JPH10502365 | [Title of Invention] Helicobacter protein and vaccine |
JP2022137076 | ANTI-CD33 ANTIBODY AND METHOD FOR USING THE SAME |
JP2022514499 | AntiIL-27Antibodies and their use |
ZHANG DINGPENG (US)
WO2022174114A1 | 2022-08-18 | |||
WO2018152326A1 | 2018-08-23 | |||
WO2022020105A1 | 2022-01-27 |
US20190010242A1 | 2019-01-10 |
CLAIMS What is claimed: 1. A fusion protein-therapeutic moiety, comprising: a fusion protein of Formula I: R1-R2-R3 (I), wherein; R1 is at least one protein of interest (POI) binder (POIB); R2 is a linker of the formula R4-R5 or R5-R4, wherein: R4 is an IgG Fc region; and R5 is a protease-sensitive linking means; and R3 is a transferrin receptor binding (TRB) means, and optionally, wherein a linkage between R2 and R3 is a glycine-rich linker; and a therapeutic moiety conjugated to the fusion protein. 2. The fusion protein-therapeutic moiety of claim 1, wherein the therapeutic agent is conjugated to the POIB. 3. The fusion protein-therapeutic moity of claim 1 or 2, wherein a linkage between R2 and R3 is a glycine-rich linker selected from the group consisting of SEQ ID NO: 9, 10, 11, 12, 13, 14, 15 and 16. 4. The fusion protein-therapeutic moiety of any one of claims 1-3, wherein the therapeutic agent is conjugated using a bioconjugation reaction. 5. The fusion protein-therapeutic moiety of claim 4, wherein the bioconjugation reaction comprises a maleimide-based cysteine reaction. 6. The fusion protein-therapeutic moiety of any one of claims 1-5, wherein the protease-sensitive linking means comprises a cathepsin-cleavable peptide. 7. The fusion protein-therapeutic moiety of claim 6 wherein the cathepsin-cleavable peptide is selected from the group consisting of FK, VA, VK, SEQ ID NO: 7, 8, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144 and 145. 8. The fusion protein-therapeutic moiety of any one of claims 1-7, wherein the TRB binding means is H7 or M16. 9. The fusion protein-therapeutic moiety of any one of claims 1-8, wherein the fusion protein of Formula I is a homodimer. 10. The fusion protein-therapeutic moiety of any one of claims 1-9, wherein the fusion protein of Formula I is a heterodimer linked to a fusion protein of Formula II: R4’-R3’ (II): wherein: R4’ is IgG Fc region; and R3’ is a transferrin receptor binding (TRB) means; optionally, wherein a linkage between R3’ and R4’ is a protease-sensitive linking means; and optionally, wherein a therapeutic moiety is conjugated to the fusion protein of Formula II. 11. The fusion protein-therapeutic moiety of any one of claims 1-11, wherein the TRB comprises an antibody or polypeptide. 12. The fusion protein-therapeutic moiety of any one of claims 1-12, wherein the TRB is selected from the group consisting of SEQ ID NOs: 3, 4, and 5. 13. The fusion protein-therapeutic moiety of any one of claims 1-12, wherein the POIB comprises an antibody. 14. The fusion protein-therapeutic moiety of any one of claims 1-13, wherein the POIB binds to an extracellular domain of a transmembrane protein. 15. The fusion protein-therapeutic moiety of any one of claims 1-14, wherein the POIB binds to an extracellular domain of CD20, CD30, CD22, CD33, CD79b, CD19, HER2, or Trop2. 16. The fusion protein-therapeutic moiety of any one of claims 1-15, wherein the therapeutic moiety comprises an anticancer agent or a glucocorticoid receptor modulator (GRM). 17. The fusion protein-therapeutic moiety of claim 16, wherein the anticancer agent comprises monomethyl auristatin A, monomethyl auristatin F, mertansine, calicheamicin, SN-38, deruxtecan, or exatecan. 18. The fusion protein-therapeutic moiety of claim 16, wherein the GRM comprises dexamethasone or budesonide. 19. A nucleic acid sequence encoding the fusion protein-therapeutic moiety of any one of claims 1-18. 20. A method for delivering a therapeutic moiety to a subject, the method comprising administering the fusion protein-therapeutic moiety of any one of claims 1-18 to the subject. 21. The fusion protein-therapeutic agent of any one of claims 1-18 for use in delivering a therapeutic moiety to a subject. 22. A homodimer of a fusion protein of Formula III: R1-R2-R3 (III): wherein: R1 is at least one protein of interest (POI) binder (POIB); R2 is a linker of the formula R4-R5 or R5-R4, wherein: R4 is IgG Fc region; and R5 is a protease-sensitive linking means; and R3 is a transferrin receptor binding (TRB) means, and optionally, wherein a linkage between R2 and R3 is a glycine-rich linker, and wherein a therapeutic moiety is conjugated to the fusion protein. 23. The homodimer of claim 22, additionally comprising a disulfide bond between cysteine amino acids in R4 of separate fusion proteins. 24. A homodimer of a fusion protein of Formula I: R1-R6-R3 (I): wherein: R1 is at least one protein of interest (POI) binder (POIB); R6 is a dimerization domain; and R3 is a transferrin receptor binding (TRB) means; and optionally, wherein a linkage between R1 and R6, or between R6 and R3, is a protease- sensitive linking means. 25. A pharmaceutical composition, comprising the fusion protein-therpeutic moiety or homodimer of a fusion protein of any one of claims 1-18 or 22-24. |
[00381] In some embodiments, a bispecific modulator can be a heterodimer between a fusion protein R1-R2-R3 (R1 is POIB; R2 is R4-R5 or R5-R4 with R4 as antibody Fc region, and R5 as protease-sensitive linker; R3 is TRB, as above) and a fusion protein R3-R4 or R4-R3, where R3 is a TRB and R4 is an antibody Fc region. In some embodiments, optionally, there can be a protease-sensitive linking means between R3 and R4, or R3-R4 or R4-R3. [00382] In some embodiments, a bispecific modulator disclosed herein can have the formula R1-R6-R3, where R1 is a protein of interest binder (POIB), R6 is a dimerization means, and R3 is a transferrin receptor binding (TRB) means. In some embodiments, R6 can be a moiety that links R1 and R3 (e.g., a linking means that connects R1 and R3). In some embodiments, R6 can be an amino acid linker. In some embodiments, the amino acid linker can be protease sensitive. In some embodiments, R6 can be a dimerization domain that can form a dimer with another copy of R6 through a covalent (e.g., cysteine-containing Fc antibody region or other dimerization domain) or non-covalent bond. In some embodiments, R6 can be a combination of a linking molecule as above (e.g., that can be protease sensitive) and a dimerization domain. [00383] In some embodiments, a bispecific modulator disclosed herein can have the formula R1-R6-R3, where R1 is a protein of interest binder (POIB), R6 is a dimerization means, and R3 is a transferrin receptor binding (TRB) means. In some embodiments, R6 can be a moiety that links R1 and R3, and contains a dimerization domain. The dimerization domain can form a dimer with another copy of R6 through a covalent (e.g., cysteine-containing Fc antibody region) or non-covalent bond. In some embodiments, the dimerization domain can be an Fc antibody region that has one or more cysteines. In some embodiments, or optionally, a linkage between R1 and R6, or between R6 and R3, can be a protease-sensitive linking means. [00384] In some embodiments, a first component of a bispecific modulator can be R1-R6 or R6-R1, where R1 can be a POIB, and R6 can be a dimerization domain as above and, optionally, also a protease sensitive amino acid linker. A second component of the bispecific modulator can be R3-R7, where R3 can be a TRB means, and R7 can be a multimerization domain as above and, optionally, also a protease sensitive amino acid linker. A bispecific modulator can be formed when the multimerization domain (e.g., dimerization domain) of the first component forms convalent or non-covalent bonding with the multimerization domain of the second component. [00385] In some embodiments, the bispecific modulators disclosed herein are designed to target cell-surface molecules (e.g., molecules of interest, such as proteins of interest) on tumor cells. In some embodiments, these cell-surface molecules can regulate growth of the cells. In some embodiments, targeting of these cell-surface molecules can kill the tumor cells. In some embodiments, the cell-surface molecule targeted by the bispecific modulators can be epidermal growth factor receptor (EGFR). In some embodiments, these bispecific modulators have 10-50- fold better IC 50 for tumor/cancer cells than other treatments. [00386] In some embodiments, the bispecific modulators disclosed herein are used to internalize and degrade multi-pass transmembrane proteins (i.e., transmembrane proteins that span the membrane multiple times and create multiple extracellular domains). In some embodiments, CD20 is a protein targeted using these bispecific modulators. [00387] In some embodiments, a bispecific modulator can be one polypeptide chain. In some embodiments, a bispecific modulator can be two polypeptide chains. In some embodiments of a two-polypeptide configuration, two binders are encoded in two polypeptide chains. In some embodiments, the two polypeptide chains can be linked or connected by a dimerizing domain. In some embodiments, the two polypeptide chains can be linked or connected by a knob-in-hole Fc. Targeted Receptor Degradation by TransTAC [00388] In some embodiments, cell-surface molecules (i.e., molecules of interest, such as proteins of interest) that a bispecific modulator targets can be internalized by the bispecific modulator. In some embodiments, the internalized molecule may not be degraded or may be minimally degraded inside the cell. In some embodiments, the bispecific modulators disclosed herein are modified to more efficiently degrade targeted proteins and to more efficiently deliver a therapeutic agent to a cell in such a way that the agent can be effective. In some embodiments, the bispecific modulators are modified to contain amino acid sequences sensitive to proteases (e.g., see FIG.35). The proteases can be endosomal or lysosomal proteases. In some embodiments, a peptide linker that is a target for cathepsin proteases can be used. When the linker is cleaved by the protease, the molecule of interest is released from the bispecific modulator. [00389] In some embodiments, the linkers are sensitive to cleavage by cathepsins. In some embodiments, the cathepsins can be cathepsin A, B, C, D, E, F, G, H, K, L1, L2, O, S, W or Z. [00390] In some examples, the molecule of interest can be released from bispecific modulators other than by inclusion of a protease-sensitive linker (e.g., pH-dependent binding of TRB). [00391] Cleavage of the linker inside the cell (e.g., in endosomes) can release the molecule of interest from the internalizing receptor or membrane protein and increase the likelihood that the molecule of interest will be degraded. In some embodiments, the protease-sensitive peptide linker can be positioned such that cleavage of the bispecific modulators by the protease releases or dissociates the targeted protein from the bispecific modulator, allowing the targeted protein to be more completely degraded. [00392] In some embodiments, bispecific modulator is R1-R2-R3, as described earlier, and where R2 can be R4-R5 or R5-R4 (R5 is protease-sensitive linking means), the protease- sensitive linking means can be located between a POIB (R1) and an Fc region from an antibody (R4), as in R1-R5-R4-R3. In some embodiments, the protease-sensitive linking means can be located between an Fc region from an antibody (R4) and a TRB (R3), as in R1-R4-R5-R3. In some embodiments, release of the targeted protein from the bispecific modulator can be accomplished by incorporating low pH-sensitive amino acid regions into the bispecific modulators. In some embodiments, when the bispecific modulator is inside an endosome, the low pH environment can release/dissociate the targeted protein from the bispecific modulator such that the targeted protein is more efficiently degraded. [00393] In some embodiments, a linker can be sensitive to the low pH present in endosomes. In some embodiments, the low pH can cause cleavage of the linker. [00394] In some embodiments, the transferrin receptor binding means (TRB) can bind to the transferrin dependent on pH. For example, the TRB may have less affinity for transferrin receptor at lower pH found in endosomes. The lower affinity may result in the TRB releasing the transferrin receptor. This release can facilitate degradation of the protein of interest bound to the POIB. Such a TRB can be “M16” as shown in FIG.35. [00395] In some embodiments, the protease-sensitive linker can be located between the first moiety (targets a molecule of interest) and the second moiety (binds to an internalizing receptor or membrane protein). In some embodiments, the linker can be localized closer to the first moiety than to the second moiety. [00396] In some embodiments, the protease sensitive linking means can include Gly-Phe-Leu- Gly (GFLG; SEQ ID NO: 118). In some embodiments, the peptide linker can include a Valine- Arginine (VR) and/or Phenylalanine-Lysine (FK) sequence. In some embodiments, the peptide linker can be a GFLG (SEQ ID NO: 118), 3xGFLG (GFLGGFLGGFLG; SEQ ID NO: 119), GFLGVA (SEQ ID NO: 120), GFLGVK (SEQ ID NO: 121), GFLGVR (SEQ ID NO: 122), GFLGGFLG (SEQ ID NO: 123), FK, VA, EVA, or VK linker (FIG.35). In some embodiments, the peptide linker can be GGFLGGVRGVDG (SEQ ID NO: 7) or GSGSGGEVRGVDG (SEQ ID NO: 8). In some embodiments, the peptide linker can be GFLGGVR (SEQ ID NO: 144) or GGGEVRG (SEQ ID NO: 145). [00397] In experiments, (FIG.57A-B) a yeast-displayed peptide library was used to identify peptides not known to be sensitive to cathepsin cleavage. These peptides can be from a combination of small motifs found in SEQ ID NOs: 144 and 145. In some embodiments, these peptides can be GRLVGFD (SEQ ID NO: 124), GRLVGFG (SEQ ID NO: 125), RMLVGFV (SEQ ID NO: 126), RRLYAFL (SEQ ID NO: 127), VFRLLMF (SEQ ID NO: 128), LVGVLLF (SEQ ID NO: 129), VKLYGLG (SEQ ID NO: 130), TWRVDLY (SEQ ID NO: 131), EQLYLYA (SEQ ID NO: 132), KLFLMIF (SEQ ID NO:133 ), NFVIILF (SEQ ID NO: 134), MSLLIGV (SEQ ID NO: 135), VRLLSLQ (SEQ ID NO: 136), STLMWNV (SEQ ID NO: 137), VRFLAAA (SEQ ID NO: 138), HGWSFHE (SEQ ID NO: 139), ENLYFQG (SEQ ID NO: 140), VVMMFLH (SEQ ID NO: 141), VFRLLMF (SEQ ID NO:142 ), or VGALVWL (SEQ ID NO: 143). [00398] Other sequences can be used. [00399] In some embodiments, any combination of these peptide linkers and/or valine- citrulline (VC) linker and/or glutamate-valine-arginine (EVR) linker can be used. Therapeutic Moieties [00400] In some embodiments, a therapeutic agent or therapeutic moiety can be associated with a bispecific modulator. In some embodiments, the therapeutic agent can be conjugated to (e.g., covalently attached) to the bispecific modulator. [00401] In embodiments, a therapeutic agent or therapeutic moiety can be conjugated to the the bispecific modulator with a drug-antibody ratio (DAR) of 1, 2, 3, 4, 5, 6, 7, 8, or 9. In examples, the therapeutic agent or therapeutic moiety can be conjugated to the the bispecific modulator with an average DAR of 2 or 4. In examples the therapeutic agent or therapeutic moiety can be conjugated to the the bispecific modulator with an average DAR of 2. In examples, the therapeutic agent or therapeutic moiety can be conjugated to the the bispecific modulator with an average DAR of 4. In examples, the therapeutic agent or therapeutic moiety can be conjugated to the hinge region of a bispecific modulator. [00402] Various types of therapeutic moieties can be used. In some embodiments, the therapeutic moiety can be a small molecule (e.g., ≤ 1,000 daltons). In some embodiments, the therapeutic moiety can be a large molecule (e.g., protein, polypeptide, nucleic acid, polysaccharide, and the like). In some embodiments, the therapeutic moiety can be a biologic (e.g., antibodies). [00403] In some embodiments, the therapeutic moiety can be any agent that can be used in the context of a conventional antibody-drug conjugate (ADC). In some embodiments, therapeutic agent can be any used in gemtuzumab ozogamicin, brentuximab vedotin, tsastuzumab emtansine, inotuzumab ozogamicin, polatuzumab vedotin, enfortumab vedotin, trastuzumab deruxtecan, sacituzumab govitecan, belantamab mafodotin, moxetumomab pasudotox, loncastuximab tesirine, tisotumab vedotin-tftv, mirvetuximab soravtansine, and the like. [00404] In some embodiments, the therapeutic moiety can be an anticancer agent or a glucocorticoid receptor modulator (GRM). [00405] In some embodiments, the anticancer agent can be a microtubulin inhibitor (e.g., monomethyl auristatin E or MMAE; monomethyl auristatin F or MMAF, mertansine and the like), a DNA binder (e.g., calicheamicin and the like), topoisomerase 1 inhibitors (e.g., SN-38, exatecan, deruxtecan and the like). In some embodiments, the GRM can be dexamethasone, budesonide, and the like. [00406] In some embodiments, the therapeutic moiety can be a small RNA, like an siRNA. In some embodiments, the therapeutic moiety can be a diabody, Fab, scFv, bicyclic peptide, and the like. Functionalizing Antibody-Drug Conjugates Using TransTAC [00407] In some embodiments, the bispecific modulators disclosed herein are used to internalize and degrade antibody drug conjugate (ADC) therapeutics. ADCs that target non- internalizing or slowly-internalizing receptors or internalizing membrane proteins may not be efficacious because the therapeutic small molecules are not efficiently released from the ADC in endosomal or lysosomal environments. [00408] Herein is disclosed that bispecific modulators can be used to target non-internalizing or slowly-internalizing receptors or internalizing membrane proteins and deliver ADC therapeutics that are efficacious. In some embodiments, the drugs are conjugated to the bispecific modulators. The bispecific modulators can cause cell internalization of the drugs and release of active forms of the drug inside the cell. In some embodiments, protease-sensitive linkers can be used to facilitate this process. In some embodiments, the receptor targeted is CD20. In some embodiments, ADCs are delivered to treat B-cell malignancies. [00409] In some embodiments, the therapeutic moiety can be conjugated to the part of the bispecific modulator that binds to a protein of interest (POI). In some embodiments, the bispecific modulator contains that has a conjugated therapeutic moiety has a protease-sensitive linker or protease-sensitive linking means located between the part of the bispecific modulator to which the therapeutic moiety is conjugated (e.g., the protein of interest binder) and the part of the bispecific modulator that binds to an internalizing receptor (e.g., transferrin receptor). [00410] In some embodiments, conjugation of the therapeutic moiety to the bispecific modulator can use a chemical reaction(s). In some embodiments, the conjugation can use bioconjugation. Bioconjugation reactions can be of different types. Common types of bioconjugation reactions can include coupling of cysteine, lysine and tyrosine amino acids. Common types of bioconjugation reactions can include modification of tryptophan amino acids and of the N- and C-terminus of a protein. In some embodiments, the bioconjugation reaction can use a linker to connect the therapeutic moiety to the bispecific modulator. [00411] In some embodiments, a strategy is used to attach a specific site of a therapeutic moiety to a specific site on the bispecific modulator. In some embodiments, this can be done by installing a unique functional group onto a protein portion and use a bioorthogonal reaction to couple the therapeutic moiety to the bispecific modulator. In some embodiments, these reactions can use modification of ketone and aldehydes, Staudinger ligation with organic azides, copper- catalyzed Huisgen cycloaddition of azides, or strain promoted Huisgen cycloaddition of azides. Other types of reactions can be used. [00412] In some embodiments, the bioconjugation reaction can be a maleimide-based cysteine reaction. Antibodies [00413] Unique recombinant monoclonal antibodies are disclosed which can be part of the bispecific modulators disclosed herein. In embodiments, an antibody may be used in the first and/or second moiety of the bispecific modulators discloses herein. [00414] “Recombinant” as it pertains to polypeptides (such as antibodies) or polynucleotides refers to a form of the polypeptide or polynucleotide that does not exist naturally, a non-limiting example of which can be created by combining polynucleotides or polypeptides that would not normally occur together. “Polypeptide” as used herein can encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, can refer to “polypeptide” herein, and the term “polypeptide” can be used instead of, or interchangeably with any of these terms. “Polypeptide” can also refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis. As to amino acid sequences, one of skill in the art will readily recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds, deletes, or substitutes a single amino acid or a small percentage of amino acids in the encoded sequence is collectively referred to herein as a "conservatively modified variant". In some embodiments the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants of the antibodies disclosed herein can exhibit increased cross-reactivity in comparison to an unmodified antibody. [00415] For example, a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in an immunoglobulin polypeptide is replaced with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members. [00416] Some embodiments also feature antibodies that have a specified percentage identity or similarity to the amino acid or nucleotide sequences of the antibodies described herein. For example, “homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence, which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. For example, the antibodies can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher amino acid sequence identity when compared to a specified region or the full length of any one of the antibodies described herein. For example, the antibodies can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher nucleic acid identity when compared to a specified region or the full length of any one of the antibodies described herein. Sequence identity or similarity to the nucleic acids and proteins of the present invention can be determined by sequence comparison and/or alignment by methods known in the art, for example, using software programs known in the art, such as those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. For example, sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment or visual inspection can be utilized to determine percent sequence identity or similarity for the nucleic acids and proteins of the present invention. [00417] Aspects of the invention provide isolated antibodies. The term “isolated” as used herein with respect to cells, nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the macromolecule. The term “isolated” can also refer to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. For example, an “isolated nucleic acid” can include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. “Isolated” can also refer to cells or polypeptides which are isolated from other cellular proteins or tissues. Isolated polypeptides can include both purified and recombinant polypeptides. [00418] As used herein, an “antibody” or “antigen-binding polypeptide” can refer to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen binding fragment or a single chain thereof. For example, “antibody” can include any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to the antigen. Non-limiting examples a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein. As used herein, the term "antibody" can refer to an immunoglobulin molecule and immunologically active portions of an immunoglobulin (Ig) molecule, i.e., a molecule that contains an antigen binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts with" can refer to the antibody reacting with one or more antigenic determinants of the desired antigen and does not react with other polypeptides. [00419] The terms “antibody fragment” or “antigen-binding fragment”, as used herein, is a portion of an antibody such as F(ab′)2, F(ab)2, Fab′, Fab, Fv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. The term “antibody fragment” can include aptamers (such as spiegelmers), minibodies, and diabodies. The term “antibody fragment” can also include any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. Antibodies, antigen-binding polypeptides, variants, or derivatives described herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab′ and F(ab′)2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, dAb (domain antibody), minibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies. [00420] A “single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (V H ) and light chains (V L ) of immunoglobulins. A single chain Fv ("scFv") polypeptide molecule is a covalently linked VH:VL heterodimer, which can be expressed from a gene fusion including VH- and VL-encoding genes linked by a peptide-encoding linker. (See Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883). In some aspects, the regions are connected with a short linker peptide of ten to about 25 amino acids. The linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N- terminus of the V H with the C-terminus of the V L , or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. A number of methods have been described to discern chemical structures for converting the naturally aggregated, but chemically separated, light and heavy polypeptide chains from an antibody V region into an scFv molecule, which will fold into a three-dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Patent No.5,091,513; No.5,892,019; No.5,132,405; and No.4,946,778, each of which are incorporated by reference in their entireties. [00421] Antibody molecules obtained from humans fall into five classes of immunoglobulins: IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε) with some subclasses among them (e.g., γ1-γ4). Certain classes have subclasses as well, such as IgG 1 , IgG 2 , IgG 3 and IgG 4 and others. The immunoglobulin subclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc. are well characterized and are known to confer functional specialization. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight approximately 53,000-70,000. The four chains are joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region. Immunoglobulin or antibody molecules described herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of an immunoglobulin molecule. [00422] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can be bound with either a kappa or lambda light chain. For example, the light and heavy chains are covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells, or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain. [00423] Both the light and heavy chains are divided into regions of structural and functional homology. The terms “constant” and “variable” are used functionally. The variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3) confer biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. The term "antigen-binding site," or "binding portion" can refer to the part of the immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches within the V regions of the heavy and light chains, referred to as "hypervariable regions," are interposed between more conserved flanking stretches known as "framework regions," or "FRs". Thus, the term "FR" can refer to amino acid sequences which are naturally found between, and adjacent to, hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions," or "CDRs." [00424] The six CDRs present in each antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain as the antibody assumes its three-dimensional configuration in an aqueous environment. The remainder of the amino acids in the antigen-binding domains, the FR regions, show less inter- molecular variability. The framework regions largely adopt a β-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the β-sheet structure. The framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen-binding domain formed by the positioned CDRs provides a surface complementary to the epitope on the immunoreactive antigen, which promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and the framework regions, respectively, can be readily identified for a heavy or light chain variable region by one of ordinary skill in the art, since they have been previously defined (See, “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)). [00425] Where there are two or more definitions of a term which is used and/or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of the term “complementarity determining region” (“CDR”) to describe the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. This region has been described by Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and by Chothia et al., J. Mol. Biol.196:901-917 (1987), which are incorporated herein by reference in their entireties. The CDR definitions according to Kabat and Chothia include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth in the table below as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody. [00426] Kabat et al. defined a numbering system for variable domain sequences that is applicable to any antibody. The skilled artisan can unambiguously assign this system of “Kabat numbering” to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). [00427] In addition to table above, the Kabat number system describes the CDR regions as follows: CDR-H1 begins at approximately amino acid 31 (i.e., approximately 9 residues after the first cysteine residue), includes approximately 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the fifteenth residue after the end of CDR-H1, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at approximately the thirty third amino acid residue after the end of CDR-H2; includes 3- 25 amino acids; and ends at the sequence W-G-X-G, where X is any amino acid. CDR-L1 begins at approximately residue 24 (i.e., following a cysteine residue); includes approximately 10-17 residues; and ends at the next tryptophan residue. CDR-L2 begins at approximately the sixteenth residue after the end of CDR-L1 and includes approximately 7 residues. CDR-L3 begins at approximately the thirty third residue after the end of CDR-L2 (i.e., following a cysteine residue); includes approximately 7-11 residues and ends at the sequence F or W-G-X-G, where X is any amino acid. [00428] As used herein, the term "epitope" can include any protein determinant that can specifically bind to an immunoglobulin, a scFv, or a T-cell receptor. The variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. For example, the VL domain and VH domain, or subset of the complementarity determining regions (CDRs), of an antibody combine to form the variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of the Y. Epitopic determinants can consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. For example, antibodies can be raised against N- terminal or C-terminal peptides of a polypeptide. More specifically, the antigen-binding site is defined by three CDRs on each of the VH and VL chains (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3). [00429] As used herein, the terms "immunological binding," and "immunological binding properties" can refer to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (K d ) of the interaction, wherein a smaller K d represents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen- binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both the "on rate constant" (Kon) and the "off rate constant" (K off ) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See Nature 361: 186-87 (1993)). The ratio of Koff /Kon allows the cancellation of all parameters not related to affinity, and is equal to the equilibrium binding constant, KD. (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). An antibody of the invention can specifically bind to an epitope when the equilibrium binding constant (K D ) is ≤1 ^M, ≤10 μΜ, ≤ 10 nM, ≤ 10 pM, or ≤ 100 pM to about 1 pM, as measured by kinetic assays such as radioligand binding assays or similar assays known to those skilled in the art, such as BIAcore or Octet (BLI). For example, in some embodiments, the KD is between about 1E-12 M and a KD about 1E-11 M. In some embodiments, the K D is between about 1E-11 M and a K D about 1E-10 M. In some embodiments, the KD is between about 1E-10 M and a KD about 1E-9 M. In some embodiments, the KD is between about 1E-9 M and a KD about 1E-8 M. In some embodiments, the K D is between about 1E-8 M and a K D about 1E-7 M. In some embodiments, the K D is between about 1E-7 M and a K D about 1E-6 M. For example, in some embodiments, the K D is about 1E-12 M while in other embodiments the KD is about 1E-11 M. In some embodiments, the K D is about 1E-10 M while in other embodiments the K D is about 1E-9 M. In some embodiments, the K D is about 1E-8 M while in other embodiments the K D is about 1E-7 M. In some embodiments, the KD is about 1E-6 M while in other embodiments the KD is about 1E-5 M. In some embodiments, for example, the KD is about 3 E-11 M, while in other embodiments the K D is about 3E-12 M. In some embodiments, the K D is about 6E-11 M. “Specifically binds” or “has specificity to,” can refer to an antibody that binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope. For example, an antibody is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. [00430] For example, the antibody can be monovalent or bivalent, and can comprise a single or double chain. Functionally, the binding affinity of the antibody is within the range of 10 −5 M to 10 −12 M. For example, the binding affinity of the antibody is from 10 −6 M to 10 −12 M, from 10 −7 M to 10 −12 M, from 10 −8 M to 10 −12 M, from 10 −9 M to 10 −12 M, from 10 −5 M to 10 −11 M, from 10 −6 M to 10 −11 M, from 10 −7 M to 10 −11 M, from 10 −8 M to 10 −11 M, from 10 −9 M to 10 −11 M, from 10 −10 M to 10 −11 M, from 10 −5 M to 10 −10 M, from 10 − M to 10 −10 M, from 10 −7 M to 10 −10 M, from 10 −8 M to 10 −10 M, from 10 −9 M to 10 −10 M, from 10 −5 M to 10 −9 M, from 10 −6 M to 10 −9 M, from 10 −7 M to 10 −9 M, from 10 −8 M to 10 −9 M, from 10 −5 M to 10 −8 M, from 10 −6 M to 10 −8 M, from 10 −7 M to 10 −8 M, from 10 −5 M to 10 −7 M, from 10 −6 M to 10 −7 M, or from 10 −5 M to 10 −6 M. [00431] Those skilled in the art will recognize that one can determine, without undue experimentation, if a human monoclonal antibody has the same specificity as a human monoclonal antibody of the invention by ascertaining whether the former prevents the latter from specifically binding. For example, if the human monoclonal antibody being tested competes with the human monoclonal antibody of the invention, as shown by a decrease in binding by the human monoclonal antibody of the invention, then the two monoclonal antibodies bind to the same, or to a closely related, epitope. [00432] Another way to determine whether a human monoclonal antibody has the specificity of a human monoclonal antibody of the invention is to pre-incubate the human monoclonal antibody of the invention with an epitope, with which it is normally reactive, and then add the human monoclonal antibody being tested to determine if the human monoclonal antibody being tested is inhibited in its ability to bind the epitope. If the human monoclonal antibody being tested is inhibited then, it has the same, or functionally equivalent, epitopic specificity as the monoclonal antibody of the invention. Screening of human monoclonal antibodies of the invention can be also carried out by utilizing epitopes and determining whether the test monoclonal antibody is able to neutralize polypeptides containing the epitope. [00433] Various procedures known within the art can be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof. (See, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). [00434] Antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, can be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No.8 (April 17, 2000), pp.25-28). [00435] The term “monoclonal antibody” or “mAb” or “Mab” or “monoclonal antibody composition”, as used herein, can refer to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. For example, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs contain an antigen binding site that can immunoreact with a specific epitope of the antigen characterized by a unique binding affinity for it. [00436] Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro. Nucleic Acids, Vectors and Cells Expressing Bispecific Modulators [00437] Also disclosed are nucleic acids encoding all or part of the bispecific modulators described herein. Also disclosed are various vectors (e.g., plasmids, viral, and the like) that include the nucleic acids. Also disclosed are various cells (e.g., prokaryotic, eukaryotic) that contain nucleic acids or vectors and can express the fusion proteins. Methods [00438] Disclosed herein are methods for administrating the bispecific modulators and conjugated therapeutic moieties described herein to a subject. In various embodiments, the bispecific modulators can internalize membrane proteins of interest (e.g., cellular receptors or other membrane proteins) and selectively degrade and/or modulate these proteins. In embodiments, the conjugated therapeutic moiety is removed from the bispecific modulator such that it is active in the cell. In some embodiments, the bispecific modulators can target CAR receptors on CAR-T cells or other receptors. In some embodiments, the methods are used to treat toxicities in subjects who have received CAR-T cell infusion for treatment of cancer (e.g., toxicities due to cytokine release). In some embodiments, the methods are used to improve efficacy of CAR-T cells that have been administered to a subject to treat cancer. In some embodiments, the bispecific modulators to which a therapeutic moiety is conjugated can be used to treat cancer or immune disorders. [00439] In some embodiments, membrane proteins on cancer cells can be targeted to degrade and/or modulate the proteins (e.g., epidermal growth factor receptor or EGFR, Programmed death-ligand or PD-L1). In some examples, bispecific modulators can be used to improve anti- tumor responses in this way. [00440] In some embodiments, the reagents and methods disclosed herein can be used with cells that are not cancer cells. [00441] In some embodiments, the methods can be used to functionalize antibody drug conjugates. Therapeutic Preparations [00442] Aspects of the invention are drawn towards therapeutic preparations. As used herein, the term “therapeutic preparation” can refer to any compound or composition that can be used or administered for therapeutic effects (e.g., bispecific modulators). As used herein, the term “therapeutic effects” can refer to effects sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. [00443] Embodiments as described herein can be administered to a subject in the form of a pharmaceutical composition or therapeutic preparation prepared for the intended route of administration. Such compositions and preparations can comprise, for example, the active ingredient(s) and a pharmaceutically acceptable carrier. Such compositions and preparations can be in a form adapted to oral, subcutaneous, parenteral (such as, intravenous, intraperitoneal), intramuscular, rectal, epidural, intratracheal, intranasal, dermal, vaginal, buccal, ocularly, or pulmonary administration, such as in a form adapted for administration by a peripheral route or is suitable for oral administration or suitable for parenteral administration. Other routes of administration are subcutaneous, intraperitoneal and intravenous, and such compositions can be prepared in a manner well-known to the person skilled in the art, e.g., as described in “Remington's Pharmaceutical Sciences”, 17. Ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, Pa., U.S.A., 1985 and more recent editions and in the monographs in the “Drugs and the Pharmaceutical Sciences” series, Marcel Dekker. The compositions and preparations can appear in conventional forms, for example, solutions and suspensions for injection, capsules and tablets, in the form of enteric formulations, e.g., as disclosed in U.S. Pat. No.5,350,741, and for oral administration. [00444] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. [00445] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition can be sterile and can be fluid to the extent that easy syringeability exists. In embodiments, it can be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyethylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by using a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by using surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it can be useful to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. [00446] Sterile injectable solutions can be prepared by incorporating the compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, examples of useful preparation methods are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. [00447] Oral compositions can include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Oral formula of the drug can be administered once a day, twice a day, three times a day, or four times a day, for example, depending on the half-life of the drug. [00448] Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition administered to a subject. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel ® (sodium starch glycolate) , or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. [00449] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as known in the art. [00450] In embodiments, administering can comprise the placement of a pharmaceutical composition into a subject by a method or route which results in at least partial localization of the composition at a desired site such that desired effect is produced. [00451] For example, the pharmaceutical composition can be administered by bolus injection or by infusion. A bolus injection can refer to a route of administration in which a syringe is connected to the IV access device and the medication is injected directly into the subject. The term “infusion” can refer to an intravascular injection. [00452] Embodiments as described herein can be administered to a subject one time (e.g., as a single injection, bolus, or deposition). Alternatively, administration can be once or twice daily to a subject for a period of time, such as from about 2 weeks to about 28 days. Administration can continue for up to one year. In embodiments, administration can continue for the life of the subject. It can also be administered once or twice daily to a subject for period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof. [00453] In embodiments, compositions as described herein can be administered to a subject chronically. “Chronic administration” can refer to administration in a continuous manner, such as to maintain the therapeutic effect (activity) over a prolonged period of time. [00454] A specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the particular antibodies, variant or derivative thereof used, the patient's age, body weight, general health, sex, and diet, and the time of administration, rate of excretion, drug combination, and the severity of the particular disease being treated. Judgment of such factors by medical caregivers is within the ordinary skill in the art. The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art. [00455] A therapeutically effective amount of a reagent or therapeutic composition of the invention can be the amount needed to achieve a therapeutic objective. As noted herein, this can be a binding interaction between the reagent or therapeutic composition and its target that, in certain cases, interferes with the functioning of the target. The amount required to be administered will furthermore depend on the binding affinity of the reagent or therapeutic composition for its specific target and will also depend on the rate at which an administered reagent or therapeutic composition is depleted from the free volume other subject to which it is administered. The dosage administered to a subject (e.g., a patient) of the binding polypeptides described herein is about 0.1 mg/kg to 100 mg/kg of the patient's body weight, between 0.1 mg/kg and 20 mg/kg of the patient's body weight, or 1 mg/kg to 10 mg/kg of the patient's body weight. Human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of reagent or therapeutic composition of the disclosure may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation. Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention can be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight. Common dosing frequencies can range, for example, from twice daily to once a week. [00456] Where fragments (e.g., antibody fragments) are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. (See, e.g., Marasco et al, Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The formulation can also contain more than one active compound as necessary for the specific indication being treated, for example, those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine (e.g., IL-15), chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. [00457] The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions. Sustained-released preparations can be prepared. [00458] The pharmaceutical or therapeutic carrier or diluent employed can be a conventional solid or liquid carrier. Nonlimiting examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid or lower alkyl ethers of cellulose. Nonlimiting examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water. Similarly, the carrier or diluent can include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax. [00459] When a solid carrier is used for oral administration, the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form or it can be in the form of a troche or lozenge. The amount of solid carrier will vary widely but can be from about 25 mg to about 1 g. [00460] When a liquid carrier is used, the preparation can be in the form of a syrup, emulsion, soft gelatin capsule or sterile injectable liquid such as an aqueous or non-aqueous liquid suspension or solution. [00461] The composition and/or preparation can also be in a form suited for local or systemic injection or infusion and can, as such, be formulated with sterile water or an isotonic saline or glucose solution. The compositions can be in a form adapted for peripheral administration only, except for centrally administrable forms. The compositions and/or preparations can be in a form adapted for central administration. [00462] The compositions and/or preparations can be sterilized by conventional sterilization techniques which are well known in the art. The resulting aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with the sterile aqueous solution prior to administration. The compositions and/or preparations can contain pharmaceutically and/or therapeutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents and the like, for instance sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. Embodiments [00463] Disclosed below in numbered paragraphs are example embodiments disclosed herein. [00464] 1. A bispecific modulator as disclosed herein. [00465] 2. The bispecific modulator of embodiment 1, comprising: [00466] a. a first moiety comprising an antigen or epitope that can be bound by a chimeric antigen receptor (CAR); and [00467] b. a second moiety that can bind to an internalizing receptor or membrane protein on a cell. [00468] 3. The bispecific modulator of embodiment 1, comprising: [00469] a. a first moiety comprising an antibody that can bind to a CAR; and [00470] b. a second moiety that can bind to an internalizing receptor or membrane protein on a cell. [00471] 4. The bispecific modulator of one of embodiments 2 or 3, wherein the second moiety comprises an antibody. [00472] 5. The bispecific modulator of one of embodiments 2 or 3, wherein binding of the first moiety by the CAR and binding of the second moiety by the internalizing receptor or membrane protein on the cell causes internalization of the CAR into the cell. [00473] 6. The bispecific modulator of embodiment 5, wherein binding of the first moiety by the CAR and binding of the second moiety by the internalizing receptor or membrane protein on the cell causes internalization of the CAR into the cell. [00474] 7. The bispecific modulator or embodiment 5, wherein binding of the first moiety by the CAR and binding of the second moiety by the internalizing receptor or membrane protein on the cell causes internalization of CAR into the cell and degradation of the CAR. [00475] 8. A molecule with at least two moieties, comprising: [00476] a. a first moiety that can bind to a molecule of interest on a cell; and [00477] b. a second moiety that can bind to an internalizing molecule on a cell. [00478] 9. The molecule of embodiment 29, wherein: [00479] a. the first moiety comprises an antibody, antibody fragment, ligand, peptide, small molecule, or apatamer; and [00480] b. the second moiety comprises an antibody, antibody fragment, ligand, peptide, a small molecule, or an apatamer. [00481] 10. The molecule of embodiment 8, wherein the molecule of interest on the cell is not the same as the internalizing molecule on the cell. [00482] 11. The molecule of embodiment 8, wherein the first moiety and the second moiety comprise one polypeptide. [00483] 12. The molecule of embodiment 9, wherein the internalizing molecule on the cell comprises an internalizing receptor. [00484] 13. The molecule of embodiment 12, wherein the internalizing molecule can be internalized by clathrin-mediated endocytosis. [00485] 14. The molecule of embodiment 12, wherein the internalizing molecule can be internalized by clathrin-independent endocytosis. [00486] 15. The molecule of embodiment 12, wherein the internalizing molecule comprises transferrin receptor. [00487] 16. The molecule of embodiment 12, wherein the internalizing molecule comprises a G-protein coupled receptor (GPCR), receptor tyrosine kinase (RTK) or transmembrane receptor (TMR). [00488] 17. The molecule of embodiment 16, wherein the GPCR comprises an adrenoceptor, chemokine receptor, or coagulation receptor. [00489] 18. The molecule of embodiment 16, wherein the RTK comprises a colony stimulating factor receptor, epidermal growth factor receptor, tyrosine kinase receptor, fibroblast growth factor receptor, insulin-like growth factor receptor, platelet-derived growth factor receptor, or transforming growth factor receptor. [00490] 19. The molecule of embodiment 16, wherein the TMR comprises a folate receptor, interleukin receptor (e.g., IL-2 receptors), low density lipoprotein receptor, or transferrin receptor. [00491] 20. The molecule of embodiment 12, wherein the internalizing molecule comprises a transferrin receptor (TfR). [00492] 21. The molecule of embodiment 20, wherein the transferrin receptor comprises transferrin receptor 1 (TfR1) or transferrin receptor 2 (TfR2). [00493] 22. The molecule of embodiment 9, wherein the ligand for the internalizing molecule comprises at least a part of a natural-occurring ligand that can be bound by the receptor. [00494] 23. The molecule of embodiment 22, wherein the ligand for the internalizing molecule comprises at least a part of transferrin, cholesterol, low-density lipoprotein, and epidermal growth factor. [00495] 24. The molecule of embodiment 9, wherein the ligand for the internalizing molecule can be bound by a G-protein coupled receptor (GPCR), receptor tyrosine kinase (RTK) or transmembrane receptor (TMR). [00496] 25. The molecule of embodiment 22, wherein the ligand for the internalizing molecule can be bound by a transferrin receptor. [00497] 26. The molecule of embodiment 22, wherein the ligand for the internalizing molecule comprises transferrin protein or a portion of a transferrin protein. [00498] 27. The molecule of embodiment 9, wherein the second moiety comprises a Fab, scFv, single-domain antibody, nanobody, monobody, DARPin or affibody. [00499] 28. The molecule of embodiment 27, wherein the second moiety comprises an H7 scFv. [00500] 29. The molecule of embodiment 27, wherein the second moiety comprises H7 Fab or an engineered H7 antibody variant. [00501] 30. The molecule of embodiment 9, wherein the second moiety can bind to a transferrin receptor, cholesterol receptor, low-density lipoprotein receptor, or epidermal growth factor receptor. [00502] 31. The molecule of embodiment 9, wherein the second moiety can bind to a G-protein coupled receptor (GPCR), receptor tyrosine kinase (RTK), or transmembrane receptor (TMR). [00503] 32. The molecule of embodiment 9, wherein the second moiety can bind to transferrin receptor (TfR). [00504] 33. The molecule of embodiment 8, wherein the molecule of interest comprises a protein. [00505] 34. The molecule of embodiment 33, wherein the protein comprises a membrane protein. [00506] 35. The molecule of embodiment 33, wherein the protein comprises an integral membrane protein. [00507] 36. The molecule of embodiment 33, wherein the protein comprises an extracellular protein. [00508] 37. The molecule of embodiment 36, wherein the extracellular protein is found in an external environment. [00509] 38. The molecule of embodiment 36, wherein the extracellular protein is selected from the group consisting of an autoantibody, a cytokine, an enzyme, and combinations thereof. [00510] 39. The molecule of embodiment 33, wherein the protein comprises a transmembrane protein. [00511] 40. The molecule of embodiment 39, wherein the transmembrane protein has one (1) or more transmembrane domains. [00512] 41. The molecule of embodiment 8, wherein the molecule of interest can bind a hormone, cytokine, growth factor, neurotransmitter, lipophilic signaling molecule (e.g., prostaglandin) or cell recognition molecule (e.g., integrin, selectin). [00513] 42. The molecule of embodiment 8 wherein the molecule of interest comprises a receptor. [00514] 43. The molecule of embodiment 42, wherein the receptor comprises a G-protein coupled receptor (GPCR). [00515] 44. The molecule of embodiment 42, wherein the receptor comprises a receptor tyrosine kinase (RTK) or transmembrane receptor (TMR). [00516] 45. The molecule of embodiment 39, wherein the receptor comprises a ligand-gated ion channel-linked molecule, a transporter, an enzyme-linked molecule, or a G-protein-linked receptor. [00517] 46. The molecule of embodiment 45, wherein the ligand-gated ion channel linked molecule provides for movement of Na+, K+, Ca2+, or Cl- to move across a plasma membrane of a cell. [00518] 47. The molecule of embodiment 45, wherein the enzyme-linked molecule comprises a receptor tyrosine kinase, tyrosine-kinase-associated receptor (e.g., enzymes that associate with cytokines), receptor-like tyrosine phosphatase (e.g., that remove phosphate groups from tyrosines of intracellular proteins), receptor serine/threonine kinase, receptor guanylyl cyclase, or histidine-kinase-associated receptor. [00519] 48. The molecule of embodiment 8, wherein the molecule of interest comprises a chimeric antigen receptor (CAR), receptor tyrosine kinase (e.g., EGFR), a molecule to which a checkpoint inhibitor can bind (e.g., PD-L1), or lineage-specific marker (e.g., CD20). [00520] 49. The molecule of embodiment 8, wherein the molecule of interest comprises a CAR, EGFR, CD20 or PD-L1. [00521] 50. The molecule of embodiment 9, wherein the first moiety comprises an amino acid sequence to which a receptor can bind. [00522] 51. The molecule of embodiment 50, wherein the first moiety comprises a ligand for the receptor. [00523] 52. The molecule of embodiment 51, wherein the ligand comprises an ectodomain of CD19 and the receptor comprises a CAR specific for CD19. [00524] 53. The molecule of embodiment 50, wherein the receptor comprises a chimeric antigen receptor (CAR), T-cell receptor (TCR) or B-cell receptor (BCR). [00525] 54. The molecule of embodiment 9, wherein the first moiety comprises an scFv, Fab, single-domain antibody, nanobody, monobody, DARPin or affibody. [00526] 55. The molecule of embodiment 8 or 9, additionally comprising a peptide linker that can be cleaved by a protease. [00527] 56. The molecule of embodiment 55, wherein the protease comprises an endosomal/lysosomal protease. [00528] 57. The molecule of embodiment 56, wherein the protease comprises cathepsin. [00529] 58. The molecule of embodiment 55, wherein the peptide linker is located between the first moiety and the second moiety on a polypeptide that comprises the first moiety and the second moiety. [00530] 59. The molecule of embodiment 58, wherein the peptide linker is closer to the first moiety than to the second moiety. [00531] 60. The molecule of embodiment 55, wherein the peptide linker comprises Gly-Phe- Leu-Gly (GFLG). [00532] 61. The molecule of embodiment 55, wherein the peptide linker comprises Valine- Arginine (VR) and/or Phenylalanine-Lysine (FK). [00533] 62. The molecule of embodiment 55, wherein the peptide linker comprises a GS, GFLG, 3xGFLG, GFLG-VA, GFLG-VK, GFLG-VR, GFLG-GFLG, FK, VA, EVR, VK linker or combinations thereof (FIG.35). [00534] 63. The molecule of embodiment 55, wherein the second moiety comprises an scFv, Fab, single-domain antibody, nanobody, monobody, DARPin or affibody. [00535] 64. The molecule of embodiment 63, wherein the scFV comprises H7. [00536] 65. The molecule of embodiment 55, wherein cleavage of the peptide linker by the protease can provide for trapping and/or degradation of the molecule of interest inside a cell into which the molecule with at least two moieties is internalized. [00537] 66. The molecule of embodiment 8 or 9, comprising an amino acid sequence SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, or 79-117 or an amino acid sequence at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identical thereto. [00538] 67. The molecule of embodiment 8 or 9, wherein a nucleotide sequence encoding the molecule comprises SEQ ID NO: 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, or 77 or a nucleotide sequence at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent identical thereto. [00539] 68. The molecule of any one of embodiments 8-67, additionally comprising a therapeutic molecule or drug conjugated to the molecule. [00540] 69. The molecule of embodiment 68, wherein the therapeutic molecule or drug comprises an anticancer agent. [00541] 69. A bispecific modulator, comprising: [00542] a. a first antibody or antibody fragment that binds to a transferrin receptor (TfR) on a cell surface; and [00543] b. a second antibody or antibody fragment that binds to a transmembrane protein on the cell surface that is not TfR. [00544] 70. A bispecific modulator, comprising: [00545] a. a transferrin protein or part of a transferrin protein that binds to a TfR on a cell surface; and [00546] b. an antibody or antibody fragment that binds to a transmembrane protein on the cell surface that is not TfR. [00547] 71. The bispecific modulator of embodiment 69 or 70, wherein the first antibody or antibody fragment and second antibody or antibody fragment (embodiment 69), or transferrin/part of transferrin and the antibody or antibody fragment (embodiment 70), are part of one polypeptide. [00548] 72. The bispecific modulator of embodiment 69 or 70, wherein the first antibody or antibody fragment and second antibody or antibody fragment, or transferrin/part of transferrin and the antibody or antibody fragment, are more than one polypeptide. [00549] 73. The bispecific modulator of embodiment 72, wherein the more than one polypeptide comprises two polypeptide chains connected by a dimerizing domain. [00550] 74. The bispecific modulator of embodiment 73, wherein the dimerizing domain comprises a knob-in-hole Fc. [00551] 75. The bispecific modulator of embodiment 69 or 70, additionally comprising a peptide linker that can be cleaved by an endosomal/lysosomal protease. [00552] 76. A nucleic acid(s) encoding the molecule of any one of embodimens 8-68, or bispecific modulator of any one of embodimens 69-75. [00553] 77. A vector comprising the nucleic acid of embodiment 76. [00554] 78. A cell comprising the vector of embodiment 77. [00555] 79. A method for treating a toxicity associated with CAR-T therapy, or for increasing the efficacy of immune checkpoint and targeted cancer therapy, comprising administering the molecule of any one of embodiments 8-68, or bispecific modulator of any one of embodiments 69-75 to a subject.
EXAMPLES [00556] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results. EXAMPLE 1 – Constructing TransTACs (bispecific modulators) for downregulating EGFR [00557] An anti-EGFR affibody-Fc-Tf TransTAC was expressed and validated by SDS-PAGE (FIG.10, Left). Zero to 60 nM of TransTAC was incubated with a MCF10A EGFR overexpression cell line for 12, 36 or 68 hrs. A drastic dose-dependent decrease of EGFR level was observed by flow cytometry with an IC 50 < 6 nM at 68 hrs (FIG.10, Right). We observed the same dose-dependent decrease of EGFR with A549, an adenocarcinomic lung cancer cell line (FIG.11A). A time course experiment was then performed with 30, 60, or 90 nM of TransTAC on A549 cells, which showed EGFR was effectively downregulated with a half-life of <30 min at 90 nM TransTAC. Additionally, we observed TransTAC-mediated killing of the MCF10A- EGFR cells (FIG.11B). These data demonstrate the effectiveness of the construct. [00558] Notably, TransTAC downregulated more than 99% of cell surface EGFR at equilibrium, while recently reported LYTACs only reached 70-80% downregulation. In addition, the kinetics are also different. TransTAC-driven EGFR internalization has a half-life of <30 min while LYTACs’ are around 10-20 hrs. These distinctions are due to the different endocytosis kinetics of the carrier proteins used in the studies. [00559] Additional data (FIG.12) show that using A549 cells that express EGF receptor (EGFR) on the cell surface, TransTAC targeting EGFR internalized the receptor to a greater degree than an EGFR-specific antibody alone. EXAMPLE 2 – Constructing TransTACs (bispecific modulators) for downregulating anti- CD19 CAR [00560] Data show that transferrin fusion proteins with antibodies specific for CD19, EGFR and HER2 express well in expi293 cells (FIG.13). [00561] FIG.14 shows data indicating that a knob-in-hole version of TransTAC targeting of an anti-CD19 chimeric antigen receptor internalized the receptor. [00562] In FIG.15 are shown data measuring surface CAR levels in cells using anti-myc- biotin/Streptavidin 647, similar to the study shown in FIG.14. The data in FIG.15 show that a homodimeric TransTAC targeting of the CAR effectively internalized the CAR. In studies as shown in FIG.16, cells expressing an anti-CD19 CAR were incubated with K562 cells (expressing CD19) with a TransTAC bispecific modulator. Cell activation was measured. The data showed that the TransTAC bispecific modulator began to show inhibitory activity at concentrations below 10 nm. These data indicate that the TransTAC molecules effectively internalized CAR and downregulated CAR-T cell activity. [00563] The data in FIG.17 are similar to those shown in FIG.16. The data in FIG.12 show that TransTAC improved the IC50 for inhibiting cell activation to about 10-20 nM. [00564] The data in FIG.18 show that a TransTAC molecule with a CD19NT.1 variant ectodomain blocks CAR-T activation with an IC50 of about 800 pM. The data show that, in absence of the K562 cells which express CD19, the TransTAC molecule has no effect on the Jurkat cells. EXAMPLE 3 – TransTAC molecules containing protease sites to increase target degradation [00565] FIG.19A-B is a schematic of molecules used in these studies (A) and results obtained with the molecules (B). TransTAC1.0 for the figures in this Example is TransTAC0.4 as shown in FIG.44A. [00566] FIG.19C illustrates fluorescence microscopy results of the targeted CAR on the cells in FIG.19B. [00567] FIG.20A-B is a schematic of molecules used in these studies (A) and Western blot results of the targeted CAR and actin control (B). [00568] FIG.20C shows a graph of the data from FIG.20B. [00569] FIG.20D-E shows Western blot results of of additional molecules used in these studies (D) and a graph of the data (E). [00570] FIG.20F-G shows a TransTAC molecule that contains GFLG linkers (F) and Western blot data using the molecule (G). [00571] FIG.20H shows a graph of the data from FIG.20G. [00572] FIG.20I shows results from other cathepsin-sensitive TransTAC molecules. [00573] FIG.21A-B shows results indicating that the molecules shown in FIG.20A inhibited activation in Jukat cells (A) and inhibited interferon gamma (IFN-γ) release from primary T cells (B) in an experiment in which cells expressing an anti-CD19 CAR receptor were incubated with CD19-positive A375 cells. [00574] FIG.22A shows results indicating that adding the indicated molecules shown in FIG 20A stopped human primary anti-CD19 CAR-T cells from killing CD19-positive A375 target cells. The A375 cells express a nucleus mCherry for fluorescence microscopy. The results show that removing the molecules led to reactivation of the CAR-T cells and killing of the CD19- positive A375 target cells. The photographs show the red fluorescence channel of the fluorescence microscope. [00575] FIG.22B shows results indicating that removal of the indicated molecules let to reactivation of the CAR-T cells and killing of the CD19-positive A375 target cells. The photographs show an overlay of the red fluorescence channel and the white-field channel. EXAMPLE 4 – TransTAC molecules targeting epidermal growth factor receptor (EGFR) [00576] FIG.23A-B shows a schematic diagram of molecules used in these studies (A). The molecules contain antibodies, affibodies and TransTAC molecules specific for EGFR. Also shown are results showing reduction of EGFR levels on the surface of A549 cells using the molecules (B). [00577] FIG.23C-D show results on inhibition of cell proliferation using the molecules of FIG.53A. EXAMPLE 5 – TransTAC molecules targeting CD20 [00578] These data show an example approach using a TransTAC molecule having an antibody specific for CD20 and a molecule that binds to the transferrin receptor (transferrin or antibody). [00579] FIG.24B-C shows a schematic diagram of molecules used in these studies (B) and results obtained from their use (C). [00580] FIG.24D shows a graph of the normalized data from FIG.24C. [00581] The data show that the TransTAC molecules are more rapidly internalized/degraded than the anti-CD20 Rituximab antibody, that targets CD20 alone. EXAMPLE 6 – Reversible control of receptor functions [00582] FIG.25A shows cells expressing a CAR. The cells in the left panel have been contacted with an CD19m-Fc antibody (the CD19NT.1 variant). The cells in the right panel have been contacted with a TransTAC molecule that binds to the CAR. Nuclei of the cells in both panels have been stained with DAPI. The cells have also been stained with an anti-CD3z antibody that stains the CAR. Immunofluoresce from the anti-CD3z antibody is localized to the surface of cells in the left panel. Anti-CD3z antibody fluorescence is localized to the cell cytoplasm in cells of the right panel. The data show that the TransTAC molecule caused internalization of the CAR. [00583] FIG.25B shows reversibility of CAR internalization by TransTAC. In the first bar in the graph, cell-surface CAR-specific immunofluorescence for cells that have not been contacted by TransTAC is relatively high (about 1.0). In the second bar of the graph, the cells have been contacted by TransTAC and cell-surface CAR-specific immunofluorescence is low (about 0.2). In the third bar of the graph, the cells have been contacted by TransTAC, but TransTAC then was removed. After 24 hours, cell-surface CAR-specfic immunofluorescence has increased to similar levels as cells not exposed to TransTAC (about 1.0). [00584] These data show that TransTAC-enabled internalization of a CAR receptor serves as a reversible CAR-T cell off-switch. This can be used to moderate any toxicities associated with CAR-T therapy. [00585] FIG.26 shows that TransTAC inhibited/stopped interferon gamma production. [00586] As discussed elsewhere, FIG.22A demonstrates that TransTAC stopped primary human anti-CD19 CAR-T cells from killing CD19-positive A375 target cells. The data show that removing the TransTAC led to reactivation of the CAR-T cells and killing of the CD19- positive A375 target cells. FIG.22B shows that removal of the TransTAC molecules led to reactivation of CAR-T cells and killing of the targets. EXAMPLE 7 – Targeted membrane protein degradation [00587] FIG.27A-B show expression of the transferrin receptor on various cells, as indicated by fluorescent antibodies. The data indicate that transferrin receptor can be expressed at higher levels on the surface of tumor cells as compared to non-tumor cells. [00588] FIG.28 shows examples of cell-surface proteins that can be targeted by TransTAC. [00589] FIG.29A shows that TransTAC molecules (DP81 and DP174) decrease the amount of EGFR in the cells. [00590] FIG.29B shows that a TransTAC molecule can degrade EGFR. The data show that TransTAC-mediated degradation of EGFR was sensitive to bafilomycin (inhibitor of autophagosome-lysosome fusion) and to MG132 (proteosome inhibitor). These data show that TransTAC-induced EGFR degradation was mediated by the lysosomal pathway. [00591] FIG 30A shows an approach to treating lung cancer using TransTAC. The high expression of TfR in cancer cells enables targeting specificity. [00592] FIG.30B shows that an EGFR TransTAC molecule can inhibit PC9 cancer cells (lung adenocarcinoma). [00593] FIG.30C-D show that an EGFR TransTAC molecule could inhibit PC9 cancer cells. [00594] FIG.31 shows that an anti-CAR TransTAC molecule can decrease the amount of CAR in these cells. [00595] FIG.32A shows that anti-PD-L1 TransTAC molecules (DP186, DP187) can decrease the amount of PD-L1 in these cells. [00596] FIG.32B illustrates data demonstrating that anti-PD-L1 TransTAC molecules can decrease the amount of PD-L1 in cells. [00597] FIG.33 shows that anti-CD20 TransTAC molecules (DP209S, DP210, DP213) can decrease the amount of CD20 in cells. [00598] FIG.34A-C and 34D show example TransTAC molecules that include protease- sensitive linkers and example data obtained with the molecules. [00599] FIG.35 shows example data obtained with TransTAC molecules containing various protease-sensitive linkers. [00600] FIG.36A-C show example TransTAC molecules that include an antibody fragment specific for transferrin binding and example data obtained with the molecules. [00601] FIG.37A-B show examples of TransTAC molecules and example data obtained with the molecules. EXAMPLE 8 – TransTAC for cancer [00602] Targeted therapy with tyrosine kinase inhibitors is a standard treatment for lung cancer with EGFR mutations. We reasoned that co-targeting an EGFR and a TfR receptor on lung cancer cells could lead to EGFR inhibition while maintaining high tumor specificity (FIG.30A). We generated an anti-EGFR affibody*H7*GFLG-VR TransTAC. Incubation of the molecule with a human lung adenocarcinoma A549 cell line led to >90% EGFR degradation (FIG.30B). Notably, treatment of a non-tumorigenic HEK cell line engineered to overexpress EGFR with the TransTAC molecules resulted in much less EGFR degradation, highlighting the tumor- specificity of the technology. This specificity results from higher TfR expression in tumor cells (FIG.27A-B). EXAMPLE 9 – Functionalizing antibody drug conjugates [00603] Antibody-drug conjugates (ADCs) and protein degraders are two distinct drug modalities that have shown potential in their respective fields. These experiments demonstrate how combining these two modalities can lead to a new class of drugs that can target non-self- internalizing membrane proteins to deliver and release drug payloads. [00604] Antibody-drug conjugates (ADCs) have revitalized targeted chemotherapy by selectively targeting overexpressed receptors on the surface of tumors and delivering cytotoxic payloads within those cells. ADCs can efficiently internalize and be transported through the endosomal-lysosomal pathway to release the payload. Therefore, non- or slow-internalizing receptors or recycling receptors that undergo minimal lysosomal degradation cannot effectively serve as targets for ADCs. In some instances, ADCs may not function optimally if they use slowly- or non-internalizing receptors (e.g., CD20) on tumor cells like B cell malignancies. Such receptors may not provide efficient internalization, and/or transport through the endosomal- lysosomal pathway, used for payload release. [00605] Here, we constructed "degrader-based ADCs" (DDCs). DDCs link small molecule drug payloads to an antibody degrader that specifically targets a membrane protein of interest. Upon binding to the target protein, the DDC is internalized and co-trafficked with the target protein to the lysosomes for degradation, releasing all drug payloads linked to the degraders and leading to a potent cytotoxic effect. [00606] In an example, we developed a CD20 DDC and demonstrated its therapeutic potential. In vitro studies on a Raji lymphoma cell line revealed an IC50 of <100 pM, while in vivo studies showed excellent half-life and efficacy in a tumor xenograft model. These results highlight the potential of DDCs as a new class of targeted therapeutics for cancer treatment. [00607] We sought to use TransTAC molecules to redirect receptors to the endosomal/lysomal pathway, using a TransTAC-based antibody-drug conjugate for targeting CD20 in B-cell malignancies (FIG.38). [00608] To do this, we made TransTAC molecules that had either a Fab or a scFv of the Rituximab antibody for binding to CD20. Mutations were introduced into the Fc domain to remove Fcγ binding. A cysteine was introduced into the Fc domain in the scFv-based TransTAC, and into the light chain of the Fab-based TransTAC, for site-specific conjugation of drug payloads. Both molecules led to CD20 degradation, demonstrating the ability of the molecule to enter the endosomal/lysosomal pathway. [00609] We then generated TransTAC molecules conjugated to monomethylauristatin F (MMAF). TransTAC-MMAF molecules were made using a modified on-resin maleimide- cysteine bioconjugation protocol. Intact protein LC-MS experiments showed a drug-antibody ratio (DAR) of ~2 for all TransTAC-MMAFs and controls . These ADCs were then incubated with Raji cells, which is a human Burkitt's lymphoma cell line with high CD20 expression. While the no H7-MMAF controls showed little cytotoxic effects, the TransTAC-based ADCs had very potent anti-tumor effects with an IC50 of ~400 pM for the scFv-based molecule and <100 pM for the Fab-based molecule (FIG.39, FIG.40A, B and C, and FIG.41). [00610] Furthermore, the treatment will show selective killing of the Raji cancer cells while keeping minimal cytotoxicity to the healthy B cells. The cytotoxicity can be selective for CD20- overexpressing cells, as the molecules may not result in significant cytotoxic effects. [00611] The stability, tissue distribution, and clearance of TransTAC ADCs in mice can be studied. The results can show that the molecules were stable in circulation and can exhibit specific enrichment to Raji cells. In vivo efficacy studies can be conducted to assess the ability of TransTAC ADCs to kill tumors in tumor-bearing mice. The results can show a significant reduction in tumor growth with minimal toxicity in normal tissues. This can indicate the potential for use of TransTAC ADCs as a treatment option for relapsed/refractory B cell malignancies. [00612] Results of additional studies related to antibody drug conjugates are shown in FIG. 83A-G. EXAMPLE 10– Transferrin receptor upregulation in cancer cell lines, primary tumors and activated T cells [00613] In additional studies, we measured cell-surface TfR levels using flow cytometry on five non-tumorigenic cell lines, including HEK293T (embryonic kidney), MCF10A (mammary epithelial), HFF-1 (foreskin fibroblast), MCR-5 (lung fibroblast), and LF-1 (fetal lung fibroblast), and 10 cancer cell lines, including Hela (cervical cancer); Raji (lymphoma); Jurkat and K562 (leukemias); MDA-MB-231 and MCF-7 (breast cancers); PC9 and A549 (lung cancers), and PC9 cells with EGFR drug-resistant mutants. We observed that cancer cell lines expressed 2-26 fold more TfR at the cell surface compared to non-tumorigenic cell lines (FIG. 42C). Among the cancer cell lines, leukemia cell lines Jurkat and K562 exhibited the highest TfR expression. Our findings demonstrate upregulation of TfR in cancer cell lines. [00614] Since cell lines have been modified to be immortalized, there is a possibility of changes in protein expression. Therefore, we further showed TfR expression is upregulated in primary tumors compared to primary healthy tissues, by performing a transcriptomics analysis of TFRC, the gene for TfR1. Microarray transcriptomics data of TFRC in primary healthy tissues and tumors was obtained from the MERAV database. Paired tissue analysis for healthy vs. tumor samples was performed using a custom python script. TFRC expression is statistically significantly increased in cancers overall (p = 3.98e-89) and in 14 out of 19 specific tissues, including breast, lung, pancreas, liver, bladder, skin, esophagus, thyroid, testes, stomach, salivary gland, kidney, central nervous system, and female reproductive system tumors (FIG.42D). These findings provide further evidence to support TfR as a cancer-upregulated target. To investigate whether TfR could also be a potential target for immune cell modulation, the DICE dataset was analyzed, which contains gene expression profiles of human immune cells isolated from blood samples of healthy donors. While most immune cells express low level of TfRs, an approximately 6-fold higher TfR expression in activated CD4 and CD8 T cells was observed compared to inactivated T cells, a level comparable to the TfR levels in some malignant tissues, indicating TfR can be a target for modulating activated T cells (FIG.42E). These findings indicate that TfR is not only upregulated in tumors but also in activated T cells, highlighting its value as a cell-surface receptor for both cancer and immune modulation. Our transcriptomic analysis provides a detailed comparison of TfR expression in specific tissues, serving as a roadmap for future selection of disease indications for our technology and beyond. EXAMPLE 11 – Targeted protein endosomal trapping with early versions of TransTAC designs [00615] In these experiments, we used Jurkat cells expressing an N-terminal myc epitope tag CAR for measuring cell-surface CAR levels (FIG.43B). Initially, we attempted to use the ectodomain of native CD19 as the CAR binding component but observed significant protein aggregation in SDS-PAGE gel (FIG.47B, lane 1-2). We then evaluated a small panel of CD19 ectodomain variants that were developed earlier using yeast surface display (Klesmith, Justin R., et al. "Retargeting CD19 chimeric antigen receptor T cells via engineered CD19-fusion proteins." Molecular pharmaceutics 16.8, 2019: 3544-3558) and showed they expressed and behaved well (FIG.47A-B). Ultimately, we selected mutant CD19NT.1, which exhibited better expression, to be used in CAR-TransTAC. [00616] We tested the concept that a molecule containing two TfR ligands would be more effective than one ligand in driving targeted CAR internalization, as TfR is a homodimeric receptor and requires binding of two transferrins (TF) to fully prime the TfR dimer for its physiological functions. Thus, we created two versions of TransTACs: v0.1, a knob-in-hole Fc construct with a single TF for binding TfR and one CD19NT.1 for CAR, and v0.2, a Fc fusion with two TFs and two CD19NT.1s (FIG.43A). We also designed a control molecule lacking the TF ligand. These CAR-TransTACs were recombinantly expressed in 293expi cells, purified by protein A resin, and then incubated with myc-CAR-Jurkat cells. After 18-24 hours, we measured the cell surface CAR levels using an anti-myc antibody. We found that treatment with both v0.1 and v0.2 significantly decreased the cell-surface CAR levels, with v0.1 exhibiting a Dmax of 60% and v0.2 exhibiting a Dmax of 80% (FIG.43C). Interestingly, a hook effect was observed with v0.1, but not v0.2. In contrast, treatment with the control CD19NT.1-Fc protein did not result in a decrease in cell surface CAR levels. These findings showed that CAR-TransTACs can effectively internalize CAR from the cell surface via a TF-dependent mechanism, and that in some embodiments a dimeric TransTAC is more effective than a monomer. [00617] However, despite the ability to efficiently remove CAR from the cell surface, TransTACv0.2 did not result in CAR degradation, shown by whole-cell lysate western blots (FIG.43D, 48A-B), indicating that the internalized receptor was trapped inside cells and was not degraded . [00618] To understand the subcellular destination of the internalized CAR by v0.2, we stably expressed CAR-GFP and mCherry tagged to different endosomal and lysosomal markers, including Rab5+ or EEA+ (EEs), Rab7+ (LEs), Rab11+ (REs), and Lamp1+ (lysosomes), in a Hela cell line (FIG.43I, J, FIG 49A-D). Fluorescence microscopy imaging of v0.2-treated cells showed co-localization of CAR-GFP with Rab11, indicating that the internalized CAR trafficked to the REs (FIG.43I, white arrow). Therefore, TransTACv0.2 effectively removes POIs from the cell membrane by trapping them in the recycling endosomal compartments in a target cell. EXAMPLE 12 –Degrader engineering by rationally rewiring intracellular trafficking pathways of the internalized protein complex [00619] Experiments related to intracellular trafficking are described in and are described with more details in this example. [00620] Additional studies were to develop a next-generation TransTAC that not only traps the POI, but also leads to its degradation. This can be partiularly beneficial for cancer-associated targets, as degradation can allow more sustained inhibition of protein functions. [00621] To direct the target protein to degradation, we tested whether the POI (protein of interest) needs to disengage from the recycling Tf/TfR complex in the EE, which is where sorting into degradative or recycling pathways occurs (FIG.42A, 43E, F). Proteases localized in the endosomes, such as the cysteine protease cathepsins, can be utilized to separate the POI from the Tf/TfR complex. Therefore, we incorporated a cathepsin B-sensitive Gly-Phe-Leu-Gly (GFLG) linker into TransTACs, either between the Fc domain and the Tf ligand (v0.3), or between CD19NT.1 and the Fc domain (v0.4) (FIG.43A, 48A). Indeed, this linker modification altered the intracellular trafficking of CAR-GFP. With TransTACv0.4, a significant portion of the receptor now co-localizing with Rab7 (late endosome) and Lamp1 (lysosomes) (FIG.43L, 49D-F). Furthermore, using western blotting, we observed that approximately 50% of CAR was degraded (FIG.48C). Comparing to v0.4, the degradation with v0.3 was lower (FIG.48C), possibly because CD19NT.1 remained linked to the Fc domain after separating from Tf/TfR, which could mediate recycling via the FcRn pathways. Overall, our studies indicated that the incorporation of a protease-cleavable linker in TransTACs leads to the degradation of POIs. [00622] Our next goal was to improve efficiency of degradation and expand our understanding of proteolysis in endosomes. Traditionally, protein degradation was believed to occur primarily in the acidic lysosomes or LEs and little was known about proteolytic activity in the EE. To look for optimal protease substrates in EEs, we conducted a small-scale linker screening, thinking that protease activity in EEs would correlate with degradation efficiency. We screened a panel of 14 linkers containing single or combined cathepsin B cleavage motifs (Poreba, Marcin. "Protease‐ activated prodrugs: strategies, challenges, and future directions." The FEBS Journal 287.10, 2020: 1936-1969), such as GFLG, Gly-Gly-Phe-Gly (GGFG), Phe-Lys (FK), Val-Ala (VA), Val- Lys (VK), and Val-Arg (VR), in both TransTACv0.4 and v1.0, using western assays (FIG.48D- E). Overall, we found that incorporating dipeptide motifs VK, VR, and FK helped to improve cleavage activity, comparing to a GFLG sequence. Based on these results, we selected a GFLG- VR linker and an EVR linker for developing later generations of TransTACs. We used the EVR linker instead of VR to prepare for in vivo studies since previous research has found that including a glutamic acid improves the stability of linker in mouse serum. [00623] Subsequently, we took another step in optimizing TransTACs by substituting the TF ligand with an anti-TfR single-chain Fv (scFv) called H7, a TF-competitive antibody identified by phage display (FIG.43A) (Goenaga, Anne-Laure, et al. "Identification and characterization of tumor antigens by using antibody phage display and intrabody strategies." Molecular immunology 44.15, 2007: 3777-3788; Tillotson, Benjamin J., et al. "Engineering an anti- transferrin receptor ScFv for pH-sensitive binding leads to increased intracellular accumulation." PLoS One 10.12, 2015: e0145820). This substitution aimed to reduce RE sorting, a step that mayincrease degradation efficiency, as proteins sorted to the RE cannot be trafficked to LE/lysosomes for degradation (FIG.43G, H). The logic was that certain molecular features of the TF/TfR complex are involved in RE sorting and that using a synthetic antibody binder like H7 could alter this sorting decision and redirect the complex's intracellular trafficking after iron release in the EE. [00624] Based on this, we generated two versions of TransTACs: v0.5 containing the H7 binder but no cleavable linker, and v1.0 containing both the H7 and the cleavable linker (FIG. 43A). Cells treated with v0.5 showed that CAR-GFP predominantly colocalized with EE markers Rab5 and EEA (FIG.43G, J, 49A,D). Pearson colocalization coefficients of the corresponding markers are statistically different from cells treated with TransTACv0.2 that contains the TF as the anti-TfR ligand (FIG.43K). This result validated that replacing TF with an anti-TfR antibody can reduce RE trafficking. Moreover, re-trafficking led to a significant improvement in degradation efficiency. With TransTACv1.0, we observed over 80% degradation of CAR in both western and fluorescence microscopy assays (FIG.43D, I, J; FIG.49A-C). In addition to improving degradation, the H7 substitution also increased the yield of the protein by approximately sevenfold, making the expression level of TransTACs similar to that of conventional antibodies. Taken together, our rational protein engineering efforts have successfully developed a novel protein design, TransTACv1.0, as a potent molecular degrader for CAR, which is fully recombinant and expresses robustly. CAR-TransTACv1.0 represents the first recombinant protein degrader made to target a synthetic receptor. EXAMPLE 13 – Reversible control of primary CAR-T cell functions with CAR-TransTAC [00625] In additional studies, we investigated the use of TrasTAC as OFF switches to fine tune CAR-T cell activity and to manage associated toxicities, such as those that can manifest as cytokine release syndrome (CRS) caused by overactivation of CAR-T cells (FIG.51A). [00626] As a proof of concept, we showed CAR-TransTACv0.4 can effectively inhibit human primary CAR-T cells. [00627] We isolated primary CD8+ T cells from human PBMCs and generated anti-CD19 CAR-T cells through lenti-viral transduction. For the tumor cells, we used an adherent melanoma cell line, A375, which has been engineered to express CD19 and a nuclear mCherry to facilitate live cell imaging. We observed CAR-TransTAC v0.4 potently inhibited IFN-γ secretion, with an IC50 of approximately 0.4 nM and a Dmax of 88% (FIG.51B, C). The molecules also effectively blocked the tumor-killing activities (FIG.51B, D). Furthermore, the inhibition was reversible, as the removal of the TransTAC resumed tumor killing activities of primary CAR-T cells (FIG. 51B, E). [00628] To gain a better understanding of the factors that influence the varying performances of the CAR-TransTACs, and to determine the general structure-function activity (SAR) relationships of TransTACs, we generated and tested four TransTACv0.5 variants, v0.6-v0.9, each containing one or two copies of CD19NT.1 or H7 in different geometries (FIG.45B). Our findings revealed that having two H7s was better than having two CD19NT.1s in enhancing CAR internalization (FIG.45C, D). This highlights the importance of dual binding to a dimeric TfR, rather than having two anti-POI binders, in creating a potent TransTAC. It also demonstrated that TransTAC-mediated CAR internalization was not the result of CAR crosslinking. Additionally, we observed significant differences in the internalization efficiency for different geometries of the molecules (FIG.45C), indicating that the tertiary complex structure plays a role in influencing TransTAC efficiency. Furthermore, we generated an Fc-H7 molecule as a competitor for TfR binding and observed a dose-dependent decrease of CAR internalization in the presence of the competitor in solution with TransTACv0.5 treatment (FIG. 45D). This observation further indicates that TransTACs function through a TfR-dependent mechanism. [00629] As CAR clustering can lead to low-level spontaneous CAR-T cell activation, we hypothesized that our dimeric CAR OFF-switch molecules, by inducing CAR clustering, may have influenced their inhibition effects on CAR-T cells. Therefore, we developed CAR OFF switches containing only one CD19NT.1 domain and compared them to the dimeric variants. The study indicated CD19NT.1 monomer had a significant 100-fold decrease in potency compared to the CD19NT.1-Fc dimers. This finding highlights the importance of avidity for CD19NT.1-Fc in achieving effective CAR-T cell inhibition. As CAR-T/tumor interactions involve multiple CAR/antigen interactions at the immunological synapse, a molecule with multiple copies appears better to effectively compete with tumor antigens for CAR binding. To further understand the role of multivalency, we also generated a tetrameric variant, which showed similar potency in CAR-T cell inhibition compared to the dimer. [00630] CAR-TransTACs did not rely on competition with tumor CD19 for its effects and therefore did not require a dimeric format to be effective. We observed that a monomeric CD19NT.1-based TransTAC (a domain to which CAR can bind fused to an Fc region)exhibited robust performance and even outperformed the dimeric variant for blocking CAR-Jurkat cells. [00631] This study has developed two types of protein-based CAR OFF switches based on distinct mechanisms: CD19NT.1-Fc acts as an "antigen trap" to block the interaction of tumor CD19 with CAR-T cells, relying on avidity for effective competition; in contrast, CAR- TransTACs remove the CAR from the cell surface. Both types of molecules have unique potential applications in the clinic. These protein switches represent the first non-genetic approaches to regulate CAR-T cells. Unlike genetic-engineering based methods, such as split- CAR, protein-based CAR OFF-switches are readily adaptable to a variety of CAR-T cell therapies, both approved and in-development. EXAMPLE 14 – Expansion of TransTAC-addressable targets [00632] In additional studies, we interrogated the generalizability of TransTACs. To date, all biologics-based degraders have been developed to target single-pass membrane proteins. We sought to expand the scope of targets by including both single-pass membrane targets such as epidermal growth factor receptor (EGFR) and programmed death-ligand 1 (PDL1), as well as a multi-pass membrane protein, cluster of differentiate 20 (CD20) (FIG.44A). These targets have diverse functions and regulatory pathways and are found on a wide range of cancer and immune cells. [00633] Our first target is programmed death-ligand 1 (PD-L1), an immune checkpoint receptor ligand, downregulation of which can enhance anti-tumor T cell activity. PD-L1 targeting using monoclonal antibodies have seen moderate success in the clinic, thus new mechanisms to target this protein could be highly valuable. A PD-L1-TransTAC was created using a fragment antigen binding (Fab) or single-chain variable fragment (scFv) of atezolizumab as the PDL1 binding domain. Up to 98% PD-L1 degradation was observed in MDA-MB-231 breast cancer cells treated with PD-L1-TransTACs, while control groups lacking H7 or containing TransTACv0.2 and v0.4 with a TF ligand showed no or little PD-L1 degradation (FIG.44B; FIG.50A). [00634] Next, we aimed to target epidermal growth factor receptor (EGFR), a receptor tyrosine kinase that plays an important role in the development and progression of various types of cancers such as lung and brain. An affibody (Friedman, Mikaela, et al. "Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding affibody molecule." Journal of molecular biology 376.5, 2008: 1388-1402) was used to bind EGFR and create an EGFR-TransTAC. A549 lung carcinoma cells treated with EGFR-TransTACv1.0s containing GFLG-VR or EVR linkers showed up to 80-90% reduction of EGFR, whereas control groups exhibited little to no degradation (FIG.44C, FIG.50B). Different linkers in v1.0 resulted in varying degrees of EGFR degradation, but all were lower than TransTACs with GFLG-VR or EVR, while v0.2 had no effect (FIG.50B). These results were consistent with the observations with the CAR-TransTAC variants, validating the importance of those modifications made to improve TransTAC. [00635] Cluster of differentiate 20 (CD20) is a B cell-specific surface marker with four transmembrane domains and an unknown function. Knocking down cell surface CD20s with a degrader can be valuable. A CD20-TransTAC was created using a Fab format of Rituximab, the first clinically approved CD20 antibody, to bind CD20. Treatment of Raji cells, a human B lymphoblastoid cell line, with the resulting CD20-TransTAC resulted in up to 97% reduction of CD20, while control groups led to no or significantly less degradation (FIG.44D, FIG.50C). [00636] Together, the successful generation of degraders against all four targets demonstrates the modularity and generality of the TransTAC design. High potencies were observed for all four targets studied, all reaching >80% in various cellular systems, which demonstrates the efficiency of targeted degradation using the TransTAC degrader designs. EXAMPLE 15 – Kinetics, structure-activity relationship (SAR), mechanism, and in vivo characterization of TransTACs [00637] We conducted further characterizations of the degraders to understand their underlying mechanisms and SARs. [00638] First, we studied the kinetics of TransTAC-mediated protein internalization by measuring the time-course change of cell-surface CAR levels (FIG.45A). We observed a rapid elimination of CAR from the cell surface, with only 17% remaining after 10 minutes and 13% after 20 minutes of treatment with TransTACv1.0-GFLG-VR. Furthermore, this response was long-lasting, with 10% of CAR observed at the cell surface after 3 hours with v1.0. This fast and sustained protein downregulation highlights TransTACs as a promising research tool for knocking down cell surface proteins as an alternative to genetic methods, offering temporal resolution of membrane protein regulation. [00639] To further understand how the number of binders and geometry of TransTACs influence its behavior, we generated and tested four CAR-TransTACv0.5 variants, v0.6-v0.9, each containing one or two copies of CD19NT.1 or H7 (FIG.45B). Our findings revealed that having two H7s was better than having two CD19NT.1s in enhancing CAR internalization (v0.6 vs. v0.7, FIG.45C). This highlights the importance of dual binding to a dimeric TfR, rather than having two anti-POI binders, in creating a potent TransTAC. It also indicated that TransTAC- mediated CAR internalization was not the result of CAR crosslinking. Additionally, we observed significant differences in the internalization efficiency for molecules in different geometries, indicating that the tertiary complex structure plays a role in influencing TransTAC efficiency (v0.8 vs. v0.9, FIG.45C). Furthermore, we generated an Fc-H7 molecule as a competitor for TfR binding and observed a dose-dependent decrease of CAR internalization in the presence of the competitor in solution with TransTACv0.5 treatment (FIG.45D) This observation further validates that TransTACs function through a TfR-dependent mechanism. These SAR analyses offer valuable insights to guide future TransTAC designs. [00640] We next investigated the cellular mechanism underlying TransTAC-mediated protein degradation. Two primary pathways involved in the degradation of cellular proteins were tested: the lysosomal pathway and the proteosome pathway. A549 cells were treated either with bafilomycin, a vacuolar proton pump inhibitor that inhibits lysosomal acidification, or MG132, a proteasomal inhibitor. We observed 1 µM bafilomycin prevented TransTAC-mediated EGFR degradation, whereas 1 µM MG132 had a much less significant effect (FIG.45E). These results show that intact lysosomal function was essential for TransTAC-mediated protein degradation. [00641] To determine whether TfR level remains consistent or reduced with TransTAC treatment, we characterized whole-cell TfR expression using western blotting assay with a PD- L1-TransTAC. No change in TfR level was observed, which is in clear contrast to the loss of PD-L1 in the same assay (FIG.45F). This result validates our hypothesis that the POI was separated from TfR before being routed to degradation, while TfR is recycled. [00642] Lastly, we asked whether TransTACs would be well tolerated and have similar antibody clearance to IgGs in vivo. We intraperitoneally injected 5 or 7 mg/kg (body weight) CD20 TransTAC or 5 mg/kg IgG control into nude mice (FIG.45G). No significant weight changes were observed with either the TransTACs or the control (FIG.45H). Western blotting analysis of plasma antibody levels revealed that the TransTAC remained in plasma up to 10 d after injection with a half-life of approximately 10 d, which is longer than the tested control IgG and comparable to the reported half-life of IgGs in mice (FIG.45I). It was known that the scFv- H7 antibody cross-reactive with mouse TfR. Together, these results demonstrate that TransTACs are well-tolerated and have favorable pharmacokinetics and are not being rapidly cleared despite cross-reactivity with mouse cells. EXAMPLE 16 – Targeting drug resistant small cell lung cancer with EGFR-TransTACs [00643] Additionally, cancers evolve rapidly to evade therapy, often developing drug-resistant mutations that lead to treatment failure and disease recurrence. The C797S mutation of EGFR, in particular, poses a substantial challenge in the treatment of non-small cell lung cancer (NSCLC), which accounts for 85% of all lung cancer cases. Emerging in roughly 10-26% of NSCLC patients following treatment with the third-generation EGFR tyrosine kinase inhibitors (TKI) Osimertinib, the C797S mutation affects a critical residue, C797, which forms covalent bonds with irreversible TKIs. Consequently, existing TKI therapies become ineffective against the disease. [00644] EGFR-TransTACs, which can induce targeted degradation of EGFR in TfR- upregulated cancer cells, can target EGFR driven lung cancer patients including the C797S- mutant population (FIG.46A). Three lung cancer cell lines were used: PC9-wildtype (WT), PC9 GR4, and PC9 GR4 C797S. PC9-WT cell is a lung adenocarcinoma cell line with a deletion in exon 19 (Del 19) of the EGFR gene, sensitive to all three-generations of TKIs. PC9-GR4 is a gefitinib-resistant, osimertinib sensitive cell line carrying the T790M mutation (Del 19/T790M), generated through a previously established drug selection protocol. Finally, PC9 GR4 C797S (Del 19/T790M/C797S) is a CRISPR-engineered cell line harboring an additional C797S mutation, making it further resistant to Osimertinib. [00645] We generated several EGFR-affibody-based TransTAC variants (FIG.46B) and evaluated their dose-dependent inhibition efficiency first on PC9-WT cells using a 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT cell viability assay. TransTACv1.0 and v0.5 variants led to dose-dependent inhibition of the cells, with IC50s in the low-nM range, whereas the control affibody-Fc fusion and v0.2 showed no or little response (FIG.46C). Consistently, more than 90% maximal EGFR degradation was observed in PC9-WT cells and PC9 GR4 C797S cells with TransTACv1.0 treatments, whereas the affibody-Fc control led to no degradation (FIG.46D). The same TransTAC molecules only induced approximately 40-50% EGFR degradation in an engineered EGFR-overexpressing HEK293 cell line, which expresses 3- 10x less TfRs compared to the three PC9 cell lines (FIG.52A). This result indicates that the efficiency of TransTAC-mediated protein degradation is positively correlated with TfR expression levels, highlighting the potential advantage of TransTAC technology's cancer- specificity. [00646] We next compared EGFR-TransTACs with first-, second-, and third-generation EGFR TKIs gefitinib, afatinib, and osimertinib. The sensitivity of the three cell lines to these TKIs was consistent with previous reports (FIG.46E, 52B). PC9-WT cells showed sensitivity to all three TKIs, with IC50s ranging from <0.1 to 33 nM. PC9-GR4 cells were less sensitive to afatinib and osimertinib, with IC50s of 168 and 207 nM, respectively, and showed full resistance to gefitinib. PC9-GR4-C797S cells did not respond to any of the three inhibitors. [00647] Different from the TKIs, TransTACv1.0s effectively inhibited all the three cell lines, exhibiting IC50s in the low or sub-nM range (FIG.46E, 52B). In particular, TransTACv1.0- GFLG-VR had an IC50 of 2 nM and TransTACv1.0-EVR had an IC50 of 8 nM against the PC9 GR4 C797S cells. To evaluate the off-tumor toxicities, a healthy cell human fibroblast cell line (HFF-1) was included in the assay. Neither the TransTACs or TKIs showed significant inhibition until the concentration of the molecules reached the high nM or µM range (FIG.46E, 52B). [00648] To further compare the efficacy and specificity of TransTACs with standard care therapies, we performed a co-culture assay of normal and cancer cells and monitored the effects of drugs using live cell fluorescence imaging (FIG.46F-G). PC9 and PC9 GR4 C797S cells were engineered to express GFP and the HFF-1 cells expressed mCherry. The cells were mixed in a 1:10 ratio and treated with TransTACs or TKIs. Consistent with the MTT assay results, TransTACs demonstrated high efficacy against both PC9-WT and PC9 GR4 C797S cells. In contrast, the TKIs inhibited PC9 WT cancer cells but not the PC9 GR4 C797S cells. The UT or affibody-Fc control molecule showed no effect on either cells. [00649] Additionally, TransTACs were also compared with chemotherapy drugs. Unlike TransTACs, which didn’t kill the HFF1 healthy cells, a combination of carboplatin and paclitaxel chemotherapy were cytotoxic to both cancer and normal cells (FIG.46F-G). This is consistent with previous notions that chemotherapy often has high off-tumor toxicities, despite it being the first-line therapy for many cancer types. [00650] Furthermore, the results of live cell imaging were validated by conducting flow cytometry analysis to determine the ratio of GFP/mCherry positive cells after treatments, which reflects the relative drug cytotoxicity to cancer cells versus healthy cells (FIG.52C). Our analysis indicated that TransTACs inhibited both PC9 WT and the GR4 CS cells, showing a near-zero cancer/healthy cell ratio, indicating high cancer targeting potency and specificity. In contrast, the TKIs were much less effective against the GR4 CS cells, and the cancer cells dominated the entire population of the cell mixtures. [00651] Taken together, these findings demonstrated that the EGFR TransTAC molecules can target lung cancer cells harboring the EGFR C797S mutation. Furtheremore, the comparison with current standard of cares demonstrates superior on-tumor efficacy and specificity of TransTACs. EXAMPLE 17 – Bispecific modulators for delivery of therapeutic agents [00652] We set out to develop a CD20 DDC for targeting B cell lymphoma. In a preliminary analysis, FIG 54A-C shows an example of the efficiency and potency of delivering a drug or therapeutic agent using a bispecific modulator. We chose CD20 as the target due to its significance in B-cell malignancies and autoimmune diseases and the lack of successful ADCs targeting CD20. A phase in development of a CD20 DDC development of a degrader that would drive trafficking of CD20 through the endosomal-lysosmal pathway. Elsewhere herein, we describe Transferrin-receptor mediated protein TArgeting Chimeras" (TransTACs, also called bispecific modulators herein). TransTACs capitalize on the transferrin receptor (TfR), functioning in cellular iron transport, to induce membrane protein degradation. TfR was selected for two reasons. First, TfR is overexpressed in cancer cells since these rapidly dividing cells require an elevated level of iron to satisfy their metabolic needs, making it an appealing target for tumor cell therapy. Second, TfR has a rapid internalization rate of approximately 500 molecules per cell per second, facilitating highly efficient targeted endocytosis of the proteins. [00653] Elsewhere herein, we demonstrated that hetero-bispecific TransTAC molecules, with one arm binding to TfR and the other to a target protein, were exceptionally successful in degrading a variety of protein families, encompassing receptor tyrosine kinase, immune checkpoint receptor ligand, synthetic receptor, and multi-pass membrane protein. Specifically, a CD20-targeting TransTAC, engineered based on the Fab domain of rituximab, can induce up to 97% CD20 degradation. Preliminary animal testing indicated that CD20 TransTAC is well- tolerated in mice and exhibits a half-life exceeding that of standard IgG controls. One feature of a TransTAC molecule is inclusion of a cathepsin sensitive linker, which can be processed in early endosomes. Afterwards, one half of the molecule with the TfR will recycle, while the other half of the molecule together with the target antigen will go to the late endosome/lysosome for targeted degradation. [00654] Initial studies determined an EC50 for the CD20 TransTAC, studies degradation kinetics, and showed that the process is reversible (after removing the TransTAC from cells, levels of CD20 on cells recovered). Degradation of CD20 by the TransTAC was demonstrated to occur in multiple cells expressing CD20. [00655] Furthermore, we developed variants of the CD20 TransTAC. Here, we employed a single-chain variable fragment (scFv) as the binding domain instead of the Fab version of Rixtuximab. This allowed the TransTAC to be expressed in a single chain format, simplifying the expression platforms. This scFv version of the degrader displayed a similarly potent degradation of CD20. Collectively, these results validated CD20 TransTAC as a robust and modular, fully recombinant design of degraders for targeting CD20 (FIG.55A-F, FIG.55G and elsewhere herein). [00656] Building on these CD20 degraders, we embarked on the design of a CD20 DDC and investigated parameters that determine their efficacy. We constructed two primary categories of degraders. In the first category, we introduced a cysteine into each of the two Fc monomers of the scFv-based degrader. We then employed a maleimide-based cysteine bioconjugation reaction to link the antibody to a MMAF drug payload via a non-cleavable linker. In the second category, we introduced a cysteine into an amino acid within the constant domain of the two light chains of the Fab-based degrader. Again, a maleimide-based cysteine bioconjugation reaction was used to link the antibody to a MMAF drug payload, utilizing a non-cleavable linker. We screened varying reaction conditions including reduction agent, reaction time, and MMAF concentration to identify an optimal condition that resulted in an average drug-antibody ratio (DAR) of 2 for the preparation of all molecules. [00657] Given the design of a TransTAC degrader, the cathepsin-sensitive linker between the binder and the IgG1 Fc may be processed in the early endosome. This design leads to contrasting outcomes for the two drug types. For the first type, the conjugated MMAF is anticipated to follow the TfR mainly through the recycling pathway. In contrast, for the second type, the conjugated MMAF is likely to stay with CD20 and follow the late endosome/lysosome pathway, resulting in its degradation and subsequent release there. This process, in turn, generates effective cytotoxicities. [00658] Our data show this (FIG.56A-B). As anticipated, unconjugated antibodies or TransTACs did not cause cytotoxicity. The CD20 antibody (rituximab) ADC or the first type of DDC resulted in minimal cytotoxicity, with IC50s within the 100-10 nM range. In contrast, the second type of DDC, which guides the molecule towards lysosomal degradation, produced an improvement in IC50 by more than 100-fold, generating an impressively potent drug with a 100 pM IC50. These findings indicate that a DDC can significantly augment the efficiency of drug internalization and release. Our results also suggest that lysosome-mediated DDC degradation is a factor for achieving this enhanced effect. Furthermore, by introducing additional drug molecules to the hinge region of the ADC, resulting in proteins with a DAR of 4, we observed an additional enhancement in DDC potency. This indicates that DDC efficacy can be further improved by optimizing drug labeling ratios. [00659] We proceeded to evaluate these molecules within an in vivo B cell lymphoma model (FIG.56C-F). Nude mice were irradiated, and GFP-labeled Raji cells were introduced subcutaneously into the flank of female nude mice. To determine the optimal dosage, we initially tested two doses and found that a dosage of 1 mg/kg DDC exhibited a stronger antitumor response compared to 0.3 mg/kg, and was thus selected for the larger-scale animal study. No higher doses were examined. TransTAC or controls were administered once the tumors had reached an approximate size of 100 mm 3 . Over time, tumor size and survival were closely monitored. Upon sacrificing the mice, tumors were harvested, processed, and run on western blots to ascertain on-target protein degradation. [00660] We observed a statistically significant reduction in tumor size and slower tumor growth with DDCs compared to PBS control or rituximab ADC control. Furthermore, a significant survival advantage was noted. On-target CD20 degradation was observed in all seven DDC-treated mice. A CD20 degradation range of 28-93% was observed, while no degradation was apparent in the PBS or rituximab treated conditions. These findings demonstrated that CD20 DDCs can target tumors in vivo. EXAMPLE 18 – A schematic illustration of the design principles we have discovered for enhancing degradation efficiency of TransTACs [00661] FIG.42B is an illustration of an example TransTAC degrader. Generally, TransTACs are recombinant proteins consisting of anti-POI binders and anti-TfR binders for bridging POI and TfR in close proximity on the cell surface. We found many formats for TransTACs can efficiently eliminate the target proteins from the cell surface as outlined in FIG.43A and 48A. Therefore, all confer effectiveness as modulators of membrane proteins. [00662] However, we discovered at least three example design principles to make TransTACs efficient internalizers and degraders: (1) a dimeric TransTAC drives more efficient protein internalization than a monomeric heterobispecific TransTAC; (2) a cathepsin B-sensitive linker facilitates lysosomal trafficking of the POI; and (3) an antibody binder for targeting TfR, rather than a native transferrin (TF) ligand, could reduce trafficking of the POI to the recycling endosomes (REs) and hence enhance degradation efficiency [00663] Using specific variants of the molecules, we could choose to induce endosomal trapping or lysosomal degradation of the targets, offering customizable possibilities for modular manipulation of membrane proteins. EXAMPLE 19 – Identification of peptides not known to be sensitive to cathepsin cleavage [00664] A yeast-displayed peptide library was used to identify peptides not known to be sensitive to cathepsin cleavage. These peptides can be from a combination of small motifs found in SEQ ID NOs: 144 and 145. The studies were performed at pH 4.4 (FIG.57A) and at pH 6.4 (FIG.57B). These peptides include GRLVGFD (SEQ ID NO: 124), GRLVGFG (SEQ ID NO: 125), RMLVGFV (SEQ ID NO: 126), RRLYAFL (SEQ ID NO: 127), VFRLLMF (SEQ ID NO: 128), LVGVLLF (SEQ ID NO: 129), VKLYGLG (SEQ ID NO: 130), TWRVDLY (SEQ ID NO: 131), EQLYLYA (SEQ ID NO: 132), KLFLMIF (SEQ ID NO:133 ), NFVIILF (SEQ ID NO: 134), MSLLIGV (SEQ ID NO: 135), VRLLSLQ (SEQ ID NO: 136), STLMWNV (SEQ ID NO: 137), VRFLAAA (SEQ ID NO: 138), HGWSFHE (SEQ ID NO: 139), ENLYFQG (SEQ ID NO: 140), VVMMFLH (SEQ ID NO: 141), VFRLLMF (SEQ ID NO:142 ), or VGALVWL (SEQ ID NO: 143). ***** EQUIVALENTS Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention.