Activatable ror binding agents and uses thereof

Abstract

The present disclosure provides activatable ROR binding agents (e.g, activatable antibodies, including monospecific and multispecific antibodies such as bispecific antibodies) and uses thereof.

Description

ACTIVATABLE ROR BINDING AGENTS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 514,782, filed July 20, 2023, the disclosure of which is incorporated by reference herein in its entirety.SEQUENCE LISTING

[0002] This application contains an electronic Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “ 14529- 148-228 SEQ LISTING. xml”, was created on July 18, 2024, and is 85,072 bytes in size.1. FIELD

[0003] The present disclosure relates generally to activatable binding agents, such as activatable antibodies (including fragments thereof) that bind to tyrosine-protein kinase transmembrane receptor (ROR) including human ROR, and methods of use thereof.2. BACKGROUND

[0004] Various tumors can demonstrate cell-surface expression of tyrosine-protein kinase transmembrane receptor (ROR) antigens, as described in greater detail in Gentile, et al.(Cancer Res,' 71(8) April 15, 2011), Rebagay, et al. (Front. Oncol., 18 April 2012), Zhang, et al. (American Journal of Pathology, Vol. 181, No. 6, December 2012), Henry, et al.(Oncotarget, Vol. 6, No. 37 2015), Zhang, et al. (PLoS ONE 7(3): e31127.), and Bainbridge, et al. PLoS ONE 9(7): el02695.), each herein incorporated by reference in their entirety. In addition, ROR expression may not be expressed, or only demonstrate limited expression, in normal, i.e. non-cancerous, tissue as described in Balakrishnan et al. (Clin Cancer Res. 2017 Jun 15; 23(12): 3061-3071), herein incorporated in its entirety. Thus ROR antigens can be used as a tumor-specific marker in certain tumors. Examples of tumors and cancers with demonstrated ROR expression include, but are not limited to, pancreatic cancer, ovarian cancer, breast cancer, lung cancer, gastric cancer, melanoma, Ewing sarcoma, chronic lymphocytic leukemia, mantle cell lymphoma, and B-ALL, as described in Gohil et al. (Oncoimmunology . 2017; 6(7): el326437.), herein incorporated in its entirety. Other cancers include, but are not limited to, hematological cancer, prostate cancer, colon cancer, renal cancer, and uterine cancer. ROR antigen binding agents thus have therapeutic potential in treatment of cancer.

[0005] Activatable binding agents (e.g., activatable antibodies) are only capable of binding their targets in certain contexts (e.g., in the protease-rich tumor microenvironment), and thus are useful for precision / context-dependent target-binding. However, the process of developing activatable binding agents is slow, labor intensive, and costly.

[0006] There is, therefore, a need for activatable ROR antigen binding agents.

[0007] Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.3. SUMMARY

[0008] The present disclosure provides activatable ROR binding agents, including activatable human ROR binding agents. Such agents include activatable antibodies that bind to ROR, for example, monospecific or multispecific (e.g., bispecific) antibodies that bind to ROR.

[0009] Such activatable binding agents, in some embodiments, are activatable antibodies that comprise: a masking peptide, an antibody light chain variable (VL) region, and an antibody heavy chain variable (VH) region; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:29, 30 or 31 (see Table 2), and wherein the antibody light chain variable region and the antibody heavy chain variable region comprise the CDRs described herein (e.g., Table 1).

[0010] Such activatable binding agents, in some embodiments, are activatable antibodies that comprise: (a) a polypeptide comprising a masking peptide and an antibody light chain variable region; and (b) an antibody heavy chain variable region; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:29, 30 or 31 (see Table 2), and wherein the antibody light chain variable region and the antibody heavy chain variable region comprise the CDRs described herein (e.g., Table 1).

[0011] Such activatable binding agents, in some embodiments, are activatable antibodies that comprise: (a) a polypeptide comprising, from N-terminus to C-terminus, a masking peptide and an antibody light chain variable region; and (b) an antibody heavy chain variable region; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:29, 30 or 31 (see Table 2), and wherein the antibody light chain variable region and the antibody heavy chain variable region comprise the CDRs described herein (e.g., Table 1).

[0012] Such activatable binding agents, in some embodiments, are activatable antibodies that comprise: (a) a polypeptide comprising, from N-terminus to C-terminus, a masking peptide and an antibody light chain variable region; and (b) an antibody heavy chain variable region; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:29,30 or 31 (see Table 2), and wherein the antibody light chain variable region and the antibody heavy chain variable region are as described herein (e.g., Table 1). In certain embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:35, 37 or 39 (see Tables 4-6). In certain embodiments, the polypeptide is as described herein (e.g., any one of Tables 4-6) or comprises the amino acid sequence of SEQ ID NO:34, 36 or 38 (see Tables 4-6).

[0013] Such activatable binding agents, in some embodiments, are activatable antibodies that comprise: (a) a polypeptide comprising, from N-terminus to C-terminus, a masking peptide and an antibody light chain; and (b) an antibody heavy chain; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:29, 30 or 31 (see Table 2), and wherein the antibody light chain and the antibody heavy chain are as described herein (e.g., Table 3) or comprise the antibody light chain and the antibody heavy chain amino acid sequences described herein (e.g., Table 3). In certain embodiments, the polypeptide is as described herein (e.g., any one of Tables 4-6) or comprises the amino acid sequence of SEQ ID NO:34, 36 or 38 (see Tables 4-6).

[0014] Such activatable binding agents, in some embodiments, are activatable antibodies that bind to the same epitope of ROR (e.g., human ROR) as an antibody comprising the CDRs described herein (e.g., Table 1) and comprise: (a) a polypeptide comprising, from N- terminus to C-terminus, a masking peptide and an antibody light chain variable region; and (b) an antibody heavy chain variable region; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:29, 30 or 31 (see Table 2).

[0015] Such activatable binding agents, in some embodiments, bind to the same epitope of ROR (e.g., human ROR) as an antibody comprising a heavy chain variable region and a light chain variable region described herein (e.g., Table 1) and comprise: (a) a polypeptide comprising, from N-terminus to C-terminus, a masking peptide and an antibody light chain variable region; and (b) an antibody heavy chain variable region; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:29, 30 or 31 (see Table 2).

[0016] The present disclosure also provides nucleic acids encoding an activatable ROR binding agent provided herein (e.g., an activatable antibody or fragment thereof, such as an activatable antigen-binding fragment), vectors comprising one or more of such nucleic acids, and cells comprising the nucleic acid, the vector, or both (such as cells expressing the binding agent).

[0017] The present disclosure also provides compositions comprising an activatable ROR binding agent. Such compositions, in some embodiments, include activatable antibodies thatbind to ROR, for example, monospecific or multispecific (e.g., bispecific) antibodies that bind to ROR.

[0018] Such compositions, in some embodiments, include activatable antibodies that comprise: (a) a polypeptide comprising, from N-terminus to C-terminus, a masking peptide and an antibody light chain variable region; and (b) an antibody heavy chain variable region; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:29, 30 or 31 (see Table 2), and wherein the antibody light chain variable region and the antibody heavy chain variable region comprise the CDRs described herein (e.g., Table 1).

[0019] Such compositions, in some embodiments, are activatable antibodies that comprise: (a) a polypeptide comprising, from N-terminus to C-terminus, a masking peptide and an antibody light chain variable region; and (b) an antibody heavy chain variable region; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:29, 30 or 31 (see Table 2), and wherein the antibody light chain variable region and the antibody heavy chain variable region are as described herein (e.g., Table 1). In certain embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:35, 37 or 39 (see Tables 4-6). In certain embodiments, the polypeptide is as described herein (e.g., any one of Tables 4-6) or comprises the amino acid sequence of SEQ ID NO:34, 36 or 38 (see Tables 4-6).

[0020] Such compositions, in some embodiments, are activatable antibodies that comprise: (a) a polypeptide comprising, from N-terminus to C-terminus, a masking peptide and an antibody light chain; and (b) an antibody heavy chain; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:29, 30 or 31 (see Table 2), and wherein the antibody light chain and the antibody heavy chain are as described herein (e.g., Table 3) or comprise the antibody light chain and the antibody heavy chain amino acid sequences described herein (e.g, Table 3). In certain embodiments, the polypeptide is as described herein (e.g., any one of Tables 4-6) or comprises the amino acid sequence of SEQ ID NO:34, 36 or 38 (see Tables 4-6).

[0021] Such compositions, in some embodiments, include activatable antibodies that bind to essentially the same epitope of ROR (e.g., human ROR) as an antibody comprising the CDRs described herein (e.g., Table 1) and comprise: (a) a polypeptide comprising, from N- terminus to C-terminus, a masking peptide and an antibody light chain variable region; and (b) an antibody heavy chain variable region; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:29, 30 or 31 (see Table 2).

[0022] Such compositions, in some embodiments, include activatable antibodies that bind to essentially the same epitope of ROR (e.g., human ROR) as an antibody comprising a heavychain variable region and a light chain variable region described herein (e.g., Table 1) and comprise: (a) a polypeptide comprising, from N-terminus to C-terminus, a masking peptide and an antibody light chain variable region; and (b) an antibody heavy chain variable region; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:29, 30 or 31 (see Table 2).

[0023] The present disclosure also provides compositions comprising the nucleic acids encoding an activatable ROR binding agent provided herein (e.g., an activatable antibody or fragment thereof, such as an activatable antigen-binding fragment), vectors comprising one or more nucleic acids, or cells comprising the nucleic acid, the vector, or both (such as cells expressing the activatable binding agent).

[0024] The present disclosure further provides various uses of the present activatable binding agents and compositions, including, for example, methods for treating a disease or disorder in a subject with an activatable ROR binding agent or a composition provided herein. Such compositions include activatable antibodies that bind to ROR, for example, monospecific or multispecific (e.g., bispecific) antibodies that bind to ROR (e.g., human ROR).

[0025] The present disclosure further provides methods of making an activatable antibody or fragment thereof that binds to ROR, comprising culturing a cell described herein and expressing the activatable antibody or fragment thereof.4. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 shows a schematic of an exemplary selection process for self-blocking peptides, as further illustrated in Example 1.

[0027] FIGs. 2A-2F provide exemplary results of the tested antibodies’ binding to antigens, as further illustrated in Example 1. FIG. 2A shows the binding of tested antibodies (P0 and Ml) to RORl-Fc measured by ELISA. FIG. 2B shows the binding of tested antibodies (P0, M2, and M3) to RORl-Fc measured by ELISA. FIG. 2C shows the binding of tested antibodies (P0, Ml, M2, and M3) to ROR2-Fc measured by ELISA. FIG. 2D shows the binding of tested antibodies (P0, Ml, M2, and M3) to HT-29 measured by FACS. FIG. 2E shows the binding of tested antibodies (P0, Ml, M2, and M3) to H226 measured by FACS. FIG. 2F shows the binding of tested antibodies (P0, Ml, M2, and M3) to 293F measured by FACS.

[0028] FIGs. 3A-3B provide exemplary differential scanning fluorimetry (DSF) results of the tested antibodies (Ml, M2, and M3), as further illustrated in Example 2. FIG. 3A plots fluorescence, while FIG. 3B plots d(fluorescence) / dT.

[0029] FIGs. 4A-4D provide exemplary capillary electrophoresis sodium dodecyl sulfate (CE-SDS) results of the tested antibodies (P0: FIG. 4A, Ml: FIG. 4B, M2: FIG. 4C, and M3: FIG. 4D), as further illustrated in Example 2.

[0030] FIGs. 5A-5D provide exemplary capillary isoelectric focusing (cIEF) results of the tested antibodies (P0: FIG. 5A, Ml: FIG. 5B, M2: FIG. 5C, and M3: FIG. 5D), as further illustrated in Example 2.

[0031] FIG. 6 provides exemplary hydrophobic interaction chromatography (HIC) results of the tested antibodies (P0, Ml, M2, and M3), as further illustrated in Example 2.

[0032] FIGs. 7A-7D provide exemplary accelerated stability assessment results under 40 °C incubation of the tested antibodies (P0: FIG. 7A, Ml: FIG. 7B, M2: FIG. 7C, and M3: FIG. 7D), as further illustrated in Example 2.

[0033] FIGs. 8A-8D provide exemplary accelerated stability assessment results after freezing and thawing (3 rounds: FT3; and 6 rounds: FT6) of the tested antibodies (P0: FIG. 8A, Ml: FIG. 8B, M2: FIG. 8C, and M3: FIG. 8D), as further illustrated in Example 2.

[0034] FIGs. 9A-9D provide exemplary accelerated stability assessment results at low pH of the tested antibodies (P0: FIG. 9A, Ml: FIG. 9B, M2: FIG. 9C, and M3: FIG. 9D), as further illustrated in Example 2.

[0035] FIGs. 10A-10D provide exemplary accelerated stability assessment results under oxidation of the tested antibodies (P0: FIG. 10A, Ml: FIG. 10B, M2: FIG. 10C, and M3: FIG. 10D), as further illustrated in Example 2.

[0036] FIGs. 11A-11C provide exemplary plasma or serum stability results of the tested antibodies, as further illustrated in Example 2. FIG. HA plots the plasma stability shown by the total antibody concentrations. FIG. 11B plots the serum stability shown by the total antibody concentrations. FIG. 11C plots the stability shown by the activated antibody concentrations.

[0037] FIG. 12 provides an exemplary western blotting result showing human IgG from mice injected with 3 mg / kg or 10 mg / kg of P0 or M2, as further illustrated in Example 4.

[0038] FIGs. 13A-13C provide exemplary western blotting results showing human IgG from mice injected with 10 mg / kg of Ml (FIG. 13A), M2 (FIG. 13B), or M3 (FIG. 13C), as further illustrated in Example 4.

[0039] FIG. 14 plots an exemplary intratumoral receptor occupancy (RO) result of mice 14 hours or 96 hours after dosing with 10 mg / kg P0, Ml, M2, or M3, as further illustrated in Example 5.

[0040] FIGs. 15A-15B provide exemplary RO flow cytometry plots of mice 14 hours after dosing with 10 mg / kg an isotype control, P0, Ml, M2, or M3, as further illustrated in Example 5. FIG. 15A provides plots relating to an isotype control, P0, and Ml, while FIG. 15B provides plots relating to M2 and M3.

[0041] FIGs. 16A-16B provide exemplary RO flow cytometry plots of mice 96 hours after dosing with 10 mg / kg an isotype control, P0, Ml, M2, or M3, as further illustrated in Example 5. FIG. 16A provides plots relating to an isotype control, P0, and Ml, while FIG. 16B provides plots relating to M2 and M3.

[0042] FIG. 17 provides exemplary IHZ and immunofluorescence (IF) staining result, as further illustrated in Example 6. White arrow heads show ROR1 staining oustside of islets, while white arrows show ROR1 staining inside islets.

[0043] FIGs. 18A-18D provide exemplary IHZ staining results, as further illustrated in Example 6 FIG. 18A shows IHZ staining of ROR on HT-29 and MCF7 cells. FIG. 18B shows IHZ staining of ROR and insulin in normal human pancreas samples #3 and #5. FIG. 18C shows IHZ staining of ROR and insulin in normal human pancreas samples #6 and #7. FIG. 18D shows IHZ staining of ROR and insulin in normal human pancreas samples #8 and #9. In the PO+insulin panels, white arrow heads show ROR1 staining in pancreas, while white arrows show ROR1 staining inside islets. In the other insulin panels, white arrows show pancreatic islets.5. DETAILED DESCRIPTION

[0044] The present disclosure is based, at least in part, on novel activatable ROR binding agents and their properties. Such agents include activatable antibodies (e.g., monospecific or multispecific, including bispecific) that bind to ROR, including activatable antibodies that bind to human ROR. In certain aspects, such activatable binding agents are useful in compositions and in methods for treating a disease or disorder such as cancer.

[0045] As it would be understood, the section or subsection headings as used herein is for organizational purposes only and are not to be construed as limiting and / or separating the subject matter described.5.1. ROR antigens

[0046] The activatable ROR binding agents described herein bind specifically to one or more ROR antigens.

[0047] As used herein, “ROR antigens” refer to members of the tyrosine-protein kinase transmembrane receptor (ROR) family, including members ROR1 and ROR2. In certainembodiments, the activatable ROR binding agent has antigen binding sites that specifically bind to R0R1 only. In other embodiments, the activatable ROR binding agent has antigen binding sites that specifically bind to R0R2 only. In preferred embodiments, the activatable ROR binding agent has antigen binding sites that are cross-reactive and specifically bind to both R0R1 and R0R2.

[0048] Thus, in one embodiment, the term “ROR” or “ROR antigen” as used herein refers to ROR1 (e.g., human ROR1). In another embodiment, the term “ROR” or “ROR antigen” as used herein refers to ROR2 (e.g., human ROR2). In a preferred embodiment, the term “ROR” or “ROR antigen” as used herein refers to both ROR1 and ROR2 (e.g., both human ROR1 and human ROR2).

[0049] ROR1 and ROR2 proteins typically consist of at least four protein domains: three extracellular domains — Ig-like, FZ, and Kringle domains — as well as the intracellular Protein Kinase domain. In some embodiments, the activatable ROR binding agent has antigen binding sites that specifically bind to the extracellular portion of the ROR antigen. In certain embodiments, the activatable ROR binding agent has antigen binding sites that specifically bind to the Ig-like domain. In other embodiments, the activatable ROR binding agent has antigen binding sites that specifically bind to the FZ domain. In still other embodiments, the activatable ROR binding agent has antigen binding sites that specifically bind to the Kringle domain. In particular embodiments, the activatable ROR binding agent has antigen binding sites that specifically bind to at least a portion of a single ROR domain. In particular embodiments, the activatable ROR binding agent has antigen binding sites that specifically bind to at least a portion of more than one ROR domain, such as the junction between a first and a second ROR domain. The ROR domains can refer to ROR1 domains or ROR2 domains.

[0050] In specific embodiments, the ROR antigen is human. UniProt accession #Q01973 describes a canonical human ROR1 protein, including its sequences and domain features, and is hereby incorporated by reference in its entirety. SEQ ID NO:40 provides the full-length ROR1 protein sequence (see Table 7). With reference to the full-length sequence from the N- terminus to C-terminus, the Ig-Like domain is defined as amino acids 42-147, the FZ domain as amino acids 165-299, and the Kringle domain as amino acids 312-391. UniProt accession #Q01974 describes a canonical human ROR2 protein, including its sequences and domain features, and is hereby incorporated by reference in its entirety. SEQ ID NO:41 provides the full-length ROR2 protein sequence (see Table 7). With reference to the full-length sequencefrom the N-terminus to C-terminus, the Ig-Like domain is defined as amino acids 55-145, the FZ domain as amino acids 169-303, and the Kringle domain as amino acids 316-394.

[0051] Various tumors can demonstrate cell-surface expression of ROR antigens, as described in greater detail in Gentile, et al. (Cancer Res,' 71(8) April 15, 2011), Rebagay, et al. (Front. Oncol., 18 April 2012), Zhang, et al. (American Journal of Pathology, Vol. 181, No. 6, December 2012), Henry, et al. (Oncotarget, Vol. 6, No. 37 2015), Zhang, et al. (PLoS ONE 7(3): e31127.), and Bainbridge, et al. PLoS ONE 9 T. el02695.), each herein incorporated by reference in their entirety. In addition, ROR expression may not be expressed, or only demonstrate limited expression, in normal, i.e. non-cancerous, tissue as described in Balakrishnan et al. Clin Cancer Res. 2017 Jun 15; 23(12): 3061-3071), herein incorporated in its entirety. Thus ROR antigens can be used as a tumor-specific marker in certain tumors. Examples of tumors and cancers with demonstrated ROR expression include, but are not limited to, pancreatic cancer, ovarian cancer, breast cancer, lung cancer, gastric cancer, melanoma, Ewing sarcoma, chronic lymphocytic leukemia, mantle cell lymphoma, and B-ALL, as described in Gohil et al. (Oncoimmunology . 2017; 6(7): el326437.), herein incorporated in its entirety. Other cancers include, but are not limited to, hematological cancer, prostate cancer, colon cancer, renal cancer, and uterine cancer.5.2. Definitions

[0052] Techniques and procedures described or referenced herein include those that are generally well understood and / or commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3d ed. 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010); and Antibody Engineering Vols 1 and 2 (Kontermann and Diibel eds., 2d ed. 2010). Unless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control.

[0053] As used herein, the term “binding agent” or a grammatical equivalent thereof refers to a molecule (e.g., antibody) with one or more antigen-binding sites that binds anantigen. In some embodiments, an activatable ROR binding agent as described herein is an activatable antibody (including an activatable antibody fragment, such as an activatable antigen-binding fragment or an activatable epitope-binding fragment) or other activatable peptide-based molecule as well as a conjugate of an activatable antibody, activatable antibody fragment, or activatable peptide-based molecule (e.g., an activatable antibody-drug conjugate) that binds to ROR, such as human ROR.

[0054] In some embodiments, an “activatable” binding agent refers to a binding agent that exhibits a first level of binding to a target when in an inhibited, masked, and / or uncleaved state, and exhibits a second level of binding to the target in an uninhibited, unmasked, and / or cleaved state, where the second level of target binding is greater than the first level of target binding. In some embodiments, access to the target by the activatable binding agent is greater after cleavage within the cleavable moiety (e.g., by one or more proteases).

[0055] The terms “antibody,” “immunoglobulin,” and “Ig” are used interchangeably herein, and are used in the broadest sense and specifically cover, for example polyclonal antibodies, monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full-length monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, recombinantly produced antibodies, single domain (e.g., VHH) antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human versions of antibodies having full-length heavy and / or light chains. VHH as used herein refers to a domain antibody derived from a variable region of a heavy chain only antibody. Exemplary single domain antibodies include, but are not limited to, antibodies naturally devoid of light chains such as those from Camelidae species (e.g., llama), single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, and bovine. VHH can also be derived from other species besides Camelidae that may produce heavy chain antibodies naturally devoid of light chain. Antibodies also include antibody fragments (and / or polypeptides that comprise antibody fragments) that retain ROR binding characteristics. Non-limiting examples of antibody fragments include antigen-binding regions and / or effector regions of the antibody, e.g., Fab, Fab’, F(ab’)2, Fv, scFv, (scFv)2, single chain antibody molecule, dual variable domain antibody, single variable domain, linear antibody, V region, a multispecific antibody formed from antibody fragments, F(ab)2, Fd, Fc,diabody, di-diabody, disulfide-linked Fvs (dsFv), single-domain antibody (e.g., nanobody) or other fragments (e.g., fragments consisting of the variable regions of the heavy and light chains that are non-covalently coupled). In general terms, a variable (V) region domain may be any suitable arrangement of immunoglobulin heavy (VH) and / or light (VL) variable domains. For example, antibodies also include tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, and an antibody heavy chain monomer. Thus, for example, the V region domain may be dimeric and contain VHH-VHH, VH-VH, VH-VL, or VL-VL dimers that bind ROR. If desired, the VH and VL may be covalently coupled either directly or through a linker to form a single chain Fv (scFv). For ease of reference, scFv proteins are referred to herein as included in the category “antibody fragments.” Another form of an antibody fragment is a peptide comprising one or more complementarity determining regions (CDRs) of an antibody. CDRs (also termed “minimal recognition units” or “hypervariable regions”) can be obtained by constructing polynucleotides that encode one or more CDRs of interest. Such polynucleotides are prepared, for example, by using the polymerase chain reaction to synthesize the variable region using mRNA of antibody-producing cells as a template (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology, 2: 106 (1991); Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166, Cambridge University Press (1995); and Ward et al, “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137, Wiley- Liss, Inc. (1995)). Antibody fragments may be incorporated, for example, into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, variable domains of new antigen receptors (v-NAR), and bis-single chain Fv regions (see, e.g., Hollinger and Hudson, Nature Biotechnology, 23(9): 1126-1136, 2005). In some embodiments, antibodies comprising a VH and / or VL further contain a light chain and / or a heavy chain constant region, such as one or more constant regions, including one or more IgGl, IgG2, IgG3 and / or IgG4 constant regions. In some embodiments, antibodies can include epitope-binding fragments of any of the above. The antibodies described herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) of immunoglobulin molecule.

[0056] The term “monospecific” when used in reference to a binding agent (e.g., an antibody) as used herein denotes a binding agent that has one or more binding sites each of which binds to the same epitope of the same antigen.

[0057] The term “multispecific” when used in reference to a binding agent (e.g., an antibody) means that the binding agent is able to specifically bind to at least two distinct epitopes, for example two binding sites each formed by a pair of an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL) or each formed by a pair of VHH domains binding to different antigens or to different epitopes on the same antigen. Such a bispecific binding agent (e.g., an antibody) may have a 1+1 format (comprising one binding site for a first antigen or epitope and one binding site for a second antigen or epitope). Other bispecific binding agent (e.g., an antibody) formats may be 2+1 or 1+2 formats (comprising two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or 2+2 format (comprising two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). When a bispecific binding agent (e.g., an antibody) comprises two antigen-binding sites, each may bind to a different epitope. Such a bispecific binding agent (e.g., an antibody) may bind to two different epitopes on the same antigen (e.g., epitopes on ROR).

[0058] The terms “identical” or percent “identity” in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two nucleic acids or polypeptides are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, or 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the amino acid sequences that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a target protein or an antibody. In someembodiments, identity exists over a region of the nucleotide sequences that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments the sequences are substantially identical over the full-length of the sequences being compared, such as a nucleotide sequence encoding a protein of interest.

[0059] A “conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue having a side chain with similar chemical characteristics. Families of amino acid residues having similar side chains have been generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. Generally, conservative substitutions in the sequences of the polypeptides, soluble proteins, and / or antibodies of the disclosure do not abrogate the binding of the polypeptide, soluble protein, or antibody containing the amino acid sequence, to the target binding site. Methods of identifying amino acid conservative substitutions which do not eliminate binding are well-known in the art.

[0060] The term “polypeptide” refers to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can include (e.g., be interrupted by) non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as linkage to or conjugation with (directly or indirectly) a moiety such as a labeling component or a drug (e.g., toxin). Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art. It is understood that, because the polypeptides of this disclosure can be based upon antibodies or other members of the immunoglobulin superfamily, in some embodiments, the polypeptides can occur as single chains or dimers of single chains.

[0061] As used herein, an “antigen” is a moiety or molecule that contains an epitope to which a binding agent (e.g., an antibody) can bind. As such, an antigen can be bound by anantibody. In some embodiments, the antigen, to which a binding agent (e.g., an antibody) described herein binds, is ROR (e.g., human ROR), or a fragment thereof, including a fragment that comprises one or more domains of ROR.

[0062] As used herein, an “epitope” is a term in the art and refers to a localized region of an antigen to which an antibody can bind. An epitope can be a linear epitope or a conformational, non-linear, or discontinuous, epitope. In the case of a polypeptide antigen, for example, an epitope can be contiguous amino acids of the polypeptide (a “linear” epitope) or an epitope can comprise amino acids from two or more non-contiguous regions of the polypeptide (a “conformational,” “non-linear” or “discontinuous” epitope), e.g., human ROR. It will be appreciated by one of skill in the art that, in general, a linear epitope may or may not be dependent on secondary, tertiary, or quaternary structure. For example, in some embodiments, an antibody binds to a group of amino acids regardless of whether they are folded in a natural three-dimensional protein structure. In other embodiments, an antibody requires amino acid residues making up the epitope to exhibit a particular conformation (e.g., bend, twist, turn or fold) in order to recognize and bind the epitope.

[0063] An antibody binds “an epitope” or “essentially the same epitope” or “the same epitope” as a reference antibody, when the two antibodies recognize identical, overlapping or adjacent epitopes in a three-dimensional space. The most widely used and rapid methods for determining whether two antibodies bind to identical, overlapping or adjacent epitopes in a three-dimensional space are competition assays, which can be configured in a number of different formats, for example, using either labeled antigen or labeled antibody. In some assays, the antigen is immobilized on a 96-well plate, or expressed on a cell surface, and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured using radioactive, fluorescent or enzyme labels.

[0064] “Epitope binning” is the process of grouping antibodies based on the epitopes they recognize. More particularly, epitope binning comprises methods and systems for discriminating the epitope recognition properties of different antibodies, using competition assays combined with computational processes for clustering antibodies based on their epitope recognition properties and identifying antibodies having distinct binding specificities.

[0065] As used herein, the terms “specifically binds,” “specifically recognizes,” “immunospecifically binds,” “selectively binds,” “immunospecifically recognizes” and “immunospecific” are analogous terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., epitope) as such binding is understood by one skilled in the art. In some embodiments, “specifically binds” means, for instance that a polypeptide ormolecule interacts more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to the epitope, protein, or target molecule than with alternative substances, including related and unrelated proteins. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides, generally with lower affinity as determined by, e.g., immunoassays, BIACORE™, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), the OctetQK384 system (ForteBio, Menlo Park, CA), or other assays known in the art. In some embodiments, an antibody or antigen-binding domain binds to or specifically binds to an antigen when it binds to the antigen with higher affinity than to any cross-reactive antigen as determined using experimental techniques, such as radioimmunoassays (RIAs) and enzyme linked immunosorbent assays (ELISAs). Typically a specific or selective reaction will be at least twice background signal or noise and may be more than 10 times background. See, e.g., Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989) for a discussion regarding binding specificity. In some embodiments, the extent of binding of an antibody or antigen-binding domain to a “non-targef ’ protein is less than about 10% of the binding of the antibody or antigen-binding domain to its particular target antigen, for example, as determined by fluorescence activated cell sorting (FACS) analysis or RIAs. In some embodiments, molecules that specifically bind to an antigen bind to the antigen with a Ka that is at least 2 logs, 2.5 logs, 3 logs, 4 logs or greater than the Ka when the molecules bind to another antigen. In some embodiments, molecules that specifically bind to an antigen do not cross react with other proteins. In another specific embodiment, molecules that specifically bind to an antigen do not cross react with other non-ROR proteins. In some embodiments “specifically binds” means, for instance, that a polypeptide or molecule binds a protein or target with a KD of about 0. ImM or less, but more usually less than about 1 pM. In some embodiments, “specifically binds” means that a polypeptide or molecule binds a target with a KD of at least about 0.1 pM or less, at least about 0.01 pM or less, or at least about InM or less. Because of the sequence identity between homologous proteins in different species, specific binding can include a polypeptide or molecule that recognizes a protein or target in more than one species. Likewise, because of homology within certain regions of polypeptide sequences of different proteins, specific binding can include a polypeptide or molecule that recognizes more than one protein or target. It is understood that, in some embodiments, a polypeptide or molecule that specifically binds a first target may or may not specifically bind a second target. As such, “specific binding” does not necessarily require (although it can include) exclusive binding, e.g., binding to a single target. Thus, a polypeptide or molecule can, in some embodiments, specifically bind more than one target. In some embodiments,multiple targets can be bound by the same antigen-binding site on the polypeptide or molecule. For example, an antibody can, in certain instances, comprise two identical antigenbinding sites, each of which specifically binds the same epitope on two or more proteins. In certain alternative embodiments, an antibody can be bispecific and comprise at least two antigen-binding sites with differing specificities. Generally, but not necessarily, reference to “binding” means “specific binding”.

[0066] “Binding affinity” generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., a binding agent such as an antibody) and its binding partner (e.g., an antigen such as ROR). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a binding molecule X for its binding partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure. In one embodiment, the “KD” or “KD value” may be measured by biolayer interferometry (BLI) using, for example, the OctetQK384 system (ForteBio, Menlo Park, CA). Alternatively, the KD may also be measured in a radiolabeled antigen-binding assay (RIA), for example, performed with the Fab version of an antibody of interest and its antigen (Chen, et al.. (1999) J. Mol Biol 293:865- 881) or using surface plasmon resonance (SPR) assays by BIACORE™, using, for example, a BIACORE™-2000 or a BIACORE™-3000 (BIACORE™, Inc., Piscataway, NJ). An “on- rate” or “rate of association” or “association rate” or “kon,” as well as an “off-rate” or “rate of dissociation” or “dissociation rate” or “koir,” can also be determined with the same SPR or BLI techniques described above using, for example, the OctetQK384 system (ForteBio, Menlo Park, CA) or a BIACORE™-2000 or a BIACORE™-3000 (BIACORE™, Inc., Piscataway, NJ), respectively.

[0067] The term “compete” or any grammatical variation thereof when used in the context of ROR binding agents (e.g., antibodies) means binding agents that compete for the same epitope or binding site on a target, which includes competition between such binding agents as determined by an assay in which the binding agent under study prevents or inhibits the specific binding of a reference molecule (e.g., a reference ligand, or reference antigenbinding protein, such as a reference antibody) to a common antigen (e.g., ROR). Numeroustypes of competitive binding assays can be used to determine if a test binding agent competes with a reference molecule for binding to ROR (e.g., human ROR). Examples of assays that can be employed include solid phase direct or indirect radioimmunoassay (RIA); solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., (1983) Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., (1986) J. Immunol. 137:3614-3619 or Cheung, et al, (1990) Virology 176:546-552); solid phase direct labeled assay; solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (see, e.g., Morel et al., (1988) Molec. Immunol. 25:7-15); and direct labeled RIA (Moldenhauer et al., (1990) Scand. J. Immunol. 32:77-82). Typically, such an assay involves the use of a purified antigen (e.g., ROR, such as human ROR) bound to a solid surface or cells bearing either of an unlabelled test antigenbinding protein (e.g., test ROR antibody) or a labeled reference antigen-binding protein (e.g., reference ROR antibody). Competitive inhibition may be measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen-binding protein. Usually, the test antigen-binding protein is present in excess. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and / or antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference for antibodies steric hindrance to occur (e.g., similar epitope or overlapping epitope). Usually, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 20%, for example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some instance, binding is inhibited by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more.

[0068] As used herein, the term “constant region” or “constant domain” is a well-known antibody term of art and refers to an antibody portion, for example, a carboxyl terminal portion of a light and / or heavy chain which is not directly involved in binding of an antibody to an antigen but which can exhibit various effector functions, such as interaction with an Fc receptor. The term includes the portion of an immunoglobulin molecule having a generally more conserved amino acid sequence relative to an immunoglobulin variable domain.

[0069] Antibody “effector functions” refer to those biological activities attributable to the Fc region (e.g., a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0070] The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is often defined to stretch from an amino acid residue at position Cys226 (according to the EU numbering system), or from Pro230 (according to the EU numbering system), to the carboxyl-terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. An exemplary Fc region sequence is provided below (CH2 domain = bold text; CH3 domain = underline text):CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGOPREPQVYTLPPSRDELTKNOVSLTCLVKGFYPSDIAVEWESNGOPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMHEALHNHYTQKSLSLSPG K (SEQ ID NO:42).

[0071] A “functional Fc region” possesses an “effector function” of a native sequence Fc region. Exemplary “effector functions” include Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis (such as antibody-dependent cellular phagocytosis, z.e., ADCP); down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain) and can be assessed using various assays as disclosed.

[0072] A “native sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature, and not manipulated, modified, and / or changed (e.g., isolated, purified, selected, including or combining with other sequences such as variable region sequences) by a human. Native sequence human Fc regions include a native sequence human IgGl Fc region (non- A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region as well as naturally occurring variants thereof.

[0073] A “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification (e.g.,substituting, addition, or deletion), preferably one or more amino acid substitution(s). In some embodiments, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, for example, from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region described herein can possess at least about 80% homology with a native sequence Fc region and / or with an Fc region of a parent polypeptide, or at least about 90% homology therewith, for example, at least about 95% homology therewith. The variant Fc region herein described herein may have a loss of an effector function (e.g., silent Fc). An exemplary variant Fc region (“silent Fc”) sequence is provided below (CH2 domain = bold text with amino acid changes underlined; CH3 domain = underline text):CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALKAPIE KTISKAKGOPREPQVYTLPPSRDELTKNOVSLTCLVKGFYPSDIAVEWESNGOPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMHEALHNHYTQKSLSLSPG K (SEQ ID NO:43).

[0074] As used herein, the term “heavy chain” when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids, and a carboxy -terminal portion includes one or more constant regions. The “heavy chain” can refer to any distinct types, e.g., for example, alpha (a), delta (5), epsilon (a), gamma (y) and mu (p), based on the amino acid sequence of the constant domain, which give rise to IgA, IgD, IgE, IgG and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgGl, IgG2, IgG3 and IgG4.

[0075] As used herein, the term “light chain” when used in reference to an antibody can refer to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids, and a carboxy -terminal portion includes a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two distinct types, e.g., kappa (K) or lambda (X) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art.

[0076] In one embodiment, a “chain” (e.g., a heavy chain or a light chain) is itself a molecule (e.g., a polypeptide). In another embodiment, a “chain” (e.g., a heavy chain or alight chain) is part of a molecule (e.g., a polypeptide), for example, is directly or indirectly conjugated to the remaining part of the molecule (such as polypeptide).

[0077] The terms “antigen-binding fragment,” “antigen-binding domain,” “antigenbinding region,” and similar terms refer to that portion of an antibody, which comprises the amino acid residues that interact with an antigen and confer on the binding fragment, domain, or region its specificity and affinity for the antigen (e.g., the CDRs). “Antigen-binding fragment” as used herein includes “antibody fragment,” which comprises a portion of an antibody including one or more CDRs, such as the antigen-binding or variable region of the antibody.

[0078] Antibodies described herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc.), camelized antibodies, Fab fragments, F(ab’) fragments, disulfide-linked Fvs (sdFv), anti -idiotypic (anti- id) antibodies, and epitope-binding fragments of any of the above.

[0079] In some embodiments, antibodies described herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, including molecules that contain one or more antigen-binding sites that bind to an ROR antigen.

[0080] Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule. In some embodiments, antibodies described herein are IgG antibodies (e.g., human IgG), or a class (e.g., human IgGl, IgG2, IgG3 or IgG4) or a subclass thereof.

[0081] In some embodiments, an antibody is a 4-chain antibody unit comprising two heavy (H) chain / light (L) chain pairs. In further embodiments, the amino acid sequences of the H chains are identical and the amino acid sequences of the L chains are identical. In other embodiments, the amino acid sequences of the H chains are different from each other. Additionally or alternatively, the amino acid sequences of the L chains are different from each other. For example, an antibody comprises a first H / L chain pair and a second H / L chain pair, wherein the first H / L chain pair binds to an ROR antigen and the second H / L chain pair binds to another ROR antigen or a non-ROR antigen. In some embodiments, an antibody is a 2-chain antibody unit comprising a VHH-VHH pair. In further embodiments, the amino acid sequences of the VHH are identical. In other embodiments, the amino acid sequence of the VHH are different from each other. For example, an antibody comprises afirst VHH and a second VHH, wherein the first VHH binds to an ROR antigen and the second VHH binds to another ROR antigen or a non-ROR antigen. In some embodiments, the H and / or L chains comprise constant regions, for example, human constant regions. In some embodiments, the L chain constant region of such antibodies is a kappa or lambda light chain constant region, for example, a human kappa or lambda light chain constant region. In some embodiments, the H chain constant region of such antibodies comprises a gamma heavy chain constant region, for example, a human gamma heavy chain constant region. In some embodiments, such antibodies comprise IgG constant regions, for example, human IgG constant regions (e.g., IgGl, IgG2, IgG3, and / or IgG4 constant regions).

[0082] An antibody or fragment thereof may preferentially bind to ROR, such as human ROR, meaning that the antibody or fragment thereof binds ROR with greater affinity than it binds to a control protein (e.g., unrelated control proteins such as hen egg white lysozyme) and / or binds human ROR with greater affinity than it binds to an unrelated control protein. For example, the antibody or fragment thereof may specifically recognize and bind ROR or a portion thereof. “Specific binding” means that the antibody or fragment thereof binds to ROR with an affinity that is at least 5, 10, 15, 20, 25, 50, 100, 250, 500, 1000, or 10,000 times greater than the affinity for an unrelated control protein (e.g., hen egg white lysozyme). In some embodiments, the antibody or fragment thereof may bind ROR substantially exclusively (e.g., is able to distinguish ROR from other known polypeptides, for example, by virtue of measurable differences in binding affinity). In some embodiments, an ROR binding agent (e.g., an antibody) may react with ROR sequences other than human ROR sequences. In other embodiments, an ROR binding agent (e.g., an antibody) does not react with nonhuman ROR sequences.

[0083] The term “variable region” or “variable domain” refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain, has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and is used in the binding and specificity of each particular antibody for its particular antigen. The variable region of the heavy chain may be referred to as “VH ” The variable region of the light chain may be referred to as “VL ” The term “variable” refers to the fact that certain segments of the variable regions differ extensively in sequence among antibodies. The V region mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable regions. Instead, the V regions consist of less variable (e.g., relatively invariant) stretches called frameworkregions (FRs) of about 15-30 amino acids separated by shorter regions of greater variability (e.g., extreme variability) called “hypervariable regions” or alternatively called “complementarity determining regions (CDRs).” The variable regions of heavy and light chains each comprise four frameworks (FR1, FR2, FR3 and FR4), largely adopting a P sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the P sheet structure. The hypervariable regions in each chain are held together in close proximity by the frameworks and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, (1991)). The constant regions are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). The variable regions differ extensively in sequence between different antibodies. The variability in sequence is concentrated in the CDRs while the less variable portions in the variable region are referred to as framework regions (FR). The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with its antigen. In specific embodiments, the variable region is a human variable region.

[0084] The term “hypervariable region,” “HVR,” “HV,” “complementarity determining region,” or “CDR” when used herein refers to the regions of an antibody variable region that are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies comprise six hypervariable regions: three in the VH (Hl or VH CDR1, H2 or VH CDR2, and H3 or VH CDR3), and three in the VL (LI or VL CDR1, L2 or VL CDR2, and L3 or VL CDR3). A number of hypervariable region delineations are in use and are encompassed herein. The Kabat CDRs are based on sequence variability and are the most commonly used (see, e.g., Kabat et al. , Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia refers instead to the location of the structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35 A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by OxfordMolecular’s AbM antibody modeling software (see, e.g., Martin, in Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag). The “contact” hypervariable regions are based on an analysis of the available complex crystal structures. The residues from each of these hypervariable regions or CDRs are noted below.

[0085] A universal numbering system has been developed and widely adopted, ImMunoGeneTics (IMGT®) Information System (Lefranc el al.. Dev. Comp. Immunol. 27(l):55-77 (2003)). IMGT is an integrated information system specializing in immunoglobulins (IG), T cell receptors (TR) and major histocompatibility complex (MHC) of human and other vertebrates. Herein, the CDRs are referred to in terms of both the amino acid sequence and the location within the light or heavy chain. As the “location” of the CDRs within the structure of the immunoglobulin variable domain is conserved between species and present in structures called loops, by using numbering systems that align variable domain sequences according to structural features, CDR and framework residues and are readily identified. This information can be used in grafting and replacement of CDR residues from immunoglobulins of one species into an acceptor framework from, typically, a human antibody. An additional numbering system (AHon) has been developed by Honegger and Pliickthun, J. Mol. Biol. 309: 657-670 (2001). Correspondence between the numbering system, including, for example, the Kabat numbering and the IMGT unique numbering system, is well known to one skilled in the art (see, e.g., Kabat, supra, Chothia and Lesk, supra, Martin, supra, Lefranc et al., supra) and is also illustrated below. Various systems known in the art or described herein represent different ways of delineating CDRs, and when they are used to define the same antibody, they are often considered equivalent. An Exemplary system, shown herein, combines Kabat and Chothia. The residues from each of these hypervariable regions or CDRs are exemplified in the table below.Exemplary CDRs According to Various Numbering Systems

[0086] Hypervariable regions may comprise “extended hypervariable regions” as follows: 24-36 or 24-34 (LI), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 or 26- 35A (Hl), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. As usedherein, the terms “hypervariable region,” “HVR,” “HV,” “complementarity determining region,” or “CDR” are used interchangeably.

[0087] “Polynucleotide” or “nucleic acid,” as used interchangeably herein, refers to polymers of nucleotides of any length and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. A cell that produces a binding molecule of the present disclosure may include a parent hybridoma cell, as well as bacterial and eukaryotic host cells into which nucleic acids encoding the antibodies have been introduced. Unless specified otherwise, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5’ end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5’ direction. The direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5’ to the 5’ end of the RNA transcript are referred to as “upstream sequences”; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3 ’ to the 3 ’ end of the RNA transcript are referred to as “downstream sequences.”

[0088] The term “vector” refers to a substance that is used to carry or include a nucleic acid sequence, including for example, in order to introduce a nucleic acid sequence into a host cell. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and / or inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more nucleic acid molecules are to be co-expressed (e.g., both an antibody heavy and light chain or an antibody VH and VL) both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter.The introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficient amount to produce a desired product (e.g., an activatable ROR binding agent as described herein), and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

[0089] The term “pharmaceutically acceptable” as used herein means being approved by a regulatory agency of the federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.

[0090] “Excipient” means a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, encapsulating materials or additives such as absorption accelerators, antioxidants, binders, buffers, carriers, coating agents, coloring agents, diluents, disintegrating agents, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, perfumes, preservatives, propellants, releasing agents, sterilizing agents, sweeteners, solubilizers, wetting agents and mixtures thereof. The term “excipient” can also refer to a diluent, adjuvant (e.g., Freunds’ adjuvant (complete or incomplete)) or vehicle. In some embodiments, excipients are pharmaceutically acceptable excipients. Examples of pharmaceutically acceptable excipients include buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., fewer than about 10 amino acid residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or nonionic surfactants, such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington’s Pharmaceutical Sciences (18th ed. 1990). In one embodiment, each component is “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue ororgan of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio. See, e.g., Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, pharmaceutically acceptable excipients are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. In some embodiments, a pharmaceutically acceptable excipient is an aqueous pH buffered solution.In some embodiments, excipients are sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is an exemplary excipient when a composition (e.g., a pharmaceutical composition) is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, particularly for injectable solutions. An excipient can also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like. Oral compositions, including formulations, can include standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Compositions, including pharmaceutical compounds, may contain a prophylactically or therapeutically effective amount of an activatable ROR binding agent (e.g., an activatable antibody), for example, in isolated or purified form, together with a suitable amount of excipient so as to provide the form for proper administration to the subject (e.g., patient). The formulation should suit the mode of administration.

[0091] An “effective amount” is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate the symptoms and / or underlying cause, prevent or delay the occurrence of symptoms and / or their underlying cause, and / or improve or remediate the damage that results from or is associated with a disease, disorder, or condition. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount.

[0092] The term “therapeutically effective amount” as used herein refers to the amount of an agent (e.g., an antibody described herein or any other agent described herein) that is sufficient to reduce and / or ameliorate the severity and / or duration of a given disease, disorder or condition, and / or a symptom related thereto. A therapeutically effective amount of an agent, including a therapeutic agent, can be an amount necessary for (i) reduction, delay or amelioration of the advancement or progression of a given disease, disorder, or condition, (ii) reduction, delay or amelioration of the recurrence, development or onset of a given disease, disorder or conditions, and / or (iii) to improve or enhance the prophylactic or therapeutic effect of another therapy (e.g., a therapy other than the administration of an agent described herein). A “therapeutically effective amount” of a substance / molecule / agent of the present disclosure (e.g., an activatable ROR antibody) may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule / agent, to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the substance / molecule / agent are outweighed by the therapeutically beneficial effects. In certain embodiments, the term “therapeutically effective amount” refers to an amount of an agent effective to “treat” a disease, disorder, or condition, in a subject or mammal.

[0093] The term “treating” or any grammatical variation thereof refers to reducing and / or ameliorating the severity and / or duration of a given disease, disorder or condition, and / or a symptom related thereto, such as (i) reduction, delay or amelioration of the advancement or progression of a given disease, disorder, or condition, (ii) reduction, delay or amelioration of the recurrence, development or onset of a given disease, disorder or conditions, and / or (iii) to improve or enhance the prophylactic or therapeutic effect of another therapy (e.g., a therapy other than the administration of an agent described herein).

[0094] A “prophylactically effective amount” is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of a disease, disorder or condition, or reducing the likelihood of the onset (or reoccurrence) of a disease, disorder, or condition or associated symptom(s).

[0095] The full therapeutic or prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount may be administered in one or more administrations.

[0096] The terms “about” and “approximately” mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less variation of a given value or range.

[0097] As used herein, comparative terms as used herein, such as reduce, decrease, increase, or any grammatical variation thereof, can refer to certain variation from the reference. In some embodiments, such variation can refer to about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 1 fold, or about 2 fold, or about 3 fold, or about 4 fold, or about 5 fold, or about 10 fold, or about 20 fold, or about 30 fold, or about 40 fold, or about 100 fold or higher than the reference. In some embodiments, such variation can refer to about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of the reference.

[0098] As used in the present disclosure and claims, the singular forms “a”, “an” and “the” include plural forms unless the context clearly dictates otherwise.

[0099] In some embodiments, the terms “first,” “second,” “third,” “fourth” and similar in a component name are used to distinguish and identify more than one component sharing certain identity in their names. For example, “first antibody” and “second antibody” are used to distinguish two antibodies.

[0100] It is understood that wherever embodiments are described herein with the term “comprising” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. It is also understood that wherever embodiments are described herein with the phrase “consisting essentially of’ otherwise analogous embodiments described in terms of “consisting of’ are also provided.

[0101] The term “between” as used in a phrase as such “between A and B” or “between A-B” refers to a range including both A and B.

[0102] The term “and / or” as used in a phrase such as “A and / or B” herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0103] The term “optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances wherein the circumstance occurs, and the instances wherein the circumstance does not occur.5.3. ROR Binding Agents

[0104] In some embodiments, the present disclosure provides activatable ROR binding agents. Such agents include activatable antibodies (e.g., monospecific or multispecific, including bispecific) that bind to ROR. Exemplary activatable antibodies include polyclonal, monoclonal, humanized, human, bispecific, and heteroconjugate antibodies, as well as variants thereof having increased or decreased affinity or other properties.

[0105] In some embodiments, the activatable ROR binding agents (e.g., activatable antibodies) of the present disclosure are context-dependent (e.g., are activated (are only capable of binding ROR) in certain contexts (such as in the protease-rich tumor microenvironment)). In some embodiments, the activatable ROR binding agents (e.g., activatable antibodies) of the present disclosure provide improved safety over more traditional, non-activatable binding agents (e.g, antibodies) (e.g., show reduced toxicity, do not induce significant alterations to the weights of many organs, do not alter liver histopathology, hematology, and / or blood biochemistry, etc.). In some embodiments, the activatable ROR binding agents (e.g., activatable antibodies) of the present disclosure have improved pharmacokinetic properties as compared to more traditional, non-activatable binding agents (e.g., antibodies) (e.g., have longer in vivo half-lives).

[0106] In some embodiments, described herein are activatable ROR binding agents (e.g., activatable antibodies) that bind to ROR, including an ROR polypeptide, an ROR polypeptide fragment, an ROR peptide or an ROR epitope. In some embodiments, the activatable ROR binding agents are human or humanized antibodies (e.g., comprising human constant regions) that bind ROR, including an ROR polypeptide, an ROR polypeptide fragment, an ROR peptide or an ROR epitope. In some embodiments, an activatable ROR binding agent (e.g., an activatable antibody), such as an activatable human ROR binding agent, can bind to ROR expressed on the surface of a mammalian (e.g., human) cell, including an ROR expressing tumor cell. In some embodiments, an activatable ROR binding agent (e.g., an activatable antibody) binds an ROR extracellular epitope exposed on a cell such as a tumor cell. In some embodiments, described herein is an activatable ROR binding agent (e.g., an activatable antibody) that binds to ROR, such as human ROR or a portion thereof. In some embodiments, ROR is a human ROR. In a specific embodiment, ROR is ROR1. In anotherspecific embodiment, ROR is ROR2. In another specific embodiment, ROR is ROR1 and ROR2. In some embodiments, an activatable ROR binding agent is an activatable human ROR binding agent (e.g., an activatable antibody that binds to human ROR).

[0107] In some embodiments, the activatable ROR binding agent (e.g., an activatable antibody) provided herein binds to ROR (e.g., human ROR) with a dissociation constant (KD) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure, including by RIA, for example, performed with the Fab version of an antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81); by biolayer interferometry (BLI) or surface plasmon resonance (SPR) assays by OCTET®, using, for example, an OCTET®Red96 system, or by BIACORE®, using, for example, a BIACORE®TM-2000 or a BIACORE®TM-3000. An “on-rate” or “rate of association” or “association rate” or “kon” may also be determined with the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above using, for example, the OCTET®Red96, the BIACORE®TM-2000, the BIACORE®TM-3000 system, the BIACORE®TM-8K, or the BIACORE®TM-8K+ system.

[0108] In various aspects, the present disclosure also relates to activatable binding polypeptides (i.e., activatable antibodies) that bind to ROR e.g., human ROR), including activatable antibodies comprising any of the anti-ROR antibodies described herein, antigen binding fragments of the activatable anti-ROR antibodies, and / or derivatives of the activatable anti-ROR antibodies. In some embodiments, the activatable anti-ROR antibodies described herein may have improved safety profiles. For example, the activatable anti-ROR antibodies described herein may have better safety margin, for example not binding to pancreas e.g., human pancreatic islets) when in inactive form, or have weaker binding to pancreas e.g., human pancreatic islets) when in inactive form relative to when in active form. The binding to pancreas e.g., human pancreatic islets) may be measured by any suitable method, for example, IHZ, immunohistochemistry (IHC), or immunofluorescence (IF) staining.

[0109] In some embodiments, an activatable antibody of the present disclosure comprises: (a) a masking moiety (MM); (b) a cleavable moiety (CM); and (c) a target binding moiety (TBM). In some embodiments, the MM is any of the masking moieties described herein. In some embodiments, the CM is any of the cleavable moieties described herein. In some embodiments, the TBM is any of the target binding moieties described herein e.g., a target binding moiety (TBM) comprising an antibody light chain variable region and / or anantibody heavy chain variable region, such as a VH and / or VL of any of the anti-ROR antibodies described herein). In some embodiments, the MM interferes with and / or inhibits the binding of the activatable antibody to its target (e.g., human ROR) when the CM is not cleaved. In some embodiments, the activatable antibody is capable of binding to its target (e.g., human ROR) when the CM is cleaved.

[0110] In some embodiments, a polypeptide comprising, for example from its N terminus to its C terminus, an MM and a CM is referred to herein as a masking peptide.

[0111] In some embodiments, the activatable antibody comprises: (a) a polypeptide comprising, from N-terminus to C-terminus, a masking moiety (MM), a cleavable moiety (CM), and a target binding moiety (TBM), where the MM is any of the masking moieties described herein, the CM is any of the cleavable moieties described herein, and where the TBM comprises an antibody light chain variable region (VL); and (b) an antibody heavy chain variable region (VH).

[0112] In some embodiments, the activatable antibody comprises: (a) a polypeptide comprising, from N-terminus to C-terminus, a masking moiety (MM), a cleavable moiety (CM), and a target binding moiety (TBM), where the MM is any of the masking moieties described herein, the CM is any of the cleavable moieties described herein, and where the TBM comprises an antibody heavy chain variable region (VH); and (b) an antibody light chain variable region (VL) .

[0113] In some embodiments, the activatable antibody comprises: a polypeptide comprising, from N-terminus to C-terminus, a masking moiety (MM), a cleavable moiety (CM), and a target binding moiety (TBM), where the MM is any of the masking moieties described herein, the CM is any of the cleavable moieties described herein, and where the TBM comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL).

[0114] The term “activatable binding polypeptide”, “ABP”, or “activatable antibody” includes a polypeptide that comprises a target binding moiety (TBM), a cleavable moiety (CM), and a masking moiety (MM). In some embodiments, the TBM comprises an amino acid sequence that binds to a target. In some embodiments, the TBM comprises an antigen binding domain (ABD) of an antibody or antibody fragment thereof (e.g., any of the antibodies or antigen binding fragments described herein). In some embodiments, the antigen binding domain comprises a heavy chain variable region comprising one, two, or three of the heavy chain variable region CDRs described herein, and a light chain variable region comprising one, two, or three of the light chain variable region CDRs described herein (e.g.,one, two, or three of the heavy chain variable region CDR sequences, and / or one, two, or three of the light chain variable region CDR sequences as shown in Table 1, including all six CDRs of any of the exemplary antibodies as shown in Table 1) . In some embodiments, the antigen binding domain comprises a heavy chain variable region comprising any of the heavy chain variable region sequences described herein, and a light chain variable region comprising any of the light chain variable region sequences described herein (e.g., a heavy chain variable region sequence and / or a light chain variable region sequence as shown in Table 1). In some embodiments, the TBM (e.g., comprising an ABD) comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), wherein the VH and VL forms a binding domain that binds to the target in the absence of the MM. In some embodiments, the VH and VL are covalently linked, e.g., in an scFv. In some embodiments, the VH and VL are not covalently linked. In some embodiments, the VH and VL form a Fab fragment. In some embodiments, the VH is linked to an antibody heavy chain constant region, and the VL is linked to an antibody light chain constant region.

[0115] In some embodiments, the activatable antibody comprises a polypeptide comprising the structure, from N-terminus to C-terminus, of: masking moiety (MM)- cleavable moiety (CM)-VL, and the activatable antibody further comprises a second polypeptide comprising a VH (e.g., a Fab fragment). In some embodiments, the activatable antibody comprises a polypeptide comprising the structure, from N-terminus to C-terminus, of: masking moiety (MM)-cleavable moiety (CM)-VL-VH (e.g., an scFv). In some embodiments, the activatable antibody comprises a polypeptide comprising the structure, from N-terminus to C-terminus, of: masking moiety (MM)-cleavable moiety (CM)-VH, and the activatable antibody further comprises a second polypeptide comprising a VL (e.g., a Fab fragment). In some embodiments, the activatable antibody comprises a polypeptide comprising the structure, from N-terminus to C-terminus, of: masking moiety (MM)- cleavable moiety (CM)-VH-VL (e.g., an scFv).

[0116] The CM generally includes an amino acid sequence that is cleavable, for example, serves as the substrate for an enzyme and / or a cysteine-cysteine pair capable of forming a reducible disulfide bond. As such, when the terms “cleavage”, "cleavable”, "cleaved" and the like are used in connection with a CM, the terms encompass enzymatic cleavage, e.g., by a protease (for example, MMP9), as well as disruption of a disulfide bond between a cysteinecysteine pair via reduction of the disulfide bond that can result from exposure to a reducing agent.

[0117] The MM generally refers to an amino acid sequence that, when the CM of the activatable antibody is intact (e.g., uncleaved by a corresponding enzyme, and / or containing an unreduced cysteine-cysteine disulfide bond), the MM interferes with or inhibits binding of the TBM to its target. In some embodiments, the MM interferes with or inhibits binding of the TBM to its target so efficiently that binding of the TBM to its target is extremely low and / or below the limit of detection (e.g., binding cannot be detected in an ELISA or flow cytometry assay). The amino acid sequence of the CM may overlap with or be included within the MM. It should be noted that for sake of convenience "ABP" or “activatable antibody” are used herein to refer to an ABP or activatable antibody in both their uncleaved (or "native") state, as well as in their cleaved state, depending on the context. It will be apparent to the ordinarily skilled artisan that in some embodiments a cleaved ABP may lack an MM due to cleavage of the CM, e.g., by a protease, resulting in release of at least the MM (e.g., where the MM is not joined to the ABP by a covalent bond (e.g., a disulfide bond between cysteine residues)). Exemplary ABPs are described in more detail below.

[0118] In some embodiments, the masking moiety (MM) interferes with, obstructs, reduces the ability of, prevents, inhibits, or competes with the target binding moiety for binding to its target (e.g, an “inactive activatable antibody). In some embodiments, the masking moiety (MM) interferes with, obstructs, reduces, prevents, inhibits, or competes with the target binding moiety for binding to its target only when the polypeptide has not been activated (e.g , activated by a change in pH (increased or decreased), activated by a temperature shift (increased or decreased), activated after being contacted with a second molecule (such as a small molecule or a protein ligand) , etc. ). In some embodiments, activation induces cleavage of the polypeptide within the cleavage moiety. In some embodiments, activation induces conformation changes in the polypeptide (e.g., displacement of the masking moiety (MM)), leading to the masking moiety no longer preventing the activatable antibody from binding to its target. In some embodiments, the masking moiety (MM) interferes with, obstructs, reduces the ability of, prevents, inhibits, or competes with the target binding moiety for binding to its target only when the cleavable moiety (CM) has not been cleaved by one or more proteases that cleave within the cleavable moiety (CM). In some embodiments, the masking moiety (MM) has a masking efficiency of at least about 2.0 (e.g., at least about 2.0, at least about 3.0, at least about 4.0, at least about 5.0, at least about 6.0, at least about 7.0, at least about 8.0, at least about 9.0, at least about 10, at least about 25, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 300, at least about 400, at least about 500, etc. ) prior to activation. In someembodiments, masking efficiency is measured as the difference in affinity of an activatable antibody comprising the masking moiety (MM) for binding its target (before activation) relative to the affinity of a polypeptide lacking the masking moiety for binding its target (e.g., the difference in affinity for a target antigen (such as ROR) of an activatable antibody comprising a masking moiety (MM) (before activation) relative to a parental antibody lacking the masking moiety (MM), or the difference in affinity for a target antigen (such as ROR) of an activatable antibody comprising a masking moiety (MM) (before activation) relative to the affinity for the target antigen of the activatable antibody after activation). In some embodiments, the masking efficiency is measured by dividing the ECso for binding of an activatable antibody comprising a masking moiety (MM) (before activation) by the ECso of the parental antibody (e.g., by measuring ECso by ELISA). In some embodiments, masking efficiency is measured as the difference in affinity of an activatable antibody comprising the masking moiety (MM) for binding its target before activation relative to the affinity of the activatable antibody comprising the masking moiety (MM) for binding its target after activation (c.g, the difference in affinity for a target antigen (such as ROR) of an activatable antibody before activation relative to the activatable antibody after activation). In some embodiments, the masking moiety (MM) binds to the target binding moiety (TBM), and prevents the activatable antibody from binding to its target (e.g., an “inactive” activatable antibody). In some embodiments, the masking moiety (MM) has a dissociation constant for binding to the target binding moiety (TBM) that is greater than the dissociation constant of the target binding moiety (TBM) for its target.

[0119] In some embodiments, the masking moiety (MM) does not interfere with, obstruct, reduce the ability of, prevent, inhibit, or compete with the target binding moiety (TBM) for binding to its target after the activatable antibody has been activated (e.g., activated by treatment with one or more proteases that cleave within the cleavable moiety (CM), activated by a change in pH (increased or decreased), activated by a temperature shift (increased or decreased), activated after being contacted with a second molecule (such as an enzyme or a protein ligand), etc.). In some embodiments, the masking moiety (MM) does not interfere with, obstruct, reduce the ability of, prevent, inhibit, or compete with the target binding moiety (TBM) for binding its target after the cleavable moiety (CM) has been cleaved by one or more proteases that cleave within the cleavable moiety (CM). In some embodiments, the masking moiety (MM) has a masking efficiency of at most about 1.75 (e.g., at most about 1.75, at most about 1.5, at most about 1.4, at most about 1.3, at most about 1.2, at most about 1.1, at most about 1.0, at most about 0.9, at most about 0.8, at mostabout 0.7, at most about 0.6, or at most about 0.5, etc.) after activation (e.g., the relative affinity of the activatable antibody after activation as compared to the affinity of a parental antibody).

[0120] In some embodiments, activatable antibodies of the present disclosure are context- dependent (e.g., are activated (are only capable of binding their targets) in certain contexts (such as in the protease-rich tumor microenvironment)). In some embodiments, the activatable antibodies of the present disclosure provide improved safety over more traditional, non-activatable antibodies (e.g., show reduced toxicity, do not induce significant alterations to the weights of many organs, do not alter liver histopathology, hematology, and / or blood biochemistry, etc.). In some embodiments, activatable antibodies of the present disclosure have improved pharmacokinetic properties as compared to more traditional, non- activatable antibodies (e.g., have longer in vivo half-lives).

[0121] In some embodiments, the present disclosure relates to activatable antibodies that bind to human ROR when in active form (e.g., the activatable antibodies are active after cleavage in the cleavable moiety (e.g., with one or more proteases), but inactive prior to cleavage in the cleavable moiety (e.g., with one or more proteases)). Also provided herein are one or more activatable antibodies that compete or cross-compete for binding to human ROR with one or more of the ROR-targeting activatable antibodies and / or anti-ROR antibodies described herein.

[0122] In some embodiments, the activatable antibodies bind to human, cynomolgus monkey, mouse, rat, and / or dog ROR with a KD of about 500 nM or more when in inactive form. In some embodiments, the activatable antibodies bind to human, cynomolgus monkey, mouse, rat, and / or dog ROR with a KD of about 500 nM or less when in active form (e.g., about 500 nM or less, about 450 nM or less, about 400 nM or less, about 350 nM or less, about 300 nM or less, about 250 nM or less, about 200 nM or less, about 150 nM or less, about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 10 nM or less, about 9 nM or less, about 8 nM or less, about 7 nM or less, about 6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2 nM or less, about 1 nM or less, about 0.5 nM or less, about 0.1 nM or less, etc. ) In some embodiments, the activatable antibodies bind to human, cynomolgus monkey, mouse, rat, and / or dog ROR with a KD of about 10 nM or less when in active form. In some embodiments, the activatable antibodies bind to human ROR with a KD of about 5 nM or less when in active form. In some embodiments, the activatable antibodies bind to human,cynomolgus monkey, mouse, rat, and / or dog ROR with a KD of about 2 nM or less when in active form. In some embodiments, the activatable antibodies bind to human ROR with a KD of about 1 nM or less when in active form. In some embodiments, the activatable antibodies bind to human and cynomolgus monkey R0R1 IgG like domain with a KD of about 2 nM or less (e.g., about 1.5 nM or less, about 1.2 nM or less, or about 1.1 nM or less) when in active form. In some embodiments, the activatable antibodies bind to human and cynomolgus monkey R0R2 IgG like domain with a KD of about 2 nM or less (e.g., about 1.5 nM or less, about 1.2 nM or less, about 1.1 nM or less, about 1.0 nM or less, about 0.9 nM or less, or about 0.8 nM or less) when in active form. In some embodiments, the activatable antibodies bind to mouse ROR1 IgG like domain with a KD of about 2 nM or less (e.g., about 1.5 nM or less, about 1.2 nM or less, about 1.1 nM or less, about 1.0 nM or less, about 0.9 nM or less, or about 0.8 nM or less) when in active form. In some embodiments, the activatable antibodies bind to mouse ROR2 IgG like domain with a KD of about 100 nM or less (e.g., about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, or about 40 nM or less) when in active form. In some embodiments, the activatable antibodies bind to rat ROR1 IgG like domain with a KD of about 2 nM or less (e.g., about 1.5 nM or less, about 1.2 nM or less, about 1.1 nM or less, about 1.0 nM or less, about 0.9 nM or less, or about 0.8 nM or less) when in active form. Methods of measuring the KD of an activatable antibody may be carried out using any method known in the art, including for example, by surface plasmon resonance, an ELISA, isothermal titration calorimetry, a filter binding assay, an EMSA, etc. In some embodiments, the KD is measured by an ELISA. In some embodiments, the KD is measured by biolayer interferometry (BLI). In other embodiments, the KD is measured by surface plasmon resonance (SPR) (see e.g., Example 3 below).

[0123] In some embodiments, the activatable antibodies remain intact in its inactive form in circulation about 24 hours, 48 hours, or 96 hours after in vivo administration.

[0124] In some embodiments, the activatable antibodies are capable of inhibiting tumor cell growth and / or proliferation. In some embodiments, the tumor cell growth and / or proliferation is inhibited by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%) when contacted with the activatable antibodies relative to corresponding tumor cells not contacted with the activatable antibodies (or relative to corresponding tumor cells contacted with an isotype control antibody). In some embodiments, the activatable antibodies are capable ofreducing tumor volume in a subject when the subject is administered the activatable antibodies. In some embodiments, the activatable antibodies are capable of reducing tumor volume in a subject by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%) relative to the initial tumor volume in the subject (e.g., prior to administration of the activatable antibodies; as compared to a corresponding tumor in a subject administered an isotype control antibody). Methods of monitoring tumor cell growth / proliferation, tumor volume, and / or tumor inhibition are known in the art.

[0125] In some embodiments, the activatable antibodies have therapeutic effect on a cancer. In some embodiments, the activatable antibodies reduce one or more signs or symptoms of a cancer. In some embodiments, a subject suffering from a cancer goes into partial or complete remission when administered the activatable antibodies.

[0126] Masking moieties (MMs)

[0127] In some embodiments, the present disclosure relates to activatable antibodies comprising a masking moiety (MM). In some embodiments, the masking moiety (MM) comprises an amino acid sequence according to Formula (I) : XmCXnCXo (SEQ ID NO: 45) , where m is from 2-10, n is from 3-10, and o is from 1-10, where each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. In some embodiments, X is not W, M, and / or C. In some embodiments, each X in Xmof formula (I) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P and / or each X in Xnof formula (I) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In some embodiments, the MM comprises a polypeptide encoded by a polynucleotide sequence according to Formula (II): (NNI<)niTGY(NNI<)nTGY(NNI<)o (SEQ ID NO: 46) , wherein each N is independently A, G, T, or C, wherein each K is independently T or G, and wherein each Y is independently T or C. In some embodiments, m is 2, n is 8 and o is 2. In some embodiments, m is 6, n is 8 and o is 2.

[0128] In some embodiments, the masking moiety (MM) comprises an amino acid sequence according to Formula (III): ZmCZnCZo (SEQ ID NO: 47), where m is from 2-10, n is from 3-10, and o is from 1-10, and each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In some embodiments, m is 6, n is 8 and o is 2.

[0129] In some embodiments, m is from 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9,3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6- 7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10. In some embodiments, m is from 6-8. In some embodiments, m is 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, m is 6.

[0130] In some embodiments, n is from 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8,4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10. In some embodiments, n is from 6-8. In some embodiments, n is 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n is 6. In some embodiments, n is 8.

[0131] In some embodiments, o is from 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5,5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10. In some embodiments, o is from 1-2. In some embodiments, o is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, o is 2.

[0132] In some embodiments, the masking moiety (MM) comprises an amino acid sequence according to Formula (IV): (Xe)C(Xs)C(X2) (SEQ ID NO: 48), where each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0133] In some embodiments, the masking moiety (MM) comprises an amino acid sequence according to Formula (V): (Ze)C(Zs)C(Z2) (SEQ ID NO: 49), where each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

[0134] In some embodiments, an activatable antibody comprises a masking moiety (MM) comprising a sequence selected from the group consisting of XmCYYFDPPSHCXX (SEQ ID NO: 50), XmCPFFFPAALCXX (SEQ ID NO: 51), and XmCHFADFVPYCXX (SEQ ID NO: 52), where m is from 2-10, and where each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0135] In some embodiments, an activatable antibody comprises a masking moiety (MM) comprising the sequence ADCPFFFPAALCSQ (SEQ ID NO: 73), EVGSYADCPFFFPAALCSQ (SEQ ID NO: 53), HDHHFDCYYFDPPSHCPA (SEQ ID NO: 54), EVGSYHDHHFDCYYFDPPSHCPA (SEQ ID NO: 55), ADPFSVCHFADFVPYCYY (SEQ ID NO: 56), or EVGSYADPFSVCHFADFVPYCYY (SEQ ID NO: 57).

[0136] In some embodiments, the masking moiety (MM) comprises an amino acid sequence selected from ADCPFFFPAALCSQ (SEQ ID NO: 73),EVGSYADCPFFFPAALCSQ (SEQ ID NO: 53), HDHHFDCYYFDPPSHCPA (SEQ ID NO: 54), EVGSYHDHHFDCYYFDPPSHCPA (SEQ ID NO: 55), ADPFSVCHFADFVPYCYY (SEQ ID NO: 56), or EVGSYADPFSVCHFADFVPYCYY (SEQ ID NO: 57).

[0137] In some embodiments, any of the masking moieties (MMs) described herein may further comprise one or more additional amino acid sequences (e.g., one or more polypeptide tags). Examples of suitable additional amino acid sequence may include, without limitation, purification tags (such as his-tags, flag-tags, maltose binding protein and glutathione-S- transferase tags), detection tags (such as tags that may be detected photometrically (e.g., red or green fluorescent protein, etc. )), tags that have a detectable enzymatic activity (e.g., alkaline phosphatase, etc. ), tags containing secretory sequences, leader sequences, and / or stabilizing sequences, protease cleavage sites (e.g., furin cleavage sites, TEV cleavage sites, Thrombin cleavage sites), and the like. In some embodiments, the one or more additional amino acid sequences are at the N-terminus of the masking moiety (MM). In some embodiments, the additional amino acid sequence comprises or consists of the sequence EVGSY (SEQ ID NO: 58).

[0138] In some embodiments, the masking moiety binds to the target binding moiety (TBM) and inhibits the activatable antibody from binding to its target before activation (e.g., before treatment with one or more proteases that cleave within the cleavable moiety (CM), before undergoing a (local) change in pH (increased or decreased), before a temperature shift (increased or decreased), before being contacted with a second molecule (such as a small molecule or a protein ligand), etc. ), but does not bind to the TBM and / or inhibit the activatable antibody from binding to its target after activation (e.g., after treatment with one or more proteases that cleave within the cleavable moiety (CM), after undergoing a (local) change in pH (increased or decreased), after a temperature shift (increased or decreased), after being contacted with a second molecule (such as a small molecule or a protein ligand), etc.). In some embodiments, the masking moiety (MM) inhibits binding of an activatable antibody to its target when the CM is not cleaved, but does not inhibit binding of the activatable antibody to its target when the CM is cleaved. In some embodiments, the masking moiety (MM) has a dissociation constant for binding to the TBM that is greater (e.g., at least about 1.5-fold greater, at least about 2-fold greater, at least about 2.5-fold greater, at least about 3-fold greater, at least about 3.5-fold greater, at least about 4-fold greater, at least about 4.5-fold greater, at least about 5-fold greater, at least about 10-fold greater, at leastabout 100-fold greater, at least about 500-fold greater, etc.) than the dissociation constant of the activatable antibody for its target (when in active form).

[0139] Cleavable moieties (CMs)

[0140] In some embodiments, the present disclosure relates to activatable antibodies comprising a cleavable moiety (CM). In some embodiments, the cleavable moiety (CM) is cleaved and / or disrupted by treatment with one or more proteases that cleave within the cleavable moiety (CM), by a change in pH (increased or decreased), by a temperature shift (increased or decreased), and / or by contact with a second molecule (such as a small molecule or a protein ligand), etc.)

[0141] In some embodiments, the cleavable moiety (CM) comprises at least a first cleavage site (CS1) (e.g., a first protease cleavage site). In some embodiments, the first cleavage site is a first protease cleavage site. Any suitable protease cleavage site recognized and / or cleaved by any protease (e.g., a protease that is known to be co-localized with a target of an activatable antibody comprising the CM) known in the art may be used, including, for example, a protease cleavage site recognized and / or cleaved by urokinase-type plasminogen activator (uPA); matrix metalloproteinases (e.g., MMP-1, MMP-2, MMP-3, MMP-7, MMP- 8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP- 17, MMP-19, MMP-20, MMP-23, MMP-24, MMP-26, and / or MMP-27); Tobacco Etch Virus (TEV) protease; plasmin; Thrombin; PSA; PSMA; ADAMS / ADAMTS (e.g., ADAM 8, ADAM 9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, and / or AD AMTS5); caspases (e.g., Caspase-1, Caspase-2, Caspase-3, Caspase- 4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase- 10, Caspase-11, Caspase-12, Caspase-13, and / or Caspase-14); aspartate proteases (e.g., RACE and / or Renin) ; aspartic cathepsins (e.g., Cathepsin D and / or Cathepsin E); cysteine cathepsins (e.g., Cathepsin B, Cathepsin C, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V / L2, and / or Cathepsin X / Z / P); cysteine proteinases (e.g., Cruzipain, Legumain, and / or Otubain-2); KLKs (e g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10,KLKl l, KLK13, and / or KLK14); metallo proteainases (e.g., Meprin, Neprilysin, PSMA, and / or BMP-1); serine proteases (e.g., activated protein C, Cathepsin A, Cathepsin G, Chymase, and / or coagulation factor proteases (such as FVIIa, FIXa, FXa, FXIa, FXIIa)); elastase; granzyme B; guanidinobenzoatase; HtrAl; human neutrophil elastase; lactoferrin; marapsin; NS3 / 4A; PACE4; tPA; tryptase; type II transmembrane serine proteases (TTSPs) (e.g., DESCI, DPP -4, FAP, Hepsin, Matriptase-2, MT-SPl / Matriptase, TMPRSS2, TMPRSS3 and / or TMPRSS4); etc. In some embodiments, the first protease cleavage site is a cleavage site for a protease selected fromuPA, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, TEV protease, plasmin, Thrombin, Factor X, PSA, PSMA, Cathepsin D, Cathepsin K, Cathepsin S, ADAMIO, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE. In some embodiments, the first protease cleavage site is a cleavage site for a protease selected from uPA, MMP-2, MMP-9, and / or TEV protease. In some embodiments, the protease cleavage comprises an amino acid sequence selected from SGRSA (SEQ ID NO: 59), PLGLAG (SEQ ID NO: 44), and ENLYFQG (SEQ ID NO: 60).

[0142] In some embodiments, the cleavable moiety (CM) further comprises a first linker (LI). In some embodiments, the first linker (LI) is N-terminal to the first cleavage site (CS1) (e.g., a first protease cleavage site). In some embodiments, the cleavable moiety (CM) comprises a structure, from N-terminus to C-terminus, of: Ll-(CSl).

[0143] Any suitable linker (e.g., a flexible linker) known in the art may be used, including, for example: glycine polymers (G)n, where n is an integer of at least 1 (e.g., at least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc.); glycine-serine polymers (GS)n, where n is an integer of at least 1 (e.g., at least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc.) such as GGGGS (SEQ ID NO: 61), SGGS (SEQ ID NO: 62), GGSG (SEQ ID NO: 63), GGSGG (SEQ ID NO: 64), GSGSG (SEQ ID NO: 65), GSGGG (SEQ ID NO: 66), GGGSG (SEQ ID NO: 67), and / or GSSSG (SEQ ID NO: 68)); glycine-alanine polymers; alanineserine polymers; and the like. Linker sequences may be of any length, such as from about 1 amino acid (e.g., glycine or serine) to about 20 amino acids (e.g., 20 amino acid glycine polymers or glycine-serine polymers), about 1 amino acid to about 15 amino acids, about 3 amino acids to about 12 amino acids, about 4 amino acids to about 10 amino acids, about 5 amino acids to about 9 amino acids, about 6 amino acids to about 8 amino acids, etc. In some embodiments, the linker is any of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0144] In some embodiments, the cleavable moiety (CM) further comprises at least a second linker (e.g., at least a second, at least a third, at least a fourth, at least a fifth, etc.). In some embodiments, the cleavable moiety (CM) further comprises a second linker (L2). The second linker (L2) may be any suitable linker described above. In some embodiments, the first (LI) and second (L2) linkers are the same. In some embodiments, the first (LI) and second (L2) linkers are different. In some embodiments, the at least second linker (L2) is C- terminal to the second cleavage site (CS1). In some embodiments, the cleavable moiety(CM) comprises a structure, from N-terminus to C-terminus, of: LI- (CS1) -L2. In further embodiments, the CM comprises GGGPLGLAGSGGS (SEQ ID NO: 69).

[0145] Exemplary MM-CM sequences

[0146] In some embodiments, an activatable antibody of the present disclosure comprises a masking peptide, from N-terminus to C-terminus, of: (MM) - LI- (CS1) -L2. In some embodiments, a polypeptide of the present disclosure comprises the structure, from N- terminus to C-terminus, of: (MM) - LI- (CS1) -L2-(TBM).

[0147] In some embodiments, an activatable antibody comprises an amino acid sequence according to Formula (VI), EVGSY(X2)C(X8)C(X2)GGGPLGLAGSGGS (SEQ ID NO: 70), where each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. In further embodiments, the masking peptide comprises EVGSYADCPFFFPAALCSOGGGPLGLAGSGGS (SEQ ID NO:29).

[0148] In some embodiments, an activatable antibody comprises an amino acid sequence according to Formula (VII), EVGSY(X6)C(X8)C(X2)GGGPLGLAGSGGS (SEQ ID NO: 71), where each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. In some embodiments, an activatable antibody comprises an amino acid sequence according to Formula (VIII), EVGSY(Z6)C(Z8)C(Z2)GGGPLGLAGSGGS (SEQ ID NO: 72), where each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In further embodiments, the masking peptide comprisesEVGSYHDHHFDCYYFDPPSHCPAGGGPLGLAGSGGS (SEQ ID NO:30). In further embodiments, the masking peptide comprises EVGSYADPFSVCHFADFVPYCYYGGGPLGLAGSGGS (SEQ ID NO:31).

[0149] In some embodiments, an activatable binding polypeptide (i.e., activatable antibody) of the present disclosure comprises: (a) a masking moiety (MM), (b) a cleavable moiety, and (c) a target binding moiety. In some embodiments, the masking moiety (MM) binds to the target binding moiety (TBM) of the activatable antibody and reduces or inhibits binding of the activatable binding moiety to ROR (e.g., human ROR), as compared to the binding of a corresponding binding polypeptide lacking the masking moiety to ROR (e.g., human ROR) and / or as compared to the binding of a parental antibody to ROR (e.g., human ROR).

[0150] In some embodiments, an “activatable” binding polypeptides refers to a binding polypeptide that exhibits a first level of binding to ROR when in an inhibited, masked, and / or uncleaved state, and exhibits a second level of binding to ROR in an uninhibited, unmasked,and / or cleaved state, where the second level of ROR binding is greater than the first level of ROR binding. In some embodiments, access to ROR by the activatable binding polypeptide is greater after cleavage within the cleavable moiety (e.g., by one or more proteases).

[0151] In some embodiments, an activatable antibody of the present disclosure is generally considered to be an “activatable” binding polypeptide when binding affinity of the polypeptide to ROR (e.g., human ROR) increases by at least about 2-fold (e.g., at least about 2-fold, at least about 2.5-fold, at least about 3, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 25- fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 250- fold, at least about 500-fold, at least about 750-fold, or at least about 1000-fold, or more) after activation of the activatable antibody as compared to prior to activation of the activatable antibody (e.g., after activation by treatment with one or more proteases that cleave within the cleavable moiety (CM), after activation by a change in pH (increased or decreased), after activation by a temperature shift (increased or decreased), after activation by being contacted with a second molecule (such as a small molecule), etc.). In some embodiments, an activatable antibody of the present disclosure is generally considered “activatable” if the ECso of the activatable antibody decreases by at least about 2-fold (e.g., at least about 2-fold, at least about 2.5-fold, at least about 3, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8- fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10- fold, at least about 25-fold, at least about 50-fold, at least about 75-fold, at least about 100- fold, at least about 250-fold, at least about 500-fold, at least about 750-fold, or at least about 1000-fold, or more) after “activation” (e.g., as measured by an ELISA or FACS assay; see the examples below). In some embodiments, an activatable antibody of the present disclosure is generally considered “activatable” if the ECso of the polypeptide decreases by at least about 2-fold after treatment with a protease that cleaves within the cleavable moiety (CM).

[0152] In some embodiments, when the masking moiety (MM) is bound to the target binding moiety (TBM) of the activatable antibody, the KD of the activatable antibody for ROR is about 2 (e.g., about 2, about 2.5, about 3, about 3.5 about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 25, about 50, about 75, about 100, about 250, about 500, about 750, or about 1000 ormore) times greater than when the masking moiety (MM) is not bound to the target binding moiety (TBM) (e.g., after “activation” of the activatable antibody (such as after protease treatment to cleave within the cleavable moiety (CM))) and / or than the KD of the parental antibody for ROR.

[0153] In some embodiments, when the masking moiety is bound to the target binding moiety of the activatable antibody, the KD of the activatable antibody for ROR is reduced by at least about 25% (e.g., at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%) relative to when the masking moiety is not bound to the target binding moiety (e.g., after “activation” of the activatable antibody (such as after protease treatment to cleave within the cleavable moiety (CM))) and / or relative to the KD of the parental antibody for ROR.

[0154] In some embodiments, the masking moiety sterically hinders binding of the activatable antibody to ROR and / or allosterically hinders binding of the activatable antibody to ROR. In some embodiments, the masking moiety does not comprise an amino acid sequence of a natural binding partner of the activatable antibody and / or parental antibody.

[0155] In some embodiments, the dissociation constant of the masking moiety for the target binding moiety is greater than the dissociation constant for the activatable antibody for ROR (when activated). In some embodiments, the dissociation constant of the masking moiety for the target binding moiety is about 2 (e.g., about 2, about 2.5, about 3, about 3.5 about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 25, about 50, about 75, about 100, about 250, about 500, about 750, or about 1000 or more) times greater than the dissociation constant for the activatable antibody for ROR (when activated). In some embodiments, the dissociation constant of the masking moiety for the target binding moiety is about equal to the dissociation constant for the activatable antibody for ROR (when activated).

[0156] In various aspects, provided herein are activatable ROR binding agents (e.g., activatable antibodies) that comprise: a masking peptide, an antibody light chain variable (VL) region, and an antibody heavy chain variable (VH) region; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:29, 30 or 31 (see Table 2), and wherein the antibody light chain variable region and the antibody heavy chain variable region are as described herein (for example, comprise the CDRs described herein (e.g., Table 1)).

[0157] In various aspects, provided herein are activatable ROR binding agents (e.g., activatable antibodies) that comprise: (a) a polypeptide comprising a masking peptide and anantibody light chain variable region; and (b) an antibody heavy chain variable region; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:29, 30 or 31 (see Table 2), and wherein the antibody light chain variable region and the antibody heavy chain variable region are as described herein (for example, comprise the CDRs described herein (e.g., Table 1)).

[0158] In various aspects, provided herein are activatable ROR binding agents (e.g., activatable antibodies) that comprise: (a) a polypeptide comprising, from N-terminus to C- terminus, a masking peptide and an antibody light chain variable region; and (b) an antibody heavy chain variable region; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:29, 30 or 31 (see Table 2), and wherein the antibody light chain variable region and the antibody heavy chain variable region are as described herein (for example, comprise the CDRs described herein (e.g., Table 1)).

[0159] In certain embodiments, an activatable ROR binding agent described herein comprises two antibody light chain variable regions, and a masking peptide is directly or indirectly conjugated to (e.g., conjugated to the N-terminus of) each antibody light chain variable region. In certain embodiments, an activatable ROR binding agent described herein comprises more than two antibody light chain variable regions, and a masking peptide is directly or indirectly conjugated to (e.g., conjugated to the N-terminus of) each antibody light chain variable region. In certain embodiments, an activatable ROR binding agent described herein comprises two antibody light chain variable regions, and a masking peptide is directly or indirectly conjugated to (e.g., conjugated to the N-terminus of) only one of the antibody light chain variable regions. In certain embodiments, an activatable ROR binding agent described herein comprises more than two antibody light chain variable regions, and a masking peptide is directly or indirectly conjugated to (e.g., conjugated to the N-terminus of) only one of the antibody light chain variable regions.

[0160] In one embodiment, the activatable ROR binding agents (e.g., activatable antibodies) provided herein comprise: a masking peptide, an antibody light chain variable region, and an antibody heavy chain variable region; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:29.

[0161] In one embodiment, the activatable ROR binding agents (e.g., activatable antibodies) provided herein comprise: a masking peptide, an antibody light chain variable region, and an antibody heavy chain variable region; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:30.

[0162] In one embodiment, the activatable ROR binding agents (e.g., activatable antibodies) provided herein comprise: a masking peptide, an antibody light chain variable region, and an antibody heavy chain variable region; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO: 31.

[0163] In one embodiment, the activatable ROR binding agents (e.g., activatable antibodies) provided herein comprise: (a) a polypeptide comprising a masking peptide and an antibody light chain variable region; and (b) an antibody heavy chain variable region; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:29.

[0164] In one embodiment, the activatable ROR binding agents (e.g., activatable antibodies) provided herein comprise: (a) a polypeptide comprising a masking peptide and an antibody light chain variable region; and (b) an antibody heavy chain variable region; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:30.

[0165] In one embodiment, the activatable ROR binding agents (e.g., activatable antibodies) provided herein comprise: (a) a polypeptide comprising a masking peptide and an antibody light chain variable region; and (b) an antibody heavy chain variable region; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:31.

[0166] In one embodiment, the activatable ROR binding agents (e.g., activatable antibodies) provided herein comprise: (a) a polypeptide comprising, from N-terminus to C- terminus, a masking peptide and an antibody light chain variable region; and (b) an antibody heavy chain variable region; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:29.

[0167] In one embodiment, the activatable ROR binding agents (e.g., activatable antibodies) provided herein comprise: (a) a polypeptide comprising, from N-terminus to C- terminus, a masking peptide and an antibody light chain variable region; and (b) an antibody heavy chain variable region; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:30.

[0168] In one embodiment, the activatable ROR binding agents (e.g., activatable antibodies) provided herein comprise: (a) a polypeptide comprising, from N-terminus to C- terminus, a masking peptide and an antibody light chain variable region; and (b) an antibody heavy chain variable region; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:31.

[0169] In one embodiment, the activatable ROR binding agents (e.g., activatable antibodies) provided herein comprise: (a) a polypeptide comprising, from N-terminus to C-terminus, a masking peptide and an antibody light chain; and (b) an antibody heavy chain; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:29.

[0170] In one embodiment, the activatable ROR binding agents (e.g., activatable antibodies) provided herein comprise: (a) a polypeptide comprising, from N-terminus to C- terminus, a masking peptide and an antibody light chain; and (b) an antibody heavy chain; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:30.

[0171] In one embodiment, the activatable ROR binding agents (e.g., activatable antibodies) provided herein comprise: (a) a polypeptide comprising, from N-terminus to C- terminus, a masking peptide and an antibody light chain; and (b) an antibody heavy chain; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:31.

[0172] In preferred embodiments, a masking peptide described herein comprises, from N- terminus to C-terminus, a masking moiety (MM) and a cleavable moiety (CM), and optionally further comprises one, two, or more linkers.

[0173] The CM generally includes an amino acid sequence that is cleavable, for example, serves as the substrate for an enzyme and / or a cysteine-cysteine pair capable of forming a reducible disulfide bond. As such, when the terms “cleavage,” “cleavable,” “cleaved” and the like are used in connection with a CM, the terms encompass enzymatic cleavage, e.g., by a protease, as well as disruption of a disulfide bond between a cysteine-cysteine pair via reduction of the disulfide bond that can result from exposure to a reducing agent.

[0174] The MM generally refers to an amino acid sequence that, when the CM of the activatable antibody is intact (e.g., uncleaved by a corresponding enzyme, and / or containing an unreduced cysteine-cysteine disulfide bond), the MM interferes with or inhibits binding of the antibody light chain variable region and / or heavy chain variable region (as the case may be) to its target. In some embodiments, the MM interferes with or inhibits binding of the antibody light chain variable region and / or heavy chain variable region (as the case may be) to its target so efficiently that binding of the antibody light chain variable region and / or heavy chain variable region (as the case may be) to its target is extremely low and / or below the limit of detection (e.g., binding cannot be detected in an ELISA or flow cytometry assay). The amino acid sequence of the CM may overlap with or be included within the MM.

[0175] In some embodiments, the masking moiety sterically hinders binding of the activatable binding agent to its target and / or allosterically hinders binding of the activatable binding agent to its target. In some embodiments, the masking moiety does not comprise an amino acid sequence of a natural binding partner of activatable binding agent.

[0176] In some embodiments, a binding agent of the present disclosure is generally considered to be an “activatable” binding agent when binding affinity of the agent to its target increases (e.g., by at least about 2-fold, at least about 2.5-fold, at least about 3, at least about3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5- fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, at least about 750-fold, or at least about 1000-fold, or more) after activation of the binding agent as compared to prior to activation of the binding agent (e.g., after activation by treatment with one or more proteases that cleave within the cleavable moiety (CM), after activation by a change in pH (increased or decreased), after activation by a temperature shift (increased or decreased), after activation by being contacted with a second molecule (such as a small molecule or a protein ligand), etc.). In some embodiments, a binding agent of the present disclosure is generally considered “activatable” if the ECso of the binding agent decreases (e.g., by at least about 2- fold, at least about 2.5-fold, at least about 3, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 25- fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 250- fold, at least about 500-fold, at least about 750-fold, or at least about 1000-fold, or more) after “activation” (e.g., as measured by an ELISA or FACS assay). In some embodiments, a binding agent of the present disclosure is generally considered “activatable” if the ECso of the binding agent decreases (e.g., by at least about 2-fold) after treatment with a protease that cleaves within the cleavable moiety (e.g., as measured by an ELISA or FACS assay).

[0177] In some embodiments, when the masking moiety is bound to the antibody light chain variable region and / or heavy chain variable region (as the case may be) of an activatable binding agent, the KD of the activatable binding agent for its target is greater (c.g, about 2, about 2.5, about 3, about 3.5 about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 25, about 50, about 75, about 100, about 250, about 500, about 750, or about 1000 or more times greater) than when the masking moiety is not bound to the antibody light chain variable region and / or heavy chain variable region (as the case may be) (e.g., after “activation” of the activatable binding agent (such as after protease treatment to cleave within the cleavable moiety)) and / orthan the KD of the parental antibody for the target. Methods of measuring affinity are known in the art.

[0178] In some embodiments, when the masking moiety is bound to the antibody light chain variable region and / or heavy chain variable region (as the case may be) of an activatable binding agent, the KD of the activatable binding agent for its target is reduced (e.g., by at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%) relative to when the masking moiety is not bound to the antibody light chain variable region and / or heavy chain variable region (as the case may be) (e.g., after “activation” of the activatable binding polypeptide (such as after protease treatment to cleave within the cleavable moiety)) and / or relative to the KD of the parental antibody for the target. Methods of measuring affinity are known in the art.

[0179] In some embodiments, the dissociation constant of the masking moiety for the antibody light chain variable region and / or heavy chain variable region (as the case may be) is greater (e.g., about 2, about 2.5, about 3, about 3.5 about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 25, about 50, about 75, about 100, about 250, about 500, about 750, or about 1000 or more times greater) than the dissociation constant for the activatable binding agent for the target (when in activated form). In some embodiments, the dissociation constant of the masking moiety for the antibody light chain variable region and / or heavy chain variable region (as the case may be) is about equal to the dissociation constant for the activatable binding agent for the target (when in activated form). In some embodiments, the masking moiety binds to the antibody light chain variable region and / or heavy chain variable region (as the case may be), and prevents the activatable binding agent from binding to its target only when the activatable binding agent has not been activated (e.g., not activated by treatment with one or more proteases that cleave within the cleavable moiety (CM), not activated by a change in pH (increased or decreased), not activated by a temperature shift (increased or decreased), not activated after being contacted with a second molecule (such as a small molecule or a protein ligand), etc.). In some embodiments, activation induces cleavage of the activatable binding agent within the cleavage moiety. In some embodiments, activation induces conformation changes in the activatable binding agent (e.g., displacement of the masking moiety), leading to the masking moiety no longer preventing the binding agent from binding to its target.

[0180] In some embodiments, the activatable ROR binding agents (e.g., activatable antibodies) described herein comprise a VH region, VL region, VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2, and / or VL CDR3 of any one of the antibodies described herein, such as the amino acid sequence of a VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 depicted in Table 1. Accordingly, in some embodiments, an activatable ROR binding agent (e.g., an activatable antibody) described herein comprises any one, any two, and / or all three heavy chain CDRs and / or any one, any two, and / or all three light chain CDRs depicted in Table 1. In some embodiments, an activatable ROR binding agent (e.g., an activatable antibody) described herein comprises any one, any two, and / or all three heavy chain CDRs and any one, any two, and / or all three light chain CDRs depicted in Table 1.

[0181] In some embodiments, an activatable ROR binding agent (e.g., an activatable antibody) comprises a VH region, which comprises a VH CDR1, a VH CDR2, and / or a VH CDR3, and / or a VL region, which comprises a VL CDR1, a VL CDR2, and / or a VL CDR3, of any one of the binding agents described herein (see, e.g., Table 1). Accordingly, in some embodiments, an activatable ROR binding agent (e.g., an activatable antibody) described herein comprises any one, any two, and / or all three heavy chain CDRs and / or any one, any two, and / or all three light chain CDRs from Table 1.

[0182] In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises (i) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:25 and / or (ii) a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:26.

[0183] In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:25 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:26. CDR sequences can be determined according to well-known numbering systems or a combination thereof. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDR sequences are determined according to a combination of any two or more of the above-mentioned numbering systems, for example, a combination of Kabat and Chothia. Various exemplary CDR numbering systems are described and illustrated above in Section 5.2.

[0184] In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises (a) a VH region comprising a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 12, 13, 18, and 27; a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 19, and 24; and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, 20, and 28; and / or (b) a VL region comprising a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21; a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22; and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 17, and 23.

[0185] In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:3; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NON, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:6.

[0186] In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:7, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:8, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:9; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:6.

[0187] In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:3; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NON, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:6.

[0188] In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence ofSEQ ID NO: 14, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 17.

[0189] In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:20; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:21, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:22, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:23.

[0190] In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:24, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:3; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NON, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:6.

[0191] In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:27, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:24, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:28; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NON, a VL CDR2 comprising the amino acid sequence of SEQ ID NON, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 17.

[0192] In some embodiments, the activatable antibody further comprises one or more framework regions of SEQ ID NOs:25 and / or 26. In some embodiments, the activatable antibody or fragment thereof further comprises a framework 1 (FR1), a framework 2 (FR2), a framework 3 (FR3) and / or a framework 4 (FR4) sequence as set forth in SEQ ID NO: 25 or 26. In some embodiments, the activatable antibody provided herein is a humanized antibody. Framework regions described herein are determined based upon the boundaries of the CDR numbering system. In other words, if the CDRs are determined by, e.g., Kabat, IMGT, or Chothia, then the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format, from the N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. For example, FR1 is defined as the amino acid residues N-terminal to the CDR1 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR2 is defined as the amino acid residues between CDR1 and CDR2 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR3 is defined as the amino acid residues between CDR2 and CDR3 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, and FR4 is defined as the amino acid residues C-terminal to the CDR3 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system.

[0193] In some embodiments, activatable ROR binding agents (e.g., activatable antibodies such as monospecific or bispecific antibodies), including activatable human ROR binding agents, described herein comprise a VH region or VH domain. Additionally or alternatively, in some embodiments, activatable ROR binding agents (e.g., activatable antibodies such as monospecific or bispecific antibodies), including activatable human ROR binding agents, described herein comprise a polypeptide comprising, from N-terminus to C- terminus, a masking peptide described herein (e.g, Table 2), and a VL region or VL domain. In some embodiments, activatable ROR binding agents (e.g., activatable antibodies such as monospecific or bispecific antibodies), including activatable human ROR binding agents, described herein have a combination of (i) a VH domain or VH region; and (ii) a polypeptide comprising, from N-terminus to C-terminus, a masking peptide described herein (e.g., Table 2), and a VL domain or VL region.

[0194] In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:25. In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a polypeptide comprising, from N-terminus to C-terminus, a masking peptide comprising the SEQ ID NO:29, 30 or 31, and a VL comprising the amino acid sequence of SEQ ID NO:26. In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:25 and a polypeptide comprising, from N-terminus to C-terminus, a masking peptide comprising the SEQ ID NO:29, 30 or 31, and a VL comprising the amino acid sequence of SEQ ID NO:26.

[0195] In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH comprising the amino acid sequence of SEQ IDNO:25. In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a polypeptide comprising, from N-terminus to C-terminus, a masking peptide comprising the amino acid sequence of SEQ ID NO:29 and a VL comprising the amino acid sequence of SEQ ID NO:26. In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:25 and a polypeptide comprising, from N-terminus to C- terminus, a masking peptide comprising the amino acid sequence of SEQ ID NO:29 and a VL comprising the amino acid sequence of SEQ ID NO:26.

[0196] In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:25. In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a polypeptide comprising, from N-terminus to C-terminus, a masking peptide comprising the amino acid sequence of SEQ ID NO:30 and a VL comprising the amino acid sequence of SEQ ID NO:26. In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:25 and a polypeptide comprising, from N-terminus to C- terminus, a masking peptide comprising the amino acid sequence of SEQ ID NO:30 and a VL comprising the amino acid sequence of SEQ ID NO:26.

[0197] In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:25. In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a polypeptide comprising, from N-terminus to C-terminus, a masking peptide comprising the amino acid sequence of SEQ ID NO:31 and a VL comprising the amino acid sequence of SEQ ID NO:26. In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:25 and a polypeptide comprising, from N-terminus to C- terminus, a masking peptide comprising the amino acid sequence of SEQ ID NO:31 and a VL comprising the amino acid sequence of SEQ ID NO:26.

[0198] In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:25. In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a polypeptide comprising, from N-terminus to C-terminus, a masking peptide comprising the amino acid sequence of SEQ ID NO:29 and a VL comprising the amino acid sequence of SEQ ID NO:26, wherein the polypeptide comprises the aminoacid sequence of SEQ ID NO: 34. In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:25 and a polypeptide comprising, from N-terminus to C-terminus, a masking peptide comprising the amino acid sequence of SEQ ID NO:29 and a VL comprising the amino acid sequence of SEQ ID NO:26, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 34.

[0199] In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:25. In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a polypeptide comprising, from N-terminus to C-terminus, a masking peptide comprising the amino acid sequence of SEQ ID NO:30 and a VL comprising the amino acid sequence of SEQ ID NO:26, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:25 and a polypeptide comprising, from N-terminus to C-terminus, a masking peptide comprising the amino acid sequence of SEQ ID NO:30 and a VL comprising the amino acid sequence of SEQ ID NO:26, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 36.

[0200] In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:25. In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a polypeptide comprising, from N-terminus to C-terminus, a masking peptide comprising the amino acid sequence of SEQ ID NO:31 and a VL comprising the amino acid sequence of SEQ ID NO:26, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:25 and a polypeptide comprising, from N-terminus to C-terminus, a masking peptide comprising the amino acid sequence of SEQ ID NO:31 and a VL comprising the amino acid sequence of SEQ ID NO:26, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 38.

[0201] In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:25. In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a polypeptide comprising, from N-terminus to C-terminus, amasking peptide comprising the amino acid sequence of SEQ ID NO:29 and a VL comprising the amino acid sequence of SEQ ID NO:26, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:25 and a polypeptide comprising, from N-terminus to C-terminus, a masking peptide comprising the amino acid sequence of SEQ ID NO:29 and a VL comprising the amino acid sequence of SEQ ID NO:26, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 35.

[0202] In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:25. In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a polypeptide comprising, from N-terminus to C-terminus, a masking peptide comprising the amino acid sequence of SEQ ID NO:30 and a VL comprising the amino acid sequence of SEQ ID NO:26, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:25 and a polypeptide comprising, from N-terminus to C-terminus, a masking peptide comprising the amino acid sequence of SEQ ID NO:30 and a VL comprising the amino acid sequence of SEQ ID NO:26, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 37.

[0203] In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:25. In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a polypeptide comprising, from N-terminus to C-terminus, a masking peptide comprising the amino acid sequence of SEQ ID NO:31 and a VL comprising the amino acid sequence of SEQ ID NO:26, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) comprises a VH comprising the amino acid sequence of SEQ ID NO:25 and a polypeptide comprising, from N-terminus to C-terminus, a masking peptide comprising the amino acid sequence of SEQ ID NO:31 and a VL comprising the amino acid sequence of SEQ ID NO:26, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 39.

[0204] In certain embodiments, the activatable ROR binding agent (e.g., an activatable antibody of a fragment thereof) provided herein comprises amino acid sequences with certainpercent identity (such as at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or as at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or higher) relative to any antibody or fragment thereof provided herein, for example, a CDR, VH or VL in Table 1, or any full-length antibody chain as disclosed herein (e.g., the amino acid sequences depicted in Tables 3-6). In some embodiments, the activatable ROR binding agent (e.g., an activatable antibody of a fragment thereof) provided herein comprises CDRs of any antibody or fragment thereof provided herein, for example in Table 1. In further embodiments, the activatable ROR binding agent provided herein comprises amino acid sequences with certain percent identity (such as at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or as at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or higher) relative to any antibody or fragment thereof provided herein, for example, a VH or VL in Table 1, or any full-length antibody chain as disclosed herein (e.g., the amino acid sequences depicted in Tables 3-6).

[0205] The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A. 87:2264 2268 (1990), modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A. 90:5873 5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 215:403 (1990). BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, word length=12 to obtain nucleotide sequences homologous to a nucleic acid molecule described herein.BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score 50, word length=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul etal., Nucleic Acids Res. 25:3389 3402 (1997). In some embodiments, the percent identity between two sequences is calculated by dividing the number of residue(s) varied (excluding or including conservative amino acid substitution(s)or degenerate nucleotide substitution(s)) between the two sequences in the alignment with the residue number of any one of the following: (i) full length of the shorter sequence, (ii) full length of the longer sequence, (iii) mean length of the two sequences, (iv) total length of the non-gap portion of the alignment, (v) length of the alignment excluding overhangs, or (vi) length of the alignment including overhangs. Overhangs as used herein with respect to a sequence alignment refer to either or both ends of the alignment where residues of one sequence are considered as aligning to no residues (e.g., gap) in the other sequence. Alternatively, PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CAB IOS 4: 11-17 (1998). Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0206] In some embodiments, the activatable binding agent (e.g., an activatable antibody) provided herein contains substitutions (e.g, conservative substitutions), insertions, or deletions relative to the reference sequence, but the activatable binding agent comprising that sequence retains the ability to bind to ROR. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in a reference amino acid sequence. In some embodiments, substitutions, insertions, or deletions occur in regions outside the CDRs (e.g, in the FRs and / or constant regions).

[0207] In some embodiments, the position of one or more CDRs along the VH (e.g, CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) region of an activatable ROR binding agent (e.g., an activatable antibody), including an activatable human ROR binding agent, described herein may vary by one, two, three, four, five, or six amino acid positions so long as binding to ROR (e.g., human ROR) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). For example, in some embodiments, the position defining a CDR of Table1 may vary by shifting the N-terminal and / or C-terminal boundary of the CDR by one, two, three, four, five, or six amino acids, relative to the current CDR position, so long as binding to ROR (e.g., human ROR) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). Additionally or alternatively, in some embodiments, the length of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) region of an activatable ROR binding agent (e.g., an activatable antibody), including an activatable human ROR binding agent, described herein may vary (e.g., be shorter or longer) by one, two, three, four, five, or more amino acids, so long as binding to ROR (e.g., human ROR) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). For example, in some embodiments, a VH and / or VL CDR1, CDR2, and / or CDR3 described herein may be one, two, three, four, five or more amino acids shorter than one or more of the CDRs described by SEQ ID NOS: 1-24 and 27-28, so long as binding to ROR (e.g., human ROR) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). In other embodiments, a VH and / or VL CDR1, CDR2, and / or CDR3 described herein may be one, two, three, four, five or more amino acids longer than one or more of the CDRs described by SEQ ID NOS: 1-24 and 27-28, so long as binding to ROR (e.g., human ROR) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). In some embodiments, the amino terminus of a VH and / or VL CDR1, CDR2, and / or CDR3 described herein may be extended or shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs described by SEQ ID NOS: 1-24 and 27-28, so long as binding to ROR (e.g., human ROR) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). Additionally or alternatively, in some embodiments, the carboxy terminus of a VH and / or VL CDR1, CDR2, and / or CDR3 described herein may be extended or shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs described by SEQ ID NOS: 1-24 and 27-28, so long as binding to ROR (e.g., human ROR) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). Any method known in the art can be used to ascertain whether binding to ROR (e.g., human ROR) is maintained.

[0208] In other embodiments, the activatable ROR binding agents (e.g., activatable antibodies), including activatable human ROR binding agents, presented herein that bind toROR, further comprise conservative sequence modifications. With respect to polypeptides that are activatable ROR binding agents (e.g., activatable antibodies), such as activatable human ROR binding agents, conservative sequence modifications include conservative amino acid substitutions that include ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Thus, in some embodiments, a predicted nonessential amino acid residue in an activatable ROR binding agent is replaced with another amino acid residue from the same side chain family. Methods of identifying amino acid conservative substitutions which do not eliminate antigen binding and nucleotides encoding thereof are well-known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)). In some embodiments, the conservative sequence modifications described herein modify the amino acid sequences of the activatable ROR binding agents (e.g., activatable antibodies), including activatable human ROR binding agents, by 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99%. In some embodiments, the amino acid sequence modifications refer to at most 1, 2, 3, 4, 5, or 6 amino acid substitutions to the CDRs, such as those described in Table 1. Thus, for example, each such CDR may contain up to 5 conservative amino acid substitutions, for example up to (not more than) 4 conservative amino acid substitutions, for example up to (not more than) 3 conservative amino acid substitutions, for example up to (not more than) 2 conservative amino acid substitutions, or no more than 1 conservative amino acid substitution. In some embodiments, an activatable ROR binding agent (e.g., an activatable antibody), including an activatable human ROR binding agent, contains one or more, including six, CDRs having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the CDRs of the antibody depicted in Table 1.

[0209] In some embodiments, an activatable ROR binding agent (e.g., an activatable antibody), including an activatable human ROR binding agent, contains a VH and a VL comprising CDRs identical to those of the antibody depicted in Table 1. In some embodiments, the amino acid sequence modifications do not include any modification within an SDR. In some embodiments, the amino acid sequence modifications do not include any modification within a CDR (such as CDR1, CDR2, CDR3, or any combination thereof). Additionally or alternatively, the amino acid sequence modifications are in the framework, constant region, and / or fragment crystallizable region (Fc).

[0210] In some embodiments, the activatable antibody or fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:25, and / or a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:26, and the binding of the activatable antibody or fragment thereof to ROR (e.g., human ROR) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0211] In some embodiments, functional epitopes can be mapped, e.g., by combinatorial alanine scanning, to identify amino acids in the ROR protein that are necessary for interaction with activatable ROR binding agents (such as activatable antibodies) provided herein. In some embodiments, conformational and crystal structure of activatable ROR binding agents (such as activatable antibodies) bound to ROR may be employed to identify the epitopes. In some embodiments, the present disclosure provides an activatable antibody that specifically binds to the same epitope as any of the activatable ROR binding agents (such as activatable antibodies or fragments thereof) provided herein.

[0212] For example, in some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) binds to the same epitope as an anti -ROR antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:25 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:26. In some embodiments, the activatable ROR binding agent provided herein (e.g., an activatable antibody) binds to the same epitope as an anti-ROR antibody comprising a VH comprising the amino acid sequence of SEQ ID NO:25, and a VL comprising the amino acid sequence of SEQ ID NO:26.

[0213] In some embodiments, the activatable ROR binding agent provided herein (e.g, an activatable antibody) specifically binds to ROR competitively with any one of the anti- ROR antibodies or fragments thereof described herein.

[0214] In some embodiments, the activatable ROR binding agent provided herein (e.g, an activatable antibody) specifically binds to ROR competitively with an anti-ROR antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:25 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:26. In some embodiments,the activatable ROR binding agent provided herein (e.g., an activatable antibody) specifically binds to ROR competitively with an anti-ROR antibody comprising a VH comprising the amino acid sequence of SEQ ID NO:25, and a VL comprising the amino acid sequence of SEQ ID NO:26.

[0215] In some embodiments, the activatable ROR binding agent comprises six CDRs as listed in one column of Table 1. In some embodiments, the activatable ROR binding agent comprises three CDRs of the heavy chain variable region as set forth in SEQ ID NO: 25 and three CDRs of the light chain variable regions as set forth in SEQ ID NO: 26. In some embodiments, the activatable ROR binding agent comprises the heavy chain variable region as set forth in SEQ ID NO: 25 and the light chain variable regions as set forth in SEQ ID NO: 26.

[0216] In some embodiments, the activatable binding agents are superior in developability based on a known assay in the art, for example, various chromatographic methods, including size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), and standup monolayer adsorption chromatography (SMAC). In some embodiments, the binding agents are superior in developability based on measurement of monomer percentage, solubility, and / or antibody aggregation or precipitation.

[0217] In some embodiments, activatable ROR binding agents (e.g., activatable antibodies such as monospecific or bispecific antibodies), including activatable human ROR binding agents, described herein comprise a heavy chain having a combination of (i) a VH described herein, such as in Table 1; and (ii) one or more heavy chain constant domains (e.g., CHI, Hinge, CH2, and / or CH3). In further embodiments, the carboxyl terminus (C-terminus) of the VH is conjugated directly or indirectly to the amino terminus (N-terminus) of the one or more heavy chain constant domains. In a specific embodiment, the heavy chain comprises the amino acid sequence of SEQ ID NO:32. In a specific embodiment, the heavy chain comprises the amino acid sequence of SEQ ID NO:32, except for (i) a D356E substitution and / or an L358M substitution, and / or (ii) a K213R substitution or a K214R substitution.

[0218] In some embodiments, activatable ROR binding agents (e.g., activatable antibodies such as monospecific or bispecific antibodies), including activatable human ROR binding agents, described herein comprise a polypeptide comprising, from N-terminus to C- terminus, a masking peptide described herein (e.g., Table 2) and a light chain having a combination of (i) a VL domain described herein, such as in Table 1; and (ii) a light chain constant domain (CL). In further embodiments, the C terminus of the VL is conjugated directly or indirectly to the N-terminus of the CL. In further embodiments, the maskingpeptide is conjugated directly or indirectly to the N-terminus of the light chain. In a specific embodiment, the light chain comprises the amino acid sequence of SEQ ID NO:33. In a specific embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 34. In a specific embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 36. In a specific embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 38.

[0219] In some embodiments, activatable ROR binding agents (e.g., activatable antibodies such as monospecific or bispecific antibodies), including activatable human ROR binding agents, described herein comprise (a) a heavy chain having a combination of (i) a VH described herein, such as in Table 1, and (ii) one or more heavy chain constant domains (e.g., CHI, Hinge, CH2, and / or CH3); and (b) a polypeptide comprising, from N-terminus to C- terminus, a masking peptide described herein (e.g., Table 2) and a light chain having a combination of (i) a VL described herein, such as in Table 1, and (ii) a light chain constant domain (CL). In further embodiments, the C-terminus of the VH is conjugated directly or indirectly to the N-terminus of the one or more heavy chain constant domains. In further embodiments, the C terminus of the VL is conjugated directly or indirectly to the N-terminus of the CL. In further embodiments, the masking peptide is conjugated directly or indirectly to the N-terminus of the light chain. In specific embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:32. In specific embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:32, except for (i) a D356E substitution and / or an L358M substitution, and / or (ii) a K213R substitution or a K214R substitution. In specific embodiment, the light chain comprises the amino acid sequence of SEQ ID NO:33. In specific embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 34. In specific embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 36. In specific embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 38. In a specific embodiment, the heavy chain comprises the amino acid sequence of SEQ ID NO:32, and the light chain comprises the amino acid sequence of SEQ ID NO:33. In a specific embodiment, the heavy chain comprises the amino acid sequence of SEQ ID NO:32, and the polypeptide comprises the amino acid sequence of SEQ ID NO: 34. In a specific embodiment, the heavy chain comprises the amino acid sequence of SEQ ID NO:32, and the polypeptide comprises the amino acid sequence of SEQ ID NO: 36. In a specific embodiment, the heavy chain comprises the amino acid sequence of SEQ ID NO:32, and the polypeptide comprises the amino acid sequence of SEQ ID NO: 38. In a specific embodiment, the heavy chain comprises the amino acid sequence of SEQ ID NO:32, exceptfor (i) a D356E substitution and / or an L358M substitution, and / or (ii) a K213R substitution or a K214R substitution, and the light chain comprises the amino acid sequence of SEQ ID NO:33. In a specific embodiment, the heavy chain comprises the amino acid sequence of SEQ ID NO:32, except for (i) a D356E substitution and / or an L358M substitution, and / or (ii) a K213R substitution or a K214R substitution, and the polypeptide comprises the amino acid sequence of SEQ ID NO: 34. In a specific embodiment, the heavy chain comprises the amino acid sequence of SEQ ID NO:32, except for (i) a D356E substitution and / or an L358M substitution, and / or (ii) a K213R substitution or a K214R substitution, and the polypeptide comprises the amino acid sequence of SEQ ID NO: 36. In a specific embodiment, the heavy chain comprises the amino acid sequence of SEQ ID NO:32, except for (i) a D356E substitution and / or an L358M substitution, and / or (ii) a K213R substitution or a K214R substitution, and the polypeptide comprises the amino acid sequence of SEQ ID NO: 38.

[0220] In some embodiments, CDRs of a parental ROR antibody as used herein are disclosed in US Patent Application Publication No. US20210155692A1, which is incorporated by reference in its entirety. In some embodiments, a parental ROR antibody as used herein is disclosed in US Patent Application Publication No. US20210155692A1

[0221] In some embodiments, provided herein is an activatable ROR binding protein comprising any one of the activatable anti-ROR antibodies described herein. In some embodiments, the activatable ROR binding protein is an activatable antibody comprising two heavy chains and two light chains. In some embodiments, the activatable ROR binding protein is an activatable antibody comprising two heavy chains comprising a same VH region and two light chains comprising a same VL region.

[0222] In some embodiments, the activatable ROR binding protein is a monoclonal antibody, including a mouse, chimeric, humanized or human antibody. In some embodiments, the activatable anti-ROR antibody is an antibody fragment, e.g., an scFv. In some embodiments, the activatable ROR binding protein is a fusion protein comprising the activatable anti-ROR antibody provided herein. In other embodiments, the activatable ROR binding protein is a multispecific activatable antibody comprising the activatable anti-ROR antibody or fragment thereof provided herein.

[0223] Also provided herein are activated activatable binding agents (z.e., activatable binding agents in an activated form). In some embodiments, the activatable binding agents (in an inactivated form) are as disclosed herein. In further embodiments, the masking peptide of the activatable binding agents (in an inactivated form) comprises a masking moiety (MM) and a cleavable moiety (CM), wherein the CM is located between the MM and a light chainvariable region. In yet further embodiments, the masking peptide of the activatable binding agents (in an inactivated form) comprises, from N-terminus to C-terminus, an MM, a CM, and a light chain variable region. In some embodiments, the CM comprises, from N-terminus to C-terminus, a first optional linker, a cleavage site, and a second optional linker. In further embodiments, the activated form of activatable binding agents have been activated from the inactivated form by contacting the inactivated form with a protease (for example, MMP9), cleaving the cleavage site. Accordingly, in some embodiments, the activated activatable binding agents (ie., the activatable binding agents in an activated form) comprise (a) a polypeptide comprising, from N-terminus to C-terminus, amino acid residues (if any) of the cleavage site after the protease cleavage (such as, zero, or one, or two, or three, or four, or more C-terminal amino acid(s) of the cleavage site), the second optional linker, and the light chain variable region and (b) an antibody heavy chain variable region; wherein the masking peptide before protease cleavage comprises the amino acid sequence of SEQ ID NO:29, 30 or 31 (see Table 2), and wherein the antibody light chain variable region and the antibody heavy chain variable region are described herein (for example, comprise the CDRs described herein (e.g., Table 1)).

[0224] Other exemplary activatable ROR binding molecules are described in more detail in the following sections. In some embodiments, the activatable anti-ROR antibody or antigen-binding protein according to any of the above embodiments may incorporate any of the features, singly or in combination, as described in Sections 5.3.1 to 5.3.4 below.Table 1: Antibody CDRs, VH and VL66NAI-1540625138vlTable 2: Masking PeptideTable 3: Parental Antibody PO67NAI-1540625138V1Table 4: Antibody MlTable 5: Antibody M268NAI-1540625138V1Table 6: Antibody M369NAI-1540625138V1Table 7: Human ROR1 and ROR2 Sequences70NAI-1540625138V15.3.1. Antibody Fragments

[0225] Even though the term “antibody” is sometimes used in a phrase of “antibody or fragment thereof’ herein, it should be understood the term “antibody” as used herein also includes various antibody fragments, such as an antigen-binding fragment or epitope-binding fragment. Thus, when the term “antibody” is used alone without being followed by “fragment thereof’ or similar terms, it should be understood the term “antibody” includes an antibody fragment, such as an antigen-binding fragment or epitope-binding fragment. Antibodies provided herein include, but are not limited to, immunoglobulin molecules and immunologically active portions of immunoglobulin molecules.

[0226] Variants and derivatives of antibodies include antibody functional fragments that retain the ability to bind to an antigen. Antibody fragments include but are not limited to those described in Section 5.2 above. Exemplary functional fragments include Fab fragments (e.g., an antibody fragment that contains the antigen-binding domain and comprises a light chain and part of a heavy chain bridged by a disulfide bond); Fab’ (e.g., an antibody fragment containing a single antigen-binding domain comprising an Fab and an additional portion of the heavy chain through the hinge region); F(ab’)2 (e.g., two Fab’ molecules joined by interchain disulfide bonds in the hinge regions of the heavy chains; the Fab’ molecules may be directed toward the same or different epitopes); a bispecific Fab (e.g., a Fab molecule having two antigen-binding domains, each of which may be directed to a different epitope); a single chain comprising a variable region, also known as, scFv (e.g., the variable, antigenbinding determinative region of a single light and heavy chain of an antibody linked together by a chain of, e.g., 10-25 amino acids); a disulfide-linked Fv, or dsFv (e.g., the variable, antigen-binding determinative region of a single light and heavy chain of an antibody linked together by a disulfide bond); a bispecific scFv (e.g., an scFv or a dsFv molecule having two antigen-binding domains, each of which may be directed to a different epitope); a diabody (e.g., a dimerized scFv formed when the VH domain of a first scFv assembles with the VL domain of a second scFv and the VL domain of the first scFv assembles with the VH domain of the second scFv; the two antigen-binding regions of the diabody may be directed towards the same or different epitopes); a triabody (e.g., a trimerized scFv, formed in a manner similar to a diabody, but in which three antigen-binding domains are created in a single complex; the three antigen-binding domains may be directed towards the same or different epitopes); and a tetrabody (e.g., a tetramerized scFv, formed in a manner similar to a diabody, but in which four antigen-binding domains are created in a single complex; the four antigenbinding domains may be directed towards the same or different epitopes).

[0227] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al.. 1992, J. Biochem. Biophys. Methods 24: 107-17; and Brennan et aL, 1985, Science 229:81-83). However, these fragments can now be produced directly by recombinant host cells. For example, Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coli, yeast or insect cells, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab’-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab’)2 fragments (Carter et al., 1992, Bio / Technology 10:163-67). According to another approach, F(ab’)2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab’)2 fragments with increased in vivo half-life comprising salvage receptor binding epitope residues are described in, for example, U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In certain embodiments, an antibody is a single chain Fv fragment (scFv) (see, e.g., WO 93 / 16185; U.S. Pat. Nos. 5,571,894 and 5,587,458). Fv and scFv have intact combining sites that are devoid of constant regions; thus, they may be suitable for reduced nonspecific binding during in vivo use. scFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an scFv (See, e.g., Borrebaeck ed., supra). The antibody fragment may also be a “linear antibody,” for example, as described in the references cited above. Such linear antibodies may be monospecific or multi-specific, such as bispecific.5.3.2. Humanized Antibodies

[0228] The present disclosure provides humanized activatable antibodies that bind ROR, including human ROR. Humanized activatable antibodies of the present disclosure may comprise one or more CDRs from a VH and / or VL disclosed herein, such as those as shown in Table 1. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanized antibodies that bind ROR may be produced using techniques known to those skilled in the art (Zhang et al. , Molecular Immunology , 42(12): 1445-1451, 2005; Hwang et aL, Methods, 36(1): 35-42, 2005; Dall’Acqua et al., Methods, 36(1): 43-60, 2005; Clark, Immunology Today, 21(8): 397-402, 2000, and U.S. Pat. Nos. 6,180,370; 6,054,927; 5,869,619; 5,861,155; 5,712,120; and 4,816,567).

[0229] In some cases, the humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the six CDRs of a VH and a VL of the parent non-human antibody (e.g., rodent) are grafted onto a human antibody framework. For example, Padlan et al. (FASEB J. 9: 133-139, 1995) determined that only about one third of the residues in the CDRs actually contact the antigen, and termed these the “specificity determining residues,” or SDRs. In the technique of SDR grafting, only the SDR residues are grafted onto the human antibody framework (see, e.g., Kashmiri et al.. Methods 36: 25-34, 2005).

[0230] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies can be important to reduce antigenicity. For example, according to the so-called “best-fit” method, the sequence of the variable domain of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent may be selected as the human framework for the humanized antibody (Sims et al. (1993) J. Immunol. 151 :2296; Chothia e / a / . (1987) J. Mol. Biol. 196:901). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al. (1992) roc. Natl. Acad. Sci. USA, 89:4285; Presta e / o / . (1993) J. Immunol., 151 :2623). In some cases, the framework is derived from the consensus sequences of the most abundant human subclasses, VL6 subgroup I (VL6I) and VH subgroup III (VHIII). In another method, human germline genes are used at the source of the framework regions.

[0231] In an alternative paradigm based on comparison of CDRs, called Superhumanization, framework homology is irrelevant. The method consists of comparison of the non-human sequence with the functional human germline gene repertoire. Those genes encoding the same or closely related canonical structures to the murine sequences are then selected. Next, within the genes sharing the canonical structures with the non-human antibody, those with highest homology within the CDRs are chosen as framework donors. Finally, the non-human CDRs are grafted onto these frameworks (see, e.g., Tan et al, J. Immunol. 169: 1119-1125, 2002).

[0232] It is further generally desirable that antibodies be humanized with retention of their affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, Protein Eng. 13: 819-824, 2000), Modeller (Sali and Blundell, J. Mol. Biol. 234: 779-815, 1993), and Swiss PDB Viewer (Guex and Peitsch, Electrophoresis 18: 2714-2713, 1997). Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, e.g., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, framework residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0233] Another method for antibody humanization is based on a metric of antibody humanness termed Human String Content (HSC). This method compares the mouse sequence with the repertoire of human germline genes and the differences are scored as HSC. The target sequence is then humanized by maximizing its HSC rather than using a global identity measure to generate multiple diverse humanized variants. See, e.g., Lazar et al., Mol. Immunol. 44: 1986-1998, 2007.

[0234] In addition to the methods described above, empirical methods may be used to generate and select humanized antibodies. These methods include those that are based upon the generation of large libraries of humanized variants and selection of the best clones using enrichment technologies or high throughput screening techniques. Antibody variants may be isolated from phage, ribosome and yeast display libraries as well as by bacterial colony screening (see, e.g., Hoogenboom, Nat. Biotechnol. 23: 1105-1116, 2005; Dufner et al., Trends Biotechnol. 24: 523-529, 2006; Feldhaus et al, Nat. BiotechnoL 21 : 163-70, 2003; Schlapschy et al., Protein Eng. Des. SeL 17: 847-60, 2004).

[0235] In the framework library approach, a collection of residue variants are introduced at specific positions in the framework followed by selection of the library to select the framework that best supports the grafted CDR. The residues to be substituted may include some or all of the “Vernier” residues identified as potentially contributing to CDR structure (see, e.g., Foote and Winter, J. Mol. Biol. 224: 487-499, 1992), or from the more limited set of target residues identified by Baca et al. (J. Biol. Chem. 272: 10678-10684, 1997).

[0236] In framework shuffling, whole frameworks are combined with the non-human CDRs instead of creating combinatorial libraries of selected residue variants (see, e.g.,Dall’ Acqua et al., Methods 36: 43-60, 2005). The libraries may be screened for binding in a two-step selection process, first humanizing VL, followed by VH. Alternatively, a one-step framework shuffling process may be used. Such a process has been shown to be more efficient than the two-step screening, as the resulting antibodies exhibited improved biochemical and physico-chemical properties including enhanced expression, increased affinity and thermal stability (see, e.g., Damschroder et al., Mol. Immunol. 44: 3049-60, 2007).

[0237] The “humaneering” method is based on experimental identification of essential minimum specificity determinants (MSDs) and is based on sequential replacement of nonhuman fragments into libraries of human frameworks and assessment of binding. It begins with regions of the CDR3 of non-human VH and VL chains and progressively replaces other regions of the non-human antibody into the human frameworks, including the CDR1 and CDR2 of both VH and VL. This methodology typically results in epitope retention and identification of antibodies from multiple sub-classes with distinct human V-segment CDRs. Humaneering allows for isolation of antibodies that are 91-96 % homologous to human germline gene antibodies. See, e.g., Alfenito, Cambridge Healthtech Institute’s Third Annual PEGS, The Protein Engineering Summit, 2007.

[0238] The "human engineering" method involves altering a non-human antibody or antibody fragment, such as a mouse or chimeric antibody or antibody fragment, by making specific changes to the amino acid sequence of the antibody so as to produce a modified antibody with reduced immunogenicity in a human that nonetheless retains the desirable binding properties of the original non-human antibodies. Generally, the technique involves classifying amino acid residues of a non-human (e.g., mouse) antibody as “low risk”, “moderate risk”, or “high risk” residues. The classification is performed using a global risk / reward calculation that evaluates the predicted benefits of making a particular substitution (e.g., for immunogenicity in humans) against the risk that the substitution will affect the resulting antibody’s folding and / or are substituted with human residues. The particular human amino acid residue to be substituted at a given position (e.g., low or moderate risk) of a non-human (e.g., mouse) antibody sequence can be selected by aligning an amino acid sequence from the non-human antibody’s variable regions with the corresponding region of a specific or consensus human antibody sequence. The amino acid residues at low or moderate risk positions in the non-human sequence can be substituted for the corresponding residues in the human antibody sequence according to the alignment. Techniques for making human engineered proteins are described in greater detail inStudnicka et aL, Protein Engineering, 7: 805-814 (1994), U.S. Pat. Nos. 5,766,886, 5,770,196, 5,821,123, and 5,869,619, and WO 93 / 11794.5.3.3. Antibody Variants

[0239] Modifications of the activatable antibodies that bind to ROR described herein are contemplated. For example, it may be desirable to optimize the binding affinity and / or other biological properties of the antibody, including but not limited to specificity, thermostability, expression level, effector functions, glycosylation, reduced immunogenicity, or solubility. Thus, it is contemplated that variants of the activatable antibodies that bind to ROR described herein can be prepared and are included in the present disclosure. In some embodiments, antibody variants are antibodies with amino acid sequence variations as compared with the original antibody, for example, substitution, deletion, or insertion of one or more amino acid(s), as described above. For example, variations may be a substitution, deletion, or insertion of one or more codons encoding the antibody or polypeptide that results in a change in the amino acid sequence (e.g., a conservative substitution) as compared with the original antibody or polypeptide. Sites of interest for substitutional mutagenesis include the CDRs, FRs and / or constant regions. For example, antibody variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA, and / or by synthesis of the desired antibody or polypeptide. Those skilled in the art who appreciate that amino acid changes may alter post-translational processes of the antibody.Chemical Modifications

[0240] Other exemplary modifications include chemical modifications, for example, by the covalent attachment of any type of molecule to the antibody. Antibody derivatives may include antibodies that have been chemically modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, or conjugation to one or more immunoglobulin domains (e.g., Fc or a portion of an Fc). Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Additionally, the antibody may contain one or more non-classical amino acids.

[0241] In some embodiments, an activatable antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion ofglycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

[0242] When the activatable antibody provided herein is fused to an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in the binding molecules provided herein may be made in order to create variants with certain improved properties.

[0243] In other embodiments, when the activatable antibody provided herein is fused to an Fc region, antibody variants provided herein may have a carbohydrate structure that lacks fucose attached (directly or indirectly) to said Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., U.S. Pat. Publ. Nos. 2003 / 0157108 and 2004 / 0093621. Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: U.S. Pat. Publ. No. 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; U.S. Pat. Publ. No. 2003 / 0115614; U.S. Pat. Publ. No. 2002 / 0164328; U.S. Pat. Publ. No. 2004 / 0093621; U.S. Pat. Publ. No.2004 / 0132140; U.S. Pat. Publ. No. 2004 / 0110704; U.S. Pat. Publ. No. 2004 / 0110282; U.S. Pat. Publ. No. 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; W02005 / 053742; W02002 / 031140; Okazaki et al. J. Mol. Biol. 336: 1239- 1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lecl3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Pat. Publ. No. 2003 / 0157108; and WO 2004 / 056312, and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and W02003 / 085107).

[0244] The activatable binding molecules comprising an activatable antibody provided herein are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region is bisected by GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function. Examples of such variants are described, e.g., in WO 2003 / 011878 (Jean-Mai ret c / a / .); U.S. Pat. No. 6,602,684 (Umana et al.) and U.S. Pat. Publ. No. 2005 / 0123546 (Umana et al ). Variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such variants may have improved CDC function. Such variants are described, e.g., in WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0245] In molecules that comprise the present activatable antibody and an Fc region, one or more amino acid modifications may be introduced into the Fc region, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0246] In some embodiments, the present application contemplates variants that possess some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the activatable binding molecule in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the activatable binding molecule lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 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). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat ’I Acad. Sci. USA 95:652-656 (1998). Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano- Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, M.S. et al., Blood 101 : 1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al, Int’l. Immunol. 18(12): 1759-1769 (2006)).

[0247] Binding molecules with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).

[0248] Certain variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

[0249] In some embodiments, a variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues). In some embodiments, alterations are made in the Fc region that result in altered (e.g., either improved or diminished) Clq binding and / or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0250] Binding molecules with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). Those molecules comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (US Patent No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94 / 29351 concerning other examples of Fc region variants.

[0251] In some embodiments, it may be desirable to create cysteine engineered antibodies, in which one or more residues of an antibody are substituted with cysteine residues. In some embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein.

[0252] Other known covalent modifications of antibodies are included within the scope of the present disclosure. Covalent modifications include reacting targeted amino acid residues of an antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the antibody. Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the a-amino groups of lysine, arginine, and histidine side chains (see, e.g., Creighton, Proteins: Structure and Molecular Properties 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C- terminal carboxyl group.

[0253] The activatable antibody that binds to ROR of the present disclosure may also be modified to form chimeric molecules comprising the activatable antibody that binds to ROR fused or conjugated to another, heterologous polypeptide or amino acid sequence or a small molecule compound, for example, an immune activator (such as a cytokine), an epitope tag (see, e.g., Terpe, Appl. Microbiol. Biotechnol. 60:523-33 (2003)) or the Fc region of an IgG molecule (see, e.g., Aruffo, Antibody Fusion Proteins 221-42 (Chamow and Ashkenazi eds., 1999)).

[0254] Also provided herein are fusion proteins comprising the activatable antibody that binds to ROR of the disclosure and a heterologous polypeptide. In some embodiments, the heterologous polypeptide to which the antibody is genetically fused or chemically conjugated is useful for targeting the antibody to cells having cell surface-expressed ROR. Genetically fused or chemically conjugated antibodies are described in more detail in sections below.In vitro Affinity Maturation

[0255] In some embodiments, antibody variants having an improved property such as affinity, stability, or expression level as compared to a parent antibody may be prepared by in vitro affinity maturation. Like the natural prototype, in vitro affinity maturation is based onthe principles of mutation and selection. Libraries of antibodies are displayed on the surface of an organism (e.g., phage, bacteria, yeast, or mammalian cell) or in association (e.g., covalently or non-covalently) with their encoding mRNA or DNA. Affinity selection of the displayed antibodies allows isolation of organisms or complexes carrying the genetic information encoding the antibodies. Two or three rounds of mutation and selection using display methods such as phage display usually results in antibody fragments with affinities in the low nanomolar range. Affinity matured antibodies can have nanomolar or even picomolar affinities for the target antigen.

[0256] Phage display is a widespread method for display and selection of antibodies. The antibodies are displayed on the surface of Fd or M13 bacteriophages as fusions to the bacteriophage coat protein. Selection involves exposure to antigen to allow phage-displayed antibodies to bind their targets, a process referred to as “panning.” Phage bound to antigen are recovered and used to infect bacteria to produce phage for further rounds of selection. For review, see, for example, Hoogenboom, Methods. Mol. Biol. 178: 1-37 (2002); and Bradbury and Marks, J. Immunol. Methods 290:29-49 (2004).

[0257] In a yeast display system (see, e.g., Boder etal., Nat. Biotech. 15:553-57 (1997); and Chao et al., Nat. Protocols 1 :755-68 (2006)), the antibody may be fused to the adhesion subunit of the yeast agglutinin protein Aga2p, which attaches to the yeast cell wall through disulfide bonds to Agalp. Display of a protein via Aga2p projects the protein away from the cell surface, minimizing potential interactions with other molecules on the yeast cell wall. Magnetic separation and flow cytometry are used to screen the library to select for antibodies with improved affinity or stability. Binding to a soluble antigen of interest is determined by labeling yeast with biotinylated antigen and a secondary reagent such as streptavidin conjugated to a fluorophore. Variations in surface expression of the antibody can be measured through immunofluorescence labeling of either the hemagglutinin or c-Myc epitope tag flanking the single-chain antibody (e.g., scFv). Expression has been shown to correlate with the stability of the displayed protein, and thus antibodies can be selected for improved stability as well as affinity (see, e.g., Shusta et al., J. Mol. Biol. 292:949-56 (1999)). An additional advantage of yeast display is that displayed proteins are folded in the endoplasmic reticulum of the eukaryotic yeast cells, taking advantage of endoplasmic reticulum chaperones and quality-control machinery. Once maturation is complete, antibody affinity can be conveniently “titrated” while displayed on the surface of the yeast, eliminating the need for expression and purification of each clone. A theoretical limitation of yeast surface display is the potentially smaller functional library size than that of other display methods;however, a recent approach uses the yeast cells’ mating system to create combinatorial diversity estimated to be 1014in size (see, e.g., U.S. Pat. Publ. No. 2003 / 0186374; and Blaise et al., Gene 342:211-18 (2004)).

[0258] In ribosome display, antibody-ribosome-mRNA (ARM) complexes are generated for selection in a cell-free system. The DNA library coding for a particular library of antibodies is genetically fused to a spacer sequence lacking a stop codon. This spacer sequence, when translated, is still attached to the peptidyl tRNA and occupies the ribosomal tunnel, and thus allows the protein of interest to protrude out of the ribosome and fold. The resulting complex of mRNA, ribosome, and protein can bind to surface-bound ligand, allowing simultaneous isolation of the antibody and its encoding mRNA through affinity capture with the ligand. The ribosome-bound mRNA is then reverse transcribed back into cDNA, which can then undergo mutagenesis and be used in the next round of selection (see, e.g., Fukuda et al., Nucleic Acids Res. 34:el27 (2006)). In mRNA display, a covalent bond between antibody and mRNA is established using puromycin as an adaptor molecule (Wilson et al., Proc. Natl. Acad. Sci. USA 98:3750-55 (2001)).

[0259] As these methods are performed entirely in vitro, they provide two main advantages over other selection technologies. First, the diversity of the library is not limited by the transformation efficiency of bacterial cells, but only by the number of ribosomes and different mRNA molecules present in the test tube. Second, random mutations can be introduced easily after each selection round, for example, by non-proofreading polymerases, as no library must be transformed after any diversification step. In some embodiments, mammalian display systems may be used.

[0260] Diversity may also be introduced into the CDRs of the antibody libraries in a targeted manner or via random introduction. The former approach includes sequentially targeting all the CDRs of an antibody via a high or low level of mutagenesis or targeting isolated hot spots of somatic hypermutations (see, e.g., Ho et al, J. Biol. Chem. 280:607-17 (2005)) or residues suspected of affecting affinity on experimental basis or structural reasons. Diversity may also be introduced by replacement of regions that are naturally diverse via DNA shuffling or similar techniques (see, e.g., Lu et al., J. Biol. Chem. 278:43496-507 (2003); U.S. Pat. Nos. 5,565,332 and 6,989,250). Alternative techniques target hypervariable loops extending into framework-region residues (see, e.g., Bond et al, J. Mol. Biol. 348:699- 709 (2005)) employ loop deletions and insertions in CDRs or use hybridization-based diversification (see, e.g., U.S. Pat. Publ. No. 2004 / 0005709). Additional methods of generating diversity in CDRs are disclosed, for example, in U.S. Pat. No. 7,985,840. Furthermethods that can be used to generate antibody libraries and / or antibody affinity maturation are disclosed, e.g., in U.S. Pat. Nos. 8,685,897 and 8,603,930, and U.S. Publ. Nos. 2014 / 0170705, 2014 / 0094392, 2012 / 0028301, 2011 / 0183855, and 2009 / 0075378, each of which is incorporated herein by reference.

[0261] Screening of the libraries can be accomplished by various techniques known in the art. For example, antibodies can be immobilized onto solid supports, columns, pins, or cellulose / poly (vinylidene fluoride) membranes / other filters, expressed on host cells affixed to adsorption plates or used in cell sorting, or conjugated to biotin for capture with streptavidin-coated beads or used in any other method for panning display libraries.

[0262] For reviews of in vitro affinity maturation methods, see, e.g., Hoogenboom, Nature Biotechnology 23 : 1105-16 (2005); Quiroz and Sinclair, Revista Ingeneria Biomedia 4:39-51 (2010); and references therein.

[0263] An antibody internalization assay may be used to determine receptor-mediated endocytosis when binding to an antibody. In some embodiments, the efficacy of certain antibody-based therapeutics depends on antibody internalization process. In some embodiments, an antibody internalization assay examines the rate and extent of antibody internalization in order to evaluate the antibody’s ability of delivering treatments to sites or cells of interest. A non-limiting exemplary assay is briefly described below. Target cells of interest are seeded at an appropriate seeding density (e.g., in a 96-well U-bottom plate), and a tested antibody is labelled with a signal reporting reagent, for example, fluorescent compounds, Horseradish peroxidase (HRP) reagent, radiolabeled compounds, or biotin. Then the tested antibody and the target cells are incubated at an appropriate molar ratio. Following the incubation, unbound antibodies are removed by wash. The cells can be left on ice or incubated at 37°C for a period of time to facilitate internalization. The cells may then be incubated for a period of time in the presence of a stop reagent to inhibit internalization. Subsequently, the cells are washed and incubated with the signal developing reagent. The final signal can be studied using plate reader or imaging instrument and an analytical software. For example, mean fluorescence intensity (MFI) of the cells can be measured using a flow cytometer, and MFI reduction can represent antibody internalization, antibody dissociation or a combination of both. Cell imaging can be scanned and acquired to analyze the signal intensity, size and shape. Alternatively, the cells are lysed, releasing internalized antibody. This antibody is then captured in a microtiter well plate coated with specific antigen against which the antibody was raised. Bound antibody in the well is detected using an alkaline phosphatase or HRP-conjugated secondary antibody and a chromogenic substrate.Alternative detectable labels for the antibody and means for detecting internalized labeled antibody will be obvious to those skilled in the art upon this disclosure. Any methods known in the art to determine antibody internalization can be used in the present disclosure.5.3.4. Other Binding Agents Comprising the Antibodies

[0264] In some embodiments, the activatable antibody or fragment thereof provided herein is a part of a larger activatable binding agent. Non-limiting exemplary activatable binding agents comprising the activatable antibody or fragment provided herein are described below.

[0265] The present disclosure provides activatable ROR binding agents (e.g., activatable antibodies) with a masking peptide. Such activatable ROR binding agents (e.g., activatable antibodies) are also useful for the preparation of conjugates, including immunoconjugates and antibody-drug conjugates (ADCs), comprising any one of the activatable ROR binding agents (e.g., activatable antibodies), such as activatable human ROR binding agents, of the present disclosure, including those directly or indirectly linked to another agent such as a drug and / or an immune activator (such as a cytokine). For example, activatable ROR binding agents (e.g., activatable antibodies), such as activatable human ROR binding agents, of the present disclosure may be covalently bound by a synthetic linker to one or more agents such as drugs and / or immune activators (such as cytokines).

[0266] If desired, an activatable ROR binding agent (e.g., an activatable antibody), including an activatable human ROR binding agent, is linked or conjugated (directly or indirectly) to a moiety with effector function, such as cytotoxic activity (e.g., a chemotherapeutic moiety or a radioisotope), immune recruitment or modulating activity. Moi eties that are linked or conjugated (directly or indirectly) include drugs that are cytotoxic (e.g., toxins such as aurostatins) or non-cytotoxic, e.g., signal transduction modulators such as kinases. Moieties that promote immune recruitment can include other antigen-binding agents, such as viral proteins that bind selectively to cells of the innate and / or adaptive immune system. Alternatively or in addition, an activatable ROR binding agent (e.g., an activatable antibody), including an activatable human ROR binding agent, is optionally linked or conjugated (directly or indirectly) to a moiety that facilitates isolation from a mixture (e.g, a tag) or a moiety with reporter activity (e.g., a detection label or reporter protein). It will be appreciated that the features of an activatable ROR binding agent (e.g., an activatable antibody), including an activatable human ROR binding agent, described herein extend also to a polypeptide comprising an activatable ROR binding agent fragment.

[0267] In some embodiments, an activatable ROR binding agent (e.g., an activatable antibody), including an activatable human ROR binding agent, is linked or conjugated (directly or indirectly) to an additional agent. In some embodiments, the additional agent is a drug, resulting in an ADC.

[0268] In some embodiments, activatable ROR binding agents (e.g., activatable antibodies), including activatable human ROR binding agents, described herein are conjugated or recombinantly linked (directly or indirectly) to a therapeutic agent (e.g., a cytotoxic agent or a cytokine) or to a diagnostic or detectable agent. The conjugated or recombinantly linked antibodies can be useful, for example, for treating or preventing a disease, disorder or condition such as an ROR-mediated disease, disorder or condition. The conjugated or recombinantly linked activatable ROR binding agents (e.g., activatable antibodies) can be useful, for example, for monitoring or prognosing the onset, development, progression, and / or severity of an ROR-mediated disease, disorder or condition.

[0269] Such diagnosis and detection can be accomplished, for example, by coupling an activatable ROR binding agent (e.g., an activatable antibody) to detectable substances including, for example: enzymes, including, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, including, but not limited to, streptavidin / biotin or avidin / biotin; fluorescent materials, including, but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, di chi orotriazinyl amine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials, including, but not limited to, luminol; bioluminescent materials, including, but not limited to, luciferase, luciferin, or aequorin; chemiluminescent material, including, but not limited to, an acridinium based compound or a HALOTAG; radioactive materials, including, but not limited to, iodine (131I,125I,123I, and121I), carbon (14C), sulfur (35S), tritium (3H), indium (115In,113In,112In, andH 1In), technetium ("Tc), thallium (201Ti), gallium (68Ga and67Ga), palladium (103Pd), molybdenum ("Mo), xenon (133Xe), fluorine (18F),153Sm,177LU,159Gd,149Pm,140La,175Yb,166Ho,90Y,47Sc,186Re,188Re,142Pr,105Rh,97RU,68Ge,57Co,65Zn,85Sr,32P,153Gd,169Yb,51Cr,54Mn,75Se,113Sn, or117Sn; positron emitting metals using various positron emission tomographies; and non-radioactive paramagnetic metal ions.

[0270] Also described herein are activatable ROR binding agents e.g., activatable antibodies) that are recombinantly linked or conjugated (covalent or non-covalent conjugations, directly or indirectly) to a heterologous protein or polypeptide or fragment thereof, for example, to a polypeptide (e.g., of about 10, about 20, about 30, about 40, about50, about 60, about 70, about 80, about 90, or about 100 amino acids) to generate fusion proteins, as well as uses thereof. In particular, described herein are fusion proteins comprising an antigen-binding fragment of an activatable ROR binding agent (e.g., an activatable antibody), including an activatable human ROR binding agent, described herein (e.g., comprising CDR1, CDR2, and / or CDR3 of VH and / or VL) and a heterologous protein, polypeptide, or peptide. In some embodiments, the heterologous protein, polypeptide, or peptide that an activatable ROR binding agent (e.g., an activatable antibody) is linked to is useful for targeting the ROR binding agent to a particular cell (e.g., an ROR-expressing cell, including a tumor cell). Other non-limiting heterologous protein, polypeptide, or peptide that an activatable ROR binding agent (e.g., an activatable antibody) is linked to can be useful as an internalization signal or engaging a tumor cell with an immune cell.

[0271] Moreover, activatable ROR binding agents (e.g., activatable antibodies), including activatable human ROR binding agents, described herein can be linked (directly or indirectly) to marker or “tag” sequences, such as a peptide, to facilitate purification. In some embodiments, the marker or tag amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (see, e.g., QIAGEN, Inc.), among others, many of which are commercially available. For example, as described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-24, hexa-histidine provides for convenient purification of a fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin (“HA”) tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767-78), and the “FLAG” tag.

[0272] Methods for linking or conjugating (directly or indirectly) moieties (including polypeptides) to antibodies are well known in the art, any one of which can be used to make an antibody-drug conjugate or fusion protein described herein.

[0273] In some embodiments, an activatable ROR binding agent (e.g., an activatable antibody) described herein is a fusion protein. The term “fusion protein” as used herein refers to a polypeptide that comprises an amino acid sequence of a binding agent (e.g., an antibody) and an amino acid sequence of a heterologous polypeptide or protein (e.g., a polypeptide or protein not normally a part of the antibody). In certain embodiments, the fusion protein retains the biological activity of an activatable ROR binding agent. In certain embodiments, the fusion protein comprises an activatable ROR antibody VH region, VL region, VH CDR (one, two or three VH CDRs), and / or VL CDR (one, two or three VL CDRs), wherein the fusion protein binds to an ROR epitope, an ROR fragment and / or an ROR polypeptide. In some embodiments, a fusion protein comprises a VH or heavy chain ofan activatable ROR antibody, a VL or light chain of an activatable ROR antibody, separated by a linker, such as a cleavable linker.

[0274] Fusion proteins may be generated, for example, through the techniques of geneshuffling, motif-shuffling, exon-shuffling, and / or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to alter the activities of activatable ROR binding agents (e.g., activatable antibodies), including activatable human ROR binding agents, as described herein, including, for example, ROR binding agents with higher affinities and lower dissociation rates (see, e.g., U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458; Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33;Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson et al., 1999, J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-13). In some embodiments, activatable ROR binding agents, including activatable human ROR binding agents, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion, or other methods prior to recombination. A polynucleotide encoding an activatable ROR binding agent described herein may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

[0275] Activatable ROR binding agents (e.g., activatable antibodies), including activatable human ROR binding agents, described herein may also be attached to solid supports, which are useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0276] Activatable ROR binding agents (e.g., activatable antibodies), including activatable human ROR binding agents, described herein can also be linked or conjugated (directly or indirectly) to a second antibody to form an antibody heteroconjugate.

[0277] The linker may be a “cleavable moiety” facilitating release of the linked or conjugated agent in a cell, but non-cleavable linkers are also contemplated herein. Linkers for use in conjugates (e.g., ADCs) of the present disclosure include, without limitation, acid labile linkers (e.g., hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers comprising amino acids, for example, valine and / or citrulline such as citrulline-valine or phenylalanine-lysine), photolabile linkers, dimethyl linkers, thioether linkers, or hydrophilic linkers designed to evade multidrug transporter-mediated resistance.

[0278] Conjugates of an antibody and agent, including wherein the agent is a drug for the preparation of ADC, may be made using a variety of bifunctional protein coupling agents.

[0279] The present disclosure further contemplates conjugates of antibodies and agents, including wherein the agent is a drug for the preparation of ADC, may be prepared using any suitable methods as disclosed in the art (see, e.g., Bioconjugate Techniques (Hermanson ed., 2d ed. 2008)).

[0280] Conventional conjugation strategies for antibodies and agents, including wherein the agent is a drug for the preparation of ADC, have been based on random conjugation chemistries involving the s-amino group of Lys residues or the thiol group of Cys residues, which results in heterogeneous conjugates. Recently developed techniques allow sitespecific conjugation to antibodies, resulting in homogeneous loading and avoiding conjugate subpopulations with altered antigen-binding or pharmacokinetics. These include engineering of “thiomabs” comprising cysteine substitutions at positions on the heavy and light chains that provide reactive thiol groups and do not disrupt immunoglobulin folding and assembly or alter antigen binding (see, e.g., Junutula et al.. 2008, J. Immunol. Meth. 332: 41-52; and Junutula et aL, 2008, Nature Biotechnol. 26:925-32). In another method, selenocysteine is co-translationally inserted into an antibody sequence by recoding the stop codon UGA from termination to selenocysteine insertion, allowing site specific covalent conjugation at the nucleophilic selenol group of selenocysteine in the presence of the other natural amino acids (see, e.g., Hofer et al., 2008, Proc. Natl. Acad. Sci. USA 105: 12451-56; and Hofer et al., 2009, Biochemistry 48(50): 12047-57).

[0281] In some embodiments, an activatable ROR binding agent (e.g, an activatable antibody), including an activatable human ROR binding agent, described herein is conjugated to an agent, for example, an immune activator or a cytotoxic agent. In some embodiments, an activatable ROR binding agent (e.g, an activatable antibody), including an activatable human ROR binding agent, disclosed herein can be optionally conjugated with one or more cytotoxic agent(s) disclosed herein or known in the art in order to generate an ADC. In some embodiments, the cytotoxic agent is a chemotherapeutic agent. In some embodiments, the cytotoxic agent is an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof. In some embodiments, the cytotoxic agent is a radioisotope to produce a radioconjugate or a radioconjugated agent. Conjugates of a polypeptide or molecule and one or more small molecule toxins. Conjugates of a polypeptide or molecule and cytotoxic agent are made using a variety of bifunctional protein-coupling agents.

[0282] In other embodiments, an activatable ROR binding agent (e.g., an activatable antibody), including an activatable human ROR binding agent, described herein is conjugated to a drug such as a signal transduction modulator, a pro-apoptotic agent, a mitotic inhibitor, an anti-tumor antibiotic, an immunomodulating agent, a nucleic acid for gene therapy, an alkylating agent, an anti -angiogenic agent, an anti-metabolite, a boron-containing agent, a chemoprotective agent, a hormone agent, an anti-hormone agent, a corticosteroid, a photoactive therapeutic agent, an oligonucleotide, a radionuclide agent, a radiosensitizer, a topoisomerase inhibitor, such as camptothecin or an analog thereof, and a tyrosine kinase inhibitor.

[0283] Activatable ROR binding agents (e.g., activatable antibodies), including activatable human ROR binding agents, described herein may be monospecific, bispecific, trispecific or of greater multispecificity. Such agents may include monospecific or multispecific activatable antibodies. Multispecific antibodies, such as bispecific antibodies, are monoclonal antibodies that have binding specificities for at least two different targets (e.g., antigens) or two different epitopes on the same target (e.g., a bispecific antibody directed to ROR with a first binding domain for a first epitope of an ROR, and a second binding domain for a second epitope of ROR). In some embodiments, the monospecific and multispecific (e.g., bispecific) antibodies can be constructed based on the sequences of the antibodies described herein, e.g., the CDR sequences listed in Table 1. In some embodiments, the multispecific antibodies described herein are bispecific antibodies. In some embodiments, bispecific antibodies are mouse, chimeric, human or humanized antibodies.

[0284] In some embodiments, one of the binding specificities of the multispecific antibody is for ROR and the other is for any other target (e.g., antigen). In some embodiments, a multispecific (e.g., bispecific) antibody can comprise more than one target (e.g., antigen) binding domain, in which different binding domains are specific for different targets (e.g. , a first binding domain that binds to ROR and a second binding domain that binds another target (e.g., antigen).

[0285] In some embodiments, multispecific (e.g., bispecific) antibody molecules can bind than one (e.g., two or more) epitopes on the same target (e.g., antigen).

[0286] Methods for making multispecific antibodies are known in the art, such as, by coexpression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (see, e.g., Milstein and Cuello, 1983, Nature 305:537-40). Forfurther details of generating multispecific antibodies (e.g., bispecific antibodies), see, for example, Bispecific Antibodies (Kontermann ed., 2011).

[0287] Exemplary structures of multispecific antibodies are known in the art and are further described in Weidle et al., 2013, Cancer Genomics & Proteomics 10: 1-18; Brinkman et al., 2017, MABS, 9:2, 182-212; Godar etal., 2018, Expert Opinion on Therapeutic Patents, 28:3, 251-276; and Spiess et al., 2015, Mol. Immunol. 67 95-106.

[0288] For example, bispecific antibody molecules can be classified into different structural groups: (i) bispecific immunoglobulin G (BsIgG); (ii) IgG appended with an additional antigen-binding moiety; (iii) bispecific antibody fragments; (iv) bispecific fusion proteins; and (v) bispecific antibody conjugates. As a non-limiting example, BsIgG formats can include crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-in- holes common LC, knobs-in-holes assembly, charge pair, Fab-arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, rA-body, and / or orthogonal Fab.

[0289] In some embodiments, BsIgG comprises heavy chains that are engineered for heterodimerization. For example, heavy chains can be engineered for heterodimerization using a “knobs-into-holes” strategy, a SEED platform, a common heavy chain (e.g., in rA- bodies), and use of heterodimeric Fc regions. Strategies are known in the art to avoid heavy chain pairing of homodimers in BsIgG, including knobs-into-holes, duobody, azymetric, charge pair, HA-TF, SEEDbody, and differential protein A affinity.

[0290] Another bispecific antibody format is IgG appended with an additional antigenbinding moiety. For example, monospecific IgG can be engineered to have bispecificity by appending an additional antigen-binding unit onto the monospecific IgG, e.g., at the N- or C- terminus of either the heavy or light chain. Exemplary additional antigen-binding units include single domain antibodies (e.g., variable heavy chain or variable light chain), engineered protein scaffolds, and paired antibody variable domains (e.g., single chain variable fragments or variable fragments). Non-limiting examples of appended IgG formats include dual variable domain IgG (DVD-Ig), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv- (L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv- IgG, IgG-2scFv, scFv4-Ig, zybody, and DVI-IgG (four- in-one). See Spiess et al. Mol. Immunol. 67(2015):95-106. In some embodiments, an exemplary antibody format is a B- Body format for monospecific or multispecific (e.g., bispecific antibodies) as described in e.g. WO 2018 / 075692 and U.S. Pat. Publ. No. 2018 / 0118811.

[0291] Bispecific (Bs) antibody (BsAb) fragments are a format of bispecific antibody molecules that lack some or all of the antibody constant domains. For example, some BsAblack an Fc region. In embodiments, bispecific antibody fragments include heavy and light chain regions that are connected by a peptide linker that permits efficient expression of the BsAb in a single host cell. Non-limiting examples of bispecific antibody fragments include, but are not limited to, nanobody, nanobody- HAS, BiTE, Diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab’)2, F(ab’)2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HC Ab, scDiabody-Fc, Diabody-Fc, tandem scFv-Fc, and intrabody.

[0292] Bispecific fusion proteins include antibody fragments linked to other proteins. For example, bispecific fusion proteins can be linked to other proteins to add additional specificity and / or functionality. In some embodiments, the dock-and-lock (DNL) method can be used to generate bispecific antibody molecules with higher valency. For example, bispecific antibody fusions to albumin binding proteins or human serum albumin can be extend the serum half-life of antibody fragments. In embodiments, chemical conjugation, e.g., chemical conjugation of antibodies and / or antibody fragments, can be used to create BsAb molecules. An exemplary bispecific antibody conjugate includes the CovX-body format, in which a low molecular weight drug is conjugated site-specifically to a single reactive lysine in each Fab arm or an antibody or fragment thereof. In some embodiments, the conjugation improves the serum half-life.

[0293] Methods of production of multispecific antibodies, including bispecific antibodies, are known in the art. For example, multispecific antibodies, including bispecific antibodies, can be produced by separate expression of the component antibodies in different host cells and subsequent purification / assembly or by expression of the component antibodies in a single host cell. Purification of multispecific (e.g., bispecific) antibody molecules can be performed by various methods known in the art, including affinity chromatography.

[0294] In some embodiments, activatable ROR binding agents (e.g., activatable antibodies), including activatable human ROR binding agents, disclosed herein can be provided in any antibody format disclosed herein or known in the art. As a non-limiting example, in some embodiments, the activatable ROR binding agents (e.g, activatable antibodies), including activatable human ROR binding agents, can be selected from Fabs-in- tandem-lg (FIT-lg); DVD-lg; hybrid hybridoma (quadroma or tetradoma); anticalin platform (Pieris); diabodies; single chain diabodies; tandem single chain Fv fragments; TandAbs, Trispecific Abs (Affimed); Darts dual affinity retargeting (Macrogenics); Bispecific Xmabs (Xencor); Bispecific T cell engagers (Bites; Amgen; 55kDa); Triplebodies; Tribody = Fab- scFv Fusion Protein multifunctional recombinant antibody derivates (CreativeBiolabs);Duobody platform (Genmab); dock and lock platform; knobs-into-holes (KIH) platform; Humanized bispecific IgG antibody (REGN1979) (Regeneron); Mab2 bispecific antibodies (F-Star); DVD-lg = dual variable domain immunoglobulin (Abbott); kappa-lambda bodies; TBTI = tetravalent bispecific tandem Ig; and CrossMab (Roche).

[0295] In some embodiments, a multispecific (e.g., bispecific) antibody disclosed herein comprises an ROR binding domain and one or more additional binding domains that bind to one or more targets that are not ROR. In some embodiments, a multispecific (e.g., bispecific) antibody disclosed herein comprises an ROR binding domain that comprises the VH and / or VL amino acid sequences as disclosed herein, such as those of Table 1.

[0296] In some embodiments, described herein is a multispecific (e.g., bispecific) antibody comprising a binding domain which binds to ROR that comprises VH and VL CDRs disclosed herein, such as those as set forth in Table 1.

[0297] In some embodiments, the activatable ROR binding agent is a bispecific antibody comprising a first binding domain and a second binding domain. In some embodiments, the first binding domain comprises six CDRs as listed in one column of Table 1. In some embodiments, the first binding domain comprises three CDRs of the heavy chain variable region as set forth in SEQ ID NO: 25 and three CDRs of the light chain variable regions as set forth in SEQ ID NO: 26. In some embodiments, the first binding domain comprises the heavy chain variable region as set forth in SEQ ID NO: 25 and the light chain variable regions as set forth in SEQ ID NO: 26.

[0298] In another aspect, the activatable antibody or antigen-binding fragment thereof provided herein can be part of an engineered cell surface receptor such as a chimeric antigen receptor (CAR). Typically, a CAR comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain.

[0299] In some embodiments, provided herein is a CAR comprising an extracellular domain that comprises one or more activatable antibody or fragment thereof provided herein. In some embodiments, the extracellular domain of a CAR provided herein comprises VH and VL CDRs disclosed herein, such as those as set forth in Table 1.

[0300] The CARs of the present disclosure comprise a transmembrane domain that can be directly or indirectly fused to the extracellular antigen-binding domain. The transmembrane domain may be derived either from a natural or from a synthetic source. As used herein, a “transmembrane domain” refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. Transmembrane domains compatible for use in the CARs described herein may be obtained from a naturally occurring protein.Alternatively, it can be a synthetic, non-naturally occurring protein segment, e.g., a hydrophobic protein segment that is thermodynamically stable in a cell membrane. Transmembrane domains are classified based on the three dimensional structure of the transmembrane domain. For example, transmembrane domains may form an alpha helix, a complex of more than one alpha helix, a beta-barrel, or any other stable structure capable of spanning the phospholipid bilayer of a cell.

[0301] The CARs of the present disclosure comprise an intracellular signaling domain. The intracellular signaling domain is responsible for activation of at least one of the normal effector functions of the immune effector cell expressing the CARs. The term “effector function” refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus the term “cytoplasmic signaling domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire cytoplasmic signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the cytoplasmic signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term cytoplasmic signaling domain is thus meant to include any truncated portion of the cytoplasmic signaling domain sufficient to transduce the effector function signal.

[0302] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, the CAR comprises an intracellular signaling domain consisting essentially of a primary intracellular signaling domain of an immune effector cell. “Primary intracellular signaling domain” refers to cytoplasmic signaling sequence that acts in a stimulatory manner to induce immune effector functions.

[0303] Many immune effector cells require co-stimulation, in addition to stimulation of an antigen-specific signal, to promote cell proliferation, differentiation and survival, as well as to activate effector functions of the cell. In some embodiments, the CAR comprises at least one co-stimulatory signaling domain. The term “co-stimulatory signaling domain,” as used herein, refers to at least a portion of a protein that mediates signal transduction within a cell to induce an immune response such as an effector function.

[0304] The CARs of the present disclosure may comprise a hinge domain that is located between the extracellular antigen-binding domain and the transmembrane domain. A hinge domain is an amino acid segment that is generally found between two domains of a proteinand may allow for flexibility of the protein and movement of one or both of the domains relative to one another. Any amino acid sequence that provides such flexibility and movement of the extracellular antigen-binding domain relative to the transmembrane domain of the effector molecule can be used.

[0305] The CARs of the present disclosure may comprise a signal peptide (also known as a signal sequence) at the N-terminus of the polypeptide. In general, signal peptides are peptide sequences that target a polypeptide to the desired site in a cell.

[0306] Other engineered transmembrane receptors comprising the antibody or fragment provided herein are also included in the present disclosure.5.4. Nucleic Acids, Vectors and Cells

[0307] Additionally provided are a nucleic acid encoding an activatable ROR binding agent (e.g., activatable antibody or antibody fragment) or a fusion polypeptide as disclosed herein, a nucleic acid complementary thereto; a vector comprising a nucleic acid as disclosed herein; and a cell comprising any one or more of: an activatable ROR binding agent as disclosed herein, a nucleic acid as disclosed herein, or a vector as disclosed herein. In some embodiments, the cell expresses the activatable ROR binding agent. In some embodiments, the cell replicates the nucleic acid or the vector. In some embodiments, provided are the materials for generating activatable ROR binding agents, e.g., activatable human ROR binding agents, and fragments thereof. For example, an isolated cell may produce an activatable ROR binding agent (e.g., activatable antibody or antibody fragment). In this regard, a cell (e.g., an isolated cell) may produce an activatable antibody or fragment thereof comprising a VH and a VL as disclosed herein. In some embodiments, polynucleotides described herein may comprise one or more nucleic acid sequences encoding an activatable ROR binding agent (e.g., activatable antibody or antibody fragment). In some embodiments, the polynucleotide is an isolated and / or recombinant polynucleotide. In various aspects, the isolated polynucleotide comprises a nucleotide sequence that encodes a VH and / or a VL, wherein the VH and the VL comprise complementarity determining regions (CDRs) identical to CDRs as disclosed herein.

[0308] As used herein, the term “complementary” refers to specific binding between polynucleotides based on the sequences of the polynucleotides. As used herein, a first polynucleotide and a second polynucleotide are complementary if they bind to each other in a hybridization assay under stringent conditions, e.g., if they produce a given or detectable level of signal in a hybridization assay. Portions of polynucleotides are complementary toeach other if they follow conventional base-pairing rules, e.g., A pairs with T (or U) and G pairs with C, although small regions (e.g., fewer than about 3 bases) of mismatch, insertion, or deleted sequence may be present. The term “stringent assay conditions” refers to conditions that are compatible to produce binding pairs of nucleic acids, e.g., probes and target mRNAs, of sufficient complementarity to provide for the desired level of specificity in the assay while being generally incompatible to the formation of binding pairs between binding members of insufficient complementarity to provide for the desired specificity. The term “stringent assay conditions” generally refers to the combination of hybridization and wash conditions.

[0309] In some embodiments, one or more vectors (e.g., expression vectors) may comprise one or more polynucleotides for expression of the one or more polynucleotides in a suitable host cell. Such vectors are useful, for example, for amplifying the polynucleotides in host cells to create useful quantities thereof, and for expressing activatable binding agents, such as activatable antibodies or antibody fragments, using recombinant techniques.

[0310] In some embodiments, one or more vectors are expression vectors wherein one or more polynucleotides are operatively linked to one or more polynucleotides comprising expression control sequences. Autonomously replicating recombinant expression constructs such as plasmid and viral DNA vectors incorporating one or more polynucleotides encoding activatable antibody sequences that bind ROR are specifically contemplated. Expression control DNA sequences include promoters, enhancers, and operators, and are generally selected based on the expression systems in which the expression construct is to be utilized. Promoter and enhancer sequences are generally selected for the ability to increase gene expression, while operator sequences are generally selected for the ability to regulate gene expression. Expression constructs may also include sequences encoding one or more selectable markers that permit identification of host cells bearing the construct. Expression constructs may also include sequences that facilitate, and preferably promote, homologous recombination in a host cell. In some embodiments, expression constructs of the can also include sequences necessary for replication in a host cell.

[0311] Exemplary expression control sequences include promoter / enhancer sequences, e.g., cytomegalovirus promoter / enhancer (Lehner et al., J. Clin. Microbiol., 29: 2494-2502, 1991; Boshart et al., Cell, 41 : 521-530, 1985); Rous sarcoma virus promoter (Davis et al., Hum. Gene Ther., 4: 151, 1993); Tie promoter (Korhonen et al., Blood, 86(5): 1828-1835, 1995); simian virus 40 promoter; DRA (downregulated in adenoma; Alrefai et al., Am. J. Physiol. Gastrointest. Liver Physiol., 293: G923-G934, 2007); MCT1 (monocarboxylatetransporter 1; Cuff el al.. Am. J. Physiol. Gastrointet. Liver Physiol., G977-G979. 2005); and Mathl (mouse atonal homolog 1; Shroyer et aL, Gastroenterology, 132: 2477-2478, 2007), for expression in mammalian cells, the promoter being operatively linked upstream (e.g., 5’) of a polypeptide coding sequence. In another variation, the promoter is an epithelial-specific promoter or endothelial-specific promoter. Polynucleotides may also optionally include a suitable polyadenylation sequence (e.g., the SV40 or human growth hormone gene polyadenylation sequence) operably linked downstream (e.g., 3’) of the polypeptide coding sequence.

[0312] If desired, the one or more polynucleotides also optionally comprise nucleotide sequences encoding secretory signal peptides fused in frame with the polypeptide sequences. The secretory signal peptides direct secretion of the antibody polypeptides by the cells that express the one or more polynucleotides, and are cleaved by the cell from the secreted polypeptides. The one or more polynucleotides may further optionally comprise sequences whose only intended function is to facilitate large scale production of the vector. One can manufacture and administer polynucleotides for gene therapy using procedures that have been described in the literature for a variety of transgenes. See, e.g., Isner et al., Circulation, 91 : 2687-2692, 1995; and Isner et al. , Human Gene Therapy, 7: 989-1011, 1996.

[0313] In some embodiments, polynucleotides may further comprise additional sequences to facilitate uptake by host cells and expression of the antibody or fragment thereof (and / or any other peptide). In some embodiments, a “naked” transgene encoding an antibody or fragment thereof described herein (e.g., a transgene without a viral, liposomal, or other vector to facilitate transfection) is employed.

[0314] The polynucleotides of the disclosure can be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand. In some embodiments, the polynucleotide is in the form of cDNA. In some embodiments, the polynucleotide is a synthetic polynucleotide.

[0315] The present disclosure further relates to variants of the polynucleotides described herein, wherein the variant encodes, for example, fragments, analogs, and / or derivatives of the binding molecules of the disclosure. In certain embodiments, the present disclosure provides a polynucleotide comprising a polynucleotide having a nucleotide sequence at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and in some embodiments, at least about 96%, 97%, 98% or 99% identical to a polynucleotide encoding the binding molecule of thedisclosure. As used herein, the phrase “a polynucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence can include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence. These mutations of the reference sequence can occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

[0316] The polynucleotide variants can contain alterations in the coding regions, noncoding regions, or both. In some embodiments, a polynucleotide variant contains alterations which produce silent substitutions, additions, or deletions, but does not alter the properties or activities of the encoded polypeptide. In some embodiments, a polynucleotide variant comprises silent substitutions that results in no change to the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code). Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host (z.e., change codons in the human mRNA to those preferred by a bacterial host such as E. colt). In some embodiments, a polynucleotide variant comprises at least one silent mutation in a non-coding or a coding region of the sequence.

[0317] In some embodiments, a polynucleotide variant is produced to modulate or alter expression (or expression levels) of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to increase expression of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to decrease expression of the encoded polypeptide. In some embodiments, a polynucleotide variant has increased expression of the encoded polypeptide as compared to a parental polynucleotide sequence. In some embodiments, a polynucleotide variant has decreased expression of the encoded polypeptide as compared to a parental polynucleotide sequence.

[0318] Any suitable vectors may be used to introduce one or more polynucleotides that encode an antibody or fragment thereof into the host. Exemplary vectors that have been described include replication deficient retroviral vectors, including but not limited tolentivirus vectors (Kim et aL, J. Virol., 72(1): 811-816, 1998; Kingsman & Johnson, Scrip Magazine, October, 1998, pp. 43-46); parvoviral vectors, such as adeno-associated viral (AAV) vectors (U.S. Pat. Nos. 5,474,9351; 5,139,941; 5,622,856; 5,658,776; 5,773,289; 5,789,390; 5,834,441; 5,863,541; 5,851,521; 5,252,479; Gnatenko etal., J. Invest. Med., 45: 87-98, 1997); adenoviral (AV) vectors (U.S. Pat. Nos. 5,792,453; 5,824,544; 5,707,618; 5,693,509; 5,670,488; 5,585,362; Quantin et al., Proc. Natl. Acad. Sci. USA, 89: 2581-2584, 1992; Stratford Perricaudet et al., J. Clin. Invest., 90: 626-630, 1992; and Rosenfeld et al., Cell, 68: 143-155, 1992); an adenoviral adeno-associated viral chimeric (U.S. Pat. No. 5,856,152) or a vaccinia viral or a herpesviral vector (U.S. Pat. Nos. 5,879,934; 5,849,571; 5,830,727; 5,661,033; 5,328,688); Lipofectin mediated gene transfer (BRL); liposomal vectors (U.S. Pat. No. 5,631,237); and combinations thereof. Any of these expression vectors can be prepared using standard recombinant DNA techniques described in, e.g., Sambrook et al. , Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y. (1994). Optionally, viral vectors are rendered replication-deficient by, e.g., deleting or disrupting select genes required for viral replication.

[0319] Other non-viral delivery mechanisms contemplated include calcium phosphate precipitation (Graham and Van Der Eb, Virology, 52: 456-467, 1973; Chen and Okayama, Mol. Cell Biol., 7: 2745-2752, 1987; Rippe et al, Mol. Cell Biol., 10: 689-695, 1990) DEAE- dextran (Gopal, Mol. Cell Biol., 5: 1188-1190, 1985), electroporation (Tur-Kaspa et al., Mol. Cell Biol., 6: 716-718, 1986; Potter et al., Proc. Nat. Acad. Sci. USA, 81 : 7161-7165, 1984), direct microinjection (Harland and Weintraub, J. Cell Biol., 101 : 1094-1099, 1985), DNA- loaded liposomes (Nicolau and Sene, Biochim. Biophys. Acta, 721 : 185-190, 1982; Fraley et al., Proc. Natl. Acad. Sci. USA, 76: 3348-3352, 1979; Feigner, Sci Am., 276(6): 102-6, 1997; Feigner, Hum Gene Ther., 7(15): 1791-3, 1996), cell sonication (Fechheimer et al., Proc. Natl. Acad. Sci. USA, 84: 8463-8467, 1987), gene bombardment using high velocity microprojectiles (Yang et al., Proc. Natl. Acad. Sci USA, 87: 9568-9572, 1990), and receptor- mediated transfection (Wu and Wu, J. Biol. Chem., 262: 4429-4432, 1987; Wu and Wu, Biochemistry, 27: 887-892, 1988; Wu and Wu, Adv. Drug Delivery Rev., 12: 159-167, 1993).

[0320] A vector (or the antibody or fragment thereof or a nucleic acid as discussed herein) may be entrapped in a liposome. See, e.g., Ghosh and Bachhawat, In: Liver diseases, targeted diagnosis and therapy using specific receptors and ligands, Wu G, Wu C ed., New York: Marcel Dekker, pp. 87-104 (1991); Radler et al., Science, 275(5301): 810-814, (1997).Also contemplated are various commercial approaches involving “lipofection” technology. In some embodiments, the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al., Science, 243: 375-378, 1989). In some embodiments, the liposome is complexed or employed in conjunction with nuclear nonhistone chromosomal proteins (HMG-1) (Kato et al., J. Biol. Chem., 266: 3361-3364, 1991). In some embodiments, the liposome is complexed or employed in conjunction with both HVJ and HMG-1. Such expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo. In some embodiments, an activatable ROR binding agent (e.g., an activatable antibody), including an activatable human ROR binding agent, is included in the liposome to target the liposome to cells (such as a tumor cell) expressing ROR on their surface.

[0321] A cell may comprise one or more polynucleotides or one or more vectors, e.g., the cell is transformed or transfected with one or more polynucleotides encoding an activatable ROR binding agent (e.g., an activatable antibody), including an activatable human ROR binding agent, or the one or more vectors comprising the one or more polynucleotides. In some embodiments, cells express an activatable ROR binding agent (e.g., an activatable antibody), including an activatable human ROR binding agent, containing one or more, including six CDRs having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the CDRs of the antibody depicted in Table 1. In some embodiments, the cell expresses an activatable ROR binding agent (e.g., an activatable antibody), including an activatable human ROR binding agent, containing the VH and the VL comprising CDRs identical to those of the antibody depicted in Table 1. The cells may be prokaryotic cells, such as Escherichia coli (see, e.g., Pluckthun et al., Methods Enzymol., 178: 497-515, 1989), or eukaryotic cells, such as an animal cell (e.g., a myeloma cell, Chinese Hamster Ovary (CHO) cell, or hybridoma cell), yeast (e.g., Saccharomyces cerevisiae), an insect cell, or a plant cell (e.g., a tobacco, corn, soybean, or rice cell). Use of mammalian host cells may provide for translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) that may be desirable to confer optimal biological activity on recombinant expression products. Similarly, polypeptides (e.g., activatable ROR binding agents (e.g., activatable antibodies), including activatable human ROR binding agents) may be glycosylated or non-glycosylated and / or have been covalently modified to include one or more water soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.

[0322] Methods for introducing DNA or RNA into host cells are well known and include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts. Such host cells are useful for amplifying polynucleotides and also for expressing polypeptides encoded by the polynucleotides. In this regard, a process for the production of an activatable ROR binding agent (e.g., an activatable antibody) may comprise culturing a host cell and isolating the activatable ROR binding agent. Transferring a naked DNA expression construct into cells can be accomplished using particle bombardment, which depends on the ability to accelerate DNA coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al.. Nature, 327: 70-73, 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al., Proc. Natl. Acad. Sci USA, 87: 9568-9572, 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads. A host cell may be isolated and / or purified. A host cell also may be a cell transformed in vivo to cause transient or permanent expression of the polypeptide in vivo. A host cell may also be an isolated cell transformed ex vivo and introduced post-transformation, e.g., to produce the polypeptide in vivo for therapeutic purposes. The definition of host cell explicitly excludes a transgenic human being.5.5. Methods of Making

[0323] Antibodies that bind ROR may be obtained by any suitable method, such as (but not limited to) immunization with whole cells comprising ROR and collection of antibodies, recombinant techniques, or screening libraries of antibodies or antibody fragments using ROR extracellular domain epitopes. Monoclonal antibodies may be generated using a variety of known techniques (see, for example, Coligan et al. (eds.), Current Protocols in Immunology, 1 :2.5.12.6.7 (John Wiley & Sons 1991); Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKeam, and Bechtol (eds.) (1980); Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press (1988); and Picksley et al., “Production of monoclonal antibodies against proteins expressed in E. colip in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford University Press 1995)). One exemplary technique for generating monoclonal antibodies comprises immunizing an animal with a human ROR antigen and generating a hybridoma from spleen cells taken from the animal. A hybridoma may produce a monoclonal antibody or antibody fragment that binds ROR.

[0324] In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in, for example, Antibody Phage Display: Methods and Protocols, P.M. O’Brien and R. Aitken, eds, Humana Press, Totawa N.J., 2002. In principle, synthetic antibody clones are selected by screening phage libraries containing phage that display various fragments of antibody variable region (Fv) fused to phage coat protein. Such phage libraries are screened for against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from the non-binding clones in the library. The binding clones are then eluted from the antigen, and can be further enriched by additional cycles of antigen adsorption / elution.

[0325] Variable domains can be displayed functionally on phage, either as single-chain Fv (scFv) fragments, in which VH and VL are covalently linked through a short, flexible peptide, or as Fab fragments, in which they are each fused to a constant domain and interact non-covalently, as described, for example, in Winter et al., Ann. Rev. Immunol, 12: 433-455 (1994).

[0326] Repertoires of VH and VL genes can be separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be searched for antigen-binding clones as described in Winter et al., supra. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned to provide a single source of human antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning the unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro as described, for example, by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

[0327] Screening of the libraries can be accomplished by various techniques known in the art. For example, ROR (e.g., an ROR polypeptide, fragment or epitope) can be used to coat the wells of adsorption plates, expressed on host cells affixed to adsorption plates or used in cell sorting, or conjugated to biotin for capture with streptavidin-coated beads, or used in any other method for panning display libraries. The selection of antibodies with slow dissociation kinetics (e.g., good binding affinities) can be promoted by use of long washes and monovalent phage display as described in Bass et al., Proteins, 8: 309-314 (1990) and in WO92 / 09690, and a low coating density of antigen as described in Marks et aL, BiotechnoL, 10: 779-783 (1992).

[0328] ROR binding agents (e.g., antibodies) can be obtained by designing a suitable antigen screening procedure to select for the phage clone of interest followed by construction of a full length activatable ROR binding agent (e.g., an activatable antibody) clone using VH and / or VL sequences (e.g., the Fv sequences), or various CDR sequences from VH and VL sequences, from the phage clone of interest and suitable constant region (e.g., Fc) sequences described in Kabat et al.. Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.

[0329] Likewise, human antibodies that bind ROR may be generated by any of a number of techniques including, but not limited to, Epstein Barr Virus (EBV) transformation of human peripheral blood cells (e.g., containing B lymphocytes), in vitro immunization of human B cells, fusion of spleen cells from immunized transgenic mice carrying inserted human immunoglobulin genes, isolation from human immunoglobulin V region phage libraries, or other procedures as known in the art and based on the disclosure herein.Methods for obtaining human antibodies from transgenic animals are further described, for example, in Bruggemann et al., Curr. Opin. BiotechnoL, 8: 455 58, 1997; Jakobovits etal., Ann. N. Y. Acad. Sci., 764: 525 35, 1995; Green et al., Nature Genet., 7: 13-21, 1994;Lonberg et al., Nature, 368: 856-859, 1994; Taylor et al., Int. Immun. 6: 579-591, 1994; and U.S. Pat. No. 5,877,397.

[0330] For example, human antibodies that bind ROR may be obtained from transgenic animals that have been engineered to produce specific human antibodies in response to antigenic challenge. For example, WO 98 / 24893 discloses transgenic animals having a human Ig locus, wherein the animals do not produce functional endogenous immunoglobulins due to the inactivation of endogenous heavy and light chain loci. Transgenic non-primate mammalian hosts capable of mounting an immune response to an immunogen, wherein the antibodies have primate constant and / or variable regions, and wherein the endogenous immunoglobulin encoding loci are substituted or inactivated, also have been described. WO 96 / 30498 discloses the use of the Cre / Lox system to modify the immunoglobulin locus in a mammal, such as to replace all or a portion of the constant or variable region to form a modified antibody molecule. WO 94 / 02602 discloses non-human mammalian hosts having inactivated endogenous Ig loci and functional human Ig loci. U.S. Pat. No. 5,939,598 discloses methods of making transgenic mice in which the mice lack endogenous heavy chains and express an exogenous immunoglobulin locus comprising one or more xenogeneicconstant regions. Using a transgenic animal, such as a transgenic animal described herein, an immune response can be produced to a selected antigenic molecule, and antibody producing cells can be removed from the animal and used to produce hybridomas that secrete human- derived monoclonal antibodies. Immunization protocols, adjuvants, and the like are known in the art, and are used in immunization of, for example, a transgenic mouse as described in WO 96 / 33735. The monoclonal antibodies can be tested for the ability to inhibit or neutralize the biological activity or physiological effect of the corresponding protein.

[0331] In some embodiments, an activatable ROR binding agent described herein comprises a non-antibody protein scaffold. Non-limiting examples of such a non-antibody protein scaffold include a fibronectin scaffold, an anticalin, an adnectin, an affibody, a DARPin, a fynomer, an affitin, an affilin, an avimer, a cysteine-rich knottin peptide, or an engineered Kunitz-type inhibitor. Methods for generating such non-antibody protein scaffolds are well known in the art, any one of which can be used to generate an activatable ROR binding agent comprising a non-antibody protein scaffold (see, e.g., Simeon and Chen, Protein Cell, 9(1):3-14 (2018); Yang et al. , Annu Rev Anal Chem (Palo Alto Calif). 10(l):293-320 (2017)).

[0332] A variety of methods for producing antibodies from polynucleotides are generally well-known. For example, basic molecular biology procedures are described by Maniatis et al. , Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York, 1989 (see also Maniatis et al., 3rd ed., Cold Spring Harbor Laboratory, New York, 2001). Additionally, numerous publications describe techniques suitable for the preparation of antibodies by manipulation of DNA, creation of expression vectors, and transformation and culture of appropriate cells (see, e.g., Mountain and Adair, Chapter 1 in Biotechnology and Genetic Engineering Reviews, Tombs ed., Intercept, Andover, UK, 1992); and Current Protocols in Molecular Biology, Ausubel ed., Wiley Interscience, New York, 1999).

[0333] An activatable ROR binding agent (e.g., an activatable antibody), including an activatable human ROR binding agent, is produced using any suitable method, e.g., isolated from an immunized animal, recombinantly or synthetically generated, or genetically- engineered, including as described above. Antibody fragments derived from an antibody are obtained by, e.g., proteolytic hydrolysis of an antibody. For example, papain or pepsin digestion of whole antibodies yields a 5S fragment termed F(ab’)2 or two monovalent Fab fragments and an Fc fragment, respectively. F(ab)2 can be further cleaved using a thiol reducing agent to produce 3.5S Fab monovalent fragments. Methods of generating antibody fragments are further described in, for example, Edelman et al., Methods in Enzymology, 1 :422 Academic Press (1967); Nisonoff et aL, Arch. Biochem. Biophys., 89: 230-244, 1960; Porter, Biochem. J., 73: 119-127, 1959; U.S. Pat. No. 4,331,647; and by Andrews, S.M. and Titus, J. A. in Current Protocols in Immunology (Coligan et al., eds), John Wiley & Sons, New York (2003), pages 2.8.1 2.8.10 and 2.10A.1 2.10A.5.

[0334] An activatable ROR binding agent (e.g., an activatable antibody), including an activatable human ROR binding agent, can be genetically engineered. For example, an activatable ROR binding agent (e.g., an activatable antibody), including an activatable human ROR binding agent, comprises, for example, a variable region domain generated by recombinant DNA engineering techniques. In this regard, a variable region is optionally modified by insertions, deletions, or changes in the amino acid sequence of the antibody to produce an antibody of interest, including as described above. Polynucleotides encoding CDRs of interest are prepared, for example, by using polymerase chain reaction to synthesize variable regions using mRNA of antibody producing cells as a template (see, for example, Courtenay Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge University Press 1995); Ward et al., “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles and Applications, Birch et al, (eds.), page 137 (Wiley Liss, Inc. 1995); and Larrick et al. , Methods: A Companion to Methods in Enzymology, 2: 106-110, 1991). Current antibody manipulation techniques allow construction of engineered variable region domains containing at least one CDR and, optionally, one or more framework amino acids from a first antibody and the remainder of the variable region domain from a second antibody. Such techniques are used, e.g., to humanize an antibody or to improve its affinity for a binding target. Exemplary methods for generating humanized antibodies are described in sections above too.

[0335] In some embodiments, the methods of making an activatable ROR binding agent (e.g., an activatable antibody) described herein comprises culturing a cell described herein and expressing the activatable ROR binding agent (e.g., activatable antibody).

[0336] In some embodiments, the methods of making an activatable ROR binding agent (e.g., an activatable antibody) described herein further comprises testing the activatable ROR binding agent (e.g., activatable antibody) for the ability to maintain an activatable phenotype e,g., the ability to maintain an activatable phenotype while soluble or the ability to maintain an activatable phenotype when expressed in mammalian cells.

[0337] In some embodiments, the methods of making an activatable ROR binding agent (e.g., an activatable antibody) described herein comprises one, two, three, or more steps asdescribed in the Examples below. In some embodiments, the methods of making an activatable ROR binding agent (e.g., an activatable antibody) described herein are as described in the Examples below.5.6. Pharmaceutical Compositions

[0338] In one aspect, the present disclosure further provides a composition, such as a pharmaceutical composition, comprising at least one of the following: an activatable binding agent provided herein (e.g., one activatable antibody or antigen-binding fragment thereof of the present disclosure), a nucleic acid provided herein, a vector provided herein, or a cell provided herein. In some embodiments, a pharmaceutical composition comprises a therapeutically effective amount of an activatable ROR binding agent provided herein (e.g., an activatable antibody or antigen-binding fragment thereof provided herein) and a pharmaceutically acceptable excipient. In some embodiments, a pharmaceutical composition comprises a therapeutically effective amount of a nucleic acid provided herein (such as a nucleic acid encoding an antibody or antigen-binding fragment thereof provided herein) and a pharmaceutically acceptable excipient. In some embodiments, a pharmaceutical composition comprises a therapeutically effective amount of a vector provided herein (such as a vector comprising a nucleic acid as disclosed herein and expressing an activatable ROR binding agent as disclosed herein) and a pharmaceutically acceptable excipient. In some embodiments, a pharmaceutical composition comprises a therapeutically effective amount of a cell provided herein (such as a cell comprising a nucleic acid encoding an activatable antibody or antigen-binding fragment thereof provided herein and / or expressing an activatable antibody or antigen-binding fragment thereof provided herein) and a pharmaceutically acceptable excipient.

[0339] In some embodiments, pharmaceutical compositions provided herein are prepared for storage by mixing the binding agents, nucleic acids, vectors, or cells provided herein having the desired degree of purity with optional physiologically acceptable excipients (see, e.g., Remington, Remington’s Pharmaceutical Sciences (18th ed. 1980)) in the form of aqueous solutions or lyophilized or other dried forms.

[0340] The binding agents, nucleic acids, vectors, or cells of the present disclosure may be formulated in any suitable form for delivery to a target cell / tissue, e.g., as microcapsules or macroemulsions (Remington, supra, Park et al., 2005, Molecules 10: 146-61; Malik et al., 2007, Curr. Drug. Deliv. 4: 141-51), as sustained release formulations (Putney and Burke,1998, Nature Biotechnol. 16: 153-57), or in liposomes (Maclean et aL, 1997, Int. J. Oncol. 11 :325-32; Kontermann, 2006, Curr. Opin. Mol. Ther. 8:39-45).

[0341] The binding agents, nucleic acids, vectors, or cells provided herein can also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions. Such techniques are disclosed, for example, in Remington, supra.

[0342] Various compositions and delivery systems are known and can be used with the binding agents, nucleic acids, vectors or cells as described herein, including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or antigen-binding fragment thereof, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-32), construction of a nucleic acid as part of a retroviral or other vector, etc. In another embodiment, a composition can be provided as a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release (see, e.g., Langer, supra, Sefton, 1987, Crit. Ref. Biomed. Eng. 14:201-40; Buchwald et al., 1980, Surgery 88:507-16; and Saudek et al., 1989, N. Engl. J. Med. 321 :569-74). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of a prophylactic or therapeutic agent (e.g., an antibody or antigen-binding fragment thereof as described herein) or a composition provided herein (see, e.g., Medical Applications of Controlled Release (Langer and Wise eds., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., 1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61-126; Levy et al., 1985, Science 228: 190-92; During et al., 1989, Ann. Neurol. 25:351-56; Howard et al., 1989, J. Neurosurg. 71 : 105-12; U.S. Pat. Nos. 5,679,377; 5,916,597; 5,912,015;5,989,463; and 5,128,326; WO 99 / 15154 and WO 99 / 20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, polyethylene glycol), polylactides (PLA), poly(lactide- co-glycolides) (PLGA), and poly orthoesters. In one embodiment, the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable.

[0343] In yet another embodiment, a controlled or sustained release system can be placed in proximity of a particular target tissue, for example, the nasal passages or lungs, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release Vol. 2, 115-38 (1984)). Controlled release systems are discussed, for example, by Langer, 1990, Science 249: 1527-33. Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more activatable antibody or antigen-binding fragment thereof as described herein (see, e.g., U.S. Pat. No. 4,526,938, WO 91 / 05548 and WO 96 / 20698, Ning et al., 1996, Radiotherapy & Oncology 39: 179-89; Song et al., 1995, PDA J. of Pharma. Sci. & Tech. 50:372-97; Cleek el al., 1997, Pro. IntT. Symp. Control. Rel. Bioact. Mater. 24:853-54; and Lam et al., 1997, Proc. IntT. Symp. Control Rel. Bioact. Mater. 24:759-60).5.7. Methods of Uses

[0344] In another aspect, methods of treating a disease or disorder are provided, the methods comprising administering an activatable ROR binding agent (e.g., activatable antibody) as described herein to a subject to treat the disease or disorder. In preferred embodiments, the disease or disorder is associated with ROR expression (e.g., overexpression of ROR). In specific embodiments, such activatable ROR antigen binding agents are useful in the treatment of ROR expressing cancers, including cancers that express an ROR1 antigen, cancers that express an ROR2 antigen, and / or cancers that express both an ROR1 antigen and an ROR2 antigen.

[0345] In some embodiments, an activatable antibody of the present disclosure may be used to treat a variety of cancers. The cancer may be a cancer from the bladder, blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), bone, bone marrow, brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), breast, colon, esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), gastrointestine, gum, head, kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia, renal cell carcinoma), liver, lung, nasopharynx, neck, ovary, prostate (adenocarcinoma, sarcoma, castrate resistant prostate cancer), skin, stomach (carcinoma, lymphoma, leiomyosarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma), tongue, or uterus. In someembodiments, the cancer may be a neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma); fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone;ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma (reticulum cell sarcoma); hodgkin's disease; hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; hairy cell leukemia; myxoma; rhabdomyoma; fibroma; squamous cell carcinomas of the head and neck; laryngeal and hypopharyngeal cancer; nasal cavity and paranasal sinus cancer; nasopharyngeal cancer; salivary gland cancer; oral; orppharyngeal cancer; bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma, non-small cell lung cancer); alveolar (bronchiolar) carcinoma; bronchial adenoma; chondromatous hamartoma; colorectal cancer; gastrointestinal stromal tumors; carcinoids; Turcot Syndrome; gastric cancer; gastroesophageal junction adenocarcinoma; pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma); small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma); large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); metastatic breast cancer; ductal carcinoma in situ; invasive ductal carcinoma; tubular carcinoma; mucinous carcinoma; lobular carcinoma in situ; triple negative breast cancer; bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma, urothelial carcinoma); clear cell carcinoma; hepatoma (hepatocellular carcinoma); angiosarcoma; hepatocellular adenoma; hemangioma; osteogenic sarcoma (osteosarcoma); malignant fibrous histiocytoma; malignant giant cell tumor chordoma; osteochrondroma (osteocartilaginous exostoses); benign chondroma; chondromyxofibroma; osteoid osteoma; giant cell tumors; medullary thyroid cancer; differentiated thyroid cancer; papillary thyroid cancer; follicular thyroid cancer; hurthle cell cancer; anaplastic thyroid cancer; skull (osteoma, hemangioma, granuloma,xanthoma, osteitis deformans); meninges (meningioma, meningiosarcoma, gliomatosis); spinal cord (neurofibroma, meningioma, glioma, sarcoma); uterus (clear); cervix (cervical carcinoma, pre-tumor cervical dysplasia); ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell tumors, Sertoli— Leydig cell tumors, dysgerminoma, malignant teratoma); vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma); vagina (clear cell carcinoma, squamous cell carcinoma); botryoid sarcoma (embryonal rhabdomyosarcoma); fallopian tubes (carcinoma); non-Hodgkin's lymphoma [malignant lymphoma]; Karposi's sarcoma; moles dysplastic nevi; angioma; dermatofibroma; keloids; psoriasis; neuroblastoma.; adrenocortical carcinoma; pheochromocytomas; paragangliomas; merkel cell carcinoma; pancreatic neuroendocrine and carcinoid tumors; neuroendocrine tumors; carcinoid tumors; pancreatic cancers; gastroesophageal; clear cell renal cell carcinoma; and primary peritoneal cancer.

[0346] An activatable antibody of the present disclosure may be administered to a subject per se or in the form of a pharmaceutical composition for the treatment of, e.g., cancer.

[0347] In another aspect, an activatable ROR binding agent (e.g., activatable antibody) as described herein may be used in a method of treating a subject with cancer in combination with one or more additional therapies. The additional therapies that may be used in combination with an activatable ROR antigen binding agent (e.g., activatable antibody) described herein include but are not limited to: (i) surgery; (ii) radiotherapy; (iii) endocrine therapy; (iv) immunotherapy (including adjuvant therapy and cell therapy such as CAR T-cell therapy); and (v) chemotherapy, including cytotoxic agents and chemotherapeutic agents.

[0348] Any therapy that has an activity against a cancer may be used in combination with an activatable ROR antigen binding molecule (e.g., activatable antibody) provided herein. Examples of such agents for cancer treatment can be found, for instance, at www.cancer.gov / about-cancer / treatment / drugs and in publicly available sources such as Cancer Principles and Practice of Oncology by V. T. Devita and S. Hellman (editors), 11thedition (2018), Lippincott Williams & Wilkins Publishers. A person of ordinary skill in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the type of cancer involved.

[0349] In certain embodiments, the additional therapy is a radiotherapy including, for example, gamma radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, and systemic radioactive isotopes. Radiotherapy may comprise radiation or associated administration of radiopharmaceuticals. The source of radiation maybe either external or internal to the subject being treated (radiation treatment may, for example, be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Exemplary radioactive elements include, e.g., radium, cesium- 137, iridium- 192, americium- 241, gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131, and indium-i l l.

[0350] In certain embodiments, the additional therapy is an immunotherapy. Immunotherapy (also called biological response modifier therapy, biologic therapy, biotherapy, immune therapy, or biological therapy) is treatment that uses parts of the immune system to fight disease. Immunotherapy can help the immune system recognize cancer cells, or enhance a response against cancer cells. Immunotherapies include active and passive immunotherapies. Active immunotherapies, including immunotherapeutic agents, stimulate the body's own immune system (e.g., vaccines) while passive immunotherapies, including immunotherapeutic agents, generally use immune system components created outside of the body (e.g., antibodies), antibodies conjugated with drugs, toxins, or radionuclides, and targeted therapeutics.

[0351] Exemplary immunotherapeutic agents include immune checkpoint inhibitors. In some embodiments, the immune checkpoint inhibitor used in methods of treatment can totally or partially reduce, inhibit, interfere with, or modulate one or more checkpoint proteins which regulate T-cell activation or function. Numerous checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86; and PD-1 with its ligands PD-L1 and PD-L2 (Pardoll, Nature Reviews Cancer, 2012, 12, 252-264). Immune checkpoint inhibitors include antibodies or are derived from antibodies.

[0352] In certain embodiments, the checkpoint inhibitor is an 0X40 (CD 134) agonist. In some embodiments, the checkpoint inhibitor is an anti-OX40 antibody. In some embodiments, the anti-OX40 antibody is anti-OX-40. In some embodiments, the anti-OX40 antibody is MEDI6469.

[0353] In certain embodiments, the checkpoint inhibitor is a CD40 agonist. In some embodiments, the checkpoint inhibitor is an anti-CD40 antibody. In some embodiments, the anti-CD40 antibody is CF-870,893.

[0354] In certain embodiments, the checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the CTLA-4 inhibitor is an anti-CTLA-4 antibody. Examples of anti CTLA 4 antibodies include, but are not limited to, those described in US Patent Nos: 5,811,097; 5,811,097; 5,855,887; 6,051,227; 6,207,157; 6,682,736; 6,984,720; and 7,605,238. In some embodiments, the anti-CTLA-4 antibody is tremelimumab (also known as ticilimumab or CP- 675,206). In some embodiments, the anti-CTLA-4 antibody is ipilimumab (also known asI l lMDX-010 or MDX-101). Ipilimumab is a fully human monoclonal IgG antibody that binds to CTLA-4. Ipilimumab is marketed under the trade name Yervoy™.

[0355] In certain embodiments, the checkpoint inhibitor is a PD-1 / PD-L1 inhibitor. Examples of PD-1 / PD-L1 inhibitors include, but are not limited to, those described in US Patent Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149, and PCT Patent Application Publication Nos. W02003042402, WO2008156712, W02010089411, W02010036959, WO2011066342, WO2011159877, WO2011082400, and WO2011161699.

[0356] In certain embodiments, the checkpoint inhibitor is a PD-1 inhibitor. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody. In some embodiments, the anti- PD-1 antibody is BGB-A317, nivolumab (also known as ONO-4538, BMS-936558, or MDX1 106) or pembrolizumab (also known as MK-3475, SCH 900475, or lambrolizumab). In some embodiments, the anti-PD-1 antibody is ni...

Claims

WHAT IS CLAIMED IS:

1. An activatable antibody or fragment thereof that binds to ROR, wherein the activatable antibody or fragment thereof comprises: a masking peptide, an antibody light chain variable (VL) region, and an antibody heavy chain variable (VH) region; wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:29, 30 or 31, and wherein the VH region comprise a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:25, and the VL region comprises a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:26; and optionally wherein the activatable antibody or fragment thereof comprises a polypeptide comprising, from N-terminus to C-terminus, the masking peptide and the antibody VL region.

2. The activatable antibody or fragment thereof of claim 1, wherein the VH region comprises:(1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 12, 13, 18, and 27;(2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 19, and 24; and(3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, 20, and 28; and the VL region comprises:(1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21;(2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22; and(3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 17, and 23.

3. The activatable antibody or fragment thereof of claim 1, wherein:(i) the VH region comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a VHCDR3 comprising the amino acid sequence of SEQ ID NO:3; and the VL region comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO:4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6;(ii) the VH region comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO:7, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:8, and a VH CDR3 comprising the amino acid sequence of SEQ ID NOV; and the VL region comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6;(iii) the VH region comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:3; and the VL region comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NON, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6;(iv) the VH region comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and the VL region comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 17;(v) the VH region comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:20; and the VL region comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO:21, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:22, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:23;(vi) the VH region comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:24, and a VH CDR3 comprising the amino acid sequence of SEQ ID NON; and the VL region comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NON, a VL CDR2 comprising the amino acid sequence of SEQ ID NON, and a VL CDR3 comprising the amino acid sequence of SEQ ID NON; or(vii) the VH region comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO:27, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:24, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:28; and the VL region comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO:4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 17.

4. The activatable antibody or fragment thereof of any one of claims 1-3, wherein the activatable antibody or fragment thereof further comprises a framework 1 (FR1), a framework 2 (FR2), a framework 3 (FR3) and / or a framework 4 (FR4) sequence.

5. The activatable antibody or fragment thereof of any one of claims 1-4, wherein the activatable antibody or fragment thereof further comprises human framework sequences, optionally a framework 1 (FR1), a framework 2 (FR2), a framework 3 (FR3) and / or a framework 4 (FR4) sequence as set forth in SEQ ID NO: 25 or 26.

6. The activatable antibody or fragment thereof of any one of claims 1-5, wherein the VH comprises the amino acid sequence of SEQ ID NO:25 and the VL comprises the amino acid sequence of SEQ ID NO:26.

7. The activatable antibody or fragment thereof of any one of claims 1-6, wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:29.

8. The activatable antibody or fragment thereof of claim 7, wherein the activatable antibody or fragment thereof comprises the polypeptide, and the polypeptide comprises the amino acid sequence of SEQ ID NO:34.

9. The activatable antibody or fragment thereof of any one of claims 1-6, wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:30.

10. The activatable antibody or fragment thereof of claim 9, wherein the activatable antibody or fragment thereof comprises the polypeptide, and wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:36.

11. The activatable antibody or fragment thereof of any one of claims 1-6, wherein the masking peptide comprises the amino acid sequence of SEQ ID NO:31.

12. The activatable antibody or fragment thereof of claim 11, wherein the activatable antibody or fragment thereof comprises the polypeptide, and wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:38.

13. The activatable antibody or fragment thereof of any one of claims 1-12, wherein the activatable antibody or fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:32.

14. The activatable antibody or fragment thereof of any one of claims 1-13, wherein the activatable antibody is a monoclonal antibody.

15. The activatable antibody or fragment thereof of any one of claims 1-14, wherein the activatable antibody is a humanized, human or chimeric antibody.

16. The activatable antibody or fragment thereof of any one of claims 1-15, which is a Fab, Fab’, F(ab’)2, Fv, scFv, (scFv)2, single chain antibody molecule, dual variable region antibody, single variable region antibody, linear antibody, V region, or a multispecific antibody formed from antibody fragments.

17. The activatable antibody or fragment thereof of any one of claims 1-16, which is conjugated or recombinantly fused to a diagnostic agent, detectable agent or therapeutic agent.

18. The activatable antibody or fragment thereof of claim 17, wherein the therapeutic agent is a chemotherapeutic agent, cytotoxin, or drug.

19. The activatable antibody or fragment thereof of any one of claims 1-18, wherein the activatable antibody is a multispecific antibody.

20. The activatable antibody or fragment of claim 19, wherein the multispecific antibody is a bispecific antibody.

21. An activatable binding agent that binds to essentially the same epitope as an activatable antibody or fragment thereof of any one of claims 1-20.

22. The activatable binding agent of claim 21, which is an activatable antibody or fragment thereof.

23. The activatable binding agent of claim 22, wherein the activatable antibody is a multispecific antibody.

24. An activatable binding agent that competes for binding to human ROR with an activatable antibody or fragment thereof of any one of claims 1-20.

25. The activatable binding agent of claim 24, wherein the activatable binding agent is an activatable antibody or fragment thereof.

26. The activatable binding agent of claim 25, wherein the activatable antibody is a multispecific antibody.

27. A polynucleotide encoding the activatable antibody or fragment thereof of any one of claims 1-20 or the activatable binding agent of any one of claims 22-23 and 25-26.

28. One or more vectors comprising one or more polynucleotides of claim 27 or a complementary polynucleotide.

29. A cell comprising any one or more of the activatable antibody or fragment thereof of any one of claims 1-20, the activatable binding agent of any one of claims 21-26, the polynucleotide of claim 27, or the one or more vectors of claim 28.

30. A pharmaceutical composition that comprises a pharmaceutically acceptable excipient and any one or more of: the activatable antibody or fragment thereof of any one of claims 1-20, the activatable binding agent of any one of claims 21-26, the polynucleotide of claim 27, the one or more vectors of claim 28, or the cell of claim 29.

31. A method for treating a disease or disorder in a subject comprising administering to the subject the activatable antibody or fragment thereof of any one of claims1-20 or the activatable binding agent of any one of claims 21-26, or the pharmaceutical composition of claim 30.

32. The method of claim 31, wherein the disease or disorder is an ROR-expressing cancer.

33. The method of claim 31 or 32, wherein the subject is a human subject.

34. A method of making an activatable antibody or fragment thereof that binds to ROR, comprising culturing the cell of claim 29 and expressing the activatable antibody or fragment thereof.

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