Multiple specificity-binding protein degradation platform and method of use

Abstract

The present invention provides a multispecific antibody for targeted degradation of cell surface proteins, a pharmaceutical composition containing the antibody, and a method for in vitro reducing the level of cell surface proteins in a cell or tissue sample. [Solution] A multispecific antibody is provided 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 ZNRF3, and the multispecific antibody has a binding affinity of less than 1 nM to RNF43 or ZNRF3.

Description

[Technical Field]

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

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

[0003] field This disclosure relates to a multispecific antibody platform for targeted degradation of cell surface proteins. This disclosure relates to a multispecific (e.g., bispecific or triplicate) binding molecule, such as a multispecific antibody that targets at least one transmembrane E3 ubiquitin ligase protein and at least one cell surface protein, for example, a cell surface protein intended for degradation, and to a method of using the same. [Background technology]

[0004] background Various platforms have been constructed to target the degradation of target proteins. For example, PROTACs (protein degradation-targeting chimeras) are small molecule constructs intended to target cytoplasmic proteins to the 26S proteasome for degradation. They may include a domain that binds to ubiquitin E3 ligase, a domain that binds to the protein to be degraded (i.e., the target protein), and a linker molecule that connects these two binding domains. However, because PROTACs target cytoplasmic protein domains, this approach cannot be used for certain membrane-bound protein targets or cell surface protein targets. (See, for example, Ahn et al., ChemRxiv, doi.org / 10.26434 / chemrxiv.12736778.v 1, published July 30, 2020). LYTAC (lysosome-targeted chimeric) constructs are multivalent constructs that, for example, bind to a target membrane protein and include ligands such as mannose-6-phosphate that bind to the cation-independent mannose-6-phosphate receptor (CI-M6PR). By linking the targeted membrane protein to CI-M6PR, the protein is guided to lysosomes for degradation. See the same literature. However, unlike PROTAC, LYTAC requires chemical conjugation to, for example, mannose-6-phosphate, and this conjugation may need to be controlled during manufacturing to avoid product heterogeneity. Furthermore, since CI-M6PR is expressed in almost all tissues, this method may not be useful for selectively degrading proteins in specific tissues. A bispecific antibody that binds to RNF43, a cell surface E3 ubiquitin ligase, and PD-L1, a cell surface immune checkpoint protein, 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 in tumor cell lines exposed to this bispecific antibody was demonstrated in vitro.WO2021 / 176034 describes a bispecific single-stranded (VHH) "anti-tag" antibody capable of degrading similarly transiently expressed tagged cell surface proteins in HEK293T cells transiently expressing tagged E3 ligase. However, the general applicability of this approach, including the degradation efficiency of endogenous cell surface proteins in cells expressing endogenous E3 ligase and its in vivo efficacy, as well as the optimal attributes of a platform utilizing antibodies against cell surface ligases, remain unknown. [Overview of the project]

[0005] overview This disclosure relates to an improved approach to targeted degradation of membrane-bound proteins 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 ligases and multispecificity binding proteins such as multispecificity (e.g., bispecificity or tripspecificity) antibodies that bind to cell surface proteins intended for degradation. These multispecificity binding proteins use transmembrane E3 ubiquitin ligases to, for example, target the cell surface protein of interest to lysosomes for degradation. Zinc and ring finger 3 (ZNRF3) and its homolog, ring finger 43 (RNF43), are exemplary transmembrane E3 ubiquitin ligases that typically 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 in certain types of cancer, for example (see, e.g., Figure 1C). Therefore, tumor cells derived from various cancers may express relatively high levels of RNF43 and ZNRF3 compared to other cells, which in some embodiments may enable selective degradation of target proteins in tumor cells. Various other transmembrane E3 ubiquitin ligases can also be used as targets for multispecific binding proteins herein. Optimal attributes of multispecific antibodies (including, for example, optimized binding affinity and multispecific format) are also provided herein. In some embodiments, the multispecific binding proteins herein are referred to as PROTABs ("protein degradation targeting antibodies").

[0006] In addition to providing a general platform for multispecific binding proteins that target cell surface proteins for transmembrane E3 ubiquitin ligases and disruption, this disclosure also provides various antibody heavy chain variable regions and antibody light chain variable regions that target RNF43 or ZNRF3, which may be useful in constructing such multispecific antibodies and binding proteins. The binding proteins described herein may, in some embodiments, be used therapeutically, for example, to degrade proteins contributing to disease progression in cells where the Wnt pathway is activated and transmembrane E3 ubiquitin ligases are expressed, for example. Certain cancers, such as colorectal cancer (CRC), are characterized by overexpression of RNF43 and ZNRF3. Therefore, multispecific binding proteins described herein that target one or both of these ligases may be selectively targeted to cancer cells due to their overexpression of these proteins. The binding proteins described herein may also be used in vitro, for example, in cell culture or tissue assays, to degrade specific membrane proteins, thereby reducing their levels and knocking down their associated signaling pathways. For example, certain E3 ubiquitin ligases (e.g., RNF130, RNF149, and RNF167) are expressed in hematopoietic cells, and in some embodiments, multispecific binding proteins that target these 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 that target these ligases can be used in some embodiments to target testicular cells in vivo or in vitro.

[0007] The present invention provides, among other things, a multispecific binding protein 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 a transmembrane E3 ubiquitin ligase. In some embodiments, the multispecific binding protein reduces the level of the second cell surface protein on the cell surface 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 cell surface 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 cell surface in vitro when measured by flow cytometry or a luminescence assay. In some cases, the multispecific binding protein reduces the level of the second cell surface protein on the cell surface 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 proteins described herein are multispecific antibodies. For example, in some embodiments, the protein is a bispecific or triplicate antibody, e.g., 1+1 FabIgG, 1+1 FvIgG, 2+1 FvIgG, 2+1 FabIgG, 1-arm FvIgG, or 1-arm FabIgG. In some such cases, the protein comprises an IgG Fc region having at least one knob-into-hole modification.

[0009] In some embodiments, the transmembrane E3 ubiquitin ligase lacks catalytic activity, and the absence of catalytic activity is determined by a cell surface degradation assay. In other embodiments, the transmembrane E3 ubiquitin ligase possesses catalytic activity, and the presence of catalytic activity is determined by a cell surface degradation assay.

[0010] In some embodiments, the multispecific binding proteins described herein bind 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 proteins described herein bind 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, ZNRF3, or both, such that its protein binds to RNF43, ZNRF3, or both RNF43 and ZNRF3, and optionally its 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) including (a) CDR-H1, (b) CDR-H2, and (c) CDR-H3, and a light chain variable domain (VL) including (d) CDR-L1, (e) CDR-L2, and (f) CDR-L3; or a multispecific antibody that binds to RNF43 or ZNRF3 and comprises VH and VL.

[0012] 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, RNF43-5It contains one of the following heavy chain complementarity-determining regions: 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 well as a heavy chain variable domain (VH) containing CDR-H2 and / or CDR-H3.

[0013] 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, RNF43-5It contains a heavy-chain variable region (VH) comprising one of the following CDR-H1, CDR-H2, and CDR-H3: 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] 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, RNF43-5It contains one of the following light chain complementarity-determining regions: 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 well as a light chain variable domain (VL) containing CDR-L2 and / or CDR-L3.

[0015] 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, RNF43-5It includes a light chain variable region (VL) containing one of the following CDR-L1, CDR-L2 and / or CDR-L3: 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] 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, RNF43-5Any 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, comprising: (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 (a) light-chain complementarity-determining region 1 (CDR-L1), CDR-L2 and CDR-L3.,

[0017] In any of the above cases, the heavy-chain CDR and / or the light-chain CDR can be Kabat CDR, Chothia CDR or McCallum CDR. The amino acid sequences and DNA sequences of the relevant VH and VL of the antibodies listed above are provided in the following Sequence Listing together with SEQ ID NOs: 33 to 444.

[0018] 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, RNF43-5The antibody contains a heavy chain variable region (VH) that is at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical in amino acid sequence to one of the following: 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 in some cases, the amino acid sequences of CDR-H1, CDR-H2, and / or CDR-H3 of that VH are 100% identical to the amino acid sequence of the selected antibody.

[0019] 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, RNF43-5The antibody contains a light chain variable region (VL) containing at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical amino acid sequence to one of the following: 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 sequences of CDR-H1, CDR-H2, and / or CDR-H3 of the VH are 100% identical to the amino acid sequence of the selected antibody.

[0020] 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, RNF43-5It comprises a VH comprising the amino acid sequence of 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] 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, RNF43-5The VL contains the amino acid sequence of one of the following light chain variable regions (VLs): 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 stated above, the amino acid sequences and DNA sequences of the VH and VL of the antibodies listed above are those of sequence numbers 33-444 in the sequence listing below.

[0022] In some embodiments, the multispecific binding protein binds to ZNRF3 and / or RNF43 and has a binding affinity to 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 50 nM to 10 nM, or 10 nM to 1 nM, or 1 nM to 0.5 nM, or 0.5 nM to 0.05 nM, or 0.05 nM to 0.01 nM. In some embodiments, the protein is a multispecific antibody containing the heavy chain variable region and / or 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 the binding of ZNRF3 or RNF43 to FZD and LRP6.

[0023] In some embodiments, the multispecific binding proteins described herein may be chimeric multispecific antibodies containing a heavy chain variable region or a light chain variable region, or multispecific antibodies containing a humanized variable region. In some cases, the protein contains a wild-type human Fc region. In some cases, the Fc region is an IgG1, IgG2, IgG3, or IgG4 Fc region, and may be either a wild-type human Fc region or a human Fc region with one or more manipulated mutations. In some cases, the protein contains an Fc region with effector function. In some cases, the protein contains a human IgG1 Fc region containing a LALAPG mutation or an N297 substitution, e.g., N297G or N297Q, and / or that 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, growth factor receptor, cytokine, mucin, Siglec receptor or immune checkpoint regulator, or 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 includes the heavy chain and light chain variable regions of an anti-HER2 antibody, e.g., 4D5, 7C2, or 2C4, or an anti-IGF1R antibody, e.g., cisutumumab, ganitumumab, darotuzumab, figtumumab, lobatumumab, teprotumumab, or istilatumumab.

[0025] This disclosure also relates to isolated nucleic acids or sets of nucleic acids (e.g., including two or more nucleic acids, each encoding a portion of a multimeric protein, such as a light chain, a heavy chain, or a half-antibody) that encode multispecific binding proteins as described herein. This disclosure also relates to host cells containing such nucleic acids or sets of nucleic acids. This disclosure also encompasses methods for producing multispecific binding proteins as described herein, the methods comprising culturing host cells containing the nucleic acids or sets of nucleic acids encoding the protein under conditions suitable for the expression of the protein. Such methods may further include recovering the protein from the host cells. This disclosure further encompasses multispecific binding proteins produced by the methods described herein.

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

[0027] This disclosure also includes, for example, drugs for use in the treatment of cancer, autoimmune symptoms, inflammatory symptoms, neurodegenerative symptoms or infections of a subject, and / or drugs for use in reducing levels of cell surface proteins in subjects that require a reduction in levels of cell surface proteins (which may in some cases have cancer, autoimmune symptoms, inflammatory symptoms, neurodegenerative symptoms or infections), and / or multispecific binding proteins and related pharmaceutical compositions for use in increasing the immune response in subjects such as subjects having cancer, autoimmune symptoms, inflammatory symptoms, neurodegenerative symptoms or infections. 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] This disclosure also relates to the use of multispecific binding proteins or pharmaceutical compositions herein in the manufacture of agents for treating cancer, autoimmune symptoms, inflammatory symptoms, neurodegenerative symptoms or infections of a subject; and / or agents for reducing levels of cell surface proteins in subjects requiring a reduction in levels of cell surface proteins (where the subject may have cancer, autoimmune symptoms, inflammatory symptoms, neurodegenerative symptoms or infections); and / or agents for increasing the immune response in subjects such as subjects having cancer, autoimmune symptoms, inflammatory symptoms, neurodegenerative symptoms or infections. 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 mutation is present.

[0029] This disclosure also relates to methods for treating cancer, autoimmune conditions, inflammatory conditions, neurodegenerative conditions, or infections in subjects requiring treatment of cancer, autoimmune conditions, inflammatory conditions, neurodegenerative conditions, or infections, the methods comprising administering an effective amount of the multispecific binding protein or pharmaceutical composition specified herein to the subject. This disclosure further relates to methods for reducing cell surface protein levels in subjects requiring a reduction in cell surface protein levels, the methods comprising administering an effective amount of the multispecific binding protein or pharmaceutical composition specified herein to the subject, the subject optionally having cancer, autoimmune conditions, inflammatory conditions, neurodegenerative conditions, or infections. This disclosure also further relates to methods for increasing immune responses in subjects requiring an increase in immune responses, the methods comprising administering an effective amount of the multispecific binding protein or pharmaceutical composition specified herein to the subject, the subject optionally having cancer, autoimmune conditions, inflammatory conditions, neurodegenerative conditions, or infections. In some embodiments, (a) the subject has a mutation in RNF43 and the multispecific binding protein does not bind to RNF43 or activate RNF43, or (b) the subject has a mutation in ZNRF3 and the multispecific binding protein does not bind to ZNRF3 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 contains the 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, wherein (a) if the subject has a mutation in RNF43, the multispecific binding protein does not bind to RNF43 or activate RNF43, and (b) if the subject has a mutation in ZNRF3, the multispecific binding protein does not bind to ZNRF3 or activate ZNRF3.

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

[0031] This disclosure also includes kits comprising multispecific binding proteins, nucleic acids, nucleic acid sets, or host cells as described herein, the kit further comprising one or more reagents for expressing or purifying the multispecific binding proteins and / or for in vitro incubation of the protein with a cell or tissue sample to reduce the level of cell surface proteins in the sample. This disclosure also includes methods for in vitro reducing the level of cell surface proteins in a cell or tissue sample, which include incubating the sample with the multispecific binding proteins described herein. [Brief explanation of the drawing]

[0032] [Figure 1A-1F]Figures 1A–1F show that cell surface ligases driven by oncogenic Wnt can be reused for targeted degradation. Figure 1A) Schematic diagram illustrating the role of RNF43 in regulating the cell surface abundance of the frizzled (FZD) receptor via ubiquitination. Figure 1B) Expression of RNF43 (left panel) and ZNRF3 (right panel) in primary human adenoma 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 RNF43 expression in various cancer subtypes. Note the 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 results in decreased cell surface expression of FZD, but the ligase-deficient mutant delta-RING RNF43 does not result in this decrease. Figure 1E) Schematic diagram of strategies used to chemically dimerize RNF43 and / or ZNRF3 on the non-native cell surface using A / C heterodimerizers along with receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). Figure 1F) Immunoblot of tagged HER2 after immunoprecipitation of tagged RNF43 in transiently transfected HEK293T cells treated with A / C heterodimerizers. [Figure 2A-2E]Figures 2A–2E show antibody-mediated dimerization of gD-tagged cell surface RNF43 or ZNRF3 ligases against HER2. Figure 2A) schematically shows dimerization of N-terminal gD-tagged RNF43 or ZNRF3 ligases to target protein using anti-gD / anti-target protein (POI) bispecific antibodies. 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 cells 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 was 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 bispecific antibody shown. Downstream regulation of the HER2 pathway using p-Erk is shown in SKBr3. Actin was used as a loading control. [Figure 3A-3E]Figures 3A–3E illustrate the mechanisms of cell surface clearance and receptor degradation during 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 HER2 gene deletion, after treatment with the bispecific antibodies shown against gD / HER2 or CD3 / HER2. Tubulin was used as a loading control. Figure 3C) Immunoblot of ubiquitin in HEK293T transiently transfected with gD-tagged RNF43, ZNRF3, or control ("mock") after treatment with the three different bispecific antibodies shown against gD and HER2, following immunoprecipitation of HER2. 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, exhibiting superior (WT RING) or insufficient (delta-RING) ligase activity, after treatment with the bispecific antibodies shown against gD / HER2. Actin was used as a loading control. Figure 3E) Western blots of HER2 in HEK293T transiently transfected with gD-tagged RNF43, ZNRF3, or a control, after treatment with the bispecific antibody and inhibitors shown against gD / HER2 (bortezomib = proteasome inhibitor; E1 activating enzyme inhibitor, BafA = lysosomal inhibitor). Actin was used as a loading control. (T = trastuzumab (4D5), P = pertuzumab (2C4), 7 = 7C2) [Figures 4A-4F]Figures 4A–4F show the phenotypic effects of cell surface ligase-mediated receptor degradation. Figure 4A) Immunoblot of tagged IGF1R after immunoprecipitation of tagged RNF43 in transiently transfected HEK293T cells treated with AC heterodimerizer. Tubulin was 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 bispecific antibodies shown against gD and various IGF1R epitopes. Tubulin was used as a loading control. Figure 4C) Clonal proliferation assay of HT55 cells transfected with dox-inducible CRISPR-Cas9 and IGF1R-targeted sgRNA or untargeted control (NTC) sgRNA. Figure 4D) In ​​vivo tumor growth of HT55 cells transfected with dox-inducible CRISPR-Cas9 and IGF1R-targeted sgRNA or untargeted control (NTC) sgRNA. Figure 4E) Clonal proliferation 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). [Figures 5A-5F]Figures 5A–5F provide characterizations of RNF43 and ZNRF3-targeted antibodies and related bispecific antibodies. Figure 5A) SPR binding of antibodies found from rat or rabbit immunization against RNF43 or ZNRF3. Figures 5B and 5C) Cross-blocking analysis of subsets of ZNRF3 (Figure 5B) and RNF43 (Figure 5C)-binding antibodies. Figure 5D) Time course of cisutumumab / hSC 37.39 bispecific antibodies driving IGF-1R cell surface clearance at concentrations of 0–10 μg / mL, as measured by tracking IGF1R MFI at 1–24 hours. Figures 5E and 5F) IGF1R clearance using a panel of ligase / cisutumumab bispecific antibodies binding to human ZNRF3 ("hu ZNRF3"; Figure 5E) or human RNF43 ("hu RNF43"; Figure 5F). For each ligase, the percentage clearance is plotted against monovalent affinity. [Figure 6A-6C] Figures 6A–6C illustrate the characterization of the degradation capabilities of novel cell surface ligase antibodies. Figures 6A and 6B) Mean fluorescence intensity (MFI) of cell surface IGF1R in gD-ZNRF3-transfected SW1417 or HT29 cells after treatment with various antibodies against IGF1R / ZNRF3 (Figure 6A) or various antibodies against IGF1R / NIST (Figure 6B). Figure 6C) Western blot of IGF1R in parental SW1417 cells after treatment with the bispecific antibodies shown. Actin was used as a loading control. [Figures 7A-7F]Figures 7A to 7F illustrate the characterization of the degradation capabilities of novel formats of cell surface ligase antibodies. Figures 7A and 7B show schematic diagrams of the antibodies tested. Figures 7C to 7F show flow cytometry analysis of IGF1R to evaluate antibody activity in HT29 cells overexpressing the relevant ligases. Figure 7C shows the results for cizutumumab / anti-RNF43 constructs having one or two cizutumumab (anti-IGFR; "cixu") binding domains, Figure 7D shows the results for cizutumumab / anti-RNF43 constructs having one or two RNF43 binding domains, Figure 7E shows the results for istilatuzumab / anti-RNF43 constructs having one or two istilatuzumab (anti-IGFR; "istira") binding domains, and Figure 7F shows the results for istilatuzumab / anti-RNF43 constructs having one or two RNF43 binding domains. [Figures 8A-8D] Figures 8A to 8D illustrate the characterization of the additional novel format degradation capabilities of cell surface ligase antibodies. Figure 8A) Schematic diagrams of the tested FvIgG or FabIbG format antibodies are shown. Figure 8B) The activity of the FvIgG format in HT29 cells overexpressing the relevant ligase is evaluated using IGF1R flow cytometry analysis. Figures 8C and 8D) The activity of the FabIgG format in HT29 cells overexpressing the relevant ligase is evaluated using IGF1R flow cytometry analysis (Figure 8C = cizutumumab / anti-RNF43; Figure 8D = istilatuzumab / anti-RNF43). [Figure 9] Figure 9 illustrates the characterization of the degradation capabilities of EGFR-targeted multispecific antibodies. EGFR flow cytometry analysis was used to evaluate the activity of multispecific antibodies in HT29 cells overexpressing relevant ligases. [Figures 10A-10F]Figures 10A–10F illustrate the characterization of the degradation capabilities of the novel cell surface ligase antibodies. Figure 10A) Schematic diagram 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 an additional E3 ligase with similar structural features to RNF43 and ZNRF3, including SP and TM. Figure 10C) FACS plot showing transfection of gD-ZNRF3 and detection of gD on the cell surface using an anti-gD antibody (conjugated with APC) in HEK293T cells (FITC positive). Figure 10D) Cell surface MFI of the gD signal from the gD-tagged ligase shown, transiently transfected into HEK293T cells. gD is detected using an anti-gD APC conjugated antibody. Figures 10E and 10F) Western blots of HER2 in HEK293T cells transiently transfected with the illustrated dox-inducible gD-tagged ligases after treatment with anti-HER2 / gD bispecific antibodies. Western blots for gD and tubulin were used as transfection evaluation and loading controls, respectively. Figure 10E shows data for the ligases RNF13, RNF43, RNF128, RNF130, RNF133, RNF148, and RNF149. Figure 10F shows data for the ligases RNF150, RNF167, ZNRF3, LNX1, RSPRY1, SYVN1, and TRIM7. [Figure 11A-11B]Figures 11A and 11B show the activation of the Wnt / β-catenin signaling pathway via a TCF reporter by the ZNRF3 antibody. Figure 11A) The rat clonal ZNRF3 bivalent antibody induced slight activation of Wnt / β-catenin in HEK293 cells in the presence of 100 ng / mL recombinant Wnt3a. Untreated cells, cells treated with DMSO, and cells treated with 100 ng / mL Wnt3a were used as negative controls. Cells treated with 2 μM GSK3 beta or various concentrations of RSPO3 (500 ng / μL, 10 ng / μL, 0.2 ng / μL) in combination with 100 ng / mL Wnt3a were used as positive controls. Figure 11B) The rabbit clonal ZNRF3 bivalent antibody induced slight activation of Wnt / β-catenin. The positive and negative controls used are listed above. The data shown are from two independent experiments (n=2). [Figures 12A-12F] Figures 12A–12F show the kinetics of bispecific antibody-mediated surface IGF1R-HibiT clearance. (Figures 12A–12C) The percentage of residual cell surface IGF1R-HibiT was assessed at 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. 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, and 10 μg / mL Istira-RNF43 Fv-IgG (Figure 12F) showed a "hook effect". [Figure 13A-13C]Figures 13A to 13C show cytotoxicity and cell surface clearance evaluated by multiplexing the HiBiT extracellular detection assay with the LDH or CellTiter-Glo® assay. Figure 13A) IGF1R-HibiT remained on the cell surface after 48 hours of treatment with 1 μg / mL cixu / ZNRF3 bispecific antibody. Figure 13B) Lactate dehydrogenase released by HT29 cells treated with 1 μg / mL cixu / ZNRF3 bispecific antibody for 48 hours was measured using the LDH cytotoxicity assay. No toxicity was observed under these treatment conditions. Figure 13C) CellTiter-Glo® assay luminescence signal (RLU) was measured after detection of IGF1R-HiBiT on the cell surface using the HiBiT extracellular detection system. Similar to the LDH assay, no toxicity was observed under these treatment conditions. [Figure 14A-14B] Figures 14A and 14B show the detection of lysis levels of total IGF1R-HibiT. Figure 14A) The amount of residual IGF1R-HibiT on the surface of HT29 cells is detected using the HiBiT extracellular system. The lysis detection indicates the total level of IGF1R-HibiT (extracellular and intracellular). Both systems measure luminescence signals. The 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 used as a positive control. Cixu / NIST, Cixu / Cixu, and UT were used as negative controls. Figure 14B) Western blot showing the total amount of IGF1R-HibiT or IGF1R in WT HT29 cells after 24 hours of treatment with the novel format and bispecific antibody. ProIGF1R-HiBiT / IGF1R is a precursor to IGF1R-HibiT / IGF1R. β-actin is the internal control. [Figures 15A-15G]Figures 15A–15G show various exemplary formats for constructing multispecific antibodies compatible with the embodiments herein: the “2+1 FabIgG” format (Figure 15A), the “1-arm FvIgG” or “OA FvIgG” format (Figure 15B), and the “1-arm FabIgG” or “OA FabIgG” format (Figure 15C). Figures 15D–15E show two different free-choice versions of the trispecific anti-EGFR-RNF43-ZnRF3 antibody, and Figures 15F–15G show two different free-choice versions of the trispecific anti-IGF1R-RNF43-ZnRF3 antibody. [Figure 16] Figure 16 shows that a combination of bispecific antibodies targeting both RNF43 / IGF1R and ZNRF3 / IGF1R is more effective in removing IGF1R from the cell surface than either bispecific antibody alone. [Figure 17] Figure 17 shows that RNF43 is uniformly expressed throughout the tumor mass in the transplanted mouse APC mutant colorectal cancer model. [Figures 18A-18B] Figures 18A and 18B show Western blots illustrating the total amount of IGF1R in WT LS180 cells after 24 hours of treatment with various bispecific antibodies (Figure 18A). ProIGF1R is a precursor of IGF1R. Tubulin was used as a loading control. Degradation percentages are summarized across various cell lines (Figure 18B). [Figures 19A-19B]Figures 19A and 19B show Western blot analysis of lysates derived from HT29 cells subjected to the described treatments: IGF1 stimulation (+IGF1; 50 ng / ml; 5 min), untreated (-), 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) (19A). IGF1R ubiquitination was assessed by immunoblotting IP samples with an antibody against ubiquitin. Immunoprecipitation levels and total IGF1Rβ levels were assessed using an antibody against IGF1Rβ, with α-tubulin used as a loading control (Figure 19A). Western blot analysis of lysates derived from parental HEK293T cells or HEK293T cells expressing ZNRF3 wild-type (WT) or delta-RING mutant (ΔRING) with N-terminal gD tagging and C-terminal FLAG tagging, after incubation with HA epitope-tagged antibody agarose conjugate or agarose-TUBE2. Total IGF1R levels and co-precipitated IGF1R levels were evaluated by immunoblotting using an antibody against IGF1Rβ. ZNRF3 ubiquitination and expression levels were evaluated using an antibody against the C-terminal FLAG tag, with α-tubulin used as a loading control (Figure 19B). [Figure 20] Figure 20 shows Western blots of IGF1R in HEK293T transiently transfected with gD-tagged ZNRF3, which exhibits either superior ligase activity (WT RING) or insufficient ligase activity (delta-RING), or in controls treated with the bispecific antibodies shown against gD / cyzutumumab. Tubulin was used as a loading control. [Figure 21A-21C]Figures 21A–21C show the distribution of RNF43 expression plotted against the presence of RNF43 mutations. Particular emphasis is placed on the SW48 CRC strain showing elevated RNF43 expression and frameshift variants within the RING domain (Figure 21A). Cell surface expression of RNF43 is shown for the non-expressing RKO strain compared to the RING domain mutant SW48 (Figure 21B). Western blot of IGF1R in SW48 after treatment with the bispecific antibodies shown against RNF43 / IGF1R or ZNRF3 / IGF1R (Figure 21C). [Figures 22A-22C] Figures 22A–22C show schematic diagrams 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-terminus truncation (N-term) or RING indels. Western blot of IGF1R in HT29 cells transiently transfected with gD-tagged ZNRF3, exhibiting superior ligase ability, after treatment with the bispecific antibody shown (Figure 22C), with N-terminus knockout or RING deficient. [Figure 23] Figure 23 shows Western blots of IGF1R in DLD1 after treatment with the bispecific antibody and inhibitors shown against gD / IGF1R (bortezomib = proteasome inhibitor; E1 activator; BafA = lysosomal inhibitor). Tubulin was used as a loading control, and ubiquitin was used to verify inhibition of the proteasome and E1 activator. Lysosomal inhibition was confirmed using LC3B. [Figure 24] Figure 24 shows a Western blot of IGF1R in DLD1 after 24 hours of processing with the novel format, and the bispecific antibody is shown in three different exemplary formats for constructing bispecific antibodies that conform to the embodiments herein: the “2+1 FabIgG” format (Figure 15A), the “1-arm FvIgG” or “OA FvIgG” format. [Figures 25A-25C] Figures 25A–25C show Western blots of IGF1R, pIGF1R, and downstream components of the IGF1R signaling axis (including pAKT and pS6) in SW48 cells treated with various antibodies shown (Figure 25A). Note the near-complete inhibition of pAKT signaling during ligase-mediated degradation of IGF1R. Figure 25C shows clonal proliferation of SW48 cells 14 days after treatment with the antibody shown (Figure 25B) and licor-based quantification in (Figure 25B). [Figures 26A-26D] Figures 26A–26D show clonal proliferation assays of dox-inducible gD-ZNRF3-transfected HT29 cells after treatment with various anti-Citxu / ZNRF3 or control bispecific antibodies. Licor-based quantifications for Figure 26A (results for gD-ZNRF3 WT-FLAG) and Figure 26C (results for gD-ZNRF3 delta-RING-FLAG variant) are shown in Figures 26B and 26D, respectively. [Figure 27] Figure 27 shows Western blots of PD-L1 in SW48 cells treated with various antibodies shown. Note the significant degradation observed when using the ZNRF3 / PD-L1 antibody. [Figures 28A-28B] Figures 28A and 28B illustrate the characterization of the degradation capabilities of novel cell surface ligase antibodies. FACS plots show cell surface expression of gD-RNF43, RNF13, and RNF128 in stable HT29 cells (Figure 28A). gD cell surface detection using anti-gD antibody (conjugated with APC). Figure 28B: Cell surface MFI of the gD signal from the gD-tagged ligase shown, stably incorporated 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 treated with the dox-inducible gD-tagged ligase shown, after treatment with an anti-IGF1R / gD bispecific antibody. Western blots for gD and tubulin were used as transfection evaluation and loading controls, respectively. [Figure 30] Figure 30 shows that bispecific antibodies designed to tether endogenous RNF43 or ZNRF3 to IGF1R induce targeted degradation of IGF1R in a SW48 in vivo xenograft model. [Figure 31] Figure 31 shows a heatmap illustrating the expression of the described cell surface ligase across normal tissue (source GTEX) as described in Example 18. The data has been z-score normalized. [Figure 32] Figure 32 shows Western blot analysis of lysate from doxycycline-treated HT29 cells containing the described doxycycline-inducible gD-ligase-FLAG expression construct after 24-hour incubation with the gD*IGF1R(Cixu) bispecific PROTAB. Endogenous IGF1Rβ and exogenous gD-ligase-FLAG protein levels were detected. Data are representative of three independent experiments. [Figure 33] Figure 33 shows further Western blot analysis of lysate from doxycycline-treated HT29 cells containing the described doxycycline-inducible gD-ligase-FLAG expression construct after 24-hour incubation with the gD*IGF1R(Cixu) bispecific PROTAB. Endogenous IGF1Rβ and exogenous gD-ligase-FLAG protein levels were detected. Data are representative of three independent experiments. [Figure 34] Figure 34 shows Western blot analysis of lysate from doxycycline-treated HT29 cells containing the described doxycycline-inducible gD-ligase-FLAG expression construct after 24-hour incubation with the gD*HER2(4D5) bispecific PROTAB. Endogenous HER2 and exogenous gD-ligase-FLAG protein levels were detected. Data are representative of two independent experiments. [Figure 35]Figure 35 shows Western blot analysis of lysate from doxycycline-treated HT29 cells containing the described doxycycline-inducible gD-ligase-FLAG expression construct after 24-hour incubation with the 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. [Figure 36] Figure 36 shows further Western blot analysis of lysate from doxycycline-treated HT29 cells containing the described doxycycline-inducible gD-ligase-FLAG expression construct after 24-hour incubation with the 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 lysate from doxycycline-treated HT29 cells containing the described doxycycline-inducible gD-ligase-FLAG expression construct after 24-hour incubation with the 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. [Figure 38A-38L]Figures 38A–38L show the cell surface clearance (Figures 38A–38F) and degradation of EpCAM in doxycycline-treated HT29 cells containing the described doxycycline-inducible gD-ligase expression constructs (Figures 38G–38J) after 24-hour incubation with 1 μg / ml and 10 μg / ml gD-EpCAM or control gD-NIST bispecific PROTAB. Figures 38A–38F show %EpCAM clearance as assessed by FACS, and Figures 38G–38L show EpCAM degradation as 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, while 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, and gD-IGF1R PROTAB, respectively, compared to other HT-29-gD-ligases (data not shown). [Figure 39A-39B]Figures 39A and 39B show HER2 degradation in SW48 cells after treatment with a bivalent antibody or a PROTAB antibody. Figure 39A shows the level of HER2 degradation by Western blot analysis in SW48 cells, either 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) over 48 hours. Figure 39B shows the level of HER2 degradation by Western blot analysis in SW48 cells, either 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 over 48 hours. Alpha-tubulin levels were used as a loading control. The data represent two independent experiments. [Modes for carrying out the invention]

[0033] I. Definition When used in accordance with this disclosure, the following terms should be understood to have the meanings set forth below, unless otherwise indicated.

[0034] "Transmembrane E3 ubiquitin ligase" refers to a class of proteins that include at least one transmembrane region and possess E3 ubiquitin ligase activity. A transmembrane E3 ubiquitin ligase has "catalytic activity" as used herein if it can facilitate the final transfer of ubiquitin from a ubiquitin conjugate enzyme (E2) to a substrate in order to alter its substrate function. In some embodiments, catalytic activity may be measured in a cell surface assay (see, for example, Example 18 below).

[0035] "Target protein," "protein targeted for degradation," or "degradation target protein," etc., refers to a protein intended to be guided into lysosomes for degradation by the molecules described herein. In some embodiments herein, such a protein intended for degradation is a "cell surface protein." As used herein, "cell surface protein" broadly refers to a protein located on the cell surface, either as a transmembrane protein having an extracellular domain, or as a protein otherwise localized on the cell surface (e.g., a protein bound to a transmembrane protein).

[0036] Proteins such as transmembrane E3 ubiquitin ligases and degradation targets described herein may originate from any vertebrate, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats), or domestic mammals (e.g., dogs, cats, horses, livestock, e.g., cattle, 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 transmembrane E3 ubiquitin ligases and degradation target proteins. The protein is “multispecific” because it binds to at least two target proteins (i.e., transmembrane E3 ubiquitin ligases and degradation targets). In some embodiments, the binding protein is an antibody, such as a bispecific or multispecific antibody, or a bispecific or multispecific protein comprising an antibody or antibody fragment and optionally another specific protein-binding domain.

[0038] As used herein, the term "antibody" refers to a molecule comprising at least heavy chain complementarity-determining regions (CDRs) 1, 2, and 3, and at least light chain CDRs 1, 2, and 3, which can bind to an antigen. The term is used in its broadest sense and encompasses a wide range of 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, as long as they exhibit the desired binding activity.

[0039]

[0001] “Isolated” antibodies are those that have been separated from the components of their natural environment. In some embodiments, antibodies are purified to a purity higher than 95% or 99%, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0040] An "antigen" is the target of an antibody, that is, the molecule to which the antibody specifically binds. The term "epitope" refers to the site on an antigen to which an antibody binds, whether proteinaceous or nonproteinaceous. Protein epitopes can be formed from a continuous sequence of amino acids (linear epitopes) or they can contain discontinuous amino acids that are spatially close together, for example, due to the folding of the antigen (i.e., by the tertiary folding of a proteinaceous antigen) (structural epitopes). Linear epitopes are usually still bound by antibodies even after the proteinaceous antigen is exposed to a denaturing agent, whereas structural epitopes are usually destroyed by treatment with a denaturing agent.

[0041] "Affinity" refers to the sum of the 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 the 1:1 interaction between members of a binding pair (e.g., between an antibody and an antigen). Generally, the affinity of molecule X for its partner Y is given by the dissociation constant (K). D Affinity can be expressed by ( ). Affinity can be measured by methods known in the art, including those described herein.

[0042] In this disclosure, the terms “binds,” “binding,” “specific binding,” and similar terms, when referring, for example, to a protein and its ligand or an antibody and its antigenic target, mean that the binding affinity is strong enough that the interaction between 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) of 1 μM or less. D ) requires a K of 100 nM or less D It is often necessary.

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

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

[0045] The term "light chain" refers to a polypeptide that includes at least a light chain variable region, with or without a leader sequence. In some embodiments, the light chain includes at least a portion of the light chain constant region. The term "full-length light chain" refers to a polypeptide that includes both the light chain variable region and the 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, which is a region that determines antigen-binding specificity, e.g., “complementarity-determining regions” (CDRs), whose sequence is hypervariable. Generally, an antibody contains six CDRs; three in the VH region (CDR-H1 or heavy-chain CDR1, CDR-H2, CDR-H3) and three in the VL region (CDR-L1, CDR-L2, CDR-L3). Illustrative CDRs as used herein include: (a) "Chothia CDR": A hypervariable loop 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 CDR": CDRs present 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": Antigen contact present in 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, the CDR shall be determined according to Kabat et al., as previously stated. Those skilled in the art will understand that the notation of the CDR may also be determined according to McCallum or any other scientifically recognized nomenclature system.

[0048] The term "framework" or "FR" refers to the residues of the variable region that are not part of the complementarity-determining region (CDR). The variable region FR generally consists of four FRs: FR1, FR2, FR3, and FR4. Thus, the CDR and FR sequences generally appear 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. For the purposes of this specification, "acceptor human framework" is a framework comprising 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 human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or human consensus framework may include the same amino acid sequence or may include a modification of the amino acid sequence. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is sequence-identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0049] The term "variable region" or "variable domain" interchangeably refers to the domains of the heavy or light chain of an antibody that are involved in the binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). For example, Kindt et al., Kuby Immunology, 6. thSee ed., WH Freeman and Co., page 91 (2007). The variable domain may include heavy chain (HC)CDR1-FR2-CDR2-FR3-CDR3 with or without all or part of FR1 and / or FR4; and light chain (LC)CDR1-FR2-CDR2-FR3-CDR3 with or without all or part of FR1 and / or FR4. That is, the variable domain may lack part of FR1 and / or FR4, as 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 specific antigen using the VH or VL domain of the antibody that binds to that antigen. For example, see Portolano et al., J.Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0050] The “constant region” of the antibody light and heavy chains refers to the additional sequence portion outside the FR and CDR and the variable region. A particular antibody fragment 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 a variable heavy chain 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 a variable light chain domain or light chain variable region, followed by a constant light chain (CL) domain.

[0051] The term “Fc region” as used herein is used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes the native sequence Fc region and the variant Fc region. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, antibodies produced by host cells by the expression of a particular nucleic acid molecule encoding a full-length heavy chain may contain the full-length heavy chain or a cleaved 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, numbered according to the EU index). Therefore, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (Lys447) in the Fc region may or may not be present. Thus, for example, "full-length IgG1" includes IgG1 having Gly446 and Lys447, or not having Lys447, or not having either Gly446 or Lys447. The amino acid sequence of the heavy chain containing the Fc region is shown herein without the C-terminal glycine-lysine dipeptide unless otherwise indicated. In one embodiment, the heavy chain containing the Fc region as specified herein, included in the antibody according to the present invention, may include Gly446 and Lys447 (numbered by the EU index). In one embodiment, the heavy chain containing the Fc region as specified herein, included in the antibody according to the present invention, may include Gly446 (numbered by the EU index). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also known 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 that may be caused by the Fc region of an antibody, and this varies depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cell-mediated cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0053] The "class" of an antibody refers to the type of constant domain or constant region held by its heavy chain. There are five main 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 a particular embodiment, an antibody is an IgG1, IgG2, IgG3, or IgG4 isotype. The heavy chain constant domains corresponding to 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] An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to an antigen (i.e., RNF43, ZnRF3, gD, or a 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; chain antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAb); and multispecific antibodies formed from antibody fragments. For a review of a particular antibody fragment, 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 natural antibody, or, in the case of an IgG antibody, an antibody having a heavy chain containing an Fc region as defined herein.

[0056] As used herein, the term "multispecificity" refers to a molecule that can bind to more than one different target or antigen, for example, two or three or more different targets or antigens. As used herein, the term "bispecificity" refers to a molecule, such as a binding protein or antibody, that can specifically bind to two different targets or antigens. As used herein, a "multispecific" antibody or a "bispecific" antibody may include appropriate full-length heavy and full-length light chains for binding to two different antigens, or may include appropriate antibody fragments for binding to two different antigens. There are various different platforms for producing multispecific binding proteins that conform to this disclosure.

[0057] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies constituting that population are identical and / or bind to the same epitope, except for any variant antibodies that may exist (usually in small amounts), such as those containing naturally occurring mutations or arising during the production of the monoclonal antibody preparation. Each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen, in contrast to polyclonal antibody preparations, which typically contain various antibodies against various determinants (epitopes). Therefore, the modifier “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies according to the present invention may be produced by a variety of methods, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a 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 part of the heavy chain and / or light chain originates from a specific origin or species, while the remaining parts of that heavy chain and / or light chain originate from a different origin or species.

[0059] A "humanized" antibody refers to a chimeric antibody that contains amino acid residues derived from non-human CDRs and amino acid residues derived from human FRs. In certain embodiments, a humanized antibody contains at least one, usually two, substantially all of the variable domains, where all or substantially all of the CDRs correspond to the variable domains of the non-human antibody, and all or substantially all of the FRs correspond to the variable domains of the human antibody. A humanized antibody may, in some cases, contain at least a portion of the antibody constant region derived from a human antibody. The "humanized form" of an antibody, for example, a non-human antibody, refers to an antibody that has undergone humanization.

[0060] A "human" antibody is an antibody produced by a human or human cell, or an antibody that has an amino acid sequence corresponding to the amino acid sequence of an antibody of non-human origin that utilizes a sequence encoding a human antibody or other human antibody. This definition of a human antibody explicitly excludes humanized antibodies that contain residues that bind to non-human antigens.

[0061] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents. A "naked antibody" refers to an antibody that is not conjugated to a heterologous portion (e.g., a cytotoxic portion) or a radiolabeled molecule. Naked antibodies may be present in pharmaceutical compositions.

[0062] The terms “nucleic acid molecule” or “polynucleotide” include any compound and / or substance containing polymers of nucleotides. Each nucleotide consists 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. Often, nucleic acid molecules are described by a sequence of bases, thereby representing the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is usually represented from 5' to 3'. In this specification, the term nucleic acid molecule includes, for example, deoxyribonucleic acid (DNA), including complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules may be linear or cyclic. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, nucleic acid molecules described herein may include naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone links or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for the direct expression of the antibodies of the present invention in vitro and / or in vivo, for example, in a host or patient. Such DNA (e.g., cDNA) vectors or RNA (e.g., mRNA) vectors may be modified or unmodified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, so that the mRNA can be injected into a target in vivo to produce an antibody (see, e.g., Stadler et al, Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or EP2101823B1).

[0063] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from their natural environment. Isolated nucleic acids typically include nucleic acid molecules found within cells that contain nucleic acid molecules, but these nucleic acid molecules are either located outside of chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0064] "An isolated nucleic acid encoding an antibody" means one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof) as used herein, including nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules located at one or more positions within a host cell.

[0065] The term “vector,” as used herein, refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures, as well as vectors incorporated into the genome of a host cell into which they are introduced. Certain vectors can direct the expression of a operably linked nucleic acid. Such vectors are referred to herein as “expression vectors.”

[0066] The terms “host cell,” “host cell line,” and “host cell culture” refer to such cells, including the offspring of cells into which exogenous nucleic acids have been introduced, and which are used interchangeably. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their offspring, regardless of the number of passages. The offspring may not have nucleic acid content that is exactly the same as that of the parent cells and may contain mutations. Mutant offspring having the same function or biological activity as those screened or selected in the original transformed cells are included herein.

[0067] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after aligning the sequences for the purpose of alignment and introducing gaps if necessary to achieve maximum percent sequence identity, and excluding any conservative substitutions from being considered part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the scope 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 appropriate parameters for aligning the sequences, including any algorithm necessary to achieve maximum alignment with respect to the full length of the sequences being compared. Alternatively, the percentage identity value can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code is filed in the user documentation of the U.S. Copyright Office (Washington DC, 20559), registered under U.S. Copyright Registration No. TXU510087, and listed in WO2001 / 007611.

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

[0069] "To reduce" means the ability to bring about an overall decrease of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In some embodiments, reducing or inhibiting may 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, reducing may refer to a decrease in the "level" (i.e., quantity or concentration) of a cell surface protein on a cell, for example. For example, protein degradation may result in a decrease in the level of that protein observed in a cell or tissue sample, such as by qualitative analysis of flow cytometry or fluorescence staining.

[0070] A multispecific binding protein that "blocks the binding" of a 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 native ligands such as frizzled (FZD) and LRP6. In some embodiments, multispecific binding proteins do not block such binding. Such inhibition can occur through any mechanism, including direct interference with ligand binding, for example, due to overlapping binding sites on the ligase for the 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 alter ligand affinity.

[0071] As used herein, the term “about” refers to a number, whether expressly indicated or not, including, for example, integers, fractions, and percentages. Generally, the term “about” refers to a range of numbers that a person skilled in the art would consider equivalent to (e.g., having the same function or result as) the stated value (e.g., + / - 5 to 10%). When terms such as “at least” and “about” precede a list of numbers or ranges, those terms qualify all the values ​​or ranges provided in that list. In some cases, the term “about” may include numbers rounded to the nearest significant figure.

[0072] Unless otherwise defined, scientific and technical terms used in connection with this invention shall have the meanings that are ordinarily understood by those skilled in the art. Furthermore, unless the context specifically requires otherwise, singular terms shall include plural forms, and plural terms shall include singular forms.

[0073] In this application, the use of “or” means “and / or” unless otherwise stated. In the context of multiple dependent claims, the use of “or” selectively refers only to more than one preceding independent or dependent claim. Also, terms such as “element” or “component” include both elements and components containing one unit and element, and components containing more than one subunit, unless otherwise specifically stated.

[0074] Exemplary techniques used in relation to 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)).

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

[0076] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical composition or preparation other than the active ingredient, and that component is non-toxic to the target. pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0077] Unless otherwise specified, “individual” or “subject” is human. Where specified, “individual” or “subject” may include non-human mammals or non-human mammals (e.g., “mammalian subject” or “non-human mammalian subject”). Mammals include, but are not limited to, domestic animals (e.g., cattle, 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 may also be specified (e.g., birds or fish).

[0078] As used herein, “treatment” (and its grammatical variations such as “treat” or “treating”) refers to a clinical intervention in an attempt to alter the natural course of a disease in an individual being treated, which may be carried out for preventive purposes or in the course of clinicopathology. Desired effects of treatment include, but are not limited to, preventing the onset or recurrence of the disease, reducing symptoms, reducing the direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, restoring or alleviating the disease state, and achieving remission or improving prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of the disease or to slow the progression of the disease.

[0079] The "effective amount" of a substance, such as a pharmaceutical composition, refers to the amount that is effective in the dosage and duration required to achieve the desired therapeutic or preventive outcome.

[0080] The terms "cancer" and "cancerous" typically refer to or describe a physiological condition in mammals characterized by uncontrolled cell growth / proliferation. Examples of cancer include, but are not limited to, carcinomas, lymphomas (e.g., Hodgkin lymphoma and non-Hodgkin lymphoma), blastomas, sarcomas, and leukemias. More specific examples of such cancers 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, liver carcinoma, leukemia, and other lymphoproliferative disorders, as well as various types of head and neck cancers.

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

[0082] In some embodiments, the transmembrane E3 ubiquitin ligase is 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 containing an amino acid sequence selected from one of SEQ ID NOs. (See the 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 to both of two transmembrane E3 ubiquitin ligases, for example, RNF43 and ZNRF3. For instance, the multispecific binding protein may contain two different antibody fragments, one recognizing RNF43 and the other recognizing ZNRF3, thereby enabling binding to both ligases.

[0083] As shown in Figure 1C, for example, RNF43 is expressed in several cancer cell lines. Both RNF43 and ZNRF3 are members of the Wnt signaling pathway, which is activated in several 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 target tumor. For example, the constructs herein may be useful in reducing the levels of target cell surface proteins, high levels of which are known to promote cancer growth. Furthermore, certain target cells, such as cancer cells, may express high levels of transmembrane E3 ubiquitin ligases such as RNF43 or ZNRF3, which may allow multispecific binding proteins to preferentially bind to those target cells compared to other normal or unaffected cells expressing lower levels of that ligase. Therefore, 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 targets such as cancer.

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

[0085] In some embodiments, multispecific binding proteins may also block the ability of transmembrane E3 ubiquitin ligases to bind to normal ligands. For example, in some embodiments, these proteins may block the ability of FZD or LRP6 to bind to RNF43 or ZNRF3. In other embodiments, multispecific binding proteins do not block the binding of transmembrane E3 ubiquitin ligases to their endogenous ligands.

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

[0087] The multispecific binding proteins of this disclosure can recognize a second cell surface protein, such as a transmembrane E3 ubiquitin ligase, in addition to the transmembrane E3 ubiquitin ligase, which can be targeted for degradation by proximity to the transmembrane E3 ubiquitin ligase. A wide variety of cell surface proteins exist that can be targeted for degradation by the multispecific binding proteins 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, as well as MUC21 and MUC22. Mucins such as MUC1 may be overexpressed in certain cancers. Examples of Siglec (sialic acid-binding immunoglobulin 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, and 4-1BB. Examples of specific cell surface proteins that can be targeted by the multispecific binding proteins described 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 multispecific binding proteins as described herein include, for example, HER2, IGF1R, EGFR, FZLD5, EpCAM, and PD-L1. Depending on the specific target cell surface protein selected, the multispecific binding proteins as described herein include, for example, anti-HER2 antibodies such as 4D5, 7C2, or 2C4, or antibodies or antibody fragments derived from anti-IGF1R antibodies such as cixutumumab, ganitumab, dalotuzumab, figitumumab, robatumumab, teprotumumab, or istilatumumab.

[0088] Reducing the cell surface levels of these or other proteins may be useful in the treatment of, for example, cancer. However, this multispecific binding protein is useful in a wide variety of situations where it is actually necessary to reduce the level of a specific protein on the cell surface by inducing its degradation. For example, selectively reducing the level of a specific cell surface protein in an in vitro cell culture or tissue sample may be useful in various experimental settings to study the effects of that protein on a particular mechanism. Therefore, the cell surface proteins targeted for disruption can include a wide variety of cell surface proteins.

[0089] A. Exemplary multispecificity binding protein format In certain embodiments, the multispecificity binding proteins provided herein are multispecific antibodies, e.g., bispecificity antibodies or triplicity antibodies. In other embodiments, the multispecificity binding proteins may comprise one or more antigen-binding fragments derived from an antibody that bind to other protein domains, e.g., ligand-binding domains derived from non-antibody proteins. A “multispecificity antibody” is generally a monoclonal antibody that has binding specificity to at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain embodiments, a multispecificity antibody has three or more binding specificities. Multispecificity antibodies can be prepared as full-length antibodies or antibody fragments. A multispecificity molecule that binds to two sites or targets is “bispecific,” and a multispecificity molecule that binds to three sites or targets is “triplicity.” Thus, bispecificity binding proteins herein, such as bispecificity antibodies, bind to both transmembrane E3 ubiquitin ligases and cell surface proteins. Triple-specific binding proteins, such as triple-specific antibodies, can be constructed to bind to, for example, a cell surface protein and one or more transmembrane E3 ubiquitin ligases, one or more cell surface proteins and one transmembrane E3 ubiquitin ligase, or one cell surface protein and one transmembrane E3 ubiquitin ligase, but to bind to any two sites of the cell surface protein or ligase.

[0090] For example, in some embodiments, the multispecific binding proteins described herein may be engineered to bind to RNF43 and one or more second cell surface proteins. In other embodiments, the protein may be engineered to bind to ZNRF3 and one or more second cell surface proteins. Alternatively, the protein may be engineered to bind to both RNF43 and ZNRF3 and one or more second cell surface proteins. In yet another embodiment, the multispecific binding proteins described herein may 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] Methods for producing multispecific antibodies include, but are not limited to, the recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)) and the "knob-in-hole" operation (see, for example, U.S. Patent No. 5,731,168 and Atwell et al., J.Mol.Biol.270:26 (1997)). Multispecific antibodies can also be produced using techniques such as: manipulating the electrostatic steering effect to create antibody Fc heterodimer molecules (see, e.g., WO2009 / 089004); crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent 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 WO2011 / 034605); using common light chain techniques to avoid light chain mispairing problems (see, e.g., WO98 / 50431); and using "diabody" techniques to produce bispecific antibody fragments (see, e.g., Hollinger et al.) They can also be prepared by the use of single-stranded Fv(sFv) dimers (see, for example, Gruber et al., J.Immunol., 152:5368 (1994)); as well as by the preparation of trispecific antibodies, such as those described in, for example, Tutt et al. J.Immunol. 147:60 (1991).

[0092] For example, engineered antibodies having three or more antigen-binding sites, including "Octopus antibody," or DVD-Ig are also included herein (see, e.g., WO2001 / 77342 and WO2008 / 024715). Other examples of multispecific antibodies having three or more antigen-binding sites can be found in WO2010 / 115589, WO2010 / 112193, WO2010 / 136172, WO2010 / 145792 and WO2013 / 026831. Bispecific antibodies or their antigen-binding fragments also include "dual-acting FAb" or "DAF" having 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, e.g., US2008 / 0069820 and WO2015 / 095539).

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

[0094] Various further molecular formats for multispecific antibodies are known in the art and are included herein (see, for example, Spiess et al., Mol Immunol 67(2015) 95-106).

[0095] Similarly, certain types of multispecific antibodies included herein are bispecific antibodies designed to simultaneously bind to a surface antigen on a target cell, for example, 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, where one binding specificity is to the cell surface protein to be degraded, and the other binding specificity is to 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 mobile linker (see, e.g., WO2004 / 106381, WO2005 / 061547, WO2007 / 042261 and WO2008 / 119567, Nagarsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011)); diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and their derivatives, e.g., tandem diabodies ("TandAb"; Kipriyanov et al., J Mol Biol 293, 41-56 (1999)); and "DART" (Dual Affinity Retargeting) molecules based on the diabody format but featuring a C-terminal disulfide crosslink for additional stabilization (Johnson et al., J Mol Biol Examples include, but are not limited to, so-called triomabs (Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)), which are whole molecules of hybrid mouse / rat IgG molecules. Specific 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) may be adapted to bind to targets such as those described herein instead of T cells, for example, by replacing the CD3-binding antibody fragment with an antibody fragment that binds to a cell surface ligase.

[0097] In some embodiments, the multispecific antibodies herein may have a symmetric "1+1 FabIgG" or "1+1 FvIgG" bispecific format, comprising two Fv domains that recognize different targets linked to an interacting Fc region, or two Fab regions that recognize different targets linked to an interacting Fc region. Some further examples of bispecific antibody formats herein are provided in Figures 15A–15C: 2+1 FabIgG, 1-arm FvIgG, and 1-arm FabIgG, respectively. These formats are, for example, asymmetric. Examples of tripspecific antibodies that bind to cell surface protein targets and 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 include an interacting Fc region containing 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, substitutions of one or more amino acids on a CH3 or CH2 region on one Fc domain to amino acids larger than those naturally occurring at that position, and substitutions of one or more adjacent amino acids on a partner Fc domain to amino acids smaller than those normally occurring. (See, for example, U.S. Patent No. 5,731,168 and Atwell et al., J.Mol.Biol.270:26 (1997), as well as, for example, WO2009 / 089004, WO2012 / 106587 and U.S. Patent Publication No. 2009 / 0182127). In some embodiments, knob-into-hole modifications may 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 include a substitution of T366 in human IgG1 Fc with a larger amino acid such as W (and optionally one of S354C or Y349C), and the accompanying “hole mutation” in the partner Fc may include substitutions of T366, L368, and Y407 with smaller amino acids (e.g., T366S, L368A, and Y407V) (and optionally Y349C or S354C). (See, for example, Carter, P. et al., Immunotechnol. 2 (1996) 73.) Numbering follows EU index numbering.

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

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

[0100] In certain embodiments, multispecificity-binding proteins, such as multispecificity antibodies provided herein, may be further modified to include additional non-proteinoid moieties known and readily available in the art. Suitable sites for antibody derivatization 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 homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in production due to its stability in water. The polymer may have any molecular weight and may be branched or not. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same molecule or different molecules. In general, the number and / or type of polymers used for derivatization may be determined based on considerations including, but not limited to, specific properties or functions of the antibody being improved, regardless of whether the antibody derivative is used in a process such as under defined conditions.

[0101] B. Exemplary anti-RNF43 and anti-ZnRF3 antibodies useful in constructing multispecific binding molecules In one aspect, the Disclosure also provides antibodies that specifically bind to RNF43 and / or ZnRF3, which may be useful in constructing the multispecific binding proteins relating to the Disclosure. These include 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, and RNF43-17, which are mouse, rat, or rabbit antibodies as described in the Examples below. 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,This includes 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 their antibodies.

[0102] In some embodiments, the multispecific binding proteins described herein are (a) 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,The multispecific binding protein comprises a heavy chain variable domain (VH) containing heavy chain complementarity-determining region 1 (CDR-H1), CDR-H2 and / or CDR-H3 of any one of the antibodies listed above: 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) containing heavy chain complementarity-determining region 1 (CDR-H1), CDR-H2 and CDR-H3 of any one of the antibodies listed above.

[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,It comprises a light chain variable domain (VL) containing light chain complementarity-determining region 1 (CDR-L1), CDR-L2 and / or CDR-L3 of any one of the following antibodies: 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) a light chain variable domain (VL) containing light chain complementarity-determining region 1 (CDR-L1), CDR-L2 and CDR-L3 of any one of those antibodies. In some embodiments, the multispecific binding protein includes all of the above-mentioned anti-RNF43 antibody or anti-ZNRF3 antibody CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3.

[0104] 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, RNF43-5It contains a VH that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequence to one of the heavy chain variable regions (VHs) among RNF43-15, RNF43-2, RNF43-21, RNF43-13, RNF43-19, ZNRF3-331, ZNRF3-333, or ZNRF3-332.

[0105] 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, RNF43-5It contains a light chain variable region (VL) that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequence to one of the following VHs: RNF43-15, RNF43-2, RNF43-21, RNF43-13, RNF43-19, ZNRF3-331, ZNRF3-333, or ZNRF3-332.

[0106] In some cases, the multispecificity binding protein 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 the antibodies, and a VL 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 VL of any one of the antibodies.

[0107] 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, RNF43-5Contains a VH (Variable Helical Region) amino acid sequence of any one of the following: 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 the 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, R 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 -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 contain a VL containing the amino acid sequence of one of these light chain variable regions (VL). In some cases, the above protein contains both the VH and 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, SEQ ID NOs. 33 to 444.

[0108] In further embodiments, a multispecific binding protein can be constructed using one or more amino acid sequences provided in SEQ ID NOs: 1 to 32 in the following sequence listing.

[0109] In some embodiments, the multispecific binding proteins, including the above-mentioned CDR or VH / VL, have binding affinities to ZNRF3 or RNF43 of less than 50 nM, less than 10 nM, less than 1 nM, less than 0.5 nM, less than 0.05 nM, or 50 nM to 10 nM, 10 nM to 1 nM, 1 nM to 0.5 nM, 0.5 nM to 0.05 nM, or 0.05 nM to 0.01 nM. In some cases, the affinity is measured using a BIACORE® surface plasmon resonance assay, for example, as described in the examples below.

[0110] In some embodiments, the multispecific binding protein binds to both ZNRF3 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 cell surface proteins targeted for their ligase and degradation. In some embodiments, it may have a format as shown in FIGS. 15A-15G, FIGS. 7A-B or FIG. 8A.

[0111] In some embodiments, the multispecific binding protein comprising the above CDR 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 having one or more amino acid substitutions (e.g., the LALAPG mutation or substitution at N297, e.g., N297G or N297Q) to reduce, for example, 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 optionally selected constant regions or Fc regions are further discussed below.

[0112] C. Conjugates Comprising Multispecific Binding Proteins In other embodiments, the multispecific binding proteins herein can be further conjugated (chemically linked) to one or more other molecules, such as labels or therapeutic agents. A variety of radioisotope labels are available for the production of radiolabeled conjugates. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212, P 32 Pb 212 Examples include radioactive isotopes of iodine, iodine, and lu. When radioactive conjugates are used for detection, they may include radioactive atoms for scintigraphy studies (e.g., tc99m or I123) or spin labels for nuclear magnetic resonance (NMR) imaging (also known as MRI) (e.g., iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron). 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, a multispecific binding protein is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more of the therapeutics described above. The antibody is typically linked to one or more therapeutics using a linker. An overview of ADC technology, including examples of therapeutics, drugs, and linkers, is presented in Pharmacol Review 68:3-19 (2016). Other promising conjugated therapeutic agents include, but are not limited to, diphtheria A chain, unbound active fragments of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modesine A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitors, curcin, crotin, sapaonaria officinalis inhibitors, geronin, mitogellin, restrictosin, phenomycin, enomycin, and trichothecenes, as well as enzymatically active toxins or their fragments.

[0114] Conjugates of proteins and cytotoxic agents can be prepared using various bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), difunctional derivatives of imide esters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, lysine 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 the conjugation of radioactive nucleotides to antibodies. See WO94 / 11026. Linkers may be “cleavable linkers” that facilitate the release of cytotoxic drugs within cells. For example, acid-unstable linkers, peptidase-sensitive linkers, photosensitive linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) may be used.

[0115] The immunoconjugates or ADCs described herein are, but are not limited to, commercially available conjugates prepared with crosslinking 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).

[0116] D. Antibody variant 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 in certain aspectsAmino 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 intended. For example, it may be desirable to alter the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from residues in the amino acid sequence of the antibody and / or insertions into residues in the amino acid sequence of the antibody and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be performed to reach the final construct, insofar as the final construct has the desired properties (e.g., antigen binding).

[0117] a) Substitution, insertion, and deletion variants In a particular embodiment, antibody variants having one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include the CDR and framework regions of the variable region. Conservative substitutions are shown under the heading "Preferred Substitutions" in Table A. More substantial substitutions are provided under the heading "Exemplary Substitutions" in Table A, as further described below with respect to amino acid side chain classes. Amino acid substitutions may be introduced into the antibody of interest, for example, in the variable region and / or constant region, and the product may be screened for desired activity, such as retention / improvement of antigen binding, reduction of immunogenicity, or improvement of ADCC or CDC. [Table A]

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

[0119] Non-conservative substitution involves exchanging a member of one class with a member of another class.

[0120] Certain substitution variants involve substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Generally, the resulting variants, selected for further study, will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody, and / or will substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity-mature antibodies, which can be readily produced using phage display-based affinity maturation methods, such as those described herein. Briefly, one or more CDR residues are mutated, the variant antibody is displayed on a phage, and screened for specific biological activity (e.g., binding affinity).

[0121] For example, modifications (e.g., substitutions) may be made in the CDR to improve antibody affinity. Such modifications may be made in CDR "hot spots," i.e., residues encoded by codons that frequently mutate during the somatic cell maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or residues that come into contact with the antigen, and the resulting variant VH or VL is tested for binding affinity. Affinity maturation through the construction of a secondary library and subsequent re-selection 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 several embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by one of various methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide directional mutagenesis). A secondary library is then constructed. This library is then screened to identify any antibody variant with the desired affinity. Another method for introducing diversity is a CDR-directed approach, which randomizes several CDR residues (e.g., 4-6 residues at a time). CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are particularly often targeted.

[0122] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, provided that such alterations do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative alterations that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made in a CDR. Such alterations may, for example, be outside the antigen-contact residue within the CDR. In certain variant VH and VL sequences provided above, each CDR is either 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 by Cunningham and Wells (1989) Science, 244:1081-1085. This method identifies one residue or group of a target residue (e.g., charged residues such as arg, asp, his, lys, and glu) and substituted it with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine if the antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions that are functionally sensitive to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact residues and adjacent residues can be targeted or excluded as candidate substitutions. Variants can be screened to determine if they contain desired properties.

[0124] Amino acid insertions include amino-terminus and / or carboxyl-terminus 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 terminal insertion is an antibody with an N-terminal methionyl residue. Other insertion variants of antibody molecules include the fusion of the N-terminus or C-terminus of an antibody to an enzyme (e.g., ADEPT for antibody-targeted enzyme prodrug therapy) or to a polypeptide that increases the serum half-life of the antibody.

[0125] b) Glycosylated variants In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree of glycosylation of the antibody. The addition or deletion of glycosylation sites to an antibody can be conveniently achieved by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0126] If the antibody contains an Fc region, the oligosaccharide attached to it may be modified. Natural antibodies produced by mammalian cells typically contain branched oligosaccharides commonly attached to Asn297 of the CH2 domain of the Fc region by N-bonding. See, for example, Wright et al. TIBTECH 15:26-32 (1997). 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 branched oligosaccharide structure. In some embodiments, modification of the oligosaccharide in the antibody of the present invention may be performed to produce antibody variants having certain improved properties.

[0127] In one embodiment, an antibody variant is provided having a non-fucosylated oligosaccharide, i.e., an oligosaccharide structure lacking fucose (directly or indirectly) attached to the Fc region. Such a non-fucosylated oligosaccharide (also referred to as "afucosylated" oligosaccharide) is, in particular, an N-linked oligosaccharide lacking a fucose residue attached to the first GlcNAc in the stem of a branched oligosaccharide structure. In one embodiment, an antibody variant is provided having an increased proportion of non-fucosylated oligosaccharides in the Fc region compared to the native antibody or the parent antibody. For example, the proportion of non-fucosylated 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). The percentage of non-fucosylated oligosaccharides is the (average) amount of fucose-less oligosaccharides relative to the total of all oligosaccharides attached to Asn297 (e.g., complex, hybrid, and high-mannose structures), as measured by MALDI-TOF mass spectrometry, for example, as described in WO2006 / 082515. Asn297 refers to the asparagine residue located at approximately position 297 (EU numbering of Fc region residues) within the Fc region, however, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence changes in the antibody. Antibodies with an increased percentage of non-fucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and / or improved effector function, particularly improved ADCC function. See, for example, U.S. Patent Application Publication 2003 / 0157108; U.S. Patent Application Publication 2004 / 0093621.

[0128] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13CHO cells with insufficient protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. 2003 / 0157108; and WO2004 / 056312, particularly 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.) Examples include cells in which GDP-fucose synthesis or transporter protein activity is reduced or eliminated (see, for example, U.S. Patent Application Publication No. 2004259150, U.S. Patent Application Publication No. 2005031613, U.S. Patent Application Publication No. 2004132140, and U.S. Patent Application Publication No. 2004110282).

[0129] In a further embodiment, antibody variants are provided, for example, bisected oligosaccharides in which a branched oligosaccharide attached to the Fc region of the antibody is 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] Antibody variants are also provided that have at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087;WO1998 / 58964; and WO1999 / 22764.

[0131] c) Fc region variant In certain embodiments, modifications to one or more amino acids may be introduced into the Fc region of an antibody provided herein, thereby enabling the creation of an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that includes amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0132] In some embodiments, the antibodies described herein have effector functions. In other embodiments, the antibodies described herein lack effector functions. In certain embodiments, the present invention intends to provide antibody variants having some, but not all, effector functions, which are desirable candidates for applications where the half-life of the antibody in vivo is important, but certain effector functions (e.g., complement-dependent cell-mediated cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / loss of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that an antibody lacks FcγR binding (and therefore 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. FcR expression 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 evaluating the ADCC activity of the target molecule are described in U.S. Patent No. 5,500,362 (see, for example, 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, for example, ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA) and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)).Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the target molecule may be evaluated in vivo in animal models, such as those 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 lacks CDC activity because it cannot bind to C1q. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To evaluate complement activation, a CDC assay 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 determination can also be performed using methods known in the field (see, e.g., Petkova, S B et al., Int'l.Immunol.18(12):1759-1769(2006); WO2013 / 120929 Al).

[0133] Antibodies with reduced effector function include those containing one or more substitutions 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 those having two or more substitutions 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 exhibiting improved or reduced binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312 and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001)).

[0135] In a particular embodiment, the antibody variant comprises an Fc region having one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333 and / or 334 (residues in 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, e.g., substitutions at positions 234 and 235 (residues in EU numbering) of the Fc region. In one embodiment, those substitutions are L234A and L235A (LALA). In certain embodiments, the antibody variant further comprises D265A and / or P329G in the Fc region derived from the human IgG1 Fc region. In one embodiment, those substitutions are L234A, L235A and P329G (LALAPG) in the Fc region derived from the human IgG1 Fc region. (See, for example, WO2012 / 130831) In another embodiment, those substitutions are L234A, L235A and D265A (LALA-DA) in the Fc region derived from the human IgG1 Fc region.

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

[0138] In some embodiments, modifications resulting in changes (i.e., either improvement or reduction) to C1q binding and / or complement-dependent cell injury (CDC) occur within the Fc region, as described, for example, in U.S. Patent No. 6,194,551, WO 99 / 51642 and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0139] Antibodies exhibiting 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)) are described in US2005 / 0014934 (Hinton et al.). These antibodies contain an Fc region with one or more substitutions within the Fc region that improve the binding of the Fc region to FcRn. Examples of such Fc variants include substitutions in one or more of the 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, for example, substitutions in Fc region residue 434 (see, for example, U.S. Patent No. 7,371,826; Dall'Acqua, WF, et al. J. Biol. Chem. 281 (2006) 23514-23524).

[0140] The Fc region residues crucial for the mouse Fc-mouse FcRn interaction have been identified by site-directed mutagenesis (see, for example, Dall'Acqua, WF, et al. J.Immunol 169(2002) 5171-5180). Residues I253, H310, H433, N434, and H435 (EU index numbering) are involved in this 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 have been found to be critical for the interaction between human Fc and 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 its 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) reported and investigated various mutants of residues 248-259, 301-317, 376-382, and 424-437.

[0141] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., substitutions at positions 253 and / or 310 and / or 435 (residues in EU numbering) of the Fc region. In certain embodiments, the antibody variant includes an Fc region having amino acid substitutions at positions 253, 310 and 435. In one embodiment, these substitutions are I253A, H310A and H435A in the Fc region derived from the human IgG1 Fc region. See, for example, 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, e.g., substitutions at positions 310 and / or 433 and / or 436 (residues in 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, these substitutions are H310A, H433A and Y436A in the Fc region derived from the human IgG1 Fc region. (See, for example, WO2014 / 177460Al).

[0143] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that increase FcRn binding, e.g., substitutions at positions 252 and / or 254 and / or 256 (residues in EU numbering) of the Fc region. In certain embodiments, the antibody variant includes an Fc region having amino acid substitutions at positions 252, 254 and 256. In one embodiment, these substitutions are M252Y, S254T and T256E in the Fc region derived from the human IgG1 Fc region. For other examples of variants of the Fc region, 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 an antibody as reported herein may be a complete C-terminus ending with the amino acid residue PGK. The C-terminus of the heavy chain may be a shortened C-terminus from which one or two C-terminal amino acid residues have been removed. In one preferred embodiment, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. In one embodiment of all the embodiments reported herein, an antibody comprising a heavy chain containing a C-terminal CH3 domain as specified herein contains a C-terminal glycine-lysine dipeptide (G446 and K447, amino acid positions in EU index numbering). In one embodiment of all the embodiments reported herein, an antibody comprising a heavy chain containing a C-terminal CH3 domain as specified herein contains a C-terminal glycine residue (G446, amino acid position in EU index numbering).

[0145] d) Cysteine-modified antibody variant In certain embodiments, it may be desirable to produce cysteine-modified antibodies, such as THIOMA® antibodies in which one or more residues of the antibody are substituted with cysteine ​​residues. In certain embodiments, the substituted residues are located in accessible sites on the antibody. By substituting these residues with cysteine, a reactive thiol group is positioned in an accessible site on the antibody, and by using this reactive thiol group, the antibody can be conjugated to other parts, such as a drug moiety or a linker drug moiety, as further described herein, to produce an immunoconjugate. Cysteine-modified antibodies can be produced, for example, as described in U.S. Patents 7,521,541, 8,30,930, 7,855,275, 9,000,130, or WO2016040856.

[0146] e) Antibody derivatives

[0147] In certain embodiments, the antibodies provided herein may be further modified to include additional non-proteinoid moieties known in the art and readily available. Suitable sites for antibody derivatization 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 homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in production due to its stability in water. The polymer may have any molecular weight and may be branched or not. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same molecule or different molecules. In general, the number and / or type of polymers used for derivatization may be determined based on considerations including, but not limited to, specific properties or functions of the antibody being improved, regardless of whether the antibody derivative is used in therapeutic applications such as under defined conditions.

[0148] E. Recombination methods and compositions The proteins described herein may be prepared 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 antibodies are provided.

[0149] For natural antibodies or natural antibody fragments, 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 encode the amino acid sequence containing the VL and / or VH of the antibody (e.g., the light chain and / or heavy chain). These nucleic acids may be present on the same expression vector or on different expression vectors.

[0150] In the case of a bispecific antibody having a heterodimer heavy chain requiring four nucleic acids, one is for the first light chain, one for the first heavy chain containing the first heteromonomer Fc region polypeptide, one for the second light chain, and one for the second heavy chain containing the second heteromonomer Fc region polypeptide. These four nucleic acids can be contained in one or more nucleic acid molecules or expression vectors. Such nucleic acids encode amino acid sequences containing the first VL and / or the first VH containing the first heteromonomer Fc region and / or the second VL and / or the second VH containing the second heteromonomer Fc region of the antibody (e.g., the first and / or second light chains and / or the first and / or second heavy chains of the antibody). These nucleic acids can reside on the same expression vector or on different expression vectors, and typically 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 EU index numbering, one heteromonomer heavy chain 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, isolated nucleic acids encoding antibodies or other components of multispecific binding proteins used in methods such as those reported herein are provided.

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

[0153] In the case of recombinant antibody production, for example, the nucleic acid encoding the antibody, as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody), or they can be produced by recombinant methods or 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 function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199 and 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, antibodies may be isolated from bacterial cell paste in a soluble fraction and further purified.

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

[0156] Host cells suitable for the expression of (glycosylated) antibodies can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified, and these 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, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES™ technology for antibody production in transgenic plants).

[0158] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 cell line (COS-7); human fetal kidney cell lines (e.g., 293 or 293T cells, as described in Graham, F. Let 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 tumor cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor cells (MMT 060562); and TRI cells (e.g., Mather, JP et al., Annals). These include cells such as those described in NYAcad.Sci.383(1982)44-68; MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells (Urlaub, G. et al., Proc. Natl. Acad.Sci. USA 77(1980)4216-4220), including DHFR-CHO cells; 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 cells are eukaryotic cells, such as Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells).

[0160] F. Pharmaceutical Compositions In a further embodiment, a pharmaceutical composition is provided comprising one of the multispecific binding proteins provided herein for use in, for example, any of the therapeutic methods described below. In one embodiment, the pharmaceutical composition comprises one of the proteins provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises one of the antibodies provided herein and at least one additional therapeutic agent, such as those described below.

[0161] Pharmaceutical compositions of proteins as described herein are prepared by mixing such antibodies of desired purity in the form of lyophilized compositions or aqueous solutions with one or more freely selected pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the doses and concentrations used, and include buffers, e.g., histidine, phosphoric acid, citrate, 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; alkylparabens, e.g., methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); and low molecular weight (less than about 10 residues) polypeptides. Examples of pharmaceutically acceptable carriers herein include, but are not limited to, cytoplasmic drugs; proteins, e.g., serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, e.g., sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, e.g., polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoproteins such as rHuPH20 (HYLENEX®, Halozyme, Inc.). Certain exemplary sHASEGP and methods of use, including rHuPH20, are described in U.S. Patent Application Publications 2005 / 0260186 and 2006 / 0104968.In one embodiment, sHASEGP is combined with one or more additional glycosaminoglycansases (e.g., chondroitinases).

[0162] An example of a lyophilized antibody composition is described in U.S. Patent No. 6,267,958. Examples of aqueous antibody compositions are described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter of which comprises a histidine-acetate buffer.

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

[0164] Pharmaceutical compositions for sustained release can be prepared. A preferred example of a sustained-release preparation is a semipermeable matrix of a solid hydrophobic polymer containing an antibody, which may be in the form of a molded article, such as a film or microcapsule.

[0165] Pharmaceutical compositions used for in vivo administration are generally sterilized. Sterilization can be easily achieved, for example, by filtration through a sterile filtration membrane.

[0166] G. Treatment methods and routes of administration Any of the multispecific binding proteins provided herein may be used in therapeutic methods. For example, they may be useful in any therapeutic method where it is beneficial to reduce the level of a particular cell surface protein in a target by targeting that protein for lysosomal degradation, and it is beneficial to do so in cells that readily express transmembrane E3 ubiquitin ligases.

[0167] In one embodiment, a multispecific binding protein is provided for use as a drug. In a further embodiment, a multispecific binding protein is provided for use in the treatment of diseases such as cancer. Examples of cancer include, but are not limited to, carcinomas, lymphomas (e.g., Hodgkin lymphoma and non-Hodgkin lymphoma), blastomas, sarcomas and leukemias. More specific examples of such cancers 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, liver carcinoma, leukemia and other lymphoproliferative disorders, as well as various types of head and neck cancers, autoimmune symptoms, inflammatory symptoms, neurodegenerative symptoms or infections.

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

[0169] In a further embodiment, the present invention provides multispecific binding proteins for use in reducing the levels of target cell surface proteins that are targeted by the multispecific binding proteins. In some cases, the target may have a disease such as cancer, autoimmune symptoms, inflammatory symptoms, neurodegenerative symptoms, or infections.

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

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

[0172] In a further embodiment, the present invention provides a method for reducing the level of a target cell surface protein targeted by a multispecific binding protein. In some cases, the target may have a disease such as cancer, autoimmune symptoms, inflammatory symptoms, neurodegenerative symptoms, or infectious diseases. For example, in some embodiments, the multispecific binding proteins described herein may be used to reduce the level of cell surface proteins on specific cell types by targeting E3 ubiquitin ligases that are 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 these ligases may be used in some embodiments to reduce the level of cell surface proteins on those cells. Furthermore, the transmembrane E3 ubiquitin ligases RNF133 and RNF148 are expressed on testicular cells, and multispecific binding proteins that target these ligases may be used in some embodiments to reduce the level of cell surface proteins on those cells.

[0173] In a further embodiment, the present invention provides a pharmaceutical composition comprising any of the multispecific binding proteins provided herein for use, for example, in any of the therapeutic methods described above. In one embodiment, the pharmaceutical composition comprises any of the multispecific binding proteins provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises any of the multispecific binding molecules provided herein and at least one additional therapeutic agent, such as those described below.

[0174] In other embodiments of the uses and treatment methods described herein, subjects may have mutations in the RNF43 or ZNRF3 proteins. For example, mutations in the RNF43 protein have been found in certain cancers, particularly colorectal cancer and endometrial cancer. See, for example, Y.J. van Herwaarden et al., Histopathology 78:749-758 (2021). In such cases, multispecific binding proteins containing an anti-RNF43 component may not be very effective in reducing the levels of the targeted cell surface protein. However, multispecific binding proteins containing an anti-ZNRF3 component can readily result in a reduction in the levels of the targeted cell surface protein. (See, for example, Example 29 and Figures 21-22 below). Therefore, in some embodiments, if a subject has been previously determined to have an RNF43 mutation or a ZNRF3 mutation, for example, if the subject has a cancer containing cancer cells with a mutation in RNF43 or ZNRF3, the subject may be administered a multispecific binding protein that does not bind to or activate the RNF43 or ZNRF3 protein. Therefore, for example, if the subject has an RNF43 mutation, the multispecific binding protein offered to the subject may contain an anti-ZNRF3 component, and vice versa. Therefore, 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, (a) if the subject has a mutation in RNF43, the multispecific binding protein will not bind to or activate RNF43, and (b) if the subject has a mutation in ZNRF3, the multispecific binding protein will not bind to or activate ZNRF3. Such mutations can be identified at the protein level, for example, 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.Since such mutations can be present in certain cancers, in some such forms, the subject may be a cancer subject.

[0175] The proteins described herein may be administered alone or used in combination therapy. For example, combination therapy may include 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 described above includes both combined administration (where two or more therapeutic agents are contained in the same or separate pharmaceutical composition) and separate administration, in which case the antibody of the present invention may be administered before, concurrently with, and / or after the administration of the additional therapeutic agents.

[0176] The multispecific binding proteins of the present invention (and any additional therapeutic agents) may be administered by any preferred means, including parenteral, intrapulmonary, and intranasal, and, if desired, intra-focal administration in the case of local treatment. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Dosage may be carried out by any preferred route, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term.

[0177] H. Kits and products and other methods of use In another aspect of this disclosure, the multispecificity binding molecules described herein may 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 instances where it may be beneficial to assay the behavior of cell or tissue samples in which the levels of specific cell surface signaling molecules have been artificially reduced. By using such multispecificity binding molecules, a relatively simple method for modulating the levels of such proteins may be provided.

[0178] Accordingly, the Disclosure also encompasses a method for reducing the level of cell surface proteins in a cell or tissue sample in vitro, the method comprising incubating the sample with a multispecific binding protein. The Disclosure also encompasses a kit for this purpose. The kit may include the multispecific binding protein, along with instructions for use, appropriate buffers and / or labeling molecules, as may be used. The kit may also include nucleic acids, vectors or host cells containing polynucleotides encoding the multispecific binding protein, so that the multispecific binding protein may be produced for use in such a manner, for example. [Examples]

[0179] Examples The following are examples of the methods and compositions of the present invention. It is understood that various other embodiments may be carried out based on the general description provided above.

[0180] Example 1: Development and characterization of rat anti-human cell surface ligase rat B cell antibody or rabbit B cell antibody Rats and rabbits were selected for the production of cell surface ligase antibodies because they offer certain advantages over alternative methods (e.g., screening of phage libraries). For example, antibodies obtained from animals may have higher specificity and better pharmacokinetic properties than phage-derived antibodies, potentially leading to better therapeutic candidates. Sprague Dawley rats (Charles River, Hollister, CA) were immunized with a priming dose of 100 μg of human (RNF43 or ZNRF3) protein solubilized in a detergent, mixed with MPL + TDM adjuvant (Sigma-Aldrich, St. Louis, MO), CFA (Sigma-Aldrich, St. Louis, MO), or a combination of TLR agonists: 50 μg of MPL (Sigma-Aldrich), 20 μg of R848 (Invivogen, San Diego, CA), 10 μg of PolyI:C (Invivogen), and 10 μg of CpG (Invivogen) separated into multiple sites. New Zealand white rabbits were also immunized with a mixture of the same protein solubilized in a surfactant, mixed with CFA (Sigma-Aldrich, St. Louis, MO). For additional protein booster immunization, the rats and rabbits were administered half the priming dose of protein diluted in PBS. This was administered to the rats and rabbits every two weeks. Polyclonal antiserum from these rats and rabbits was purified and tested by ELISA for binding to human RNF43 and human ZNRF3. In the case of rats, multiple lymph nodes were collected three days after the last immunization, showing detectable FACS reactivity to human RNF43 or human ZNRF3.These rat-derived IgM-negative B cells were purified from whole lymphocytes using magnetic separation (Miltenyi Biotec, San Diego, CA) and stained with anti-rat IgM antibody (Jackson ImmunoResearch, West Grove, PA), anti-rat CD45RA (Biolegend, San Diego, CA), anti-rat CD8a (Biolegend, San Diego, CA), and labeled human RNF43-Alex633 or human ZNRF3-Alex633 using the Lightning-Link Alex 633 Antibody Labeling Kit (Novus, Centennial, CO). Rat B cells exhibiting minimal rat IgM expression while simultaneously binding to human RNF43 or human ZNRF3 protein were selected and placed in 96-well plates containing feeder cells and cytokine-supplemented culture medium using a FACSAria III sorting machine (BD, Franklin Lakes, NJ). In rabbits, blood was collected 3 days after the last immunization showing detectable FACS reactivity to human RNF43 and human ZNRF3. These rabbit-derived IgG-positive B cells 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 using the Lightning-Link Alex 633 Antibody Labeling Kit (Novus, Centennial, CO). Rabbit B cells showing maximum rabbit IgG expression while simultaneously binding to human RNF43 or human ZNRF3 protein were sorted and placed in 96-well plates containing cytokine-supplemented culture medium with feeder cells using a FACSAria III sorter (BD, Franklin Lakes, NJ). Seven days after sorting, the supernatant was screened by ELISA for human RNF43 or human ZNRF3.The supernatant showing binding to human RNF43 or human ZNRF3 was 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 FACS binding of RNF43 or ZNRF3 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: Preparation of recombinant antibodies DNA encoding the heavy chain and light chain variable domains of the antibody was synthesized by gene synthesis. The synthesized gene fragments were inserted into mammalian expression vectors containing the corresponding heavy chain constant domain or light chain constant domain. Species and isotypes included human IgG1 and mouse IgG2a. Several variable domain sequences were edited to remove apparent unpaired cysteine ​​residues and NX[S / T]N-glycosylation motifs. Recombinant antibodies were produced by transient transfection of Expi293 cells with mammalian expression vectors encoding the antibody heavy and light chains. The heavy and light chains were encoded on separate vectors and transfected using a 1:1 ratio of heavy chain expression vector to light chain expression vector. Antibodies were purified from the cell culture supernatant by affinity chromatography. In some cases, the antibodies underwent an additional purification step based on SEC.

[0182] Using the knob-into-hole technique (Ridgway et al., Prot Eng. 1996), bispecific antibodies were constructed using human IgG1 or mouse IgG2a scaffolds typically containing mutations that reduce effector function (e.g., L234A, L235A, P329G, and / or N297G). After expressing and purifying the antibodies containing either the knob or the hole, they were assembled into a bispecific format essentially as previously described (Williams et al., Biotechnol Prog., 2015). In some cases, mutations were introduced to enable bispecific expression in single cells with appropriate light chain pairing (Dillon et al., mAbs, 2017).

[0183] Example 3: Affinity and epitope determination of recombinant antibody 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 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 domains to the captured antibodies was analyzed using single-cycle kinetics at 25°C 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 domains 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 at 30 μl / min for 30 seconds. Data were 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 sub-nanomolar affinity, as shown in Tables 1-4. Cell surface ligase antibodies with sub-nanomolar affinity may, in certain cases, offer advantages in degradation efficiency correlated with the durability of the ternary complex, for example, as shown in Figure 5D. [Table 1] TIFF2026102613000004.tif159170 [Table 2] TIFF2026102613000006.tif76170 [Table 3] [Table 4]

[0184] Example 4: HTP Epitope Binning Antibodies produced by immunizing animals were reformatted to the 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 antibody was immobilized on a CMD 200 M SPR sensor prism (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 competitive analysis based on SPR. Immobilized antibodies were first exposed to 100 nM recombinant human-to-human RNF43 or ZNRF3 extracellular domains using HBS-EP running buffer (10 mM HEPES, 150 mM NaCl, 0.05% Tween 20, pH 7.4, 1 mM EDTA), and then exposed to 10 ug / ml antibody in solution. 10 mM glycine / HCl pH 1.7 was used as the regeneration buffer. The results of epitope binning 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: Evaluation 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 those modified to express N-terminally tagged ligases. N-terminal tags included epitope tags (gD) or HiBit tags (Promega) for quantification of tagged proteins using the Nano-glo HiBit Extracellular Detection System (Promega). Cell lines were pooled or cloned and, in some cases, included ligase expression on doxycycline-inducible promoters. Cells were plated in 96-well plates with 50,000–120,000 cell wells and grown in ATCC recommended medium (+ / - doxycycline) in the presence of various antibody concentrations. After 24 hours (or the period described), the medium was removed, the cells were washed with PBS (150 μL), and detached from the well surface by adding 100 μL of Millipore (Accutase) at 37°C for 10 minutes. Accutase activity was quenched by adding 100 μL of RPMI + 10% heat-inactivated fetal bovine serum containing penicillin, streptomycin, and glutamine, the cells were centrifuged at 1200 rpm for 4 minutes, and the supernatant was discarded. The cells were resuspended in 150 μL of FACS buffer (PBS containing 0.5% BSA and 0.05% sodium azide) at 4°C for 10 minutes, centrifuged, and the supernatant was discarded. The cells were incubated with 40 μL of staining antibody (final concentration 2 μg / ml) at 4°C for 45 minutes, washed twice with 150 μL of FACS buffer, and resuspended in 40 μL of FACS buffer for analysis. Examples of staining antibodies include xIGF-1R mIgG1 APC (1H7, ebioscience catalog #17-8849-42) and xRNF43 37.17APC (in-house produced reagent). Examples of background / negative control staining antibodies include NISTMab hIgG1APC and xRagweed mIgG1 APC (both in-house produced reagents).

[0186] Flow cytometry analysis was performed using a BD FACSCelesta® flow cytometer with the High Throughput Sampler (HTS) in high-throughput mode. Each sample was initially resuspended twice in a resuspension volume of 10 μl. Then, 10 μl of sample was aspirated from each well and passed through the cytometer at a flow rate of 180 μl / min. Between each sample, the system was washed with 400 μl (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 was used as a measure of IGF-1R levels on the cell surface.

[0187] Example 6: Gene-induced dimerization of ligases to cell surface receptors The effect of HER2-mediated chemically induced dimerization of RNF43 or ZNRF3 was evaluated using the iDimerize system (Takara Bio). Briefly, HEK293T cells were reverse-transfected with the 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 hours, cells were left untreated or treated with a 500 nM A / C heterodimerizing agent (Takara Bio; REF 635057) to induce ligase-targeted dimerization, and the cells were incubated for a further 24 hours. Next, the cells were dissolved 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 lysate was subjected to HA immunoprecipitation (IP) by incubation with 25 μl of Pierce HA epitope-tagged antibody agarose conjugate (2-2.2.14) (Thermo Scientific; REF 26182) at 4°C for 3 hours. 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 blotting and immunoprecipitation HT29, HEK293T, and SW1417 cells were plated in 6-well dishes (Corning, cat #3516) at a density of 500,000 cells per well and allowed to adhere overnight. Cells were treated for 40 hours with bispecific antibodies diluted to 1 ug / mL in RPMI-1640 medium supplemented with FBS, L-glutamine, and Pen / Strep. Where applicable, cells were pretreated with doxycycline (500 ng / ml), bortezomib (200 nM, Selleck, cat # S1013), E1 inhibitor (MLN7243, cat # S8341, 100 nM), or bafilomycin A (Tocris-Cat. No. 1334, 50 nM). After 24–48 hours of incubation, cells were lysed in RIPA buffer supplemented with a phosphatase / protease inhibitor (Thermo Fisher, Cat #78442) on ice for 20 minutes. The lysates were clarified and normalized, and 20ug of each sample was run on 4–12% Bis-Tris gel (Invitrogen, Cat #WG1403) at 90V for approximately 2 hours using BOLT MOPS SDS electrophoresis buffer. The gels were transferred to a nitrocellulose membrane using the iBlot 2 system with iBlot2 Nitrocellulose Regular stack (Invitrogen, Cat #IB23001) and transferred using protocol P0 (1 min at 20V, 4 min at 23V, and 2 min at 25V). The blots were blocked with 5% milk in TBST and blocked at room temperature for 1 hour. The blots were transferred to 5% milk in TBST containing antibodies against IGF1R-beta (Cell signaling, #3027) and vinculin (Cell signaling, cat #13901), and incubated overnight at 4°C. Each blot was washed four times with TBST for 5 minutes, and then incubated at room temperature for 1 hour in 5% milk containing IRDye secondary antibody (Li-Cor, Cat #926-32211). Each blot was washed four times for 5 minutes, and color development was performed 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 regulate the turnover of Wnt ligand receptors FZD and LRP on the cell surface (Figure 1A). This is achieved 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 datasets of various colorectal cancer patients in whom 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 RNF43 expression in CRCs compared to other tumor types and normal colon (Figure 1C). Importantly, in-situ hybridization of Rnf43 in a mouse colon cancer model demonstrated broad and homogeneous ligase expression within the entire tumor mass (Figure 17). The canonical roles of RNF43 and ZNRF3 were investigated using transfection with inducible plasmids that conditionally expressed either ligase in HPAF-II cells upon doxycycline (dox) addition. Overexpression of Dox-mediated WT RNF43 resulted in ligase-dependent cell surface clearance of the FZD receptor (Figure 1D). To explore the potential for degradation of RNF43 and ZNRF3 beyond their native substrates, namely FZD and LRP5 / 6, we utilized the idimerize system. HEK293T cells were transfected with the pBind plasmid to induce constitutive co-expression of RNF43-DmrA-HA and HER2-DmrC-FLAG. 24 hours after the addition of the AC heterodimerizer, increased binding between RNF43 and HER2 was detected by immunoblotting of HER2 following immunoprecipitation of RNF43 (Figure 1E, left panel). This increased binding of the ligase to the target was functional as it resulted in HER2 target degradation (Figure 1E, right panel).

[0190] Example 9: Expression of tagged RNF43 ligase and tagged ZNRF3 ligase and dimerization with HER2 The inventors further evaluated the ability of both cell surface ligases to degrade the receptor when associated with bispecific antibodies. They constructed ligases tagged with gD at the N-terminus and dimerized these ligases to HER2 using bispecific antibodies targeting gD with three different HER2 epitopes (4D5, 7C2, and 2C4) (Figures 2A-B). Flow cytometry analysis of the cell surface expression of both ligases (Figure 2C) confirmed that gD-based dimerization via either HER2 epitope resulted in HER2 degradation. This was dependent on ligase expression (Figure 2D). The inventors further verified 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 decreased pERK activity (Figure 2E).

[0191] Example 10: Antibody-dependent cell surface clearance Bispecific antibodies targeting three different HER2 epitopes (4D5, 7C2, and 2C4) (Fendly et al. Canc Res 1990) and either a gD epitope tag or an unrelated antigen (human CD3) were constructed 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. The various antibodies exhibited 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 that observed with the 2C4 / gD bispecific antibody. To understand the effect of cell surface clearance on HER2 degradation, we examined HER2 levels by Western blotting. Similar to what was observed by flow cytometry, HER2 degradation required ligase dimerization to HER2. 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 lead to receptor degradation and that ligase dimerization is required (Figure 3B). To verify this claim, the inventors investigated the ubiquitination status of HER2 by immunoprecipitation. Ubiquitin smears were detected in HER2 after treatment with various HER2 / gD bispecific antibodies only in the presence of gD-tagged ligases (Figure 3C). Furthermore, deletion of the RING domain from any of the ZNRF3s prevented HER2 degradation after ligase dimerization, although not to the same extent as RNF43, indicating that ligase activity is necessary for HER2 degradation (Figure 3D). Conversely, inhibition of E1 ubiquitin-activating enzymes also rescued degradation, indicating that this depends on lysosomal activity rather than proteasome activity (Figure 3E).

[0192] Example 11: Cell surface ligase-mediated degradation is applicable to multiple receptors. Using chemoinducible dimerization, we investigated the ability of RNF43 to degrade additional cell surface receptors, particularly IGF1R, which are receptor tyrosine kinases, similar to HER2. Similar to what was observed with HER2, when HEK293T cells transfected with a pBind plasmid co-expressing RNF43-DmrA-HA and IGF-1R-DmrC-FLAG were treated with an AC heterodimerizer, binding between the two receptors increased in a dose-dependent manner (Figure 4A left panel). Consequently, this increased ligase-target binding led to dose-dependent degradation of IGF-1R (Figure 4A right panel). Bispecific antibodies were constructed using variable regions targeting IGF-1R and gD, and their ability to degrade IGF1R in HT-29 cells expressing doxycycline-inducible gD-tagged ZNRF3 was evaluated. Western blot analysis of HT-29 cells treated with the illustrated antibody at 1 ug / ml was performed 24 hours after treatment. For most bispecific antibodies, gD-ZnRF3 dimerization resulted in IGF1R degradation equivalent to the decrease in protein expression observed in conditional knockouts using sgRNAs specific to CRISPr Cas9 and IGF1R (Figure 4B). The phenotypic results of ligase-mediated degradation of cell surface receptors were further evaluated using HT55 colon cancer cells. These cells are IGF1R-dependent for survival both in vitro (Figure 4C) and in vivo (Figure 4D). Consistent with the viability deficit observed after gene-mediated IGF-1R knockout, HT55 cells treated for 7 days with bispecific antibodies targeting IGF-1R and gD showed a decrease in clonal proliferation dependent on ligase expression (Figures 4E, 4F).

[0193] Example 12: Kinetics of clearance from the cell surface Bispecific antibodies were constructed using variable regions targeting IGF-1R (cisutumumab) and RNF43 (hSC37.39, US20170073430A1). Flow cytometry was used to evaluate their ability to degrade IGF-1R in HT-29 cells via doxycycline-induced RNF43 overexpression (Figure 5D). One hour after antibody administration, cells treated with higher levels of bispecific antibody (≥0.1 ug / ml) showed slightly higher levels of cell surface IGF1R. By 3 hours, this trend reversed due to a decrease in cell surface IGF1R, and by 8 hours, this trend saturated. Continuous treatment up to 96 hours did not show further clearance of the target.

[0194] Example 13: Evaluation of bispecific antibodies for RNF43 and ZNRF3 As described above, subsets of the discovered RNF43 and ZNRF3 antibodies were reformatted into bispecific antibodies targeting their respective ligases and IGF1R (cisutumumab) (i.e., "PROTABs"). The activity of these ligases was evaluated by flow cytometry by their ability to drive IGF1R cell surface clearance from HT29 cells via doxycycline-inducible expression of the corresponding ligases (Figure 6A). Ligase-targeting antibodies with high monovalent affinity (over approximately 1 nM) to these ligases showed saturated levels of IGF1R clearance when paired with cisutumumab, while low-affinity antibodies showed decreased activity (Figure 6B). Further evaluation of the ZNRF3-IGF-1R PROTABs was performed in SW1417 cells expressing 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 blotting confirmed cytolytic degradation of IGF-1R in SW1417 cells after treatment with RNF43-IGF-1R antibody (Figure 6C).

[0195] While not bound by theory, the correspondence between ligase affinity and degradation efficiency is assumed to be due to the durability of the ternary complex between IGF1R and the antibody-ligase. Biochemical estimations suggested that at least four ubiquitins must be delivered to the target to enable efficient degradation, and therefore a long-lived complex is required to allow for multiple catalytic cycles. The durability of the entire complex was determined by the binding affinity of both sides of the bispecific antibody. For ligase antibodies much stronger than 1 nM, the affinity of cisutumumab (approximately 5 nM) was the major determinant in the half-life of the complex, and therefore the degradation efficiency saturated. For ligase antibodies weaker than 1 nM, we observed a decrease in clearance corresponding to the affinity of the ligase arm. Interestingly, no strong epitope dependence was observed with the ligase antibodies, so for RNF43 and ZNRF3, the specific geometry of the interaction did not strongly influence the degradation efficiency. This is potentially different from what the inventors observed with HER2 antibodies paired with anti-gD Tag antibodies (Figures 2A-2B), where changes in the epitopes of HER2-targeting antibodies are thought to affect degradation efficiency. While not bound by theory, the inventors hypothesize that this is due to a relative subtle distinction between epitopes on a single extracellular protease-associated domain of the ligase and an overall change in epitopes targeting different domains of HER2. Therefore, in the case of bispecific ligase antibodies with other (non-cyzutumumab) arms, the affinity at which this saturation occurs may vary in accordance with the affinity of that arm.

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

[0197] Example 15: Effect of binding distance on target degradation The inventors hypothesized that the distance between the ligase and the target could affect the efficiency of target clearance. To investigate this phenomenon, bispecific antibodies in one-arm FabIgG (OA-FabIgG) and one-arm FvIgG (OA-FvIgG) formats were constructed (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 expression of the corresponding ligase induced by doxycycline (Figures 8B-8C). Both the OA-FvIgG and OA-FvIgG formats increased degradation compared to the corresponding 1+1 bispecific antibodies. Similar results were observed with both cizutumumab and istilatumab as anti-IGF1R antibodies in the case of the Fab-IgG format. Furthermore, the results shown in Figure 8 suggest that the degradation efficiency can be fine-tuned or optimized by changing the position of each antigen-binding domain, for example, by positioning the ligase antigen-binding domain either externally (Fv in OA-FvIgG or Fab in OA-FabIgG) or internally.

[0198] Example 16: Combination of bond valency and shortest distance The Fab of cisutumumab was positioned internally to produce 2+1 Fab-IgG and FvIgG antibodies containing 2×RNF37.39 and 1× cisutumumab. Evaluation of these antibodies in the cell surface clearance assay described in Example 5 above revealed that they cleared IGF1R more efficiently than the corresponding conventional knob-in-hole (1+1) bispecific antibodies. This data was further validated by examining total IGF1R levels using Western blot analysis with DLD1 (Figure 24).

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

[0200] Example 18: Identification and Characterization of Novel Cell Surface Ligases To identify the putative cell surface ligases, the domain structures of RNF43 and ZNRF3 were evaluated (Figure 10A). This revealed the presence of an N-terminal signal peptide (SP) associated with membrane integration. Therefore, a list of known E3 ubiquitin ligases was evaluated using both UNIPROT and Signal-P software to identify ligases possessing SPs. Of the identified proteins, we evaluated the following 15 ligases, including both single-pass and multi-pass transmembrane domain ligases: RNF13, RNF43, RNF128, RNF130 (with a double transmembrane domain, Figure 10B), RNF133, RNF148, RNF149, RNF150, RNF167, ZNRF3, ZNRF4, LYNX1, RSPRY1, SYVN1 (with a quintuple transmembrane domain, Figure 10B), and TRIM7.

[0201] Doxycycline-inducible pBind plasmids containing IRES-eGFP encoding all 15 ligases with an N-terminal gD tag were constructed (Figure 10B). Cell surface presentation of each ligase was evaluated using FACS analysis. Briefly, HEK293T cells were transiently transfected with each ligase plasmid. After 24 hours, cells were treated with 1 ug / ml doxycycline for a further 24 hours to induce ligase expression. Live cells were then prepared for FACS analysis by staining with an anti-gD primary antibody followed by an APC secondary antibody. IRES-eGFP signaling was examined to assess doxycycline induction (Figure 10C). Median APC 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, as well as HEK293T cells constitutively expressing gD-tagged Fzd8, a known cell surface protein, were also evaluated.

[0202] In parallel with FACS analysis, the degradation capacity 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 gD+HER2(7C2) bispecific antibody for 24 hours. The cells were then lysed and prepared for Western blot analysis to examine the total level of HER2 upon bispecific antibody treatment (Figures 10E and 10F). Evaluation of these cell surface ligases in standard cytodegradation assays reveals that numerous cell surface ligases can dimerize to HER2, potentially leading to efficient degradation of HER2.

[0203] Additional experiments were conducted as follows. Figure 10D shows that RNF128, RNF130, RNF133, RNF149, and RNF150 showed detectable cell surface expression, while the expression of RNF13, RNF148, and RNF167 was substantially low. To evaluate whether cell surface expression broadly enabled the degradation of non-natural targets, the inventors conducted gD * Cells were treated with IGF1R PROTAB, and IGF1R levels were monitored. As shown in Figure 32, Western blot analysis confirmed that degradation was driven by co-localization of validated cell surface E3 ubiquitin ligases to IGF1R. Conversely, ligases with cell surface expression undetectable by flow cytometry failed to induce targeted degradation, as shown in Figure 33. We also created bispecific antibodies against gD targeting either HER2 or programmed death ligand 1 (PD-L1), two therapeutically valid cancer targets. Similar to IGF1R, degradation of HER2 and PD-L1 occurred upon association with various ligases, as shown in Figures 34 and 35, but degradation was not detected when using ligases with undetectable cell surface expression, as shown in Figures 36 and 37.

[0204] Figure 31 shows that some of the newly identified cell surface E3 ligases exhibited distinct tissue expression patterns, suggesting 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, suggesting these ligases could be used to target hematopoietic-derived cells, while RNF133 and RNF148 expression was primarily limited to the testes, suggesting these ligases could be used to target testicular cells.

[0205] The catalytic activity of E3 ubiquitin ligases is considered essential for facilitating the final transfer of ubiquitin from ubiquitin conjugate enzymes (E2) to substrates, thereby altering their substrate function. To test the importance of catalytic activity, catalytic and inactive versions of each individual ligase can be used in degradation assays to assess whether catalytic activity is necessary for each ligase to mediate the degradation of cell surface receptors. Preliminary data suggest that certain ligases may require catalytic activity while others do not. Cell surface expression of newly identified ligases was verified using separate cell lines. Each individual ligase was transfected into HT29 cells selected with puromycin for stable incorporation. Stable lines were treated with 1 ug / ml doxycycline for 24 hours to induce ligase expression. Live cells were then prepared for FACS analysis by staining with anti-gD primary antibody followed by APC secondary antibody. As outlined, the percentage of APC-positive cells was also quantified from three independent experiments for each sample (Figure 27A, B).

[0206] Example 19: Cell surface clearance of various substrates We will create bispecific antibodies that target various structurally different cell surface proteins, including gD epitopes and exemplary receptor tyrosine kinases such as HER2, IGF1R, and EGFR, as well as multi-pass transmembrane domain proteins such as FZLD5, proteins with large extracellular and intracellular domains such as EpCAM, and proteins with immunoglobulin domains including short intracellular domains such as PD-L1. A 10ug / ml antibody is added to cells overexpressing ligases tagged with an N-terminal gD tag. Western blot analysis reveals that the addition of the bispecific antibody drives the degradation of the target antigen. An example using HT29 cells that stably express various cell surface ligases is shown in Figure 29. Dimerization of several ligases to IGF1R using the gD / IGF1R antibody results in significant receptor degradation.

[0207] Example 20: Evaluation of Wnt signaling pathway activation by TOPBrite TCF reporter assay We evaluated the activation of Wnt / β-Catenine by rat or rabbit multispecific antibodies targeting ZNRF3 using a Wnt-dependent reporter Nano-Glo Dual® luciferase system (Promega) containing a control promoter expressing sea urchin luciferase and a TCF promoter expressing firefly luciferase. 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 following day, 0.1 mg / mL of rat or rabbit cloned ZNRF3 antibody and 100 ng / mL of recombinant human Wnt3a (Abcam) were added to the cells. 2 μM of GSK 3β inhibitor or 500, 10, and 0.2 ng / μL of RSPO3 were used as positive controls in the presence of 100 ng / mL of Wnt3a. Cells treated with DMSO or 100 ng / mL of Wnt3a were used 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 and gently mixed. 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 after incubation for 10 minutes using the same settings, the sea urchin luminescence signal was measured. The relative ratio was calculated from the ratio of firefly to sea urchin luminescence and then further normalized by taking the ratio of the untreated sample. The results for the ZNRF3 antibody are shown in Figures 11A-11B.

[0208] Example 21: Evaluation of IGF1R cell surface clearance using NanoLuc® luciferase complementation assay HiBiT tags were introduced to the N-terminus of IGF1R using a CRISPR-Cas9 system on HT29 cells. A single clone with the highest NanoLuc® luciferase luminescence readout value was selected and validated 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 varying antibody concentrations. After 24 hours (or the period described), cells were washed once with 100 μL of PBS and then supplemented with 100 μL of fresh medium. Detection reagents were prepared by diluting LgBiT protein in a 1:100 ratio and substrate in a 1:50 ratio in the desired volume of detection buffer supplied in the detection kit. 100 μL of detection reagent was then added to cells in 100 μL of fresh medium. For the Nano-Glo® HiBiT extracellular detection system, which detects IGF1R at the surface level, detection was performed by incubation at room temperature for 10 minutes while gently mixing using a plate shaker. % clearance = (untreated sample RLU - treated sample RLU) / untreated sample RLU * Percent IGF1R clearance was calculated using the formula 100. RNF43 and ZNRF3 multispecific antibodies were also evaluated in this cell line (Tables 5-7). A subset of the samples was run in two separate runs, in which case both results are included. [Table 5] TIFF2026102613000010.tif252170TIFF2026102613000011.tif42170 [Table 6] [Table 7]

[0209] Tables 5-7 show the percentage 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 novel format antibodies, including Fv-IgG and 2+1 triplicate formats, induced cell surface IGF1R-HibiT clearance in HT29 at a concentration of 1 μg / mL.

[0210] Example 22: Monitoring of IGF1R surface clearance kynetics using 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 antibodies and novel format antibodies were added to the cells and incubated at various time points: 0, 4, 8, and 24 hours. The IGF1R-HibiT luminescence signal was detected using the HibiT extracellular system as described in the previous examples. Percent IGF1R was expressed using the formula %IGF1R=(1-(untreated sample RLU-treated sample RLU) / untreated sample RLU) * The calculation was performed using a value of 100. Various multispecific antibodies showed different degradation rates (Figures 12A-12F).

[0211] Example 23: Multiplex NanoLuc Luciferase Complementary 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 medium containing lactate dehydrogenase was collected for the LDH cytotoxicity assay (Promega), and HiBiT extracellular detection reagent was added. Approximately 2–5 μL of this medium 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 the control sample to maximize LDH detection. The reaction was incubated at room temperature for 30–60 minutes, and the luminescence signal was measured using a GloMax® Discover Microplate Reader with an integration time of 1 second. % 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 minutes, followed by luminescence detection using a GloMax® Discover Microplate Reader with an integration time of 1 second (Promega). Various multispecific antibodies showed variable effects on targeted degradation, but did not affect cell death as measured by the LDH assay or cell number as measured by Cell Titer-glo (Figures 13A–13C).

[0212] Example 24: Evaluation of IGF1R degradation using 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 incubation for 24 hours, the cells were incubated with HibiT lysis detection reagent to determine the total amount of IGF1R-HibiT. The lysis detection reagent was prepared by diluting LgBiT protein in a 1:100 ratio and substrate in a 1:50 ratio in the desired volume of detection buffer supplied in the detection kit. After 20 minutes of incubation with the lysis detection reagent, the luminescence signal was measured using a GloMax® Discover Microplate Reader with an integration time of 1 second. Percent IGF1R was calculated using the formula described in the previous example (Figure 14A). Western blotting 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 described (1. Cixu-RNF43 Fv-Ig, 2. Cizutumumab / RNF43.hSC37.39, 3. Cixu / NIST, 4. Cixu / Cixu) at 1 μg / mL for 24 hours. Untreated cells (5. UT) were used as a control. 20 μg of total protein was loaded into each lane, and the target protein was probed with an anti-IGF1 receptor antibody (Abcam EPR19322) that recognizes both pro-IGF1R (approx. 200 kDa) and IGF1R (approx. 95 kDa). β-actin is shown as a protein loading control (Figure 14B). Western blot analysis shows that the HiBit strain primarily possesses intracellular, non-degradable pro-IGF1R, which is only partially degraded in the soluble Hibit assay. Extracellular mature IGF1R is more abundant in WT HT-29 cells and is efficiently degraded by ligase bispecific antibodies, as shown by Western blotting.

[0213] Example 25: Triple-specific antibody for enhancing the degradation of IGF1R and EGFR Preliminary data showed that a combination of two conventional knob-in-hole bispecific antibodies (RNF43 / Cixu and ZNRF3 / Cixu bispecific antibodies) resulted in increased clearance. For example, to facilitate manufacturing and administration to the target, it may be advantageous to produce a single molecule with all desired antigen-binding domains, and therefore the 2+1 FabIgG format can be used. Figures 15D-15G show an exemplary trispecific antibody degrader using the 2+1 FabIgG format. Trispecific antibodies targeting RNF43 (RNF43.37.39), ZNRF3 (ZNRF3-6), and IGF1R (cisutumumab) or EGFR (cetuximab) are produced. The antibody format is Fab-IgG / IgG, in which the receptor tyrosine kinase antibody is positioned internally and the ligase antibodies are alternately positioned at two external positions. This arrangement can optimize proximity to each anti-ligase antigen-binding domain and further improve degradation efficiency. The Fab-IgG semiantibody incorporates an LC pairing mutation (Dillon et al. mAbs, 9:2, 213-230, 2017) that enables efficient pairing of LC arms. This FabIgG and IgG semiantibody 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 antibody.

[0214] Example 26: Monitoring of IGF1R surface clearance after combination of bispecific antibodies. The activity of multiple bispecific antibodies showed saturation at less than 100% clearance for HER2, EGFR, and IGF1R. We hypothesized that simultaneous treatment of a single target with two different ligases might improve clearance due to different rate-limiting steps. To investigate 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 day 2, 1 μg / mL of a single bispecific antibody (targeting IGF1R and ZNRF3 or RNF43) or a combination of two bispecific antibodies (the first targeting IGF1R and RNF43, and the second 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 as described in the previous example. Percent IGF1R was calculated using the formula %IGF1R=(1-(RLU in untreated sample + RLU in treated sample) / RLU in untreated sample). * Calculations were performed using a value of 100. Two different combinations of bispecific antibodies resulted in more efficient IGF1R clearance than the saturation level of either individual bispecific antibody (Figure 16).

[0215] Example 27: Degradation of IGF1R across multiple CRC strains As described above, subsets of the discovered RNF43 and ZNRF3 antibodies were reformatted into bispecific antibodies targeting their respective ligases and IGF1R (cisutumumab). The activity of these ligases was evaluated by their ability to drive whole-cell degradation of IGF1R in a large panel of DLD1 cells and colon cancer cell lines (Figure 18A, B). Notably, KM-12, RKO, GP2D, and JHH7 cells did not exhibit recognizable ligase expression and therefore served as negative controls (Figure 18B).

[0216] Example 28: Evaluation of target ubiquitination during treatment with divalent / bispecific antibody To evaluate the effect of bispecific antibody treatment on the ubiquitination of the target (IGF1R), parental HEK293T cells or HEK293T cells with 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 a 10 cm dish 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 hours of doxycycline induction, cells were subjected to medium change (+doxycycline) and treated with gDx cyzutumumab (IGF1R) bispecific antibody (0.5 μg / ml). After treating cells with a bispecific antibody at 37°C for 2 hours, cells were dissolved 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 clarified lysates were divided equally and incubated with either 20 μl / sample of pre-washed Pierce HA epitope-tagged antibody agarose conjugate (2-2.2.14) (Thermo Scientific; REF 26182) for nonspecific pull-down evaluation, or 20 μl / sample of pre-washed agarose-TUBE 2 beads (Life Sensors; REF UM402) to concentrate polyubiquitinated proteins. The lysate-bead mixture was incubated at 4°C for 2 hours and 30 minutes. 30 μl of the prepared pre-incubation pull-down sample was used as the input sample. After incubation, the beads were washed and prepared for Western blot analysis (Figure 19B).

[0217] Furthermore, IGF1R ubiquitination after IGF1 stimulation (R&D systems; REF 291-G1), bivalent antibody treatment (cidtumumab X cidtumumab), control bispecific antibody treatment (cidtumumab X NIST), or ligase-based bispecific antibody treatment (cidtumumab X RNF43-35; cidtumumab X ZNRF3-55) were evaluated in HT29 cells. Briefly, 20 million cells were plated in a 15 cm dish. 48 hours after plating, the cells were subjected to serum starvation. After 22 hours of serum starvation, the cells were subjected to bivalent or bispecific antibody treatment (1 μg / ml) simultaneously with a medium change (serum starvation medium). The cells were incubated at 37°C for 2 hours and 15 minutes. For IGF1 stimulation, the cells were treated with 50 ng / ml IGF1 5 minutes before cell lysis. After incubation, the 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 clarified lysates were divided equally and incubated with 1.87 μg / sample of pre-washed Dynabeads Protein G (ThermoFisher Scientific; REF 10004D) at a dose of 25 μl / sample, conjugated with either rabbit (DA1E) mAb IgG XP isotype control (cell signaling; REF 3900) for nonspecific bait protein precipitation or IGF-1 receptor β (D23H3) XP rabbit mAb (cell signaling; REF 9750) for IGF1Rβ immunoprecipitation. The lysate-bead mixture was incubated at 4°C for 24 hours. For the input sample, 60 μl was used from the prepared pre-incubation pull-down sample. After incubation, the beads were washed and prepared for Western blot analysis (Figure 19A).

[0218] Example 29: Verification of ligase activity requirements across cell lines and targets To support the claims made above regarding HER2 in Example 10, the inventors investigated 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, the inventors utilized a naturally occurring RNF43 mutation in a small subset of colon cancer. SW48 was selected because it has a frameshift deletion within the RING domain of RNF43 (Figure 21A) but maintains cell surface ligase expression (Figure 21B). Since dimerization with the RNF43 / IGF1R bispecific antibody in this cell line did not affect IGF1R levels, it was demonstrated that the RING and c-terminus of RNF43 are required for ligase activity. Notably, when ZNRF3 / IGF1R antibodies were used, ZNRF3 remained capable of degrading IGF1R. To further support this claim, we generated endogenous ligase knockouts for both RNF43 and ZNRF3 in HT29 cells (Figure 22A). As expected, RNF43 / IGF1R treatment of RNF43 KOs did not affect IGF1R levels, but ZNRF3 / IGF1R treatment was still able to degrade its receptor. Importantly, RING knockouts did not affect cell surface expression of either ligase (Figure 22A FACS panel), but degradation was also rescued, demonstrating that the presence of the RING / C-terminus is necessary for the complete degradation ability of either ligase (Figure 22B).

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

[0220] Example 30: Biological consequences of degradation on cell signaling and proliferation The biological effects of degradation on receptor binding were evaluated by focusing on the modulation of IGF1R signaling. SW48 cells treated with the ZNRF3 / IGF1R bispecific antibody exhibited significant IGF1R degradation. Importantly, this degradation also affected further downstream signaling, as evidenced by the complete loss of AKT activity (phosphorylation of AKT) and the reduction of S6 phosphorylation, two key 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 resulted in more significant pathway inhibition than classical blocking antibodies. This reduction in IGF1R signaling was also functionally transformed, as evidenced by the reduced cell proliferation in SW48 cells treated with ZNRF3 / IGF1R (Figure 25B, C). Consistent with the above, ZNRF3 RING-deficient HT29 cells did not affect cell proliferation, but WT ZNRF3 dimerized to IGF1R resulted in slower proliferation in these cells, indicating that the ligase domain was necessary to influence cell proliferation (Figure 26A-D).

[0221] Example 31: Endogenous degradation of additional substrates We constructed bispecific antibodies 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 almost complete degradation of PD-L1, demonstrating the breadth of degradation across multiple substrates.

[0222] The invention described above has been explained in some detail by illustrations and examples to clarify its understanding, but these explanations and examples should not be construed as limiting the scope of the invention. All patent and scientific literature disclosures cited herein are expressly incorporated by reference in their entirety.

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

[0224] Approximately 10 6 SW48 cells were resuspended in PBS basal medium and mixed with 50% Matrigel (Corning) to a final volume of 200 μl. This mixture was subcutaneously injected 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 calipers, and tumor volume was calculated as 0.523 × length × width × width. Animals were humanely euthanized according to the following criteria: persistent distress or clinical signs of pain, significant weight loss (>20%), and 2,500 mm². 3 If the tumor size exceeds this limit, or if the tumor has ulcerated. The maximum tumor size permitted by the Institutional Animal Care and Use Committee (IACUC) is 3,000 mm². 3 And in all experiments, this limit was not exceeded. The tumor was approximately 400 mm. 3 Once the target was reached, the animals were randomized and given a single intraperitoneal injection of PROTAB. 72 hours after injection, all mice were euthanized, and the tumors were collected for further processing.

[0225] The intraluminal implantation technique has been previously described (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 of buprenorphine. A blunt-ended hemostat (Micro-Mosquito, No. 13010 - 12, Fine Science Tools) was inserted approximately 1 cm into the anus. The hemostat was tilted towards the mucosa, slightly opened, and a single mucosal fold was grasped by closing the hemostat up to the first incision mark. The hemostat was retracted from the anus to expose the grasped mucosal fold. A 10 μL solution containing 50,000 cells mixed with 50% Matrigel (Corning) in PBS was injected directly into the colonic mucosa. After inverting the prolapse, the hemostat was released.

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

[0227] The lower part of Figure 30 shows mice left untreated (-), or the Cixu bivalent antibody, NIST * IGF1R (Cixu) or ZNRF3 - 55 *Western blot analysis of SW48 in vivo tumor lysates from mice treated with IGF1R (Cixu) bispecific PROTAB for 72 hours is shown. 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 the ZNRF3-based IGF1R PROTAB drove substantial degradation of IGF1R. Interestingly, IGF1R degradation was partially suppressed at the highest dose (Figure 30), suggesting a hook effect 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. Sequences The following table provides exemplary sequences referred to in this disclosure. [Table] TIFF2026102613000014.tif253170TIFF2026102613000015.tif254170TIFF2026102613000016.tif253170TIFF2026102613000017.tif254170TIFF2026102613000018.tif254170TIFF2026102613000019.tif254170TIFF2026102613000020.tif253170TIFF2026102613000021.tif254170TIFF2026102613000022.tif253170TIFF2026102613000023.tif254170TIFF2026102613000024.tif253170TIFF2026102613000025.tif254170TIFF2026102613000026.tif254170TIFF2026102613000027.tif254170TIFF2026102613000028.tif254170TIFF2026102613000029.tif254170TIFF2026102613000030.tif254170TIFF2026102613000031.tif254170TIFF2026102613000032.tif254170TIFF2026102613000033.tif254170TIFF2026102613000034.tif254170TIFF2026102613000035.tif254170TIFF2026102613000036.tif254170TIFF2026102613000037.tif254170TIFF2026102613000038.tif254170TIFF2026102613000039.tif254170TIFF2026102613000040.tif254170TIFF2026102613000041.tif254170TIFF2026102613000042.tif254170TIFF2026102613000043.tif254170TIFF2026102613000044.tif254170TIFF2026102613000045.tif254170TIFF2026102613000046.tif254170TIFF2026102613000047.tif254170TIFF2026102613000048.tif254170TIFF2026102613000049.tif254170TIFF2026102613000050.tif254170TIFF2026102613000051.tif254170TIFF2026102613000052.tif254170TIFF2026102613000053.tif254170TIFF2026102613000054.tif254170TIFF2026102613000055.tif254170TIFF2026102613000056.tif254170TIFF2026102613000057.tif254170TIFF2026102613000058.tif254170TIFF2026102613000059.tif254170TIFF2026102613000060.tif254170TIFF2026102613000061.tif254170TIFF2026102613000062.tif254170TIFF2026102613000063.tif254170TIFF2026102613000064.tif254170TIFF2026102613000065.tif254170TIFF2026102613000066.tif254170TIFF2026102613000067.tif254170TIFF2026102613000068.tif254170TIFF2026102613000069.tif254170TIFF2026102613000070.tif254170TIFF2026102613000071.tif254170TIFF2026102613000072.tif254170TIFF2026102613000073.tif254170TIFF2026102613000074.tif254170TIFF2026102613000075.tif254170TIFF2026102613000076.tif254170TIFF2026102613000077.tif254170TIFF2026102613000078.tif254170TIFF2026102613000079.tif254170TIFF2026102613000080.tif254170TIFF2026102613000081.tif254170TIFF2026102613000082.tif254170TIFF2026102613000083.tif254170TIFF2026102613000084.tif254170TIFF2026102613000085.tif255170TIFF2026102613000086.tif255170TIFF2026102613000087.tif255170TIFF2026102613000088.tif255170TIFF2026102613000089.tif254170TIFF2026102613000090.tif254170TIFF2026102613000091.tif254170TIFF2026102613000092.tif254170TIFF2026102613000093.tif254170TIFF2026102613000094.tif84170. Sequence Listing SEQUENCE LISTING <110> GENENTECH, INC. <120> MULTISPECIFIC BINDING PROTEIN DEGRADER PLATFORM AND METHODS OF USE <130> 01164-0011-00PCT <150> US 63 / 145,336 <151> 2021-02-03 <150> US 63 / 217,470 <151> 2021-07-01 <150> US 63 / 274,288 <151> 2021-11-01 <160> 459 <170> PatentIn version 3.5 <210> 1 <211> 1419 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: gD.gD.mIgG2a.LALAPG.AV-Hole (heavy chain (HC) DNA including signal peptide (SP)) <400> 1 atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattcagag 60 gttcagctgg tggagtctgg cggtggcctg gtgcagccag ggggctcact ccgtttgtcc 120 tgtgccgctt ctggctactc catcacctcc gactttgcct ggaactgggt ccgtcaggcc 180 ccgggtaagg gcctggaatg ggttggatac attagttact ctggaaccac tagctataac 240 cctagcctga agtcccgtat cactataagt cgcgacaatt ccaaaaacac attctacctg 300 cagatgaaca gcctgcgtgc tgaggacact gccgtctatt attgtgctcg agaaaactac 360 tatggccgtt ctcacgttgg gtacttcgac gtctggggtc aaggaaccct ggtcaccgtc 420 tcgagtgcca aaacaacagc cccatcggtc tatccactgg ctcctgtgtg tggagataca 480 actggctcct cggtgactct aggatgcctg gtcaagggtt atttccctga gccagtgacc 540 ttgacctgga actctggatc cctgtccagt ggtgtgcaca ccttcccagc tgtcctgcag 600 tctgacctct acaccctcag cagctcagtg actgtaacgt cgagcacctg gcccagccag 660 tccatcacct gcaatgtggc ccacccggca agcagcacca aggtggacaa gaaaattgag 720 cccagaggac ccacaatcaa gccctgtcct ccatgcaaat gcccagcacc taacgccgcg 840. ggtggaccat ccgtcttcat cttccctcca aagatcaagg atgtactcat gatctccctg agccccatag tcacatgtgt ggtggtggat gtgagcgagg atgacccaga tgtccagatc 900 agctggtttg tgaacaacgt ggaagtacac acagctcaga cacaaaccca tagagaggat tacaacagta ctctacgcgt ggtcagtgcc ctccccatcc agcaccagga ctggatgagt ggcaaggagt tcaaatgcaa ggtcaacaac aaagacctcg gagcgcccat cgagagaacc 1080 atctcaaaac ccaaagggtc agtaagagct ccacaggtat atgtcttgcc tccaccagaa 1140 gaagagatga ctaagaaaca ggtcactctg acctgcgctg tcacagactt catgcctgaa 1200 gacatttacg tggagtggac caacaacggg aaaacagagc taaactacaa gaacactgaa 1260 ccagtcctgg actctgatgg ttcttacttc atggttagca agctgagagt ggaaaagaag 1320 aactgggtgg aaagaaatag ctactcctgt tcagtggtcc acgagggtct gcacaatcac 1380 cacacgacta agagcttctc ccggactccg ggtaaatga 1419 <210> 2 <211> 714 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: gD.gD.mIgG2a.LALAPG.AV-Hole (light chain (LC) DNA including SP) <400> 2 atgggatggt catgtatcat cctttttcta gtagcaactg caactggagt acattcagat 60 atccagatga cccagtcccc gagctccctg tccgcctctg tgggcgatag ggtcaccatc 120 acctgccgtg ccagtgcgtc tgtcgacagc tatggtaaca gcttcatcca ctggtatcag 180 cagaaaccag gaaaagctcc gaaactactg atttaccggg cctcggacct ggagtctgga 240 gtcccttctc gcttctctgg atccggttct gggacggatt tcactctgac catcagcagt 300 ctgcagccag aagacttcgc aacttattac tgtcagcaaa attacgcgga tccgttcaca 360 tttggacagg gtaccaaggt ggagatcaag cgcgctgatg ctgcaccaac tgtatccatc 420 ttcccaccat ccagtgagca gttaacatct ggaggtgcct cagtcgtgtg cttcttgaac 480 aacttctacc ccaaagacat caatgtcaag tggaagattg atggcagtga acgacaaaat 540 ggcgtcctga acagttggac tgatcaggac agcaaagaca gcacctacag catgagcagc 600 accctcacgt tgaccaagga cgagtatgaa cgacataaca gctatacctg tgaggccact 660 cacaagacat caacttcacc cattgtcaag agcttcaaca ggaatgagtg ttga 714 <210> 3 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: gD.gD.mIgG2a.LALAPG.AV-Hole heavy chain (mature sequence) <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Phe Ala Trp Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Gly Tyr Ile Ser Tyr Ser Gly Thr Thr Ser Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Phe Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Asn Tyr Tyr Gly Arg Ser His Val Gly Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Ala 115 120 125 Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser 130 135 140 Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr 180 185 190 Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala 195 200 205 His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly 210 215 220 Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Ala 225 230 235 240 Ala Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val 245 250 255 Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val 260 265 270 Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val 275 280 285 Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser 290 295 300 Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met 305 310 315 320 Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Gly Ala 325 330 335 Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro 340 345 350 Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln 355 360 365 Val Thr Leu Thr Cys Ala Val Thr Asp Phe Met Pro Glu Asp Ile Tyr 370 375 380 Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr 385 390 395 400 Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Val Ser Lys Leu 405 410 415 Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser 420 425 430 Val Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser 435 440 445 Arg Thr Pro Gly Lys 450 <210> 4 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: gD.gD.mIgG2a.LALAPG.AV-Hole light chain (mature sequence) <400> 4 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ala Ser Val Asp Ser Tyr 20 25 30 Gly Asn Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asp Leu Glu Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Tyr 85 90 95 Ala Asp Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln 115 120 125 Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr 130 135 140 Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln 145 150 155 160 Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg 180 185 190 His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro 195 200 205 Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 210 215 <210> 5 <211> 1389 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: xRNF43.hSC37.39.mIgG2a.LALAPG.AV-Hole.AX (HC DNA with SP) <400> 5 atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattcacag 60 gtgcagctgg tgcagtccgg cgccgaggtg aagaagcccg gcgcctccgt gaaggtgtcc 120 tgcaaggcct ccggctacac cttcaccacc tacaccatcc actgggtgag gcaggccccc 180 ggccagggcc tggagtggat gggctacatc aaccccaggt ccggctacac cgagtacaac 240 cagaagttcc aggacagggt gaccatgacc agggacacct ccacctccac cgtgtacatg 300 gagctgtcct ccctgaggtc cgaggacacc gccgtgtact actgcgccag gtcctacgag 360 ttctggggcc agggcaccac cgtgaccgtc tcgagtgcca aaacaacagc cccatcggtc 420 tatccactgg ctcctgtgtg tggagataca actggctcct cggtgactct agatgcctg 480 gtcaagggtt atttccctga gccagtgacc ttgacctgga actctggatc cctgtccagt 540 ggtgtgcaca ccttcccagc tgtcctgcag tctgacctct acaccctcag cagctcagtg 600 actgtaacgt cgagcacctg gcccagccag tccatcacct gcaatgtggc ccaccccggca 660 aggcagcacca aggtggacaa gaaattgag cccagaggac ccaaatcaa gccctgtcct 720 ccatgcaaat gcccagcacc taacgccgcg ggtggaccat ccgtcttcat cttccctcca 780 aagatcaagg atgtactcat gatctccctg agccccatag tcacatgtgt ggtggtggat 840 gtgagcgagg atgacccaga tgtccagatc agctggtttg tgaacaacgt ggagtacac 900 960 ctcccccatcc agcaccagga ctggatgagt ggcaaggagt tcaaatgcaa ggtcaacaac 1020 aaagacctcg gagcgcccat cgagagaacc atctcaaaac ccaaagggtc agtaagagct 1080 ccacaggtat atgtcttgcc tccaccagaa gaagagatga ctaagaaaca ggtcactctg 1140 acctgcgctg tcacagactt catgcctgaa gacatttacg tggagtggac caacaacggg 1200 aaaacagagc taaactacaa gaacactgaa ccagtcctgg actctgatgg ttcttacttc 1260 atggttagca agctgagagt ggaaaagaag aactgggtgg aaagaaatag ctactcctgt 1320 tcagtggtcc acgagggtct gcacaatcac cacacgacta agagcttctc ccggactccg 1380 ggtaaatga 1389 <210> 6 <211> 701 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: xRNF43.hSC37.39.mIgG2a.LALAPG.AV-Hole.AX (LC DNA with SP) <400> 6 atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattcagag 60 atcgtgatga cccagtcccc cgccaccctg tccgtgtccc ccggcgagag ggccaccctg 120 tcctgcaagg cctcccagaa cgtgggcatc aacgtggcct ggtatcagca gaagcccggc 180 caggccccca gggccctgat ctactccgcc tcctacaggt actccggcat ccccgccagg 240 ttctccggct ccggctccgg caccgagttc accctgacca tctcctccct gcagtccgag 300 gactcgccg tgtactactg ccaccagtac aagacctacc cctacacctt cggcggcggt 360 accaagctgg agatcaagcg cgctgatgct gcaccaactg tatccatctt cccaccatcc 420 agtgagcagt taacatctgg aggtgcctca gtcgtgtgct tcttgaacaa cttctacccc 480 aaagacatca atgtcaagtg gaagattgat ggcagtgaac gacaaaatgg cgtcctgaac 540 agttggactg atcaggacag caaagacagc acctacagca tgagcagcac cctcacgttg 600 accaaggacg agtatgaacg acataacagc tatacctgtg aggccactca caagacatca 660 acttcaccca ttgtcaagag cttcaacagg aatgagtgtt g 701 <210> 7 <211> 443 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: xRNF43.hSC37.39.mIgG2a.LALAPG.AV-Hole.AX mature HC <400> 7 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Pro Arg Ser Gly Tyr Thr Glu Tyr Asn Gln Lys Phe 50 55 60 Gln Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Tyr Glu Phe Trp Gly Gln Gly Thr Thr Val Thr Val Ser 100 105 110 Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys 115 120 125 Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly 130 135 140 Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr 165 170 175 Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser 180 185 190 Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys 195 200 205 Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys 210 215 220 Cys Pro Ala Pro Asn Ala Ala Gly Gly Pro Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr 245 250 255 Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser 260 265 270 Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His 275 280 285 Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile 290 295 300 Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn 305 310 315 320 Asn Lys Asp Leu Gly Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys 325 330 335 Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu 340 345 350 Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Ala Val Thr Asp Phe 355 360 365 Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu 370 375 380 Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr 385 390 395 400 Phe Met Val Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg 405 410 415 Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His 420 425 430 Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys 435 440 <210> 8 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: xRNF43.hSC37.39.mIgG2a.LALAPG.AV-Hole.AX mature LC <400> 8 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Asn Val Gly Ile Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ala Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Lys Thr Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <210> 9 <211> 1410 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: NIST.mIgG2a.LALAPG.AV-Hole (HC DNA with SP) <400> 9 atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattcacag 60 gtgaccctga gggagtccgg ccccgccctg gtgaagccca cccagaccct gaccctgacc 120 tgcaccttct ccggcttctc cctgtccacc gccggcatgt ccgtgggctg gatcaggcag 180 ccccccggca aggccctgga gtggctggcc gacatctggt gggacgacaa gaagcactac 240 aacccctccc tgaaggacag gctgaccatc tccaaggaca cctccaagaa ccaggtggtg 300 ctgaaggtga ccaacatgga ccccgccgac accgccacct actactgcgc cagggacatg 360 atcttcaact tctacttcga cgtgtggggc cagggcacca ccgtgaccgt ctcgagtgcc 420 aaaacaacag ccccatcggt ctatccactg gctcctgtgt gtggagatac aactggctcc 480 tcggtgactc taggatgcct ggtcaagggt tatttccctg agccagtgac cttgacctgg 540 aactctggat ccctgtccag tggtgtgcac accttcccag ctgtcctgca gtctgacctc 600 tacaccctca gcagctcagt gactgtaacg tcgagcacct ggcccagcca gtccatcacc 660 tgcaatgtgg cccacccggc aagcagcacc aaggtggaca agaaaattga gcccagagga 720 cccacaatca agccctgtcc tccatgcaaa tgcccagcac ctaacgccgc gggtggacca 780 tccgtcttca tcttccctcc aaagatcaag gatgtactca tgatctccct gagccccata 840 gtcacatgtg tggtggtgga tgtgagcgag gatgacccag atgtccagat cagctggtttt 900 gtgaacaacg tggaagtaca cacagctcag acacaaaccc atagagagga ttacaacagt 960 actctacgcg tggtcagtgc cctccccatc cagcaccagg actggatgag tggcaaggag 1020 ttcaaatgca aggtcaacaa caagacctc ggagcgccca tcgagagaac catctcaaaa 1080 cccaaagggt cagtaagagc tccacaggta tatgctttgc ctccaccaga agaagagatg 1140 actaagaaac aggtcactct gacctgcgct gtcacagact tcatgcctga agacatttac 1200 gtggagtgga ccaacaacgg gaaaacagag ctaaactaca agaacactga accagtcctg 1260 gactctgatg gttcttactt catggttagc aagctgagag tggaaaagaa gaactgggtg 1320 gaaagaaata gctactcctg ttcagtggtc cacgagggtc tgcacaatca ccacacgact 1380 aagagcttct cccggactcc gggtaaatga 1410 <210> 10 <211> 699 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: NIST.mIgG2a.LALAPG.AV-Hole (LC DNA with SP) <400> 10 atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattcagac 60 atccagatga cccagtcccc ctccaccctg tccgcctccg tgggcgacag ggtgaccatc 120 acctgctccg cctcctccag ggtgggctac atgcactggt atcagcagaa gcccggcaag 180 gcccccaagc tgctgatcta cgacacctcc aagctggcct ccggcgtgcc ctccaggttc 240 tccggctccg gctccggcac cgagttcacc ctgaccatct cctccctgca gccgacgac 300 ttcgccacct actactgctt ccagggctcc ggctacccct tcaccttcgg cggcggtacc 360 aaggtggaga tcaagcgcgc tgatgctgca ccaactgtat ccatcttccc accatccagt 420 gagcagttaa catctggagg tgcctcagtc gtgtgcttct tgaacaactt ctaccccaaa 480 gacatcaatg tcaagtggaa gattgatggc agtgaacgac aaaatggcgt cctgaacagt 540 tggactgatc aggacagcaa agacagcacc tacagcatga gcagcaccct cacgttgacc 600 aaggacgagt atgaacgaca taacagctat acctgtgagg ccactcacaa gacatcaact 660 tcacccattg tcaagagctt caacaggaat gagtgttga 699 <210> 11 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: NIST.mIgG2a.LALAPG.AV-Hole (HC mature) <400> 11 Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ala 20 25 30 Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys His Tyr Asn Pro Ser 50 55 60 Leu Lys Asp Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Asp Met Ile Phe Asn Phe Tyr Phe Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val 115 120 125 Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr 130 135 140 Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr 145 150 155 160 Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser 180 185 190 Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala 195 200 205 Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile 210 215 220 Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Ala Ala Gly Gly 225 230 235 240 Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile 245 250 255 Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp 260 265 270 Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His 275 280 285 Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg 290 295 300 Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys 305 310 315 320 Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Gly Ala Pro Ile Glu 325 330 335 Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr 340 345 350 Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu 355 360 365 Thr Cys Ala Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp 370 375 380 Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Tyr Phe Met Val Ser Lys Leu Arg Val Glu 405 410 415 Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His 420 425 430 Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro 435 440 445 Gly <210> 12 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: NIST.mIgG2a.LALAPG.AV-Hole (LC mature) <400> 12 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Arg Val Gly Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Ala Asp Ala Ala Pro 100 105 110 Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly 115 120 125 Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn 130 135 140 Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn 145 150 155 160 Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser 165 170 175 Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr 180 185 190 Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe 195 200 205 Asn Arg Asn Glu Cys 210 <210> 13 <211> 2088 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: HER2.4D5VH-mIgG2aCH1-4D5VH.mIgG2a.LALAPG.Knob (HC DNA with SP) <400> 13 atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattcagaa 60 gttcagctgg tggagtctgg cggtggcctg gtgcagccag ggggctcact ccgtttgtcc 120 tgtgcagctt ctggcttcaa cattaaagac actatatatac actgggtgcg tcaggccccg 180 ggtaagggcc tggaatgggt tgcaaggatt tatcctacga atggttatac tagatatgcc 240 gatagcgtca agggccgttt cactataagc gcagacacat ccaaaaacac agcctacctg 300 cagatgaaca gcctgcgtgc tgaggacact gccgtctatt attgttctag atggggaggg 360 gacggcttct atgctatgga ctactggggt caaggaaccc tggtcaccgt ctcgagtgcc 420 aagaccaccg cccccagcgt gtaccccctg gcccccgtgt gcggcgacac caccggcagc 480 agcgtgaccc tgggctgcct ggtgaagggc tacttccccg agcccgtgac cctgacctgg 540 aacagcggca gcctgagcag cggcgtgcac accttccccg ccgtgctgca gagcgacctg 600 tacaccctga gcagcagcgt gaccgtgacc agcagcacct ggcccagcca gagcatcacc 660 tgcaacgtgg cccaccccgc cagcagcacc aaggtggaca agaagatcga gcccagaggc 720 cccaccatca agcccgaggt gcagctggtg gagagcggcg gcggcctggt gcagcccggc 780 ggcagcctga gactgagctg cgccgccagc ggcttcaaca tcaaggacac ctacatccac 840 tgggtgagac aggcccccgg caagggcctg gagtgggtgg ccagaatcta ccccaccaac 900 ggctacacca gatacgccga cagcgtgaag ggcagattca ccatcagcgc cgacaccagc 960 aagaacaccg cctacctgca gatgaacagc ctgagagccg aggacaccgc cgtgtactac 1020 tgcagcagat ggggcggcga cggcttctac gccatggact actggggcca gggcaccctg 1080 gtgaccgtct cgagtgccaa aacaacagcc ccatcggtct atccactggc tcctgtgtgt 1140 ggagatacaa ctggctcctc ggtgactcta ggatgcctgg tcaagggtta tttccctgag 1200 ccagtgacct tgacctggaa ctctggatcc ctgtccagtg gtgtgcacac cttcccagct 1260 gtcctgcagt ctgacctcta caccctcagc agctcagtga ctgtaacgtc gagcacctgg 1320 cccagccagt ccatcacctg caatgtggcc caccggcaa gcagcaccaa ggtggacaag 1380 aaaattgagc ccagaggacc caaatcaag ccctgtcctc catgcaaatg cccagcacct 1440 aacgccgcgg gtggaccatc cgtcttcatc ttccctccaa agatcaagga tgtactcatg 1500 atctccctga gccccatagt cacatgtgtg gtggtggatg tgagcgagga tgacccagat 1560 gtccagatca gctggtttgt gaacaacgtg gagatcaca cagctcagac acaaacccat 1620 agagaggatt aaacagtac tctacgcgtg gtcagtgccc tccccatcca gcaccaggac 1680 1740 gagagaacca tctcaaaacc caaagggtca gtaagagctc cacaggtata tgtcttgcct 1800 ccaccagaag aagagatgac taagaaacag gtcactctgt ggtgcatggt cacagacttc 1860 atgcctgaag acatttacgt ggagtggacc aacaacggga aaacagagct aaactacaag 1920 aacactgaac cagtcctgga ctctgatggt tcttacttca tgtacagcaa gctgagagtg 1980 gaaaagaaga actgggtgga aagaaatagc tactcctgtt cagtggtcca cgagggtctg 2040 cacaatcacc acacgactaa gagcttctcc cggactccgg gtaaatga 2088 <210> 14 <211> 702 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: HER2.4D5VH-mIgG2aCH1-4D5VH.mIgG2a.LALAPG.Knob (LC DNA with SP) <400> 14 atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattcagat 60 atccagatga cccagtcccc gagctccctg tccgcctctg tgggcgatag ggtcaccatc 120 acctgccgtg ccagtcagga tgtgaatact gctgtagcct ggtatcaaca gaaaccagga 180 aaagctccga aactactgat ttactcggca tccttcctct actctggagt cccttctcgc 240 ttctctggat ccagatctgg gacggatttc actctgacca tcagcagtct gcagccggaa 300 gacttcgcaa cttattactg tcagcaacat tatactactc ctcccacgtt cggacagggt 360 accaagctgg agatcaagcg cgctgatgct gcaccaactg tatccatctt cccaccatcc 420 agtgagcagt taacatctgg aggtgcctca gtcgtgtgct tcttgaacaa cttctacccc 480 aaagacatca atgtcaagtg gaagattgat ggcagtgaac gacaaaatgg cgtcctgaac 540 agttggactg atcaggacag caaagacagc acctacagca tgagcagcac cctcacgttg 600 accaaggacg agtatgaacg acataacagc tatacctgtg aggccactca caagacatca 660 acttcaccca ttgtcaagag cttcaacagg aatgagtgtt ga 702 <210> 15 <211> 676 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: HER2.4D5VH-mIgG2aCH1-4D5VH.mIgG2a.LALAPG.Knob (HC mature) <400> 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val 115 120 125 Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr 130 135 140 Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr 145 150 155 160 Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser 180 185 190 Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala 195 200 205 Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile 210 215 220 Lys Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 225 230 235 240 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys 245 250 255 Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 260 265 270 Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp 275 280 285 Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr 290 295 300 Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 305 310 315 320 Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp 325 330 335 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro 340 345 350 Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser 355 360 365 Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr 370 375 380 Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro 385 390 395 400 Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val 405 410 415 Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His 420 425 430 Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro 435 440 445 Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Ala Ala 450 455 460 Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu 465 470 475 480 Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser 485 490 495 Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu 500 505 510 Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr 515 520 525 Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser 530 535 540 Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Gly Ala Pro 545 550 555 560 Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln 565 570 575 Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val 580 585 590 Thr Leu Trp Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val 595 600 605 Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu 610 615 620 Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg 625 630 635 640 Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val 645 650 655 Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg 660 665 670 Thr Pro Gly Lys 675 <210> 16 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: HER2.4D5VH-mIgG2aCH1-4D5VH.mIgG2a.LALAPG.Knob (LC mature) <400> 16 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <210> 17 <211> 2082 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: HER2.2C4VH-mIgG2aCH1-2C4VH.mIgG2a.LALAPG.Knob (HC DNA with SP) <400> 17 atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattcagag 60 gtgcagctgg tggagagcgg cggcggcctg gtgcagccag gcggcagcct gcgcctgagc 120 tgcgccgcca gcggcttcac cttcaccgac tacaccatgg actgggtgcg ccaggcccca 180 ggcaagggcc tggagtgggt ggccgacgtg aacccaaaca gcggcggcag catctacaac 240 cagcgcttca agggccgctt caccctgagc gtggaccgca gcaagaacac cctgtacctg 300 cagatgaaca gcctgcgcgc cgaggacacc gccgtgtact actgcgcccg caacctgggc 360 ccaagcttct acttcgacta ctggggccag ggcaccctgg tgaccgtctc gagtgccaag 420 accaccgcc ccagcgtgta ccccctggcc cccgtgtgcg gcgacaccac cggcagcagc 480 gtgaccctgg gctgcctggt gaagggctac ttccccgagc ccgtgaccct gacctggaac 540 agcggcagcc tgagcagcgg cgtgcacacc ttccccgccg tgctgcagag cgacctgtac 600 accctgagca gcagcgtgac cgtgaccagc agcacctggc ccagccagag catcacctgc 660 aacgtggccc accccgccag cagcaccaag gtggacaaga agatcgagcc cagaggcccc 720 accatcaagc ccgaggtgca gctggtggag agcggcggcg gcctggtgca gcccggcggc 780 agcctgagac tgagctgcgc cgccagcggc ttcaccttca ccgactacac catggactgg 840 gtgagacagg cccccggcaa gggcctggag tgggtggccg acgtgaaccc caacagcggc 900 ggcagcatct acaaccagag attcaagggc agattcaccc tgagcgtgga cagaagcaag 960 aacaccctgt acctgcagat gaacagcctg agagccgagg acaccgccgt gtactactgc 1020 gccagaaacc tgggccccag cttctacttc gactactggg gccagggcac cctggtgacc 1080 gtctcgagtg ccaaaacaac agccccatcg gtctatccac tggctcctgt gtgtggagat 1140 acaactggct cctcggtgac tctaggatgc ctggtcaagg gttatttccc tgagccagtg 1200 accttgacct ggaactctgg atccctgtcc agtggtgtgc acaccttccc agctgtcctg 1260 cagtctgacc tctacaccct cagcagctca gtgactgtaa cgtcgagcac ctggcccagc 1320 cagtccatca cctgcaatgt ggccacccg gcaagcagca ccaggtgga caagaaaatt 1380 gagcccagag gacccacaat caagccctgt cctccatgca aatgcccagc acctaacgcc 1440 gcgggtggac catccgtctt catctccct ccagatca aggatgtact catgatctcc 1500 ctgagcccca tagtcacatg tgtggtggtg gatgtgagcg aggatgaccc agatgtccag 1560 atcagctggt ttgtgaacaa cgtggaagta cacacagctc agacacaac ccatagagag 1620 gattacaca gtacttacg cgtggtcagt gccctcccca tccagcacca ggactggatg 1680 agtggcaagg agttcaatg caggtcaac aaaaagacc tcggagcgcc catcgagaga 1740 accatctcaa aacccaagg gtcagtaaga gctccacagg tatatgtct gcccacca 1800 gagaagaga tgactaagaa acaggtcact ctgtggtgca tggtcacaga cttcatgcct 1860 gagacattt acgtggagtg gaccaac gggaaacag agctaacta gaccaac 1920 gaaccagtcc tggactctga tggttcttac ttcatgtaca gcaagctgag agtggaaaag 1980 aagaactggg tggaaagaaa tagctactcc tgttcagtgg tccacgaggg tctgcacaat 2040 caccacacga ctaagagctt ctcccggact ccgggtaaat ga 2082 <210> 18 <211> 702 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: HER2.2C4VH-mIgG2aCH1-2C4VH.mIgG2a.LALAPG.Knob (LC DNA with SP) <400> 18 atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattcagac 60 atccagatga cccagagccc aagcagcctg agcgccagcg tgggcgaccg cgtgaccatc 120 acctgcaagg ccagccagga cgtgagcatc ggcgtggcct ggtatcagca gaagccaggc 180 aaggccccaa agctgctgat ctacagcgcc agctaccgct acaccggcgt gccaagccgc 240 ttcagcggca gcggcagcgg caccgacttc accctgacca tcagcagcct gcagccagag 300 gactcgcca cctactactg ccagcagtac tacatctacc catacacctt cggccagggt 360 accaagctgg agatcaagcg cgctgatgct gcaccaactg tatccatctt cccaccatcc 420 agtgagcagt taacatctgg aggtgcctca gtcgtgtgct tcttgaacaa cttctacccc 480 aaagacatca atgtcaagtg gaagattgat ggcagtgaac gacaaaatgg cgtcctgaac 540 agttggactg atcaggacag caaagacagc acctacagca tgagcagcac cctcacgttg 600 accaaggacg agtatgaacg acataacagc tatacctgtg aggccactca caagacatca 660 acttcaccca ttgtcaagag cttcaacagg aatgagtgtt ga 702 <210> 19 <211> 674 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: HER2.2C4VH-mIgG2aCH1-2C4VH.mIgG2a.LALAPG.Knob (HC mature) <400> 19 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser 180 185 190 Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys 210 215 220 Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 225 230 235 240 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp 245 250 255 Tyr Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 260 265 270 Val Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg 275 280 285 Phe Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu 290 295 300 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 305 310 315 320 Cys Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln 325 330 335 Gly Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val 340 345 350 Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr 355 360 365 Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr 370 375 380 Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val 385 390 395 400 Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser 405 410 415 Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala 420 425 430 Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile 435 440 445 Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Ala Ala Gly Gly 450 455 460 Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile 465 470 475 480 Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp 485 490 495 Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His 500 505 510 Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg 515 520 525 Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys 530 535 540 Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Gly Ala Pro Ile Glu 545 550 555 560 Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr 565 570 575 Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu 580 585 590 Trp Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp 595 600 605 Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val 610 615 620 Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu 625 630 635 640 Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His 645 650 655 Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro 660 665 670 Gly Lys <210> 20 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: HER2.2C4VH-mIgG2aCH1-2C4VH.mIgG2a.LALAPG.Knob (LC mature) <400> 20 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Serves Trp Thr Asp Gln Asp Serves Lys Asp Serves Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <210> 21 <211> 2076 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: HER2.7C2VH-mIgG2aCH1-7C2VH.mIgG2a.LALAPG.Knob (HC DNA with SP) <400> 21 atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acatcacag 60 gtccaactgc agcagcctgg ggctgaactg gtgaggctg gagcttcagt gaagctgtcc 120 tgcaggctt ctggctactc cttcaccggc tactggatga actggttga gcagaggcct 180 ggacaaggcc ttgagtggat tggcatgatt catccttccg atagtgaaat tagggcaaat 240 cagaaattca gggacaaggc cacattgact gtagacaagt cctccaccac agcctacatg 300 caactcagca gcccgacatc tgaggactct gcggtctatt actgtgcacg agggacttac 360 gacggaggct ttgaatactg gggccaaggc accactctca cagtctcgag tgccaagacc 420 accgccccca gcgtgtaccc cctggccccc gtgtgcggcg acaccaccgg cagcagcgtg 480 accctgggct gcctggtgaa gggctacttc cccgagcccg tgaccctgac ctggaacagc 540 ggcagcctga gcagcggcgt gcacaccttc cccgccgtgc tgcagagcga cctgtacacc 600 ctgagcagca gcgtgaccgt gaccagcagc acctggccca gccagagcat cacctgcaac 660 gtggcccacc ccgccagcag caccaaggtg gacaagaaga tcgagcccag aggccccacc 720 atcaagcccc aggtgcagct gcagcagccc ggcgccgagc tggtgagacc cggcgccagc 780 gtgaagctga gctgcaaggc cagcggctac agcttcaccg gctactggat gaactggctg 840 aagcagagac ccggccaggg cctggagtgg atcggcatga tccaccccag cgacagcgag 900 atcagagcca accagaagtt cagagacaag gccaccctga ccgtggacaa gagcagcacc 960 accgcctaca tgcagctgag cagccccacc agcgaggaca gcgccgtgta ctactgcgcc 1020 agaggcacct acgacggcgg cttcgagtac tggggccagg gcaccaccct gaccgtctcg 1080 agtgccaaaa caacagcccc atcggtctat ccactggctc ctgtgtgtgg agatacaact 1140 ggctcctcgg tgactctagg atgcctggtc aagggttatt tccctgagcc agtgaccttg 1200 acctggaact ctggatccct gtccagtggt gtgcacacct tcccagctgt cctgcagtct 1260 gacctctaca ccctcagcag ctcagtgact gtaacgtcga gcacctggcc cagccagtcc 1320 atcacctgca atgtggccca cccggcaagc agcaccaagg tggacaagaa aattgagccc 1380 agaggaccca caatcaagcc ctgtcctcca tgcaatgcc cagcaccta cgccgcgggt 1440 ggaccatccg tctcatctt ccctccaag atcagatg tactcatgat ctccctgagc 1500 cccatagtca catgtgtgt ggtgtgtg agcgaggatg acccagatgt ccagatcagc 1560 tggttttgtga acaacgtgga agtacacaca gctcagacac aaacccatag agaggattac 1620 aacagtactc tacgcgtggt cagtgccctc cccatccagc accaggactg gatgagtgc 1680 aaggagttca atgcaggt spaceaa gacctcggag cgcccatcga gagaaccac 1740 tcaaaccca aagggtcagt aagagctcca caggtatatg tcttgccctc accagaagaa 1800 gagatgacta agaacaggt cactgtgg tgcatggtca cagactcat gcctgaagac 1860 atttacgtgg agtgaccaa caacgggaaa acgagcta actacagaa cactgaacca 1920 gtcctggact ctgatggttc ttactcatg tacagcaagc tgagagtgga aagaagaac 1980 tgggtggaaa gaaatagcta ctcctgttca gtggtccacg agggtctgca caatcaccac 2040 acgactaaga gcttctcccg gactccgggt aaatga 2076 <210> 22 <211> 717 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: HER2.7C2VH-mIgG2aCH1-7C2VH.mIgG2a.LALAPG.Knob (LC DNA with SP) <400> 22 atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattcagac 60 atcgtgctga cccagagccc agccagcctg gtggtgagcc tgggccagcg cgccaccatc 120 agctgccgcg ccagccagag cgtgagcggc agccgcttca cctacatgca ctggtatcag 180 cagaagccag gccagccacc aaagctgctg atcaagtacg ccagcatcct ggagagcggc 240 gtgccagccc gcttcagcgg cggcggcagc ggcaccgact tcaccctgaa catccaccca 300 gtggaggagg acgacaccgc cacctactac tgccagcaca gctgggagat cccaccatgg 360 accttcggcg gcggtaccaa gctggagatc aagcgcgctg atgctgcacc aactgtatcc 420 atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg 480 aacaacttct accccaaaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa 540 aatggcgtcc tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc 600 agcaccctca cgttgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc 660 actcacaaga catcaacttc acccattgtc aagagcttca acaggaatga gtgttga 717 <210> 23 <211> 672 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: HER2.7C2VH-mIgG2aCH1-7C2VH.mIgG2a.LALAPG.Knob (HC mature) <400> 23 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Trp Met Asn Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Ser Asp Ser Glu Ile Arg Ala Asn Gln Lys Phe 50 55 60 Arg Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Thr Tyr Asp Gly Gly Phe Glu Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr 180 185 190 Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro 210 215 220 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 225 230 235 240 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 245 250 255 Trp Met Asn Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 260 265 270 Gly Met Ile His Pro Ser Asp Ser Glu Ile Arg Ala Asn Gln Lys Phe 275 280 285 Arg Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Thr Thr Ala Tyr 290 295 300 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 305 310 315 320 Ala Arg Gly Thr Tyr Asp Gly Gly Phe Glu Tyr Trp Gly Gln Gly Thr 325 330 335 Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro 340 345 350 Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly 355 360 365 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn 370 375 380 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 385 390 395 400 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr 405 410 415 Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser 420 425 430 Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro 435 440 445 Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Ala Ala Gly Gly Pro Ser 450 455 460 Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu 465 470 475 480 Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro 485 490 495 Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala 500 505 510 Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val 515 520 525 Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe 530 535 540 Lys Cys Lys Val Asn Asn Lys Asp Leu Gly Ala Pro Ile Glu Arg Thr 545 550 555 560 Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu 565 570 575 Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Trp Cys 580 585 590 Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn 595 600 605 Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp 610 615 620 Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys 625 630 635 640 Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly 645 650 655 Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys 660 665 670 <210> 24 <211> 219 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: HER2.7C2VH-mIgG2aCH1-7C2VH.mIgG2a.LALAPG.Knob (LC mature) <400> 24 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Val Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Gly Ser 20 25 30 Arg Phe Thr Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Lys Tyr Ala Ser Ile Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Gly Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Asp Asp Thr Ala Thr Tyr Tyr Cys Gln His Ser Trp 85 90 95 Glu Ile Pro Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 115 120 125 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 130 135 140 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 145 150 155 160 Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 180 185 190 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 195 200 205 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 210 215 <210> 25 <211> 1440 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: IGF-1R.xIGF-1RcixutumumabVH.mIgG2a.LALAPG.Knob (HC DNA with SP) <400> 25 atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattcagag 60 gtgcagctgg tgcagagcgg cgccgaggtg aagaagcccg gcagcagcgt gaaggtgagc 120 tgcaaggcca gcggcggcac cttcagcagc tacgccatca gctgggtgag acaggccccc 180 ggccagggcc tggagtggat gggcggcatc atccccatct tcggcaccgc caactacgcc 240 cagaagttcc agggcagagt gaccatcacc gccgacaaga gcaccagcac cgcctacatg 300 gagctgagca gcctgagaag cgaggacacc gccgtgtact actgcgccag agcccccctg 360 agattcctgg agtggagcac ccaggaccac tactactact actacatgga cgtgtggggc 420 aagggcacca ccgtgaccgt ctcgagtgcc aaaacaacag ccccatcggt ctatccactg 480 gctcctgtgt gtggagatac aactggctcc tcggtgactc taggatgcct ggtcaagggt 540 tatttccctg agccagtgac cttgacctgg aactctggat ccctgtccag tggtgtgcac 600 accttcccag ctgtcctgca gtctgacctc tacaccctca gcagctcagt gactgtaacg 660 tcgagcacct ggcccagcca gtccatcacc tgcaatgtgg cccacccggc aagcagcacc 720 aaggtggaca agaaaattga gcccagagga cccacaatca agccctgtcc tccatgcaaa 780 tgcccagcac ctaacgccgc gggtggacca tccgtcttca tcttccctcc aaagatcaag 840 gatgtactca tgatctccct gagccccata gtcacatgtg tggtggtgga tgtgagcgag 900 gatgacccag atgtccagat cagctggttt gtgaacaacg tggaagtaca cacagctcag 960 acacaaaccc atagagagga ttacaacagt actctacgcg tggtcagtgc cctccccatc 1020 cagcaccagg actggatgag tggcaaggag ttcaaatgca aggtcaacaa caaagacctc 1080 ggagcgccca tcgagagaac catctcaaaa cccaaagggt cagtaagagc tccacaggta 1140 tatgctttgc ctccaccaga agaagagatg actaagaaac aggtcactct gtggtgcatg 1200 gtcacagact tcatgcctga agacatttac gtggagtgga ccaacaacgg gaaaacagag 1260 ctaaactaca agaacactga accagtcctg gactctgatg gttcttactt catgtacagc 1320 aagctgagag tggaaaagaa gaactgggtg gaaagaaata gctactcctg ttcagtggtc 1380 cacgagggtc tgcacaatca ccacacgact aagagcttct cccggactcc gggtaaatga 1440 <210> 26 <211> 699 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: IGF-1R.xIGF-1RcixutumumabVH.mIgG2a.LALAPG.Knob (LC DNA with SP) <400> 26 atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattcaagc 60 agcgagctga cccaggaccc cgccgtgagc gtggccctgg gccagaccgt gagaatcacc 120 tgccagggcg acagcctgag aagctactac gccacctggt accagcagaa gcccggccag 180 gcccccatcc tggtgatcta cggcgagaac aagagaccca gcggcatccc cgacagattc 240 agcggcagca gcagcggcaa caccgccagc ctgaccatca ccggcgccca ggccgaggac 300 gaggccgact actactgcaa gagcagagac ggcagcggcc agcacctggt gttcggcggc 360 ggcaccaagc tgaccgtgct tggccagccc aagtccactc ccactctcac cgtgtttcca 420 ccctcgagtg aggagctcaa ggaaaacaaa gccacactgg tgtgtctgat ttccaacttt 480 tccccgagtg gtgtgacagt ggcctggaag gcaaatggta cacctatcac ccagggtgtg 540 gacacttcaa atcccaccaa agagggcaac aagttcatgg cgagcagctt cctacatttg 600 acatcggacc agtggagatc tcacaacagt tttacctgtc aagttacaca tgaaggggac 660 actgtggaga agagtctgtc acgtgctgac tgtctctga 699 <210> 27 <211> 460 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: IGF-1R.xIGF-1RcixutumumabVH.mIgG2a.LALAPG.Knob (HC mature) <400> 27 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Pro Leu Arg Phe Leu Glu Trp Ser Thr Gln Asp His Tyr 100 105 110 Tyr Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val 115 120 125 Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val 130 135 140 Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys 145 150 155 160 Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu 165 170 175 Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr 180 185 190 Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln 195 200 205 Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp 210 215 220 Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys 225 230 235 240 Lys Cys Pro Ala Pro Asn Ala Ala Gly Gly Pro Ser Val Phe Ile Phe 245 250 255 Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val 260 265 270 Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile 275 280 285 Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr 290 295 300 His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro 305 310 315 320 Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val 325 330 335 Asn Asn Lys Asp Leu Gly Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro 340 345 350 Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu 355 360 365 Glu Glu Met Thr Lys Lys Gln Val Thr Leu Trp Cys Met Val Thr Asp 370 375 380 Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr 385 390 395 400 Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser 405 410 415 Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu 420 425 430 Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His 435 440 445 His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys 450 455 460 <210> 28 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: IGF-1R.xIGF-1RcixutumumabVH.mIgG2a.LALAPG.Knob (LC mature) <400> 28 Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30 Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Ile Leu Val Ile Tyr 35 40 45 Gly Glu Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Lys Ser Arg Asp Gly Ser Gly Gln His 85 90 95 Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ser Thr Pro Thr Leu Thr Val Phe Pro Pro Ser Ser Glu Glu Leu Lys 115 120 125 Glu Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asn Phe Ser Pro Ser 130 135 140 Gly Val Thr Val Ala Trp Lys Ala Asn Gly Thr Pro Ile Thr Gln Gly 145 150 155 160 Val Asp Thr Ser Asn Pro Thr Lys Glu Gly Asn Lys Phe Met Ala Ser 165 170 175 Ser Phe Leu His Leu Thr Ser Asp Gln Trp Arg Ser His Asn Ser Phe 180 185 190 Thr Cys Gln Val Thr His Glu Gly Asp Thr Val Glu Lys Ser Leu Ser 195 200 205 Arg Ala Asp Cys Leu 210 <210> 29 <211> 1410 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: NIST.mIgG2a.LALAPG.Knob (HC DNA with SP) <400> 29 atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattcacag 60 gtgaccctga gggagtccgg ccccgccctg gtgaagccca cccagaccct gaccctgacc 120 tgcaccttct ccggcttc cctgtccacc gccggcatgt ccgtgggctg gatcaggcag 180 ccccccggca aggccctgga gtggctggcc gacatctggt gggacgacaa gaagcactac 240 aacccctccc tgaaggacag gctgaccatc tccaaggaca cctccaagaa ccaggtggtg 300 ctgaaggtga ccaacatgga ccccgccgac accgccacct actactgcgc cagggacatg 360 atcttcaact tctacttcga cgtgtggggc cagggcacca ccgtgaccgt ctcgagtgcc 420 aaaaacag ccccatcggt ctatccactg gctcctgtgt gtggagatac aactggctcc 480 tcggtgactc taggatgcct ggtcaagggt tatttccctg agccagtgac cttgacctgg 540 aactctggat ccctgtccag tggtgtgcac accttcccag ctgtcctgca gtctgacctc 600 tacaccctca gcagctcagt gactgtaacg tcgagcacct ggcccagcca gtccatcacc 660 tgcaatgtgg cccacccggc aagcagcacc aaggtggaca agaaaattga gcccagagga 720 cccacaatca agccctgtcc tccatgcaaa tgcccagcac ctaacgccgc gggtggacca 780 tccgtcttca tcttccctcc aaagatcaag gatgtactca tgatctccct gagccccata 840 gtcacatgtg tggtggtgga tgtgagcgag gatgacccag atgtccagat cagctggtttt 900 gtgaacaacg tggaagtaca cacagctcag acacaaaccc atagagagga ttacaacagt 960 actctacgcg tggtcagtgc cctccccatc cagcaccagg actggatgag tggcaaggag 1020 ttcaaatgca aggtcaacaa caagacctc ggagcgccca tcgagagaac catctcaaaa 1080 cccaaagggt cagtaagagc tccacaggta tatgctttgc ctccaccaga agaagagatg 1140 actaagaaac aggtcactct gtggtgcatg gtcacagact tcatgcctga agacatttac 1200 gtggagtgga ccaacaacgg gaaaacagag ctaaactaca agaacactga accagtcctg 1260 gactctgatg gttcttactt catgtacagc aagctgagag tggaaaagaa gaactgggtg 1320 gaaagaaata gctactcctg ttcagtggtc cacgagggtc tgcacaatca ccacacgact 1380 aagagcttct cccggactcc gggtaaatga 1410 <210> 30 <211> 699 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: NIST.mIgG2a.LALAPG.Knob (LC DNA with SP) <400> 30 atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattcagac 60 atccagatga cccagtcccc ctccaccctg tccgcctccg tgggcgacag ggtgaccatc 120 acctgctccg cctcctccag ggtgggctac atgcactggt atcagcagaa gccggcaag 180 gcccccaagc tgctgatcta cgacacctcc aagctggcct ccggcgtgcc ctccaggttc 240 tccggctccg gctccggcac cgagttcacc ctgaccatct cctccctgca gccgacgac 300 ttcgccacct actactgctt ccagggctcc ggctacccct tcaccttcgg cggcggtacc 360 aaggtggaga tcaagcgcgc tgatgctgca ccaactgtat ccatcttccc accatccagt 420 gagcagttaa catctggagg tgcctcagtc gtgtgcttct tgaacaactt ctaccccaaa 480 gacatcaatg tcaagtggaa gattgatggc agtgaacgac aaaatggcgt cctgaacagt 540 tggactgatc aggacagcaa agacagcacc tacagcatga gcagcaccct cacgttgacc 600 aaggacgagt atgaacgaca taacagctat acctgtgagg ccactcacaa gacatcaact 660 tcacccattg tcaagagctt caacaggaat gagtgttga 699 <210> 31 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: NIST.mIgG2a.LALAPG.Knob (HC mature) <400> 31 Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ala 20 25 30 Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys His Tyr Asn Pro Ser 50 55 60 Leu Lys Asp Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Asp Met Ile Phe Asn Phe Tyr Phe Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val 115 120 125 Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr 130 135 140 Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr 145 150 155 160 Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser 180 185 190 Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala 195 200 205 Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile 210 215 220 Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Ala Ala Gly Gly 225 230 235 240 Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile 245 250 255 Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp 260 265 270 Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His 275 280 285 Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg 290 295 300 Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys 305 310 315 320 Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Gly Ala Pro Ile Glu 325 330 335 Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr 340 345 350 Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu 355 360 365 Trp Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp 370 375 380 Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu 405 410 415 Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His 420 425 430 Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro 435 440 445 Gly Lys 450 <210> 32 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: NIST.mIgG2a.LALAPG.Knob (LC mature) <400> 32 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Arg Val Gly Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Ala Asp Ala Ala Pro 100 105 110 Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly 115 120 125 Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn 130 135 140 Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn 145 150 155 160 Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser 165 170 175 Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr 180 185 190 Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe 195 200 205 Asn Arg Asn Glu Cys 210 <210> 33 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: RNF43-104 (variable heavy chain (VH) DNA) <400> 33 caggtgcagc tgaaggagtc aggacctggc ctggtgcagc cctcacagac cctgtctctc 60 acctgcagtg tctctgggtt ttcattaacc agcttccatg tgcactgggt tcgacagcct 120 ccaggaaaag gtctggagtg gatgggatta atgtggagta atggagacac atcatataat 180 tcagatctca aatcccgact gagcatcagc agggacacct ccaagagtca agtcttctta 240 aaaatgcaca gtctgcagac tgaagacaca gccacttact actgtgccag aacagatgta 300 taccacggat tcggtgctat ggatgcctgg ggtcagggaa cttcagtcac tgtctcctca 360 <210> 34 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Synthetic: RNF43-104 (variable light chain (VL) DNA) <400> 34 gacatccaga tgacacagtc tccgtcattt ctgtctgcat ctcttggaaa cagcatcacc 60 atcacttgcc atgccagtca gttcatcaag ggttggttag cctggtacca acaaaagtca 120 gggaatgctc ctgaactgtt aatttataag tcatctagcc tgcattcagg ggttccatca 180 agattcagtg gcagcggatc tggaacagat tatattctca ctatcagcaa cctacagcct 240 gaagatattg ccacttatta ctgtcagcat tatcaaggct ttccgctcac gttcggttct 300 gggaccaagc tggagatcaa a 321 <210> 35 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: RNF43-104 (VH protein) <400> 35 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Ser Gly Phe Ser Leu Thr Ser Phe 20 25 30 His Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Leu Met Trp Ser Asn Gly Asp Thr...

Claims

1. A multispecific binding protein 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 a transmembrane E3 ubiquitin ligase.

2. The multispecific binding protein according to claim 1, wherein the multispecific binding protein reduces the level of the second cell surface protein on the cell surface compared to the level observed in the absence of the multispecific binding protein.

3. The multispecific binding protein according to claim 2, wherein the multispecific binding protein reduces the level of the second cell surface protein on the cell surface in vitro compared to the level observed in the absence of the multispecific binding protein.

4. The multispecific binding protein according to claim 3, wherein the multispecific binding protein reduces the level of the second cell surface protein on the surface of a cell as measured by flow cytometry or a luminescence assay.

5. The multispecific binding protein according to claim 2, 3, or 4, wherein the multispecific binding protein reduces the level of the second cell surface protein on the cell surface in vivo compared to the level observed in the absence of the multispecific binding protein.

6. The multispecific binding protein according to any one of claims 1 to 5, wherein the multispecific binding protein is a multispecific antibody.

7. The multispecific antibody according to claim 6, which is a bispecific or triplicate antibody, for example, 1+1 FabIgG, 1+1 FvIgG, 2+1 FvIgG, 2+1 FabIgG, 1-arm FvIgG, or 1-arm FabIgG.

8. The multispecific binding protein according to claim 6 or 7, wherein the protein comprises an IgG Fc region having at least one knob-into-hole modification.

9. The multispecific binding protein according to any one of claims 1 to 8, wherein the transmembrane E3 ubiquitin ligase does not have catalytic activity, and the lack of catalytic activity is determined in a cell surface degradation assay.

10. The multispecific binding protein according to any one of claims 1 to 8, wherein the transmembrane E3 ubiquitin ligase has catalytic activity, and the presence of catalytic activity is determined in a cell surface degradation assay.

11. The multispecific binding protein according to any one of claims 1 to 10, wherein the transmembrane E3 ubiquitin ligase is RNF43, ZnRF3, RNF13, RNF128, RNF130, RNF133, RNF148, RNF149, RNF150, RNF167, ZnRF4, RSPRY1, SYVN1, LNX1 isoform 2, or TRIM7 isoform 3.

12. The multispecific binding protein according to any one of claims 1 to 10, wherein the transmembrane E3 ubiquitin ligase is RNF43, RNF128, RNF130, RNF133, RNF149, RNF150, or ZnRF3.

13. The multispecific binding protein according to any one of claims 1 to 10, wherein the transmembrane E3 ubiquitin ligase is RNF130, RNF133, RNF149, or RNF150.

14. The multispecific binding protein according to any one of claims 1 to 10, wherein the transmembrane E3 ubiquitin ligase is RNF130, RNF149, or RNF167.

15. The multispecific binding protein according to any one of claims 1 to 10, wherein the transmembrane E3 ubiquitin ligase is RNF133 or RNF148.

16. The multispecific binding protein according to any one of claims 1 to 10, wherein 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.

17. The multispecific binding protein according to any one of claims 1 to 10 or 16, wherein 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) including (a) CDR-H1, (b) CDR-H2 and (c) CDR-H3 and a light chain variable domain (VL) including (d) CDR-L1, (e) CDR-L2 and (f) CDR-L3; or a multispecific antibody that binds to RNF43 or ZnRF3 and comprises VH and VL.

18. The protein is 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, RNF43-5, RNF43-15, RNF43-2,The multispecificity-binding protein according to claim 16 or 17, comprising a heavy chain variable domain (VH) containing one heavy chain complementarity-determining region 1 (CDR-H1), CDR-H2 and / or CDR-H3 from among RNF43-16, RNF43-14, RNF43-17, RNF43-22, RNF43-9, RNF43-21, RNF43-13, RNF43-19, ZnRF3-331, ZnRF3-333 or ZnRF3-332.

19. The multispecific binding protein is 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, RNF43-5, RNF43-15,A multispecific binding protein according to any one of claims 16 to 18, comprising a heavy chain variable region (VH) containing one of CDR-H1, CDR-H2, and CDR-H3 from among 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.

20. The protein is 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, RNF43-5, RNF43-15, RNF43-2,The multispecificity binding protein according to claim 16 or 17, comprising a light chain variable domain (VL) containing one light chain complementarity determining region 1 (CDR-L1), CDR-L2 and / or CDR-L3 from among RNF43-16, RNF43-14, RNF43-17, RNF43-22, RNF43-9, RNF43-21, RNF43-13, RNF43-19, ZnRF3-331, ZnRF3-333 or ZnRF3-332.

21. The multispecific binding protein is 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, RNF43-5, RNF43-15,A multispecific binding protein according to any one of claims 16-17 or 20, comprising a light chain variable region (VL) containing CDR-L1, CDR-L2, and CDR-L3 of any one of 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.

22. The protein is 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, RNF43-5, RNF43-15, RNF43-2,A multispecific binding protein according to any one of claims 16 to 21, comprising (a) a heavy chain variable domain (VH) including heavy chain complementarity determination region 1 (CDR-H1), CDR-H2, and CDR-H3, and (b) a light chain variable domain (VL) including light chain complementarity determination region 1 (CDR-L1), CDR-L2, and CDR-L3, of any one of RNF43-16, RNF43-14, RNF43-17, RNF43-22, RNF43-9, RNF43-21, RNF43-13, RNF43-19, ZnRF3-331, ZnRF3-333, or ZnRF3-332, respectively.

23. The multispecific binding protein according to any one of claims 18 to 22, wherein the heavy chain CDR and / or the light chain CDR is Kabat CDR, Chothia CDR, or McCallum CDR.

24. The multispecific binding protein is 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, RNF43-5, RNF43-15,A multispecific binding protein according to any one of claims 16 to 23, comprising a heavy chain variable region (VH) having an amino acid sequence identical to at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of any one of 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.

25. The multispecific binding protein is 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, RNF43-5, RNF43-15,A multispecific binding protein according to any one of claims 16 to 24, comprising a light chain variable region (VL) having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical amino acid sequence to one of 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.

26. The multispecific binding protein is 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, RNF43-5, RNF43-15,A multispecific binding protein according to any one of claims 16 to 25, comprising a VH containing the amino acid sequence of one heavy chain variable region (VH) from among 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.

27. The multispecific binding protein is 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, RNF43-5, RNF43-15,A multispecific binding protein according to any one of claims 16 to 26, comprising a VL containing the amino acid sequence of one of the light chain variable regions (VLs) among 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.

28. The multispecific binding protein according to any one of claims 16 to 27, wherein the protein has a binding affinity for ZnRF3 or RNF43 of less than 50 nM, less than 1 nM, less than 1 nM, less than 0.5 nM, less than 0.05 nM, or 50 nM to 10 nM, or 10 nM to 1 nM, or 1 nM to 0.5 nM, or 0.5 nM to 0.05 nM, or 0.05 nM to 0.01 nM.

29. The multispecific binding protein according to any one of claims 16 to 28, wherein the protein is a multispecific antibody comprising the heavy chain variable region and / or 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.

30. The multispecific binding protein according to any one of claims 16 to 29, wherein 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 the binding of ZnRF3 or RNF43 to FZD and LRP6.

31. The multispecific binding protein according to any one of claims 1 to 30, wherein the protein is a multispecific antibody containing a chimeric heavy chain variable region or a light chain variable region, or the variable region is humanized.

32. The multispecific binding protein according to any one of claims 1 to 7 or 9 to 31, wherein the protein comprises a wild-type human Fc region.

33. The multispecific binding protein according to claim 32, wherein the Fc region is an IgG1, IgG2, IgG3, or IgG4 Fc region.

34. The multispecific binding protein according to any one of claims 1 to 33, wherein the protein includes an Fc region having an effector function.

35. The multispecific binding protein according to any one of claims 1 to 32, wherein the protein comprises a human IgG1 Fc region containing a LALAPG mutation or a substitution at position N297, for example, N297G or N297Q, and / or the Fc region lacks effector function.

36. The multispecific binding protein according to any one of claims 1 to 35, wherein the 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 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.

37. The multispecific binding protein according to claim 36, wherein the second cell surface protein is HER2, EGFR, or IGF1R.

38. The multispecific binding protein according to any one of claims 1 to 37, wherein the protein comprises an anti-HER2 antibody, for example, 4D5, 7C2, or 2C4, or an anti-IGF1R antibody, for example, cyclotumumab, ganitumab, darotuzumab, figtumumab, lobatumumab, teprotumumab, or istilatumumab, including its heavy chain variable region and light chain variable region.

39. An isolated nucleic acid or set of nucleic acids encoding a multispecific binding protein according to any one of claims 1 to 38.

40. A host cell comprising the nucleic acid or nucleic acid set described in claim 39.

41. A method for producing a multispecific binding protein according to any one of claims 1 to 38, comprising culturing a host cell according to claim 40 under conditions suitable for the expression of the protein.

42. The method according to claim 41, further comprising recovering the protein from the host cell.

43. A multispecific binding protein produced by the method described in claim 42.

44. A pharmaceutical composition comprising a multispecific binding protein according to any one of claims 1 to 38 and a pharmaceutically acceptable carrier.

45. A multispecific binding protein according to any one of claims 1 to 38 or a pharmaceutical composition according to claim 44, for use as a medicine.

46. A multispecific binding protein according to any one of claims 1 to 38 or a pharmaceutical composition according to claim 44, for use in the treatment of target cancer, autoimmune symptoms, inflammatory symptoms, neurodegenerative symptoms, or infections.

47. A multispecific binding protein according to any one of claims 1 to 38 or a pharmaceutical composition according to claim 44 for use in reducing the level of cell surface proteins in a subject requiring a reduction in the level of cell surface proteins, wherein the subject optionally has cancer, autoimmune symptoms, inflammatory symptoms, neurodegenerative symptoms or infectious diseases.

48. A multispecific binding protein according to any one of claims 1 to 38 or a pharmaceutical composition according to claim 44, for use in increasing the immune response in subjects such as those with cancer, autoimmune symptoms, inflammatory symptoms, neurodegenerative symptoms, or infectious diseases.

49. The multispecific binding protein according to any one of claims 46 to 48, wherein (a) the subject has a mutation in RNF43 and the multispecific binding protein does not bind to RNF43 or does not activate RNF43, or (b) the subject has a mutation in ZnRF3 and the multispecific binding protein does not bind to ZnRF3 or does not activate ZnRF3.

50. Use of a multispecific binding protein according to any one of claims 1 to 38 or a pharmaceutical composition according to claim 44 in the manufacture of a drug for treating a target cancer, autoimmune symptoms, inflammatory symptoms, neurodegenerative symptoms or infection.

51. Use of a multispecific binding protein according to any one of claims 1 to 38 or the pharmaceutical composition according to claim 44 in the manufacture of a drug for reducing the level of cell surface proteins in a subject requiring a reduction in the level of cell surface proteins, wherein the subject optionally has cancer, autoimmune symptoms, inflammatory symptoms, neurodegenerative symptoms or infection.

52. Use of a multispecific binding protein according to any one of claims 1 to 38 or a pharmaceutical composition according to claim 44 in the manufacture of a drug for increasing the immune response in subjects such as those with cancer, autoimmune symptoms, inflammatory symptoms, neurodegenerative symptoms, or infectious diseases.

53. The use according to any one of claims 50 to 52, wherein (a) the subject has a mutation in RNF43 and the multispecific binding protein does not bind to RNF43 or does not activate RNF43, or (b) the subject has a mutation in ZnRF3 and the multispecific binding protein does not bind to ZnRF3 or does not activate ZnRF3.

54. A method for treating cancer, autoimmune symptoms, inflammatory symptoms, neurodegenerative symptoms, or infections in a subject requiring treatment for cancer, autoimmune symptoms, inflammatory symptoms, neurodegenerative symptoms, or infections, comprising administering an effective amount of a multispecific binding protein according to any one of claims 1 to 38 or a pharmaceutical composition according to claim 44 to the subject.

55. A method for reducing the level of cell surface proteins in a subject requiring a reduction in the level of cell surface proteins, wherein the method comprises administering to the subject an effective amount of a multispecific binding protein according to any one of claims 1 to 38 or a pharmaceutical composition according to claim 44, wherein the subject optionally has cancer, autoimmune symptoms, inflammatory symptoms, neurodegenerative symptoms or an infection.

56. A method for increasing an immune response in a subject requiring an increased immune response, the method comprising administering to the subject an effective amount of a multispecific binding protein according to any one of claims 1 to 38 or a pharmaceutical composition according to claim 44, wherein the subject optionally has cancer, autoimmune symptoms, inflammatory symptoms, neurodegenerative symptoms or an infection.

57. The method according to any one of claims 54 to 56, wherein (a) the subject has a mutation in RNF43 and the multispecific binding protein does not bind to RNF43 or does not activate RNF43, or (b) the subject has a mutation in ZnRF3 and the multispecific binding protein does not bind to ZnRF3 or does not activate ZnRF3.

58. The method according to any one of claims 54 to 56, further comprising determining whether the subject has a mutation in RNF43 or ZnRF3 before administering the multispecific binding protein, wherein (a) if the subject has a mutation in RNF43, the multispecific binding protein does not bind to RNF43 or does not activate RNF43, and (b) if the subject has a mutation in ZnRF3, the multispecific binding protein does not bind to ZnRF3 or does not activate ZnRF3.

59. A multispecific binding protein, use, or method according to any one of claims 49, 53, 57, or 58, wherein the subject has cancer, and the cancer comprises a mutation in RNF43 or ZnRF3.

60. A method for reducing the level of cell surface proteins on the surface of hematopoietic cells in a cell sample or tissue sample in vitro or in vivo of a subject, comprising administering the cell or tissue sample or the subject, respectively, the multispecific binding protein according to claim 14.

61. A method for reducing the level of cell surface proteins on the surface of testicular cells in a cell sample or tissue sample in vitro or in vivo of a subject, comprising administering the cell or tissue sample or the subject, respectively, to the multispecific binding protein according to claim 15.

62. A kit comprising a multispecific binding protein according to any one of claims 1 to 38, or a nucleic acid or nucleic acid set according to claim 39, or a host cell according to claim 40, further comprising one or more reagents for expressing or purifying the protein and / or one or more reagents for incubating the protein with a cell or tissue sample in vitro to reduce the level of cell surface protein in the sample.

63. A method for in vitro reducing the level of cell surface proteins in a cell sample or tissue sample, comprising incubating the sample with a multispecific binding protein according to any one of claims 1 to 38 or a kit according to claim 62.

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