Degradation of surface proteins using bispecific binding agents

Abstract

To provide methods for degrading target surface proteins in the ubiquitin pathway using bispecific binding agents or immunoconjugates that bind to the target surface proteins and membrane-bound ubiquitin E3 ligase.SOLUTION: The present invention relates, inter alia, to methods of degrading target surface proteins in the ubiquitin pathway using bispecific binding agents or immunoconjugates that bind to target surface proteins and membrane-bound ubiquitin E3 ligase. The present invention also relates to methods of degrading target surface proteins in the ubiquitin pathway using engineered membrane spanning proteins that bind to target surface proteins and exhibit ubiquitin E3 ligase activity. The present disclosure also provides compositions and methods useful for making bispecific binding agents and engineered transmembrane proteins, immunoconjugates, nucleic acids encoding them, host cells genetically modified with said nucleic acids, and methods of modulating the activity of cells and / or treating various diseases, such as cancer.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 929,674, filed November 1, 2019, the entire contents of which are incorporated herein by reference.

[0002] Reference to a "Sequence Listing," a computer program listing submitted as an ASCII file in a table or appendix The 213,455 byte sequence listing (machine format: IBM-PC, MS Windows operating system) published on October 30, 2020 at 048536-670001WO_SequenceListing_ST25 is incorporated herein by reference.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under grants P41 CA196276 and R35 GM122451 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0004] The present disclosure generally relates to novel methods and agents that utilize the ubiquitin pathway to degrade surface proteins on cell surfaces. The disclosure also provides methods for producing the agents, nucleic acids encoding the agents, host cells genetically modified with the nucleic acids, methods for modulating cellular activity, and / or methods for treating various diseases, such as cancer. [Background technology]

[0005] Malignant tumor cells often express surface proteins that have proliferative or immunosuppressive effects. For example, some tumors overexpress growth factor receptors (HER2 or HER3) that stimulate proliferation. Overexpression of immune checkpoint proteins (e.g., PD-L1 and CTLA-4) suppresses the innate immune response, allowing malignant cells to proliferate and evade the host's immune system, resulting in tumor formation.

[0006] Current therapies address this problem using antibodies and antibody derivatives that specifically bind to these surface proteins and inhibit their activity during binding. However, there is a need for more effective (e.g., more durable) treatments and / or treatments that target cancer or tumor cells by enhancing the cancer-fighting response and / or countering surface proteins that have proliferative or immunosuppressive effects. The disclosure provided herein addresses these problems and provides additional solutions.

[0007] All references and patents cited herein are incorporated by reference in their entirety as if fully set forth herein. Summary of the Invention

[0008] This disclosure describes novel therapeutic methods and agents that utilize the ubiquitin pathway to promote the removal and degradation of target surface proteins. Described herein are bispecific binding agents that bind to both a target surface protein and a membrane-bound ubiquitin E3 ligase, whereby binding of the bispecific binding agent ubiquitinates the target surface protein, followed by degradation. Also described herein are E3 ligase derivatives that contain a target surface protein-binding domain, whereby the target surface protein is ubiquitinated when the E3 ligase derivative is present in the plasma membrane of a target cell.

[0009] One aspect of the present disclosure is a bispecific binding agent comprising a first binding domain that specifically binds to an E3 ligase and a second binding domain that specifically binds to an extracellular epitope on a target protein of a target cell, wherein both the E3 ligase and the target protein are membrane-bound. One embodiment is a bispecific binding agent that ubiquitinates the target protein upon binding to both the E3 ligase and the target protein. One embodiment is a bispecific binding agent wherein the target cell is a tumor cell. One embodiment is a bispecific binding agent wherein the cell is a cancer cell selected from the group consisting of breast cancer, B-cell lymphoma, pancreatic cancer, Hodgkin's lymphoma, ovarian cancer, prostate cancer, mesothelioma, lung cancer, non-Hodgkin's B-cell (B-NHL), melanoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, neuroblastoma, glioma, glioblastoma, bladder cancer, and colorectal cancer. In one embodiment, the bispecific binding agent is one in which the target protein is an immune checkpoint protein. In one embodiment, the target protein is selected from the group consisting of PD-L1, PD-1, CTLA-4, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, NKG2D, TIM-3, VISTA, and SIGLEC7. In some embodiments, the first binding domain of the bispecific binding agent specifically binds to an extracellular protein bound by an E3 ligase. In some embodiments, the first binding domain of the bispecific binding agent specifically binds to a transmembrane protein that interacts with an E3 ligase. In certain embodiments of the bispecific binding agent, degradation of the target protein reduces the proliferative capacity of the target cell. Some proteins, for example, A2aR, can modulate the immune system, i.e., promote CD8 immune responses and proliferation. In certain embodiments of the bispecific binding agents, the target protein is selected from the group consisting of HER2, CD19, CD20, PD-L1, EGFR, CTLA-4, MMP14, and CDCP1.

[0010] In other embodiments, the E3 ligase of the bispecific binding agent comprises a transmembrane protein. For example, in some embodiments, the E3 ligase comprises a transmembrane E3 ligase. In exemplary embodiments, the E3 ligase is selected from the group consisting of RNF43, RNF128 (GRAIL), ZNRF3, and MARCH11. One embodiment is a bispecific binding agent wherein the first binding domain and the second binding domain are each independently selected from the group consisting of half antibodies, single-domain antibodies, nanobodies, monospecific Fab2s, scFvs, scFv-Fcs, minibodies, IgNARs, V-NARs, hcIgGs, VhHs, camelid antibodies, and peptibodies, or wherein the first binding domain and the second binding domain together form a bispecific antibody, bispecific diabody, bispecific Fab2, bispecific camelid antibody, or bispecific peptibody. One embodiment is a bispecific binding agent comprising a bispecific antibody. One embodiment is a bispecific binding agent comprising a bispecific IgG. One embodiment is a bispecific binding agent comprising a knob-and-hole bispecific IgG. One embodiment is a bispecific binding agent wherein the first binding domain comprises a Fab and the second binding domain comprises a single-chain Fab. One embodiment is a bispecific binding agent wherein the first binding domain comprises a Fab and the second binding domain comprises an scFv. In some embodiments, the first binding domain comprises a heavy chain framework region (FR) sequence set forth in SEQ ID NO: 12 or 320 and a light chain FR sequence set forth in SEQ ID NO: 11 or 319. In some embodiments, the second binding domain comprises a heavy chain FR sequence set forth in SEQ ID NO: 12 or 320 and a light chain FR sequence set forth in SEQ ID NO: 11 or 319.

[0011] In some embodiments, the first binding domain comprises a light chain variable region CDR3 (LC-CDR3) sequence, a heavy chain variable region CDR1 (HC-CDR1) sequence, a HC-CDR2 sequence, and a HC-CDR3 sequence, each comprising a sequence set forth in Table 2. In some embodiments, the second binding domain comprises a LC-CDR3 sequence, a HC-CDR1 sequence, a HC-CDR2 sequence, and a HC-CDR3 sequence, each set forth in Table 3.

[0012] In some embodiments, the first binding domain of the bispecific binding agent comprises a heavy chain variable region (VH), wherein the VH comprises the FR sequence set forth in SEQ ID NO: 321, and the second binding domain of the bispecific binding agent comprises the heavy chain FR sequence set forth in SEQ ID NO: 12 or 320 and the light chain FR sequence set forth in SEQ ID NO: 11 or 319.

[0013] In some embodiments, the first binding domain comprises the VH-CDR1, VH-CDR2, and VH-CDR3 sequences, respectively, shown in Table 4, and the second binding domain comprises the LC-CDR3, HC-CDR1, HC-CDR2, and HC-CDR3 sequences, respectively, that comprise the sequences shown in Table 3.

[0014] One aspect of the present disclosure is a nucleic acid encoding any one of the bispecific binding agents, comprising a first binding domain that specifically binds to an E3 ligase and a second binding domain that specifically binds to an extracellular epitope on a target protein in a target cell, wherein both the E3 ligase and the target protein are membrane-bound. One embodiment is a nucleic acid further comprising a vector. One embodiment is a nucleic acid further comprising a promoter operably linked to a sequence encoding the bispecific binding agent.

[0015] One aspect of the present disclosure is a vector comprising a nucleic acid encoding any one of the bispecific binding agents described above or herein. In one embodiment, the vector further comprises a promoter operably linked to the sequence encoding the bispecific binding agent.

[0016] One aspect of the present disclosure is an immunoconjugate. In some embodiments, the immunoconjugate comprises a bispecific binding agent disclosed herein and a small molecule. In other embodiments, the immunoconjugate further comprises a linker. In certain embodiments, the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, and a dicarboxylic acid-based linker. In some embodiments, the immunoconjugate comprises DBCO-PEG4-CGS21680, where DBCO is used for linking, PEG4 is the linker, and CGS21680 is the small molecule. In some embodiments, the immunoconjugate comprises DBCO-PEG4-amine. In some embodiments, the small molecule comprises an amine, CGS21680, oxaziridine-azide, ZM241385, plerixafor, maraviroc, and aplaviroc. However, it will be apparent that the small molecule can be any suitable small molecule as deemed suitable by one of skill in the art.

[0017] One aspect of the present disclosure is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a bispecific binding agent, an immunoconjugate, or a nucleic acid described herein. In one embodiment, the bispecific binding agent binds to both the E3 ligase and the target protein, resulting in ubiquitination of the target protein. In one embodiment, the target cell is a tumor cell. In one embodiment, the cell is a cancer cell selected from the group consisting of breast cancer, B-cell lymphoma, pancreatic cancer, Hodgkin's lymphoma, ovarian cancer, prostate cancer, mesothelioma, lung cancer, non-Hodgkin's B-cell (B-NHL), melanoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, neuroblastoma, glioma, glioblastoma, bladder cancer, and colorectal cancer. In one embodiment, the target protein is an immune checkpoint protein. In one embodiment, the target protein is selected from the group consisting of PD-L1, PD-1, CTLA-4, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, NKG2D, TIM-3, VISTA, and SIGLEC7. In one embodiment, the target protein is degraded to reduce the proliferative capacity of target cells. In one embodiment, the target protein is selected from the group consisting of HER2, CD19, CD20, PD-L1, EGFR, CTLA-4, MMP14, and CDCP1.

[0018] One aspect of the present disclosure is an engineered cell comprising a cell capable of expressing a protein and a nucleic acid encoding a bispecific binding agent. In one embodiment, the cell is an engineered cell that is a B cell, a B memory cell, or a plasma cell.

[0019] One aspect of the present disclosure is a method of treating a neoplastic disease or disorder in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a bispecific binding agent of this disclosure, a nucleic acid of this disclosure, a pharmaceutical composition of this disclosure, or an engineered cell of this disclosure.

[0020] One aspect of the present disclosure is a method of making a bispecific binding agent of the present disclosure by providing a cell capable of synthesizing a protein comprising a nucleic acid encoding a bispecific binding agent of the present disclosure and inducing expression of said bispecific binding agent.

[0021] One aspect of the present disclosure is an engineered transmembrane protein for treating a neoplastic disease in which a target protein is present on the surface of a tumor cell or immune cell, the protein comprising a membrane-bound E3 ligase bound to a target protein binding domain specific for the target protein. One embodiment is an engineered transmembrane protein in which the E3 ligase and the target protein binding domain are covalently linked. One embodiment is an engineered transmembrane protein in which the E3 ligase and the target protein binding domain are expressed as a fusion protein. One embodiment is an engineered transmembrane protein in which the E3 ligase and the target protein binding domain are covalently linked by a disulfide bond. One embodiment is an engineered transmembrane protein in which the target protein binding domain is specific for a target protein selected from the group consisting of HER2, EGFR, MMP14, CDCP1, PD-L1, PD-1, CTLA-4, CD19, CD20, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, NKG2D, TIM-3, VISTA, and SIGLEC7.

[0022] One aspect of the present disclosure is a nucleic acid encoding an engineered transmembrane protein of the present disclosure. One embodiment is a nucleic acid further comprising a vector. One embodiment is a nucleic acid further comprising a promoter operably linked to the sequence encoding the engineered transmembrane protein.

[0023] One aspect of the present disclosure is a composition for treating a neoplastic disease in which a target protein is present on the surface of a tumor cell, the composition comprising a therapeutic amount of an engineered transmembrane protein of the present disclosure and a fusogenic carrier, wherein the carrier is capable of fusing with the cell membrane of the tumor cell. In one embodiment, the carrier is a fusogenic liposome.

[0024] One aspect of the present disclosure is a composition for treating a neoplastic disease in which a target protein is present on the surface of a tumor cell, the composition comprising a therapeutic amount of a nucleic acid encoding an engineered transmembrane protein of the present disclosure and a pharmaceutically acceptable carrier, wherein the carrier is capable of delivering the nucleic acid to the cytosol of the tumor cell. In one embodiment, the carrier comprises a viral particle, a liposome, or an exosome. In one embodiment, the carrier comprises a viral particle. In one embodiment, the carrier comprises a liposome. In one embodiment, the carrier comprises an exosome.

[0025] One aspect of the present disclosure is the use of a bispecific binding agent of this disclosure; a nucleic acid encoding a bispecific binding agent of this disclosure; an engineered transmembrane protein of this disclosure; a nucleic acid encoding an engineered transmembrane protein of this disclosure; a pharmaceutical composition comprising a bispecific binding agent of this disclosure, a nucleic acid encoding a bispecific binding agent of this disclosure, an immunoconjugate of this disclosure, an engineered transmembrane protein of this disclosure, or a nucleic acid encoding an engineered transmembrane protein of this disclosure; a vector encoding a bispecific binding agent of this disclosure or an engineered transmembrane protein of this disclosure; or an engineered cell comprising a nucleic acid encoding a bispecific binding agent of this disclosure, for the treatment of a neoplastic disease.

[0026] One aspect of the present disclosure is the use of a bispecific binding agent of this disclosure; a nucleic acid encoding a bispecific binding agent of this disclosure; an immunoconjugate of this disclosure; an engineered transmembrane protein of this disclosure; a nucleic acid encoding an engineered transmembrane protein of this disclosure; a pharmaceutical composition comprising a bispecific binding agent of this disclosure, a nucleic acid encoding a bispecific binding agent of this disclosure, an engineered transmembrane protein of this disclosure, or a nucleic acid encoding an engineered transmembrane protein of this disclosure; a vector encoding a bispecific binding agent of this disclosure or an engineered transmembrane protein of this disclosure; or an engineered cell comprising a nucleic acid encoding a bispecific binding agent of this disclosure, for the manufacture of a medicament for the treatment of a neoplastic disease.

[0027] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the exemplary embodiments and features described herein, further aspects, embodiments, objects, and features of the present disclosure will become more fully apparent from the drawings, detailed description, and claims. [Brief explanation of the drawings]

[0028] [Figure 1] FIG. 1 is a schematic diagram showing a bispecific binding agent of the present disclosure (here, an exemplary bispecific antibody) bound to the membrane-bound E3 ligase RNF43 and a target surface protein of interest ("POI"), and the intracellular ubiquitination of the POI.

[0029] [Figure 2] Figure 1 shows a schematic diagram of an engineered transmembrane protein with a green fluorescent protein (GFP)-binding domain and a membrane-associated E3 ligase domain, and a membrane-associated reporter construct with an intracellular NanoLuc domain and an extracellular GFP domain. Binding of the engineered transmembrane protein to the GFP domain promotes intracellular ubiquitination of the NanoLuc domain, leading to its degradation and loss of signal.

[0030] [Figure 3] FIG. 1 is a schematic diagram of a bispecific IgG antibody of the present disclosure having a "knob-into-hole" configuration.

[0031] [Figure 4A] Figure 4A shows the results of an experiment using a bispecific IgG of the present disclosure to remove and degrade PD-L1 from a triple-negative breast cancer cell line (MDA-MB-231). Figure 4A shows that PD-L1 levels are unaffected by an anti-RNF43 antibody (R3 IgG) or an anti-PD-L1 antibody (Tecentriq®), but are substantially reduced or eliminated by the use of 10 nM of a bispecific anti-RNF43 / PD-L1 IgG of the present disclosure for 24 hours. [Figure 4B]Figure 4B shows the dose response using the same cells and bispecific IgG, showing that 10 nM bispecific IgG induced the greatest degradation of PD-L1.

[0032] [Figure 5A] Figures 5A, 5B, and 5C compare the PD-L1 degradation activity of a bispecific IgG of the present disclosure with Tecentriq® (atezolizumab) in three different cancer cell lines. The agents were administered at 10 nM for 24 hours. Figure 5A shows that the bispecific IgG substantially degraded PD-L1 in MDA-MB-231 cells (a triple-negative breast cancer model), while atezolizumab did not promote degradation or downregulation of PD-L1 expression. [Figure 5B] Figure 5B shows that bispecific IgG substantially degraded PD-L1 in HCC827 cells (a non-small cell lung cancer model), whereas atezolizumab did not degrade or downregulate PD-L1 expression. [Figure 5C] Figure 5C shows that bispecific IgG substantially degraded PD-L1 in T24 cells (a model of advanced bladder cancer), whereas atezolizumab did not substantially degrade or downregulate PD-L1 expression.

[0033] [Figure 6] Figure 6 is a bar graph showing the efficacy of bispecific IgG of the present disclosure in degrading PD-L1 from a triple-negative breast cancer cell line (MDA-MB-231).

[0034] [Figure 7] FIG. 7 shows a summary of biolayer interferometry (BLI) graphs for each Ala mutant.

[0035] [Figure 8] FIG. 8 shows the correlation between percent degradation and Koff.

[0036] [Figure 9] FIG. 9 shows the correlation between percent degradation and Kd.

[0037] [Figure 10] FIG. 10 shows the correlation between percent degradation and Kon.

[0038] [Figure 11] Figure 11 shows a Western blot of anti-RNF43 alanine mutants. Mutants are distinguished by their Kd for RNF43: 12.5 nM for wild type, 40 nM for S113A, and 125 nM for F115A.

[0039] [Figure 12] FIG. 12 is a schematic diagram of a membrane-bound E3 ligase and an immune complex bound to a protein of interest (POI).

[0040] [Figure 13] FIG. 13 is an exemplary schematic diagram of the conjugation procedure to generate the immunoconjugate.

[0041] [Figure 14] FIG. 14 shows some exemplary small molecules used in this disclosure.

[0042] [Figure 15] FIG. 15 shows the dose-dependent degradation of adenosine 2a receptors (A2aR) in MOLT-4 CCR5+ cells after 24 hours of treatment with immune complex-dissolving drugs.

[0043] [Figure 16] FIG. 16 shows the amount of A2aR in MOLT-4 CCR5+ cells after 24 hours of treatment with CGS21680 (agonist). DETAILED DESCRIPTION OF THE INVENTION

[0044] Detailed Description of the Invention The present disclosure generally relates to binding agents, including bispecific binding agents, engineered transmembrane proteins, and immunoconjugates thereof. These bind to both membrane-bound ubiquitin E3 ligase and target surface proteins present on the surface of target cells. In some embodiments, the present disclosure provides bispecific binding agents that bind to both membrane-bound ubiquitin E3 ligase and target surface proteins present on the surface of target cells. In other embodiments, the present disclosure provides engineered transmembrane proteins with target surface protein binding domains based on modified membrane-bound E3 ligase.

[0045] In some embodiments, the present disclosure provides exemplary methods for generating specific types of constructs, such as bispecific IgG, bispecific IgG with a single-chain Fab in one arm, and Fab-scFV fusions. The present disclosure provides methods for testing bispecific IgG, bispecific IgG with a single-chain Fab in one arm, and Fab-scFV fusions. In some embodiments, the present disclosure demonstrates that bispecific binding agents of the present disclosure can degrade targets in a variety of clinically relevant cell lines.

[0046] In some embodiments, the present disclosure provides the synthesis and testing of engineered transmembrane proteins in the degradation of target proteins.In certain embodiments, the present disclosure demonstrates that the engineered transmembrane proteins provided herein can cause the internalization and lysosomal aggregation of target proteins.Thus, the present disclosure demonstrates that the engineered transmembrane proteins provided herein can be used to induce the proteolysis of endogenous proteins.In some embodiments, the present disclosure further provides a method for generating an AAV transfection vector for inserting the engineered transmembrane protein into target cells.

[0047] In some embodiments, the present disclosure demonstrates that the strong binding affinity of the provided binding agents to their targets is an advantage. Also provided herein are immunoconjugates comprising the bispecific binding agents of the present disclosure and engineered transmembrane proteins. In some embodiments, the present disclosure demonstrates that immunoconjugates comprising the binding agents of the present disclosure can be recruited to targets and induce their degradation.

[0048] The present disclosure also provides nucleic acids encoding the bispecific binding agents or engineered transmembrane proteins. It also provides therapeutic compositions comprising the bispecific binding agents, the engineered transmembrane proteins, and / or nucleic acids encoding the bispecific binding agents or engineered transmembrane proteins, as well as cells encoding the nucleic acids. The present disclosure also provides therapeutic methods using the bispecific binding agents or engineered transmembrane proteins, therapeutic methods using immunoconjugates, therapeutic methods using nucleic acids encoding the bispecific binding agents or engineered transmembrane proteins, or therapeutic compositions using the bispecific binding agents, engineered transmembrane proteins, immunoconjugates, and / or nucleic acids encoding the bispecific binding agents or engineered transmembrane proteins. The present disclosure also provides compositions and methods useful for producing such agents. It also provides compositions and methods useful for producing nucleic acids encoding the agents, host cells genetically modified with the nucleic acids, and methods for modulating cellular activity and / or treating various diseases, such as cancer.

[0049] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In these drawings, like symbols generally refer to like elements unless dictated by context. The exemplary alternatives set forth in the detailed description, drawings, and claims are not meant to be limiting. Other alternatives may be employed, and other changes may be made without departing from the spirit or scope of the subject matter presented herein. Obviously, these aspects, generally as described herein and illustrated in the figures, can be arranged, substituted, combined, and designed in a variety of different configurations, all of which are expressly contemplated and made a part of this application.

[0050] definition The singular forms "a," "an," and "the" include plurals unless dictated by context. For example, the term "a cell" includes one or more cells, including mixtures thereof. As used herein, "A and / or B" includes all the alternatives of "A," "B," "A or B," and "A and B."

[0051] The terms "administration" and "administering" are used interchangeably herein and refer to the delivery of a composition or formulation by a route of administration, including, but not limited to, intravenous, intraarterial, intracerebral, intrathecal, intramuscular, intraperitoneal, subcutaneous, intramuscular, and combinations thereof. This term includes, but is not limited to, administration by a medical professional and self-administration.

[0052] The terms "host cell" and "recombinant cell" are used interchangeably herein. It is understood that these terms, as well as "cell culture" and "cell line," refer not only to the particular subject cell or cell line, but also to the progeny or potential progeny of such a cell or cell line, regardless of the number of introductions. It is understood that not all progeny will be identical to the parent cell. This is because certain modifications may occur in successive generations due to mutations (e.g., deliberate or inadvertent) or environmental influences, and such progeny may not, in fact, be identical to the parent cell. However, such progeny are within the scope of the terms used herein, so long as they retain the same functionality as the original cell or cell line.

[0053] As used herein, the term "operably linked" refers to a physical or functional connection between two or more elements (e.g., polypeptide sequences or polynucleotide sequences) so that they function in their intended manner.

[0054] The term "heterologous" refers to a nucleic acid sequence or amino acid sequence that is operably linked or otherwise linked to a nucleic acid construct or chimeric polypeptide that is not operably linked or that is not naturally contiguous to each other.

[0055] The term "percent identity," as used herein in the context of two or more nucleic acids or proteins, refers to two or more sequences or subsequences that have the same nucleotides or amino acids, or a specified percentage of the same nucleotides or amino acids (e.g., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or a higher percentage identity for a particular region when aligned to maximize correspondence over a comparison window or designated region), as determined using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters, as described below, or by manual alignment and visual inspection. See, e.g., the NCBI website at ncbi.nlm.nih.gov / BLAST. This definition may also refer to or apply to the complement of a test sequence. This definition also includes sequences that contain deletions, additions, or substitutions. Sequence identity is typically calculated over a region at least about 20 amino acids or nucleotides in length, a region 10 to 100 amino acids or nucleotides in length, or the entire length of a given sequence. Sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al., Nucleic Acids Res (1984) 12:387), BLASTP, BLASTN, and FASTA (Atschul et al., J Mol Biol (1990) 215:403). Sequence identity can be measured using sequence analysis software, such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, WI 53705), using its default parameters.

[0056] The term "treatment" when used in reference to a disease or condition means at least an improvement in the symptoms associated with the condition from which an individual suffers. Here, improvement is used broadly to refer to at least a decrease in the magnitude of a parameter, e.g., a symptom, associated with the condition being treated. Treatment also includes situations in which the pathology, or at least the symptoms associated therewith, are completely inhibited, e.g., prevented from occurring, or entirely eliminated so that the host is no longer afflicted by the condition, or at least the symptoms that characterize the condition. Thus, treatment includes (i) prevention (i.e., reducing the risk of clinical progression, including, e.g., preventing the progression of a clinical condition, e.g., preventing the progression of a disease), and (ii) inhibition (i.e., preventing the progression or further progression of a clinical condition, e.g., alleviating or completely inhibiting active disease).

[0057] Throughout this specification, unless otherwise specified, a "therapeutically effective amount" of a drug is an amount sufficient to provide a therapeutic benefit in the treatment or management of cancer or to delay or minimize one or more symptoms associated with cancer. A therapeutically effective amount of a compound means an amount of a therapeutic agent that, alone or in combination with other therapeutic agents, provides a therapeutic benefit in the treatment or management of cancer. The term "therapeutically effective amount" can include an amount that improves overall treatment, reduces or avoids the symptoms or causes of cancer, or enhances the therapeutic effectiveness of other therapeutic agents. An example of an "effective amount" is an amount sufficient to contribute to the treatment, prevention, or alleviation of one or more symptoms of a disease, which may also be referred to as a "therapeutically effective amount." "Alleviating" a symptom means reducing the severity or frequency of or eliminating the symptom(s). The precise amount of a composition that comprises a "therapeutically effective amount" will depend on the purpose of the treatment, and can be determined by those skilled in the art using well-known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 2010); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (2016); Pickar, Dosage Calculations (2012); and Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0058] As used herein, a "subject" or "individual" includes animals, e.g., humans (e.g., human individuals) and non-human animals. In some embodiments, a "subject" or "individual" may be a patient being treated by a physician. Thus, a subject may be a human patient or individual who is at risk of or suspected of having a disease of interest (e.g., cancer) and / or one or more symptoms of the disease. A subject may also be an individual who has been diagnosed as being at risk for a condition of interest, either at the time of diagnosis or thereafter. The term "non-human animal" includes all vertebrates, e.g., mammals such as rodents (e.g., mice), non-human primates, sheep, dogs, cows, chickens, and non-mammals such as amphibians and reptiles.

[0059] The terms "derivative," "functional fragment thereof," or "functional variant thereof" refer to a molecule that shares a common biological activity with the wild-type molecule from which the fragment or derivative is derived. A functional fragment or variant of an antibody retains the ability to bind to essentially the same epitope as the antibody from which the functional fragment or variant is derived. For example, an antibody capable of binding to an epitope of a cell surface receptor may be truncated at the N-terminus and / or C-terminus, and retention of epitope-binding activity may be assessed using assays well known to those skilled in the art. Antibody derivatives generally do not directly resemble existing antibodies, but may further include constructs based on the general binding properties of antibodies. For example, phage-based libraries that bind to desired targets can be suitably screened to obtain binding agents, such as nanobodies (small antibody proteins) and scFv agents, that are not based on existing antibodies.

[0060] Where a range of values ​​is given, it is clear, but unless dictated otherwise by context, that values ​​between the upper and lower limits of that range, as well as any value stated in or within that specified range, are encompassed in the disclosure to the tenth of the unit of the lower limit. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are encompassed in the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0061] All ranges disclosed herein also encompass any and all possible subranges and combinations of subranges. Any specified range can be considered fully delineated, and the same range can be divided into at least two, three, four, five, ten, etc. As a non-limiting example, each range described herein can be readily divided into a lower third, middle third, upper third, etc. Furthermore, those skilled in the art will appreciate that all expressions such as "up to," "less than," etc., are inclusive of the recited numerals and refer to ranges that can be successively divided into subranges as described above. Finally, those skilled in the art will appreciate that a range includes individual components. Thus, for example, a group having 1 to 3 items refers to a group having 1 item, a group having 2 items, or a group having 3 items. Similarly, a group having 1 to 5 items refers to a group having 1 item, a group having 2 items, a group having 3 items, a group having 4 items, or a group having 5 items.

[0062] For clarity, certain features of the present disclosure are described in the context of separate embodiments, but may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure are described for brevity in the context of a single embodiment, but may also be provided separately or in any suitable subcombination. All combinations of the embodiments according to the present disclosure are specifically embraced by this disclosure and are disclosed herein as if each and every combination were individually and explicitly disclosed. Also, all subcombinations of the various embodiments and elements thereof are specifically embraced by this disclosure and are disclosed herein as if each and every subcombination were individually and explicitly disclosed.

[0063] Although features of the present disclosure may be described in the context of a single embodiment, these features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may be described herein in the context of separate embodiments for clarity, the present disclosure may also be implemented in a single embodiment. Published patent applications and other published references, literature, manuscripts, and scientific literature cited herein are all incorporated by reference herein for any purpose. In the case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative and not intended to be limiting.

[0064] ubiquitin The major pathway of protein degradation in eukaryotic cells involves ubiquitination, which targets cellular proteins for rapid proteolysis. Ubiquitination is a highly regulated post-translational process that occurs by the covalent transfer of ubiquitin to lysine residues of target proteins. The addition of ubiquitin is mediated by the cooperative action of three enzymes: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3). The ubiquitin-activating enzyme E1 activates ubiquitin in an ATP-dependent process that forms a thioester bond between the C-terminal glycine of ubiquitin and a cysteine ​​residue in the E1 active site. The activated ubiquitin is then transferred to the cysteine ​​residue of the ubiquitin-conjugating enzyme E2. The ubiquitin-protein ligase E3 then facilitates the transfer of ubiquitin from the E2 enzyme to the lysine residue of the protein substrate. Because the human genome encodes two E1 enzymes, approximately 40 E2 enzymes, and over 800 E3 ligases, E3 ligases are primarily responsible for conferring substrate specificity in proteolytic processes. Thus, engineering the substrate specificity of E3 ligases provides a way to redirect the cellular degradation machinery toward targeted proteolysis of proteins of interest.

[0065] Compositions of the present disclosure As described in more detail below, the present disclosure provides binding agents, such as the bispecific binding agents and engineered transmembrane proteins provided herein, that are useful for degrading target surface proteins present on the surface of target cells. Without being bound by any particular theory, these agents are designed to function by binding to both the target surface protein and a membrane-bound E3 ligase, resulting in the ubiquitination and degradation of the target surface protein. Engineered transmembrane proteins are also disclosed that have a membrane-bound E3 ligase domain and a target surface protein-binding domain. Without being bound by any particular theory, these agents are designed to function by binding to the target surface protein such that the target surface protein is ubiquitinated by the membrane-bound E3 ligase domain and, as a result of this binding, is degraded.

[0066] As described in the Examples herein, bispecific binding agents and engineered transmembrane proteins have been tested and validated in tumor cell lines. Without being bound by any particular theory, these novel agents appear to perform similarly in mouse models and other mammalian cells, as well as in mammalian subjects, including humans. The agents disclosed herein may be introduced into various cell types to create engineered cells for enhanced differentiation and tumor elimination. Therefore, engineered cells modified to express one or more of the agents disclosed herein are also within the scope of this disclosure.

[0067] Bispecific Binding Agents structure Bispecific binding agents of the present disclosure comprise two binding domains: one specific for a membrane-bound E3 ligase and the other specific for a target surface protein. Bispecific binding agents of the present disclosure include, but are not limited to, agents in which the E3 ligase-binding domain and the target surface protein-binding domain are independently selected from an antibody (or antibody half), nanobody, minibody, Fab fragment, single-chain variable fragment (scFv), and single-domain antibody (sdAb), or functional fragments thereof. These two binding domains may be the same or different molecules. For example, bispecific binding agents of the present disclosure include, but are not limited to, bispecific binding agents having an IgG that binds to E3 ligase and an scFv domain that binds to the target surface protein. The two binding domains of the bispecific binding agent can be bound by a covalent bond, a non-covalent interaction, or a combination thereof.

[0068] The bispecific binding agents may generally take the form of proteins, glycoproteins, lipoproteins, phosphoproteins, etc. Some bispecific binding agents of the present disclosure may take the form of bispecific antibodies or antibody derivatives. In some embodiments, the target protein-binding domain is selected from the group consisting of a half antibody, nanobody, or minibody, F(ab')2 fragment, Fab fragment, single-chain variable fragment (scFv), and single-domain antibody (sdAb), or functional fragments thereof. The two binding domains may together take the form of a bispecific antibody, bispecific diabody, bispecific camelid antibody, or bispecific peptibody, etc. Antibody derivatives need not be derived from a specific wild-type antibody. For example, well-known techniques, such as phage display, can be used to generate and select small proteins with binding domains similar to antibody complementarity-determining regions (CDRs). In some embodiments, the antigen-binding site comprises an scFv. The binding domains can also be derived from natural or synthetic ligands or receptors that specifically bind to target surface proteins, whether soluble or membrane-bound, such as, but not limited to, PD-1, EGF, etc.

[0069] The antigen-binding site can comprise a naturally occurring amino acid sequence or can be engineered, designed, or modified to provide desired and / or improved properties (e.g., binding affinity). Generally, the binding affinity of an antigen-binding site (e.g., an antibody) to a target antigen (e.g., a CD19 antigen) can be calculated by the Scatchard method described in Frankel et al., Mol Immunol (1979) 16:101-06. In some embodiments, binding affinity is measured by the antigen / antibody dissociation rate. In some embodiments, binding affinity is measured by competitive radioimmunoassay. In some embodiments, binding affinity is measured by ELISA. In some embodiments, antibody affinity is measured by flow cytometry. In some embodiments, binding affinity is measured by biolayer interferometry. An antibody that selectively binds to an antigen (e.g., CD19) will not significantly bind to other antigens if it can bind to that antigen with high affinity.

[0070] Bispecific antibodies can be produced using known methods. Embodiments of the present disclosure include "knobs-into-hole" bispecific antibodies, in which the symmetric dimerization region of the bispecific binding agent is modified to be asymmetric. For example, a knobs-into-hole bispecific IgG specific for antigen A and antibody B can be modified so that the Fc portion of the A-binding chain has one or more protrusions ("knobs") and the Fc portion of the B-binding chain has one or more cavities ("holes"). These knobs and holes are then positioned to interact with each other. This reduces homodimerization (AA and BB antibodies) and promotes heterodimerization, which is desirable for bispecific binding agents. See, for example, Y. Xu et al., mAbs (2015) 7(1):231-42. In some embodiments, the bispecific binding agent has a knobs-into-hole design. In some embodiments, the "knobs" include a T336W modification in the CH3 domain, i.e., threonine at position 336 is replaced with tryptophan. In some embodiments, the "hole" comprises one or a combination of T366S, L368A, and Y407V. In some embodiments, the "hole" comprises T366S, L368A, and Y407V. For example, as illustrated in FIG. 3. In some embodiments, the "knob" constant region comprises SEQ ID NO: 14. In some embodiments, the heavy chain Fc "knob" constant region has a histidine tag. In some embodiments, the heavy chain Fc "hole" constant region comprises SEQ ID NO: 15. In certain embodiments, an exemplary CH2-CH3 domain sequence of a knob construct with N297G is provided in SEQ ID NO: 335. In other embodiments, an exemplary CH2-CH3 domain sequence of a hole construct with N297G is provided in SEQ ID NO: 336. In some embodiments, an exemplary wild-type CH2-CH3 domain sequence is provided by SEQ ID NO: 337. In other embodiments, the "knob" and "hole" constant regions comprise sequences that are about 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to the sequences provided herein.

[0071] In some embodiments, the bispecific binding agent comprises a fusion protein having two binding domains. In some embodiments, the E3 binding domain comprises a half antibody, Fab, single-chain Fab, or scFv. In some embodiments, the E3 binding domain comprises a half IgG. In some embodiments, the target surface protein binding domain comprises a half antibody, Fab, single-chain Fab, or scFv, independent of the form of the E3 binding domain selected. In some embodiments, the E3 binding domain comprises a half antibody and the target surface protein binding domain comprises a half antibody. In some embodiments, each half antibody is a half IgG antibody. In some embodiments, each half antibody is a half knob-into-hole IgG antibody. In some embodiments, the E3 binding domain comprises a half antibody and the target surface protein binding domain comprises an scFab. In some embodiments, the E3 binding domain comprises a half antibody and the target surface protein binding domain comprises an scFv. In some embodiments, the E3 binding domain comprises an scFv and the target surface protein binding domain comprises an scFab.

[0072] In some embodiments, the bispecific binding agent comprises an FcRn receptor recognition domain that facilitates the return of the bispecific binding agent to the extracellular space once it is internalized.

[0073] Target surface proteins The bispecific binding agents disclosed herein have binding affinity for one or more target surface proteins in addition to membrane-bound E3 ligases. Target surface proteins are selected based on their involvement in immunosuppression, tumor cell evasion of immune surveillance, or tumor cell proliferation or metastasis. Surface proteins that can be targeted by the disclosed methods include proteins such as membrane steroid receptors, EGF receptors, TGF receptors, transferrin receptors, CD19, CD20, and CDCP1. Other suitable target surface proteins include proteins that inhibit attack by immune cells such as T cells, natural killer cells, and macrophages, such as PD-L1, PD-L2, CTLA-4, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, NKG2D, TIM-3, VISTA, and SIGLEC7. In some embodiments, the target surface protein is a protein overexpressed by the target cell. In some embodiments, the target surface protein is a protein that contributes to the target cell's ability to proliferate, metastasize, or evade the immune system. In some embodiments, the target surface protein is an immune checkpoint protein. In some embodiments, the target surface protein is PD-L1, PD-L2, CTLA-4, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, NKG2D, TIM-3, VISTA, or SIGLEC7. In some embodiments, the target surface protein is selected from a membrane steroid receptor, an EGF receptor, a TGF receptor, a transferrin receptor, CDCP1, CD19, and CD20.

[0074] In some embodiments, the target surface protein is a T cell receptor (TCR) polypeptide, a TCR costimulatory surface protein, CD4, CD8, or CAR-T. Bispecific binding agents with this specificity are useful for downregulating or suppressing T cells and CAR-T cells.

[0075] In some embodiments, the bispecific binding agent can bind to a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). TAAs include molecules such as proteins that are present on the surface of tumor cells and on a subset of normal cells, or that are present on many normal cells at much lower concentrations than on tumor cells. Examples include, but are not limited to, CEA, AFP, HER2, CTAG1B, and MAGEA1. In contrast, TSAs generally include molecules such as proteins that are present on the surface of tumor cells but not expressed on the surface of normal cells. Examples include, but are not limited to, tumor virus antigens and mutated proteins (known as neoantigens).

[0076] In some cases, the target surface protein-binding domain is specific for an epitope present in an antigen expressed by malignant tumor cells, such as, for example, a tumor-associated antigen or a tumor-specific antigen. The tumor-associated antigen or tumor-specific antigen can be, for example, an antigen associated with breast cancer cells, B-cell lymphoma, pancreatic cancer, Hodgkin's lymphoma cells, ovarian cancer cells, prostate cancer cells, mesothelioma, lung cancer cells, non-Hodgkin's B-cell lymphoma (B-NHL) cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, melanoma cells, chronic lymphocytic leukemia cells, acute lymphocytic leukemia cells, neuroblastoma cells, glioma, glioblastoma, bladder cancer cells, colorectal cancer cells, etc. Obviously, tumor-associated antigens can also be expressed by non-cancerous cells. In some embodiments, the antigen-binding domain is specific for an epitope present in a tissue-specific antigen. In some embodiments, the antigen-binding domain is specific for an epitope present in a disease-associated antigen.

[0077] E3 ligase Bispecific binding agents of the present disclosure also bind to membrane-bound E3 ligases. E3 ligases useful in the present disclosure include ligases found associated with the plasma membrane (cytoplasmic membrane) of target cells. These membrane-bound E3 ligases include, for example, RNF43, ZNRF3, RNF128 (GRAIL), MARCH11, and the like. Because RNF128 is characteristically expressed in T cells, the activity of bispecific binding agents that bind to RNF128 is limited to T cells and any other cells that express RNF128.

[0078] Exemplary Constructs In some embodiments, the bispecific binding agents of the present disclosure comprise an E3 ligase-binding arm and a target surface protein-binding arm provided herein, hi some embodiments, the E3 ligase-binding arm binds to an extracellular protein bound by the E3 ligase or a transmembrane protein that interacts with the E3 ligase.

[0079] In certain embodiments, the E3 ligase-binding arm comprises a light chain and a heavy chain. In some embodiments, the light chain and the heavy chain each comprise a variable region. Generally, the variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs of each chain are held in close proximity by the FRs and contribute to the formation of the antigen-binding site of the binding agent, along with the CDRs of the other chain. There are at least two techniques for determining CDRs: (1) methods based on interspecies sequence variability and (2) methods based on crystallographic studies of antigen-antibody complexes. In addition, those skilled in the art sometimes use a combination of these two methods to determine CDRs.

[0080] In some embodiments, the first binding domain comprises a heavy chain framework region sequence set forth in SEQ ID NO: 12 or 320, and a light chain framework region sequence set forth in SEQ ID NO: 11 or 319. In some embodiments, the second binding domain comprises a heavy chain framework region sequence set forth in SEQ ID NO: 12 or 320, and a light chain framework region sequence set forth in SEQ ID NO: 11 or 319. In some embodiments, the heavy chain framework region sequence and the light chain framework region sequence comprise sequences that are about 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to the sequences provided herein.

[0081] In some embodiments, the first binding domain comprises a light chain variable region CDR3 (LC-CDR3) sequence, a heavy chain variable region CDR1 (HC-CDR1) sequence, a HC-CDR2 sequence, and a HC-CDR3 sequence, each comprising a sequence shown in Table 2, or a variant thereof comprising one, two, three, or four conservative amino acid substitutions. In some embodiments, the second binding domain comprises a LC-CDR3 sequence, a HC-CDR1 sequence, a HC-CDR2 sequence, and a HC-CDR3 sequence, each comprising a sequence shown in Table 3, or a variant thereof comprising one, two, three, or four conservative amino acid substitutions.

[0082] In other embodiments, the first binding domain of the bispecific binding agent comprises a heavy chain variable region, and the second binding domain of the bispecific binding agent comprises a heavy chain FR sequence set forth in SEQ ID NO: 12 or 320 and a light chain FR sequence set forth in SEQ ID NO: 11 or 319. Such bispecific binding agents are also referred to throughout this specification as "VH binders." In one exemplary embodiment, the heavy chain variable region of the first binding domain comprises the FR sequence set forth in SEQ ID NO: 321. In some VH binder embodiments, the first binding domain comprises a VH-CDR1 sequence, a VH-CDR2 sequence, and a VH-CDR3 sequence, each comprising a sequence set forth in Table 4, or a variant thereof comprising one, two, three, or four conservative amino acid substitutions. In some embodiments of the VH binders, the second binding domain comprises an LC-CDR3 sequence, an HC-CDR1 sequence, an HC-CDR2 sequence, and an HC-CDR3 sequence, each comprising a sequence shown in Table 3, or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions.

[0083] "Conservative amino acid substitution" refers to the substitution of one amino acid residue with another amino acid residue having a similar side chain.Families of amino acid residues with similar side chains have been defined in the art, and include, for example, basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).For example, the substitution of phenylalanine with tyrosine is a conservative substitution. In certain embodiments, conservative substitutions in the sequence of a binding agent of the present disclosure do not abolish binding of a binding agent comprising this amino acid sequence to its antigen (i.e., the E3 ligase and / or target surface protein to which the binding agent binds). Methods for identifying conservative nucleotide and amino acid substitutions that do not eliminate antigen binding are well known in the art. synthesis

[0084] Bispecific binding agents are produced using recombinant DNA and protein expression technology. For example, suitable DNA sequences encoding heavy and light chain constant regions are widely available for synthesizing DNA encoding the bispecific IgG of the present disclosure. Sequences encoding selected variable regions are inserted using conventional methods, and the resulting nucleic acids encoding full-length heavy and light chains are transformed into suitable host cells and expressed. Alternatively, these nucleic acids can be expressed in cell-free expression systems that allow for better control over redox conditions, pH, folding, glycosylation, and the like.

[0085] Bispecific IgG proteins have two different complementarity-determining regions (CDRs), each specific for either the target surface protein or the membrane-bound E3 ligase. Therefore, two different heavy chains and two different light chains are required. These can be expressed in the same host cell, and the resulting product contains a mixture of homodimers and bispecific heterodimers. The homodimers can be separated from the bispecific antibody by affinity purification (e.g., using beads coated first with one antigen and then with the other), reduced to monomers, and reassociated. Alternatively, a "knob-into-hole" design can be employed. In this design, the dimerization region of the heavy chain constant region is modified so that its surface either protrudes (compared to the wild-type structure) ("knob") or forms a cavity ("hole") that allows the two modified surfaces to dimerize. The knob heavy chain and its associated light chain are then expressed in one host cell, and the hole heavy chain and its associated light chain are expressed in a different host cell, and the expressed proteins are allowed to bind. The asymmetry of these dimerization regions promotes heterodimer formation. (See, e.g., Example 1 below and Figure 3, which shows the changes compared to the wild-type.) To achieve dimerization, the two "monomers" (each consisting of a heavy chain and a light chain) are combined under reducing conditions at a mildly basic pH (e.g., about pH 8-9) to promote disulfide bond formation between the appropriate heavy chain regions. See, e.g., US8216805 and EP1870459A1, which are incorporated herein by reference.

[0086] Other methods can be used to promote heavy chain heterodimerization of the first and second polypeptide chains of a bispecific antibody. For example, in some embodiments, heavy chain heterodimerization of the first and second polypeptide chains of an engineered antibody disclosed herein can be achieved by the controlled Fab arm exchange method described in FL Aran et al., Proc Natl Acad Sci USA (2013) 110(13):5145-50.

[0087] This dimerization process can lead to exchange of light chains between different heavy chain monomers. One way to avoid this outcome is to replace the antibody binding domain with a "single-chain Fab." For example, the light chain CDRs are fused to the heavy chain CDRs by a linking polypeptide. The Fab region of an IgG (or other antibody) can also be replaced with an scFv, nanobody, etc.

[0088] The binding activity of modified antibodies of the present disclosure can be assayed by any suitable method known in the art. For example, the binding activity of modified antibodies of the present disclosure can be determined by Scatchard analysis (Munsen et al., Analyt Biochem (1980) 107:220-39). Specific binding may be assessed by techniques well known in the art, including, but not limited to, competitive ELISA, BIACORE® assay, and / or KINEXA® assay. Antibodies that bind preferentially or specifically (these are interchangeable herein) to a target antigen or target epitope are terms well understood in the art, and methods for determining such specific, or preferential, binding are also well known in the art. An antibody is said to exhibit specific, or preferential, binding if it reacts or associates more frequently, rapidly, for longer, and / or with greater affinity with a particular antigen or epitope than with another antigen or epitope. An antibody specifically, i.e., preferentially, binds to a target if it binds with greater affinity, avidity, more readily, and / or for longer than it binds to other substances. Also, an antibody specifically, i.e., preferentially, binds to a target in a sample if it binds with greater affinity, avidity, more readily, and / or for longer than it binds to other substances present in the sample. For example, an antibody that specifically, i.e., preferentially, binds to a HER2 epitope is an antibody that binds to this epitope with greater affinity, avidity, more readily, and / or for longer than it binds to other HER2 epitopes or non-HER2 epitopes. As can be understood from this definition, for example, an antibody that specifically, i.e., preferentially, binds to a first target antigen may or may not specifically, i.e., preferentially, bind to a second target antigen. Thus, specific binding and preferential binding do not necessarily require exclusive binding (although they can be inclusive).

[0089] Engineered Membrane Proteins The engineered transmembrane proteins disclosed herein have binding affinity for one or more target surface proteins and incorporate a domain with membrane-associated E3 ligase ubiquitin ligase activity. All of the target surface proteins described herein with respect to bispecific binding agents are also suitable targets for the engineered transmembrane proteins. In some embodiments, the engineered transmembrane proteins include CD19, B7H3 (CD276), BCMA, CD123, CD171, CD179a, CD20, CD213A2, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD71, CD97, CEA, CLDN6, CLECL1, CS-1, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, ephrin B2, FAP, FL In some embodiments, the engineered transmembrane protein has binding affinity for T3, GD2, GD3, GM3, GPRC5D, HER2 (ERBB2 / neu), IGLL1, IL-11Ra, KIT (CD117), MMP14, MUC1, NCAM, PAP, PDGFR-β, PRSS21, PSCA, PSMA, ROR1, SSEA-4, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2, BCMA (CD269), ALPI, citrullinated vimentin, cMet, or AxI. In some embodiments, the engineered transmembrane protein has binding affinity for PD-L1, PD-L2, CTLA-4, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, NKG2D, TIM-3, VISTA, or SIGLEC7. In some embodiments, the engineered transmembrane protein has binding affinity for a membrane steroid receptor, an EGF receptor, a TGF receptor, a transferrin receptor, CD19, or CD20. In some embodiments, the engineered transmembrane protein has binding affinity for a T cell receptor (TCR) polypeptide, a TCR costimulatory surface protein, CD4, CD8, or CAR-T.

[0090] The E3 ligase domain can be selected from any of the E3 ligases listed above as targets for bispecific binding agents. Furthermore, the selected E3 ligase does not need to be native to or expressed by the target cell, so long as it is capable of transferring ubiquitin or a linked ubiquitin chain from an endogenous E2 ubiquitin-conjugating enzyme. This allows for the use of an E3 ligase from a mammalian species different from that of the target cell, e.g., using a mouse E3 ligase in a human subject. This also prevents malignant tumor cells from circumventing the therapeutic effect by downregulating the expression of one E3 ligase. Because the engineered transmembrane proteins provided herein contain E3 ligase activity, malignant tumor cells must downregulate or suppress the expression of all endogenous E2 ubiquitin-conjugating enzymes to avoid the activity of the engineered transmembrane protein. In some embodiments, the E3 ligase domain comprises a membrane-bound E3 ligase or a functional portion thereof. In some embodiments, the membrane-bound E3 ligase is a human membrane-bound E3 ligase. In some embodiments, the membrane-bound E3 ligase is RNF43, ZNRF3, RNF128 (GRAIL), or MARCH11.

[0091] The engineered transmembrane protein comprises a binding domain specific for a selected target surface protein. This binding domain can take the form of any of the target surface protein binding domains described herein, including, for example, antibodies, nanobodies, minibodies, Fab fragments, single-chain variable fragments (scFv), and single-domain antibodies (sdAbs), or functional fragments thereof. The binding domain can also be derived from a natural or synthetic ligand or receptor, whether soluble or membrane-bound, that specifically binds to the target surface protein, including, but not limited to, PD-1, HER2, and HER3.

[0092] The binding domain and the E3 ligase domain can be expressed together as a fusion protein. Alternatively, they can be associated by a covalent bond (e.g., a disulfide bond between two cysteine ​​residues) or by non-covalent affinity binding. In some embodiments, the engineered transmembrane protein is a fusion protein. In some embodiments, the E3 ligase domain and the target surface protein binding domain of the engineered transmembrane protein are linked by a disulfide bond. An exemplary engineered transmembrane protein having a GFP-binding domain and a membrane-associated E3 ligase domain is shown in Figure 2.

[0093] In one exemplary embodiment, an engineered transmembrane protein of the present disclosure comprises an anti-GFP scFab sequence having the sequences of SEQ ID NO:2 (light chain) and SEQ ID NO:4 (heavy chain) with a binding domain shown in SEQ ID NO:3. A short linker (SEQ ID NO:5) connects the anti-GFP scFab domain and the RNF43 domain (SEQ ID NO:6). In this exemplary embodiment, the complete sequence of the engineered transmembrane protein is shown in SEQ ID NO:1.

[0094] In another exemplary embodiment, the reporter construct is assembled from a GFP domain (SEQ ID NO: 8), a transmembrane / linker domain (SEQ ID NO: 9), and a nanoluciferase domain (SEQ ID NO: 10). In this exemplary embodiment, the complete sequence of the reporter construct is shown in SEQ ID NO: 7.

[0095] immune complex The present disclosure further includes immunoconjugates comprising any of the binding agents disclosed herein. In some embodiments, the immunoconjugates of the present disclosure comprise a bispecific binding agent provided herein. In other embodiments, the immunoconjugates of the present disclosure comprise an engineered transmembrane protein disclosed herein. As used herein, the term "immunoconjugate" or "conjugate" refers to a compound or derivative thereof bound to a binding agent, such as a bispecific binding agent or an engineered transmembrane protein provided herein. The immunoconjugates of the present disclosure generally comprise a bispecific binding agent or an engineered transmembrane protein provided herein and a binding agent, such as a small molecule. In some embodiments, the immunoconjugate further comprises a linker.

[0096] A "linker" is any chemical moiety capable of covalently and stably attaching a compound (e.g., a small molecule disclosed herein) to a binding agent (e.g., a bispecific binding agent or an engineered transmembrane protein provided herein). A linker can be susceptible to or substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage under conditions under which the compound or antibody is active. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid-labile groups, photolabile groups, peptidase-labile groups, and esterase-labile groups. Linkers also include charged linkers and their hydrophilic forms described herein and well known in the art. In certain embodiments, the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, and a dicarboxylic acid-based linker. In an exemplary embodiment, the linker is a non-cleavable linker. In another exemplary embodiment, the linker is a spacer, such as PEG 4. In another embodiment, the small molecule does not dissociate from the binding agent.

[0097] Small molecules encompassed by the present disclosure can be any small molecule deemed suitable by one of skill in the art for use in, for example, targeted degradation of a protein of interest. In another exemplary embodiment, the small molecule includes, but is not limited to, an agonist, such as CGS21680. In an additional exemplary embodiment, the small molecule includes, but is not limited to, an antagonist, such as ZN241385, plerixafor, maraviroc, or aplaviroc. The small molecule can be conjugated to a binding agent, such as a bispecific binding agent or an engineered transmembrane protein, provided herein by methods well known in the art. Some exemplary conjugation methods include, but are not limited to, methionine using oxaziridine-based reagents (illustrated in FIG. 13), cysteine ​​labeled with maleimide-based or disulfide exchange reagents, lysine-reactive activated esters, incorporation of unnatural amino acids containing reactive handles for conjugation, and N- or C-terminal conjugation. Some methods use modified amino acids for reactive conjugation, such as aldehydes. Other methods include tag-based bioconjugation methods. The present disclosure provides several exemplary methods for conjugation. See, e.g., Example 6. Obviously, the present disclosure is not limited to the few examples shown herein, and other commonly known conjugation methods can also be used to generate the immunoconjugates disclosed herein.

[0098] nucleic acid molecule In one aspect, some embodiments disclosed herein relate to nucleic acid molecules comprising nucleotide sequences encoding the bispecific binding agents and engineered transmembrane proteins of the present disclosure, including expression cassettes and expression vectors comprising these nucleic acid molecules operably linked to heterologous nucleic acid sequences, such as, for example, regulatory sequences directing the in vivo expression of the engineered transmembrane protein in a host cell.

[0099] The nucleic acid molecules of the present disclosure may be of any length, and generally include nucleic acid molecules of about 5 Kb to about 50 Kb, for example, about 5 Kb to about 40 Kb, about 5 Kb to about 30 Kb, about 5 Kb to about 20 Kb, or about 10 Kb to about 50 Kb, for example, about 15 Kb to 30 Kb, about 20 Kb to about 50 Kb, about 20 Kb to about 40 Kb, about 5 Kb to about 25 Kb, or about 30 Kb to about 50 Kb.

[0100] In some embodiments, the nucleotide sequence is incorporated into an expression cassette or expression vector. As will be appreciated, an expression cassette generally comprises a construct of genetic material that includes a coding sequence and regulatory information sufficient to direct proper transcription and / or translation of the coding sequence in a recipient cell, in vivo, and / or in vitro. Generally, the expression cassette may be inserted into a vector for targeting to a desired host cell or tissue and / or individual. Thus, in some embodiments, an expression cassette of the present disclosure comprises a nucleotide sequence of a bispecific binding agent or engineered transmembrane protein operably linked to expression control elements sufficient to direct expression of the cassette in vivo. In some embodiments, the expression control elements include a promoter and / or transcriptional enhancer, and optionally, any or a combination of other nucleic acid sequences capable of effecting transcription and / or translation of the coding sequence.

[0101] In some embodiments, the nucleotide sequence is incorporated into an expression vector. Generally, transformation, i.e., a vector may be used to introduce heterologous DNA into a host cell, and includes a recombinant polynucleotide construct designed for transfer between host cells. Thus, in some embodiments, the vector may be a replicon, such as a plasmid, phage, or cosmid. Other DNA segments may be inserted into the vector to allow replication of the inserted segment. The expression vector further comprises a promoter operably linked to the recombinant polynucleotide so that the recombinant polynucleotide is expressed in a suitable cell under appropriate conditions. In some embodiments, the expression vector is an integrating vector capable of integrating into a host nucleic acid.

[0102] In some embodiments, the expression vector is a viral vector. This viral vector further comprises viral-derived nucleic acid elements that typically facilitate the introduction or incorporation of nucleic acid molecules into a cellular genome or into viral particles that mediate nucleic acid transfer. Viral particles typically contain various viral components and may also contain host cell components in addition to nucleic acids. The term "viral vector" may refer to a virus or viral particle that can transfer nucleic acids into cells or the nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements primarily derived from viruses. Retroviral vectors contain structural and / or functional genetic elements or portions thereof primarily derived from retroviruses. Lentiviral vectors are viral vectors or plasmids that contain structural and / or functional genetic elements or portions thereof, including long terminal repeats (LTRs) primarily derived from lentiviruses.

[0103] The nucleic acid sequence can be optimized for expression in the target host cell. For example, the GC content of the sequence can be adjusted to an average amount for the cellular host, for example, by calculating based on known genes expressed in a certain host cell. Methods for codon optimization are well known in the art. The codon usage in the coding sequence of the protein disclosed herein can be optimized so that about 1%, about 5%, about 10%, about 25%, about 50%, about 75%, or 100% of the codons in the coding sequence are optimal for expression in a specific host cell, thereby enhancing expression in the host cell.

[0104] Some embodiments disclosed herein relate to vectors or expression cassettes containing recombinant nucleic acid molecules encoding the proteins disclosed herein. Generally, the expression cassette contains a coding sequence and sufficient regulatory information to direct proper transcription and / or translation of the coding sequence in a recipient cell, in vivo, and / or in vitro. The expression cassette may be inserted into a vector for targeting a desired host cell and / or individual. The expression cassette may be inserted into a plasmid, cosmid, virus, self-replicating polynucleotide molecule, or bacteriophage containing one or more nucleic acid sequences in a functionally operable manner, i.e., as a linear or circular single- or double-stranded DNA or RNA polynucleotide capable of genome integration or self-replication, from any source, containing operably linked nucleic acid molecules.

[0105] Also provided herein are vectors, plasmids, or viruses that contain one or more nucleic acid molecules encoding any of the bispecific binding agents or engineered proteins disclosed herein. The nucleic acid molecules can be included, for example, in a vector and directed for expression in cells transformed / transduced with the vector. Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available or readily produced by one of ordinary skill in the art. For example, Sambrook, J., & Russell, DW (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, DW (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, FM (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, DM et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, MG et al. (1995). Viral Vectors: Gene Therapy and neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997).See The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, KB, Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, EA (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, SL et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, SC (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences BV, the disclosures of which are incorporated herein by reference. .

[0106] DNA vector can be introduced into eukaryotic cells by conventional transformation or transfection technology.Suitable methods for transforming or transfecting host cells include those described in Sambrook et al. (2012, see above) and other common molecular biology experimental procedures, such as calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape loading, ballistic transfection, nuclear poration, hydrodynamic shock and infection.

[0107] Viral vectors that can be used in the present disclosure include, for example, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, herpesvirus, simian virus 40 (SV40), and bovine papillomavirus vectors (see, e.g., Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, NY).

[0108] The exact components of the expression system are not critical. For example, the bispecific binding agents disclosed herein can be produced in eukaryotic hosts such as mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, Virginia). In selecting an expression system, it is important only that the components are compatible with each other. One of skill in the art can make such a determination. Additionally, one of skill in the art may consult P. Jones, "Vectors: Cloning Applications," John Wiley and Sons, New York, NY, 2009) for guidance in selecting an expression system.

[0109] The provided nucleic acid molecules can include naturally occurring sequences or sequences that differ from those occurring in nature but encode the same gene product due to the degeneracy of the genetic code. These nucleic acid molecules can be composed of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA produced by phosphoramidite-based synthesis), or nucleotide combinations or modifications within these types of nucleic acids. Additionally, the nucleic acid molecules can be double-stranded or single-stranded (e.g., including either the sense or antisense strand).

[0110] The nucleic acid molecule is not limited to a sequence encoding a polypeptide (e.g., an antibody). It may also include some or all of the non-coding sequences upstream or downstream of the coding sequence (e.g., the coding sequence of a bispecific binding agent or an engineered transmembrane protein). Those skilled in the art of molecular biology are familiar with procedures for isolating nucleic acid molecules. For example, the nucleic acid molecule can be generated by treating genomic DNA with restriction endonucleases or by polymerase chain reaction (PCR). In this case, the nucleic acid molecule is ribonucleic acid (RNA), and the transcript can be produced, for example, by in vitro transcription.

[0111] Recombinant Cells and Cell Cultures The nucleic acids of the present disclosure can be introduced into host cells, such as human B lymphocytes, to produce recombinant cells containing the nucleic acid molecules. Accordingly, some embodiments of the present disclosure relate to methods of making recombinant cells, the methods comprising: (a) providing a cell capable of expressing a protein; and (b) contacting the provided cell with any of the recombinant nucleic acids described herein.

[0112] Introduction of the nucleic acid molecules of the present disclosure into cells can be achieved by viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0113] Thus, in some embodiments, the nucleic acid molecule is delivered to cells using viral or non-viral delivery vehicles known in the art. For example, the nucleic acid molecule can be stably integrated into the host genome, replicated episomally, or present in the recombinant host cell as a short, circular expression vector for stable or transient expression. Thus, in some embodiments disclosed herein, the nucleic acid molecule is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid molecule is stably integrated into the genome of the recombinant cell. Stable integration can be achieved using traditional random genome recombination techniques or more precise genome editing techniques (e.g., guide RNA-directed CRISPR / Cas9, or DNA-guided endonuclease genome editing NgAgo (Natronobacterium gregoryi Argonaute), or TALENs genome editing (transcription activator-like effector nucleases)). In some embodiments, the nucleic acid molecule is present in the recombinant host cell as a short, circular expression vector for stable or transient expression.

[0114] The nucleic acid molecule can be encapsulated in a viral capsid or lipid nanoparticle. For example, introduction of the nucleic acid into cells can be achieved by viral transduction. In one non-limiting example, adeno-associated virus (AAV) is a non-enveloped virus that can be modified to deliver nucleic acids to target cells by viral transduction. Several AAV serotypes have been described, and all known serotypes are capable of infecting cells from multiple, diverse tissue types. AAV can transduce a wide range of species and tissues in vivo without toxicity and generates relatively mild innate and adaptive immune responses. One embodiment is an AAV vector encoding the engineered transmembrane protein of the present disclosure.

[0115] Lentiviral systems are also suitable for nucleic acid delivery and gene therapy via viral transduction. Lentiviral vectors offer several attractive properties as gene delivery vehicles, including (i) sustained gene delivery through stable integration of the vector into the host genome, (ii) the ability to infect both dividing and non-dividing cells, (iii) broad tissue tropism, including important gene and cell therapy target cell types, (iv) no expression of viral proteins after vector transduction, (v) the ability to deliver complex genetic elements such as polycistronic or intron-containing sequences, (vi) a safer integration site profile, and (vii) a relatively easy system for vector manipulation and production.

[0116] In some embodiments, host cells have been genetically modified (e.g., transduced, transformed, or transfected) with a vector containing a nucleic acid sequence encoding, for example, an engineered transmembrane protein described herein. The vector is either a virus-derived expression vector or a vector for homologous recombination that contains a nucleic acid sequence homologous to a portion of the host cell's genome. The host cell can be either an untransformed cell or a cell that has already been transfected with one or more nucleic acid molecules.

[0117] In some embodiments, the recombinant cell is a prokaryotic or eukaryotic cell. In some embodiments, the cell is transformed in vivo. In some embodiments, the cell is transformed ex vivo. In some embodiments, the cell is transformed ex vivo. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the animal cell is a human cell. In some embodiments, the cell is a non-human primate cell. In some embodiments, the mammalian cell is an immune cell, a neuronal cell, an epithelial cell, an endothelial cell, or a stem cell. In some embodiments, the recombinant cell is an immune system cell, e.g., a lymphocyte (e.g., a T cell or an NK cell), or a dendritic cell. In some embodiments, the immune cell is a B cell, a monocyte, a natural killer (NK) cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell, a cytotoxic T cell, or other T cell. In some embodiments, the immune system cell is a T lymphocyte.

[0118] In some embodiments, the cells are stem cells. In some embodiments, the cells are hematopoietic stem cells. In some embodiments, the cells are lymphocytes. In some embodiments of the cells, the cells are precursor T cells or regulatory T (Treg) cells. In some embodiments, the cells are CD34+ cells, CD8+ cells, or CD4+ cells. In some embodiments, the cells are CD8+ T cytotoxic lymphocyte cells selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells. In some embodiments of the cells, the cells are CD4+ T helper lymphocyte cells selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some embodiments, the cells are obtained by leukapheresis of a sample obtained from a human subject.

[0119] In other aspects, provided herein are various cell cultures and culture media comprising at least one recombinant cell disclosed herein. Generally, the culture medium can be any one of the culture media suitable for the cell cultures described herein. Techniques for transforming the various host cells and species described above are well known in the art and described in the technical and scientific literature. Accordingly, cell cultures comprising at least one recombinant cell disclosed herein are within the scope of this application. Suitable methods and systems for generating and maintaining cell cultures are well known in the art.

[0120] Pharmaceutical Composition In some embodiments, the bispecific binding agents, engineered transmembrane proteins, nucleic acids, and recombinant cells of the present disclosure can be incorporated into compositions, including pharmaceutical compositions. Such compositions typically include the bispecific binding agents, engineered transmembrane proteins, nucleic acids, and / or recombinant cells, and a pharmaceutically acceptable excipient (e.g., carrier).

[0121] The bispecific binding agent of the present disclosure can be administered using the formulations used to administer antibodies, and antibody-based therapeutic methods, or formulations based on them.The nucleic acid of the present disclosure can be administered using the formulations used to administer oligonucleotides, antisense RNA agents, and / or gene therapy, such as CRISPR / Cas9-based therapeutic methods.The engineered transmembrane protein can be administered as a nucleic acid that is expressed in target cells, or as a protein in a carrier that can fuse with the target cell membrane (for example, a fusogenic carrier, as described below).

[0122] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to be syringable. It must be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerin, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by using a coating such as lecithin to maintain the required particle size in the case of dispersions, and by using surfactants (e.g., sodium dodecyl sulfate). The activity of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars, polyalcohols (mannitol, sorbitol, etc.), or sodium chloride, are generally included in the composition. The absorption of injectable compositions can be prolonged by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0123] The active compound can be incorporated into the required amount of suitable solvent with one or a combination of the above-mentioned components, and then filtered and sterilized to prepare sterile injectable solution.Generally, the active compound is incorporated into a sterile medium containing a basic dispersion medium and the other necessary components from the above-mentioned components to prepare dispersion solution.For the sterile powder used to prepare sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying.This can obtain powder containing active ingredient and any desired additional components from the solution previously sterilized and filtered.

[0124] In some embodiments, bispecific binding agents or engineered transmembrane proteins of the present disclosure are administered by transfection or infection with nucleic acids encoding them using methods well known in the art, including, but not limited to, those described in McCaffrey et al., Nature (2002) 418:6893, Xia et al., Nature Biotechnol (2002) 20:1006-10, and Putnam, Am J Health Syst Pharm (1996) 53:151-60, erratum at Am J Health Syst Pharm (1996) 53:325.

[0125] The engineered transmembrane proteins of the present disclosure can be administered using a formulation comprising a fusogenic carrier. These are carriers that can fuse with the cell membrane of mammalian cells. Fusogenic carriers include, but are not limited to, membrane-enclosed viral particles and carriers based thereon, exosomes and microvesicles (see, for example, Y. Yang et al., J Extracellular Vessicles (2018) 7:144131), and fusogenic liposomes (see, for example, Bailey et al., US 5552155; Martin et al., US 5891468; Holland et al., US 5885613; and Leamon, US 6379698). One embodiment is a formulation comprising an engineered transmembrane protein and a fusogenic carrier.

[0126] Methods of the present disclosure Administration of bispecific binding agents For example, any one or more of the therapeutic compositions described herein, such as bispecific binding agents, engineered transmembrane proteins, nucleic acids, recombinant cells, and pharmaceutical compositions, can be administered to treat an individual with a neoplastic disease, such as cancer. In some embodiments, the bispecific binding agents, engineered transmembrane proteins, nucleic acids, recombinant cells, and pharmaceutical compositions are incorporated into therapeutic compositions for use in methods of downregulating or inactivating T cells, such as CAR-T cells.

[0127] Thus, in one aspect, provided herein are methods for inhibiting target cell activity in an individual. The methods include administering to the individual a first treatment comprising one or more bispecific binding agents, engineered transmembrane proteins, nucleic acids, recombinant cells, and pharmaceutical compositions provided herein, wherein the first treatment inhibits the target cell activity by degrading the target surface protein. For example, target cell activity may be inhibited if the proliferation of the target cell is reduced, if the pathological or pathogenic behavior of the target cell is reduced, or if the target cell is destroyed or killed. Inhibition includes a measured reduction of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the method comprises administering to the individual an effective number of recombinant cells disclosed herein, wherein the recombinant cells inhibit target cells in the individual by expression of the bispecific binding agent. Generally, the target cells of the disclosed methods can be any cell, such as, for example, acute myeloma leukemia cells, anaplastic lymphoma cells, astrocytoma cells, B-cell cancer cells, breast cancer cells, colon cancer cells, ependymoma cells, esophageal cancer cells, glioblastoma cells, bladder cancer cells, glioma cells, leiomyosarcoma cells, liposarcoma cells, liver cancer cells, lung cancer cells, mantle cell lymphoma cells, melanoma cells, neuroblastoma cells, non-small cell lung cancer cells, oligodendroglioma cells, ovarian cancer cells, pancreatic cancer cells, peripheral T-cell lymphoma cells, kidney cancer cells, sarcoma cells, gastric cancer cells, carcinoma cells, mesothelioma cells, or sarcoma cells. In some embodiments, the target cells are pathogenic cells.

[0128] The bispecific binding agents of the present disclosure are typically administered in a solution or suspension formulation by injection or infusion. In one embodiment, the bispecific binding agent is administered by direct injection into the tumor mass. In another embodiment, the bispecific binding agent is administered by systemic infusion.

[0129] Some bispecific binding agents of the present disclosure are effective at a concentration of 10 nM. Other bispecific binding agents may be most effective at higher or lower concentrations depending on the binding affinity with each ligand and the degree of expression of each ligand. However, the range of effective concentrations can be determined by those of ordinary skill in the art using the present disclosure and the experimental protocols provided herein. Similarly, the effective concentration can be used to determine the effective dosage or the range of dosages required for administration.

[0130] Depending on the disease or disorder being treated, the severity and extent of the disease, the subject's health, and the co-administration of other treatments, multiple doses may be administered. Alternatively, continuous administration may be required. However, it is expected that the bispecific binding agent will remain in close proximity to the cell so that each molecule of the bispecific binding agent can ubiquitinate multiple molecules of the target surface protein for degradation. Thus, the bispecific binding agents of the present disclosure may require lower doses or less frequent administration than therapies based on antibody competitive binding.

[0131] Administration of recombinant cells to an individual In some embodiments, the method includes administering the recombinant cells to an individual in need thereof. This administering step can be accomplished using any injection method known in the art. For example, the recombinant cells can be injected intravenously directly into the individual's bloodstream or can be otherwise administered to the individual.

[0132] The terms "administration," "transduction," and "implantation" are used interchangeably herein to refer to methods of delivering recombinant cells expressing a bispecific binding agent provided herein to an individual. In some embodiments, the methods involve administering the recombinant cells to an individual using a method or route of administration that at least partially localizes the introduced cells at a desired site to produce a desired effect. The recombinant cells or their differentiated progeny can be administered by any suitable route that delivers them to a desired location within an individual, where at least a portion of the administered cells or cellular components remain viable. The survival period of the cells after administration to an individual can be as short as a few hours, e.g., 24 hours to a few days, or as long-term engraftment of several years, or even the lifetime of the individual.

[0133] When given prophylactically, in some embodiments, the recombinant cells described herein are administered to an individual prior to any symptoms of the disease or condition being treated. Thus, in some embodiments, prophylactic administration of the recombinant stem cell population acts to prevent the onset of symptoms of the disease or condition.

[0134] When provided as a treatment, in some embodiments, the recombinant stem cells are provided at (or after) the onset of symptoms or indications of a disease or condition, eg, at the onset of a disease or condition.

[0135] When used in various embodiments described herein, an effective amount of the recombinant cells disclosed herein is at least 10 2 Cells, at least 5 x 10 2 cells, at least 10 3 Cells, at least 5 x 10 3 cells, at least 10 4 Cells, at least 5 x 10 4 cells, at least 10 5 Cells, at least 2 x 10 5 Cells, at least 3 x 10 5 Cells, at least 4 x 10 5 Cells, at least 5 x 10 5 Cells, at least 6 x 10 5Cells, at least 7 x 10 5 Cells, at least 8 x 10 5 cells, at least 9 x 10 5 cells, at least 1 x 10 6 Cells, at least 2 x 10 6 Cells, at least 3 x 10 6 Cells, at least 4 x 10 6 Cells, at least 5 x 10 6 Cells, at least 6 x 10 6 Cells, at least 7 x 10 6 Cells, at least 8 x 10 6 cells, at least 9 x 10 6 The recombinant cells may be derived from one or more donors or may be obtained from an autologous source (i.e., the human subject being treated). In some embodiments, the recombinant cells are expressed in culture prior to administration to an individual in need thereof.

[0136] In some embodiments, delivery of a composition comprising recombinant cells (i.e., a composition comprising a plurality of recombinant cells of a bispecific binding agent provided herein) to an individual by a method or route results in at least partial localization of the cell composition at a desired site. The cell composition can be administered by any suitable route that results in effective treatment in the individual. For example, upon administration, at least a portion of the composition, e.g., at least 1 x 10 cells, can be delivered to a desired location within the individual and localize to a desired site. 4 The cells are delivered to the desired site for a period of time. Administration forms include injection, infusion, instillation, etc. Injection forms include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intravesical, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcuticular, subcapsular, subarachnoid, intrathecal, intracerebrospinal, and intrasternal injection and infusion. In some embodiments, the route is intravenous administration. Cell delivery can be achieved by injection or instillation.

[0137] In some embodiments, the recombinant cells are administered systemically, that is, rather than administering the recombinant cell population directly to a target site, tissue, or organ, the recombinant cell population is administered so that it enters the individual's circulatory system and undergoes metabolic and other processing.

[0138] The effectiveness of treatment with a composition for treating a disease or disorder can be determined by a clinician skilled in the art. However, as will be apparent to those skilled in the art, treatment is considered effective if any one or all of the signs, symptoms, or markers of the disease improve or ameliorate. Efficacy can also be measured by the individual's failure to improve as assessed by the need for hospitalization or medical intervention (e.g., the progression of the disease has been halted or at least slowed). Methods for measuring these signs are well known to those skilled in the art and / or are described herein. Treatment includes any treatment of a disease in an individual or animal (including, by non-limiting example, a human or mammal), including (1) inhibiting the progression of the disease, e.g., preventing or slowing the progression of the symptoms, or (2) alleviating the disease, e.g., reducing the symptoms, and (3) preventing or reducing the likelihood of the onset of the symptoms.

[0139] As noted above, a therapeutically effective amount includes an amount of a therapeutic composition sufficient to promote a particular effect when administered to an individual suspected of or at risk of suffering from a disease. In some embodiments, an effective amount includes an amount sufficient to prevent or delay the onset of a disease symptom, alter the course of a disease symptom (e.g., but not limited to, slowing the progression of a disease symptom), or reverse a disease symptom. Obviously, in any case, the appropriate effective amount can be determined by one of ordinary skill in the art using routine experimentation.

[0140] The efficacy of a treatment, including the disclosed therapeutic compositions for treating a disease, can be determined by a clinician skilled in the art. However, a treatment is considered effective if at least any one or all of the signs or symptoms of the disease are improved or ameliorated. Efficacy can also be measured from the failure of an individual to worsen as assessed by the need for hospitalization or medical intervention (e.g., the progression of the disease has been halted or at least slowed). Methods for measuring these signs are well known to those skilled in the art and / or described herein. Treatment includes any treatment of a disease in an individual or animal (including, by non-limiting example, a human or mammal), including (1) inhibiting the disease, e.g., preventing or slowing the progression of a symptom; (2) alleviating the disease, e.g., reducing a symptom; or (3) preventing or reducing the likelihood of the onset of a symptom.

[0141] In some embodiments, the individual is a mammal. In some embodiments, the mammal is a human. In some embodiments, the individual has or is suspected of having a disease associated with cell signaling mediated by cell surface proteins. In some embodiments, the disease is cancer or a chronic infectious disease.

[0142] Systems and Kits Also provided herein are systems and kits including the bispecific binding agents, engineered transmembrane proteins, recombinant nucleic acids, recombinant cells, or pharmaceutical compositions described and provided herein, as well as instructions for making and using the same. For example, in some embodiments, systems and / or kits are provided herein that include one or more of the bispecific binding agents described herein, the engineered transmembrane proteins described herein, the recombinant nucleic acids described herein, the recombinant cells described herein, or the pharmaceutical compositions described herein. In some embodiments, the systems and / or kits of the present disclosure further include one or more syringes (including pre-filled syringes) and / or catheters used to administer any one of the provided bispecific binding agents, engineered transmembrane proteins, recombinant nucleic acids, recombinant cells, or pharmaceutical compositions to an individual. In some embodiments, the kits can include one or more additional therapeutic agents, which can be administered simultaneously or sequentially with other kit components for a desired purpose, e.g., to modulate cellular activity, inhibit target cancer cells, or treat a disease in an individual in need thereof.

[0143] Any of the above-described systems and kits may further comprise one or more additional reagents, which may be selected from a dilution buffer, a reconstitution solution, a wash buffer, a conditioning reagent, a conditioning expression vector, a negative control polypeptide, a positive control polypeptide, a bispecific binding agent, or a reagent for the in vitro production of an engineered transmembrane protein.

[0144] In some embodiments, the system or kit may further include instructions for using the components of the kit to practice the method. The instructions for practicing the method are typically recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. The instructions may be present in the kit, such as on a packaging insert, on a label on a container of the kit or its components (i.e., on or in association with the packaging therein), or the like. The instructions may be present as an electronically stored data file on a suitable computer-readable storage medium (e.g., CD-ROM, diskette, flash drive, etc.). In some cases, the actual instructions are not present in the kit, but rather a means for obtaining the instructions from a remote source (e.g., via the Internet) may be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or downloaded. The instructions, along with the means for obtaining the instructions, may be recorded on a suitable substrate.

[0145] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0146] None of the references cited herein are admitted to constitute prior art. The discussion of the references states what their authors assert, and the inventors reserve the right to challenge the accuracy and pertinence of the cited documents. Although several sources of information, including scientific journals, patent documents, and textbooks, are referenced herein, this reference does not constitute an admission that any of these documents form part of the general knowledge in the art.

[0147] The discussion of the general methods presented herein is for illustrative purposes only, and other alternative methods and substitutions will be apparent to those skilled in the art upon consideration of this disclosure, and are within the spirit and scope of the present application. [Example]

[0148] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those of skill in the art, and which are fully described in the above-cited references.

[0149] Further embodiments are disclosed in more detail in the following examples, which are provided for illustrative purposes and are not intended to limit the scope of the disclosure or claims in any way.

[0150] Example 1 Synthesis of bispecific binders and engineered transmembrane proteins This example describes experiments performed to generate each of the following constructs: a bispecific IgG, a bispecific IgG with a single-chain Fab in one arm, and a Fab-scFV fusion. The mode of action of the bispecific degrader is shown in Figure 1. Figure 2 shows an engineered transmembrane protein with an anti-GFP domain control fused to RNF43.

[0151] Expression of half IgG To express half IgG from these constructs, the following 6-day process was performed: on day 1, 7.5 x 10 per transfection 7Expi293F™ cells (ThermoFisher Scientific) were dispensed into 25.5 mL of Expi293™ growth medium in a 125 mL flask. Expi™ transfection reagent was used according to the manufacturer's protocol. First, 1.5 mL of OptiMEM™ was added to a 15 mL tube, and 30 μg of plasmid DNA was added and mixed (15 μg of heavy and light chain plasmids). 1.5 mL of OptiMEM™ was aliquoted into separate tubes for each transfection, and 81 μL of ExpiFectamine™ reagent was added to each tube for each transfection. The solution was mixed and incubated at room temperature (RT) for 5 minutes. DNA was then added to ExpiFectamine™ (final volume: 3 mL) and incubated for 20 minutes. Finally, 3 mL of the DNA and ExpiFectamine™ mixture was added to OptiMEM™ in each culture flask containing Expi293F™ cells. For each transfection, the final culture volume was 28.5 mL (+ 3 mL of 1 mM biotin for Fc fusions).

[0152] On day 2, 20 hours after the last step of day 1, ExpiFectamine™ transfection enhancer was added to each culture to bring the final culture volume to 30 mL. ExpiFectamine™ Transfection Enhancer 1 (150 μL) and ExpiFectamine™ Transfection Enhancer 2 (1.5 mL) were then added to each flask.

[0153] On day 6, the cultures were centrifuged at 4137 rpm, 4 x g, and 4°C for 20 minutes in a centrifuge. Half IgG was then purified using a standard Protein A protocol, and the final knob or whole constructs were recovered by buffer exchange into 10 nM Tris (pH 7.5), 100 mM NaCl.

[0154] Half-IgG in vitro assembly For the in vitro assembly of half-IgG constructs and for the purification of bispecific IgG, a 1:1 mixture of half-IgG knob constructs and half-IgG hole constructs (see Tables 2 and 5 below) was prepared in 10 nM Tris, 100 mM NaCl (pH: 7.5). The pH of the mixture was adjusted to approximately 8.5 by adding 20% ​​800 mM L-Arg (pH: 10). A 200-fold excess of reduced glutathione in 800 mM L-Arg (pH: 10) was added to the mixture and incubated at 37°C for 16 hours. After the 16-hour incubation, the bispecific IgG was buffer-exchanged into phosphate-buffered saline (PBS) using a 30 kDa rotary concentrator. Finally, the bispecific IgG was purified by his-tag purification.

[0155] Generation of scFab-based bispecific IgG and bispecific Fab-scFv For the generation of scFab-based bispecific IgG and bispecific Fab-scFv, a 6-day process was carried out as follows.

[0156] On day 1, 7.5 x 10 cells were transfected per transfection. 7Expi293F™ cells were aliquoted into 25.5 mL of Expi293™ growth medium in a 125 mL flask. Expi™ transfection reagent was used according to the manufacturer's protocol. First, 1.5 mL of OptiMEM™ was added to a 15 mL test tube, and 30 μg of plasmid DNA was added and mixed (15 μg of heavy and light chain plasmids). 1.5 mL of OptiMEM™ was aliquoted into separate test tubes for each transfection, and 81 μL of ExpiFectamine™ reagent was added to each tube for each transfection. The solution was mixed and incubated for 5 minutes at room temperature (RT). DNA was then added to ExpiFectamine™ (final volume: 3 mL) and incubated for 20 minutes. Finally, 3 mL of the DNA and ExpiFectamine™ mixture was added to OptiMEM™ in each culture flask containing Expi293F™ cells. For each transfection, the final culture volume was 28.5 mL (+3 mL of 1 mM biotin for Fc fusions).

[0157] On day 2, 20 hours after the last step of day 1, ExpiFectamine™ transfection enhancer was added to each culture to bring the final culture volume to 30 mL. After this, 150 μL of ExpiFectamine™ Transfection Enhancer 1 and 1.5 mL of ExpiFectamine™ Transfection Enhancer 2 were added to each flask.

[0158] On day 6, the culture was centrifuged at 4000 rpm for 20 minutes. The bispecific Fab-scFv construct was purified with Protein A, and the scFab-based bispecific IgG was his-tag purified. These final constructs were buffer-exchanged and recovered in PBS.

[0159] Generated constructs The constructs generated by the above procedure include:

[0160] [Table 1]

[0161] The constructs for the E3 ligase arms in Table 1 were made using the light chain framework region of SEQ ID NO: 11, the heavy chain Fab framework region of SEQ ID NO: 12, a bispecific IgG with a single-chain Fab in one arm of SEQ ID NO: 13, the heavy chain Fc "knob" constant region of SEQ ID NO: 14 with a His tag, and Fab-scFv construct-heavy chain scFv fusions of SEQ ID NOs: 16 and 17. The LC-CDR3, HC-CDR1, HC-CDR2, and HC-CDR3 shown in A5 of Table 2 were used in all RNF43-binding arms (also referred to as RNF43 binders). The LC-CDR3, HC-CDR1, HC-CDR2, and HC-CDR3 shown in A22 of Table 2 were used in all ZNRF3-binding arms.

[0162] Constructs for target proteins were made using the light chain constant region of SEQ ID NO: 11, the heavy chain Fab constant region of SEQ ID NO: 12, a bispecific IgG with a single-chain Fab in one arm of SEQ ID NO: 13, the heavy chain Fc "hole" constant region of SEQ ID NO: 15, and Fab-scFv construct-heavy chain scFv fusions of SEQ ID NOs: 16 and 17. The LC and HC variable regions used for the target surface proteins PD-L1, HER2, EGFR, CTLA-4, MMP14, and CDCP1 are shown in Table 5 below.

[0163] Alternative LC-CDR3, HC-CDR1, HC-CDR2, and HC-CDR3 sequences used for the E3 ligases RNF43, ZNRF3, and GRAIL (RNF128) are shown below in Table 2. An additional construct was made using the light chain constant region of SEQ ID NO: 319 and the heavy chain Fab constant region of SEQ ID NO: 320.

[0164] In another example, the Fab binding arm to RNF43 was replaced with a VH binder. The sequences for the VH framework regions are provided in SEQ ID NO: 321. The VH CDR sequences for RNF43 are provided in Table 4.

[0165] [Table 2-1] [Table 2-2]

[0166] [Table 3-1] [Table 3-2]

[0167] [Table 4]

[0168] [Table 5]

[0169] Example 2 This example describes experiments performed to test each of the following constructs: a bispecific IgG, a bispecific IgG with a single-chain Fab in one arm, and a Fab-scFv fusion.

[0170] Western blot The cell lines MDA-MB-231, HCC827, H460, and T24 were tested for PD-L1 degradation by Western blot according to the following 3-day procedure.

[0171] On day 1, cells were incubated in a 6-well plate at approximately 60-70% confluency and then treated with different concentrations of bispecific antibodies in 1 mL of fresh growth medium. The cells were then incubated for a set time (24 hours).

[0172] On day 2, 24 hours after incubation with the bispecific antibody, samples were considered ready for Western blot analysis. To do this, the cell culture medium was aspirated and the cells were washed with cold PBS. The cells were then suspended in Gibco® Versene solution and centrifuged. The supernatant was then removed, and the cell pellets were individually resuspended in 140 μL of RIPA lysis buffer + cOmplete™ protease inhibitor cocktail (Millipore Sigma, #11836170001) and transferred to Eppendorf tubes. The resuspended cells were then incubated with lysis buffer at 4°C for 30 minutes. (Lysis buffer: 5 M NaCl (3 mL), 1 M Tris-HCl (5 mL, pH 8.0), Nonidet™ P-40 (1 mL), 10% sodium deoxycholate (5 mL), 10% SDS (1 mL), ddH2O (qs to 100 mL)). The cell lysate was then centrifuged at 15,000 g for 10 minutes at 4°C, and 100 μL of the soluble fraction was removed. Protein concentration was normalized using a bicinchoninic acid assay (BCA assay, also known as Smith assay). The diluted lysate was added to 20 μL of LDL buffer + 2 μL of BME, and the solution was boiled for 10 minutes. The lysate was electrophoresed on an SDS page gel (200 V, 37 minutes), and the gel was blocked with a 20% ethanol solution. The blocked gel was transferred to a polyvinylidene fluoride (PVDF) membrane using the iBlot2® platform. The membrane was blocked for 60 minutes using the manufacturer's blocking buffer. Primary antibodies were added to 7.5 mL of blocking buffer + 0.2% Tween® 20. The ratios for anti-PD-L1 and anti-tubulin were 1:1000 and 1:2000, respectively. Finally, the membranes were gently shaken overnight at 4°C in a black box.

[0173] On day 3, the overnight buffer was removed, and the membrane was washed with 1x TBS-T (0.1% Tween® 20), covered with 1x TBS-T (0.1% Tween® 20), and shaken at room temperature for 5 minutes. The wash solution was discarded, and this washing step was repeated three more times. The secondary antibody was diluted in 8 mL of blocking buffer + 0.2% Tween® 20 (160 μL) + 0.01% SDS (8 μL). Two secondary antibodies were used: goat anti-rabbit (800 nm) and goat anti-mouse (680 nm). The membrane was incubated with the secondary antibody in the dark for 1 hour at room temperature with gentle shaking. Following this incubation, the membrane was washed with 1x TBS-T (0.1% Tween® 20), covered with 1x TBS-T (0.1% Tween® 20), and shaken at room temperature for 5 minutes. The wash solution was discarded, and this washing step was repeated three more times. The membrane was finally washed with 1x PBS to remove residual Tween® 20, and the membrane was imaged on a Li-Cor® Imaging System.

[0174] Exemplary Western blot results for the effect of the tested bispecific IgG or atezolizumab (Tecentriq®, all at 10 nM in solution) on PD-L1 levels in MDA-MB-231, HCC827, or T24 cell lines after 24 hours of treatment are shown in Figures 5A, 5B, and 5C. In summary, the tested bispecific IgG was able to degrade PD-L1 in these three different clinically relevant cell lines (MDA-MB-231, HCC827, or T24), whereas atezolizumab showed little or no degradation.

[0175] Figure 6 also shows the effect of bispecific RNF43-PD-L1 IgG on the degradation of PD-L1 from the triple-negative breast cancer cell line MDA-MB-231. From left to right, each bar represents a PBS control, a construct with 10 nM RNF43 A5 (SEQ ID NOs: 332 and 333), a Fab construct with 10 nM RNF43 A4 (SEQ ID NOs: 322 and 323), and a Fab construct with 10 nM RNF43 A6 (SEQ ID NOs: 324 and 325). All constructs were bispecific IgGs with one arm targeting RNF43 and the other binding arm to PD-L1 in Tecentriq. The PD-L1-binding variable region was the same for all constructs and was represented by SEQ ID NOs: 106 and 107. Western blots were performed as described in Example 2 of this disclosure.

[0176] Flow cytometry The cell lines MDA-MB-231, HCC827, H460, and T24 were tested for PD-L1 by flow cytometry following the two-day process described below.

[0177] On day 1, cells were incubated in a 6-well plate at approximately 60-70% confluency and then treated with different concentrations of bispecific antibodies in 1 mL of fresh growth medium. The cells were then incubated for a set time (24 hours).

[0178] On day 2, 24 hours after incubation with the bispecific antibody, the samples were considered ready for Western blot analysis. To do this, the cell culture medium was aspirated and the cells were washed with cold PBS. The cells were then suspended in Gibco® Versene solution and centrifuged. The supernatant was then removed, and the cell pellets were individually washed with 1x PBS. The cells were then blocked with PBS + 3% BSA, and biotinylated antibodies were added to the samples and incubated for 30 minutes at 4°C with shaking. The cells were then washed three times with PBS + 3% BSA. Alexa Fluor® 647 Streptavidin (ThermoFisher Scientific) was then added, and the cells were incubated for 30 minutes at 4°C with shaking. The cells were then washed three times with PBS + 3% BSA. Finally, the cells were resuspended in 200 μL of PBS and run on a flow cytometer.

[0179] Flow cytometry results show that the tested dual-specific IgGs were able to degrade PD-L1 in these three different clinically relevant cell lines (MDA-MB-231, HCC827, and T24), whereas atezolizumab showed little or no degradation.

[0180] Example 3 Engineered Membrane Proteins This example describes experiments performed on the synthesis of RNF43 engineered transmembrane proteins and reporter construct degradation studies.

[0181] Transfection and synthesis All DNA fragments were purchased from IDT and assembled using Gibson Cloning. DNA fragments with 30 bp overlaps were incubated with the cleavage vector pFUSE vector and Gibson master mix at 50°C for 30 minutes. An engineered transmembrane protein was designed based on RNF43 and anti-GFP scFab. The anti-GFP scFab sequences are provided in SEQ ID NO:2 (light chain) and SEQ ID NO:4 (heavy chain), with the binding domain shown in SEQ ID NO:3. A short linker (SEQ ID NO:5) connects the scFab domain to the RNF43 domain (SEQ ID NO:6). The complete sequence is shown in SEQ ID NO:1.

[0182] The reporter construct was assembled from a GFP domain (SEQ ID NO: 8), a transmembrane / linker domain (SEQ ID NO: 9), and a nanoluciferase domain (SEQ ID NO: 10). The complete sequence of the reporter construct is provided in SEQ ID NO: 7.

[0183] The Gibson product was transformed into XL10 integrant cells by heat shock. Transformed cells were allowed to recover for 1 hour at 37°C. Recovered cells were plated onto LB / Carbenicillin plates overnight at 37°C.

[0184] On day 2, single colonies from the overnight plates were picked and added to 5 mL of low-salt LB / Carbenicillin and incubated at 37°C until confluent. Once the cells were confluent, DNA was miniprepped and sequenced.

[0185] For the synthesis of RNF43-engineered transmembrane proteins, Hek293 or HeLa cells were transiently transfected with both the reporter GFP-Nanoluc construct and the RNF43-engineered transmembrane protein using TransIT®-293 transfection reagent. Cells were grown in 6-well plates to 60-70% confluency. DNA (2.5 μg) was incubated with 7.5 μL of TransIT-293 reagent in an Opti-membrane tube at room temperature for 20 minutes, and the DNA-TransIT mixture was added to the cells.

[0186] A stable cell line expressing the GFP-NanoLuc construct was generated using Hek293 FLP / IN cells. Because GFP-NanoLuc was already present in this cell line, the following experiments (nanoluciferase readout) were performed by transiently transfecting this cell line with the RNF43 fusion. Transient transfections were performed in 6-well plates with cells at approximately 60% confluency.

[0187] Nanoluciferase readout For the nanoluciferase readout described above, 24 hours after transfection with the appropriate construct(s), the transfected cells were split into 96-well plates and allowed to sit for 24 hours. Nano-Glo® Reagent was thawed at room temperature and mixed 1:50. An equal volume of reagent was added to the cells (100 μL), and the cells were shaken for 10 minutes at room temperature. Finally, chemiluminescence was read on a plate reader. Compared to the negative control, the reporter protein added to the anti-GFP-RNF43 fusion significantly reduced the nanoluciferase signal.

[0188] Confocal microscope For confocal microscopy and subsequent confocal fluorescence imaging, cells were transfected as described above and incubated for 48 hours. After 48 hours, cells were plated onto glass-bottom Petri dishes for 12 hours before imaging. Before imaging, the cell culture medium was replaced and LysoTracker® was added to this solution. Cells were fixed with 4% PFA and permeabilized with 0.5% Triton™-X in PBS. DAPI (4',6-diamidino-2-phenylindole) was incubated with the permeabilized cells. Finally, cells were washed three times with PBS and imaged at 100x magnification using a confocal microscope equipped with a rotating stage.

[0189] Confocal microscopy showed that soluble GFP Fab alone had no effect on GFP reporter localization, whereas the anti-GFP-RNF43 engineered transmembrane protein caused internalization and lysosomal aggregation of the GFP reporter. These data suggest that RNF43 can be used to induce proteolysis of endogenous proteins.

[0190] Example 4 This experiment was designed to generate an AAV transfection vector that would insert an engineered transmembrane protein into target cells.

[0191] AAV transfer plasmids were constructed by placing genes expressing the scFab-E3 engineered transmembrane proteins under the control of CAG, EF1, or tissue-specific promoters. HEK293T cells were transfected with an AAV helper plasmid (pHelper), a Rep-Cap plasmid (pAAV-RC1 or pAAV-RC9), and an AAV transfer plasmid at a 1:1:2 ratio. The cells were incubated at 37°C under 5% CO2 for 3 days. The cells were then harvested and lysed by sonication in PBS buffer supplemented with 0.001% Pluronic® Acid and 200 mM NaCl. The cell debris was pelleted at 3,200 x g for 15 minutes at 4°C, and the supernatant was transferred to another tube. Benzonase (50 units / mL) was added to the supernatant and further incubated at 37°C for 45 minutes. The supernatant was purified by centrifugation at 2,400 g for 10 minutes at 4°C. The recombinant AAV was then purified twice by iodixanol gradient ultracentrifugation (15%, 25%, 40%, and 60% iodixanol diluted in PBS-MK gradient buffer), and the 40% fractions were pooled and desalted using a MWCO 100 kDa centrifugal concentrator. The desalted AAV was then stored at -80°C in PBS buffer supplemented with 0.001% Pluronic® Acid and 200 mM NaCl.

[0192] For the production of exosome-associated AAV (exoAAV), a stable HEK293T cell line capable of overexpressing CD9-GFP was constructed by lentiviral transduction. HEK293T CD9-GFP cells were transfected with an AAV helper plasmid (pHelper), a Rep-Cap plasmid (pAAV-RC1 or pAAV-RC9), and an AAV transfer plasmid at a 1:1:2 ratio. The cells were then incubated in exosome-depleted medium at 37°C under 5% CO2 for 3 days, after which the cell culture medium was collected and depleted at 300xg for 5 minutes and 1000xg for 10 minutes. The supernatant was centrifuged at 20,000xg at 15°C for 1 hour and then centrifuged again at 100,000xg at 15°C for 1.5 hours. The final exoAAV product was then stored at 4°C.

[0193] In vitro targeting of AAV A HeLa cell line stably expressing the GFP-nanoluciferase (GFP-Nluc, SEQ ID NO: 7) reporter gene is constructed using the Flp-In™ system recombination system (ThermoFisher Scientific). GFP-Nluc Cells were seeded in 96-well plates at 50,000 cells / well for 24 hours. Cells were then incubated overnight at 37°C in 5% CO2 with 10% generic AAV or exoAAV. 8 The cells were transduced with 100 genome copies. The culture medium was replaced with Dulbecco / Vogt modified Eagle's minimum essential medium (DMEM) containing 10% FBS, and the cells were further incubated at 37°C under 5% CO2. 48 hours after transduction, luciferase assays and flow cytometry were performed using the same procedures as above.

[0194] Example 5 Dynamics requirements This example provides further data on the use of bispecific antibodies to degrade PD-L1 by recruiting RNF43. The results were somewhat unexpected. We concluded that there are affinity requirements for each component of a bispecific antibody that determine its merit as a degrading agent. However, it was initially hypothesized that strong binding is not ideal, and that a somewhat weaker binder would improve turnover during the degradation process, thereby increasing the amount of degradation. Surprisingly, a strong binder with a slow off-rate was actually required to induce degradation.

[0195] Alanine scanning of key residues involved in binding to PD-L1 was performed on the PD-L1 binding component Tecentriq. The mutants were expressed as Fabs to measure their kinetic parameters (Kd, Kon, Koff). These were then made into bispecific antibodies, the other of which was an anti-RNF43 A5 construct. Degradation experiments of 10 nM bispecific IgG against MDA-MB-231 cells were then performed using the same protocol as described in Example 2 above in a Western blot to quantify degradation. From these data, the amount of degradation was plotted against the different kinetic parameters. From this data, a correlation (R 2 = 0.67), which means that the slower the off-rate, the greater the amount of decomposition.

[0196] Similar experiments were then performed by alanine scanning of the RNF43 A5 binder of clone anti-RNF43. Alanine mutants of CDR H3 were generated. Binding was completely attenuated for all but two clones, with two clones retaining some binding. The two mutants that retained some binding had affinities for RNF43 of 40 nM and 125 nM (S113A and F115A, respectively). These were used in conjunction with wild-type PD-L1 binders of SEQ ID NOs: 106 and 107 to form bispecific IgGs, and the degradation experiments described above were repeated. This time, degradation was only observed with the wild-type RNF43 binder with an affinity of 12.5 nM; PD-L1 levels remained unchanged when RNF43 binding was reduced to 40 nM or 125 nM. Again, this data suggests that strong binders are required on each side of the bispecific antibody.

[0197] Figure 7 also shows the biolayer interferometry (BLI) graphs for each Ala mutant. The kinetic parameters for each alanine mutant shown in the biolayer interferometry (BLI) graphs of Figure 7 are shown in Table 6 below.

[0198] [Table 6]

[0199] Figure 8 shows the correlation between percent degradation and Koff. The slower the off-rate, the greater the degradation. Furthermore, Figure 9 shows the correlation between percent degradation and Kd. As shown, there is a slight correlation, meaning that stronger binders are associated with greater degradation. Figure 10 shows that there is no correlation between percent degradation and Kon. Figure 11 shows Western blots of anti-RNF43 alanine mutants. The mutants are distinguished by their Kd for RNF43: 12.5 nM for wild-type RNF43 A5, 40 nM for S113A, and 125 nM for F115A. The results demonstrate that after 24 hours of treatment with 10 nM of the bispecific binder, only the most potent anti-RNF43 construct degraded PD-L1.

[0200] Example 6 Here, we describe an exemplary method for conjugating a small molecule to a Fab construct. These data suggest that immune complexes containing the binding agents of the present disclosure can be recruited to targets and induce their degradation. An exemplary diagram of the antibody-drug conjugate disclosed herein is shown in Figure 12.

[0201] Cell lines. Cell lines were grown and maintained in T75 (Thermo Fisher Scientific) flasks at 37°C and 5% CO2. MOLT-4 CCR5+ cells were grown in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 2% geneticin. MOLT-4 CCR5+ cells were obtained from the NIH AIDS Reagent Program.

[0202] Antibody cloning, expression, and purification. The anti-RNF43 Fab LC S7M single mutation was introduced using Gibson Assembly. The Fab was expressed in E. coli C43(DE3) Pro+ using optimized autoinduction medium and purified by protein A affinity chromatography (Hornsby, M. et al. A High Throughput Platform for Recombinant Antibodies to Folded Proteins. Mol. Cell. Proteomics 14, 2833-2847 (2015)). Fab purity and integrity were assessed by SDS-PAGE and intact mass spectrometry. The light and heavy chain sequences of the anti-RNF43 Fab used in the antibody-drug conjugate are shown in SEQ ID NOs: 326 and 327, respectively.

[0203] Synthesis of DBCO-CGS21680. Commercially available CGS21680 (Cayman Chemical, 17126, 5 mg, 0.01 mmol) was added to a 2 mL dimethylformamide solution of 1.5 equivalents of 1-[bis(dimethylaminomethylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 6 mg, 0.015 mmol) and 4 equivalents of N,N-diisopropylethylamine (7 μL, 0.04 mmol) and stirred at room temperature for 10 min. Then, 1.5 equivalents of DBCO-PEG4-amine (BroadPharm, BP-23958 (5 mg, 0.015 mmol) was added to the reaction flask and stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography (HPLC). The final product was lyophilized and isolated as a pale yellow powder (4.8 mg, yield: 48%). Calculated ESI-HRMS [M+H + ]=1005.48; Actual value 1005.54

[0204] Conjugation of modified anti-RNF43 Fab with oxaziridine and DBCO-CGS21680. Figure 13 shows an exemplary diagram of the conjugation process. For oxaziridine conjugation, 50 μM Fab was incubated with 5 molar equivalents of oxaziridine azide in phosphate-buffered saline (PBS) at room temperature for 30 minutes (AH Christian et al., A physical organic approach to tuning reagents for selective and stable methionine bioconjugation. J. Am. Chem. Soc. 141, 12657-12662 (2019)). The reaction was quenched with 10 molar equivalents of methionine. The antibody was buffer-exchanged into PBS and desalted on a 0.5 mL Zeba 7-kDa desalting column (Thermo Fisher Scientific). 10 molar equivalents of DBCO-CGS21680 was then added and incubated overnight at room temperature. The agonist-labeled complex was desalted using a 0.5 mL Zeba 7-kDa desalting column to remove excess DBCO-CGS21680. Complete conjugation at each step was monitored by intact mass analysis using a Xevo G2-XS Mass Spectrometry (Waters). Some exemplary small molecules used for conjugation are shown in Figure 14. This is not intended to be a comprehensive list of small molecules that can be used for conjugation, and those skilled in the art will recognize that alternative small molecules can be conjugated to the antigen-binding agents provided in this disclosure based on their utility.

[0205] Degradation assay: Cells at 1 million cells / mL were treated with antibody-drug conjugates, agonists, or antagonists in complete growth medium. After 24 hours, cells were pelleted by centrifugation (300 x g, 5 minutes, 4°C). Cell pellets were lysed in RIPA buffer containing cOmplete™ mini protease inhibitor cocktail for 40 minutes on ice. Lysates were centrifuged at 16,000 x g for 10 minutes at 4°C, and protein concentrations were normalized using a BCA assay. Fourfold NuPAGE LDS sample buffer and 2-mercaptoethanol (BME) were added to the lysates. Equal amounts of lysates were loaded onto 4-12% Bis-Tris gels and run at 200V for 37 minutes. The gels were incubated in 20% ethanol for 10 minutes and transferred to polyvinylidene fluoride (PVDF) membranes. The membrane was blocked with 0.1% Tween 20 + 5% bovine serum albumin (BSA) in PBS for 30 minutes at room temperature with gentle shaking. The membrane was then co-incubated with rabbit anti-A2aR (Abcam, ab3461, 1:1000) and mouse anti-tubulin (Cell Signaling Technologies, DM1A, 1:1600) in PBS + 0.2% Tween 20 + 5% BSA overnight at 4°C with gentle shaking. The membrane was washed four times with Tris-buffered saline (TBS) + 0.1% Tween 20 and then co-incubated with HRP-anti-rabbit IgG (Cell Signaling Technologies, 7074S, 1:2000) and 680RD goat anti-mouse IgG (LI-COR, 926-68070, 1:10000) in PBS + 0.2% Tween 20 + 5% BSA for 1 hour at room temperature. The membrane was washed four times with TBS + 0.1% Tween 20 and then with PBS. The membrane was first imaged using an OdysseyCLxImager (LI-COR). SuperSignal West Pico PLUS Chemiluminescent Substrate was then added and imaged using a ChemiDoc Imager (BioRad). Band intensity was quantified using Image Studio Software (LI-COR). Exemplary results are shown in Figures 15 and 16.In particular, Figure 15 shows the degradation of adenosine 2a receptors (A2aR) in MOLT-4 CCR5+ cells after 24 hours of treatment, and Figure 16 shows the A2aR abundance after 24 hours of treatment with CGS21680 (agonist) or ZM241385 (antagonist). These data suggest that immunoconjugates can be used to recruit RNF43 to A2aR and induce its degradation at a concentration of 1 nM after 24 hours (Figure 15). Figure 16 is a control showing that treatment with only the small molecule (agonist at 100 nM) without binding to anti-RNF43 Fab has no effect on A2aR abundance.

[0206] Other targets include, but are not limited to, CXCR4, CCR5, Smoothened, CCR2, CCR9, proteinase-activated receptor 1 (PAR1), PAR2, mu opioid receptor, delta opioid receptor, kappa opioid receptor, and neurokinin receptor 1.

[0207] While certain alternatives of the present disclosure have been disclosed, it will be apparent that various modifications and combinations are possible and contemplated within the true spirit and scope of the appended claims. Accordingly, no limitation is intended to the summary and disclosure set forth herein.

[0208] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] Table 7-8 Table 7-9 Table 7-10 Table 7-11 Table 7-12 Table 7-13 Table 7-14 Table 7-15

Claims

1. 1. A bispecific binding agent for degrading PD-L1 protein present on the surface of a target, said bispecific binding agent comprising: (a) a first binding domain that specifically binds to an extracellular epitope of the RNF43 protein; and (b) a second binding domain that specifically binds to an extracellular epitope on the PD-L1 protein; wherein contact of the bispecific binding agent with the PD-L1 protein and the RNF43 protein results in degradation of the PD-L1 protein.

2. 2. The bispecific binding agent of claim 1, wherein the first binding domain binds to the RNF43 protein with a Kd of less than 100 nM.

3. 3. The bispecific binding agent of claim 2, wherein the first binding domain binds to the RNF43 protein with a Kd of 12.5 nM or less.

4. 2. The bispecific binding agent of claim 1 , wherein contact of the PD-L1 protein and the RNF43 protein with the bispecific binding agent results in internalization and lysosomal aggregation of the PD-L1 protein.

5. 10. The bispecific binding agent of claim 1, wherein the bispecific binding agent comprises a bispecific antibody or antibody derivative.

6. 2. The bispecific binding agent of claim 1, wherein the bispecific binding agent comprises a knob and hole bispecific IgG.

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