binds to a cell surface protein (see, for example, FIG. 8A, second from the left and FIG. 15B); e) one-arm FabIgG comprising a first Fab region that binds to a cell surface protein and a second Fab region that binds to a transmembrane E3 ubiquitin ligase; (see, for example, FIG. 8A, third from the left and FIG. 15C); f) one-arm FabIgG comprising a first Fab region that binds a transmembrane E3 ubiquitin ligase and a second Fab region that binds a cell surface protein (see, for example, FIG. 8A, far right and FIG. 15C); g) a 1+1 FabIgG comprising a first Fab region that binds to a transmembrane E3 ubiquitin ligase and a second Fab region that binds to a cell surface protein; and

[0099] h) a 1+1 FvIgG comprising a first Fv region that binds a transmembrane E3 ubiquitin ligase and a second Fv region that binds a cell surface protein. In any of the above, said protein may, for example, comprise at least one knobs-into-hole modification in the Fc region which in some embodiments may reduce mispairing of the two halves of the IgG molecule.

[0100] In certain embodiments, the multispecific binding proteins, such as the multispecific antibodies provided herein, may be further modified to include additional non-proteinaceous moieties that are known in the art and readily available. Suitable sites for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and when more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in a process, under defined conditions, etc.

[0101] B. Exemplary Anti-RNF43 and Anti-ZNRF3 Antibodies Useful in Constructing Multispecific Binding Molecules In one embodiment, the present disclosure also provides antibodies that specifically bind to RNF43 and / or ZNRF3 that may be useful in constructing multispecific binding proteins according to the present disclosure. These include the mouse, rat or rabbit antibodies RNF43-104, RNF43-106, RNF43-107, RNF43-108, RNF43-116, RNF43-117, RNF43-123, RNF43-126, RNF43-128, RNF43-129, RNF43-130, RNF43-136, RNF43-145, RNF43-152, RNF43-156, RNF43-168, RNF43-170, RNF43-176, RNF43-17 7, RNF43-179, RNF43-180, RNF43-181, RNF43-186, RNF43-187, RNF43-196, RNF43-200, RNF43-201, RNF43-206, RNF43-210, RNF43-213, RNF 43-217, RNF43-221, RNF43-224, RNF43-25, RNF43-31, RNF43-33, RNF43-35, RNF43-38, RNF43-41, RNF43-53, RNF43-56, RNF43-61, RNF43-6 7, RNF43-69, RNF43-71, RNF43-74, RNF43-75, RNF43-76, RNF43-80, RNF43-86, RNF43-90, ZNRF3-101, ZNRF3-117, ZNRF3-128, ZNRF3-131, Z NRF3-163, ZNRF3-17, ZNRF3-170, ZNRF3-171, ZNRF3-172, ZNRF3-179, ZNRF3-182, ZNRF3-195, ZNRF3-219, ZNRF3-222, ZNRF3-223, ZNRF3-2 31, ZNRF3-237, ZNRF3-244, ZNRF3-247, ZNRF3-253, ZNRF3-254, ZNRF3-255, ZNRF3-265, ZNRF3-269, ZNRF3-270, ZNRF3-275, ZNRF3-279, ZN RF3-287, ZNRF3-296, ZNRF3-30, ZNRF3-300, ZNRF3-301, ZNRF3-305, ZNRF3-312, ZNRF3-314, ZNRF3-322, ZNRF3-329, ZNRF3-35, ZNRF3-55,ZNRF3-6, ZNRF3-90, RNF43-1, RNF43-24, RNF43-8, RNF43-12, RNF43-20, RNF43-11, RNF43-23, RNF43-6, RNF43-5, RNF43-15, RNF43-2, RNF43-16, RNF43-14, RNF43-17, RNF43-22, RNF43-9, RNF43-21, RNF43-13, RNF43-19, ZNRF3-331, ZNRF3-333 and ZNRF3-332, or humanized versions of those antibodies.

[0102] In some embodiments, the multispecific binding proteins herein comprise: (a) the antibodies RNF43-104, RNF43-106, RNF43-107, RNF43-108, RNF43-116, RNF43-117, RNF43-123, RNF43-126, RNF43-128, RNF43-129, RNF43-130, RNF43-136, RNF43-145, RNF43-152, RNF43-156, RNF43-168, RNF43-170, RNF43-176, RNF43-177, RNF43-179, RNF43-180 , RNF43-181, RNF43-186, RNF43-187, RNF43-196, RNF43-200, RNF43-201, RNF43-206, RNF43-210, RNF43-213, RNF43-217, RNF43-221, RNF43-224, RNF 43-25, RNF43-31, RNF43-33, RNF43-35, RNF43-38, RNF43-41, RNF43-53, RNF43-56, RNF43-61, RNF43-67, RNF43-69, RNF43-71, RNF43-74, RNF43-75, RN F43-76, RNF43-80, RNF43-86, RNF43-90, ZNRF3-101, ZNRF3-117, ZNRF3-128, ZNRF3-131, ZNRF3-163, ZNRF3-17, ZNRF3-170, ZNRF3-171, ZNRF3-172, Z NRF3-179, ZNRF3-182, ZNRF3-195, ZNRF3-219, ZNRF3-222, ZNRF3-223, ZNRF3-231, ZNRF3-237, ZNRF3-244, ZNRF3-247, ZNRF3-253, ZNRF3-254, ZNRF3- 255, ZNRF3-265, ZNRF3-269, ZNRF3-270, ZNRF3-275, ZNRF3-279, ZNRF3-287, ZNRF3-296, ZNRF3-30, ZNRF3-300, ZNRF3-301, ZNRF3-305, ZNRF3-312, Z NRF3-314, ZNRF3-322, ZNRF3-329, ZNRF3-35, ZNRF3-55, ZNRF3-6, ZNRF3-90, RNF43-1, RNF43-24, RNF43-8, RNF43-12, RNF43-20, RNF43-11, RNF43-23,RNF43-6, RNF43-5, RNF43-15, RNF43-2, RNF43-16, RNF43-14, RNF43-17, RNF43-22, RNF43-9, RNF43-21, RNF43-13, RNF43-19, ZNRF3-331, ZNRF3-333 or ZNRF3-332. In some embodiments, the multispecific binding protein comprises (a) a heavy chain variable domain (VH) comprising the heavy chain complementarity determining region 1 (CDR-H1), CDR-H2 and CDR-H3 of any one of the above antibodies.

[0103] In some embodiments, the multispecific binding protein is (a) antibody RNF43-104, RNF43-106, RNF43-107, RNF43-108, RNF43-116, RNF43-117, RNF43-123, RNF43-126, RNF43-128, RNF43-129, RNF43-130, RNF43-136, RNF43-145, RNF43-152, RNF43-156, RNF43-168, RNF43-170, RNF43-176, RNF43-177, RNF43-179, RNF43-180, RNF43-181, RNF43-186, RNF43-187, RNF43-196, RNF43-200, RNF43-201, RNF43-206, RNF43-210, RNF43-213, RNF43-217, RNF43-221, RNF43-224, RNF43-25, RNF43-31, RNF43-33, RNF43-35, RNF43-38, RNF43-41, RNF43-53, RNF43-56, RNF43-61, RNF43-67, RNF43-69, RNF43-71, RNF43-74, RNF43-75, RNF43-76, RNF43-80, RNF43-86, RNF43-90, ZNRF3-101, ZNRF3-117, ZNRF3-128, ZNRF3-131, ZNRF3-163, ZNRF3-17, ZNRF3-170, ZNRF3-171, ZNRF3-172, ZNRF3-179, ZNRF3-182, ZNRF3-195, ZNRF3-219, ZNRF3-222, ZNRF3-223, ZNRF3-231, ZNRF3-237, ZNRF3-244, ZNRF3-247, ZNRF3-253, ZNRF3-254, ZNRF3-255, ZNRF3-265, ZNRF3-269, ZNRF3-270, ZNRF3-275, ZNRF3-279, ZNRF3-287, ZNRF3-296, ZNRF3-30, ZNRF3-300, ZNRF3-301, ZNRF3-305, ZNRF3-312, ZNRF3-314, ZNRF3-322, ZNRF3-329, ZNRF3-35, ZNRF3-55, ZNRF3-6, ZNRF3-90, RNF43-1, RNF43-24, RNF43-8, RNF43-12, RNF43-20, RNF43-11, RNF43-23, RNF43-6,In some embodiments, it comprises a light chain variable domain (VL) comprising the light chain complementarity determining region 1 (CDR-L1), CDR-L2 and / or CDR-L3 of any one of RNF43-5, RNF43-15, RNF43-2, RNF43-16, RNF43-14, RNF43-17, RNF43-22, RNF43-9, RNF43-21, RNF43-13, RNF43-19, ZNRF3-331, ZNRF3-333 or ZNRF3-332. In some embodiments, it comprises a light chain variable domain (VL) comprising the light chain complementarity determining region 1 (CDR-L1), CDR-L2 and CDR-L3 of any one of (a) those antibodies. In some embodiments, the multispecific binding protein comprises all of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 of one of the above anti-RNF43 or anti-ZNRF3 antibodies.

[0104] <h2 style=";text-align:left;direction:ltr">RNF 43-104、RNF43-106、RNF43-107、RNF4 3-108、RNF43-116、RNF43-117、RNF43-123、RNF43-126、RNF43-128、RNF43-1 29、RNF43-130、RNF43-136、RNF43-145、RNF43-152、RNF43-156、RNF43-168 、RNF43-170、RNF43-176、RNF43-177、RNF43-179、RNF43-180、RNF43-181、RN F43-186, RNF43-187, RNF43-196, RNF43-200, RNF43-201, RNF43-206, RNF43-210, RNF43-213, RNF43-217, RNF43-221, RNF43-224, RNF43-25, RNF43-31 RNF43-33, RNF43-35, RNF43-38, RNF43-41, RNF43-53, RNF43-56, RNF43-61, RNF43-67, RNF43-69, RNF43-71, RNF43-74, RNF43-75, RNF43-76, RNF43-8 0、RNF43-86、RNF43-90、ZNRF3-101、ZNRF3-117、ZNRF3-128、ZNRF3-131、ZN RF3-163、ZNRF3-17、ZNRF3-170、ZNRF3-171、ZNRF3-172、ZNRF3-179、ZNRF3- 182、ZNRF3-195、ZNRF3-219、ZNRF3-222、ZNRF3-223、ZNRF3-231、ZNRF3-23 7、ZNRF3-244、ZNRF3-247、ZNRF3-253、ZNRF3-254、ZNRF3-255、ZNRF3-265、Z NRF3-269、ZNRF3-270、ZNRF3-275、ZNRF3-279、ZNRF3-287、ZNRF3-296、ZNR F3-30、ZNRF3-300、ZNRF3-301、ZNRF3-305、ZNRF3-312、ZNRF3-314、ZNRF3-3 22, ZNRF3-329, ZNRF3-35, ZNRF3-55, ZNRF3-6, ZNRF3-90, RNF43-1, RNF43-24, RNF43-8, RNF43-12, RNF43-20, RNF43-11, RNF43-23, RNF43-6, RNF43-5,The heavy chain variable region (VH) of any one of RNF43-15, RNF43-2, RNF43-16, RNF43-14, RNF43-17, RNF43-22, RNF43-9, RNF43-21, RNF43-13, RNF43-19, ZNRF3-331, ZNRF3-333, or ZNRF3-332 includes a VH having an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the VH of any one of RNF43-15, RNF43-2, RNF43-16, RNF43-14, RNF43-17, RNF43-22, RNF43-9, RNF43-21, RNF43-13, RNF43-19, ZNRF3-331, ZNRF3-333, or ZNRF3-332.

[0105] <h2 style=";text-align:left;direction:ltr">RNF 43-104、RNF43-106、RNF43-107、RNF4 3-108、RNF43-116、RNF43-117、RNF43-123、RNF43-126、RNF43-128、RNF43-1 29、RNF43-130、RNF43-136、RNF43-145、RNF43-152、RNF43-156、RNF43-168 、RNF43-170、RNF43-176、RNF43-177、RNF43-179、RNF43-180、RNF43-181、RN F43-186, RNF43-187, RNF43-196, RNF43-200, RNF43-201, RNF43-206, RNF43-210, RNF43-213, RNF43-217, RNF43-221, RNF43-224, RNF43-25, RNF43-31 RNF43-33, RNF43-35, RNF43-38, RNF43-41, RNF43-53, RNF43-56, RNF43-61, RNF43-67, RNF43-69, RNF43-71, RNF43-74, RNF43-75, RNF43-76, RNF43-8 0、RNF43-86、RNF43-90、ZNRF3-101、ZNRF3-117、ZNRF3-128、ZNRF3-131、ZN RF3-163、ZNRF3-17、ZNRF3-170、ZNRF3-171、ZNRF3-172、ZNRF3-179、ZNRF3- 182、ZNRF3-195、ZNRF3-219、ZNRF3-222、ZNRF3-223、ZNRF3-231、ZNRF3-23 7、ZNRF3-244、ZNRF3-247、ZNRF3-253、ZNRF3-254、ZNRF3-255、ZNRF3-265、Z NRF3-269、ZNRF3-270、ZNRF3-275、ZNRF3-279、ZNRF3-287、ZNRF3-296、ZNR F3-30、ZNRF3-300、ZNRF3-301、ZNRF3-305、ZNRF3-312、ZNRF3-314、ZNRF3-3 22, ZNRF3-329, ZNRF3-35, ZNRF3-55, ZNRF3-6, ZNRF3-90, RNF43-1, RNF43-24, RNF43-8, RNF43-12, RNF43-20, RNF43-11, RNF43-23, RNF43-6, RNF43-5,and a light chain variable region (VL) comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the VH of any one of RNF43-15, RNF43-2, RNF43-16, RNF43-14, RNF43-17, RNF43-22, RNF43-9, RNF43-21, RNF43-13, RNF43-19, ZNRF3-331, ZNRF3-333 or ZNRF3-332.

[0106] In some cases, the multispecific binding protein comprises a VH that comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the VH of any one of the above antibodies, and a VL that comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the VL of any one of the above antibodies.

[0107] <h2 style=";text-align:left;direction:ltr">RNF 43-104、RNF43-106、RNF43-107、RNF4 3-108、RNF43-116、RNF43-117、RNF43-123、RNF43-126、RNF43-128、RNF43-1 29、RNF43-130、RNF43-136、RNF43-145、RNF43-152、RNF43-156、RNF43-168 、RNF43-170、RNF43-176、RNF43-177、RNF43-179、RNF43-180、RNF43-181、RN F43-186, RNF43-187, RNF43-196, RNF43-200, RNF43-201, RNF43-206, RNF43-210, RNF43-213, RNF43-217, RNF43-221, RNF43-224, RNF43-25, RNF43-31 RNF43-33, RNF43-35, RNF43-38, RNF43-41, RNF43-53, RNF43-56, RNF43-61, RNF43-67, RNF43-69, RNF43-71, RNF43-74, RNF43-75, RNF43-76, RNF43-8 0、RNF43-86、RNF43-90、ZNRF3-101、ZNRF3-117、ZNRF3-128、ZNRF3-131、ZN RF3-163、ZNRF3-17、ZNRF3-170、ZNRF3-171、ZNRF3-172、ZNRF3-179、ZNRF3- 182、ZNRF3-195、ZNRF3-219、ZNRF3-222、ZNRF3-223、ZNRF3-231、ZNRF3-23 7、ZNRF3-244、ZNRF3-247、ZNRF3-253、ZNRF3-254、ZNRF3-255、ZNRF3-265、Z NRF3-269、ZNRF3-270、ZNRF3-275、ZNRF3-279、ZNRF3-287、ZNRF3-296、ZNR F3-30、ZNRF3-300、ZNRF3-301、ZNRF3-305、ZNRF3-312、ZNRF3-314、ZNRF3-3 22, ZNRF3-329, ZNRF3-35, ZNRF3-55, ZNRF3-6, ZNRF3-90, RNF43-1, RNF43-24, RNF43-8, RNF43-12, RNF43-20, RNF43-11, RNF43-23, RNF43-6, RNF43-5,The heavy chain variable region (VH) comprises the amino acid sequence of any one of RNF43-15, RNF43-2, RNF43-16, RNF43-14, RNF43-17, RNF43-22, RNF43-9, RNF43-21, RNF43-13, RNF43-19, ZNRF3-331, ZNRF3-333 or ZNRF3-332. In some cases, the multispecific binding protein is selected from the group consisting of antibodies RNF43-104, RNF43-106, RNF43-107, RNF43-108, RNF43-116, RNF43-117, RNF43-123, RNF43-126, RNF43-128, RNF43-129, RNF43-130, RNF43-136, RNF43-145, RNF43-152, RNF43-156, RNF43-168, RNF43-170, RNF43-176, RNF43-180, RNF43-182, RNF43-184, RNF43-186, RNF43-188, RNF43-189, RNF43-200, RNF43-201, RNF43-202, RNF43-203, RNF43-204, RNF43-205, RNF43-206, RNF43-207, RNF43-208, RNF43-209, RNF43-300, RNF43-310, RNF43-311, RNF43-312, RNF43-313, RNF43-314, RNF43-315, RNF43-316, RNF43-317, RNF43-318, RNF43-319, RNF43-401, RNF43-402, RNF43-403, RNF43-404, RNF43-405, RNF43-406, RNF43-407, RNF43-408, RNF43-409, RNF43-501 NF43-177, RNF43-179, RNF43-180, RNF43-181, RNF43-186, RNF43-187, RNF43-196, RNF43-200, RNF43-201, RNF43-206, RNF43- 210, RNF43-213, RNF43-217, RNF43-221, RNF43-224, RNF43-25, RNF43-31, RNF43-33, RNF43-35, RNF43-38, RNF43-41, RNF43-5 3, RNF43-56, RNF43-61, RNF43-67, RNF43-69, RNF43-71, RNF43-74, RNF43-75, RNF43-76, RNF43-80, RNF43-86, RNF43-90, ZNR F3-101, ZNRF3-117, ZNRF3-128, ZNRF3-131, ZNRF3-163, ZNRF3-17, ZNRF3-170, ZNRF3-171, ZNRF3-172, ZNRF3-179, ZNRF3-182 , ZNRF3-195, ZNRF3-219, ZNRF3-222, ZNRF3-223, ZNRF3-231, ZNRF3-237, ZNRF3-244, ZNRF3-247, ZNRF3-253, ZNRF3-254, ZNRF 3-255, ZNRF3-265, ZNRF3-269, ZNRF3-270, ZNRF3-275, ZNRF3-279, ZNRF3-287, ZNRF3-296, ZNRF3-30, ZNRF3-300, ZNRF3-301,ZNRF3-305, ZNRF3-312, ZNRF3-314, ZNRF3-322, ZNRF3-329, ZNRF3-35, ZNRF3-55, ZNRF3-6, ZNR F3-90, RNF43-1, RNF43-24, RNF43-8, RNF43-12, RNF43-20, RNF43-11, RNF43-23, RNF43-6, RNF43 In some cases, the protein comprises both a VH and a VL of one of the above antibodies. The sequence listing below provides the DNA and protein sequences of the VH and VL of the above antibodies of SEQ ID NO:33 to SEQ ID NO:444.

[0108] In a further embodiment, multispecific binding proteins can be constructed using the amino acid sequences provided in one or more of SEQ ID NOs: 1-32 in the sequence listing below.

[0109] In some embodiments, a multispecific binding protein comprising the above CDRs or VH / VL has a binding affinity for ZNRF3 or RNF43 of less than 50 nM, or less than 10 nM, or less than 1 nM, or less than 0.5 nM, or less than 0.05 nM, or between 50 nM and 10 nM, or between 10 nM and 1 nM, or between 1 nM and 0.5 nM, or between 0.5 nM and 0.05 nM, or between 0.05 nM and 0.01 nM. In some cases, affinity is measured using a BIACORE® surface plasmon resonance assay, e.g., as described in the Examples below.

[0110] In some embodiments, the multispecific binding protein binds to both ZNFR3 and RNF43. For example, in some embodiments, the multispecific binding protein is a multispecific antibody that binds to both ZNFR3 and RNF43. In some embodiments, it is a trispecific antibody that binds to their ligase and the cell surface protein that is targeted for degradation. In some embodiments, it may have a format as shown in Figures 15A-15G, 7A-B, or 8A.

[0111] In some embodiments, the multispecific binding protein comprising the above CDRs or VH / VL is a rat anti-human antibody or a rabbit anti-human antibody or a mouse anti-human antibody. In other embodiments, it is humanized or chimeric. In some embodiments, the multispecific binding protein comprises a wild-type human Fc region, such as from human IgG1, IgG2, IgG3 or IgG4. In other embodiments, the protein comprises a human IgG1 Fc with one or more amino acid substitutions (e.g., a LALAPG mutation or a substitution at N297, e.g., N297G or N297Q), e.g., to reduce Fc effectors. In some embodiments, the multispecific binding protein has no effector function. In other embodiments, the multispecific binding protein has effector function. These and other optional constant regions or Fc regions are also discussed further below.

[0112] C. Conjugates Containing Multispecific Binding Proteins In other embodiments, the multispecific binding proteins herein may be further conjugated (chemically linked) to one or more other molecules, such as a label or a therapeutic agent. A variety of radioisotope labels are available for making radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212, P 32 , Pb 212 , and radioactive isotopes of Lu. When a radioactive conjugate is used for detection, it may contain a radioactive atom (e.g., tc99m or I123) for scintigraphy studies or a spin label (e.g., iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron) for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri). Exemplary therapeutic agents include cytotoxic agents, chemotherapeutic agents, drugs, growth inhibitors, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), or radioactive isotopes.

[0113] In one embodiment, the multispecific binding protein is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more of the therapeutic agents described above. The antibody is usually connected to one or more therapeutic agents using a linker. An overview of ADC technology, including examples of therapeutic agents and drugs and linkers, is provided in Pharmacol Review 68:3-19 (2016). Other potential conjugated therapeutics include enzymatically active toxins or fragments thereof, including, but not limited to, diphtheria A chain, unbound active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the trichothecenes.

[0114] Conjugates of proteins and cytotoxic agents can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bisazide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotides to antibodies. See WO94 / 11026. The linker can be a "cleavable linker" that facilitates the release of the cytotoxic drug inside the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers or disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) can be used.

[0115] The immunoconjugates or ADCs herein expressly contemplate, but are not limited to, such conjugates prepared with cross-linking reagents, including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., USA).

[0116] D. Antibody Variants In certain embodiments, the antibodies RNF43-104, RNF43-106, RNF43-107, RNF43-108, RNF43-116, RNF43-117, RNF43-123, RNF43-126, RNF43-128, RNF43-129, RNF43-130, RNF43-136, RNF43-145, RNF43-152, RNF43-156, RNF43-168, RNF43-170, RNF43-176, RNF43-177, RNF43-179, RNF43-180, RNF43-181, RNF43-186, RNF43-187, RNF43-196, RNF43-200, RNF43-201, RNF43-206, RNF43-210, RNF43-213, RNF43-217, RNF43-221, RNF43-224, RNF43-25, RNF43-31, RNF43-33, RNF43-35, RNF43-38, RNF43-41, RNF43-53, RNF43-56, RNF43-61, RNF43-67, RNF43-69, RNF43-71, RNF43-74, RNF43-75, RNF43-76, RNF43-80, RNF43-86, RNF43-90, ZNRF3-101, ZNRF3-117, ZNRF3-128, ZNRF3-131, ZNRF3-163, ZNRF3-17, ZNRF3-170, ZNRF3-171, ZNRF3-172, ZNRF3-179, ZNRF3-182, ZNRF3-195, ZNRF3-219, ZNRF3-222, ZNRF3-223, ZNRF3-231, ZNRF3-237, ZNRF3-244, ZNRF3-247, ZNRF3-253, ZNRF3-254, ZNRF3-255, ZNRF3-265, ZNRF3-269, ZNRF3-270, ZNRF3-275, ZNRF3-279, ZNRF3-287, ZNRF3-296, ZNRF3-30, ZNRF3-300, ZNRF3-301, ZNRF3-305, ZNRF3-312, ZNRF3-314, ZNRF3-322, ZNRF3-329, ZNRF3-35, ZNRF3-55, ZNRF3-6, ZNRF3-90, RNF43-1, RNF43-24, RNF43-8, RNF43-12, RNF43-20, RNF43-11, RNF43-23, RNF43-6, RNF43-5, provided herein are in certain aspects.Amino acid sequence variants of RNF43-15, RNF43-2, RNF43-16, RNF43-14, RNF43-17, RNF43-22, RNF43-9, RNF43-21, RNF43-13, RNF43-19, ZNRF3-331, ZNRF3-333 or ZNRF3-332 are contemplated. For example, it may be desirable to change the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions into and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions and substitutions can be made to arrive at the final construct, so long as the final construct has the desired characteristics (e.g., antigen binding).

[0117] a) Substitution, insertion and deletion variants In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Target sites for substitution mutagenesis include the CDRs and framework regions of the variable region. Conservative substitutions are shown under the heading of "preferred substitutions" in Table A. More substantial changes are provided under the heading of "exemplary substitutions" in Table A, as further described below with respect to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, for example, in the variable region and / or constant region, and the products can be screened for desired activity, for example, retention / improvement of antigen binding, reduction of immunogenicity, or improvement of ADCC or CDC. [Table A]

[0118] Amino acids can be classified according to common side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0119] Non-conservative substitutions will involve exchanging a member of one of these classes for a member of another class.

[0120] Certain substitutional variants involve substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant selected for further study will have an alteration (e.g., improvement) in certain biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which can be conveniently generated using, for example, phage display-based affinity maturation methods such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0121] For example, to improve antibody affinity, changes (e.g., substitutions) can be made in the CDRs. Such changes can be made in CDR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or residues that contact the antigen, and the resulting variants VH or VL are tested for binding affinity. Affinity maturation by construction of and reselection from secondary libraries is described, for example, in Hoogenboom et al. Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling or oligonucleotide-directed mutagenesis). A secondary library is then created. This library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity is the CDR-directed approach, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

[0122] In certain embodiments, substitutions, insertions or deletions may occur within one or more CDRs, so long as such changes do not substantially reduce the ability of the antibody to bind to antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the CDRs. Such changes may, for example, be outside the antigen contact residues within the CDRs. In certain variant VH and VL sequences provided above, each CDR is unaltered or contains no more than one, two or three amino acid substitutions.

[0123] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, one residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively or additionally, a crystal structure of an antigen-antibody complex may be used to identify contact points between the antibody and the antigen. Such contact and adjacent residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

[0124] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion of the N- or C-terminus of an antibody to an enzyme (e.g., for ADEPT (antibody-directed enzyme prodrug therapy) or to a polypeptide which increases the serum half-life of the antibody.

[0125] b) Glycosylation variants In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent of glycosylation of the antibody. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0126] If the antibody comprises an Fc region, the oligosaccharides attached thereto may be modified. Natural antibodies produced by mammalian cells usually comprise a branched biantennary oligosaccharide, which is generally attached to Asn297 of the CH2 domain of the Fc region by N-linkage. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to the GlcNAc of the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibody of the present invention may be made to generate antibody variants with certain improved properties.

[0127] In one embodiment, antibody variants are provided that have nonfucosylated oligosaccharides, i.e., oligosaccharide structures that lack fucose attached (directly or indirectly) to the Fc region. Such nonfucosylated oligosaccharides (also referred to as "afucosylated" oligosaccharides) are in particular N-linked oligosaccharides that lack a fucose residue attached to the first GlcNAc in the stem of the biantennary oligosaccharide structure. In one embodiment, antibody variants are provided that have an increased proportion of nonfucosylated oligosaccharides in the Fc region compared to the native or parent antibody. For example, the proportion of nonfucosylated oligosaccharides may be at least about 20%, at least about 40%, at least about 60%, at least about 80% or even about 100% (i.e., no fucosylated oligosaccharides are present). The percentage of nonfucosylated oligosaccharides is the (average) amount of oligosaccharides lacking a fucose residue relative to the sum of all oligosaccharides attached to Asn297 (e.g., complex, hybrid and high mannose structures) as measured, for example, by MALDI-TOF mass spectrometry as described in WO2006 / 082515. Asn297 refers to an asparagine residue located at about position 297 (EU numbering of Fc region residues) in the Fc region, although Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such antibodies with an increased percentage of nonfucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and / or improved effector function, particularly improved ADCC function. See, e.g., U.S. Patent Application Publication No. 2003 / 0157108; U.S. Patent Application Publication No. 2004 / 0093621.

[0128] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. 2003 / 0157108; and WO2004 / 056312, especially Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al. al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107), or cells with reduced or abolished activity of GDP-fucose synthesis or transporter proteins (see, e.g., U.S. Patent Application Publication No. 2004259150, U.S. Patent Application Publication No. 2005031613, U.S. Patent Application Publication No. 2004132140, U.S. Patent Application Publication No. 2004110282).

[0129] In further embodiments, the antibody variants are provided with bisected oligosaccharides, e.g., biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function, as described above. Examples of such antibody variants are described, for example, in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO99 / 54342; WO2004 / 065540, WO2003 / 011878.

[0130] Also provided is an antibody variant that has at least one galactose residue in the oligosaccharide attached to Fc region.Such an antibody variant can have improved CDC function.Such an antibody variant is described, for example, in WO1997 / 30087;WO1998 / 58964;and WO1999 / 22764.

[0131] c) Fc domain variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0132] In some embodiments, the antibodies herein have effector functions. In other embodiments, the antibodies herein lack effector functions. In certain embodiments, the invention contemplates antibody variants that have some, but not all, effector functions, making them desirable candidates for applications where the half-life of the antibody in vivo is important, but where certain effector functions (e.g., complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / loss of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus may lack ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII and FcγRIII. Expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be employed (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, Calif.) and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.)).Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest may be evaluated in vivo, for example in an animal model as disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, CDC assays may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations may also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO2013 / 120929 A1).

[0133] Antibodies with reduced effector function include those containing substitutions of one or more of residues 238, 265, 269, 270, 297, 327 and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0134] Certain antibody variants have been described with improved or diminished binding to FcRs. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312 and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0135] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that improve ADCC, e.g., substitutions at residues 298, 333 and / or 334 (EU numbering) of the Fc region.

[0136] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcγR binding, such as substitutions at positions 234 and 235 (EU numbering residues) of the Fc region. In one embodiment, the substitutions are L234A and L235A (LALA). In certain embodiments, the antibody variant further comprises D265A and / or P329G in the Fc region derived from a human IgG1 Fc region. In one embodiment, the substitutions are L234A, L235A and P329G (LALAPG) in the Fc region derived from a human IgG1 Fc region. (See, e.g., WO2012 / 130831) In another embodiment, the substitutions are L234A, L235A and D265A (LALA-DA) in the Fc region derived from a human IgG1 Fc region.

[0137] In some embodiments, the antibodies may have a modification at position N297 to reduce or eliminate ADCC activity, such as N297G or N297Q. In some such cases, the antibody lacks effector function.

[0138] In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0139] Antibodies with extended half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), have been described in US2005 / 0014934 (Hinton et al.). These antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of Fc region residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434, e.g., a substitution at Fc region residue 434 (see, e.g., U.S. Patent No. 7,371,826; Dall'Acqua, WF, et al. J. Biol. Chem. 281 (2006) 23514-23524).

[0140] The Fc region residues critical for mouse Fc-mouse FcRn interaction have been identified by site-directed mutagenesis (see, e.g., Dall'Acqua, WF, et al. J. Immunol 169 (2002) 5171-5180). Residues I253, H310, H433, N434 and H435 (EU index numbering) are involved in the interaction (Medesan, C., et al., Eur. J. Immunol. 26 (1996) 2533; Firan, M., et al., Int. Immunol. 13 (2001) 993; Kim, JK, et al., Eur. J. Immunol. 24 (1994) 542). Residues I253, H310 and H435 were found to be critical for the interaction of human Fc with mouse FcRn (Kim, JK, et al., Eur. J. Immunol. 29 (1999) 2819). Studies of the human Fc-human FcRn complex have shown that residues I253, S254, H435 and Y436 are critical for the interaction (Firan, M., et al., Int. Immunol. 13 (2001) 993; Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591-6604). Yeung, YA, et al. (J. Immunol. 182 (2009) 7667-7671) report and investigate various mutants of residues 248-259, 301-317, 376-382, and 424-437.

[0141] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, such as substitutions at positions 253 and / or 310 and / or 435 (residues of EU numbering) of the Fc region. In certain embodiments, the antibody variant comprises an Fc region having amino acid substitutions at positions 253, 310 and 435. In one embodiment, the substitutions are I253A, H310A and H435A in the Fc region derived from a human IgG1 Fc region. See, e.g., Grevys, A., et al., J. Immunol. 194 (2015) 5497-5508.

[0142] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, such as substitutions at positions 310 and / or 433 and / or 436 (residues of EU numbering) of the Fc region. In certain embodiments, the antibody variant comprises an Fc region having amino acid substitutions at positions 310, 433 and 436. In one embodiment, the substitutions are H310A, H433A and Y436A in the Fc region derived from human IgG1 Fc region. (See, e.g., WO2014 / 177460A1).

[0143] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that increase FcRn binding, such as substitutions at positions 252 and / or 254 and / or 256 (residues of EU numbering) of the Fc region. In certain embodiments, the antibody variant comprises an Fc region having amino acid substitutions at positions 252, 254 and 256. In one embodiment, the substitutions are M252Y, S254T and T256E in the Fc region derived from human IgG1 Fc region. For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO94 / 29351.

[0144] The C-terminus of the heavy chain of the antibody as reported herein may be a complete C-terminus terminating in the amino acid residue PGK. The C-terminus of the heavy chain may be a shortened C-terminus in which one or two of the C-terminal amino acid residues are removed. In one preferred embodiment, the C-terminus of the heavy chain is a shortened C-terminus terminating in PG. In one embodiment of all embodiments reported herein, the antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine-lysine dipeptide (G446 and K447, amino acid positions of EU index numbering). In one embodiment of all embodiments reported herein, the antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine residue (G446, amino acid position of EU index numbering).

[0145] d) Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to create cysteine ​​engineered antibodies, such as THIOMA™ antibodies, in which one or more residues of an antibody are replaced with cysteine ​​residues. In certain embodiments, the replaced residues are located at accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are placed at accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker drug moieties, as further described herein, to create immunoconjugates. Cysteine ​​engineered antibodies can be created, for example, as described in U.S. Patent Nos. 7,521,541, 8,30,930, 7,855,275, 9,000,130, or WO2016040856.

[0146] e) Antibody derivative

[0147] In certain embodiments, the antibodies provided herein may be further modified to include additional non-proteinaceous moieties that are known in the art and readily available. Suitable sites for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and when more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in therapy, such as under defined conditions.

[0148] E. Recombinant Methods and Compositions The proteins herein can be made using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. For these methods, one or more isolated nucleic acids encoding the antibody are provided.

[0149] In the case of a natural antibody or a natural antibody fragment, two nucleic acids are required, one for the light chain or its fragment and the other for the heavy chain or its fragment. Such nucleic acids code for the amino acid sequence comprising the VL and / or the amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors.

[0150] In the case of a bispecific antibody with a heterodimeric heavy chain, four nucleic acids are required: one for the first light chain, one for the first heavy chain comprising the first heteromonomeric Fc region polypeptide, one for the second light chain, and one for the second heavy chain comprising the second heteromonomeric Fc region polypeptide. The four nucleic acids may be contained in one or more nucleic acid molecules or expression vectors. Such nucleic acids encode an amino acid sequence comprising a first VL and / or an amino acid sequence comprising a first VH comprising the first heteromonomeric Fc region and / or an amino acid sequence comprising a second VL and / or an amino acid sequence comprising a second VH comprising the second heteromonomeric Fc region of the antibody (e.g., the first and / or second light chain and / or the first and / or second heavy chain of the antibody). These nucleic acids may be present on the same expression vector or on different expression vectors, and usually these nucleic acids are located on two or three expression vectors, i.e. one vector may contain more than one of these nucleic acids. An example of these bispecific antibodies is CrossMab (see, e.g., Schaefer, W. et al, PNAS, 108 (2011) 11187-1191). For example, according to the EU index numbering, one of the heteromonomer heavy chains contains a so-called "knob mutation" (T366W, and optionally one of S354C or Y349C) and the other contains a so-called "hole mutation" (T366S, L368A and Y407V, and optionally Y349C or S354C) (see, e.g., Carter, P. et al., Immunotechnol. 2 (1996) 73).

[0151] In one embodiment, an isolated nucleic acid is provided that encodes an antibody or other component of a multispecific binding protein for use in the methods as reported herein.

[0152] In one embodiment, a method of making a multispecific binding protein is provided, the method comprising culturing a host cell containing nucleic acid encoding an antibody or protein component, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody or other protein component from the host cell (or host cell culture).

[0153] For recombinant production of an antibody, nucleic acid encoding the antibody, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody), or can be produced by recombinant methods, or can be obtained by chemical synthesis.

[0154] Suitable host cells for cloning or expressing protein-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent No. 5,648,237, U.S. Patent No. 5,789,199 and U.S. Patent No. 5,840,523. (See also Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0155] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for protein-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized" to produce antibodies with partially or fully human glycosylation patterns. See Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0156] Suitable host cells for the expression of (glycosylated) antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in combination with insect cells, particularly for the transfection of Spodoptera frugiperda cells.

[0157] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0158] Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are the SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (e.g., 293 or 293T cells, as described in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells, as described in Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (e.g., Mather, JP et al., Annals NY Acad. Sci. 383 (1982) 44-68); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines, such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0159] In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (eg, a Y0, NS0, Sp20 cell).

[0160] F. Pharmaceutical Compositions In further embodiments, pharmaceutical compositions are provided comprising any of the multispecific binding proteins provided herein, e.g., for use in any of the therapeutic methods described below. In one embodiment, the pharmaceutical composition comprises any of the proteins provided herein and a pharma- ceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises any of the antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

[0161] Pharmaceutical compositions of proteins as described herein are prepared by mixing such antibodies having the desired purity, in the form of a lyophilized composition or aqueous solution, with one or more optional pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and may include buffers, such as histidine, phosphate, citric acid, acetic acid, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; The pharmacopoietins and / or pharmacopoietins may include, but are not limited to, glycines, proteins such as serum albumin, gelatin or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine, monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmacopoietins and / or pharmacopoietins may include, but are not limited to, interstitial drug dispersion agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins such as rHuPH20 (HYLENEX®, Halozyme, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one embodiment, a sHASEGP is combined with one or more additional glycosaminoglycanases (eg, chondroitinases).

[0162] Exemplary lyophilized antibody compositions are described in U.S. Patent No. 6,267,958. Aqueous antibody compositions include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter compositions including a histidine-acetate buffer.

[0163] The active ingredient may be encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, in microcapsules prepared, for example, by coacervation or interfacial polymerization, e.g., hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0164] Pharmaceutical compositions can be prepared for sustained release. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0165] Pharmaceutical compositions to be used for in vivo administration are generally sterile. Sterilization may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0166] G. Treatment Methods and Routes of Administration Any of the multispecific binding proteins provided herein can be used in therapeutic methods. For example, they can be useful in any therapeutic method in which it is beneficial to reduce the levels of a particular cell surface protein in a subject by targeting that protein for lysosomal degradation, and to do so in cells that readily express a transmembrane E3 ubiquitin ligase.

[0167] In one embodiment, the multispecific binding protein is provided for use as a medicine. In a further embodiment, the multispecific binding protein is provided for use in the treatment of diseases such as cancer. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma and leukemia. More specific examples of such cancer include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, digestive cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatoma, leukemia and other lymphoproliferative disorders, as well as various types of head and neck cancer, autoimmune conditions, inflammatory conditions, neurodegenerative conditions or infectious diseases.

[0168] In certain embodiments, the multispecific binding protein is provided for use in a method of treatment. In certain embodiments, the invention provides a multispecific binding protein for use in a method of treating an individual having a disease, such as cancer, an autoimmune condition, an inflammatory condition, a neurodegenerative condition, or an infectious disease, comprising administering to the individual an effective amount of the multispecific binding protein. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (e.g., 1, 2, 3, 4, 5, or 6 additional therapeutic agents), e.g., as described below.

[0169] In a further aspect, the invention provides a multispecific binding protein for use in reducing the level of a cell surface protein in a subject targeted by the multispecific binding protein. In some cases, the subject may have a disease such as cancer, an autoimmune condition, an inflammatory condition, a neurodegenerative condition or an infectious disease.

[0170] In a further embodiment, the present invention provides the use of the multispecific binding protein in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of a disease such as cancer, an autoimmune condition, an inflammatory condition, a neurodegenerative condition, or an infectious disease. In a further embodiment, the medicament is for use in a method of treating a disease such as cancer, an autoimmune condition, an inflammatory condition, a neurodegenerative condition, or an infectious disease, comprising administering an effective amount of the medicament to an individual having cancer. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as those described below. In a further embodiment, the medicament is for reducing the level of a cell surface protein of a subject targeted by the multispecific binding protein. In some cases, the subject may have a disease such as cancer, an autoimmune condition, an inflammatory condition, a neurodegenerative condition, or an infectious disease.

[0171] In a further embodiment, the invention provides a method for treating a disease such as cancer. In one embodiment, the method comprises administering to an individual having such cancer, autoimmune condition, inflammatory condition, neurodegenerative condition or infectious disease an effective amount of a multispecific binding protein. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below.

[0172] In a further aspect, the present invention provides a method for reducing the level of a cell surface protein in a subject that is targeted by a multispecific binding protein. In some cases, the subject may have a disease such as cancer, an autoimmune condition, an inflammatory condition, a neurodegenerative condition, or an infectious disease. For example, in some aspects, the multispecific binding protein herein may be used to reduce the level of a cell surface protein on a particular cell type by targeting an E3 ubiquitin ligase that is primarily expressed on those cell types. For example, the transmembrane E3 ubiquitin ligases RNF130, RNF149, and RNF167 are expressed on hematopoietic cells, and multispecific binding proteins that target those ligases can be used in some embodiments to reduce the level of a cell surface protein on those cells. Furthermore, the transmembrane E3 ubiquitin ligases RNF133 and RNF148 are expressed on testicular cells, and multispecific binding proteins that target those ligases can be used in some embodiments to reduce the level of a cell surface protein on those cells.

[0173] In further embodiments, the invention provides pharmaceutical compositions comprising any of the multispecific binding proteins provided herein, e.g., for use in any of the above methods of treatment. In one embodiment, the pharmaceutical composition comprises any of the multispecific binding proteins provided herein and a pharma- ceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises any of the multispecific binding molecules provided herein and at least one additional therapeutic agent, e.g., as described below.

[0174] In other embodiments of the uses and methods of treatment herein, the subject may have a mutation in the RNF43 or ZNRF3 protein. For example, in certain cancers, such as colorectal cancer and endometrial cancer, among others, the RNF43 protein has been found to be mutated. See, for example, YJ van Herwaarden et al., Histopathology 78:749-758 (2021). In such cases, a multispecific binding protein containing an anti-RNF43 component may be less effective in reducing the levels of the targeted cell surface protein. However, a multispecific binding protein containing an anti-ZNRF3 component may readily result in a reduction in the levels of the targeted cell surface protein. (See, for example, Example 29 and Figures 21-22 below). Thus, in some embodiments, if a subject has previously been determined to have an RNF43 or ZNRF3 mutation, for example, if the subject has a cancer that includes cancer cells with mutated RNF43 or mutated ZNRF3, the subject may be administered a multispecific binding protein that does not bind to or activate RNF43 or ZNRF3 protein.Thus, for example, if a subject has an RNF43 mutation, the multispecific binding protein provided to the subject may include an anti-ZNRF3 component, and vice versa.Thus, in some embodiments, the treatment method and use may also include determining whether the subject has a mutation in RNF43 or ZNRF3 before administering the multispecific binding protein, and (a) if the subject has a mutation in RNF43, the multispecific binding protein does not bind to or activate RNF43, and (b) if the subject has a mutation in ZNRF3, the multispecific binding protein does not bind to or activate ZNRF3. Such mutations may be identified, for example, at the protein level by techniques such as immunohistochemistry (IHC) or at the nucleic acid level by techniques such as reverse transcription polymerase chain reaction (RT-PCR), whole genome sequencing, exome sequencing or in situ hybridization.Such mutations may be present in certain cancers, and thus in some such embodiments, the subject may be a cancer subject.

[0175] The proteins herein may be administered alone or may be used in combination therapy. For example, combination therapy includes administering a multispecific binding protein and administering at least one additional therapeutic agent (e.g., 1, 2, 3, 4, 5, or 6 additional therapeutic agents). Such combination therapy as described above includes combined administration (when two or more therapeutic agents are contained in the same or separate pharmaceutical compositions) and separate administration, in which case administration of the antibody of the present invention may occur before, simultaneously with, and / or after administration of the additional therapeutic agent.

[0176] The multispecific binding proteins of the invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired, for localized treatment, by intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.

[0177] H. Kits and Products and Other Methods of Use In another aspect of the present disclosure, the multispecific binding molecules herein can be used in vitro, i.e., in the laboratory, to modify the levels of cell surface proteins in cell culture samples or tissue samples.For example, there are many cases in which it may be beneficial to assay the behavior of cells or tissue samples in which the levels of certain cell surface signaling molecules are artificially reduced.The use of such multispecific binding molecules can provide a relatively simple method of regulating the levels of such proteins.

[0178] Thus, the present disclosure also encompasses a method for reducing the level of cell surface proteins in a cell or tissue sample in vitro, comprising incubating the sample with a multispecific binding protein.The present disclosure also encompasses a kit for this purpose.The kit may comprise a multispecific binding protein, optionally with instructions for use, suitable buffers and / or labeling molecules.The kit may also comprise a nucleic acid, vector or host cell comprising a polynucleotide encoding the multispecific binding protein, so that the protein may be produced, for example, for use in such a method. EXAMPLES

[0179] III. Examples The following are examples of methods and compositions of the present invention. Given the general description provided above, it will be understood that other various embodiments may be practiced.

[0180] Example 1: Development and characterization of rat anti-human cell surface ligase rat B cell antibodies or rabbit B cell antibodies Rats and rabbits were chosen for the generation of cell surface ligase antibodies because they offer certain advantages over alternative methods, such as screening phage libraries. For example, antibodies obtained from animals may have higher specificity and better pharmacokinetic properties than phage-derived antibodies, and may provide better therapeutic candidates than phage-derived antibodies. Sprague Dawley rats (Charles River, Hollister, CA) were immunized with a priming dose of 100 μg detergent-solubilized human (RNF43 or ZNRF3) protein mixed with MPL+TDM adjuvant (Sigma-Aldrich, St. Louis, MO), CFA (Sigma-Aldrich, St. Louis, MO), or a combination of TLR agonists: 50 ug MPL (Sigma-Aldrich), 20 ug R848 (Invivogen, San Diego, CA), 10 ug PolyI:C (Invivogen), and 10 ug multi-site CpG (Invivogen). New Zealand White rabbits were also immunized with a mixture of the same detergent-solubilized proteins mixed with CFA (Sigma-Aldrich, St. Louis, MO). For additional protein boosts, the rats and rabbits were given half the priming dose of protein diluted in PBS. Rats and rabbits were dosed every 2 weeks. Polyclonal antisera from these rats and rabbits were purified and tested by ELISA for binding to human RNF43 and human ZNRF3. For rats, multiple lymph nodes were harvested 3 days after the last immunization that showed detectable FACS reactivity to human RNF43 or human ZNRF3.IgM-negative B cells from these rats were purified from total lymphocytes using magnetic separation (Miltenyi Biotec, San Diego, CA) and stained with anti-rat IgM antibodies (Jackson ImmunoResearch, West Grove, PA), anti-rat CD45RA (Biolegend, San Diego, CA), anti-rat CD8 a (Biolegend, San Diego, CA) and labeled human RNF43-Alex633 or human ZNRF3-Alex633 by Lightning-Link Alex 633 Antibody Labeling Kit (Novus, Centennial, CO). Rat B cells that showed minimal rat IgM expression while binding to human RNF43 or human ZNRF3 protein were sorted and placed into 96-well plates containing cytokine-supplemented medium containing feeder cells using a FACSAriaIII sorter (BD, Franklin Lakes, NJ). For rabbits, blood was collected 3 days after the last immunization that showed detectable FACS reactivity against human RNF43 and human ZNRF3. IgG-positive B cells from these rabbits were purified from whole blood using magnetic separation (Miltenyi Biotec, San Diego, CA) and stained with anti-rabbit IgG antibody (Southern Biotech, Birmingham, AL) and labeled human RNF43-Alex 633 or human ZNRF3-Alex 633 by Lightning-Link Alex 633 Antibody Labeling Kit (Novus, Centennial, CO). Rabbit B cells that showed maximum rabbit IgG expression while binding human RNF43 or human ZNRF3 protein were sorted and placed into 96-well plates containing cytokine-supplemented culture medium with feeder cells using a FACSAriaIII sorter (BD, Franklin Lakes, NJ). Seven days after sorting, the supernatants were screened by ELISA against human RNF43 or human ZNRF3.Supernatants showing human RNF43 or human ZNRF3 binding were tested by FACS for binding to human RNF43 or human ZNRF3 expressed on the surface of gD-hRNF43, or to human ZNRF3 expressed on the surface of gD-hZNR3. RNA was extracted from B cells showing RNF43 or ZNRF3 FACS binding for molecular cloning and recombinant expression. Recombinant antibodies were tested by FACS for binding to human RNF43 expressed on the surface of gD-hRNF43, or to human ZNRF3 expressed on the surface of gD-hZNR3.

[0181] Example 2: Production of recombinant antibodies DNA encoding the antibody heavy and light chain variable domains was generated by gene synthesis. The synthesized gene fragments were inserted into mammalian expression vectors containing the corresponding heavy or light chain constant domains. Species and isotypes included human IgG1 and mouse IgG2a. Some variable domain sequences were edited to remove obvious unpaired cysteine ​​residues and NX[S / T]N-glycosylation motifs. Recombinant antibodies were generated by transient transfection of Expi293 cells with mammalian expression vectors encoding the antibody heavy and light chains. Heavy and light chains were encoded on separate vectors and transfected using a 1:1 ratio of heavy and light chain expression vectors. Antibodies were purified from cell culture supernatants by affinity chromatography. In some cases, antibodies underwent an additional purification step based on SEC.

[0182] Bispecific antibodies were generated using knob-into-hole technology (Ridgway et al., Prot Eng. 1996) using human IgG1 or mouse IgG2a scaffolds that typically contain mutations that reduce effector function (e.g., L234A, L235A, P329G, and / or N297G). Abs containing either knobs or holes were expressed, purified, and then assembled into bispecific formats essentially as previously described (Williams et al., Biotechnol Prog., 2015). In some cases, mutations were introduced to allow bispecific expression in single cells with appropriate light chain pairing (Dillon et al., mAbs, 2017).

[0183] Example 3: Affinity and epitope determination of recombinant antibodies Antibodies were screened for binding to recombinant human RNF43 and ZNRF3 ECD using a Biacore® 8k instrument (GE Life Sciences). Briefly, antibodies diluted to 1 μg / ml in 1XHBSP buffer (Cytiva, BR100368) were captured on a Sensor Chip Protein A (GE Life Sciences) using a flow rate of 10 μl / min and a contact time of 60 seconds. Binding of recombinant human RNF43 and ZNRF3 extracellular domain to the captured antibodies was analyzed at 25°C using single cycle kinetics with a flow rate of 30 μl / min, a contact time of 180 seconds, and a dissociation time of 600 seconds. The concentrations of recombinant human RNF43 and ZNRF3 extracellular domain in single cycle kinetics were 0, 0.8, 4, 20, and 100 nM. Between cycles, the chip was regenerated using 10 mM glycine HCl pH 1.5 injected for 30 s at 30 μl / min. Data was evaluated using Biacore 8K Evaluation software (GE Life Sciences). Rate constants were obtained using a 1:1 binding model with the parameter RI set to 0. Multiple antibodies targeting RNF43 and ZNRF3 were shown to have subnanomolar affinities as shown in Tables 1-4. Cell surface ligase antibodies with subnanomolar affinities may offer advantages in certain cases, for example in resolution efficiency that correlates with durability of the ternary complex as shown in Figure 5D. [Table 1] TIFF2024509695000004.tif159170 [Table 2] TIFF2024509695000006.tif76170 [Table 3] [Table 4]

[0184] Example 4: HTP epitope binning Antibodies generated by immunizing animals were reformatted into a hIgG1 backbone and binned using a CFM2 / MX96 SPR system (Wasatch Microfluidics, now Carterra) equipped with DA v6.19.3, IBIS SUIT, SprintX & Carterra Epitope Tool software. 10 μg / ml of antibodies were immobilized on an SPR sensor prism CMD 200 M (Xantec Bioanalytics) by amine coupling using 10 mM sodium acetate pH 4.5 immobilization buffer. Immobilization was performed using a CFM2 instrument, and the sensor prism was then transferred to an IBIS MX96 instrument for SPR-based competition analysis. The immobilized antibodies were first exposed to 100 nM recombinant human RNF43 or ZNRF3 extracellular domain and then to 10 ug / ml antibody in solution using HBS-EP running buffer (10 mM HEPES, 150 mM NaCl, 0.05% Tween 20, pH 7.4, 1 mM EDTA). 10 mM glycine / HCl pH 1.7 was used as the renaturation buffer. The epitope binning results are shown in Figure 5b. These results show sandwich profiles of several different RNF43 and ZNRF3 indicating binding to multiple different epitopes on the antigen.

[0185] Example 5: Assessment of target cell surface clearance by flow cytometry Antibodies were evaluated for their ability to remove target antigens from the cell surface of multiple target cells. Cell lines included SW1417, HT29, and engineered HT-29 cell lines, including one engineered to express an N-terminally tagged ligase. N-terminal tags included epitope tags (gD) or HiBit tags (Promega) for quantification of tagged proteins by the Nano-glo HiBit Extracellular Detection System (Promega). Cell lines were pooled or clonal and in some cases contained ligase expression on a doxycycline-inducible promoter. Cells were plated in 96-well plates at 50,000-120,000 cells per well and grown in ATCC recommended medium (+ / - doxycycline) in the presence of various concentrations of antibody. After 24 hours (or time periods as indicated), media was removed, cells were washed with PBS (150uL) and detached from the well surface by adding Accutase (Millipore) (100uL) for 10 minutes at 37°C. Accutase activity was quenched by adding 100uL RPMI + 10% heat inactivated fetal bovine serum with penicillin, streptomycin and glutamine, cells were centrifuged at 1200rpm for 4 minutes and the supernatant was discarded. Cells were resuspended in 150uL FACS buffer (PBS with 0.5% BSA and 0.05% sodium azide) for 10 minutes at 4°C, centrifuged and the supernatant was discarded. Cells were incubated with 40uL staining antibody (final concentration 2ug / ml) for 45 minutes at 4°C, washed twice with 150uL FACS buffer and resuspended in 40uL FACS buffer for analysis. Staining antibodies include xIGF-1R mIgG1 APC (1H7, ebioscience catalog #17-8849-42) and xRNF43 37.17APC (in-house generated reagents). Background / negative control staining antibodies include NISTMab hIgG1APC and xRagweed mIgG1 APC (both in-house generated reagents).

[0186] Flow cytometry analysis was performed on a BD FACSCelesta™ flow cytometer using the High Throughput Sampler (HTS) in high throughput mode. Each sample was first resuspended twice with a resuspension volume of 10 ul. 10 ul of sample was then aspirated from each well and run through the cytometer at a flow rate of 180 ul / min. Between each sample, the system was washed with 400 ul (wash volume) of buffer. SSC, FSC and APC signals were measured. Cells were gated for single cells based on SSC and FSC profiles using Flowjo FACS analysis software. The mean fluorescence intensity (MFI) of the APC signal is used as a measure of IGF-1R levels on the cell surface.

[0187] Example 6: Gene-induced dimerization of ligase to cell surface receptors The effect of chemically induced dimerization of RNF43 or ZNRF3 with HER2 was assessed using the iDimerize system (Takara Bio). Briefly, HEK293T cells were reverse transfected with pBind plasmid to induce constitutive co-expression of RNF43-DmrA-HA and HER2-DmrC-FLAG, or ZNRF3-DmrA-HA and HER2-DmrC-FLAG. After 24 h, cells were left untreated or treated with 500 nM A / C heterodimerizer (TakaraBio; REF 635057) to induce ligase-target dimerization, and cells were incubated for another 24 h. Cells were then lysed in GST lysis buffer [25 mM Tris·HCl, pH 7.2, 150 mM NaCl, 5 mM MgCl2, 1% NP-40 and 5% glycerol, 1% Halt protease & phosphatase inhibitor cocktail]. The clarified lysates were subjected to HA immunoprecipitation (IP) by incubating the lysates with 25 μl of Pierce HA epitope tag antibody agarose conjugate (2-2.2.14) (Thermo Scientific; REF 26182) for 3 h at 4 °C. For the input sample, 20 μl from the prepared IP sample before incubation was used. After incubation, the beads were washed and prepared for Western blot analysis.

[0188] Example 7: Western Blot and Immunoprecipitation HT29, HEK293T and SW1417 cells were plated at a density of 500,000 cells per well in 6-well dishes (Corning, cat #3516) and allowed to adhere overnight. Cells were treated for 40 hours with bispecific antibodies diluted to 1ug / mL in RPMI-1640 medium supplemented with FBS, L-glutamine and Pen / Strep. Where applicable, cells were pretreated with doxycycline (500ng / ml), bortezomib (200nM, Selleck, cat# S1013), E1 inhibitor (MLN7243, cat# S8341, 100nM) or bafilomycin A (Tocris-Cat.No.1334, 50nM). After 24-48 hours of incubation, cells were lysed in RIPA buffer supplemented with phosphatase / protease inhibitors (Thermo Fisher, Cat #78442) for 20 minutes on ice. Lysates were cleared and normalized and 20ug of each sample was run on a 4-12% Bis-Tris gel (Invitrogen, Cat #WG1403) for approximately 2 hours at 90V using BOLT MOPS SDS running buffer. Gels were transferred to nitrocellulose membranes using the iBlot 2 system using an iBlot2 Nitrocellulose Regular stack (Invitrogen, Cat #IB23001)) and protocol P0 (1 minute at 20V, 4 minutes at 23V and 2 minutes at 25V). Blots were blocked with 5% milk in TBST and blocked for 1 hour at room temperature. Blots were transferred to 5% milk in TBST containing antibodies against IGF1R-beta (Cell signaling, #3027), Vinculin (Cell signaling, cat #13901) and incubated overnight at 4 degrees. Blots were washed 4 times for 5 minutes each in TBST and then incubated in 5% milk containing IRDye secondary antibody (Li-Cor, Cat #926-32211) for 1 hour at room temperature. Blots were washed 4 times for 5 minutes each and developed using the Li-Cor system.

[0189] Example 8: Characterization of cell surface ligases driven by Wnt signaling activity RNF43 and ZNRF3 are Wnt canonical targets that control the turnover of Wnt ligand receptors FZD and LRP at the cell surface (Figure 1A). This is accomplished by ubiquitination of FZD / LRP by RNF43 / ZNRF3 and the resulting cell surface clearance (Figure 1A). To evaluate the impact of oncogenic Wnt signaling on the expression of RNF43 and ZNRF3, we used publicly available gene expression profiles from various colorectal cancer patient datasets in which Wnt signaling is constitutively hyperactive. Both cell surface ligases showed elevated expression in colorectal adenoma samples compared to healthy control normal mucosa (Galamb O., et al., Dis Markers, 2008. GSE 4183; Figure 1B). Furthermore, colorectal cancer gene expression analysis derived from The Cancer Genome Atlas (TCGA) demonstrated elevated expression of RNF43 in CRC compared to other tumor types and normal colon (Figure 1C). Importantly, in situ hybridization of Rnf43 in a mouse colon cancer model demonstrated widespread and uniform expression of the ligase within the entire tumor mass (Figure 17). The canonical roles of RNF43 and ZNRF3 were verified using transfection of inducible plasmids that conditionally express either ligase in HPAF-II cells upon addition of doxycycline (dox). Dox-mediated overexpression of WT RNF43 leads to cell surface clearance of FZD receptors in a ligase-dependent manner (Figure 1D). To explore the degradative potential of RNF43 and ZNRF3 over their natural substrates, i.e., FZD and LRP5 / 6, we utilized the idimerize system. HEK293T were transfected with pBind plasmid to induce constitutive co-expression of RNF43-DmrA-HA and HER2-DmrC-FLAG. 24 h after addition of AC heterodimerizers, we detected increased binding of RNF43 to HER2 by immunoprecipitation of RNF43 followed by immunoblotting for HER2 (Fig. 1E, left panel). This increased binding of the ligase to its target was functional as it led to HER2 target degradation (Fig. 1E, right panel).

[0190] Example 9: Expression of tagged RNF43 ligase and tagged ZNRF3 ligase and dimerization with HER2 We further evaluated the ability of both cell surface ligases to degrade the receptor when associated with a bispecific antibody. We generated a ligase tagged with gD at the N-terminus and dimerized it to HER2 by using a bispecific antibody targeting three different epitopes of HER2 (4D5, 7C2 and 2C4) (Figure 2A-B) and gD. We confirmed cell surface expression of both ligases by flow cytometry (Figure 2C), and gD-based dimerization via either HER2 epitope led to degradation of HER2. This was dependent on the expression of the ligase (Figure 2D). We further validated this degradation ability in additional HER2-expressing breast cancer cell lines, including the HER2-amplified KPL4 cell line. Importantly, HER2 degradation also affected downstream signaling, as evidenced by a decrease in pERK activity (Figure 2E).

[0191] Example 10: Antibody Dependence of Cell Surface Clearance Bispecific antibodies targeting three different epitopes of HER2 (4D5, 7C2 and 2C4) (Fendly et al. Canc Res 1990) and either the gD epitope tag or an unrelated antigen (human CD3) were generated as described above. The effects of these bispecific antibodies on HT-29 cells expressing doxycycline-inducible gD-tagged ZNRF3 or RNF43 were evaluated as described above. Various antibodies showed different levels of HER2 clearance from the cell surface (Figure 3A). Monovalent binding of 2C4 / CD3 was sufficient to drive partial clearance of HER2, but substantially less than seen with the 2C4 / gD bispecific Ab. To understand the impact of cell surface clearance on HER2 degradation, we examined HER2 levels by Western blot. Similar to what was observed by flow cytometry, ligase dimerization to HER2 was required for HER2 degradation. Importantly, monovalent binding to HER2 using anti-2C4 / CD3 did not result in substantial HER2 degradation, suggesting that cell surface receptor clearance does not necessarily result in receptor degradation, but that ligase dimerization is required (Figure 3B). To substantiate this contention, we probed the ubiquitination status of HER2 by immunoprecipitation. Ubiquitin smear was detected in HER2 after treatment with various HER2 / gD bispecific antibodies only in the presence of gD-tagged ligase (Figure 3C). Furthermore, ligase activity was required for HER2 degradation, as deletion of the RING domain from either ZNRF3 prevented HER2 degradation after ligase dimerization, although not to the same extent as RNF43 (Figure 3D). Conversely, inhibition of E1 ubiquitin-activating enzyme also rescued degradation, indicating that it was dependent on lysosomal, but not proteasomal, activity (Figure 3E).

[0192] Example 11: Cell surface ligase-mediated degradation is applicable to multiple receptors Using chemically induced dimerization, we explored the degradative ability of RNF43 against additional cell surface receptors that, like HER2, are receptor tyrosine kinases, specifically IGF1R. Similar to what was observed with HER2, addition of AC heterodimerizer to HEK293T cells transfected with pBind plasmids co-expressing RNF43-DmrA-HA and IGF-1R-DmrC-FLAG increased the binding between the two receptors in a dose-dependent manner (Figure 4A, left panel). Consequently, this increase in ligase-target binding led to dose-dependent degradation of IGF-1R (Figure 4A, right panel). Bispecific antibodies were generated with variable regions targeting IGF-1R and gD and evaluated for their ability to degrade IGF1R in HT-29 cells expressing doxycycline-inducible gD-tagged ZNRF3. Western blot analysis of HT-29 cells treated with 1ug / ml of the indicated antibodies was performed 24 hours after treatment. For most bispecific antibodies, gD-ZNRF3 dimerization resulted in IGF1R degradation equivalent to the reduction in protein expression observed in conditional KOs made with CRISPr Cas9 and sgRNA specific for IGF1R (Figure 4B). The phenotypic consequences of ligase-mediated degradation of cell surface receptors were further evaluated using HT55 colon cancer cells. These cells are dependent on IGF1R for survival both in vitro (Figure 4C) and in vivo (Figure 4D). Consistent with the viability defects observed following gene-mediated KO of IGF-1R, HT55 cells treated for 7 days with bispecific antibodies targeting IGF-1R and gD showed a reduction in clonal expansion that was dependent on ligase expression (Figure 4E, 4F).

[0193] Example 12: Kinetics of clearance from the cell surface Bispecific antibodies were generated with variable regions targeting IGF-1R (cizutumumab) and RNF43 (hSC37.39, US20170073430A1) and assessed for their ability to degrade IGF-1R in HT-29 cells with doxycycline-induced RNF43 overexpression by flow cytometry (Figure 5D). One hour after antibody administration, cells treated with higher levels of bispecific antibody (0.1ug / ml or higher) showed slightly higher levels of cell surface IGF1R. By 3 hours, this trend was reversed with a decrease in cell surface IGF1R, and by 8 hours, this trend was saturated. Continuous treatment for up to 96 hours showed no further clearance of the target.

[0194] Example 13: Evaluation of RNF43 and ZNRF3 bispecific antibodies A subset of RNF43 and ZNRF3 antibodies discovered as described above were reformatted into bispecific antibodies targeting the respective ligases and IGF1R (cizutumumab) (i.e., "PROTABs"). The activity of these ligases was assessed by flow cytometry by their ability to drive cell surface clearance of IGF1R from HT29 cells upon doxycycline-inducible expression of the corresponding ligases (Figure 6A). Antibodies targeting the ligases with high monovalent affinity for their ligases (approximately greater than 1 nM) showed saturable levels of IGF1R clearance when paired with cizutumumab, whereas low affinity antibodies showed reduced activity (Figure 6B). Further evaluation of the ZNRF3-IGF-1R PROTABs was performed in SW1417 cells, which express high endogenous levels of ZNRF3 and RNF43. Flow cytometry revealed dose-dependent cell surface clearance of IGF-1R in these cells after treatment with ZNRF3-IGF-1R antibody (Figure 6A). Western blot confirmed cellular degradation of IGF-1R in SW1417 after treatment with RNF43-IGF-1R antibody (Figure 6C).

[0195] Without being bound by theory, it is hypothesized that the correspondence between ligase affinity and degradation efficiency is due to the durability of the ternary complex between IGF1R, antibody, and ligase. Biochemical estimation suggested that at least four ubiquitins must be transferred to the target to allow efficient degradation, and thus a long-lived complex is required to allow multiple catalytic cycles. The durability of the entire complex was defined by the binding affinity of both sides of the bispecific antibody. For ligase Abs much stronger than 1 nM, the affinity of cizutumumab (~5 nM) is the major determinant in the half-life of the complex and therefore the efficiency of degradation is saturated. For ligase Abs weaker than 1 nM, we observed a decrease in clearance corresponding to the affinity of the ligase arms. Interestingly, no strong epitope dependency was observed with ligase antibodies, so for RNF43 and ZNRF3, the specific geometry of the interaction did not strongly affect the degradation efficiency. This is potentially different from what we saw with the HER2 antibody paired with the anti-gD Tag antibody (Figures 2A-2B), where changes in the epitope of the HER2-targeting antibody appear to affect degradation efficiency. Again, without being bound by theory, we hypothesize that this is due to the relative fine discrimination of epitopes on a single extracellular protease-associated domain of the ligase versus the overall change in epitopes targeting different domains of HER2. Thus, for bispecific ligase antibodies with other (non-cizutumumab) arms, the affinity at which this saturation occurs may change corresponding to the affinity of that arm.

[0196] Example 14: Effect of bivalent binding on target degradation To examine the impact of receptor clustering on target clearance, antibodies targeting two copies of anti-RNF43 or anti-IGF1R were generated in a 2+1 Fab-IgG format (Figures 7A-7B). The activity of these antibodies was assessed by flow cytometry by their ability to drive cell surface clearance of IGF1R from HT29 cells upon doxycycline-induced expression of the corresponding ligase (Figures 7C-7F). Antibodies binding two copies of IGF1R and one copy of RNF43 saturated clearance at levels similar to the corresponding conventional knobs-in-hole (1+1) bispecific antibodies (Figures 7C and 7E). Antibodies binding two copies of the ligase increased clearance compared to the corresponding conventional knobs-in-hole (1+1) bispecific antibodies (Figures 7D and 7F). These antibodies also reduced the levels of cell surface RNF43, likely due to autoubiquitination in trans (data not shown).

[0197] Example 15: Effect of bond distance on target degradation We hypothesized that the distance between the ligase and the target may affect the efficiency of target clearance. To investigate this phenomenon, we generated bispecific antibodies in one-arm FabIgG (OA-FabIgG) and one-arm FvIgG (OA-FvIgG) formats (Figure 8A). The activity of these antibodies was evaluated by flow cytometry by their ability to drive cell surface clearance of IGF1R from HT29 cells upon doxycycline-induced expression of the corresponding ligase (Figures 8B-8C). Both the OA-FvIgG and OA-FvIgG formats increased degradation compared to the corresponding 1+1 bispecific antibodies. In the case of the Fab-IgG format, similar results were seen with both cizutumumab and istiratumab as anti-IGF1R antibodies. Furthermore, the results shown in Figure 8 suggest that degradation efficiency can be fine-tuned or optimized by varying the position of each antigen-binding domain, for example, by placing the ligase antigen-binding domain either in an external position (Fv in OA-FvIgG or Fab in OA-FabIgG) or in an internal position.

[0198] Example 16: Combination of bond valence and minimum distance The Fab of cizutumumab was placed in an internal position to generate 2+1 Fab-IgG and FvIgG antibodies containing 2x RNF37.39 and 1x cizutumumab. Evaluation of these antibodies in the cell surface clearance assay described in Example 5 above found that they cleared IGF1R more efficiently than the corresponding conventional knobs-in-holes (1+1) bispecific antibodies. This data was further validated using DLD1 to examine total IGF1R levels using Western blot analysis (Figure 24).

[0199] Example 17: Cell surface ligase-mediated clearance of EGFR Multispecific antibodies were generated that target EGFR (using cetuximab or D1.5 variable domains) and RNF43 or unrelated antigens. The activity of these antibodies was evaluated by flow cytometry by their ability to drive cell surface clearance of EGFR from HT29 cells due to the expression of the corresponding ligase induced by doxycycline (Figure 9). Both 1+1 bispecific antibodies targeting both ligase and EGFR resulted in efficient clearance. Consistent with the results described in Example 14, 2+1 FabIgG targeting 2xRNF43 and 1xEGFR resulted in more complete clearance.

[0200] Example 18: Identification and characterization of novel cell surface ligases To identify putative cell surface ligases, the domain structure of RNF43 and ZNRF3 was evaluated (Figure 10A). This revealed the presence of an N-terminal signal peptide (SP) associated with incorporation into the membrane. Therefore, the list of known E3 ubiquitin ligases was evaluated using both UNIPROT and Signal-P software to identify ligases with SP. Among the identified proteins, we evaluated the following 15 ligases, including both single-pass and multiple-pass transmembrane domain ligases: RNF13, RNF43, RNF128, RNF130 (with two transmembrane domains, Figure 10B), RNF133, RNF148, RNF149, RNF150, RNF167, ZNRF3, ZNRF4, LYNX1, RSPRY1, SYVN1 (with five transmembrane domains, Figure 10B) and TRIM7.

[0201] A doxycycline-inducible pBind plasmid containing IRES-eGFP encoding all 15 ligases with an N-terminal gD tag was generated (Figure 10B). Cell surface display of each ligase was assessed using FACS analysis. Briefly, HEK293T cells were transiently transfected with the respective ligase plasmid. After 24 hours, cells were treated with 1ug / ml doxycycline for an additional 24 hours to induce ligase expression. Live cells were then prepared for FACS analysis by staining using anti-gD primary antibody followed by APC secondary antibody. To assess doxycycline induction, IRES-eGFP signal was examined (Figure 10C). APC median fluorescence intensity (MFI) was also quantified from three independent experiments for each sample as outlined (Figure 10D). As assay controls, parental and mock-transfected cells were also evaluated, as well as HEK293T cells constitutively expressing gD-tagged Fzd8, a known cell surface protein.

[0202] In parallel with the FACS analysis, the degradation ability of each putative cell surface ligase was also evaluated using HEK293T transfected cells. After 24 hours of doxycycline induction, cells were treated with 10 ig / ml of gD+HER2(7C2) bispecific antibody for 24 hours. Cells were then lysed and prepared for Western blot analysis to examine the total levels of HER2 upon bispecific antibody treatment. (Figure 10E and Figure 10F.) Evaluation of these cell surface ligases in a standard cell degradation assay reveals that numerous cell surface ligases can dimerize to HER2, resulting in efficient degradation of HER2.

[0203] Additional experiments were performed as follows. Figure 10D shows that RNF128, RNF130, RNF133, RNF149 and RNF150 showed detectable cell surface expression, whereas expression of RNF13, RNF148 and RNF167 was substantially lower. To assess whether cell surface expression broadly enabled degradation of non-natural targets, we used gD * Cells were treated with IGF1R PROTAB and IGF1R levels were monitored. Western blot analysis confirmed that co-localization of a validated cell surface E3 ubiquitin ligase to IGF1R drives degradation, as shown in Figure 32. Conversely, ligases with undetectable cell surface expression by flow cytometry were unable to induce target degradation, as shown in Figure 33. We also generated bispecific antibodies targeting gD and either HER2 or programmed death ligand 1 (PD-L1), two therapeutically relevant cancer targets. Similar to IGF1R, degradation of HER2 and PD-L1 occurred upon association of various ligases, as shown in Figures 34 and 35, but no degradation was detected when ligases with undetectable cell surface expression were used, as shown in Figures 36 and 37.

[0204] Figure 31 shows that some of the newly identified cell surface E3 ligases showed distinct tissue expression patterns, providing the possibility of tissue-specific degradation based on the selection of a particular E3 ligase. For example, RNF130, RNF149 and RNF167 showed clear upregulation in the hematopoietic compartment, and these ligases could be used to target cells of hematopoietic origin, whereas the expression of RNF133 and RNF148 was mainly restricted to the testis, and these ligases could be used to target testis cells.

[0205] The catalytic activity of E3 ubiquitin ligases is thought to be essential to facilitate the final transfer of ubiquitin from the ubiquitin-conjugating enzyme (E2) to the substrate, altering its function. To test the importance of catalytic activity, catalytically active and inactive versions of each individual ligase can be used in a degradation assay to assess whether catalytic activity is necessary for each ligase to mediate the degradation of cell surface receptors. Preliminary data suggests that certain ligases may require catalytic activity, whereas certain others may not. Separate cell lines were used to verify cell surface expression of the newly identified ligases. Each individual ligase was transfected into HT29 cells that were selected with puromycin for stable integration. Stable lines were treated with 1ug / ml doxycycline for 24 hours to induce expression of the ligases. Live cells were then prepared for FACS analysis by staining using anti-gD primary antibody followed by APC secondary antibody. The percentage of APC positive cells was also quantified from three independent experiments for each sample as outlined (FIG. 27A, B).

[0206] Example 19: Cell surface clearance of various substrates Bispecific antibodies are generated that target various structurally distinct cell surface proteins, including the gD epitope and exemplary receptor tyrosine kinases HER2, IGF1R and EGFR, as well as multiple-spanning transmembrane domain proteins, such as FZLD5, proteins with large extracellular and intracellular domains, such as EpCAM, and proteins with immunoglobulin domains that contain short intracellular domains, such as PD-L1. 10ug / ml of antibodies are added to cells overexpressing ligase tagged with an N-terminal gD tag. Western blot analysis reveals that the addition of bispecific antibodies drives degradation of the target antigen. An example using HT29 cells stably expressing various cell surface ligases is shown in Figure 29. Dimerization of some ligases to IGF1R using gD / IGF1R antibodies results in significant receptor degradation.

[0207] Example 20: Evaluation of Wnt signaling pathway activation by TOPBrite TCF reporter assay The Wnt-dependent reporter Nano-Glo Dual® Luciferase System (Promega), containing a control promoter expressing Renilla luciferase and a TCF promoter expressing firefly luciferase, was used to evaluate activation of Wnt / β-Catenine by rat or rabbit multispecific antibodies targeting ZNRF3. HEK 293 cells transiently transfected with plasmids containing the control and TCF promoters were plated at 100,000 cells / well in Corning® 96-well Flat Clear Bottom White Polystyrene TC-treated microplates and incubated overnight in RPMI medium containing 10% FBS, 1% penicillin-streptomycin, 1×L-glutamine and 40 μg / mL hygromycin B (Thermo). The next day, 0.1 mg / mL rat or rabbit clone ZNRF3 antibody and 100 ng / mL recombinant human Wnt3a (Abcam) were added to the cells. 2uM GSK 3β inhibitor or 500, 10 and 0.2ng / μL RSPO3 in the presence of 100ng / mL Wnt3a were used as positive controls. DMSO or 100ng / mL Wnt3a treated cells served as negative controls. After 24 hours of incubation, the cell medium was replaced with fresh medium and an equal volume of Dual-Glo® reagent was added to each well followed by gentle mixing. After 10 minutes of incubation, the firefly luminescence signal was measured using a GloMax® Discover Microplate Reader with an integration time of 0.3 seconds (Promega). Then, an equal volume of Dual-Glo Stop&Glo® reagent was added to each well and Renilla luminescence signal was measured after 10 minutes of incubation using the same settings. Relative ratios were calculated from the ratios of firefly and Renilla luminescence and then further normalized by taking the ratio of untreated samples. The results for the ZNRF3 antibody are shown in Figures 11A to 11B.

[0208] Example 21: Assessment of IGF1R cell surface clearance by NanoLuc® luciferase complementation assay A HiBiT tag was introduced at the N-terminus of IGF1R by the CRISPR-Cas9 system on HT29 cells. A single clone with the highest NanoLuc® luciferase luminescence readout was selected and verified by Sanger sequencing. Cells were plated at 100,000 cells / well in Corning® 96-well Flat Clear Bottom White Polystyrene TC-treated microplates and incubated in ATCC recommended medium in the presence of various concentrations of antibodies. After 24 hours (or for the indicated period), cells were washed once with 100 μL of PBS and then replenished with 100 μL of fresh medium. Detection reagent was prepared by diluting LgBiT protein at a ratio of 1:100 and substrate at a ratio of 1:50 in the desired volume of detection buffer provided in the detection kit. Then, 100 μL of detection reagent was added to the cells in 100 μL of fresh medium. For the Nano-Glo® HiBiT Extracellular Detection System, which detects surface levels of IGF1R, detection was performed by incubating for 10 minutes at room temperature with gentle mixing using a plate shaker. % Clearance = (Untreated Sample RLU - Treated Sample RLU) / Untreated Sample RLU * Percent IGF1R clearance was calculated using the formula: 1 / 100. RNF43 and ZNRF3 multispecific antibodies were also evaluated in this cell line (Tables 5-7). A subset of samples was performed in two separate runs, in which case both results are included. [Table 5] TIFF2024509695000010.tif252170TIFF2024509695000011.tif42170 [Table 6] [Table 7]

[0209] Tables 5-7 show the percent surface IGF1R-HibiT clearance. Table 5 shows that the rat Cixu / RNF43 bispecific antibody induced cell surface IGF1R-HibiT clearance in HT29 at a concentration of 1 μg / mL. Table 6 shows that the rat Cixu / ZNRF3 bispecific antibody induced cell surface IGF1R-HibiT clearance in HT29 at a concentration of 1 μg / mL. Table 7 shows that the novel format antibodies, including Fv-IgG and 2+1 trispecific formats, induced cell surface IGF1R-HibiT clearance in HT29 at a concentration of 1 μg / mL.

[0210] Example 22: Monitoring IGF1R surface clearance kinetics using the NanoLuc® luciferase complementation assay HibiT tagged IGF1R HT29 cells were seeded overnight at 100,000 cells / well in Corning® 96-well Flat Clear Bottom White Polystyrene TC-treated microplates and incubated in ATCC recommended medium. On day 2, 1 μg / mL or 10 μg / mL of bispecific and new format antibodies were added to the cells and incubated for various time points: 0, 4, 8, 24 hours. The luminescence signal of IGF1R-HibiT was detected using the HibiT extracellular system as described in the previous examples. Percent IGF1R was calculated using the formula %IGF1R=(1-(RLU of untreated sample-RLU of treated sample) / RLU of untreated sample). * 100. Evaluation of various multispecific antibodies showed different degradation rates (FIGS. 12A-12F).

[0211] Example 23: Multiplex NanoLuc luciferase complementation assay with cell viability assay HiBiT-tagged IGF1R HT29 cells were seeded overnight at 100,000 cells / well in Corning® 96-well Flat Clear Bottom White Polystyrene TC-treated microplates and incubated in ATCC recommended medium. On day 2, 1 μg / mL of bispecific antibody was added to the cells and incubated for 24 hours. Cell media containing lactate dehydrogenase was collected for LDH cytotoxicity assay (Promega) followed by addition of HiBiT extracellular detection reagent. Approximately 2-5 μL of the media was diluted 300-fold with LDH storage buffer and then mixed with an equal volume of LDH detection reagent. LDH lysis reagent was added to control samples to maximize LDH detection. The reactions were incubated at room temperature for 30-60 minutes and luminescence signals were measured using a GloMax® Discover Microplate Reader with 1 second integration time. % Cytotoxicity was calculated using the following formula: % Cytotoxicity = 100 × ((experimental LDH release - medium background) / (maximum LDH release control - medium background)). After HiBiT extracellular detection, cells were washed twice with PBS and replenished with fresh medium. CellTiter-glo® reagent (Promega) was then added to the cells in equal volumes and incubated for 10-30 min, followed by luminescence detection with a 1 s integration time (Promega) using a GloMax® Discover Microplate Reader. Different multispecific antibodies showed variable effects on target degradation, but no effects were seen on cell death measured by LDH assay or cell number measured by Cell Titer-go (Figures 13A-13C).

[0212] Example 24: Assessment of IGF1R Degradation Using the NanoLuc® Luciferase Complementation Assay HibiT tagged IGF1R HT29 cells were seeded overnight at 100,000 cells / well and then treated with various bispecific and novel format antibodies at 1 μg / mL. After 24 hours of incubation, the cells were incubated with HibiT lytic detection reagent to determine the total amount of IGF1R-HibiT. The lytic detection reagent was prepared by diluting LgBiT protein at a ratio of 1:100 and substrate at a ratio of 1:50 in the desired volume of detection buffer provided in the detection kit. After 20 minutes of incubation with the lytic detection reagent, the luminescence signal was measured using a GloMax® Discover Microplate Reader with an integration time of 1 second. The percent IGF1R was calculated using the formula described in the previous example (FIG. 14A). Western blot was also used to measure the total amount of IGF1R after 24 hours of antibody treatment. HT29IGF1R-HiBiT tagged cells and wild type cells (WT) were treated with various antibodies as indicated (1. Cixu-RNF43 Fv-Ig, 2. Cizutumumab / RNF43.hSC37.39, 3. Cixu / NIST, 4. Cixu / Cixu) at 1 μg / mL for 24 h. Untreated cells (5. UT) were used as control. 20 μg of total protein is loaded in each lane and target proteins are probed with anti-IGF1 receptor antibody (Abcam EPR19322) that recognizes both pro-IGF1R (~200 kDa) and IGF1R (~95 kDa). β-actin is shown as a protein loading control (Figure 14B). Western blot analysis indicates that the HiBit line has predominantly intracellular pro-IGF1R that does not undergo degradation, with limited degradation in the soluble HiBit assay. Extracellular mature IGF1R is more abundant in WT HT-29 cells and is efficiently degraded by the ligase bispecific antibody as shown by Western blot.

[0213] Example 25: Trispecific antibodies for enhanced degradation of IGF1R and EGFR Preliminary data showed that the combination of two conventional knobs-in-holes bispecific antibodies (RNF43 / Cixu and ZNRF3 / Cixu bispecific antibodies) resulted in increased clearance. For example, for ease of manufacturing and administration to subjects, it may be advantageous to create a single molecule with all the desired antigen binding domains, so a 2+1 FabIgG format can be used. Figures 15D-15G show an exemplary trispecific antibody degrader using a 2+1 FabIgG format. Trispecific antibodies targeting RNF43 (RNF43.37.39), ZNRF3 (ZNRF3-6) and IGF1R (cizutumumab) or EGFR (cetuximab) are created. The antibody format is a Fab-IgG / IgG with the receptor tyrosine kinase antibody placed at the internal position and the ligase antibody alternating at the two external positions. This arrangement may optimize the proximity to each anti-ligase antigen binding domain, further improving degradation efficiency. The Fab-IgG half antibody incorporates an LC pairing mutation (Dillon et al. mAbs, 9:2, 213-230, 2017) that allows efficient pairing of the LC arm. The FabIgG and IgG half antibodies are assembled using methods well known to those skilled in the art. Evaluation of these antibodies in the cell surface clearance assay described in Example 5 shows that they clear IGF1R more efficiently than either of the corresponding 1+1 bispecific antibodies in cells with substantial expression of each.

[0214] Example 26: Monitoring IGF1R surface clearance following bispecific antibody combinations. The activity of multiple bispecific antibodies showed saturation with less than 100% clearance for HER2, EGFR and IGF1R. We hypothesized that by simultaneously addressing one target with two different ligases, we might be able to improve clearance due to different rate-limiting steps. To examine this hypothesis, HibiT-tagged IGF1R HT29 cells were seeded overnight at 100,000 cells / well in Corning® 96-well Flat Clear Bottom White Polystyrene TC-treated microplates and incubated in ATCC recommended medium. On the second day, 1 μg / mL of a single bispecific antibody (targeting IGF1R and ZNRF3 or RNF43) or a combination of two bispecific antibodies (the first one targeting IGF1R and RNF43, the second one targeting IGF1R and ZNRF3) was added to the cells and incubated for 24 hours. The luminescence signal of IGF1R-HibiT was detected using the HibiT extracellular system according to the description in the previous example. Percent IGF1R was calculated according to the formula: %IGF1R=(1-(RLU of untreated sample / RLU of treated sample) / RLU of untreated sample). * 100. Two different combinations of bispecific antibodies resulted in more efficient clearance of IGF1R than the saturating levels of either individual bispecific antibody (FIG. 16).

[0215] Example 27: Degradation of IGF1R across multiple CRC lines A subset of RNF43 and ZNRF3 antibodies discovered as described above were reformatted into bispecific antibodies targeting the respective ligases and IGF1R (cizutumumab). The activity of these ligases was assessed by their ability to drive whole-cell degradation of IGF1R in DLD1 cells and a large panel of colon cancer cell lines (Figure 18A, B). Of note, KM-12, RKO, GP2D and JHH7 cells did not show any appreciable ligase expression and, as a result, served as negative controls (Figure 18B).

[0216] Example 28: Assessment of target ubiquitination upon bivalent / bispecific antibody treatment To evaluate the impact of bispecific antibody treatment on target (IGF1R) ubiquitination, parental HEK293T cells or HEK293T cells harboring doxycycline-inducible constructs of ZNRF3 wild type (WT) or delta RING mutant (ΔRING) were subjected to TUBE 2 pulldown and samples were evaluated by Western blot analysis. Briefly, 10 million cells were plated in 10 cm dishes in the presence of doxycycline (1 μg / ml or 31.25 ng / ml) to induce expression of ZNRF3 WT or ZNRF3Δ RING tagged with gD at the N-terminus and FLAG at the C-terminus, respectively. After 24 h of doxycycline induction, cells were subjected to a medium change (+ doxycycline) and treated with gDxcizutumumab (IGF1R) bispecific antibody (0.5 μg / ml). After treatment with bispecific antibodies for 2 h at 37°C, cells were lysed in 500 μl of GST lysis buffer [25 mM Tris·HCl, pH 7.2, 150 mM NaCl, 5 mM MgCl2, 1% NP-40 and 5% glycerol, 1% Halt protease & phosphatase inhibitor cocktail, 50 μM PR-619, 5 mM 1,10 phenanthroline, 10 μM MG132]. The cleared lysates were aliquoted and incubated with either 20 μl / sample prewashed Pierce HA epitope tag antibody agarose conjugate (2-2.2.14) (Thermo Scientific; REF 26182) for nonspecific pull-down assessment, or 20 μl / sample prewashed agarose-TUBE 2 beads (Life Sensors; REF UM402) to enrich for polyubiquitinated proteins. The lysate-bead mixture was incubated for 2 hours and 30 minutes at 4° C. For the input sample, 30 μl was used from the prepared pre-incubated pull-down sample. After incubation, the beads were washed and prepared for Western blot analysis (FIG. 19B).

[0217] Furthermore, IGF1R ubiquitination after IGF1 stimulation (R&D systems; REF 291-G1), bivalent antibody treatment (cizutumumab x cizutumumab), control bispecific antibody treatment (cizutumumab x NIST) or ligase-based bispecific antibody treatment (cizutumumab x RNF43-35; cizutumumab x ZNRF3-55) was evaluated in HT29 cells. Briefly, 20 million cells were plated in 15 cm dishes. 48 h after plating, cells were subjected to serum starvation. After 22 h of serum starvation, cells were subjected to bivalent or bispecific antibody treatment (1 μg / ml) simultaneously with medium change (serum starvation medium). Cells were incubated at 37 °C for 2 h 15 min. For IGF1 stimulation, cells were treated with 50 ng / ml IGF1 5 min before cell lysis. After incubation, cells were scraped into 800 μl of PBS and the cell pellet was resuspended in 1.2 ml of GST lysis buffer [25 mM Tris·HCl, pH 7.2, 150 mM NaCl, 5 mM MgCl2, 1% NP-40 and 5% glycerol, 1% Halt protease and phosphatase inhibitor cocktail, 10 mM NEM, 1 mM PMSF]. The cleared lysates were aliquoted and incubated with 25 μl / sample of pre-washed Dynabeads Protein G (ThermoFisher Scientific; REF 10004D) conjugated to either rabbit (DA1E) mAb IgG XP isotype control (cell signaling; REF 3900) for non-specific bait protein precipitation or IGF-1 receptor beta (D23H3) XP rabbit mAb (cell signaling; REF 9750) for IGF1Rβ immunoprecipitation at 1.87 μg / sample. The lysate-bead mixture was incubated for 24 hours at 4°C. For the input sample, 60 μl was used from the prepared pre-incubated pull-down sample. After incubation, the beads were washed and prepared for Western blot analysis (Figure 19A).

[0218] Example 29: Validation of ligase activity requirements across cell lines and targets To support the assertion made above for HER2 in Example 10, we explored whether ligase activity is also required for the degradation of additional targets. As demonstrated in Figure 20, deletion of the RING domain from ZNRF3 prevented IGF1R degradation after ligase dimerization following treatment with various ZNRF3 / IGF1R bispecific antibodies. Furthermore, we took advantage of naturally occurring RNF43 mutations in a small subset of colon cancers. We chose SW48 because it has a frameshift deletion within the RING domain of RNF43 (Figure 21A) but maintains cell surface ligase expression (Figure 21B). Dimerization of RNF43 / IGF1R bispecific antibodies in this cell line did not affect IGF1R levels, demonstrating that the RING and c-terminus of RNF43 are required for ligase activity. Notably, when ZNRF3 / IGF1R antibody was used, ZNRF3 was still competent for IGF1R degradation. To further support this assertion, we made endogenous ligase knockouts for both RNF43 or ZNRF3 in HT29 cells (Figure 22A). As expected, RNF43 / IGF1R treatment of RNF43 KO did not affect IGF1R levels, but ZNRF3 / IGF1R treatment was still able to degrade the receptor. Importantly, RING knockout did not affect either ligase cell surface expression (Figure 22A FACS panel), but also rescued degradation, demonstrating that the presence of RING / C-terminus is necessary for the full degradation ability of either ligase (Figure 22B).

[0219] Interestingly, we found that this was dependent on both lysosomal and proteasomal activity towards IGF1R, as inhibition of E1 ubiquitin-activating enzyme also rescued the degradation (Figure 23). This differs from what was observed for HER2, where only lysosomal activity was required for the degradation of its receptor. Thus, this data suggests that the degradation pathway is defined by the target, rather than the ligase itself.

[0220] Example 30: Biological consequences of degradation on cell signaling and proliferation The biological impact of degradation on receptor binding was assessed by focusing on the modulation of IGF1R signaling. SW48 treated with ZNRF3 / IGF1R bispecific antibody exhibited significant IGF1R degradation. Importantly, this degradation also affected further downstream signaling as revealed by the complete loss of AKT activity (phosphorylation of AKT) and reduced phosphorylation of S6, two important downstream effectors of IGF1R (Figure 25A). Importantly, the reduction in AKT and S6 activity was superior in ligase-treated cells compared to bivalent IGF1R, demonstrating that degradation results in greater pathway inhibition than classical blocking antibodies. This reduction in IGF1R signaling was also functionally transformed as evidenced by the reduction in cell proliferation in SW48 cells treated with ZNRF3 / IGF1R (Figure 25B, C). Consistent with the above, ZNRF3 RING-deficient HT29 had no effect on cell proliferation, whereas WT ZNRF3 dimerized to IGF1R led to slower proliferation in these cells, so the ligase domain was required to affect cell proliferation (Figure 26A-D).

[0221] Example 31: Endogenous Degradation of Additional Substrates Bispecific antibodies are generated that target the PD-L1 receptor and the exemplary ligase antibodies described above. As shown in Figure 27, treatment of SW48 cells with ZNRF3 / PDL1 revealed significant and nearly complete degradation of PD-L1, demonstrating the breadth of degradation across multiple substrates.

[0222] The foregoing invention has been described in some detail by way of illustration and illustration, for clarity of understanding, but these illustrations and illustrations should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entireties.

[0223] Example 32. In vivo evaluation An attractive advantage of PROTAB technology is that it may overcome challenges shared by small molecule intracellular degraders (e.g., limited bioavailability and cell permeability) that can limit in vivo activity.

[0224] Approximately 10 6 SW48 cells were resuspended in PBS basal medium, mixed with 50% Matrigel (Corning) to a final volume of 200 μl, and injected subcutaneously into the left flank of NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ (NSG) (colony 005557). Mice were purchased from Jackson Laboratory. Female mice aged 6–12 weeks were used for the experiment. Tumor dimensions were measured using a vernier caliper, and tumor volume was calculated as 0.523 × length × width × width. Animals were humanely euthanized according to the following criteria: persistent clinical signs of distress or pain, significant weight loss (>20%), tumor volume exceeding 2,500 mm 3 The maximum tumor size permitted by the Institutional Animal Care and Use Committee (IACUC) is 3,000 mm. 3 This limit was not exceeded in any experiment. 3 Once tumor size reached 100%, animals were randomized to receive a single intraperitoneal injection of PROTAB. All mice were euthanized 72 hours after injection and tumors were harvested for further processing.

[0225] The intraluminal implantation technique has been described previously (de Sousa E Melo F et al. Modeling Colorectal Cancer Progression Through Orthotopic Implantation of Organoids. Methods Mol Biol 2171, 331-346 (2020)). Briefly, mice were anesthetized by isoflurane inhalation and injected intraperitoneally (i.p.) with 0.05–0.1 mg / kg buprenorphine. A blunt-ended hemostat (Micro-Mosquito, No. 13010-12, Fine Science Tools) was inserted approximately 1 cm into the anus. The hemostat was angled toward the mucosa, opened slightly, and a single mucosal fold was grasped by closing the hemostat up to the first notch. The hemostat was retracted from the anus to expose the grasped mucosa. 10 μL of a solution containing 50,000 cells mixed with 50% Matrigel (Corning) in PBS was injected directly into the colonic mucosa. After reversing the prolapse, the hemostat was released.

[0226] In vivo activity was evaluated in SW48 tumor-bearing mice (Figure 30). The top part of Figure 30 shows the in vivo activity of cixutuzumab (anti-IGF1R) based bivalent antibody, NIST * IGF1R(Cixu) bispecific antibody or ZNRF3 as described * Shows a schematic of the SW48 xenograft model used to analyze the effect of IGF1R(Cixu) bispecific PROTAB. Three weeks after SW48 implantation, animals were subjected to antibody treatment. Tumors were harvested, homogenized, and biochemically analyzed 72 hours after antibody treatment.

[0227] The lower part of Figure 30 shows mice that were left untreated (-) or treated with Cixu bivalent antibody, NIST * IGF1R(Cixu) or ZNRF3-55 *Western blot analysis of SW48 in vivo tumor lysates from mice subjected to IGF1R(Cixu) bispecific PROTAB for 72 hours. Data are representative of 4 animals per treatment group. Endogenous IGF1Rβ levels were detected. GAPDH was used as a loading control.

[0228] The results demonstrate that a single dose of ZNRF3-based IGF1R PROTAB drove substantial degradation of IGF1R. Interestingly, IGF1R degradation was partially inhibited at the highest dose (Figure 30), suggesting a hook effect that has been reported for some PROTACs (Kannt A et al. Expanding the arsenal of E3 ubiquitin ligases for proximity-induced protein degradation. Cell Chem Biol 28, 1014-1031. (2021); Khan, S et al. PROteolysis TArgeting Chimeras (PROTACs) as emerging anticancer therapeutics. Oncogene 39, 4909-4924 (2020)).

[0229] IV. Sequence The table below provides exemplary sequences referenced in this disclosure. [table] TIFF2024509695000014.tif253170TIFF2024509695000015.tif254170TIFF2024509695000016.tif253170TIFF2024509695000017.tif254170TIFF2024509695000018.tif254170TIFF2024509695000019.tif254170TIFF2024509695000020.tif253170TIFF2024509695000021.tif254170TIFF2024509695000022.tif253170TIFF2024509695000023.tif254170TIFF2024509695000024.tif253170TIFF2024509695000025.tif254170TIFF2024509695000026.tif254170TIFF2024509695000027.tif254170TIFF2024509695000028.tif254170TIFF2024509695000029.tif254170TIFF2024509695000030.tif254170TIFF2024509695000031.tif254170TIFF2024509695000032.tif254170TIFF2024509695000033.tif254170TIFF2024509695000034.tif254170TIFF2024509695000035.tif254170TIFF2024509695000036.tif254170TIFF2024509695000037.tif254170TIFF2024509695000038.tif254170TIFF2024509695000039.tif254170TIFF2024509695000040.tif254170TIFF2024509695000041.tif254170TIFF2024509695000042.tif254170TIFF2024509695000043.tif254170TIFF2024509695000044.tif254170TIFF2024509695000045.tif254170TIFF2024509695000046.tif254170TIFF2024509695000047.tif254170TIFF2024509695000048.tif254170TIFF2024509695000049.tif254170TIFF2024509695000050.tif254170TIFF2024509695000051.tif254170TIFF2024509695000052.tif254170TIFF2024509695000053.tif254170TIFF2024509695000054.tif254170TIFF2024509695000055.tif254170TIFF2024509695000056.tif254170TIFF2024509695000057.tif254170TIFF2024509695000058.tif254170TIFF2024509695000059.tif254170TIFF2024509695000060.tif254170TIFF2024509695000061.tif254170TIFF2024509695000062.tif254170TIFF2024509695000063.tif254170TIFF2024509695000064.tif254170TIFF2024509695000065.tif254170TIFF2024509695000066.tif254170TIFF2024509695000067.tif254170TIFF2024509695000068.tif254170TIFF2024509695000069.tif254170TIFF2024509695000070.tif254170TIFF2024509695000071.tif254170TIFF2024509695000072.tif254170TIFF2024509695000073.tif254170TIFF2024509695000074.tif254170TIFF2024509695000075.tif254170TIFF2024509695000076.tif254170TIFF2024509695000077.tif254170TIFF2024509695000078.tif254170TIFF2024509695000079.tif254170TIFF2024509695000080.tif254170TIFF2024509695000081.tif254170TIFF2024509695000082.tif254170TIFF2024509695000083.tif254170TIFF2024509695000084.tif254170TIFF2024509695000085.tif255170TIFF2024509695000086.tif255170TIFF2024509695000087.tif255170TIFF2024509695000088.tif255170TIFF2024509695000089.tif254170TIFF2024509695000090.tif254170TIFF2024509695000091.tif254170TIFF2024509695000092.tif254170TIFF2024509695000093.tif254170TIFF2024509695000094.tif84170.

Claims

1. A multispecific antibody that binds to at least a first cell surface target protein and a second cell surface protein, wherein the first cell surface target protein is RNF43 or ZNR3, and the multispecific antibody has a binding affinity to RNF43 or ZNR3 of less than 1 nM.

2. 2. The multispecific antibody of claim 1, which is a bispecific or trispecific antibody, optionally a 1+1 FabIgG, 1+1 FvIgG, 2+1 FvIgG, 2+1 FabIgG, 1-arm FvIgG or 1-arm FabIgG.

3. The multispecific antibody of claim 1 or 2, wherein the multispecific antibody comprises an IgG Fc region comprising at least one knob-into-hole modification.

4. The multispecific antibody of any one of claims 1 to 3, wherein the multispecific antibody binds to RNF43, ZNR3, or both RNF43 and ZNR3, and to a second cell surface protein, and optionally the multispecific antibody does not block binding between RNF43 and / or ZNR3 and FZD and / or LRP6.

5. The multispecific antibody is selected from the group consisting of antibodies RNF43-107 (VH SEQ ID NO:43, VL SEQ ID NO:44), RNF43-130 (VH SEQ ID NO:75, VL SEQ ID NO:76), RNF43-145 (VH SEQ ID NO:83, VL SEQ ID NO:84), RNF43-156 (VH SEQ ID NO:91, VL SEQ ID NO:92), RNF43-181 (VH SEQ ID NO:119, VL SEQ ID NO:120), RNF43-196 (VH SEQ ID NO:131, VL SEQ ID NO:132), RNF43-200 (VH SEQ ID NO:135, VL SEQ ID NO:136), RNF43-206 ( VH SEQ ID NO: 143, VL SEQ ID NO: 144), RNF43-224 (VH SEQ ID NO: 163, VL SEQ ID NO: 164), RNF43-31 (VH SEQ ID NO: 171, VL SEQ ID NO: 172), RNF43-35 (VH SEQ ID NO: 179, VL SEQ ID NO: 180), RNF43-41 (VH SEQ ID NO: 187, VL SEQ ID NO: 188), RNF43-61 (VH SEQ ID NO: 199, VL SEQ ID NO: 200), RNF43-67 (VH SEQ ID NO: 203, VL SEQ ID NO: 204), RNF43-71 (VH SEQ ID NO: 211, VL SEQ ID NO: 212 ), RNF43-74 (VH SEQ ID NO: 215, VL SEQ ID NO: 216), RNF43-80 (VH SEQ ID NO: 227, VL SEQ ID NO: 228), RNF43-90 (VH SEQ ID NO: 235, VL SEQ ID NO: 236), ZNRF3-101 (VH SEQ ID NO: 239, VL SEQ ID NO: 240), ZNRF3-117 (VH SEQ ID NO: 243, VL SEQ ID NO: 244), ZNRF3-163 (VH SEQ ID NO: 255, VL SEQ ID NO: 256), ZNRF3-17 (VH SEQ ID NO: 259, VL SEQ ID NO: 260), ZNRF3-172 (VH SEQ ID NO: 271, VL SEQ ID NO: 272), ZNRF3-179 (VH SEQ ID NO: 275, VL SEQ ID NO: 276), ZNRF3-182 (VH SEQ ID NO: 279, VL SEQ ID NO: 280), ZNRF3-195 (VH SEQ ID NO: 283, VL SEQ ID NO: 284), ZNRF3-223 (VH SEQ ID NO: 295, VL SEQ ID NO: 296), ZNRF3-231 (VH SEQ ID NO: 299, VL SEQ ID NO: 300), ZNRF3-247 (VH SEQ ID NO: 311, VL SEQ ID NO: 312), ZNRF3-253 (VH SEQ ID NO: 315, VL SEQ ID NO: 316),ZNRF3-254 (VH SEQ ID NO: 319, VL SEQ ID NO: 320), ZNRF3-255 (VH SEQ ID NO: 323, VL SEQ ID NO: 324), ZNRF3-265 (VH SEQ ID NO: 327, VL SEQ ID NO: 328), ZNRF3-269 (VH SEQ ID NO: 331, VL SEQ ID NO: 332), ZNRF3-270 (VH SEQ ID NO: 335, VL SEQ ID NO: 336), ZNRF3-275 (VH SEQ ID NO: 339, VL SEQ ID NO: 340), ZNRF3-287 (VH SEQ ID NO: 347, VL SEQ ID NO: 348), ZNRF3-296 (VH SEQ ID NO: 351, VL SEQ ID NO: 352), ZNRF3-30 (VH SEQ ID NO: 355, VL SEQ ID NO: 356), ZNRF3-300 (VH SEQ ID NO: 359, VL SEQ ID NO: 360), ZNRF3-301 (VH SEQ ID NO: 363, VL SEQ ID NO: 364), ZNRF3-312 (VH SEQ ID NO: 371, VL SEQ ID NO: 372), ZN ZNRF3-314 (VH SEQ ID NO: 375, VL SEQ ID NO: 376), ZNRF3-329 (VH SEQ ID NO: 283, VL SEQ ID NO: 284), ZNRF3-55 (VH SEQ ID NO: 391, VL SEQ ID NO: 392), ZNRF3-6 (VH SEQ ID NO: 395, VL SEQ ID NO: 396), ZNRF3-90 (VH SEQ ID NO: 399, VL SEQ ID NO: 400), RNF43-14 (VH SEQ ID NO: 425, VL SEQ ID NO: 426), RNF43-22 (a) a heavy chain variable domain (VH) comprising heavy chain complementarity determining region 1 (CDR-H1), CDR-H2 and CDR-H3, and (b) a light chain variable domain (VL) comprising light chain complementarity determining region 1 (CDR-L1), CDR-L2 and CDR-L3, of any one of ZNRF3-331 (VH SEQ ID NO: 429, VL SEQ ID NO: 430), or ZNRF3-331 (VH SEQ ID NO: 439, VL SEQ ID NO: 440), The multispecific antibody of any one of claims 1 to 4, wherein optionally the heavy chain CDRs and / or the light chain CDRs are Kabat, Chothia or McCallum CDRs.

6. The multispecific antibody is selected from the group consisting of RNF43-107 (VH SEQ ID NO: 43, VL SEQ ID NO: 44), RNF43-130 (VH SEQ ID NO: 75, VL SEQ ID NO: 76), RNF43-145 (VH SEQ ID NO: 83, VL SEQ ID NO: 84), RNF43-156 (VH SEQ ID NO: 91, VL SEQ ID NO: 92), RNF43-181 (VH SEQ ID NO: 119, VL SEQ ID NO: 120), RNF43-196 (VH SEQ ID NO: 131, VL SEQ ID NO: 132), RNF43-200 (VH SEQ ID NO: 135, VL SEQ ID NO: 136), RNF43-206 (VH SEQ ID NO: 137, VL SEQ ID NO: 138), RNF43-210 (VH SEQ ID NO: 139, VL SEQ ID NO: 140), RNF43-220 (VH SEQ ID NO: 141, VL SEQ ID NO: 142), RNF43-230 (VH SEQ ID NO: 143, VL SEQ ID NO: 144), RNF43-240 (VH SEQ ID NO: 145, VL SEQ ID NO: 146), RNF43-250 (VH SEQ ID NO: 147, VL SEQ ID NO: 148), RNF43-260 (VH SEQ ID NO: 149, VL SEQ ID NO: 200), RNF43-270 (VH SEQ ID NO: 280, VL SEQ ID NO: 281), RNF43-282 (VH SEQ H SEQ ID NO: 143, VL SEQ ID NO: 144), RNF43-224 (VH SEQ ID NO: 163, VL SEQ ID NO: 164), RNF43-31 (VH SEQ ID NO: 171, VL SEQ ID NO: 172), RNF43-35 (VH SEQ ID NO: 179, VL SEQ ID NO: 180), RNF43-41 (VH SEQ ID NO: 187, VL SEQ ID NO: 188), RNF43-61 (VH SEQ ID NO: 199, VL SEQ ID NO: 200), RNF43-67 (VH SEQ ID NO: 203, VL SEQ ID NO: 204), RNF43-71 (VH SEQ ID NO: 211, VL SEQ ID NO: 212) , RNF43-74 (VH SEQ ID NO: 215, VL SEQ ID NO: 216), RNF43-80 (VH SEQ ID NO: 227, VL SEQ ID NO: 228), RNF43-90 (VH SEQ ID NO: 235, VL SEQ ID NO: 236), ZNRF3-101 (VH SEQ ID NO: 239, VL SEQ ID NO: 240), ZNRF3-117 (VH SEQ ID NO: 243, VL SEQ ID NO: 244), ZNRF3-163 (VH SEQ ID NO: 255, VL SEQ ID NO: 256), ZNRF3-17 (VH SEQ ID NO: 259, VL SEQ ID NO: 260), ZNRF3-172 (VH SEQ ID NO: 261, VL SEQ ID NO: 262), 71, VL SEQ ID NO: 272), ZNRF3-179 (VH SEQ ID NO: 275, VL SEQ ID NO: 276), ZNRF3-182 (VH SEQ ID NO: 279, VL SEQ ID NO: 280), ZNRF3-195 (VH SEQ ID NO: 283, VL SEQ ID NO: 284), ZNRF3-223 (VH SEQ ID NO: 295, VL SEQ ID NO: 296), ZNRF3-231 (VH SEQ ID NO: 299, VL SEQ ID NO: 300), ZNRF3-247 (VH SEQ ID NO: 311, VL SEQ ID NO: 312), ZNRF3-253 (VH SEQ ID NO: 315, VL SEQ ID NO: 316),ZNRF3-254 (VH SEQ ID NO: 319, VL SEQ ID NO: 320), ZNRF3-255 (VH SEQ ID NO: 323, VL SEQ ID NO: 324), ZNRF3-265 (VH SEQ ID NO: 327, VL SEQ ID NO: 328), ZNRF3-269 (VH SEQ ID NO: 331, VL SEQ ID NO: 332), ZNRF3-270 (VH SEQ ID NO: 335, VL SEQ ID NO: 336), ZNRF3-275 (VH SEQ ID NO: 339, VL SEQ ID NO: 340), ZNRF3-2 87 (VH SEQ ID NO: 347, VL SEQ ID NO: 348), ZNRF3-296 (VH SEQ ID NO: 351, VL SEQ ID NO: 352), ZNRF3-30 (VH SEQ ID NO: 355, VL SEQ ID NO: 356), ZNRF3-300 (VH SEQ ID NO: 359, VL SEQ ID NO: 360), ZNRF3-301 (VH SEQ ID NO: 363, VL SEQ ID NO: 364), ZNRF3-312 (VH SEQ ID NO: 371, VL SEQ ID NO: 372), ZNRF3-314 (VH SEQ ID NO: 373, VL SEQ ID NO: 374), : 375, VL SEQ ID NO: 376), ZNRF3-329 (VH SEQ ID NO: 283, VL SEQ ID NO: 284), ZNRF3-55 (VH SEQ ID NO: 391, VL SEQ ID NO: 392), ZNRF3-6 (VH SEQ ID NO: 395, VL SEQ ID NO: 396), ZNRF3-90 (VH SEQ ID NO: 399, VL SEQ ID NO: 400), RNF43-14 (VH SEQ ID NO: 425, VL SEQ ID NO: 426), RNF43-22 (VH SEQ ID NO: 429, VL SEQ ID NO: 43 6. The multispecific antibody according to any one of claims 1 to 5, comprising a heavy chain variable region (VH) and a light chain variable region (LH) that comprise amino acid sequences at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to the heavy chain variable region (VH) and / or light chain variable region (LH), respectively, of any one of ZNRF3-331 (VH SEQ ID NO: 439, VL SEQ ID NO: 440),

7. The multispecific antibody of any one of claims 1 to 2 or 4 to 6, wherein said multispecific antibody comprises a wild-type human Fc region, optionally wherein said Fc region is an IgG1, IgG2, IgG3 or IgG4 Fc region.

8. The multispecific antibody of any one of claims 1 to 6, wherein said multispecific antibody comprises a human IgG1 Fc region comprising a LALAPG mutation or a substitution at position N297, such as N297G or N297Q, and / or said Fc region lacks effector function.

9. 9. The multispecific antibody of any one of claims 1 to 8, wherein said second cell surface protein is a receptor tyrosine kinase, a growth factor receptor, a cytokine, a mucin, a Siglec receptor or an immune checkpoint regulator, or is HER2, HER3, IGF1R, EGFR, FGFR, VEGFR, PDGFR, EpCAM, FZD, PD-L1, CTLA4, PD-1, TIM3, LAG3, TIGIT, CEACAM1, CD25, ILT-2, ILT-3, ILT-4, ILT-5, LAIR-1, PECAM-1 (CD31), PILR-alpha, SIRL-1 or SIRP-alpha.

10. An isolated nucleic acid or set of nucleic acids encoding the multispecific antibody according to any one of claims 1 to 9.

11. A host cell comprising the nucleic acid or set of nucleic acids according to claim 10.

12. 12. A method for producing a multispecific antibody according to any one of claims 1 to 9, comprising culturing a host cell according to claim 11 under conditions suitable for expression of said multispecific antibody, and optionally further comprising recovering said multispecific antibody from said host cell.

13. A pharmaceutical composition comprising the multispecific antibody of any one of claims 1 to 9 and a pharma- ceutical acceptable carrier. (i) treating cancer, an autoimmune condition, an inflammatory condition, a neurodegenerative condition, or an infectious disease in a subject; (ii) reducing the level of a second cell surface protein targeted by the multispecific antibody binding in a subject, optionally wherein the subject has cancer, an autoimmune condition, an inflammatory condition, a neurodegenerative condition or an infectious disease; or (iii) increasing the immune response, such as in a subject with cancer, an autoimmune condition, an inflammatory condition, a neurodegenerative condition, or an infectious disease; A pharmaceutical composition comprising the multispecific antibody of any one of claims 1 to 9 or the pharmaceutical composition of claim 13 for the treatment of

15. A method for reducing the level of a cell surface protein in a cell or tissue sample in vitro, comprising incubating said sample with a multispecific antibody according to any one of claims 1 to 9.

Images