PROTEOLYSIS-TARGETING ANTIBODIES AND METHODS OF USE THEREOF

Abstract

Antigen-binding molecules for targeting protein degradation are provided. The molecules typically comprise (1) TRIM21-binding characteristics, (2) the cell-permeability characteristics of a cell-permeable antibody, and (3) the antigen-binding characteristics that bind to the target protein. The TRIM21-binding characteristics typically comprise a polypeptide sequence sufficient to bind to TRIM21 and are effective in directing the target protein along the TRIM21 degradation pathway. The cell-permeability characteristics of the cell-permeable antibody typically enhance the delivery of the molecule across the cell membrane. The antigen-binding characteristics bind to the target protein and mediate binding of the molecule to the target protein. In some embodiments, the antigen-binding molecule is a multispecific antibody, e.g., a bispecific or trispecific antibody. Compositions comprising an effective amount of the antigen-binding molecule and methods of use thereof are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 375,140, ​​filed September 9, 2022, which is incorporated herein by reference in its entirety.

[0002] Reference to sequence listing The Sequence Listing submitted as a text file entitled "YU_8465_PCT_ST26.xml", created on September 11, 2023, and having a size of 131,184 bytes, is hereby incorporated by reference pursuant to 37 CFR Rule 1.52(e)(5).

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under R35 CA197574 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0004] FIELD OF THE INVENTION The field of the invention is generally that of targeting protein degradation. [Background technology]

[0005] Background of the Invention Methods for targeted disruption of protein function have revolutionized science and greatly facilitated the systematic characterization of genes. Two main approaches are currently used to disrupt protein function: DNA knockout and RNA interference, which act at the genomic and mRNA levels, respectively. Antibody-based techniques have been developed to sensitively degrade endogenous proteins in mammalian cells without prior modification of the genome or mRNA. However, "Trim-Away" typically utilizes transfection assistance such as electroporation to facilitate entry of the composition into cells, limiting its use in clinical settings (Clift, et al., Cell. 2017;171(7):1692-1706.e18. doi:10.1016 / j.cell.2017.10.033; Clift, Nat Protoc 13, 2149-2175 (2018). doi.org / 10.1038 / s41596-018-0028-3; Zeng, Nat Struct Mol Biol 28, 278-289 (2021). doi.org / 10.1038 / s41594-021-00560-2).

[0006] Thus, there remains a need in this area for improved compositions and methods. It is an object of the present invention to provide improved compositions and methods for targeting protein degradation. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Clift, et al., Cell. 2017;171(7):1692-1706.e18. doi:10.1016 / j.cell.2017.10.033 [Non-patent document 2] Clift, Nat Protoc 13, 2149-2175 (2018). doi.org / 10.1038 / s41596-018-0028-3 [Non-patent document 3] Zeng, Nat Struct Mol Biol 28, 278-289 (2021). doi.org / 10.1038 / s41594-021-00560-2 Summary of the Invention [Means for solving the problem]

[0008] Summary of the Invention Antigen-binding molecules for targeting protein degradation and methods of use thereof are provided. The molecules typically comprise (1) TRIM21-binding characteristics, (2) the cell-permeability characteristics of a cell-permeability antibody, and (3) the antigen-binding characteristics that bind to the target protein.

[0009] TRIM21-binding characteristics typically comprise a polypeptide sequence sufficient to bind to TRIM21 and, when bound to one or more antigen-binding molecules, are effective in directing the target protein along the TRIM21 degradation pathway. Preferred TRIM21-binding characteristics comprise the boundary of the CH2 and CH3 domains of an immunoglobulin constant region, more preferably the CH2 and CH3 domains of an immunoglobulin constant region (e.g., an Fc region), optionally wherein the immunoglobulin is an IgG, optionally a human IgG, e.g., an IgG1.

[0010] The cell-penetrating characteristic of a cell-penetrating antibody typically enhances the delivery of an antigen-binding molecule across a cell membrane. The cell-penetrating characteristic can be the cell-penetrating domain of an autoantibody, such as 3E10 or 5C6, or a cell-penetrating variant or humanized form thereof. In some embodiments, the antigen-binding molecule comprises the complementarity-determining region (CDR) of a cell-penetrating antibody (e.g., 3E10, 5C6, or a variant or humanized form thereof), and optionally the heavy and light chain variable regions, regardless of whether such regions are important for cell penetration. In some embodiments, the antigen-binding molecule comprises the Fc region of a cell-penetrating antibody, regardless of whether such regions are important for cell penetration.

[0011] The antigen-binding feature binds to a target protein and mediates binding of the molecule to the target protein. Some molecules feature two or more antigen-binding features that bind to different epitopes on the same target protein, different epitopes on different proteins, or a combination thereof. The antigen-binding feature can be a traditional VH and VL domain, a single-chain antibody, a single-chain variable fragment (scFv), a di-scFv, a tri-scFv, a diabody, a triabody, a teratbody, a disulfide-linked Fv (sdFv), a Fab', a F(ab')2, an Fv, a single-domain antibody fragment (sdAb), a nanobody, or a VHH. In some embodiments, the antigen-binding feature is derived from a known antibody against the target protein. In some embodiments, the antigen-binding molecule comprises the Fc region of a known antibody.

[0012] In some embodiments, the antigen-binding molecule comprises an additional sequence or conjugate. An exemplary additional sequence is the amino acid sequence of TRIM21 or a functional fragment or variant thereof. Thus, in some embodiments, the antigen-binding molecule is or comprises a TRIM21 fusion protein.

[0013] In some embodiments, the antigen-binding molecule is a multispecific antibody, e.g., a bi- or trispecific antibody. In some embodiments, the bispecific and trispecific antibody is a bispecific or trispecific IgG, IgG-scFv, IgG-dAb, scFv-Fc-scFv, knob-in-hole (KIH)-IgG, κλ-body, KIH0Fc-Fab / scFv, tandem scFv, KIH trispecific, bispecific Fc fusion (with or without N- or C-terminus, KIH).

[0014] Also provided are compositions comprising an effective amount of antigen-binding molecules.In some embodiments, the compositions comprise an effective amount of two or more different antigen-binding molecules.In some embodiments, the compositions comprise two or more different antigen-binding molecules that target the same protein, two or more different antigen-binding molecules that target different proteins, or a combination thereof.

[0015] Methods of use are also provided, including, but not limited to, targeting degradation of one or more target proteins. Such methods typically involve contacting cells with an effective amount of an antigen-binding molecule. The contacting can occur in vitro, ex vivo, or in vivo in a subject in need thereof. In some embodiments, the composition is administered by injection or infusion. In some embodiments, the subject has a disease or disorder, such as one caused by overexpression and / or abnormal expression of the target protein and / or expression of a mutant thereof. In such embodiments, the composition can be administered in an effective amount to treat the disease or disorder. [Brief explanation of the drawings]

[0016] [Figure 1] 1A-1C are autoradiograms of Western blots showing the effect of 3E10 treatment on RAD51 in HCC38 human breast cells (FIG. 1A), YUGASP human melanoma cells (FIG. 1B), and U2OS human osteosarcoma cells (FIG. 1C). [Figure 2] Figure 2 is a diagram of an exemplary chimeric antibody with the Fab portion containing cell-penetrating characteristics, the other Fab arm consisting of binding characteristics for a protein of interest, and the Fc portion containing TRIM21 binding characteristics. [Figure 3A]Figures 3A-3B demonstrate proteosome-mediated degradation of bispecific antibodies. Figure 3A is an image showing steps in which B16 cells were treated with cell-permeable 3E10 / GFP bispecific Ab in the presence or absence of a proteosome inhibitor (MG132). Figure 3B is an image of a Western blot showing the levels of bispecific antibody in the absence or presence of the proteosome inhibitor MG132. [Figure 3B] Figures 3A-3B demonstrate proteosome-mediated degradation of bispecific antibodies. Figure 3A is an image showing steps in which B16 cells were treated with cell-permeable 3E10 / GFP bispecific Ab in the presence or absence of a proteosome inhibitor (MG132). Figure 3B is an image of a Western blot showing the levels of bispecific antibody in the absence or presence of the proteosome inhibitor MG132. [Figure 4A] Figures 4A-4C show the degradation of nuclear green fluorescent protein (GFP) after treatment with the cell-permeable 3E10 / GFP bispecific antibody. Figure 4A shows an image depicting the level of fluorescence generated by the presence of a GFP control (no antibody) after incubation with the bispecific antibody. Figures 4B and 4C are a pair of plots quantifying the amount of GFP after incubation with the control (no antibody, Figure 4B) and the bispecific antibody (Figure 4C). [Figure 4B] Figures 4A-4C show the degradation of nuclear green fluorescent protein (GFP) after treatment with the cell-permeable 3E10 / GFP bispecific antibody. Figure 4A shows an image depicting the level of fluorescence generated by the presence of a GFP control (no antibody) after incubation with the bispecific antibody. Figures 4B and 4C are a pair of plots quantifying the amount of GFP after incubation with the control (no antibody, Figure 4B) and the bispecific antibody (Figure 4C). [Figure 4C]Figures 4A-4C show the degradation of nuclear green fluorescent protein (GFP) after treatment with the cell-permeable 3E10 / GFP bispecific antibody. Figure 4A shows an image depicting the level of fluorescence generated by the presence of a GFP control (no antibody) after incubation with the bispecific antibody. Figures 4B and 4C are a pair of plots quantifying the amount of GFP after incubation with the control (no antibody, Figure 4B) and the bispecific antibody (Figure 4C). DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed Description of the Invention I. Definition As used herein, the term "single-chain Fv" or "scFv" refers to a single-chain variable fragment comprising a light chain variable region (VL) and a heavy chain variable region (VH) joined in a single polypeptide chain by a linker which enables the scFv to form the desired structure for antigen binding (i.e., for the VH and VL of the single polypeptide chain to associate with each other to form the Fv). The VL and VH regions may be derived from a parent antibody or may be synthesized chemically or recombinantly.

[0018] As used herein, the term "variable region" is intended to distinguish such domains of immunoglobulins from domains broadly shared by antibodies (such as the antibody Fc domain). The variable region contains "hypervariable regions" whose residues are responsible for antigen binding. The hypervariable region comprises amino acid residues from the "complementarity determining regions" or "CDRs" (i.e., typically residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain, and residues 27-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)), and / or residues from the "hypervariable loops" (i.e., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain, and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917).

[0019] As used herein, the terms "framework region" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0020] As used herein, the term "antibody" refers to a natural or synthetic antibody that binds to a target antigen. The term includes polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, the term "antibody" also includes binding proteins, fragments, and polymers of these immunoglobulin molecules, as well as human or humanized versions of immunoglobulin molecules that bind to a target antigen.

[0021] As used herein, the term "cell-permeable antibody" refers to an immunoglobulin protein, fragment, variant thereof, or fusion protein based thereon that is transported into the cytoplasm and / or nucleus of a living mammalian cell. A "cell-permeable anti-DNA antibody" specifically binds to DNA (e.g., single-stranded and / or double-stranded DNA). In some embodiments, the antibody is transported into the cytoplasm of the cell without the aid of a carrier or conjugate. In other embodiments, the antibody is conjugated to a cell-permeable moiety, e.g., a cell-permeable peptide. In some embodiments, the cell-permeable antibody is transported into the nucleus with or without a carrier or conjugate.

[0022] As used herein, the term "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Generally, the differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and a reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). The substituted or inserted amino acid residues may or may not be those encoded by the genetic code. A variant of a polypeptide may be naturally occurring, such as an allelic variant, or may be a variant that is not known to occur naturally.

[0023] Modifications and changes can be made in the structure of the polypeptide of the present disclosure, and still obtain molecules with similar properties (e.g., conservative amino acid substitutions) to the polypeptide. For example, certain amino acids can be substituted with other amino acids in the sequence without appreciable loss of activity. Because it is the interaction ability and characteristics of a polypeptide that define its biological functional activity, certain amino acid sequence substitutions can be made in the polypeptide sequence, and still obtain polypeptides with similar properties.

[0024] When making such changes, the hydropathic index of the amino acid can be taken into consideration. The importance of the hydropathic amino acid index in imparting interactive biological function to a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted with other amino acids having similar hydropathic indexes or scores and still result in polypeptides with similar biological activity. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. These indices are isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0025] The relative hydropathic characteristics of amino acids are believed to determine the secondary structure of the resulting polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and cofactors. It is known in the art that an amino acid can be substituted with another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, substitution of amino acids whose hydropathic index is within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0026] Similar amino acid substitutions can also be made based on hydrophilicity, particularly for the creation of biologically functional equivalent polypeptides or peptides intended for use in immunological embodiments. The following hydrophilicity values ​​have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamic acid (+0.2); glycine (0); proline (-0.5±1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent, polypeptide. In such changes, substitutions of amino acids whose hydrophilicity values ​​are within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0027] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into consideration the various aforementioned characteristics are well known to those of skill in the art and include the following (original residue: exemplary substitution): (Ala:Gly, Ser), (Arg:Lys), (Asn:Gln, His), (Asp:Glu, Cys, Ser), (Gln:Asn), (Glu:Asp), (Gly:Ala), (His:Asn, Gln), (Ile:Leu, Val), (Leu:Ile, Val), (Lys:Arg), (Met:Leu, Tyr), (Ser:Thr), (Thr:Ser), (Tip:Tyr), (Tyr:Trp, Phe), and (Val:Ile, Leu). Embodiments of the present disclosure therefore contemplate functional or biological equivalents of the above-described polypeptides. In particular, embodiments of the polypeptides may include variants having about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the polypeptide of interest.

[0028] As used herein, the term "sequence identity percentage (%)" is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical to the nucleotides or amino acids in a reference nucleic acid sequence after aligning the sequences and introducing gaps as necessary to achieve the maximum sequence identity percentage.Alignment for determining sequence identity percentage can be achieved in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.Appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences to be compared, can be determined by known methods.

[0029] As used herein, the term "specifically binds" refers to the binding of an antibody to its cognate antigen (e.g., DNA) without significant binding to other antigens. Specific binding of an antibody to a target under such conditions requires the antibody to be selected for its specificity for the target. Various immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Preferably, the antibody binds to a second molecule within about 10 5 mol -1 greater than (e.g., 10 6 mol -1 , 10 7 mol -1 , 10 8 mol -1 , 10 9 mol -1 , 10 10 mol -1 , 10 11 mol -1 , and 10 12 mol -1 The antibody "specifically binds" to the antigen with an affinity constant (Ka) equal to or greater than 0.01.

[0030] As used herein, the term "monoclonal antibody" or "MAb" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for naturally occurring mutations that may be present in a small subset of antibody molecules.

[0031] As used herein, the term "subject" means any individual who is the target of administration. A subject may be a vertebrate, e.g., a mammal. Thus, a subject may be a human. The term does not denote a particular age or sex.

[0032] As used herein, the term "effective amount" means that the amount of the composition used is sufficient to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration requires only reduction or alteration, not necessarily elimination. The exact dosage will vary according to various factors, such as subject-dependent variables (e.g., age, immune system health, etc.), the disease or disorder being treated, and the route of administration and pharmacokinetics of the administered agent.

[0033] As used herein, the term "pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject without causing any undesired biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

[0034] As used herein, the term "carrier" or "excipient" refers to an organic or inorganic component, a natural or synthetic inactive component in a formulation, with which one or more active components are combined. Naturally, the carrier or excipient will be selected to minimize any degradation of the active component and to minimize any adverse side effects in the subject, as would be well known to one skilled in the art.

[0035] As used herein, the term "treating" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, condition, or disorder. This term includes active treatment, i.e., treatment specifically directed toward ameliorating a disease, condition, or disorder, and also includes causal treatment, i.e., treatment directed toward eliminating the cause of the associated disease, condition, or disorder. In addition, this term includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure the disease, condition, or disorder; preventative treatment, i.e., treatment directed toward minimizing or partially or completely inhibiting the occurrence of the associated disease, condition, or disorder; and adjunctive treatment, i.e., treatment used to supplement another specific therapy directed toward ameliorating the associated disease, condition, or disorder.

[0036] As used herein, a "targeting moiety" is a substance that can direct a particle or molecule to a receptor site in a selected cell or tissue type, can act as an adhesion molecule, or can act to couple or attach another molecule. As used herein, "directing" refers to preferentially attaching a molecule to a selected cell or tissue type. This can be used to direct cellular material, molecules, or drugs, as discussed below.

[0037] As used herein, the terms "inhibit" or "reduce" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete elimination of the activity, response, condition, or disease. It can also include, for example, a 10% reduction in the activity, response, condition, or disease compared to native or control levels. Thus, the reduction can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between compared to native or control levels.

[0038] As used herein, "fusion protein" refers to a polypeptide formed by joining two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. Fusion proteins can be formed by chemical coupling of the constituent polypeptides or can be expressed as a single polypeptide from a nucleic acid sequence encoding a single contiguous fusion protein. A single-chain fusion protein is a fusion protein having a single contiguous polypeptide backbone. Fusion proteins can be prepared using conventional techniques in molecular biology, joining two genes in frame into a single nucleic acid sequence and then expressing the nucleic acid in an appropriate host cell under conditions that produce the fusion protein.

[0039] The recitation of ranges of values ​​herein, unless otherwise indicated herein, is merely intended to serve as a shorthand method of referring individually to each separate value within the range, and each separate value is incorporated herein as if it were individually set forth herein.

[0040] Use of the term "about" is intended to describe values ​​above or below a stated value within approximately + / - 10%, in other embodiments, values ​​may range above or below a stated value within approximately + / - 5%, in other embodiments, values ​​may range above or below a stated value within approximately + / - 2%, and in other embodiments, values ​​may range above or below a stated value within approximately + / - 1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied.

[0041] All methods described herein can be performed in any suitable order unless otherwise indicated or clearly contradicted by context. Any and all examples provided herein, or the use of exemplary language (e.g., "etc.") are intended merely to better illustrate the embodiments and do not pose a limitation on the scope of the embodiments unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0042] As used herein, "as needed" or "as needed" means that the subsequently described event, circumstance, or material may or may not occur, or may or may not exist, and that the description includes instances in which the event, circumstance, or material occurs or exists, as well as instances in which it does not occur or exist.

[0043] Ranges may be expressed herein as "about" from one particular value and / or to another particular value. When such ranges are expressed, ranges from one particular value and / or to the other particular value are considered to be specifically contemplated and disclosed unless the context specifically dictates otherwise. Similarly, when values ​​are expressed as approximations, it will be understood that the use of the antecedent "about" indicates that the particular value forms another specifically contemplated embodiment that should be considered disclosed unless the context specifically dictates otherwise. It will be further understood that each of the endpoints of a range is significant both in relation to the other endpoint and independently of the other endpoint, unless the context specifically dictates otherwise. It should be understood that all individual values ​​and subranges of values ​​contained within an explicitly disclosed range are also considered to be specifically contemplated and disclosed unless the context specifically dictates otherwise. Finally, all ranges should be understood to refer both to ranges recited as ranges and as individual numerical sets from the first endpoint, inclusive, to the second endpoint, inclusive. In the latter cases, it should be understood that any of the individual numbers can be selected as one form of the quantity, value, or characteristic to which the range refers. Thus, a range describes a set of numbers or values ​​from a first, inclusive endpoint to a second, inclusive endpoint, and a single member of the set (i.e., a single number) can be selected as the quantity, value, or characteristic to which the range refers. The foregoing applies regardless of whether, in a particular instance, some or all of these embodiments are explicitly disclosed.

[0044] Any compound disclosed herein is intended to be specifically disclosed herein and should be considered as such. Furthermore, any subgroups that may be identified within this disclosure are intended to be specifically disclosed herein and should be considered as such. Consequently, it is specifically contemplated that any compound or subgroup of compounds can be specifically included or excluded for use, or can be included or excluded from a list of compounds.

[0045] Disclosed are the components used to prepare the disclosed compositions, and the compositions themselves used within the methods disclosed herein. When these and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that specific reference to each of the various individual and collective combinations and permutations of these compounds is not expressly disclosed, but each is specifically contemplated and described herein. For example, when a particular polypeptide is disclosed and discussed, and many modifications that can be made to many peptides are discussed, any and all combinations and permutations of polypeptides and possible modifications are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C is disclosed, and similarly, a class of molecules D, E, and F, as well as exemplary combined molecules A-D, then each is individually and collectively contemplated, even if not individually listed, meaning that combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered to be disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the subgroups A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application, including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, where there are various additional steps that may be performed, it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

[0046] II. Proteolytic System A. Strategies for Targeted Protein Degradation TRIM21 is a multi-domain protein, containing an N-terminal RING (SEQ ID NO: 127) domain with E3 ubiquitin ligase activity, a B-box domain, a coiled-coil dimerization domain, and a C-terminal PRYSPRY (SEQ ID NO: 125) domain. The domain structure is conserved within the TRIM protein family, and it is the C-terminal PRYSPRY (SEQ ID NO: 125) domain that contains the antibody binding site and thus directs function. The PRYSPRY (SEQ ID NO: 125) domain of TRIM21 is a globular fold containing two antiparallel β-sheet β-sandwiches connected by a flexible loop, which are subdivided into PRY and SPRY (SEQ ID NO: 126) elements. In solution, TRIM21 exists as a homodimer and forms a stable 1:1 complex with antibody, where the two PRYSPRY (SEQ ID NO: 125) domains symmetrically bind to Fc.

[0047] It has been discovered that the cell-penetrating domain of a cell-penetrating antibody can be used to enhance the delivery of other binding proteins across the cell membrane and enhance the induction of degradation of intracellular target proteins through the TRIM21 pathway. Disclosed herein are chimeric antigen-binding constructs that penetrate cells and bind to target proteins mediating their destruction through the TRIM21 pathway and the cellular proteolytic machinery.

[0048] Typically, the construct includes (1) a TRIM21-binding feature, (2) a cell-permeability feature of a cell-permeability antibody, and (3) an antigen-binding feature that binds to the target protein. This system is believed to be effective for rapid degradation of the target protein, thus compensating for the phenotype and minimizing the risk of secondary nonspecific defects accumulating over time. As discussed in more detail below, because the system utilizes an antigen-binding domain, it can be applied to a wide range of target proteins using currently available antibody sequences.

[0049] B. Design of Antigen-Binding Molecules for Targeting Proteolysis The disclosed antigen-binding molecules for targeting protein degradation typically comprise three core features: (1) a TRIM21-binding feature, (2) a cell-permeability feature of a cell-permeability antibody, and (3) an antigen-binding feature that binds to a target protein. Each of these features is discussed in more detail below. In some embodiments, the three features are derived from three different molecules, while in other embodiments, two or more features are derived from a single source. For example, in some embodiments, features (1) and (2) are derived from a cell-permeability antibody, while (3) is derived from a heterologous molecule. Similarly, in some embodiments, features (1) and (3) are derived from the same source, and (2) is derived from a heterologous cell-permeability antibody molecule. Likewise, the molecules can include one or more additional features or domains.

[0050] As discussed in more detail below, the molecules can be assembled in a myriad of different ways and can be formed from a variety of traditional and non-traditional antibody and antigen binding structures.

[0051] 1. TRIM21-binding domain TRIM21 binding characteristics most typically include an Fc domain, which is capable of binding to TRIM21 and implicating it in its proteolytic pathway.

[0052] In the case of antibodies, the variable region (V region) generally refers to a region that is present in the amino acid sequence at the N-terminus of an immunoglobulin and is highly diverse. The portion other than the variable region has a structure with low diversity, so it is called the constant region (C region). The individual variable regions of the heavy and light chains associate to form an antigen-binding site, which determines the binding properties of the antibody to the antigen.

[0053] The antigen-binding site is the site in an antibody that recognizes and binds to an antigen, forming a complementary conformation with an antigenic determinant (epitope). At the antigen-binding site, strong intermolecular interactions occur between antigenic determinants. The antigen-binding site is composed of VH and VL, which contain at least three complementarity-determining regions (CDRs). In the case of a human antibody, VH and VL each contain three CDRs. These CDRs are referred to as CDR1, CDR2, and CDR3, respectively, in order from the N-terminus.

[0054] In the constant region, the heavy chain constant region and the light chain constant region are designated as CH and CL, respectively. CH is classified into heavy chain subclasses: alpha chain, delta chain, epsilon chain, gamma chain, and mu chain. CH is composed of a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain arranged in order from the N-terminal side, and the CH2 domain and the CH3 domain together are called the Fc region. On the other hand, CL is classified into two subclasses called C lambda chain and C kappa chain. The CH2 and CH3 domains together are called the Fc region.

[0055] In the case of human IgG, in the heavy chain, the variable region corresponds to the amino acid sequence from positions 1 to 117, numbered according to the EU index of Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 1991 Fifth edition), and the constant region corresponds to the amino acid sequence downstream of position 118. In the light chain of a human antibody, the amino acid sequence from positions 1 to 107, numbered according to Kabat et al. (Kabat numbering), corresponds to the variable region, and the amino acid sequence downstream of position 108 corresponds to the constant region.

[0056] In the case of human IgG, CH1 refers to the region having the amino acid sequence of positions 118 to 215 as indicated by the EU index. Similarly, CH2 refers to the region having the amino acid sequence of positions 231 to 340 as indicated by the EU index of Kabat et al., and CH3 refers to the region having the amino acid sequence of positions 341 to 447 as indicated by the EU index of Kabat et al. Between CH1 and CH2 is a highly flexible amino acid region called the hinge region (hereinafter sometimes referred to as the hinge). The hinge region refers to the region having the amino acid sequence of positions 216 to 230 as indicated by the EU index of Kabat et al.

[0057] CL refers to the region having the amino acid sequence of positions 108 to 214 as indicated by Kabat numbering in the case of a human antibody chain, and to the region having the amino acid sequence of positions 108 to 215 in the case of a lambda chain.

[0058] The characteristics of antibodies important for interacting with TRIM21 have been previously characterized. See, for example, Foss, et al., "TRIM21—From Intracellular Immunity to Therapy," Front. Immunol., 10:2049, (2019), doi.org / 10.3389 / fimmu.2019.02049, which is specifically incorporated by reference herein in its entirety and the references cited therein. Direct binding between the antibody and TRIM21 has been demonstrated, and its affinity and mechanism of binding have been disrupted by site-directed mutagenesis, and binding studies have been combined with the solution of the crystal structure of the PRYSPRY (SEQ ID NO: 125) domain of human TRIM21 in complex with a human IgG1 Fc fragment.

[0059] The structure confirmed that two PRYSPRY (SEQ ID NO: 125) domains bind to each side of the homodimeric Fc. The TRIM21 binding site is located at the CH2-CH3 interface of Fc. This is distant from the binding sites for classical Fcγ receptors and complement factor C1q, but overlaps with those of the neonatal Fc receptor (FcRn) and viral and bacterial defense proteins. The TRIM21-IgG interaction is highly pH-independent and unaffected by removal of the biantennary N-glycan structure attached to N297 in the Fc CH2 domain. However, it is sensitive to high salt concentrations.

[0060] The core TRIM21-IgG1 interaction is formed between a protruding loop encompassing residues 429-436 in the Fc CH3 domain and a deep binding pocket formed on the surface of the PRYSPRY (SEQ ID NO: 125) domain. Residues H433, N434, H435, and Y436 (HNHY motif) at the apex of the Fc loop are inserted into the PRYSPRY (SEQ ID NO: 125) binding pocket. The residues form a hydrogen-bonding network with the base of the pocket, which is protected from solvent by shielding of hydrophobic side chains. Key interacting residues in the PRYSPRY (SEQ ID NO: 125) domain include D355, W381, W383, D452, F450, and W299.

[0061] The binding affinity between human IgG1 and recombinant human PRYSPRY (SEQ ID NO: 125) domains has been measured by both isothermal titration calorimetry and surface plasmon resonance to be in the range of 150-200 nM. However, because TRIM21 is a homodimer, its functional affinity for symmetric binding to IgG1 Fc is as low as 0.6 nM, as measured by fluorescence anisotropy. This represents a >300-fold increase compared to monomeric binding, making TRIM21 the highest affinity Fc receptor known in humans. Furthermore, the TRIM21-IgG interaction is highly conserved across species, as indicated by the fact that both human and mouse TRIM21 effectively bind IgG from a range of mammalian species.

[0062] Therefore, the disclosed molecules typically contain a functional fragment or all of an antibody Fc domain that can be bound by TRIM21 and involved in its proteolytic pathway. Such a domain typically includes at least the CH2-CH3 boundary of the Fc, more preferably the entire CH2-CH3 domain. In some embodiments, the protein targeting structure includes one or more of the hinge domain and / or CH1 domain. Although other isotypes are contemplated, in preferred embodiments, the TRIM21-binding characteristics are derived from an IgG antibody. The Fc domain can be derived from a mammal, such as mouse, rabbit, goat, horse, chicken, hamster, human, etc. In preferred embodiments, the TRIM21-binding domain is derived from a human IgG antibody, optionally a human IgG1 antibody.

[0063] 2. Cell Permeability Characteristics The cell-penetrating characteristic typically derives from or is part or all of a cell-penetrating antibody. Selected lupus erythematosus anti-DNA autoantibodies are capable of penetrating living cells. For example, Im et al., "Cell- and nuclear-penetrating anti-dsDNA autoantibodies have multiple arginines in CDR3 of VH and increase cellular level of pERK and Bcl-2 in mesangial cells", Mol Immunol 67, 377-387 (2015), Jang et al., "A nucleic acid-hydrolyzing antibody penetrates into cells via caveolae-mediated endocytosis, "localizes in the cytosol and exhibits cytotoxicity", Cell Mol. Life Sci., 66:1985-97 (2009), Lee, et al., "Cell-penetrating autoantibody induces caspase-mediated apoptosis through catalytic hydrolysis of DNA", Bioorg. Med. Chem., 15(5):2016-23 (2007), Ruiz-Arguelles, et al. al., "Penetration of anti-DNA antibodies into immature live cells." J. Autoimmun., 11(5):547-56 (1998), Song, et al., "Arginines in the CDR of anti-dsDNA autoantibodies facilitate cell internalization via electrostatic interactions", Eur. J. Immunol., 38(11):3178-90 (2008), Vlahakos, et al., "Murine Monoclonal Anti-DNA Antibodies Penetrate Cells, Bind To Nuclei, And Induce Glomerular Proliferation And Proteinuria In Vivo." J. Am. Soc. Nephrol. 2(8):1345-54 (1992), Yanase, et al., "Receptor-mediated Cellular Entry of Nuclear Localizing Anti-DNA Antibodies via Myosin 1", J. Clin. Invest., 100:25-31 (1997), Yung, et al., "Anti-DNA antibodies in the pathogenesis of lupus nephritis-The emerging mechanisms", Autoimmunity Rev., 7(4):317-21 (2008), Hansen, et al., Sci Transl Med, 4(157):157ra142 (2012), Noble, et al., Cancer Research, 2015; 75(11):2285-2291, Noble, et al., Sci Rep-Uk, 4 (2014), and Noble, et al., Nat Rev Rheumatol (2016), each of which is specifically incorporated by reference herein in its entirety.

[0064] The mechanisms of autoantibody internalization are diverse: some are taken up by cells via electrostatic interactions or FcR-mediated endocytosis, whereas others utilize mechanisms based on association with cell surface myosin or calreticulin, followed by endocytosis (Ying-Chyi et al., Eur J Immunol 38, 3178-3190 (2008); Yanase et al., J Clin Invest 100, 25-31 (1997)). 3E10 penetrates cells via an Fc-independent mechanism (as evidenced by the ability of Fc-lacking 3E10 fragments to penetrate cells), but involves the presence of the nucleotide transporter ENT2 (Weisbart et al., Sci Rep 5:12022. doi: 10.1038 / srep12022. (2015); Zack et al., J Immunol 157, 2082-2088 (1996); Hansen et al., J Biol Chem 282, 20790-20793 (2007)). In some embodiments, the antibodies utilized in the disclosed compositions and methods penetrate cells via an Fc-dependent mechanism. Thus, as introduced above, in some embodiments, the two features: (1) the TRIM21-binding feature and (2) the cell-penetrating antibody feature are the same domain: e.g., portions of the Fc domain from a cell-penetrating antibody. In other embodiments, antibodies utilized in the disclosed compositions and methods permeate cells by an Fc-independent mechanism, but involve the presence of a nucleoside transporter such as ENT2.

[0065] In some embodiments, the cell-penetrating characteristic is that of the cell-penetrating antibody from which it is derived, or a fragment or fusion protein based thereon, and thus may comprise the antigen-binding domain of the cell-penetrating antibody from which it is derived.

[0066] Although cell-permeable molecules are generally referred to herein as "cell-permeable binding proteins" or "cell-permeable antibodies," it will be recognized that fragments, variants, binding proteins and fusion proteins comprising antigen-binding fragments, e.g., scFv, di-scFv, tri-scFv, and other single-chain variable fragments, as well as other cell-permeable molecules disclosed herein, are also expressly provided for use in the compositions and methods disclosed herein, provided that they comprise the cell-permeable domain of an intact antibody.

[0067] Cell-permeable antibodies that can be used in the compositions and methods include whole immunoglobulins of any class (i.e., intact antibodies), fragments thereof, and synthetic proteins containing at least the antigen-binding variable domain of an antibody. The variable domains differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not usually uniformly distributed throughout the variable domain of an antibody. It is typically concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in both the light and heavy chain variable domains. The more highly conserved portions of the variable domain are called framework regions (FRs). Native heavy and light chain variable domains each contain four FR regions, primarily adopting a beta-sheet configuration, connected by three CDRs, which form loops that connect, and in some cases, form part of, the beta-sheet structure. The CDRs in each chain are held in close proximity by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody. Thus, an antibody typically contains at least the CDRs necessary to maintain antigen binding to a target protein or other antigen, and may also contain one or more copies of the light chain variable region (VL) and heavy chain variable region (VH) of an antibody that binds to the antigen. In some embodiments, each VH and VL contains three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0068] In some embodiments, the cell-penetrating domain comprises an antigen-binding domain (e.g., an anti-DNA antigen-binding domain) and exists in the form of a single-chain antibody, a single-chain variable fragment (scFv), a di-scFv, a tri-scFv, a diabody, a triabody, a tetrabody, a disulfide-linked Fv (sdFv), a Fab', a F(ab')2, an Fv, and a single-domain antibody fragment (sdAb).

[0069] The antibody molecule may also be a single-domain antibody. Single-domain antibodies may include antibodies whose complementarity-determining regions are portions of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain antibodies, antibodies naturally lacking light chains, single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single-domain scaffolds other than those derived from antibodies. Single-domain antibodies may be any of the single-domain antibodies currently available in the art or any future single-domain antibodies. Single-domain antibodies may be derived from any species, including, but not limited to, mouse, human, camel, llama, fish, shark, goat, rabbit, and cow. According to another aspect of the present invention, the single-domain antibody is a naturally occurring single-domain antibody known as a heavy-chain antibody lacking light chains. Such single-domain antibodies are disclosed, for example, in WO9404678. For clarity, this variable region derived from a heavy-chain antibody naturally lacking light chains is known herein as a VHH or nanobody to distinguish it from the conventional VH of four-chain immunoglobulins. Such VHH molecules may be derived from antibodies raised in Camelidae species, such as camel, llama, dromedary, alpaca and guanaco.

[0070] In some embodiments, the cell-penetrating domain is derived from or is part or all of the antibody 3E10 or 5C6. A panel of hybridomas, including 3E10 and 5C6 hybridomas, has previously been generated from the MRLmpj / lpr lupus erythematosus mouse model and evaluated for DNA binding activity (Zack, et al., J. Immunol. 154:1987-1994 (1995); Gu, et al., J. Immunol., 161:6999-7006 (1998)). Mouse 3E10 may refer to the monoclonal antibody produced by the ATCC accession number PTA 2439 hybridoma. 5C6 may refer to a monoclonal anti-DNA antibody with nucleolytic activity produced by a hybridoma derived from the MRL / lpr lupus erythematosus mouse model described in Noble et al., 2014, Sci Rep 4:5958 doi: 10.1038 / srep05958. Non-limiting examples of 3E10 and 5C6 antibody sequences are provided below and can be used in preparing the molecules disclosed herein. Thus, in some embodiments, the cell-permeable antibody domain is a 3E10 or 5C6 antibody, or a variant, fragment, and fusion protein thereof, or a humanized form thereof.

[0071] 3. Antigen-binding characteristics of target proteins The antigen-binding feature is a domain that facilitates binding to and captures the target protein for proteolysis through the TRIM21 pathway. Therefore, the antigen-binding domain is typically an intact antibody, antibody fragment, or fusion protein that binds to the target protein with high affinity and specificity. The antigen-binding domain can be of any suitable structure, provided that the target protein specifically binds to it. Antibodies that can be used in the compositions and methods include any class of whole immunoglobulin (i.e., intact antibody), fragments thereof, and synthetic proteins containing at least the antigen-binding variable domain of an antibody. The variable domain differs in sequence between antibodies and is used in the binding and specificity of each specific antibody for its specific antigen, as introduced above. Thus, the antigen-binding domain that targets the target protein typically contains at least the CDRs necessary to maintain antigen binding to the target protein, and may also contain one or more copies of the light chain variable region (VL) and heavy chain variable region (VH) of the antibody that binds to the target protein.

[0072] Thus, in some embodiments, the antigen binding characteristics targeted to the target protein are present in the form of a single chain antibody, single chain variable fragment (scFv), di-scFv, tri-scFv, diabody, triabody, tetrabody, disulfide-linked Fv (sdFv), Fab', F(ab')2, Fv, single domain antibody fragment (sdAb), nanobody, VHH, etc., as discussed above.

[0073] 4. Additional Domains and Conjugates The molecules may also include additional domains and conjugates that serve to further increase targeting for proteolysis and / or achieve alternative or additional functions or purposes.

[0074] For example, in some embodiments, the molecule comprises a TRIM21 protein, or a functional fragment or variant thereof, which can further enhance the proteolysis of the target protein. In some embodiments, the TRIM21 is human TRIM21. Sequences for TRIM21 are known in the art, for example: [ka] (SEQ ID NO: 102, UniProt Accession No. P19474 RO52_HUMAN), or a functional fragment or variant thereof having at least 70, 80, 85, 90, 95, or more sequence identity to SEQ ID NO: 102.

[0075] The conjugate can be, for example, a small molecule drug to aid in the treatment of a targeted disease or disorder.

[0076] C. Molecular Structures for Targeting Protein Degradation The three core features, including additional domains as needed, can be assembled in any manner suitable to perform the desired target proteolytic function. As such, antibodies of various structures, including whole antibodies and fusion proteins, are envisioned, provided they exhibit the core features.

[0077] In some embodiments, the molecule takes the form of a multispecific antibody. In some embodiments, the multispecific antibody is a bispecific antibody, having two binding molecules that target different epitopes. In some embodiments, the multispecific molecule includes one or more additional binding moieties (e.g., a third binding moiety, a fourth binding moiety) that target three or more different epitopes (e.g., a trispecific or tetraspecific molecule).

[0078] A multispecific antibody molecule can contain two or more antigen-binding sites, where different sites are specific for different antigens. A multispecific antibody molecule can additionally or alternatively bind to two or more (e.g., two or more) epitopes on the same antigen. For example, when two or more binding moieties are present, one can optionally be the binding domain of a cell-permeable antibody from which the cell-permeability characteristic is derived. In some embodiments, the two or more binding moieties target two or more different epitopes of the same target protein. In some embodiments, the two or more binding moieties target two or more different epitopes of different target proteins. Thus, in some embodiments, the molecule simultaneously targets two or more different proteins for degradation.

[0079] In some embodiments, the molecules are heterodimeric bi- and tri- (or higher) specific Ig antibodies and Fc fusion proteins. Exemplary structures include, but are not limited to, IgG, IgG, IgM, mono-, di-, tri-, or higher order scFv-Fc. For example, bispecific, trispecific, and multispecific formats include, but are not limited to, bispecific and trispecific IgG, IgG-scFv, IgG-dAb, scFv-Fc-scFv, knob-in-hole (KIH)-IgG, kappa lambda-body, KIH0Fc-Fab / scFv, tandem scFv, KIH trispecific, bispecific Fc fusions (with or without N- or C-terminus, KIH).

[0080] In some embodiments, a multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibody molecules can be classified into different structural groups, including having one or more of: (i) bispecific immunoglobulin G (BsIgG); (ii) IgG with an additional antigen-binding moiety appended; (iii) bispecific fusion proteins; and (iv) bispecific antibody conjugates.

[0081] BsIgG is a format that is monovalent for each antigen. Exemplary BsIgG formats include, but are not limited to, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knob-in-hole common LC, knob-in-hole assembly, charge pair, Fab arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, kappa-lambda body, and orthogonal Fab. See Spiess et al. Mol. Immunol. 67(2015):95-106. Exemplary BsIgGs include catumaxomab (Fresenius Biotech, Trion Pharma, Neopharm), which contains an anti-CD3 arm and an anti-EpCAM arm; and ertumaxomab (Neovii Biotech, Fresenius Biotech), which targets CD3 and HER2. In some embodiments, the BsIgG comprises a heavy chain engineered for heterodimerization. For example, heavy chains can be engineered for heterodimerization using the "knobs-into-holes" strategy, the SEED platform, common heavy chains (e.g., in Kk-bodies), and the use of heterodimeric Fc regions. See Spiess et al. Mol. Immunol. 67(2015):95-106. Strategies that have been used to avoid homodimeric heavy chain pairing in BsIgG include knobs-in-holes, duobodies, azymetrics, charge pairs, HA-TF, SEEDbodies, and differential Protein A affinity. See ibid. BsIgG can be produced by separate expression of antibody components in different host cells and subsequent purification / assembly into BsIgG. BsIgG can also be produced by expression of antibody components in a single host cell. BsIgG can be purified using affinity chromatography, for example, using Protein A, and sequential pH elution.

[0082] IgGs with additional antigen-binding moieties added are another format of multispecific antibody molecules, including bispecific antibody molecules. For example, bivalent monospecific IgGs and monovalent bispecific IgGs can be engineered to have bispecificity or higher specificity by adding additional antigen-binding units on the IgG, for example, at the N- or C-terminus of either the heavy or light chain. Exemplary additional antigen-binding units include single domain antibodies (e.g., variable heavy chains or variable light chains), engineered protein scaffolds, and paired antibody variable regions (e.g., single-chain variable fragments or variable fragments). See ibid. Examples of added IgG formats include dual variable domain IgG (DVD-Ig), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody, and DVI-IgG (four-in-one). See Spiess et al. Mol. Immunol. 67(2015):95-106. An example of an IgG-scFv is MM-141 (Merrimack Pharmaceuticals), which binds to IGF-1R and HER3. Examples of DVD-Igs include ABT-981 (AbbVie), which binds to IL-1 alpha and IL-1 beta, and ABT-122 (AbbVie), which binds to TNF and IL-17A.

[0083] Bispecific fusion proteins include, for example, antibody fragments (nanobodies, nanobody-HAS, BiTEs, diabodies, DARTs, TandAbs, scDiabodies, scDiabody-CH3, diabody-CH3, triplebodies, miniantibodies, minibodies, TriBi minibodies, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAbs, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, intrabodies) linked to other proteins, such as and Fc domains, to add additional functionality.

[0084] Chemical conjugation, for example, of antibodies and / or antibody fragments, can be used to create BsAb molecules. See ibid. Exemplary bispecific antibody conjugates include the CovX-body format, in which a low-molecular-weight drug is site-specifically conjugated to each Fab arm or a single reactive lysine on an antibody or its fragment. In embodiments, the conjugation improves the serum half-life of the low-molecular-weight drug. An exemplary CovX-body is CVX-241 (NCT01004822), which comprises an antibody conjugated to two short peptides that inhibit either VEGF or Ang2. See ibid.

[0085] In some embodiments, the multispecific molecule comprises a heavy chain constant region (e.g., Fc region) selected from the heavy chain constant regions of IgG1, IgG2, and IgG4, more particularly, the heavy chain constant region of human IgG1, IgG2, or IgG4. In some embodiments, one or two antigen-binding moieties (or VH thereof) are linked, e.g., covalently linked, to a heavy chain constant region (e.g., one or more of CH1, hinge, CH2, CH3, Fc region, etc.). In some embodiments, one or both antigen-binding moieties (or VL thereof) are linked, e.g., covalently linked, to a light chain constant region (e.g., CL). In some embodiments, CH3 and / or CL are further linked, e.g., covalently linked, to additional antigen-binding moieties.

[0086] In some embodiments, the heavy chain constant region (e.g., Fc region) is altered, e.g., mutated, to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function.

[0087] In some embodiments, the interface of the first and second heavy chain constant regions (e.g., Fc regions) is altered, e.g., mutated, to increase or decrease dimerization, e.g., compared to an unengineered interface. In some embodiments, dimerization of the heavy chain constant regions (e.g., Fc regions) is enhanced by providing one or more of paired cavity-protrusions ("knobs-in-holes"), electrostatic interactions, or strand exchange at the Fc interface of the first and second Fc regions, resulting in a higher ratio of heteromultimers:homomultimers, e.g., compared to an unengineered interface. In some embodiments, the heavy chain constant region (e.g., Fc region) comprises an amino acid substitution at a position selected from one or more of 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409 of the Fc region of human IgG1, numbered according to the EU numbering system. In some embodiments, the heavy chain constant region (e.g., Fc region) comprises an amino acid substitution selected from T366S, L368A, or Y407V (e.g., corresponding to a cavity or hole), or T366W (e.g., corresponding to a protrusion or knob), or a combination thereof, numbered according to the EU numbering system.

[0088] In some embodiments, the heavy chain constant region (e.g., Fc region) comprises one or more mutations that increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function compared to a naturally occurring heavy chain constant region.

[0089] In some embodiments, a multispecific antibody is formed from two or more portions, where a first portion of the molecule comprises a first heavy chain constant region (e.g., a first Fc region) and a second portion of the molecule comprises a second heavy chain constant region (e.g., a second Fc region), where the first heavy chain constant region comprises one or more mutations that increase heterodimerization of the first heavy chain constant region and the second heavy chain constant region compared to naturally occurring heavy chain constant regions, and / or where the second heavy chain constant region comprises one or more mutations that increase heterodimerization of the second heavy chain constant region and the first heavy chain constant region compared to naturally occurring heavy chain constant regions. In some embodiments, the first and second heavy chain constant regions (e.g., the first and second Fc regions) comprise one or more of paired cavity-protrusions ("knobs-in-holes"), electrostatic interactions, or strand exchange, resulting in the formation of a higher ratio of heteromultimers:homomultimers, e.g., compared to naturally occurring heavy chain constant regions. In some embodiments, the first and / or second heavy chain constant region (e.g., the first and / or second Fc region, e.g., the first and / or second IgG1 Fc region) comprises an amino acid substitution at a position selected from one or more of 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409, numbered according to the EU numbering system. In some embodiments, the first and / or second heavy chain constant region (e.g., the first and / or second Fc region, e.g., the first and / or second IgG1 Fc region) comprises an amino acid substitution selected from T366S, L368A, Y407V, or Y349C (e.g., corresponding to a cavity or hole), or T366W or S354C (e.g., corresponding to a protrusion or knob), or a combination thereof, numbered according to the EU numbering system.

[0090] Antibody molecules can be produced, for example, by recombinant expression of at least one or more components in a host system. Exemplary host systems include eukaryotic cells (e.g., mammalian cells, such as CHO cells, or insect cells, such as SF9 or S2 cells) and prokaryotic cells (e.g., E. coli). Bispecific antibody molecules can be produced by separate expression of the components in different host cells and subsequent purification / assembly. Alternatively, antibody molecules can be produced by expression of the components in a single host cell. Purification of bispecific antibody molecules can be achieved by various methods, such as affinity chromatography using, for example, protein A, and sequential pH elution. In other embodiments, affinity tags, such as histidine-containing tags, myc tags, or streptavidin tags, can be used for purification.

[0091] A recombinant fusion protein is a protein created by genetic engineering of a fusion gene. This typically involves removing the stop codon from a cDNA sequence encoding a first protein and then adding the cDNA sequence of a second protein in frame by ligation or overlap extension PCR. The DNA sequence is then expressed by cells as a single protein. The protein can be engineered to contain the complete sequences of both original proteins, or only portions of either. When the two entities are proteins, a linker (or "spacer") peptide is often also added, which makes the proteins more likely to fold independently and behave predictably.

[0092] The function of an antibody may be enhanced by coupling the antibody or fragment thereof with a therapeutic agent. Such coupling of the antibody or fragment to a therapeutic agent can be achieved by creating an immunoconjugate, or by creating a fusion protein, or by linking the antibody or fragment to a nucleic acid, such as DNA or RNA (e.g., siRNA), comprising the antibody or antibody fragment and the therapeutic agent.

[0093] In some embodiments, the antibody is modified to alter its half-life. In some embodiments, it is desirable to increase the half-life of the antibody so that it remains in the circulation or at the site of treatment for a longer period of time. For example, it may be desirable to maintain the titer of the antibody in the circulation or at the site of treatment for an extended period of time. In other embodiments, the half-life of the antibody is decreased to reduce potential side effects. Antibody fragments may have a shorter half-life than full-sized antibodies. Other methods of altering half-life are known and can be used in the methods described. For example, the antibody can be engineered with an Fc variant that extends half-life, for example, using Xtend™ antibody half-life extension technology (Xencor, Monrovia, CA).

[0094] III. Exemplary Cell-Penetrating Antibody Sequences Although generally referred to herein as "3E10" or "3E10 antibody," "5C6" or "5C6 antibody," it will be recognized that fragments and binding proteins, including antigen-binding fragments, variants, and fusion proteins, such as scFv, di-scFv, tri-scFv, and other single-chain variable fragments, and other cell-permeable nucleic acid transport molecules disclosed herein, are also encompassed by the term expressly provided for use in the compositions and methods disclosed herein. As such, antibodies and other binding proteins are also referred to herein as cell-permeable.

[0095] A.3E10 antibody In a preferred embodiment, the 3E10 antibody is transported into the cytoplasm and / or nucleus of cells without the aid of a carrier or conjugate. For example, the monoclonal antibody 3E10 and its active fragments, which are transported into the nucleus of mammalian cells in vivo without cytotoxic effects, are disclosed in U.S. Patent Nos. 4,812,397 and 7,189,396 to Richard Weisbart.

[0096] In some embodiments, the antibody may bind to and / or inhibit Rad51. See, e.g., the antibodies described in Turchick, et al., Nucleic Acids Res., 45(20): 11782-11799 (2017), WO2020 / 047344, and WO2020 / 047353, each of which is specifically incorporated by reference herein in its entirety.

[0097] The 3E10 antibody is typically monoclonal 3E10, or a variant, derivative, fragment, fusion, or humanized form thereof that binds to the same or a different epitope as 3E10.

[0098] A deposit pursuant to the requirements of the Budapest Treaty of a hybridoma cell line producing monoclonal antibody 3E10 was received on September 6, 2000, and accepted by the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, VA 20110-2209, USA, and assigned patent deposit number PTA-2439.

[0099] Therefore, the antibody may have the same or different epitope specificity as the monoclonal antibody 3E10 produced by the ATCC No. PTA 2439 hybridoma. The antibody may have the paratope of the monoclonal antibody 3E10. The antibody may be a single-chain variable fragment of 3E10 or a variant thereof, such as a conservative variant. For example, the antibody may be a single-chain variable fragment of 3E10 (3E10 Fv) or a variant thereof.

[0100] 1.3E10 array The amino acid sequence of monoclonal antibody 3E10 is known in the art. For example, the sequences of the 3E10 heavy and light chains are provided below, where single underlines indicate CDR regions identified according to the Kabat system, and in SEQ ID NOs: 12-14, 103-106, and 118, italics indicate variable regions, and double underlines indicate signal peptides. CDRs according to the IMGT system are also provided. All sequences are disclosed with and without signal peptides, and the respective heavy and light chain variable regions are explicitly disclosed both with and without constant regions. Therefore, the heavy and light chain variable regions (and their CDRs) of SEQ ID NOs: 12-14, 103-106, and 118 are also explicitly provided without their constant regions and can be used to form the binding fragments and fusion proteins provided herein, with or without alternative constant regions.

[0101] a.3E10 heavy chain In some embodiments, the heavy chain variable region of 3E10 is [ka] (SEQ ID NO: 1; Zack, et al., Immunology and Cell Biology, 72:513-520 (1994); GenBank: L16981.1 - mouse Ig rearranged light chain gene, partial cds; and GenBank: AAA65679.1 - immunoglobulin heavy chain, partial [Mus musculus]).

[0102] In some embodiments, the 3E10 heavy chain is [ka] It is expressed as:

[0103] Variants of the 3E10 antibody, which incorporate mutations into the wild-type sequence, are also known in the art, as disclosed, for example, in Zack, et al., J. Immunol., 157(5):2082-8 (1996). For example, amino acid 31 of the heavy chain variable region of 3E10 has been determined to influence the ability of antibodies and fragments thereof to penetrate the nucleus and bind to DNA (bold in SEQ ID NOS: 1, 2, and 13). The D31N mutation in CDR1 (bold in SEQ ID NOs: 2 and 13) penetrates the nucleus and binds to DNA much more efficiently than the original antibody (Zack, et al., Immunology and Cell Biology, 72:513-520 (1994); Weisbart, et al., J. Autoimmun., 11, 539-546 (1998); Weisbart, Int. J. Oncol., 25, 1867-1873 (2004)). In some embodiments, the antibody has a D31N substitution.

[0104] In some embodiments, the amino acid sequence for a preferred variant of the heavy chain variable region of 3E10 is: [ka] is.

[0105] Other heavy chain variable regions include, but are not limited to: [ka] Examples include:

[0106] In some embodiments, the 3E10 heavy chain is [ka] It is expressed as:

[0107] In some embodiments, the C-terminal serine of SEQ ID NO: 1 or 2 is absent or substituted, for example, with alanine, in the 3E10 heavy chain variable region.

[0108] In some embodiments, the 3E10 heavy chain is [ka] [ka] It is expressed as:

[0109] The complementarity determining regions (CDRs) identified by Kabat are shown underlined above and include: CDR H1.1 (original sequence): DYGMH (SEQ ID NO: 15); CDR H1.2 (with D31N mutation): NYGMH (SEQ ID NO: 16); CDR H2.1: YISSGSSTIYYADTVKG (SEQ ID NO: 17); CDR H3.1: RGLLDY (SEQ ID NO: 18).

[0110] Other variants of Kabat CHR H1.1 and H1.2 include CDR H1.3 (with a D31R mutation): RYGMH (SEQ ID NO: 107); and CDR H1.4 (with a D31K mutation) KYGMH (SEQ ID NO: 108).

[0111] Variants of Kabat CDR H2.1 include YISSGSSTIYYADSVKG (SEQ ID NO: 19) and YISSSSSTIYYADSVKG (SEQ ID NO: 42).

[0112] Additionally or alternatively, heavy chain complementarity determining region (CDR) can be defined according to the IMGT system.The complementarity determining region (CDR) identified by the IMGT system includes: CDR H1.3 (original sequence): GFTFSDYG (SEQ ID NO: 20); CDR H1.4 (with D31N mutation): GFTFSNYG (SEQ ID NO: 21); GFTFSRYG (SEQ ID NO: 109); CDR H1.4 (with D31K mutation) GFTFSKYG (SEQ ID NO: 110); CDR H2.2: ISSGSSTI (SEQ ID NO: 22) and variant ISSSSTI (SEQ ID NO: 43); CDR H3.2: ARGLLLDY (SEQ ID NO: 23).

[0113] b.3E10 light chain In some embodiments, the light chain variable region of 3E10 is [ka] is.

[0114] The amino acid sequence for the light chain variable region of 3E10 is: [ka] But it's possible.

[0115] In some embodiments, the 3E10 light chain is [ka] [ka] It is expressed as:

[0116] Other 3E10 light chain sequences are known in the art, see, e.g., Zack, et al., J. Immunol., 15;154(4):1987-94 (1995); GenBank: L16981.1 - Mouse Ig rearranged light chain gene, partial cds; GenBank: AAA65681.1 - Immunoglobulin light chain, partial [Mus musculus].

[0117] The complementarity determining regions (CDRs) identified by Kabat are shown underlined and include: CDR L1.1: RASKSVSTSSYSYMH (SEQ ID NO: 24); CDR L2.1: YASYLES (SEQ ID NO: 25); CDR L3.1: QHSREFPWT (SEQ ID NO: 26).

[0118] Variants of Kabat CDR L1.1 include RASKSVSTSSYSYLA (SEQ ID NO: 27), RASKTVSTSSYSYMH (SEQ ID NO: 44), and CDR L1.2: RASKSVDTSSYSYMH (SEQ ID NO: 113).

[0119] A variant of Kabat CDR L2.1 is YASYLQS (SEQ ID NO: 28).

[0120] A variant of Kabat CDR L3.1 is CDR L3.3:QHSNEFPWT (SEQ ID NO: 114).

[0121] Additionally or alternatively, heavy chain complementarity determining regions (CDRs) can be defined according to the IMGT system. The complementarity determining regions (CDRs) identified by the IMGT system include CDR L1.2 KSVSTSSYSY (SEQ ID NO: 29) and variant CDR L1.2 KTVSTSSYSY (SEQ ID NO: 45) and KSVDTSSYSY (SEQ ID NO: 115); CDR L2.2: YAS (SEQ ID NO: 30); CDR L3.2: QHSREFPWT (SEQ ID NO: 26); CDR L3.3: QHSNEFPWT (SEQ ID NO: 114).

[0122] In some embodiments, the C-terminus of the sequence of SEQ ID NO: 7 or 8 further comprises an arginine in the 3E10 light chain variable region.

[0123] 2. Humanized 3E10 In some embodiments, the antibody is a humanized antibody. Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically obtained from an "import" variable domain. Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule.

[0124] Exemplary 3E10 humanized sequences are discussed in WO2015 / 106290, WO2016 / 033324, WO2019 / 018426, and WO / 2019 / 018428 and are provided below.

[0125] a. Humanized 3E10 heavy chain variable region In some embodiments, the humanized 3E10 heavy chain variable domain comprises: [ka] [ka] Includes.

[0126] b. Humanized 3E10 light chain variable region In some embodiments, the humanized 3E10 light chain variable domain comprises: [ka] [ka] Includes.

[0127] c. Cell penetration and nuclear localization The disclosed compositions and methods typically utilize antibodies that maintain the ability to penetrate the cell, and optionally, the nucleus.

[0128] Mutations in 3E10 that prevent its ability to bind to DNA can render the antibody unable to penetrate the nucleus. Therefore, typically, the disclosed variants and humanized forms of the antibody maintain the ability to bind to nucleic acids, particularly DNA. In addition, 3E10 scFv has previously been shown to be able to penetrate living cells and nuclei in an ENT2-dependent manner, and the efficiency of uptake is impaired in ENT2-deficient cells (Hansen, et al., J. Biol. Chem. 282, 20790-20793 (2007)). Therefore, in some embodiments, the disclosed variants and humanized forms of the antibody maintain the ability to penetrate the nucleus of cells in an ENT-dependent, preferably ENT2-dependent, manner.

[0129] As discussed in WO2019 / 152806 and WO2019 / 152808, some humanized 3E10 variants were found to penetrate the cell nucleus more efficiently than the original murine 3E10(D31N) di-scFv, while others were found to have lost the ability to penetrate the nucleus. In particular, variants 10 and 13 penetrated the nucleus much better than the murine antibody.

[0130] A potential bipartite nuclear localization signal (NLS) in humanized 3E10 VL has been identified, with the following sequence: [ka] may include part or all of

[0131] An exemplary consensus NLS is [ka] (where (X) = any residue, but preferably a basic residue (R or K) (SEQ ID NO: 91), or a variant thereof having at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 percent sequence identity to SEQ ID NO: 53.

[0132] Thus, in some embodiments, particularly where nuclear import is important, the disclosed antibodies can comprise the sequence of any one of SEQ ID NOs: 88-91, or fragments and variants thereof (e.g., 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% amino acid sequence identity to any one of SEQ ID NOs: 88-91) that can translocate to the nucleus of a cell.

[0133] The presence of an NLS indicates that 3E10 can cross the nuclear envelope via the nuclear import pathway. In some embodiments, the NLS improves import by interacting with one or more members of the import pathway. Thus, in some embodiments, the NLS can bind to importin-β, importin-β / importin-α heterodimer, or a combination thereof.

[0134] 3. Nucleic acid binding The disclosed compositions and methods optionally utilize antibodies that maintain the ability to bind to nucleic acids, such as DNA, RNA, or a combination thereof.

[0135] Molecular modeling (Pymol) of 3E10 revealed a putative nucleic acid binding pocket (NAB1), the sequence of which is shown below with underlining. [ka]

[0136] In some embodiments, the disclosed antibodies comprise part or all of the underlined NAB1 sequence. In some embodiments, the antibodies comprise variant sequences with altered nucleic acid binding ability. In some embodiments, mutations (e.g., substitutions, insertions, and / or deletions) in NAB1 improve antibody binding to nucleic acids such as DNA, RNA, or combinations thereof. In some embodiments, the mutations are conservative substitutions. In some embodiments, the mutations increase the cationic charge of the NAB1 pocket.

[0137] As discussed and exemplified herein, mutation of residue 31 of CDR1 from aspartic acid to asparagine increased the cationic charge of this residue and enhanced nucleic acid binding and delivery in vivo (3E10-D31N).

[0138] Additional exemplary variants include mutation of residue 31 of CDR1 from aspartic acid to arginine (3E10-D31R), which modeling indicates an expansion of the cationic charge, or to lysine (3E10-D31K), which modeling indicates a change in the direction of the charge. Thus, in some embodiments, a 3E10 binding protein includes a D31R or D31K substitution.

[0139] Additional exemplary variants include mutations of arginine (R) 96 to asparagine (N) and / or serine (S) 30 to aspartic acid (D), alone or in combination with D31N, D31R, or D31K. Variants with a mutation of arginine (R) 96 to asparagine (N) may reduce cell penetration, so in some embodiments, the antibody does not have an R96N mutation.

[0140] All sequences disclosed herein having residues corresponding to 3E10 D31 or N31 are expressly disclosed with the D31R or D31K or N31R or N31K substitution.

[0141] Molecular modeling (Pymol) of 3E10 revealed a putative nucleic acid binding pocket (NAB1). Mutation of residue 31 of CDR1 from aspartic acid to asparagine increased the cationic charge of this residue and enhanced nucleic acid binding and delivery in vivo (3E10-D31N).

[0142] Mutation of residue 31 of CDR1 from aspartic acid to arginine (3E10-D31R) further extended the cationic charge, whereas mutation to lysine (3E10-D31K) changed the direction of the charge.

[0143] NAB1 amino acids predicted from molecular modeling are underlined in the heavy and light chain sequences above.

[0144] All sequences disclosed herein having a residue corresponding to R96 are expressly disclosed with the R96N substitution.

[0145] All sequences disclosed herein having a residue corresponding to S30 are expressly disclosed with S30D.

[0146] Any of the substitutions can be included in any combination, so sequences with two or three substitutions in any combination of residues 31, 30, and 96 are expressly provided.

[0147] In certain embodiments, the sequence has 31N, 31K, or 31R alone or in combination with 30D without the R96N substitution, so that in some embodiments the residue corresponding to 96 is not N, and in more specific embodiments remains R.

[0148] B.5C6 antibody sequence mAb 5C6 inhibits BRCA2 (-) In certain embodiments, the recombinant antibody is an MRL / MpJ-Fas autoantibody that selectively inhibits the growth of DLD1 cells. lpr It has the same or similar epitope specificity as the murine-produced IgG2a-k mAb 5C6. This can be achieved by producing a recombinant antibody containing the paratope of monoclonal antibody 5C6.

[0149] 1.5C6 light chain variable region The amino acid sequence for the kappa light chain variable region (VL) of mAb 5C6 is [ka] is.

[0150] Complementarity determining regions (CDRs) are underlined and include: CDR L1: RASKSVSTSGYSYMH (SEQ ID NO: 95); CDR L2: LVSNLES (SEQ ID NO: 96); CDR L3: QHIRELDTF (SEQ ID NO: 97).

[0151] 2.5C6 heavy chain variable region The amino acid sequence for the heavy chain variable region (VH) of mAb 5C6 is: [ka] is.

[0152] Complementarity determining regions (CDRs) are underlined and include: CDR H1: SYTMS (SEQ ID NO: 99); CDR H2: TISSGGGSTYYPDSVKG (SEQ ID NO: 100); CDR H3: RAYSKRGAMDY (SEQ ID NO: 101).

[0153] C. Fragments, Variants, and Fusion Proteins The anti-DNA antibody may be composed of an antibody fragment or fusion protein comprising a variable heavy and / or variable light chain amino acid sequence that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the variable heavy and / or light chain amino acid sequence of 3E10, 5C6, or a humanized form thereof.

[0154] The cell-permeable antibody may be composed of an antibody fragment or fusion protein containing one or more CDRs that are at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of the CDR of 3E10, 5C6, or a variant or humanized form thereof. The percent identity of two amino acid sequences can be determined by BLAST protein comparison. In some embodiments, the antibody contains one, two, three, four, five, or all six of the CDRs of the preferred variable domains described above.

[0155] Preferably, the antibody comprises one each of heavy chain CDR1, CDR2, and CDR3 in combination with one each of light chain CDR1, CDR2, and CDR3.

[0156] The predicted complementarity-determining regions (CDRs) of the light chain variable sequences for 3E10 and 5C6 are provided above. See also GenBank: AAA65681.1 - immunoglobulin light chain, partial [Mus musculus] and GenBank: L34051.1 - mouse Ig rearranged kappa chain mRNA V region. The predicted complementarity-determining regions (CDRs) of the heavy chain variable sequence for 3E10 are provided above. See also, for example, Zach, et al., Immunology and Cell Biology, 72:513-520 (1994), GenBank accession number AAA65679.1. Zach, et al., J. Immunol. 154 (4), 1987-1994 (1995) and GenBank: L16982.1 - mouse Ig rearranged H chain gene, partial cds.

[0157] All sequences disclosed herein having a residue corresponding to 3E10 D31 or N31 are expressly disclosed with a D31R or D31K or N31R or N31K substitution therein. Thus, in some embodiments, a 3E10 binding protein is a variant of any of the foregoing or following sequences, wherein the amino acid residue corresponding to residue 31 of the 3E10 heavy chain is substituted with arginine (R) or lysine (K).

[0158] Also included are biologically active antibody fragments, which, whether attached to other sequences or not, contain insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acid residues, provided that the activity of the fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment.

[0159] Techniques can also be adapted for the production of single-chain antibodies specific to the antigenic proteins of the present disclosure. Methods for producing single-chain antibodies are well known to those skilled in the art. Single-chain antibodies can be created by fusing heavy and light chain variable domains together using a short peptide linker, thereby reconstituting an antigen-binding site on a single molecule. Single-chain antibody variable fragments (scFvs) have been developed in which the C-terminus of one variable domain is tethered to the N-terminus of the other variable domain via a 15-25 amino acid peptide or linker without significantly disrupting antigen binding or binding specificity. The linker is selected to allow the heavy and light chains to bind together in their proper conformational orientation.

[0160] Cell-permeable antibodies can be modified to improve their therapeutic potential. For example, in some embodiments, the cell-permeable antibody is conjugated to another antibody specific for a second therapeutic target in the cytoplasm and / or nucleus of target cells. For example, the cell-permeable antibody can be a fusion protein containing 3E10 or 5C6 Fv and a single-chain variable fragment of a monoclonal antibody that specifically binds to the second therapeutic target. In other embodiments, the cell-permeable antibody is a bispecific antibody having a first heavy chain and a first light chain derived from 3E10 or 5C6, and a second heavy chain and a second light chain derived from a monoclonal antibody that specifically binds to the second target.

[0161] Bispecific antibodies and other binding proteins having a first heavy chain and a first light chain derived from 3E10 and a second heavy chain and a second light chain derived from a monoclonal antibody that specifically binds to a second target are discussed in Weisbart, et al., Mol. Cancer Ther., 11(10):2169-73 (2012), and Weisbart, et al., Int. J. Oncology, 25:1113-8 (2004), and U.S. Patent Application No. 2013 / 0266570, which are specifically incorporated by reference in their entireties.

[0162] Bivalent single-chain variable fragments (di-scFvs) can be engineered by linking two scFvs. This can be done by generating a single peptide chain with two VH and two VL regions, resulting in a tandem scFv. ScFvs can also be designed with a linker peptide that is too short (approximately 5 amino acids) to fold the two variable regions together and allow the scFvs to dimerize. This type is known as a diabody. Diabodies have been shown to have dissociation constants up to 40-fold lower than those of corresponding scFvs, meaning they have much higher affinity for their targets. Even shorter linkers (1 or 2 amino acids) have led to the formation of trimers (triabodies or tribodies). Tetrabodies have also been produced. They exhibit even higher affinity for their targets than diabodies. In some embodiments, the cell-permeable antibody may contain two or more linked single-chain variable fragments of 3E10 or 5C6 (e.g., di-scFv, 3E10 tri-scFv), or conservative variants thereof. In some embodiments, the antibody is a diabody or triabody. Sequences for single and two or more linked single-chain variable fragments of 3E10 are provided in WO2017 / 218825 and WO2016 / 033321.

[0163] 1. Linker The term "linker" as used herein includes, but is not limited to, peptide linkers. Peptide linkers can be of any size, provided they do not interfere with epitope binding by the variable regions. In some embodiments, the linker comprises one or more glycine and / or serine amino acid residues. Monovalent single-chain antibody variable fragments (scFvs) comprise a structure in which the C-terminus of one variable domain is tethered to the N-terminus of the other variable domain via a peptide or linker, typically 15-25 amino acids long. The linker is selected to enable the heavy and light chains to bind together in their proper conformational orientation. Linkers in diabodies, triabodies, etc. typically contain shorter linkers than those of monovalent scFvs discussed above. Bivalent, trivalent, and other multivalent scFvs typically contain three or more linkers. The linkers can be the same or different in length and / or amino acid composition. Thus, the number of linkers, linker composition, and linker length can be determined based on the desired valency of the scFv, as is known in the art. Linkers can enable or drive the formation of bivalent, trivalent, and other multivalent scFvs.

[0164] For example, the linker can contain 4 to 8 amino acids. In certain embodiments, the linker contains the amino acid sequence GQSSRSS (SEQ ID NO: 31). In other embodiments, the linker contains 15 to 20 amino acids, e.g., 18 amino acids. In certain embodiments, the linker contains the amino acid sequence GQSSRSSSGGGSSGGGGS (SEQ ID NO: 32). Other flexible linkers include, but are not limited to, the amino acid sequences Gly-Ser, Gly-Ser-Gly-Ser (SEQ ID NO: 33), Ala-Ser, Gly-Gly-Gly-Ser (SEQ ID NO: 34), (Gly-Ser) (SEQ ID NO: 35), (Gly-Ser) (SEQ ID NO: 36), and (Gly-Gly-Gly-Gly-Ser) (SEQ ID NO: 37).

[0165] Other exemplary linkers include, for example: [ka] Examples include:

[0166] 2. Exemplary scFv Sequences Exemplary murine 3E10 scFv sequences, including mono-, di-, and tri-scFvs, are disclosed in WO2016 / 033321, WO2017 / 218825, WO2019 / 018426, and WO / 2019 / 018428, and are provided below. Cell-permeable antibodies for use in the disclosed compositions and methods include exemplary scFvs, as well as fragments and variants thereof.

[0167] The amino acid sequence for scFv 3E10(D31N) is [ka] [ka] is.

[0168] scFv Protein Domain Annotation for SEQ ID NO: 38 The AGIH sequence increases solubility (amino acids 1 to 4 of SEQ ID NO: 38) Vk variable region (amino acids 5 to 115 of SEQ ID NO: 38) The first (6 aa) of the light chain CH1 (amino acids 116-121 of SEQ ID NO: 38) (GGGGS)3 (SEQ ID NO: 37) linker (amino acids 122 to 136 of SEQ ID NO: 38) VH variable region (amino acids 137 to 252 of SEQ ID NO: 38) Myc tag (amino acids 253 to 268, SEQ ID NO: 38) His6 tag (amino acids 269 to 274 of SEQ ID NO: 38)

[0169] Amino acid sequence of 3E10 di-scFv(D31N) Di-scFv 3E10(D31N) is a di-single chain variable fragment containing 2x the heavy and light chain variable regions of 3E10, in which the aspartic acid at position 31 of the heavy chain is mutated to asparagine. The amino acid sequence for di-scFv 3E10(D31N) is: [ka] is.

[0170] Annotation of di-scFv protein domains for SEQ ID NO: 39 The AGIH sequence increases solubility (amino acids 1 to 4 of SEQ ID NO: 39) Vk variable region (amino acids 5 to 115 of SEQ ID NO: 39) The first (6 aa) of the light chain CH1 (amino acids 116-121 of SEQ ID NO: 39) (GGGGS)3 (SEQ ID NO: 37) linker (amino acids 122 to 136 of SEQ ID NO: 39) VH variable region (amino acids 137 to 252 of SEQ ID NO: 39) A linker between Fv fragments consisting of the first 13 amino acids of human IgG CH1 (amino acids 253 to 265 of SEQ ID NO: 39) Swivel sequence (amino acids 266 to 271 of SEQ ID NO: 39) Vk variable region (amino acids 272 to 382 of SEQ ID NO: 39) The first (6 aa) of the light chain CH1 (amino acids 383-388 of SEQ ID NO: 39) (GGGGS)3 (SEQ ID NO: 37) linker (amino acids 389 to 403 of SEQ ID NO: 39) VH variable region (amino acids 404 to 519 of SEQ ID NO: 39) Myc tag (amino acids 520 to 535 of SEQ ID NO: 39) His6 tag (amino acids 536 to 541 of SEQ ID NO: 39)

[0171] Amino acid sequence for tri-scFv Tri-scFv 3E10(D31N) is a tri-single chain variable fragment containing 3x the heavy and light chain variable regions of 310E, in which the aspartic acid at position 31 of the heavy chain is mutated to asparagine. The amino acid sequence for tri-scFv 3E10(D31N) is: [ka] [ka] is.

[0172] Tri-scFv Protein Domain Annotation for SEQ ID NO: 40 The AGIH sequence increases solubility (amino acids 1 to 4 of SEQ ID NO: 40) Vk variable region (amino acids 5 to 115 of SEQ ID NO: 40) The first (6 aa) of the light chain CH1 (amino acids 116-121 of SEQ ID NO: 40) (GGGGS)3 (SEQ ID NO: 37) linker (amino acids 122 to 136 of SEQ ID NO: 40) VH variable region (amino acids 137 to 252 of SEQ ID NO: 40) A linker between Fv fragments consisting of the first 13 amino acids of human IgG CH1 (amino acids 253 to 265 of SEQ ID NO: 40) Swivel sequence (amino acids 266 to 271 of SEQ ID NO: 40) Vk variable region (amino acids 272 to 382 of SEQ ID NO: 40) The first (6 aa) of the light chain CH1 (amino acids 383-388 of SEQ ID NO: 40) (GGGGS)3 (SEQ ID NO: 37) linker (amino acids 389 to 403 of SEQ ID NO: 40) VH variable region (amino acids 404 to 519 of SEQ ID NO: 40) Human IgG C H a linker between Fv fragments consisting of the first 13 amino acids of 1 (amino acids 520 to 532 of SEQ ID NO: 40) Swivel sequence (amino acids 533 to 538 of SEQ ID NO: 40) Vk variable region (amino acids 539 to 649 of SEQ ID NO: 40) The first (6 aa) of the light chain CH1 (amino acids 650-655 of SEQ ID NO: 40) (GGGGS)3 (SEQ ID NO: 37) linker (amino acids 656 to 670 of SEQ ID NO: 40) VH variable region (amino acids 671 to 786 of SEQ ID NO: 40) Myc tag (amino acids 787 to 802 of SEQ ID NO: 40) His6 tag (amino acids 803 to 808 of SEQ ID NO: 40)

[0173] WO2016 / 033321 and Noble, et al., Cancer Research, 75(11):2285-2291 (2015) show that di-scFv and tri-scFv have some improved additional activity compared to their monovalent counterparts. Subsequences corresponding to the different domains of exemplary fusion proteins are also provided above. Those skilled in the art will recognize that exemplary fusion proteins or domains thereof can be used to construct the fusion proteins discussed in more detail above. For example, in some embodiments, a di-scFv comprises a first scFv comprising a Vk variable region (e.g., amino acids 5-115 of SEQ ID NO: 39, or a functional variant or fragment thereof) linked to a VH variable domain (e.g., amino acids 137-252 of SEQ ID NO: 39, or a functional variant or fragment thereof) linked to a second scFv comprising a Vk variable region (e.g., amino acids 272-382 of SEQ ID NO: 39, or a functional variant or fragment thereof). In some embodiments, a tri-scFv comprises a di-scFv linked to a VH variable domain (e.g., amino acids 671-786 of SEQ ID NO: 40, or a functional variant or fragment thereof) linked to a third scFv domain comprising a Vk variable region (e.g., amino acids 539-649 of SEQ ID NO: 40, or a functional variant or fragment thereof).

[0174] The Vk variable region can be linked to the VH variable domain, for example, by a linker (e.g., (GGGGS)3 (SEQ ID NO: 37) alone or in combination with (6 aa) of the light chain CH1 (amino acids 116-121 of SEQ ID NO: 39). Other suitable linkers are discussed above and known in the art. The scFv can be linked by a linker (e.g., the first 13 amino acids (253-265) of human IgG CH1 of SEQ ID NO: 39) alone or in combination with a swivel sequence (e.g., amino acids 266-271 of SEQ ID NO: 39). Other suitable linkers are discussed above and known in the art.

[0175] Thus, a di-scFv can comprise amino acids 5-519 of SEQ ID NO: 39. A tri-scFv can comprise amino acids 5-786 of SEQ ID NO: 40. In some embodiments, the fusion protein comprises an additional domain. For example, in some embodiments, the fusion protein comprises a solubility-enhancing sequence (e.g., amino acids 1-4 of SEQ ID NO: 39). Thus, in some embodiments, a di-scFv can comprise amino acids 1-519 of SEQ ID NO: 39. A tri-scFv can comprise amino acids 1-786 of SEQ ID NO: 40. In some embodiments, the fusion protein comprises one or more domains that enhance the purification, isolation, capture, identification, separation, etc. of the fusion protein. Exemplary domains include, for example, a Myc tag (e.g., amino acids 520-535 of SEQ ID NO: 39) and / or a His tag (e.g., amino acids 536-541 of SEQ ID NO: 39). Thus, in some embodiments, a di-scFv can comprise the amino acid sequence of SEQ ID NO: 39. A tri-scFv can comprise the amino acid sequence of SEQ ID NO: 40. Other substitutable and additional domains are discussed in more detail above.

[0176] Exemplary 3E10 humanized Fv sequences are discussed in WO2016 / 033324: [ka] .

[0177] Exemplary 3E10 humanized di-scFv sequences are discussed in WO2019 / 018426 and WO / 2019 / 018428, [ka] [ka] [ka] [ka] [ka] [ka] Includes.

[0178] 3. Additional Arrays Additional sequences that may be used in the construction of cell-permeable antigen binding proteins, antibodies, fragments and fusion proteins include, but are not limited to: [ka] [ka] [ka] Examples include:

[0179] IV. Targets and Exemplary Antibodies for Proteolysis of Target Proteins Also provided are examples of antibodies from which antigen-binding characteristics can be derived to bind to proteins targeted for proteolysis. Such antibodies can be, for example, commercially available antibodies or other antibodies whose antigen-binding domains (e.g., CDRs, heavy and light chain variable domains, etc.) are known.

[0180] In some embodiments, the antibodies target, for example, DNA repair and / or DNA damage response factors, oncogenes like RAS and MYC, anti-apoptotic / pro-survival factors such as BCL-2pro, growth and transcription factors such as the androgen receptor, dual homeobox 4, also known as DUX4 (misexpression of which is the cause of facioscapulohumeral muscular dystrophy (FSHD)), mutant huntingtin protein, amyloid protein, tau protein, etc.

[0181] Specific exemplary target proteins include, but are not limited to, 4E-BP1, 5-hydroxyuridine, A1BG, AAT, ABCB1, ABCB1, ABCB5, ABCC4, ABCG2, ABCG5, ABL1, ABL2, ACLY, ACP5, ACSS1, ACTA2, ACTH, ACTR3, ACVR1, ADAM10, ADAMTS1, ADAP1, ADAR, ADD1, ADIPOQ, ADORA2A, ADRB2, AEBP2, AFAP1L2, AFP, AGR2, AGT, AHR, AIB1, AIF, AIM2, AKT1, AK T1S1, AKT2, Akt3, AKT3, ALB, ALCAM, AlCAM, ALCAM, ALDH1A1, ALDH2, ALDH6A1, ALDOA, ALK, ALK / p80, ALPI, ALPL, ALPP, AMACR, AMBRA1, AMY1A, ANAPC1, AN APC10, ANAPC11, androgen receptor, ANPEP, ANXA5, AOF1, AP2M1, APAF1, APBA2, APBB1IP, APC2, APEX1, APOA1, APOA4, APOA5, Apoa5(ab), Apoa5(bd), ApoB, ApoE , APOL1, ApoM, ApoM, APP, AQP2, AR, ARF1, ARFGAP1, ARG1, ARHGAP42, ARHGDIA, ARNTL, ASF1A, ASF1B, ASGR2, ASH2L, ASS1, ATF2, ATF3, ATF4, ATG10, ATG13 , ATG14L, ATG16L1, ATG2A, ATG3, ATG4A, ATG4B, ATG4C, ATG5, ATG7, ATL1, ATM, ATP2A1, ATP2C1, ATPIF1, ATR, ATRX, ATXN1, AUP1, AURKA, AURKA, AURKB, AX IN1, AXL, B2M, B3GAT1, B7H3, B7H4, BACE1, BAD, BAG1, BAK1, BATF, BAX, BCAT2, BCL10, BCL11B, BCL-2, BCL2L10, BCL2L2, BCL6, BCL9L, BCR, BDH1, BDNF, BECN1, beta-amyloid, beta-actin, BHMT, BID, BIN1, BIRC5, BLK, BLNK, BMI1, BMP2, BMP4, BMP7, BMPR1A, BMPR2, BMX, BNIP3, BNIP3L, BNP, Bpifa2, BPTF, BRAF,BRCA1、BRD2、BRIP1、BSA、BTK、BTLA、BTN1A1、BTN2A2、BTN3A1、BTRC、BUB1、C17ORF53、C1QA、C1QC、C3C、CA1、CA9、CALB2、カルサイクリン、CALD1、カルネキシン、CALR、カルレティキュリン、CAMK2G、CAMK4、cAMP、CAPN1、CAPN2、CARM1、CASP3、CASP6、CASP-7、CASP8、CASP9、CAV2、CBX1、CBX2、CBX3、CBX4、CBX5、CBX6、CBX7、CBX8、C-CBL、CCL2、CCL4、CCNA2、CCNB1、CCNB1、CCND1、CCND1、CCNE1、CCT2、CCT2、CD10、CD100、CD101、CD102、CD102、CD104、CD104、CD105、CD106、CD107b、CD108、CD109、CD110、CD112、CD113、CD114、CD118、CD119、CD11a、CD11C、CD11D、CD120B、CD122、CD124、CD125、CD127、CD129、CD130、CD131、CD132、CD133、CD134、CD137、CD14、CD140a、CD141、CD143、CD147、CD148、CD15、CD152、CD154、CD155、CD156、CD158D、CD158E1、CD16、CD161、CD163、CD163、CD166、CD167、CD168、CD169、CD16B、CD170、CD171、CD172A、CD172G、CD174、CD177、CD179A、CD18、CD181、CD182、CD183、CD19、CD191、CD192、CD193、CD195、CD196、CD197、CD1A、CD1B、CD1C、CD2、CD20、CD200、CD201、CD202B、CD203C、CD204、CD205、CD207、CD208、CD209、CD21、CD213A1、CD215、CD217、CD218A、CD22、CD222、CD223、CD224、CD226、CD227、CD230、CD232、CD239、CD24、CD243、CD247、CD248、CD256、CD267、CD268、CD269、CD27、CD274、CD275、CD276、CD282、CD283、CD289、CD299、CD3、CD30、CD300A、CD300C、CD301、CD302、CD303、CD305、CD306、CD307A、CD307B、CD307C、CD307E、CD31、CD314、CD315、CD319、CD322、CD327、CD328、CD329、CD33、CD334、CD334、CD335、CD336、CD339、CD34、CD344、CD351、CD352、CD353、CD354、CD355、CD36、CD360、CD361、CD363、CD364、CD367、CD368、CD369、CD37、CD370、CD38、CD39、CD3D、CD3E、CD3G、CD4、CD40、CD42A、CD42B、CD43、CD44、CD44、CD45、CD45R、CD45R、CD46、CD48、CD49B、CD49C、CD49D、CD49E、CD5、CD50、CD53、CD53、CD54、CD57、CD59、CD6、CD61、CD63、CD64、CD66A、CD66B、CD68、CD69、CD7、CD72、CD74、CD79B、CD8、CD80、CD83、CD84、CD85G、CD86、CD87、CD89、CD89、CD8A、CD9、CD93、CD94、CD96、CD97、CD99、CDC2、CDC25C、CDC27、CDC37、CDCP1、CDH1、CDH11、CDH17、CDH2、CDH5、CDK1、CDK2、CDK5、CDK9、CDKN1B、CDKN1C、CDKN2A、CDKN2A / P16、CDX1、CDX2、CEA、CEACAM5、CEBPA、CEBPB、CER1、CFHR5、CFLAR、CGA、CGB、CHD3、CHD4、CHGA、CHIT1、CHK2、CHRM3、CHRM5、CHRNA10、CHRNA2、CHRNA3、CHRNA4、CHRNA5、CHRNA6、CHRNA7、CHRNB2、CHRNB3、CHRNB4、CHRND、CHRNE、CHUK、CIB1、CIDEC、CIRBP、c-Jun、CK1、CK17、CK5、CK7、C-Kit、CKM、CLDN6、クレンブテロール、CLGN、CLL1、CLOCK、C-MYC、CNN1、CNN3、COL1A1、COL1A2、COL3A1、COTL1、COX4I1、CPV-NS1、CPV-VP2、CREB1, c-Rel, CRK, CRKL, CRP, CRTC1, CRTC2, CRTC3, CRYAB, CSF1, CSF1R, CSF2, CSF3, CSK, CSNK2A2, CSNK2B, CSPG4, CST3, CTCF, cTnI, CTNNA1, CTNNB1, CTNNBL1, CTSD, CTTN, CXCL16, CYCS, CYLD, CYP1A1, CYP3A4, cytokeratin (pan), cytokeratin 18, cytokeratin 19, cytokeratin 5, cytokeratin 5, cytokeratin 8, cytokeratin (pan), DAPK3, D APP1, DAXX, DCN, DCN, DCTN4, DCX, DDR1, DDR2, DDX1, DDX20, DDX39B, DDX3X, DDX4, DDX5, DDX58, delta Np63 alpha, desmin, DFFA, DFFB, DHX58, DIS3L2, DKK1, DKK3, DLK1, DLL4, DNAL4, DNM1L, DNMT1, DNMT3A, DNMT3B, DNMT3L, DNTT, DOC2, DSG3, DUX4, dynamin-1, dynamin-2, E2F1, E7, EDA2R, EEF2, EGF, EGFR, EGFR mutations Body, EGR1, EHMT2, EhpB1, EhpB6, EIF2A, EIF2AK2, EIF2AK3, EIF4B, EIF4E, EIF5, EIF5A, ELANE, ELK1, EMD, ENO2, eNOS, EP300, EPCAM, EphA1, Epha10, EphA 2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB2, EphB3, EphB4, EphB4, EphB6, EPN1, EPO, ER, ER-alpha, ERBB2, ERBB3, ERBB3, ERBB4, ERCC1, ERK2, ER K3, ERN1, ESR1, ESRRA, ETS1, ETV1, ETV4, ETV5, EZH1, EZH2, EZR, F2R, F8, FABP2, FABP4, FADD, FAK, FAP, FAS, FBLN2, FBLN5, FBXL10, FCER1A, FCER2, FCGR T, FER, FES, FGB, FGF2, FGF4, FGFR1, FGFR3, FGFR4, FGG, FGL1, FGR, fibulin 5, Flag, FLI1, FLT1, FLT3, FLT4, FMR1, FN1, FOLR1, FOS, FOXA2, FOXC2, FOXD3,FOXD3、FOXM1、FOXO1、FOXP1、FOXP2、FOXP3、FRK、FSHR、FTL、FUK、FUT4、FYN、FZD5、 G6PD, GAB1, GABBR2, GABPA, GAD2, GAPDH, GATA1, GATA3, GATA4, GATA5, GATA6, GCG, GCK, GFAP, GFI1, GFP, GFPT1, GH1, GITR, GKAP, GLI1, GLP, GLRA1, GLRB, glucose-6-phosphate isomerase, GLUL, GNAS, GNL3, GOT2, GPC3, GPNMB, GRIA2, GRIA3, GRIK2, GRIK3, GRIK4, GRIK5, GRIN2A, GRIN2B, GRIN3B, GRK2, GRM1, GRM2, GR M3, GRM5, GRM6, GRM7, GRM8, GSC, GSK3 alpha, GSK3B, GSN, GST, GSTM1, GSTP1, GUCY1A3, GYS1, GZMB, HAND1, HAS1, HAS2, HAS3, HAUSP, HAVCR1, HCK, HDAC10, HDAC2, HDAC3, HDAC4, HDAC6, HDAC9, HER-2, HEXA, HFE, HH3, HIF1A, HIST2H3C(27Ac), HIST2H4A(20Me), HIST2H4A(20Me), HIST2H4A(20Me3), HK1, HK2, HLA-B, HLA-DRA, HOXA9, HOXB4, HPRT, HPRT1, HPS1, HRP, HSF1, HSF4, HSP27, HSP60, HSP70, HSP90AA1, HSP90AB1, HSP90AB1, HSPA5, HSPB2, HTR3A, HTR3B, HTRA2, human albumin, human IgG, human IgG (Fc specific), human IgM, human P16, human Splunc2, huntingtin, ICAM1, ID2, IDH1, IDH2, IFN-gamma, IGF1R-beta, IGF2, IGF2BP3, IGFBP2, IGFB P7, IghA1, IGHM, IGLC2, IHOG, IKBKB, IKBKE, IL-10, IL10RB, IL-16, IL-1a, IL1B, IL1R1, IL1RAPL1, IL-2, IL28A, IL2RA, IL34, IL3RA, IL-6, IL-8, ILK, INCENP, influenza A virus nucleoprotein, influenza B virus nucleoprotein, INHA (inhibin alpha), IRAK3, IRAK4, ISL1, ITGA2B, ITGA4, ITGA5, ITGAM, ITGB1, ITGB4, ITK, JAK2,JAK3, JARID2, JUP, KARS, KAT2B, KAT6B, KAT7, KAT7, KBTBD8, KCND2, KCNQ1, KDM1A, KDM2A KDM3A, KDM4A, KDM4B, KDM5B, KDM6A, KDR, KEAP1, KHDRBS2, Ki67, KID, KIR3DL1, KIT, KLF 1, KLF15, KLF2, KLF4, KLF6, KLHL1, KLHL11, KLHL12, KLHL13, KLHL21, KLHL22, KLHL25, KL K3, KMT2A, KMT2C, KMT2D, KMT5A, KPNA2, KRT10, KRT13, KRT15, ​​KRT18, KRT19, KRT20, KSHV K8a, KSHV ORF26, KSHV ORF45, KSHV ORF62, KSHV ORF8, LAL, LAMB1, Lck, LCN1, LDLR, LEF1, LGALS1, LGR5, LHCGR, LHX2, LILRA1, LILRA2, LILRA3, LILRA 5 LILRA6, LILRB1, LILRB2, LILRB5, LIN28, LMNA, LMNB2, LMO2, LPA, LPL, LPlunc1, LPP, LRP1, LRP12. LRP1B, LRP3, LRP4, LRP5, LRP6, LRP8, LSD1 / AOF2, LTBR, ​​LYN, Lilac-1, MAP1LC3A, MAP1LC3B, MAP2. MAP2K2, MAP2K3, MAP2K4, MAP2K6, MAP2K7, MAP3K14, MAP3K2, MAP3K5, MAP3K7, MAP4K4, MAP4K4, MAPK1 0, MAPK11, MAPK14, MAPK3, MAPK8, MAPK9, MARK3, MATK, MATN1, MB, MBP, MCAM, MCL-1, MCM2, MCM3AP, MC P-1, MDM2, MDM4, MECP2, MEF2A, MEF2C, MELK, MEN1, MER, MESP1, MESP2, MET, MET, MIB1, MITF MLANA, MLH1, MLL, MLXIPL, MMEL1, MMP1, MMP14, MMP2, MMP3, MMP9, MOB1A, Lplunc1, Splunc2 TUG, MPL, MPS1, MRPL42, MSH2, MSH6, MSI1, MSI2, MSI2, MSLN, MSN, MSTN, MSX1, MTA1, MTHFR, mTORMUC16, MUC2, MUC5AC, MUC5B, MUM1, MuRF1, MUSK, MYC, MYCN, myeloperoxidase, MYF6, MYL2, MYL3, MYLK, MYOD1, myoglobin, myostatin, MYST1, NAA10, NACC1, NAGR1, NANOG, NAPSA, NBN, NCAM1, NCK1, NCOA3, NCOR1, NEDD8, NEFH, NEFL, NEFM, NES, Neurod, NeuroD, Neuropilin-1, NFE2L2, NFKB1, NFKB2, NFKBIA, NFKBIB, NF-κB p65, NGFR, NKX2.2, NKX2.5, NKX3A, NME1, NME2, NOS1, NOS2, Notch3, NOX4, NPC1, NPT, NQO1, NR1I2, NR2C2, NR3C1, NR6A1, NRCAM, NRP1, NS1, NT5E, NTRK2, NTRK3, nucleophosmin, Oct4, OLIG2, ONECUT3, OTUD4, OTX2, OVA, OX40, P16 (mouse and human), P2RX7, P2RY1, P2RY12, P2RY13, P2RY14, P2RY2, P2RY4, P2RY8, p44 / 42 MAPK(Erk1 / 2), p53, p63α, PAK2, PAK3, PAPLN, PAR1, PAR4, PARL, PARP, PAX2, PAX3, PAX4, PAX5, PAX6, PAX8, PBEF1, PBK, PCDH9, PCK2, PCNA, PC T, PD1, PDE1B, PDGFRA, PDGFRB, PDGFRβ, PDK1, PDK2, PDPK1, PDX1, PEG10, PELP1, PER3, PGA5, PGC-1alpha, PGR, PGRMC1, PHB, PHC1, Phospho-4E-BP1( Ser65), Phospho-Er alpha (Tyr-537), Phospho-NLRC4 (Ser-533), PIDD1, PIGR, PIK3CA, PIK3CG, PIK3R1, PIK3R4, PINCH, PINK1, Pirh2, PIWIL4, PKHD1, PKN1, PLA2G12A, PLA2G7, PLAGL1, PLCG1, PLCG2, PLD2, PLIN2, PLK1, PLXNA1, PMAIP1, PMS2, PODXL, POLR2A, POMC, PON1, POU3F2, PPARA, PPARG,<h2 style=";text-align:left;direction:ltr">PPARGC1B、PPM1A、PPP1A、PPP1CA、PPP1CB、PPP1R1B、PPP2R4、PPY、PR、PRAK、 PRDM1、PRDM14、PRDM4、PRDM4、PRDM5、PRK2、PRKAA1、PRKAA2、PRKAB2、PRKACA PRKACG、PRKAG1、PRKAG3、PRKDC、PRKN、PRMT6、PROM1、プロテイナーゼ3、PROZ、PSA (KLK3)、PSAP、PSG1、PSIP1、PSMA、PSMB8、PSMC3、PTEN、PTH、PTH1R、PTK6、PTK 7、PTP4A2、PTPN1、PTPN11、PTPN14、PTPN6、PTPRC、PTPRD、PTPRF、PTPRM、PTP RU、PYCARD、PYK2、Rab10、RAB11FIP1、Rab13、Rab1b、Rab25、RAB27A、RAB27B、 Rab3a, RAB4A, Rab5a, Rab6b, Rab8, Rab8, RAD18, RAD21, RAD23A, RAD50, RAD52, RAF1, RAG1, RAG2, RALA, RALB, RAN, RANGAP1, RAP1A, RAP1GAP, RAS, RB, RB1CC1、RBBP7、RBFOX2、RBFOX3、RBL2、RBP4、REG1A、RET、RF1、RHOF、RICTOR 、Ring1、RIPK1、RIPK4、RND3、RNLS、ROCK1、RON、ROP1、ROR1、ROR2、RPA1、RPL1 8A、RPS6KA2、RPS6KB1、RPTOR、RRM1、RSK1、RSK2、R-スポンジン1、RTN3、RUBCN、RU NX1、RUNX3、S100A1、S100A10 / P11、S100B、SAA1、SAG2、SALL4、SARS-CoV-2、S ARS-Cov2-NP1、SARS-Cov2-NP1、SARS-Cov2-NP2、SARS-CoV-2-NP3、SARS-E2、SARS-M、SATB2、SATB2、SCGB1A1、SCGB2A2、SDC1、SDHB、SEC31A、SELL、SERP INA、SERPINA1、SERPINA3、SERPINA7、SERPINE1、SETD2、SETD7、SETDB1、SFTP C、SH3GL1、SHC1、SHH、SIRT1、SIRT2、SIRT3、SIRT4、SIRT6、SIRT7、SK2、SKP1、SKP2, SLC22A1, SLC27A2, SLC27A5, SLC2A4, SLINGSHOT-1L, SMAD1, SMAD2, S.S MAD3, SMAD4, SMAD5, SMAD6, SMARCA1, SMC1, SMCP, SMN1, SMYD2, SNAI1, SNAI 2. SNCA, SNCG(SND1 / P100, SOD1, SOD2, SORL1, SOX10, SOX1 1, SOX2, SOX9, SP10, SP17, SPI1, SPIB, SPP1, SRA, SRA, SRC, SRY, SST, SSTR2 STAT3, STAT5A, STAT5B, STAT6, STK11, STYK1, SUZ12, SYCP3, SYK, SY N1, SYN1, SYP, SYT1, T, TAB2, TARDBP, TBC1D4, TBCC, TBP, TBX5, TCF3, TC F4, TCL1A, TEC, TERF2, TERT, TET2, TFAP2A, TFAP2B, TFAP2C, TFF2, TFRC, TG Flint1, TGFb1, TGFBR3, TH, THAP1, THAP11, THBS1, TRX, THPO, THY1 TIA1, TIE1, TIGIT, TIM3, TIP60, TLL1, Tlr2, TLR9, TNF-ルファ, TNFRSF10B. TNFRSF10D, TNFRSF11A, TNFRSF11B, TNFRSF12A, TNFRSF18, TNFRSF19, TNFR SF25, TNFRSF6B, TNFSF11, TNFSF13B, TNK1, TNNI2, TOP2A, TP53BP1, TRADD. TRAF2, TRAFD1, TRIM25, TRIM29, Trim5a, TRIP6, TrkA, TSHB, TSLPR TTF1, TTR, TUBA4A, TUBA8, TUBB1, TUBB2A, TUBB3, TUBE1, TWF1, TWIST1, TW IST2, Tyk2, TYRO3, UBB, UBE1L, UBE2C, UBE2I, UCP2, UCP3, UFD1L, UHRF1, ULB P1, ULBP2, ULK2, UTF1, VAV1, VAV2, VCAM1, VEGFA, VIL1, VIM, ビメンチン, VIMP, V ISTA, VP2, VTN, WAS, WDFY3, WDR5, WDR66, WHSC2, WIF1, WNT1, WNT10B, WNT3A.These include WNT5A, WT1, WTAP, XBP1, XIAP, XRCC5, XRCC6, XRN2, YAP1, YES1, YWHAB, YWHAQ, ZAP70, ZBTB16, ZBTB7B, ZEB1, ZFP42, and ZFP91.

[0182] Antibodies that bind to each of the aforementioned target proteins are known in the art and can be used in constructing the disclosed constructs and antibodies. For example, in some embodiments, the antigen-binding characteristics comprise the CDRs, and optionally the heavy and light variable regions, of an antibody that binds to one of the aforementioned proteins.

[0183] V. Pharmaceutical Compositions The compositions can be used therapeutically in combination with a pharmaceutically acceptable carrier.

[0184] Compositions containing molecules for targeting proteolysis, such as bispecific antibodies, are preferably used for therapeutic use in combination with a suitable pharmaceutical carrier. Such compositions comprise an effective amount of the composition and a pharmaceutically acceptable carrier or excipient.

[0185] The composition may be formulated for local, regional, or systemic administration in a suitable pharmaceutical carrier. Remington's Pharmaceutical Sciences, 15th Edition by E. W. Martin (Mark Publishing Company, 1975) discloses typical carriers and methods for preparation. The molecule may also be encapsulated in suitable biocompatible particles formed from biodegradable or non-biodegradable polymers, or proteins, or liposomes for cell targeting. Such systems are well known to those skilled in the art. In some embodiments, the molecule is encapsulated in nanoparticles.

[0186] Injectable preparations may be presented in unit dosage form, for example, in ampoules or multi-dose containers, with added preservatives as needed. The compositions may take the form of sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which, in certain embodiments, may be isotonic with the subject's blood. Examples of non-aqueous solvents include polypropylene glycol, polyethylene glycol, vegetable oils such as olive oil, sesame oil, coconut oil, peanut oil, mineral oil, injectable organic esters such as ethyl oleate, or fixed oils, including synthetic mono- or diglycerides. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, 1,3-butanediol, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, and electrolyte replenishers (such as those based on Ringer's dextrose). The material may be in solution, emulsion, or suspension (e.g., incorporated into particles, liposomes, or cells). Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to make the formulation isotonic. Trehalose, typically in an amount of 1 to 5%, may be added to the pharmaceutical composition. The pH of the solution is preferably about 5 to about 8, and more preferably about 7 to about 7.5.

[0187] Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, and surfactants. Carrier formulations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. Those skilled in the art can easily determine various parameters for preparing and formulating the compositions without resorting to undue experimentation.

[0188] The compositions, alone or in combination with other suitable components, can also be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and air. For administration by inhalation, the compounds are delivered in the form of aerosol spray presentation from a pressurized pack or nebulizer by using a suitable propellant.

[0189] In some embodiments, a pharmaceutically acceptable carrier is included with formulation ingredients such as salts, carriers, buffers, emulsifiers, diluents, excipients, chelating agents, preservatives, solubilizers, or stabilizers.

[0190] Further carriers include sustained-release preparations, such as semipermeable materials of solid hydrophobic polymers containing molecules, where the matrix is ​​in the form of shaped particles, such as films, liposomes, or microparticles. Implantation includes inserting implantable drug delivery systems, such as microspheres, hydrogels, polymer reservoirs, cholesterol matrices, polymer systems, such as matrix erosion and / or diffusion systems, and non-polymer systems. Inhalation includes administering the composition by aerosol in an inhaler, either alone or attached to an absorbable carrier. For systemic administration, it may be preferable for the composition to be encapsulated in liposomes.

[0191] The compositions can be delivered in a manner that allows for tissue specificity, for example, using invasive devices, such as vascular or urinary catheters, and interventional devices, such as stents with drug delivery capabilities and configured as expandable devices or stent grafts.

[0192] The formulation may be delivered using a biodegradable implant by diffusion or by degradation of the polymer matrix. In certain embodiments, the administration of the formulation may be designed to provide continuous exposure to the composition for a certain period of time, for example, for several hours, days, weeks, months, or years. This may be achieved, for example, by repeated administration of the formulation or by a sustained-release or controlled-release delivery system in which the composition is delivered over an extended period of time without repeated administration.

[0193] Other suitable delivery systems include timed-release, delayed-release, sustained-release, or controlled-release delivery systems. Such systems can avoid repeated administration in many cases, increasing convenience for patients and physicians. Many types of release delivery systems are available and known to those skilled in the art. These include, for example, polymer-based systems such as polylactic acid and / or polyglycolic acid, polyanhydrides, polycaprolactones, copolyoxalates, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and / or combinations thereof. Microcapsules of the aforementioned polymers containing nucleic acids are described, for example, in U.S. Pat. No. 5,075,109. Other examples include lipid-based non-polymer systems, including sterols, such as cholesterol, cholesterol esters, and fatty acids or neutral lipids, such as mono-, di-, and triglycerides; hydrogel release systems; liposome-based systems; phospholipid-based systems; silastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; or partially fused implants. The formulation may be, for example, as a microsphere, hydrogel, polymer reservoir, cholesterol matrix, or polymer system. In some embodiments, the system may allow for sustained or controlled release of the composition, for example, by controlling the diffusion or erosion / degradation rate of the formulation.

[0194] The composition can be formulated for pulmonary or mucosal administration.Administration can include delivering the composition to the mucous membrane of the lung, nose, oral cavity (sublingual, buccal), vagina or rectum.The term aerosol as used herein refers to any preparation of atomized mist of particles, which can be in solution or suspension, regardless of whether it is produced using a propellant.Aerosol can be produced by standard techniques, for example, ultrasonication or high pressure treatment.

[0195] For administration via the upper respiratory tract, the formulation may be formulated into a solution, e.g., buffered or unbuffered, in water or isotonic saline, or a suspension, for intranasal administration as drops or a spray. Preferably, such a solution or suspension is isotonic with respect to nasal secretions and has approximately the same pH, e.g., from about pH 4.0 to about pH 7.4, or from pH 6.0 to pH 7.0. The buffer should be physiologically compatible, and examples thereof include, by way of example only, phosphate buffer.

[0196] The composition can be delivered to target cells using a particle delivery vehicle. Nanoparticles generally refer to particles with diameters in the range of 500 nm to less than 0.5 nm, preferably 50 to 500 nm, and more preferably 50 to 300 nm. Cellular internalization of polymer particles is highly dependent on their size, with nanoparticle polymer particles being internalized into cells much more efficiently than microparticle polymer particles. For example, Desai et al. demonstrated that approximately 2.5 times more nanoparticles with a diameter of 100 nm were taken up by cultured Caco-2 cells than microparticles with a diameter of 1 μm (Desai, et al., Pharm. Res., 14:1568-73 (1997)). Nanoparticles also have a greater ability to diffuse deeper into tissues in vivo.

[0197] In some embodiments, the delivery vehicle is a dendrimer.

[0198] Examples of preferred biodegradable polymers include synthetic polymers that degrade by hydrolysis, such as poly(hydroxy acids), e.g., polymers and copolymers of lactic acid and glycolic acid, other degradable polyesters, polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butyric acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates), poly(lactide-co-caprolactone), and poly(amine-co-ester) polymers, such as those described in Zhou, et al., Nature Materials, 11:82-90 (2012), and WO2013 / 082529, U.S. Published Application No. 2014 / 0342003, and PCT / US2015 / 061375.

[0199] The targeting moiety can be directly or indirectly associated, linked, conjugated, or otherwise attached to the nanoparticle or other delivery vehicle. The targeting molecule can be a protein, peptide, nucleic acid molecule, saccharide, or polysaccharide that binds to a receptor or other molecule on the surface of the targeted cell. The degree of specificity and avidity of binding to the graft can be modulated by the selection of the targeting molecule.

[0200] Examples of moieties include targeting moieties that provide delivery of molecules to specific cells. Examples of molecules that target the extracellular matrix ("ECM") include glycosaminoglycans ("GAGs") and collagen. In one embodiment, the outer surface of the polymer particle can be modified to enhance the particle's ability to interact with selected cells or tissues. The method described above, in which an adapter element conjugated to a targeting molecule is inserted into the particle, is preferred. However, in another embodiment, the outer surface of a polymer micro- or nanoparticle with a carboxy terminus can be linked to a targeting molecule with a free amine terminus.

[0201] In another embodiment, the outer surface of the particle can be treated with mannose amine, thereby mannosylating the outer surface of the particle.This treatment can make the particle bind to target cells or tissues through the mannose receptor on the surface of antigen-presenting cells.Alternatively, the surface conjugation with immunoglobulin molecules containing Fc part (targeting Fc receptor), heat shock protein part (HSP receptor), phosphatidylserine (scavenger receptor), and lipopolysaccharide (LPS) is an additional receptor target in cells or tissues.

[0202] Lecithin can be covalently attached to micro- and nanoparticles to specifically target them to mucin and mucosal cell layers.

[0203] The selection of the targeting moiety depends on the method of administration of the nanoparticle composition and the cells or tissues to be targeted. Targeting molecules can generally increase the binding affinity of particles to cells or tissues, or can target nanoparticles to specific tissues in an organ or specific cell types in a tissue. In some embodiments, the targeting moiety targets the thymus, spleen, or cancer cells.

[0204] Covalent attachment of any of the natural components of mucin, either in pure or partially purified form, to particles will reduce the surface tension of the bead-intestine interface and increase the solubility of beads in the mucin layer. Attachment of polyamino acids containing extra pendant carboxylic acid side groups, such as polyaspartic acid and polyglutamic acid, increases bioadhesion. The use of polyamino acids in the molecular weight range of 15,000 to 50,000 kDa results in chains of 120 to 425 amino acid residues attached to the particle surface. The polyamino chains increase bioadhesion by intertwining the mucin chains and by increasing the carboxylic acid charge.

[0205] VI. Method of Use Methods of use are also provided, which typically involve contacting a cell with an effective amount of the disclosed molecules to enhance proteolysis of one or more target proteins through the TRIM21 pathway.

[0206] Molecules can be homogeneous, e.g., all of the same type target the same protein for degradation. Molecules can also be heterogeneous, e.g., two or more different types target the same or different proteins for degradation.

[0207] The results indicate that the effectiveness of the TRIM21 pathway can be increased by inducing clustering of TRIM21 (Zeng, et al., "Target-induced clustering activates Trim-Away of pathogens and proteins", Nat Struct Mol Biol 28, 278-289 (2021). doi.org / 10.1038 / s41594-021-00560-2). This can be achieved by targeting multiple epitopes on the same target protein (e.g., using multispecific molecules) and / or using two or more different molecules that target different epitopes on the same target protein.

[0208] Cell penetration of certain antibodies, such as 3E10, can be enhanced by the presence of extracellular nucleic acid, and therefore, in some embodiments, the disclosed compositions are administered in combination with nucleic acid, either in the same or separate mixtures.

[0209] In some embodiments, the composition is administered in an effective amount or therapeutically effective amount sufficient to treat, inhibit, or alleviate one or more symptoms of a disease or disorder, or otherwise provide a desired pharmacological and / or physiological effect, for example, to reduce, inhibit, or reverse one or more of the pathophysiological mechanisms underlying a disease or disorder. In some embodiments, the protein targeted for degradation contributes to the underlying cause of a disease or disorder. For example, in some embodiments, the protein is overexpressed, abnormally expressed, and / or mutant compared to wild-type protein. Exemplary target proteins are discussed above, and any of them can be the subject of the disclosed methods.

[0210] The formulation of composition is carried out to suit the mode of administration.Pharmaceutically acceptable carrier is determined in part by the specific composition to be administered and by the specific method used to administer the composition.Therefore, there are a wide variety of suitable formulations of pharmaceutical compositions containing molecules.Accurate dosage varies according to various factors, such as subject-dependent variables (for example, age, immune system health, clinical symptoms, etc.).

[0211] The composition can be administered or otherwise contacted with target cells once, twice, or three times daily, once, twice, three, four, five, six, or seven times weekly, or once, twice, three, four, five, six, seven, or eight times monthly. For example, in some embodiments, the composition is administered every two or three days, or about two to about four times per week on average. Thus, in some embodiments, the composition is administered as part of a dosing regimen that includes two or more separate treatments.

[0212] Dosage regimens include a maintenance regimen in which the dosage remains the same between two or more administrations, an ascending regimen in which the dosage is increased between two or more administrations, a tapering regimen in which the dosage is decreased between two or more administrations, or a combination thereof.

[0213] In some embodiments, the initial dose may be low. Dose escalation may continue until a satisfactory biochemical or clinical response is achieved. The clinical response depends on the disease or disorder being treated and / or the desired outcome. In some embodiments, the dosage may be increased until a therapeutic effect is identified, preferably without inducing undesirable toxicity or an acceptably high dose thereof. The dosage may then be maintained or gradually reduced to a maintenance dose. The method can be used to standardize, optimize, or individualize the dose level, dosing frequency, or duration of therapy.

[0214] A. In vivo method In some embodiments, the bispecific antibody is used to treat a disease or disorder in a subject in vivo. The composition can be directly administered to the subject for in vivo therapy. In some embodiments, the bispecific antibody targets an antigen (e.g., a protein) associated with the disease or disorder being treated. For example, if the disease or disorder is caused by an overexpressed and / or mutant protein, the disclosed compositions and methods can be used to increase the effective amount of targeted degradation of the protein to treat the subject or the disease or disorder.

[0215] In general, methods for administering compounds, including antibodies, are well known in the art.The composition can be administered by many routes, including but not limited to intravenous, intraperitoneal, intraamniotic, intramuscular, subcutaneous, or topical (sublingual, rectal, intranasal, pulmonary, rectal mucosa, and vaginal), and oral (sublingual, buccal).In a preferred embodiment, the composition is injected or infused into an animal.

[0216] In some embodiments, the composition is formulated for pulmonary delivery, for example, intranasal administration or oral inhalation.The administration of the formulation can be achieved by any acceptable method that allows molecules to reach their target.Depending on the condition to be treated, administration can be localized (i.e., to a specific region, physiological system, tissue, organ, or cell type) or systemic.Compositions and methods for in vivo delivery are also discussed in WO2017 / 143042.

[0217] Method can also include administering an effective amount of composition to embryo or fetus or its pregnant mother in vivo.In some methods, composition is delivered into uterus by injecting and / or injecting into vein or artery, for example, vitelline vein or umbilical vein, or into the amniotic sac of embryo or fetus.For example, see Ricciardi, et al., Nat Commun.2018 Jun 26;9(1):2481.doi:10.1038 / s41467-018-04894-2 and WO2018 / 187493.

[0218] B. In Vitro and Ex Vivo Methods For in vitro and ex vivo methods, cells are typically contacted with the composition during culturing. For ex vivo methods, cells can be isolated from a subject and contacted with the composition ex vivo to produce cells containing molecules. In a preferred embodiment, cells are isolated from the subject to be treated or from an inbred host. Target cells can be removed from the subject before contacting with the composition.

[0219] The disclosed invention can be further understood by the following numbered sections:

[0220] 1. An antigen-binding molecule comprising (i) a TRIM21-binding characteristic, (ii) a cell-permeability characteristic of a cell-permeability antibody, and (iii) an antigen-binding characteristic that binds to a target protein.

[0221] 2. The antigen-binding molecule of paragraph 1, wherein the TRIM21-binding characteristic comprises the boundary between the CH2 and CH3 domains of the immunoglobulin constant region.

[0222] 3. The antigen-binding molecule of paragraphs 1 and 2, wherein the TRIM21-binding characteristic comprises the CH2 and CH3 domains of the immunoglobulin constant region.

[0223] 4. The antigen-binding molecule of paragraphs 2 and 3, wherein the immunoglobulin is IgG, optionally human IgG, optionally IgG1.

[0224] 5. The antigen-binding molecule according to any one of Items 1 to 4, wherein the cell-permeable characteristic of the cell-permeable antibody comprises a cell-permeable domain of an autoantibody.

[0225] 6. The antigen-binding molecule of any one of items 1 to 5, wherein the cell-permeable antibody has a cell-permeable domain of antibody 3E10 or 5C6, or a variant or humanized form thereof.

[0226] 7. The antigen-binding molecule according to any one of items 1 to 6, wherein the antigen-binding molecule that binds to the target protein is a VH and VL domain, a single-chain antibody, a single-chain variable fragment (scFv), a di-scFv, a tri-scFv, a diabody, a triabody, a tetrabody, a disulfide-linked Fv (sdFv), Fab', F(ab')2, Fv, a single-domain antibody fragment (sdAb), a nanobody, or a VHH.

[0227] 8. The antigen-binding molecule of any one of Items 1 to 7, further comprising a complementarity-determining region (CDR) of a cell-permeable antibody, and, if necessary, heavy chain and light chain variable regions.

[0228] 9. The antigen-binding molecule of any one of items 1 to 8, further comprising the sequence of TRIM21 or a functional fragment or variant thereof.

[0229] 10. The antigen-binding molecule of any one of clauses 1 to 9, wherein the molecule comprises two or more antigen-binding features that bind to different epitopes on the same protein.

[0230] 11. The antigen-binding molecule of any one of clauses 1 to 10, wherein the molecule comprises two or more antigen-binding features that bind to epitopes on different proteins.

[0231] 12. The antigen-binding molecule of any one of items 1 to 11, wherein the molecule is a multispecific antibody.

[0232] 13. The antigen-binding molecule of paragraph 12, wherein the molecule is a bi- or trispecific antibody.

[0233] 14. The antigen-binding molecule of any one of items 1 to 11, wherein the molecule is a bispecific or trispecific IgG, IgG-scFv, IgG-dAb, scFv-Fc-scFv, knob-in-hole (KIH)-IgG, κλ-body, KIH0Fc-Fab / scFv, tandem scFv, KIH trispecific, or bispecific Fc fusion (with or without N- or C-terminus, KIH).

[0234] 15. The six CDRs, and optionally heavy and light chain variable regions, of 3E10 or 5C6, optionally selected from the CDRs and / or heavy and light chain variable regions provided herein, the heavy and light chain variable regions of SEQ ID NOs: 12 and 14, respectively, or optionally variants or humanized forms thereof having at least 70% sequence identity thereto; an antigen-binding domain for the target protein, and Fc domain Item 15. The antigen-binding molecule of any one of Items 1 to 14, comprising:

[0235] 16. A composition comprising an effective amount of the antigen-binding molecule according to any one of items 1 to 15.

[0236] 17. The composition of paragraph 16, comprising any effective amount of two or more different antigen-binding molecules.

[0237] 18. The composition of clause 17, wherein the two or more different antigen-binding molecules target the same protein.

[0238] 19. The composition of paragraph 17 or 18, wherein the two or more different antigen-binding molecules target different proteins.

[0239] 20. A method for targeting degradation of one or more target proteins, comprising contacting a cell with the composition of any one of paragraphs 16-19.

[0240] 21. The method of paragraph 20, wherein the contacting occurs in vivo in a subject in need thereof.

[0241] 22. The method of paragraph 21, wherein the composition is administered to a subject in need thereof by injection or infusion.

[0242] 23. The method of paragraph 21 or 22, wherein the subject has a disease or disorder.

[0243] 24. The method of paragraph 23, wherein the disease or disorder is caused by overexpression and / or aberrant expression of the target protein, and / or expression of a mutant thereof.

[0244] 25. The method of paragraph 24, wherein the composition is administered in an effective amount to treat the disease or disorder. [Example]

[0245] Example 1 3E10 treatment of cells mediates the intracellular degradation of RAD51. material and method HCC38, YUGASP, and U2OS cells were seeded in 6-well plates and treated with the 3E10 antibody (heavy and light chains of SEQ ID NOs: 12 and 14, respectively) at the indicated concentrations for 3 days. After treatment, the cells were trypsinized, pelleted, and protein extracted. RAD51 levels were quantified by Western blot.

[0246] result A modified version of the cell-permeable antibody 3E10 mediated the intracellular degradation of RAD51, where 3E10 has moderate intrinsic binding affinity. See Figures 1A-1C. This is likely due to a pathway for degradation of intracellular antibodies bound to pathogens (and internalized by cells in antibody / pathogen complexes) by the TRIM21 protein, which has ubiquitination activity. TRIM21 binds to intracellular antibodies because it has a domain that binds to the IgG Fc domain. Upon recruitment of multiple TRIM21 molecules, the RING (SEQ ID NO: 127) domain is ubiquitinated and subsequently degraded along with the antibody-binding protein. Because RAD51 typically exists in filaments or clusters, binding of the full-length mAb 3E10 to RAD51 promoted its intracellular degradation via the ubiquitination pathway mediated by TRIM21.

[0247] Example 2 Cell-permeable bispecific antibodies that degrade target proteins of interest Bispecific antibodies were designed in which one Fab arm was 3E10 and the other arm targeted an intracellular antigen of interest (Figure 2). These were generated using a standard CrossMab format employing sequences from the parent 3E10 (heavy and light chains of SEQ ID NOs: 118 and 14, respectively) and a mAb targeting GFP (heavy and light chains of SEQ ID NOs: 119 and 120, respectively). See also anti-GFP [N86 / 38.1R], Addgene plasmid no. 114492; Andrews, et al., Elife. 2019 Jan 22;8. pii: 43322. doi: 10.7554 / eLife.43322. 10.7554 / eLife.43322; Krah, "Engineering IgG-Like Bispecific Antibodies—An Overview," Antibodies 7, no. 3: 28. doi.org / 10.3390 / antib7030028 (2018), and Klein, mAbs, 8:6, 1010-1020, DOI: 10.1080 / 19420862.2016.1197457 (2016). [ka] [ka]

[0248] Example 3 Cell-permeable bispecific antibodies are rapidly degraded by the proteosome material and method B16 cells in culture were pretreated with or without the proteasome inhibitor MG132. After 60 minutes of treatment, the cells were treated with 1 μM of the 3E10 / GFP bispecific mAb of Example 2. After 24 hours of treatment, the cells were harvested and the levels of the bispecific antibody were monitored by Western blot.

[0249] result Figures 3A-3B show the assay (Figure 3A) and results (Figure 3B) for proteosome-mediated intracellular degradation of a bispecific antibody. Use of the proteosome inhibitor MG132 rescues the signal, as shown by Western blot in Figure 3B.

[0250] Example 4 Cell-permeable bispecific antibodies mediate nucleoproteolysis of target proteins material and method 293T cells in culture were transfected with GFP-NLS mRNA. After 24 hours, the cells were treated with the 3E10 / GFP bispecific antibody described in Example 2 and analyzed by immunofluorescence imaging and flow cytometry. One Fab arm targets 3E10, and the other arm targets GFP.

[0251] result An experiment was designed to determine whether the 3E10 / GFP bispecific antibody increases degradation of the target protein. Cells were treated with the bispecific antibody of Example 1. Results show that introducing the bispecific antibody into 293T cells resulted in a decrease in fluorescence after 24 hours, indicating degradation of nuclear green fluorescent protein (GFP) levels, as demonstrated by the immunofluorescence image in Figure 4A. Results obtained using flow cytometry showed a 19% degradation of GFP levels (Figures 4B and 4C), compared to untreated controls, demonstrating targeted degradation of GFP using this bispecific antibody.

[0252] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs. Publications cited herein and the material for which they are cited are specifically incorporated by reference.

[0253] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. An antigen-binding molecule comprising (i) a TRIM21-binding characteristic, (ii) a cell-permeability characteristic of a cell-permeability antibody, and (iii) an antigen-binding characteristic that binds to a target protein.

2. The antigen-binding molecule of claim 1, wherein the TRIM21 binding characteristic comprises the boundary between the CH2 and CH3 domains of an immunoglobulin constant region.

3. The antigen-binding molecule of claims 1 and 2, wherein the TRIM21 binding characteristics comprise the CH2 and CH3 domains of an immunoglobulin constant region.

4. The antigen-binding molecule of claims 2 and 3, wherein the immunoglobulin is an IgG, optionally a human IgG, optionally an IgG1.

5. The antigen-binding molecule according to any one of claims 1 to 4, wherein the cell-penetrating feature of the cell-penetrating antibody comprises a cell-penetrating domain of an autoantibody.

6. The antigen-binding molecule of any one of claims 1 to 5, wherein the cell-penetrating feature of the cell-penetrating antibody comprises the cell-penetrating domain of antibody 3E10 or 5C6, or a variant or humanized form thereof.

7. The antigen-binding molecule of any one of claims 1 to 6, wherein the antigen-binding feature that binds to the target protein is a VH and VL domain, a single-chain antibody, a single-chain variable fragment (scFv), di-scFv, tri-scFv, diabody, triabody, tetrabody, disulfide-linked Fv (sdFv), Fab', F(ab')2, Fv, a single-domain antibody fragment (sdAb), a nanobody, or a VHH.

8. The antigen-binding molecule of any one of claims 1 to 7, further comprising the complementarity-determining regions (CDRs) of the cell-permeable antibody, and optionally heavy and light chain variable regions.

9. The antigen-binding molecule of any one of claims 1 to 8, further comprising a sequence of TRIM21, or a functional fragment or variant thereof.

10. The antigen-binding molecule of any one of claims 1 to 9, comprising two or more antigen-binding characteristics that bind to different epitopes on the same protein.

11. The antigen-binding molecule of any one of claims 1 to 10, comprising two or more antigen-binding features that bind to epitopes on different proteins.

12. The antigen-binding molecule of any one of claims 1 to 11, which is a multispecific antibody.

13. The antigen-binding molecule of claim 12, which is a bispecific or trispecific antibody.

14. The antigen-binding molecule of any one of claims 1 to 11, which is a bispecific or trispecific IgG, IgG-scFv, IgG-dAb, scFv-Fc-scFv, knobs-in-holes (KIH)-IgG, κλ-body, KIH0Fc-Fab / scFv, tandem scFv, KIH trispecific, or bispecific Fc fusion (with or without N- or C-terminal KIH).

15. the six CDRs and optionally heavy and light chain variable regions of 3E10 or 5C6 optionally selected from the CDRs and / or heavy and light chain variable regions provided herein, the heavy and light chain variable regions of SEQ ID NOs: 12 and 14, respectively, or optionally variants or humanized forms thereof having at least 70% sequence identity thereto; an antigen-binding domain for the target protein, and Fc domain The antigen-binding molecule of any one of claims 1 to 14, comprising:

16. A composition comprising an effective amount of the antigen-binding molecule of any one of claims 1 to 15.

17. 17. The composition of claim 16, comprising any effective amount of two or more different antigen-binding molecules.

18. The composition of claim 17, wherein the two or more different antigen-binding molecules target the same protein.

19. The composition of claim 17 or 18, wherein the two or more different antigen-binding molecules target different proteins.

20. 20. A method for targeting the degradation of one or more target proteins, comprising contacting a cell with a composition according to any one of claims 16 to 19.

21. 21. The method of claim 20, wherein said contacting occurs in vivo in a subject in need thereof.

22. 22. The method of claim 21, wherein the composition is administered to a subject in need thereof by injection or infusion.

23. 23. The method of claim 21 or 22, wherein the subject has a disease or disorder.

24. 24. The method of claim 23, wherein the disease or disorder is caused by overexpression and / or aberrant expression of the target protein and / or expression of a mutant thereof.

25. 25. The method of claim 24, wherein the composition is administered in an effective amount to treat the disease or disorder.

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