Receptor-mediated endocytosis for targeted internalization and degradation of membrane proteins and cargoes

Abstract

Bispecific modulators are disclosed that can bind to a protein of interest and to an internalization receptor on the cell surface. Once bound, the protein of interest can be internalized and / or degraded within the cell. TIFF2025532646000110.tif135128

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Application No. 63 / 376,389, filed September 20, 2022, and U.S. Provisional Application No. 63 / 462,828, filed April 28, 2023, the entire contents of which are incorporated herein by reference.

[0002] All patents, patent applications, and publications cited herein are incorporated by reference in their entirety, and the disclosures of these publications in their entireties are incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled in the art as of the date of the invention described and claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction of the patent document or patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.

[0004] Field Aspects of the present invention relate to compositions and methods for modulating cell surface molecules, including chimeric antigen receptors (CARs) on the surface of CAR-T cells, to reversibly control CAR receptors and receptors on cancer cells to inhibit cancer cell signaling and proliferation.

[0005] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy made at [] is named [] and is [] bytes in size. [Background technology]

[0006] background Conventional small molecule inhibitor-based therapies have drawbacks such as toxicity and limited efficacy. Examples of inhibitor characteristics that contribute to these drawbacks include binding affinity, which is a determinant of inhibitor efficacy; a therapeutic response that depends on persistent target binding; limited functionality on the target protein; overexpression of the target protein; native ligand competition; and disruption of efficacy due to the occurrence of target protein mutations that limit binding or promote resistance. Therefore, desirable therapies exhibit high efficacy with low toxicity. For example, desirable therapies include various characteristics that contribute to overall efficacy, a therapeutic response that is not entirely or largely dependent on persistent target binding, the ability to completely or substantially limit target protein function, and limited or no ability of target cells to override efficacy due to overexpression of the target protein, native ligand competition, and / or the occurrence of target protein mutations that limit binding or promote resistance.

[0007] Membrane proteins are central to numerous cellular functions and are the targets of more than half of all drugs, so developing strategies to degrade them is of great interest for both basic research and therapeutic intervention purposes.

[0008] Targeted protein degradation (TPD) is a rapidly growing field in drug discovery and pharmacology. TPD molecules complement traditional drug therapies and offer new therapeutic mechanisms to address challenging targets or enhance the therapeutic potential of currently used drugs.

[0009] Proof-of-concept strategies for membrane receptor degradation have been described. These strategies involve the use of heterobifunctional biologics to introduce specific "effector" proteins, such as membrane E3 ligases (Cotton, AD, Nguyen, DP, Gramespacher, JA, Seiple, IB, & Wells, JA. Development of Antibody-Based PROTACs for the Degradation of the Cell-Surface Immune Checkpoint Protein PD-L1. J Am Chem Soc 143, 593-598 (2021)) or lysosomal shuttling receptors (Ahn, G. et al. LYTACs that engage the asialoglycoprotein receptor for targeted protein degradation. Nat Chem Biol 17, 937-946 (2021)). However, the efficacy of these biological effectors is often limited by tissue-specific expression patterns. For example, GalNAc-LYTAC targets the hepatocyte-specific receptor asialoglycoprotein receptor (ASGPR), making it suitable only for treating liver disease or removing circulating targets (Ahn, G. et al. Nat Chem Biol 17, 937-946 (2021) (Non-Patent Document 3); Zhou, Y., Teng, P., Montgomery, NT, Li, X. & Tang, W. Development of Triantennary N-Acetylgalactosamine Conjugates as Degraders for Extracellular Proteins. ACS Cent Sci 7, 499-506 (2021) (Non-Patent Document 4)). On the other hand, methods based on RNF43 and ZNRF3 are effective in treating diseases with elevated Wnt signaling, where RNF43 and ZNRF3 are expressed at high levels.Therefore, current technologies cannot cover the entire spectrum of diseases, and the development of alternative effectors that are overexpressed in various diseases and tissues could greatly expand the range of controllable cell surface targets and increase targeting specificity. Therefore, there is a need to improve targeted proteolysis, especially membrane proteolysis, to treat a range of diseases, most notably cancer. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Cotton,AD,Nguyen,DP,Gramespacher,JA,Seiple,IB& Wells,JADevelopment of Antibody-Based PROTACs for the Degradation of the Cell-Surface Immune Checkpoint Protein PD-L1.J Am Chem Soc 143,593-598(2021) [Non-patent document 2] Ahn,G.et al.LYTACs that engage the asialoglycoprotein receptor for targeted protein degradation.Nat Chem Biol 17,937-946(2021) [Non-patent document 3] Ahn, G. et al. Nat Chem Biol 17,937-946(2021) [Non-patent document 4] Zhou, Y., Teng, P., Montgomery, NT, Li, X. & Tang, W. Development of Triantennary N-Acetylgalactosamine Conjugates as Degraders for Extracellular Proteins.ACS Cent Sci 7,499-506(2021) Summary of the Invention [Problem to be solved by the invention]

[0011] overview Disclosed herein are novel reagents and methods for modulating molecules (e.g., molecules of interest, such as proteins of interest) on the surface of cells. In some embodiments, the reagents and methods are used to control the level of CAR molecules on CAR-T cells. In some embodiments, the reagents and methods are used to reversibly control CAR-T cell activation. In some embodiments, CAR-T cell activation is controlled in vivo. In some embodiments, the reagents and methods are used to control the level of receptors (e.g., epidermal growth factor receptor or EGFR, programmed death ligand 1 or PDL1) on the surface of cancer cells to inhibit cancer cell growth or modulate an immune response. In some embodiments, the reagents and methods can be used on cells that are not cancer cells. [Means for solving the problem]

[0012] In some embodiments, bispecific modulators as described herein are disclosed. In some embodiments, an antigen capable of binding a cell surface molecule, or an antibody or antibody fragment capable of binding to a cell surface molecule, is fused to a ligand of an internalizing receptor, or an antibody or antibody fragment capable of binding to an internalizing receptor or membrane protein. In embodiments, after binding, the bispecific modulator can internalize the cell surface molecule (e.g., a molecule of interest such as a protein of interest) into the cell, and in some embodiments, can degrade the internalized cell surface molecule. In various embodiments, the bispecific modulator can target a single-transmembrane or a multi-transmembrane membrane protein.

[0013] In some embodiments, reagents and methods are disclosed for improving the degradation of internalized cell surface proteins using the bispecific modulators disclosed herein. In some embodiments, a peptide linker that is sensitive to a specific protease is inserted into the bispecific modulator. In some embodiments, the peptide linker is sensitive to a cathepsin protease.

[0014] Disclosed are nucleic acids encoding these molecules, vectors containing the nucleic acids, and cells containing the vectors and / or expressing the bispecific modulator molecules disclosed herein.

[0015] In some embodiments, a method of administering a bispecific modulator to a subject is disclosed.

[0016] Certain diagrams, charts, or flow charts are provided to facilitate a better understanding of the invention. It should be noted, however, that the drawings depict only selected embodiments of the invention and are therefore not intended to limit the scope. Additional and equally effective embodiments and applications of the invention exist. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 is a schematic diagram showing CAR-T cell therapy in patients. [Figure 2] We present an example approach to treat toxicities observed after CAR-T cell therapy with immunosuppressants. [Figure 3] An example approach to treat toxicity observed after CAR-T cell therapy using suicide genes or exclusion markers is presented. [Figure 4] We present an example approach to treat toxicity observed after CAR-T cell therapy using a reversible gene switch. [Figure 5] We demonstrate a reversible CAR-T modulation mechanism as a strategy to enhance CAR-T cell efficacy. [Figure 6]As disclosed herein, an exemplary approach is provided for controlling CAR-T cell activation using targeted CAR internalization and / or degradation (bispecific modulators comprising TransTAC molecules) (TransTAC is a Transferrin receptor-mediated TArgeting Chimera). [Figure 7] 1 is a schematic diagram of an example of TransTAC technology (e.g., a type of bispecific modulator). Extracellular proteins (e.g., membrane proteins with extracellular domains) can be selectively internalized and degraded by tethering an antibody against the target membrane protein (or a ligand of the target membrane protein) to the transferrin receptor with a bispecific modulator, e.g., a membrane protein-specific antibody-transferrin fusion protein. [Figure 8] Schematic diagram of another example of bispecific regulator / TransTAC technology, not covered in the main text. [Figure 9] Schematic diagram of another example of bispecific regulator / TransTAC technology, not covered in the main text. [Figure 10] The results show the expression of anti-EGFR affibody-Fc-Tr TransTAC molecules (left) and the effect on EGFR levels when TransTAC molecules are incubated with the MCF10A EGFR-overexpressing cell line (right). [Figure 11] 11A-B show the results of the effect on EGFR levels when TransTAC molecules are incubated with A549 cells (A) and the effect when they kill MCF10A EGFR cells (B). [Figure 12] These results demonstrate that TransTAC targeting effectively internalized the receptor. [Figure 13] An example of the results showing the expression of various TransTAC proteins in cultured cells is shown. [Figure 14]10 shows results demonstrating that TransTAC targeting of CD19-specific CAR effectively reduces levels of CAR (e.g., internalizes CAR). [Figure 15] This is an example of a result showing that TransTAC targeting of a CD19-specific CAR effectively internalized the CAR. [Figure 16] An example of results is shown showing that TransTAC targeting of a CAR specific for CD19 on Jurkat cells in the presence of K562 cells inhibited Jurkat cell activation. [Figure 17] An example of results is shown showing that TransTAC targeting of CD19-specific CAR inhibited CAR internalization / CAR-T activation. [Figure 18] These results demonstrate that the CD19-specific TransTAC molecule inhibits CAR-T cell activation in the presence of K562 cells, and that the TransTAC molecule has minimal effect on Jurkat cells in the absence of K562 cells. [Figure 19-1] Figures 19A-B are schematic diagrams showing a CARTrap molecule (a fusion of a domain capable of binding to a CAR and an Fc region), a TransTAC molecule capable of binding to a CAR and an internalizing receptor, and a dimer of a TransTAC molecule capable of binding to a CAR and an internalizing receptor (A), and the results of using these molecules at the CAR level in cells expressing a CAR (B). [Figure 19-2] Figure 19C is a fluorescence micrograph of targeted CAR on the cells of Figure 19B. [Figure 20-1]Figures 20A-B, 20C, 20D-E, 20F-G, 20H, and 20I show embodiments in which internalized CAR is not degraded, but rather is degraded by linker technology (here incorporating a cathepsin-sensitive linker). (A) Schematic of the various molecules used in this study. GFLG denotes a Gly-Phe-Leu-Gly peptide linker that is sensitive to lysosomal cathepsin proteases. (B) Western blots of targeting CAR (anti-CD3z) and actin control (β-actin) using various molecules from (A). (C) Graph of the data from (B), where CAR levels are normalized to β-actin levels. (D) Western blots similar to those shown above using other molecules from (A). (E) Graph of the normalized data from (D). (F) shows a schematic diagram of a dimer of TransTAC molecules capable of binding to CAR and an internalized receptor that also contains a GFLG linker. (G) shows a Western blot using the molecules shown in (F). (H) shows a graph of the normalized data from (F). (I) shows the results of further screening for cathepsin-sensitive TransTAC molecules with improved inhibitory potency. [Figure 20-2] See description of Figure 20-1. [Figure 20-3] See description of Figure 20-1. [Figure 20-4] See description of Figure 20-1. [Figure 20-5] See description of Figure 20-1. [Figure 20-6] See description of Figure 20-1. [Figure 21] Figures 21A-B show results demonstrating that TransTAC inhibits T cell activity more potently than CARTrap (a domain capable of binding to a CAR fused to an Fc region) in Jurkat cells (A) and primary T cells (B). [Figure 22A] Figures 22A and 22B show results demonstrating that CAR-TransTAC turns off tumor cell killing of CAR-T cells, and removal of the TransTAC molecule resumes tumor cell killing of CAR-T. [Figure 22B]See legend to Figure 22A. [Figure 23-1] Figures 23A-B show a schematic of the molecules used in the study, including the affibody-based EGFR TransTAC molecule (A). (B) shows the results of depleting EGFR from the surface of A549 cells using the molecule shown in (A). The data demonstrate that the use of the affibody-based TransTAC molecule provides superior results (approximately 10-50 fold improvement in IC50). [Figure 23-2] Figures 23C-D show cell proliferation inhibition by the molecules shown in (A) as measured using an MTT cell proliferation assay. The data demonstrate that the use of affibody-based TransTAC molecules provides favorable results (approximately 10-50 fold improvement in IC50). [Figure 24-1] Figures 24A-B show a schematic diagram of the molecules used in this study (B) and the results of Western blots measuring the internalization and degradation of the molecules (C). [Figure 24-2] FIG. 24C shows a graph of the normalized data of FIG. 54(C). [Figure 25] 25A-B show example data showing protein internalization by TransTAC and the reversibility of internalization. [Figure 26] 1 shows exemplary data demonstrating that TransTAC can inhibit IFNγ production. [Figure 27A] Figures 27A and 27B show example data of transferrin receptor expression in various cells. [Figure 27B] See legend to Figure 27A. [Figure 28] Some examples of cell surface molecules that can be regulated by TransTAC are shown. [Figure 29A] Figures 29A and 29B show example data demonstrating that TransTAC can degrade EGFR in cells and the underlying cellular machinery that mediates degradation. [Figure 29B] See legend to Figure 29A. [Figure 30-1]30A, 30B, and 30C-D show an example approach to treating lung cancer with TransTAC. [Figure 30-2] See description of Figure 30-1. [Figure 30-3] See description of Figure 30-1. [Figure 31] Illustrative data showing that TransTAC with linker variants can degrade CAR in CAR-Jurkat cells. [Figure 32A] Figures 32A and 32B provide example data showing that TransTAC can degrade PD-L1 in breast cancer cells. [Figure 32B] See legend to Figure 32A. [Figure 33] 1 is an example of data showing that TransTAC can degrade CD20 in lymphoma cells. [Figure 34-1] Figures 34A-C and 34D show examples of TransTAC molecules containing protease-sensitive linkers and example data obtained with those molecules. [Figure 34-2] See description of Figure 34-1. [Figure 35] Example data obtained with TransTAC molecules containing various protease-sensitive linkers are shown. [Figure 36] 36A-C show an exemplary TransTAC molecule comprising an antibody fragment specific for transferrin binding and exemplary data obtained with this molecule. [Figure 37] 37A-B show an example of a TransTAC molecule and example data obtained with that molecule. [Figure 38-1]Figures 38A-E and 38F-H show an example overview of TransTAC technology and TfR expression analysis. (A) Schematic of an example of TransTAC technology. TransTAC brings TfR and POI into close proximity on the cell surface, inducing co-uptake of the complex into early endosomes (EEs). Here, cathepsin enzymes cleave TransTAC, separating the POI from TfR. The POI then translocates to late endosomes (LEs) / lysosomes for degradation, and TfR is recycled back to the cell surface. (B) An example of a TransTAC protein is shown. Examples of designs for making TransTAC an efficient degrader include: (1) containing two anti-TfR binders to bind and prime TfR dimers for endocytosis; (2) containing a cathepsin B-sensitive linker between the anti-POI binder and Fc to induce endosomal cleavage to separate the POI from the recycling TfR; and (3) using an antibody binder instead of the natural TF ligand to reduce trafficking to the recycling endosome (RE). (C) Relative cell surface TfR expression levels in various non-tumorous and cancerous cell lines characterized by flow cytometry. Cancer cell lines express higher levels of TfR compared with non-tumorous cell lines. Data are representative of three independent experiments. (D) Comparison of TFRC RNA expression levels in primary tumors and normal tissues based on the MERAV database. TFRC expression is significantly higher in most tumors than in corresponding normal tissues. T-tests (Figure 38F and G) were significant for tumor vs. healthy tissue overall (p=3.98e-89) and for 14 of 19 tumor / healthy tissue pairings. Female reproductive tissues were endometrium, cervix, fallopian tube, myometrium, ovary, placenta, and uterus. Central nervous system (CNS) tissues were basal ganglia, brainstem, cerebral cortex, hippocampus, spinal cord, and supra-vestibular nuclei. Brain tissues were hypothalamus, pituitary, thalamus, ganglion, and ganglion nodule. (E) Relative TFRC RNA expression levels in naive T cells.TfR was upregulated approximately six-fold in activated CD4 and CD8 T cells compared to resting T cells, a statistically significant difference (p=1.25e-68 for CD4 T cells and 4.81e-68 for CD8 T cells, Figure 38H). [Figure 38-2] See description of Figure 38-1. [Figure 39]Figures 39A-L show examples of TransTAC degradation-inducing compound designs. (A) Schematic diagram of examples of CAR-TransTAC and control. TransTAC v0.1 contains one CD19NT.1 domain, one TF, and one Knob-in-hole (KIH) Fc. v0.2 contains two CD19NT.1 domains, two TFs, and a homodimeric Fc binding binder. v0.4 contains a cathepsin-sensitive linker between CD19NT.1 and the Fc. v0.5 contains an H7 scFv for TfR binding. v1.0 contains both the H7 and cathepsin-sensitive linker. (B) Schematic diagram of a myc-tagged anti-CD19 CAR receptor. (C) Flow cytometry measurement of cell surface CAR expression levels in CAR-Jurkat mice treated with TransTAC v0.1, v0.2, and a control. TransTAC v0.2 results in higher CAR clearance from the cell surface than v0.1, without any apparent hook effect. Data are representative of two independent experiments. (D) Characterization of total cellular CAR levels by Western blot in TransTAC-treated CAR-Jurkat cells. TransTAC v1.0 degrades approximately 80% of the total CAR. v0.2 did not show significant CAR degradation. (E-H) Schematic diagram showing that different TransTACs alter the intracellular trafficking of POI. Cleavage of the cathepsin-sensitive linker in v0.4 and v1.0 results in separation of POI from TfR, thus enhancing LE / lysosomal trafficking and degradation of POI; H7 scFv in v0.5 and v1.0 reduces trafficking of the complex to the RE, thus increasing the proportion of POI in the EE and subsequent proteolytic processing when the cleavable linker is present. (I, J) Representative fluorescence images of Hela cells co-expressing CAR-GFP (green) and the endosomal / lysosomal marker mCherry (red) treated with various TransTAC molecules. Cell nuclei are stained with Hochest (blue). In untreated (UT) or control-treated cells, CAR-GFP was localized to the plasma membrane. In v0.5 and v1.0, CAR-GFP was efficiently degraded, resulting in a significant decrease in GFP signal.In v0.2-treated cells, CAR was primarily transported to the RE, where colocalization of mCherry-Rab11 (white arrow) and CAR-GFP was observed. In v0.5-treated cells, CAR was transported to the EE, where colocalization of mCherry-Rab5 (white arrow) and CAR-GFP was observed. (K) Pearson correlation analysis of colocalization of CAR-GFP with Rab5 (EE), EEA1 (EE), and Rab11 (RE) markers. T-tests showed that colocalization of Rab5, EEA1, and Rab11 with CAR was statistically different in cells treated with v0.2 and v0.5. (L) Pearson correlation analysis of colocalization of CAR-GFP with Rab7 (LE) and Lamp1 (lysosomal) markers. T-tests showed that colocalization of Rab7 and Lamp1 with CAR was statistically significant in v0.2 vs. v0.4 and v0.5 vs. v1.0. For k and l, the number of cells used in each analysis was as follows: N = 12, N = 12, and N = 13 for the EEA1, Rab5, and Rab11 markers in v0.2, respectively. N = 10, N = 22, and N = 15 for the EEA1, Rab5, and Rab11 markers in v0.5, respectively. N = 16, N = 21, N = 13, and N = 13 for the Lamp1 marker in v0.2, v0.4, v0.5, and v1.0, respectively. [Figure 40] Figures 40A-D show examples of the development of TransTAC degradation-inducing compounds for various membrane targets. (A) Schematic diagram of the membrane proteins targeted by TransTAC in this study. These targets are either synthetic proteins or natural single- or multi-transmembrane proteins expressed on the surface of cancer or immune cells. (B) Degradation of PD-L1 by TransTAC in MDA-MB-231 breast cancer cells analyzed by Western blot. The scFv or Fab format of atezolizumab is used as the PD-L1 binding moiety. (C) Degradation of EGFR by TransTAC in A549 lung cancer cells. An affibody is used as the EGFR binding moiety. (D) Degradation of CD20 by TransTAC. The Fab portion of rituximab is used as the CD20 binding site. [Figure 41-1]Figures 41A-H and 41I show examples of structure-activity relationship (SAR) studies of TransTAC, its mechanism, and in vivo characterization. (A) Time-dependent measurement of cell surface CAR levels in TransTAC-treated CAR-Jurkats revealed the rapid kinetics of TransTAC-mediated CAR internalization. (B) Schematic diagram of CAR-TransTAC variants composed of one or two copies of anti-POI and anti-TfR binders with different protein structures. (C) Measurement of cell surface CAR levels in CAR-Jurkats treated with the CAR-TransTAC variants outlined in (B). The results highlight the influence of two and only one TfR binders (v0.5 and v0.7) and geometric shape (v0.8 and v0.9) in regulating protein internalization. Data are representative of three independent measurements. (D) Competition assay using H7-Fc fusion protein. A concentration-dependent decrease in CAR internalization was observed with H7-Fc, demonstrating that internalization is mediated through TfR. Data are representative of three independent measurements. (E) Investigation of the basal degradation pathway using TransTAC. When EGFR-TransTAC was administered to A549 cells, degradation was completely inhibited by bafilomycin, indicating that intact lysosomal function is important for degradation. (F) Measurement of total cellular TfR levels after TransTAC treatment. In MDA-MB-231 cells treated with PDL1 TransTAC, PD-L1 was degraded while TfR levels remained constant. (G) Schematic diagram of a mouse experiment to evaluate the safety and serum half-life of TransTAC by IP injection. (H) Monitoring mouse weight over time after injection of TransTAC or control IgG. Results showed no significant effect on mouse weight over time, indicating that the molecule was well tolerated. N=2 per treatment group. (I) Western blot quantification of plasma concentrations of CD20-TransTAC and IgG control over time. N=2 for each treatment group. [Figure 41-2] See description of Figure 41-1. [Figure 42]Figures 42A–G show examples of targeting TKI-resistant lung cancer cells with EGFR-TransTAC. (A) Schematic depicting the development of drug-resistant mutations in lung cancer cells and available treatment options. EGFR Del19 and L858R mutations can be targeted by first- and second-generation TKIs, and T790M can be targeted by osimertinib, but cells harboring the additional C797S mutation have no available targeted treatment options. (B) Schematic representation of EGFR TransTACs designed with different cleavable linkers and TfR binders. (C) Cell viability assay of PC9 WT cells treated with the EGFR TransTAC variants shown in (B). v0.5 and v1.0 resulted in potent cell inhibition, while the affibody-Fc control and v0.2 had no effect. Data are representative of three independent experiments. (D) Western blot showing efficient EGFR degradation mediated by TransTAC1.0s in PC9 WT and PC4 GR4 C797S cells. (E) Cell viability assays of lung cancer cells PC9 WT, PC9 GR4, and PC9 GR4 C797S, and the normal fibroblast cell line HFF-1, treated with TransTAC and TKIs. PC9 WT cells responded to all three TKIs; PC9-GR4, which contains the T790M mutation, is resistant to gefitinib; and PC9 GR4 C797S is resistant to afatinib, gefitinib, and osimertinib. All three PC9 cell lines were inhibited by EGFR-TransTAC. In the HFF-1 cell line, neither TKIs nor TransTACs showed significant toxicity. Data are representative of three independent experiments. (F) The efficacy and specificity of TransTAC was tested in a co-culture assay of PC9 WT cancer cells and HFF-1 healthy cells, comparing it to a TKI and carboplatin / paclitaxel chemotherapy combination. PC9 WT cells and HFF-1 cells express GFP and mCherry, respectively. TransTAC and TKI specifically inhibit PC9 WT cancer cells while sparing HFF-1 cells, whereas chemotherapy inhibits both cell types. (G) The same experiment as in (F) in which PC9 GR4 C797S cancer cells were co-cultured with HFF-1 healthy cells. TransTAC and chemotherapy inhibited the cancer cells, but TKI did not.Furthermore, TransTAC and TKIs did not cause cytotoxicity to HFF-1 cells, whereas chemotherapy inhibited HFF-1. [Figure 43] Figures 43A-B show the characteristics of wild-type (WT) CD19 ectodomain and Fc fusions of the variants. CD19ecto-WT-Fc aggregates on SDS-PAGE gels, whereas the yeast-derived variants do not. Of the four variants, CD19NT.1 was selected for CAR-TransTAC technology due to its high expression level. [Figure 44] Figures 44A-E show examples of different CAR degradation efficiencies mediated by TransTAC variants. (A) Schematic diagram of various generations of CAR-TransTAC and the CD19NT.1-Fc control. (B) Western blot showing that neither the control nor v0.2 caused CAR degradation. (C) Western blot showing that v0.4-GFLG, which contains a cathepsin-sensitive GFLG linker between the CD19NT.1 and Fc domains, resulted in approximately 40-50% CAR degradation. v0.3-GFLG, which contains a cleavable linker between the Fc and TF domains, did not result in significant CAR degradation, likely due to Fc-mediated CAR recycling. (D) Western blot showing different CAR degradation efficiencies depending on the linker variants in v0.4. (E) Western blot showing different CAR degradation efficiencies depending on the linker variants in v1.0. Among all variants, the linkers GFLG-VR and VR showed the highest degradation. [Figure 45-1]Figures 45A-D and 45E-F show examples of colocalization analysis of internalized CAR with various endosomal / lysosomal markers. (A-C) Representative fluorescence images of Hela cells coexpressing CAR-GFP (green) and the endosomal / lysosomal marker mCherry (red) and treated with various TransTACs or controls. EEA1: EE marker, Rab7: LE marker, Lamp1: lysosomal marker. Cell nuclei are stained with Hochest (blue). (D) Pearson correlation analysis of colocalization between CAR-GFP and five endosomal / lysosomal markers. By t-test, colocalization of CAR with Rab5, EEA1, and Rab11 was statistically different at v0.2 vs. v0.5, while colocalization of CAR with Rab7 and Lamp1 was statistically significant at v0.2 vs. v0.4 and v0.5 vs. v1.0. The numbers of cells used for analysis were as follows: for the EEA1 marker, N = 5, N = 4, N = 12, N = 15, N = 10, and N = 11 for control, UT, v0.2, v0.4, v0.5, and v1.0, respectively. For the Rab5 marker, N = 12, N = 11, N = 12, N = 15, N = 22, and N = 17 for control, UT, v0.2, v0.4, v0.5, and v1.0, respectively. For the Rab7 marker, N = 9, N = 7, N = 8, N = 12, N = 26, and N = 11 for control, UT, v0.2, v0.4, v0.5, and v1.0, respectively. For the Rab11 marker, N = 9, N = 6, N = 13, N = 13, N = 15, and N = 22 for control, UT, v0.2, v0.4, v0.5, and v1.0, respectively. For the Lamp1 marker, N = 17, N = 12, N = 16, N = 21, N = 13, and N = 13 for control, UT, v0.2, v0.4, v0.5, and v1.0, respectively. (E-F) Incorporation of a cathepsin-sensitive linker into TransTAC promotes LE / lysosome trafficking. Representative fluorescence images of HeLa cells co-expressing CAR-GFP (green) and mCherry-Rab7 or Lamp1-mCherry (red) treated with TransTAC v0.2 vs. v0.4. Cell nuclei are stained with Hochest (blue).The v0.4GFP images were collected at 1000x longer exposures than the v0.2 images to obtain sufficient GFP signal for the Pearson coefficient analysis in Figure 2i. Cells treated with v0.4 show colocalization of internalized CAR-GFP with mCherry-Rab7 and Lamp1 (white arrows). [Figure 45-2] See description of Figure 45-1. [Figure 46] Figures 46A-C show examples characterizing TransTAC degradation-inducing compounds for various membrane proteins. Different linker and geometry designs result in different degradation efficiencies. (A) Western blot showing that the control or the v0.2 and v0.4 PDL1 TransTAC variants cause minimal target degradation in MDA-MB-231 cells. (B) Western blot showing that EGFR TransTAC v0.2 causes minimal target degradation in A549 cells, while v0.4 or v1.0 with different linkers cause varying degrees of degradation, with v1.0-EVR and GFLG-VR showing the highest degradation efficiencies. (C) Western blot showing that the rituximab-scFv-Fc control does not cause significant CD20 degradation in Raji cells. [Figure 47]Figures 47A-E show an example of TransTAC regulating primary CAR-T cell activity. (A) Schematic of reversible CAR-T cell regulation using CAR-TransTAC. Removal of the CAR from the cell surface by TransTAC prevents CAR-T cells from binding to CD19+ tumor cells, inhibiting cytokine release and cytotoxicity. (B) Schematic of the setup for the primary CAR-T cell co-culture assay. Secreted IFN-γ levels are measured to determine the activation level of CAR-T cells in the presence of CD19+ A375 cells and TransTAC, and anti-tumor efficacy is determined using live-cell fluorescence microscopy. (C) Measurement of IFN-γ release from human primary CAR-T cells in the co-culture assay in (b) using an IFN-γ split luciferase assay (Promega). IFN-γ secretion is inhibited by TransTACv0.4 in a dose-dependent manner. The IC50 of TransTAC is approximately 0.4 nM. Data are representative of two independent experiments. (D) Fluorescence microscopy of mCherry-labeled A375 cells demonstrated reversible CAR-T cell-mediated A375 killing with CAR-TransTACv4. (E) Overlay of brightfield and mCherry channel images shows that CAR-T cell-mediated A375 killing resumed over time after TransTAC washout. [Figure 48] Figures 48A-C show an example of EGFR TransTAC characterization. (A) Western blot analysis shows that EGFR-TransTAC induces 40-50% target degradation in HEK293 cells overexpressing EGFR. This level is significantly lower than that in A549 and PC9 cells, likely due to lower TfR expression levels. (B) IC50 values ​​of TransTACv1.0s and the TKIs afatinib, gefitinib, and osimertinib in PC9 cells based on published data. (C) Flow cytometry analysis of PC9 (GFP) / HFF-1 (mCherry) cell ratios, reflecting the differential sensitivity of tumor cells to various treatments. Data are representative of three independent experiments. [Figure 49A](A) Cleavage of the indicated linkers by recombinant cathepsin B in yeast at pH 4.4 compared to cleavage of GFLGGVR (SEQ ID NO: 144). [Figure 49B] (B) Cleavage of the indicated linkers by recombinant cathepsin B in yeast at pH 6.4 compared to GFLGGVR (SEQ ID NO: 144). DETAILED DESCRIPTION OF THE INVENTION

[0018] Detailed Description Targeted protein degradation (TPD) is a rapidly growing field in drug discovery and pharmacology. TPD molecules complement traditional drug therapies, addressing challenging targets and enhancing the therapeutic potential of currently used drugs, providing new therapeutic mechanisms. While much of the effort in this field has focused on small molecules targeting intracellular proteins, inducing the targeted degradation of extracellular proteins offers new possibilities. Developing strategies to degrade extracellular proteins is of great interest for both basic research and therapeutic intervention purposes.

[0019] Iron is an essential element for cells, and its transport is facilitated by the transferrin receptor (TfR). As a recycling receptor, TfR undergoes rapid endocytosis, with an average internalization rate of 500 molecules per second per cell, making it one of the fastest internalizing receptors known. Furthermore, TfR is upregulated in cells with a high demand for iron, including rapidly dividing cancer cells and activated T cells. TfR expression in these cells is higher than in non-dividing or slowly dividing normal tissues. TfR can be expressed in non-cancerous cells at levels sufficient to enable the use of the reagents and methods described herein.

[0020] Herein, we exploit these characteristics of the TfR and use a protein engineering strategy to develop a novel technology for membrane protein degradation. This technology is referred to herein as the Transferrin Receptor-Mediated Targeting Chimera (TransTAC). In some embodiments, TransTAC is a heterobispecific antibody that brings a protein of interest (POI) and TfR into close proximity on the cell surface, inducing endocytosis of the POI / TfR complex and subsequent lysosomal degradation of the POI. TransTAC is effective in degrading various types of membrane proteins, including single-pass, multi-pass, native, and synthetic receptors, and demonstrated degradation efficiencies of over 80% for all targets in various cell lines. A notable feature of TransTAC is its rapid internalization of targeted drugs, occurring on a timescale of minutes, making it a valuable molecular tool for rapid knockdown of cell surface expression and providing temporal specificity for cell signaling studies that is not possible with genetic approaches. Furthermore, TransTAC molecules are fully recombinable, modular, and cancer-specific, properties that make TransTAC a versatile technology for engineering cell surface targets in a disease-specific manner.

[0021] TransTAC has broad applicability in both basic research and translational applications. Herein, we demonstrate non-limiting examples of TransTAC applications, including reversible control of CAR-T cells and targeting of drug-resistant EGFR-positive lung cancer. TransTAC is a prototype of a new molecule that regulates cell surface proteins.

[0022] TransTAC is the first bispecific antibody technology to repurpose ligand-receptor interactions for target protein internalization and degradation, greatly expanding the range of cell surface effectors suitable for such purposes.

[0023] Chimeric antigen receptor (CAR) T cells have emerged as a promising treatment for patients with hematological malignancies (Figure 1). However, in some cases, CAR-T therapy can cause side effects, such as cytokine-release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), in patients receiving CAR-T therapy.

[0024] To address these adverse events, several regulatory mechanisms have been developed to control CAR-T cells in vivo, but these strategies do not adequately meet current needs, as toxicity and mortality continue to be reported in CAR-T clinical trials.

[0025] Disclosed herein are novel, modular, and reversible strategies for modulating the activity of CAR-T cells. Generally, these methods do not require additional genetic manipulation of CAR-T cells. In some embodiments, these strategies can modulate the toxicity of CAR-T. In some embodiments, these strategies can enhance the efficacy of CAR-T cell therapy. In some embodiments, these strategies are based on the reversible internalization of CAR receptors.

[0026] In some embodiments, bispecific modulators, including, for example, transferrin receptor-mediated targeting chimeras or TransTAC molecules, can co-localize the CAR receptor with internalized cell surface proteins. In embodiments, the bispecific modulators can downregulate cell surface levels of the CAR. In embodiments, the bispecific modulators can inhibit CAR-T cell activation and / or function.

[0027] In embodiments, a bispecific modulator can have a first portion or moiety that is an antibody, antibody fragment, or surrogate antibody scaffold that specifically binds to a target molecule on a cell (e.g., a molecule of interest, such as a protein of interest like CAR, EGFR, or CD20), and a second portion or moiety (e.g., transferrin, an antibody, or antibody fragment) that can bind to an internalization molecule on the cell surface (e.g., a transferrin receptor). In embodiments, a bispecific modulator can have a first portion that is an antigen or ligand for a target protein on a cell (e.g., a CD19 antigen or a variant thereof for a CD19-specific CAR). In embodiments, a bispecific modulator can have a first portion that is an antibody or antibody fragment that can specifically bind to a target molecule on a cell (e.g., a molecule of interest). A bispecific modulator can have a second portion that binds to an internalization protein on the cell surface (e.g., a transferrin receptor). Binding of the bispecific modulator to the target molecule and the internalization protein results in internalization of the target molecule.

[0028] In some embodiments, the bispecific modulators do not require technical manipulation of the CAR-T receptor or CAR-T cells and can be applied to CAR-T therapies that are already approved or in clinical development.

[0029] In other embodiments, the bispecific modulators may be reversible, allowing for fine tuning of CAR-T cell activity, such as managing toxicity and / or rejuvenating cells for continued therapy.

[0030] In some embodiments, the bispecific modulators can be tailored to target CAR-T cells to different tumor antigens, for example, by swapping the components used in the design (i.e., the trap and / or modulator can be modularized).

[0031] Also disclosed are approaches to enhancing CAR-T efficacy. Temporary "resting" of CAR-T cells can reverse CAR-T cell exhaustion. In some embodiments, the disclosed reversible CAR modulators can enhance CAR-T cell efficacy by alternating CAR-T cells between an "active" and a "resting" state.

[0032] Approaches targeting cancer cells, including those harboring drug-resistant mutations, have also been disclosed. Cancers can rapidly evolve to evade treatment, often resulting in drug-resistant mutations that lead to treatment failure and disease recurrence. For example, the EGFR C797S mutation poses a challenge to the treatment of non-small cell lung cancer (NSCLC). The C797S mutation, which appears in approximately 10–26% of NSCLC patients after treatment with the third-generation EGFR tyrosine kinase inhibitor (TKI) osimertinib, affects the critical residue C797, which forms a covalent bond with the irreversible TKI. As a result, existing TKIs are ineffective against this disease. There is a need to develop drugs that target these drug-resistant oncogenes.

[0033] Disclosed herein is the development of EGFR TransTAC degradation-inducing compounds targeting EGFR-driven lung cancers, including those with the C797S mutation. We demonstrate (1) that EGFR TransTAC effectively degrades drug-resistant EGFR mutant proteins and suppresses cancer growth, and (2) that EGFR TransTAC specifically targets cancer cells while sparing healthy cells due to the overexpression of TfR in cancer cells. The reagents and methods disclosed herein can also be used on non-cancerous cells.

[0034] Detailed descriptions of one or more embodiments are provided herein. However, it should be understood that the present invention can be embodied in various forms. Accordingly, the specific details disclosed herein should not be construed as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art how to employ the present invention in any suitable manner.

[0035] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. In the claims and / or specification, the use of the words "a" or "an" when used in conjunction with the word "comprising" may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0036] As used herein, when phrases such as "for example," "such as," "including," and the like are used, they are understood to be followed by the phrase "and without limitation," unless expressly stated otherwise. Similarly, "an example," "exemplary," and the like are understood to be non-limiting.

[0037] The term "substantially" permits deviations from the description so long as the intended purpose is not adversely affected. Descriptive terms are understood to be modified by the word "substantially," even if the word "substantially" is not explicitly stated.

[0038] The terms "comprising," "including," "having," "involving" (and likewise "comprises," "includes," "has," "involving"), etc. are used interchangeably and have the same meaning. Specifically, each term is defined consistent with the general U.S. patent law definition of "comprising" and, therefore, is to be interpreted as open-ended, meaning "at least" and not excluding additional features, limitations, aspects, etc. Thus, for example, "a process comprising steps a, b, and c" means that the process includes at least steps a, b, and c. When the terms "a" or "an" are used, they are to be understood as "one or more," unless the context makes such an interpretation meaningless.

[0039] As used herein, the term "about" can refer to approximately, roughly, around, or in the region thereof. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. As used herein, the term "about" is used to vary a numerical value above and below the stated numerical value by a variance of 20% (higher or lower).

[0040] CAR-T cells, toxicity and toxicity control Chimeric antigen receptor (CAR) T cells have emerged as a promising treatment for patients with advanced B-cell cancers (Figure 1). However, poor control of transfused CAR T cells can limit the widespread application of this therapy due to potentially life-threatening toxicity. Toxicity has been an obstacle to the development of CAR T therapy for both hematological and solid tumors. Several deaths from CAR T therapy have been reported in recent years (Neelapu, Sattva S., et al. "Toxicity management after chimeric antigen receptor T cell therapy: one size does not fit 'all'." Nature reviews Clinical oncology 15.4(2018):218-218).

[0041] Cytokine-release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) are the two most common toxicities observed after CAR-T cell therapy.

[0042] CRS, characterized by hyperthermia, hypoxia, hypotension, and multisystem organ toxicity, occurs in 37% to 93% of patients with lymphoma and 77% to 93% of patients with leukemia. ICANS, characterized by confusion, delirium, seizures, or cerebral edema, occurs in 23% to 67% of patients with lymphoma and 40% to 62% of patients with leukemia. Severe CRS and ICANS require intensive care unit monitoring and treatment, and multiple deaths have been reported due to uncontrollable CRS or ICANS toxicity.

[0043] Three types of treatment are currently used to address these toxicities.

[0044] In some instances, patients are treated with systemic immunosuppressants (Figure 2), including corticosteroids, IL-6 receptor antibodies (e.g., tocilizumab), lymphocytotoxic anti-CD52 antibodies (e.g., alemtuzumab), and tyrosine kinase inhibitors (e.g., dasatinib) (LCK inhibitors do not inhibit already activated T cells). However, these treatments have limitations. For example, high-dose steroid treatment limits the duration of CAR T cell function and can induce hematologic hypoplasia and toxicity. Anti-IL-6 receptor antibodies have diverse biological activities and can nonspecifically inhibit the immune system (Bonifant, Challice L., et al. “Toxicity and management in CAR T-cell therapy.” Molecular Therapy-Oncolytics 3(2016):16011).

[0045] In some embodiments, patients are treated with suicide genes or exclusion markers (Figure 3), including iCasp9, anti-CD20 (e.g., rituximab), anti-EGFR (e.g., cetuximab), and the like. However, these treatments have limitations. For example, they can irreversibly and / or permanently eliminate CAR T cells from the body (Brandt, Laerke JB, et al. "Emerging approaches for regulation and control of CAR T cells: a mini review." Frontiers in Immunology 11 (2020): 326).

[0046] In some cases, CAR-T cells with switchable CAR receptors, such as split-CAR, SMaSh-CAR, and CAR PROTAC, can be used in patients (Figure 4). However, these therapies have limitations. For example, they can impair CAR-T activity, the switch can be leaky, and the switch can be immunogenic (Labanieh, Louai, et al. "Enhanced safety and efficacy of protease-regulated CAR-T cell receptors." Cell 185.10(2022):1745-1763).

[0047] However, it is known that CAR-T efficacy can be enhanced by using reversible CAR-T regulatory mechanisms (Figure 5). Constitutively expressing CAR-T cells may exhibit elevated levels of exhaustion-associated proteins. However, in some embodiments, transient "rest" can reverse the exhaustion phenotype. In some embodiments, CAR-T cells can be switched between "off" and "on" states by reversibly turning regulated CARs off and on (Weber, Evan W., et al. "Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling." Science 372.6537(2021):eaba1786; Labanieh, Louai, et al. "Enhanced safety and efficacy of protease-regulated CAR-T cell receptors." Cell 185.10(2022):1745-1763).

[0048] In some embodiments of the invention disclosed herein, bispecific modulators (e.g., TransTAC molecules) are used to regulate CAR-T cells. In some embodiments, these bispecific modulators can reversibly regulate CAR-T cells.

[0049] Bispecific regulator In some embodiments, the strategies disclosed herein for modulating molecules on cell surfaces (e.g., molecules of interest, such as proteins of interest) and / or the regulatory activity of such molecules can employ a bispecific modulator approach. In some embodiments, the bispecific modulator molecule can have at least two moieties. The first moiety can be a ligand to which the cell surface molecule can bind, or an antibody or antibody fragment capable of binding to the cell surface molecule (e.g., molecule of interest, such as a protein of interest). The second moiety can be a molecule capable of binding to an internalized receptor or membrane protein on a cell. In embodiments, the second molecule can be an antibody or antibody fragment that binds to an internalized receptor or membrane protein on a cell. In embodiments, the bispecific modulator can be a bispecific antibody.

[0050] In some embodiments, the bispecific modulator approach can modulate molecules other than those on the cell surface. In some embodiments, the bispecific modulator can bind to and internalize (and optionally degrade) proteins present in the extracellular / external environment. In some embodiments, these can be soluble proteins. In some embodiments, these proteins can include, by way of non-limiting example, autoantibodies, cytokines, enzymes, etc.

[0051] In embodiments, these two-part bispecific modulators bind, or can be bound, to a cell surface or other molecule (e.g., a molecule of interest such as a protein of interest) and can bind to an internalizing receptor or membrane protein. After such binding, the internalizing receptor or membrane protein can internalize the cell surface or other molecule into the cell (e.g., endocytosis). In embodiments, the internalized cell surface or other molecule can be degraded. In embodiments, this reduces the amount of the cell surface molecule on the cell surface. In embodiments, the internalized cell surface molecule is not functional. In some embodiments, the cell surface molecule targeted by the first part of the bispecific modulator is different from the molecule targeted by the second part.

[0052] In some embodiments, administering a bispecific modulator to a subject can be used for the targeted internalization of membrane or other proteins, hi some embodiments, administering a bispecific modulator to a subject can be used for the targeted degradation of membrane or other proteins.

[0053] In some embodiments, the addition of a bispecific modulator to a cell or administration to a patient can cause targeted internalization and / or degradation of a cell surface or extracellular protein. In some embodiments, this internalization / degradation is reversible. For example, when a cell is no longer exposed to the bispecific modulator, the membrane protein that the bispecific modulator specifically acts on is no longer internalized / degraded. Generally, the membrane protein continues to be synthesized and transported to the cell membrane. Thus, when the bispecific modulator is removed or no longer administered to the subject, the stimulus for internalizing / degrading the protein is removed. In some embodiments, a cell membrane protein that can be internalized but not degraded by a bispecific modulator can be both internalized and degraded using a bispecific modulator that also includes a protease-sensitive linker. As discussed elsewhere, the placement of a protease-sensitive linker within the bispecific modulator allows the targeted cellular protein of interest to be released intracellularly from the bispecific modulator.

[0054] In some embodiments, internalization and degradation of a cell surface or other molecule (e.g., a molecule of interest such as a protein of interest) can kill the cell (e.g., in embodiments where the cell surface molecule is required for cell viability or cell division; in some embodiments, EGFR). In some embodiments, internalization and degradation of a cell surface or other molecule does not kill the cell (e.g., in embodiments where the cell surface or other molecule is not required for cell viability or cell division; in some embodiments, CAR).

[0055] In some embodiments, internalization of a bispecific modulator or portion thereof may involve receptor-mediated endocytosis, also known as clathrin-mediated endocytosis. In some embodiments, internalization of a bispecific modulator may involve clathrin-independent endocytosis. In some embodiments, internalization of a bispecific modulator may involve phagocytosis.

[0056] In embodiments, the cell surface molecule or molecule targeted by the first moiety (e.g., a molecule of interest such as a protein of interest) can be a CAR molecule. In some embodiments, the CAR molecule can be present on a CAR-T cell. In some embodiments, a strategy for modulating CAR-T activity includes internalizing the CAR receptor with a bispecific modulator. In some embodiments, the molecule of interest targeted by the first moiety can be a cellular regulator, such as a protein that is part of an immune checkpoint pathway (e.g., PD-L1) or other signaling protein (e.g., EGFR). In some embodiments, the molecule of interest can be a marker for a particular cell type (e.g., CD20 for B cells).

[0057] In some embodiments, the molecule of interest targeted by the first moiety can be a protein. In some embodiments, the molecule of interest can be a membrane protein. The membrane protein can be an integral membrane protein. The membrane protein can be a transmembrane protein having one or more transmembrane domains. In some embodiments, the molecule of interest can be an extracellular molecule, such as an autoantibody, a cytokine, an enzyme, etc.

[0058] In some embodiments, the molecule of interest can bind to a hormone, cytokine, growth factor, neurotransmitter, lipophilic signaling molecule (e.g., prostaglandin), or cell recognition molecule (e.g., integrin, selectin). The molecule of interest can be a receptor. The receptor can be a G-protein coupled receptor (GPCR), receptor tyrosine kinase (RTK), or transmembrane receptor (TMR).

[0059] In some embodiments, the molecule of interest (e.g., a molecule of interest such as a protein of interest) can be a ligand-gated ion channel-linked receptor or an enzyme-linked receptor. Non-limiting examples of ligand-gated ion channel-linked receptors include Na + , K. + , Ca2 + , Cl - It may be a channel. Non-limiting examples of enzyme-linked receptors may be receptor tyrosine kinases, tyrosine kinase-related receptors (e.g., enzymes that associate with cytokines), receptor-like tyrosine phosphatases (e.g., that remove phosphate groups from tyrosines of intracellular proteins), receptor serine / threonine kinases, receptor guanylyl cyclases, or histidine kinase-related receptors. In some embodiments, the molecule of interest may be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA).

[0060] In some embodiments, the molecule of interest can be a transporter, hi some embodiments, the molecule of interest can be an ion transporter.

[0061] In some embodiments, the molecule targeted by the first portion of the bispecific modulator (i.e., the molecule of interest) may be a different molecule than the molecule targeted by the second portion of the bispecific modulator (e.g., an internalized receptor or membrane protein).

[0062] In some embodiments, the bispecific modulator is a single molecule. In some embodiments, the bispecific modulator can be a single polypeptide. In some embodiments, the single polypeptide can contain both the first and second portions of the bispecific modulator.

[0063] In some embodiments, the first moiety can be an antigen or epitope to which the molecule of interest can bind. In some embodiments, the antigen or epitope can bind to a receptor on a cell. In some embodiments, the antigen or epitope can be a ligand for the receptor. In embodiments, the receptor can be a chimeric antigen receptor (CAR), a T-cell receptor (TCR), or a B-cell receptor (BCR).

[0064] In embodiments, the first portion may be an antibody or antibody fragment that binds to an antigen or epitope derived from a tumor cell and / or cancer cell. In embodiments, the antigen or epitope to which the CAR can bind that the antibody or antibody fragment can bind may be CD19, B cell maturation antigen (BCMA), human epidermal growth factor 2 (HER2), etc.

[0065] In some embodiments, the first moiety, an antibody, can be an scFv, Fab, single domain antibody, nanobody, monobody, DARPin, or affibody. Antibody fragments and other molecules that can be used are described in the "Antibodies" section of this application.

[0066] In embodiments, the second moiety binds to a receptor or membrane protein on a cell. In some embodiments, the receptor or membrane protein bound by the second moiety is an internalizing receptor or membrane protein. In some embodiments, the internalizing receptor or membrane protein can mediate endocytosis. In some embodiments, endocytosis can involve clathrin-coated pits. In some embodiments, endocytosis can be clathrin-independent. In some embodiments, the second moiety can bind to a receptor that mediates phagocytosis. In embodiments, the second moiety can also be an antibody, antibody fragment, or other molecule.

[0067] In some embodiments, the first and / or second moiety can be any type of moiety capable of binding to a cell surface molecule or an extracellular molecular target, hi some embodiments, the first and / or second moiety can be a polypeptide, a ligand, an aptamer, a nanoparticle, a small molecule, etc.

[0068] In embodiments, a non-limiting list of internalized receptors or membrane proteins that can be used in bispecific modulators includes G-protein coupled receptors (GPCRs), receptor tyrosine kinases (RTKs), and transmembrane receptors (TMRs) (Xu, Yanjie, et al. "Endocytosis and membrane receptor internalization: implications of F-BAR protein Carom." Frontiers in bioscience (Landmark edition) 22 (2017): 1439). In some embodiments, GPCRs can include adrenergic receptors, chemokine receptors, coagulation receptors, etc. In embodiments, RTKs can include colony-stimulating factor receptors, epidermal growth factor receptors, tyrosine kinase receptors, fibroblast growth factor receptors, insulin-like growth factor receptors, platelet-derived growth factor receptors, transforming growth factor receptors, etc. In some embodiments, the TMR may include a folate receptor, an interleukin receptor (eg, an IL-2 receptor), a low density lipoprotein receptor, a transferrin receptor, or the like.

[0069] In embodiments, a non-limiting list of internalized receptors or membrane proteins that can be used in bispecific modulators includes G-protein coupled receptors (GPCRs), receptor tyrosine kinases (RTKs), and transmembrane receptors (TMRs) (Xu, Yanjie, et al. "Endocytosis and membrane receptor internalization: implications of F-BAR protein Carom." Frontiers in bioscience (Landmark edition) 22 (2017): 1439). In some embodiments, GPCRs can include adrenergic receptors, chemokine receptors, coagulation receptors, etc. In embodiments, RTKs can include colony-stimulating factor receptors, epidermal growth factor receptors, tyrosine kinase receptors, fibroblast growth factor receptors, insulin-like growth factor receptors, platelet-derived growth factor receptors, transforming growth factor receptors, etc. In some embodiments, the TMR may include a folate receptor, an interleukin receptor (eg, an IL-2 receptor), a low density lipoprotein receptor, a transferrin receptor, or the like.

[0070] In some embodiments, the internalized receptor or membrane protein can be a transferrin receptor. In embodiments, the ligand (e.g., second moiety) (first moiety) to which the transferrin receptor can bind can be transferrin or a fragment of transferrin. In some embodiments, the first moiety can be an antibody or antibody fragment capable of binding to the transferrin receptor.

[0071] In some embodiments, the internalized receptor or membrane protein can be a transferrin receptor (TfR). The transferrin receptor can be transferrin receptor 1 or transferrin receptor 2.

[0072] In some embodiments, the transferrin receptor can have a high endocytosis rate of about 500 molecules per second per cell and is suitable for inducing endocytosis of proteins. In some embodiments, the expression of the transferrin receptor is low in healthy tissues, but can be more highly expressed in various tumors, such as cancers of the brain, liver, breast, lung, colon, and blood, and in some activated immune cells.

[0073] In embodiments, the ligand (e.g., second moiety) that can be bound by an internalizing receptor or membrane protein can be at least a portion of a naturally occurring ligand. For example, the ligand can be at least a portion of transferrin, cholesterol, low density lipoprotein, and epidermal growth factor that can be bound by their cognate receptors.

[0074] In some embodiments, the ligand bound by the transferrin receptor can be transferrin or a fragment of transferrin. In some embodiments, the ligand that the transferrin receptor can bind can be about 80 kDa in size and can have glycosylation modifications.

[0075] In some embodiments, the second moiety can be an antibody, antibody fragment, or other molecule capable of binding to an internalizing receptor or membrane protein, hi some embodiments, the second moiety can be an scFv, Fab, single domain antibody, nanobody, monobody, DARPin, or affibody.

[0076] In some embodiments, the antibody or antibody fragment capable of binding to the transferrin receptor may be an anti-TfR1 antagonistic scFv antibody identified by phage display. This antibody may be referred to as "H7" (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).

[0077] In one embodiment, the amino acid sequence of the H7 molecule may comprise the following molecule, or may comprise a molecule that is at least 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identical to the following amino acid sequence:

[0078] H7 scFV-LC (SEQ ID NO: 1): TIFF2025532646000002.tif9159

[0079] H7 scFV-HC (SEQ ID NO: 2): TIFF2025532646000003.tif15170

[0080] H7-scFv (SEQ ID NO: 3) TIFF2025532646000004.tif25170

[0081] M16 (SEQ ID NO: 4): TIFF2025532646000005.tif25170

[0082] In some embodiments, strategies for modulating CAR-T activity include using a molecule (e.g., a bispecific modulator) that can bind to the CAR on the surface of the CAR-T cell (first moiety) and to an internalized receptor or membrane protein (second moiety: e.g., transferrin) on the same cell or an adjacent cell. The bispecific modulator can colocalize the CAR receptor to the internalized cell surface receptor or membrane protein. For example, an internalized CAR cannot be activated or does not continue to function in an activated state. In some embodiments, the bispecific modulator can downregulate the cell surface levels of the CAR and inhibit CAR-T cell function (Figure 6).

[0083] Here, we identify a novel mechanism regulating plasma membrane proteins. Endocytosis is a common mechanism controlling the recycling and degradation of membrane proteins. Among the various transmembrane proteins regulated by endocytosis, the transferrin receptor (TfR) is a well-characterized recycling receptor with a rapid internalization rate (500 molecules / cell / sec). TfR internalizes iron by binding to the iron-complexed plasma protein transferrin (Tf). It is highly expressed in various cancers and affects cancer cell proliferation, migration, invasion, apoptosis, and metastasis.

[0084] We demonstrated that extracellular proteins, particularly tumor-associated proteins of interest (POIs), can be selectively degraded by tethering the POI to TfR using an antibody-Tf fusion protein (Figure 7). We termed this technology Transferrin Receptor-Mediated Targeting Chimera (TransTAC). Upon TfR / TransTAC-mediated endocytosis, the receptor dissociates from the complex to a different local environment, the endosome (Figure 7, red square), where it is subjected to lysosomal degradation.

[0085] This method represents a new and versatile platform for protein degradation using fully recombinant biological molecules. A universal approach to membrane / extracellular protein degradation opens up endless possibilities for manipulating cellular behavior, and therefore represents an important research tool, expanding the PROTAC field's efforts to target challenging extracellular targets. The fully recombinant nature of TransTAC allows for straightforward generalization to a wide range of targets and optimization of binding properties.

[0086] TfR-based degradation improves tumor targeting specificity. TfR is used because: (1) TfR is a recycling receptor, so cellular levels of TfR can be maintained at a constant level, an important feature for a "carrier" protein; (2) Tf has been studied for iron and small molecule drug delivery, providing the necessary therapeutic potential and stability; and (3) targeting TfR further enhances tumor specificity. Taken together, TransTAC's modularity, genetic tractability, and tumor specificity make it a suitable approach for academic and translational applications.

[0087] In some embodiments, the bispecific modulators disclosed herein comprise an antigen to which the CAR can bind and a ligand for an internalizing receptor or membrane protein (e.g., the transferrin receptor). In some embodiments, the bispecific modulators disclosed herein comprise an antibody that can bind to the CAR or another molecule on the cell surface, such as EGFR, PD-L1, CD20, and a ligand for the internalizing receptor or membrane protein (e.g., a molecule of interest, such as a protein of interest).

[0088] In some embodiments, a bispecific modulator can be a fusion protein of the formula R1-R2-R3. In some embodiments, a bispecific modulator can be a fusion protein of the formula R3-R2-R1. For example, R1 or R3 can be located at the C-terminus or N-terminus of the fusion proteins disclosed herein. In some embodiments, a bispecific modulator can be a dimer (homodimer) of R1-R2-R3 or R3-R2-R1.

[0089] In some embodiments, R1 can be a protein of interest binder (POIB) or a means of a POIB. In some embodiments, the POIB or means can be an antibody. In some embodiments, the POIB means can be a portion of a molecule to which a protein of interest would normally bind (e.g., a peptide to which a CAR binds). The POIB or means can bind to a molecule of interest, such as a protein of interest, on the surface of a cell. In some embodiments, the POIB or means can bind to the extracellular domain of a transmembrane protein. In some embodiments, the POIB means can bind to the extracellular domain of a chimeric antigen receptor (CAR), a receptor tyrosine kinase, a checkpoint inhibitor-binding molecule, a lineage-specific marker, or the like. In some embodiments, the POIB or means can bind to the extracellular domain of epidermal growth factor receptor (EGFR), programmed death-ligand (PD-L1), or CD20.

[0090] In some embodiments, the POIB device is capable of binding to the extracellular domain of a B cell receptor (BCR), a human leukocyte antigen (HLA), a fibroblast growth factor receptor (FGFR), a Notch protein, or claudin 18.2.

[0091] In some embodiments, R3 can be a transferrin receptor binding means (TRB). TRB binds to a transferrin receptor on the surface of a cell. In some embodiments, TRB can be an antibody that binds to a transferrin receptor (e.g., H7 or M16). In some embodiments, TRB can be a polypeptide. In some embodiments, TRB can be a ligand or a portion of a ligand (e.g., transferrin) to which a transferrin receptor can bind. In some embodiments, a portion of transferrin that can be used as a TRB is the following (SEQ ID NO: 5): TIFF2025532646000006.tif84170

[0092] In some embodiments, R2 can be a linker of the formula R4-R5 or R5-R4. In some embodiments, R4 can be an Fc region from an antibody. In some embodiments, R4 can be an Fc region from IgG, IgM, IgA, IgE, or IgD. In some embodiments, the Fc region is (SEQ ID NO: 6). TIFF2025532646000007.tif27170

[0093] In some embodiments, the Fc region can dimerize to form homodimeric or heterodimeric structures. In some embodiments, the Fc region can have, or be modified to have, cysteine ​​amino acids capable of forming disulfide bonds. In some embodiments, dimers of R1-R2-R3 fusion proteins can form through disulfide bonds (one or more, e.g., two disulfide bonds) between cysteine ​​residues in the R2 regions of separate fusion protein molecules. In some embodiments, disulfide bonds are formed between R4 in separate fusion molecules (e.g., a disulfide-bearing Fc can be a type of dimerization domain).

[0094] In certain embodiments, the Fc region can be a variant containing amino acid substitutions that alter antigen-independent effector functions, such as the circulatory half-life of the molecule to which it is conjugated. Molecules conjugated to these Fc regions can exhibit either increased or decreased binding to FcRn, potentially resulting in increased or decreased serum half-life, compared to Fc regions lacking these substitutions. Fc variants with improved affinity for FcRn are predicted to have longer serum half-lives, making such molecules useful for methods in which a longer half-life of the conjugated molecule is desired. In contrast, Fc variants with reduced FcRn binding affinity are predicted to have shorter half-lives, making such molecules useful, for example, when a shorter circulation time may be advantageous. Fc variants with reduced FcRn binding affinity also exhibit reduced placental crossing. Furthermore, other applications in which reduced FcRn binding affinity is desirable include applications in which localization to the brain, kidney, and / or liver is desired. In one embodiment, the Fc variant binding molecule can reduce transport from the vasculature to epithelial cells in the glomeruli of the kidney.

[0095] In another embodiment, the Fc variant binding molecule may exhibit reduced transport from the brain across the blood-brain barrier (BBB) ​​into the vascular space. In one embodiment, the Fc region with altered FcRn binding comprises an Fc domain with one or more amino acid substitutions in the "FcRn-binding loop" of the Fc region. The FcRn-binding loop consists of amino acid residues 280-299 (according to EU numbering). Exemplary amino acid substitutions that alter FcRn-binding activity are disclosed in PCT Publication WO 05 / 047327, which is incorporated herein by reference. In an exemplary embodiment, the bispecific modulator disclosed herein comprises an Fc domain with one or more of the following substitutions: V284E, H285E, N286D, K290E, and S304D (EU numbering).

[0096] In some embodiments, the molecules disclosed herein may be conjugated to Fc variants containing amino acid substitutions that alter glycosylation. For example, the Fc variants may have reduced glycosylation (e.g., N-linked or O-linked glycosylation). In some embodiments, the Fc variants contain reduced glycosylation of the N-linked glycan normally found at amino acid position 297 (EU numbering). In other embodiments, the molecules may have amino acid substitutions near or within a glycosylation motif, e.g., an N-linked glycosylation motif containing the amino acid sequence NXT or NXS. In certain embodiments, the Fc variants may have amino acid substitutions at amino acid positions 228 or 299 (EU numbering). One example of an amino acid substitution that results in reduced or altered glycosylation is described in PCT Publication WO 05 / 018572, incorporated herein by reference in its entirety.

[0097] In some embodiments, the molecules disclosed herein may be modified to remove glycosylation and may be referred to as "agly" molecules. An exemplary aglycosylation is the aglycosylation of the Fc region of an IgG4 antibody, which lacks Fc effector functions and eliminates the potential for Fc-mediated toxicity to normal tissues and cells. In yet other embodiments, the molecules disclosed herein may have an altered carbohydrate chain. For example, the number of fucose residues on the N-glycan at Asn297 in the Fc region may be reduced, i.e., the molecules may be afucosylated. In some embodiments, the number of sialic acid residues on the N-glycan at Asn297 in the Fc region may be altered.

[0098] In some embodiments, the CH2 or CH3 region of an Fc antibody domain can be truncated or modified to adjust the half-life of the molecule. In some embodiments, the Fc truncation comprises CH3 or CH2 (e.g., Gehlsen, Kurt R., et al. "Pharmacokinetics of engineered human monomeric and dimeric CH2 domains." MAbs. Vol. 4. No. 4. Taylor & Francis, 2012; Ying, Tianlei, et al. "Engineered soluble monomeric IgG1 CH3 domain: generation, mechanisms of function, and implications for design of biological therapeutics." Journal of Biological Chemistry 288.35(2013):25154-25164).

[0099] In some embodiments, R4 can be a dimerization domain. A dimerization domain can be any region that can associate with another dimerization domain via covalent or non-covalent bonds to form a dimer (e.g., a homodimeric or heterodimeric bispecific modulator). In some embodiments, R4 is not an Fc region derived from an antibody.

[0100] Many protein dimerization domains are known in the art (see, for example, Dang, Dung Thanh. "Molecular Approaches to Protein Dimerization: Opportunities for Supramolecular Chemistry," Frontiers in Chemistry 10 (2022): 829312). Examples of dimerization domains include zipper motifs such as leucine zippers.

[0101] In some embodiments, dimerization may occur between regions of the bispecific modulator that are not the R4 region.

[0102] In some embodiments, R5 can be a protease-sensitive linker. In some embodiments, the protease-sensitive linker can be an amino acid sequence that can be cleaved by a protease. In some embodiments, the protease can be a protease in an endosome or lysosome. In some embodiments, the protease can be a cathepsin (e.g., cathepsin B) and the protease-sensitive linker can be a cathepsin-cleavable peptide. Some examples of protease-sensitive linkers are shown in Figure 35. In some embodiments, the protease-sensitive linker can be GGFLGGVRGVDG (SEQ ID NO: 7) or GSGSGGEVRGVDG (SEQ ID NO: 8).

[0103] In some embodiments, a bond (e.g., a linker) can be placed between various sections of the R1-R2-R3 fusion protein. In some embodiments, the bond can be located between R2 and R3. In some embodiments, the bond can be located between R1 and R2. In some embodiments, the bond can be a glycine-rich linker ("GS linker"). In some embodiments, the "GS" linker can be a combination of glycine and serine amino acids. In some embodiments, the GS linker can be GSSGGSGGSGGS (SEQ ID NO: 9). Other sequences are possible. In some embodiments, the GS linker can be SGGGG (SEQ ID NO: 10), SGGGSGGG (SEQ ID NO: 11), GSSGGSGGSGGS (SEQ ID NO: 12), GSGS (SEQ ID NO: 13), GSGGS (SEQ ID NO: 14), GSSGSS (SEQ ID NO: 15), GSSSSSS (SEQ ID NO: 16), etc. In some embodiments, the GS linker can have at least four amino acids that are glycine and / or serine. In some embodiments, other amino acids can also be part of the GS linker, so long as glycine and serine account for the majority.

[0104] In some embodiments, the bispecific modulators disclosed herein can comprise the following nucleotide and amino acid sequences, and molecules at least 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identical to the following nucleotide and amino acid sequences: Specifically, the amino acid sequences of the bispecific modulators can be labeled as follows:

[0105] The underlined Times New Roman font is the signal peptide.

[0106] In bold Times New Roman font are the anti-proteins of interest, Fab-heavy chains, scFvs, or affibodies.

[0107] The italicized Times New Roman font is the linker encoded by the restriction enzyme site construct.

[0108] The underlined, bold Times New Roman font is the GS linker.

[0109] Underlined, italicized, bold Times New Roman font is a cleavable linker;

[0110] The Courier New font is H7-scFv.

[0111] The underlined Courier New font is the Fc domain.

[0112] The bold Courier New font is from the TEV site.

[0113] The italicized Courier New font is a fragment derived from transferrin.

[0114] The underlined, bold Courier New font is the His-tag.

[0115] The Courier New font, bold italic, is light chain.

[0116] The underlined, italic, bold Courier New font is the Avi-tag.

[0117] pDP14-CD19 ETD_Hole Fc (SEQ ID NO: 17) TIFF2025532646000008.tif120170

[0118] (SEQ ID NO: 18) TIFF2025532646000009.tif50170

[0119] pDP16-EGFR-Affibody-FC-Tf (SEQ ID NO: 19) TIFF2025532646000010.tif197170

[0120] (SEQ ID NO: 20) TIFF2025532646000011.tif77170

[0121] pDP18-EGFR-Affibody (SEQ ID NO: 21) TIFF2025532646000012.tif149170

[0122] (SEQ ID NO: 22) TIFF2025532646000013.tif68170

[0123] pDP20-EGFR-Affibody-FC (SEQ ID NO: 23) TIFF2025532646000014.tif83170

[0124] (SEQ ID NO: 24) TIFF2025532646000015.tif29170

[0125] pDP22-EGFR-Affibody (SEQ ID NO: 25) TIFF2025532646000016.tif39170

[0126] (SEQ ID NO: 26) TIFF2025532646000017.tif16170

[0127] pDP24-CD19-FC-Tf (SEQ ID NO: 27) TIFF2025532646000018.tif219170

[0128] (SEQ ID NO: 28) TIFF2025532646000019.tif96170

[0129] pDP25-pFUSE-Tf-knob-Fc (SEQ ID NO: 29) TIFF2025532646000020.tif177170

[0130] (SEQ ID NO: 30) TIFF2025532646000021.tif73170

[0131] pDP32-CD19-FC-CD19 (SEQ ID NO: 31) TIFF2025532646000022.tif162170

[0132] (SEQ ID NO: 32) TIFF2025532646000023.tif73170

[0133] pDP44-CD19 NT.1-FC-Tf (SEQ ID NO: 33) TIFF2025532646000024.tif219170

[0134] (SEQ ID NO: 34) TIFF2025532646000025.tif96170

[0135] pDP49-CD19 NT.1-FC-GFLG-Tf (SEQ ID NO: 35) TIFF2025532646000026.tif224170

[0136] (SEQ ID NO: 36) TIFF2025532646000027.tif101170

[0137] pDP50-His8-CD19 NT.1-GFLG-FC-Tf (SEQ ID NO: 37) TIFF2025532646000028.tif226170

[0138] (SEQ ID NO: 38) TIFF2025532646000029.tif100170

[0139] pDP85-cd20-scfv-GFLG-FC-Tf (SEQ ID NO: 39) TIFF2025532646000030.tif243170

[0140] (SEQ ID NO: 40) TIFF2025532646000031.tif96170

[0141] pDP95-Herceptin_HC_Fc-N297G,S427C (SEQ ID NO: 41) TIFF2025532646000032.tif108170

[0142] (SEQ ID NO: 42) TIFF2025532646000033.tif40170

[0143] pDP69-His8-CD19 NT.1-2XGFLG-FC-Tf (SEQ ID NO: 43) TIFF2025532646000034.tif224170

[0144] (SEQ ID NO: 44) TIFF2025532646000035.tif101170

[0145] pDP70-His8-CD19 NT.1-3XGFLG-FC-Tf (SEQ ID NO: 45) TIFF2025532646000036.tif225170

[0146] (SEQ ID NO: 46) TIFF2025532646000037.tif100170

[0147] pDP71-His8-CD19 NT.1-GFLG-FK-FC-Tf (SEQ ID NO: 47) TIFF2025532646000038.tif224170

[0148] (SEQ ID NO: 48) TIFF2025532646000039.tif101170

[0149] pDP72-His8-CD19 NT.1-GFLG-VA-FC-Tf (SEQ ID NO: 49) TIFF2025532646000040.tif225170

[0150] (SEQ ID NO: 50) TIFF2025532646000041.tif101170

[0151] pDP73-His8-CD19 NT.1-GFLG-VK-FC-Tf (SEQ ID NO: 51) TIFF2025532646000042.tif225170

[0152] (SEQ ID NO: 52) TIFF2025532646000043.tif102170

[0153] pDP74-His8-CD19 NT.1-GFLG-VR-FC-Tf (SEQ ID NO: 53) TIFF2025532646000044.tif225170

[0154] (SEQ ID NO: 54) TIFF2025532646000045.tif101170

[0155] pDP75-His8-CD19 NT.1-GFLG-GGFG-FC-Tf (SEQ ID NO: 55) TIFF2025532646000046.tif227170

[0156] (SEQ ID NO: 56) TIFF2025532646000047.tif100170

[0157] pDP76-His8-CD19 NT.1-FK-FC-Tf (SEQ ID NO: 57) TIFF2025532646000048.tif225170

[0158] (SEQ ID NO: 58) TIFF2025532646000049.tif99170

[0159] pDP77-His8-CD19 NT.1-VA-FC-Tf (SEQ ID NO: 59) TIFF2025532646000050.tif224170

[0160] (SEQ ID NO: 60) TIFF2025532646000051.tif100170

[0161] pDP78-His8-CD19 NT.1-VK-FC-Tf (SEQ ID NO: 61) TIFF2025532646000052.tif225170

[0162] (SEQ ID NO: 62) TIFF2025532646000053.tif101170

[0163] pDP79-His8-CD19 NT.1-VR-FC-Tf (SEQ ID NO: 63) TIFF2025532646000054.tif224170

[0164] (SEQ ID NO: 64) TIFF2025532646000055.tif100170

[0165] pDP80-His8-CD19 NT.1-GGFG-FC-Tf (SEQ ID NO: 65) TIFF2025532646000056.tif225170

[0166] (SEQ ID NO: 66) TIFF2025532646000057.tif104170

[0167] pDP86-PDL1-scfv-GFLG-fc-Tf (SEQ ID NO: 67) TIFF2025532646000058.tif243170

[0168] (SEQ ID NO: 68) TIFF2025532646000059.tif95170

[0169] pDP96-CD20_HC_Fc-N297G,S427C-Tf (SEQ ID NO: 69) TIFF2025532646000060.tif238170

[0170] (SEQ ID NO: 70) TIFF2025532646000061.tif96170

[0171] pDP97-cd20-scfv-GFLG-FC-N297G, S427C-Tf (SEQ ID NO: 71) TIFF2025532646000062.tif244170

[0172] (SEQ ID NO: 72) TIFF2025532646000063.tif95170

[0173] pDP98-CD20_HC_Fc-N297G,S427C (SEQ ID NO: 73) TIFF2025532646000064.tif129170

[0174] (SEQ ID NO: 74) TIFF2025532646000065.tif46170

[0175] cd20-scFV-FC-Tf (SEQ ID NO: 75) TIFF2025532646000066.tif238170

[0176] (SEQ ID NO: 76) TIFF2025532646000067.tif95170

[0177] PD-L1-scFV-FC-Tf (SEQ ID NO: 77) TIFF2025532646000068.tif240170

[0178] (SEQ ID NO: 78) TIFF2025532646000069.tif98170

[0179] pDP124-cd20-scfv-FC-TfR-H7 (SEQ ID NO: 79) TIFF2025532646000070.tif64170

[0180] pDP125-cd20-scfv-FC-TfR-M16 (SEQ ID NO: 80) TIFF2025532646000071.tif63170

[0181] pDP126-cd20-scfv-GFLG-FC-TfR-H7 (SEQ ID NO: 81) TIFF2025532646000072.tif64170

[0182] pDP127-cd20-scfv-GFLG-FC-TfR-M16 (SEQ ID NO: 82) TIFF2025532646000073.tif62170

[0183] pDP155-His8-CD19 NT.1-2XGFLG-FC-TfR-H7 (SEQ ID NO: 83) TIFF2025532646000074.tif68170

[0184] pDP156-His8-CD19 NT.1-3XGFLG-FC-TfR-H7 (SEQ ID NO: 84) TIFF2025532646000075.tif70170

[0185] pDP157-His8-CD19 NT.1-GFLG-FK-FC-TfR-H7 (SEQ ID NO: 85) TIFF2025532646000076.tif67170

[0186] pDP158-His8-CD19 NT.1-GFLG-VA-FC-TfR-H7 (SEQ ID NO: 86) TIFF2025532646000077.tif68170

[0187] pDP159-His8-CD19 NT.1-GFLG-VK-FC-TfR-H7 (SEQ ID NO: 87) TIFF2025532646000078.tif68170

[0188] pDP160-His8-CD19 NT.1-GFLG-VR-FC-TfR-H7 (SEQ ID NO: 88) TIFF2025532646000079.tif68170

[0189] pDP161-His8-CD19 NT.1-GFLG-GGFG-FC-TfR-H7 (SEQ ID NO: 89) TIFF2025532646000080.tif68170

[0190] pDP162-His8-CD19 NT.1-FK-FC-TfR-H7 (SEQ ID NO: 90) TIFF2025532646000081.tif68170

[0191] pDP163-His8-CD19 NT.1-VA-FC-TfR-H7 (SEQ ID NO: 91) TIFF2025532646000082.tif67170

[0192] pDP164-His8-CD19 NT.1-VK-FC-TfR-H7 (SEQ ID NO: 92) TIFF2025532646000083.tif69170

[0193] pDP165-His8-CD19 NT.1-VR-FC-TfR-H7 (SEQ ID NO: 93) TIFF2025532646000084.tif69170

[0194] pDP166-His8-CD19 NT.1-GGFG-FC-TfR-H7 (SEQ ID NO: 94) TIFF2025532646000085.tif69170

[0195] pDP167-CD20_HC_Fc-N297G,S427C-TfR-H7 (SEQ ID NO: 95) TIFF2025532646000086.tif64170

[0196] pDP168-CD20-scfv-GFLG-FC-N297G, S427C-TfR-H7 (SEQ ID NO: 96) TIFF2025532646000087.tif63170

[0197] pDP169-cd20-scfv-GFLG-FK-FC-(N297G, S427C)-TfR-H7 (SEQ ID NO: 97) TIFF2025532646000088.tif63170

[0198] pDP170-cd20-scfv-GFLG-VR-FC-(N297G, S427C)-TfR-H7 (SEQ ID NO: 98) TIFF2025532646000089.tif63170

[0199] pDP171-His8-EGFR affi-FC-TfR-H7 (SEQ ID NO: 99) TIFF2025532646000090.tif49170

[0200] pDP172-His8-EGFR affi-GFLG-FC-TfR-H7 (SEQ ID NO: 100) TIFF2025532646000091.tif49170

[0201] pDP173-His8-EGFR affi-GFLG-FK-FC-TfR-H7 (SEQ ID NO: 101) TIFF2025532646000092.tif49170

[0202] pDP174-His8-EGFR affi-GFLG-VR-FC-TfR-H7 (SEQ ID NO: 102) TIFF2025532646000093.tif49170

[0203] pDP210-CD20-HC-(EVR)-FC(GRLR)-N297G-TfR-H7 (SEQ ID NO: 103) TIFF2025532646000094.tif62170

[0204] pDP213-CD20-HC-(EVR)-FC(GRLR)-N297G (SEQ ID NO: 104) TIFF2025532646000095.tif44170

[0205] pDP219-FC-TfR-H7 (SEQ ID NO: 105) TIFF2025532646000096.tif44170

[0206] pDP223-pFUSE-H7 scFV-knob-Fc-His (SEQ ID NO: 106) TIFF2025532646000097.tif45170

[0207] pDP224-pFUSE-CD 19 NT.1-knob-Fc-H7 scFV-His (SEQ ID NO: 107) TIFF2025532646000098.tif68170

[0208] pDP225-2-Hole Fc_H7 scFV (SEQ ID NO: 108) TIFF2025532646000099.tif43170

[0209] pDP226-Hole Fc_-avitag (SEQ ID NO: 109) TIFF2025532646000100.tif25170

[0210] pDP227-Knob Fc_H7 scFV-His (SEQ ID NO: 110) TIFF2025532646000101.tif46170

[0211] PD-L1 TransTAC (pDP186) (SEQ ID NO: 111) TIFF2025532646000102.tif63170CD20 TransTAC:

[0212] Heavy chain (pDP210) (SEQ ID NO: 112) TIFF2025532646000103.tif63170

[0213] Light chain (pDP118) (SEQ ID NO: 113) TIFF2025532646000104.tif20170

[0214] CD20 TransTAC-M16 version (pDP127): Heavy chain (SEQ ID NO: 114) (light chain as immediately above) TIFF2025532646000105.tif63170

[0215] EGFR TransTAC (pDP211) (SEQ ID NO: 115) TIFF2025532646000106.tif48170

[0216] CD19 CAR TransTAC (pDP160) (SEQ ID NO: 116) TIFF2025532646000107.tif67170

[0217] CD19 CAR TransTAC previous version (pDP50) (SEQ ID NO: 117) TIFF2025532646000108.tif101170

[0218] In some embodiments, the bispecific modulator may be a heterodimer of the fusion protein R1-R2-R3 (R1 is POIB; R2 is R4-R5 or R5-R4, where R4 is an antibody Fc region and R5 is a protease-sensitive linker; R3 is TRB as described above) and the fusion protein R3-R4 or R4-R3 (R3 is TRB and R4 is an antibody Fc region). In some embodiments, there may optionally be a protease-sensitive linkage between R3 and R4, or between R3-R4 and R4-R3.

[0219] In some embodiments, bispecific modulators disclosed herein may 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 may be a moiety that connects R1 and R3 (e.g., a linking means connecting R1 and R3). In some embodiments, R6 may be an amino acid linker. In some embodiments, the amino acid linker may be protease-sensitive. In some embodiments, R6 may be a dimerization domain that may form a dimer with another copy of R6 via a covalent bond (e.g., a cysteine-containing Fc antibody region or other dimerization domain) or a non-covalent bond. In some embodiments, R6 may be a combination of a binding molecule (e.g., which may be protease-sensitive) and a dimerization domain, as described above.

[0220] In some embodiments, bispecific modulators disclosed herein may 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 may be the moiety linking R1 and R3 and contains a dimerization domain. The dimerization domain may form a dimer with another copy of R6 via a covalent bond (e.g., an Fc antibody region containing a cysteine) or a non-covalent bond. In some embodiments, the dimerization domain may be an Fc antibody region having one or more cysteines. In some embodiments, or optionally, the bond between R1 and R6 or between R6 and R3 may be a protease-sensitive linkage means.

[0221] In some embodiments, the first component of the bispecific modulator can be R1-R6 or R6-R1, where R1 can be a POIB and R6 can be a dimerization means as described above and optionally a protease-sensitive amino acid linker. The 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 described above and optionally a protease-sensitive amino acid linker. A bispecific modulator can be formed when a multimerization domain (e.g., a dimerization domain) of a first component forms a covalent or non-covalent bond with a multimerization domain of a second component.

[0222] 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 cell proliferation. In some embodiments, targeting these cell surface molecules can kill tumor cells. In some embodiments, the cell surface molecule targeted by the bispecific modulator can be epidermal growth factor receptor (EGFR). In some embodiments, these bispecific modulators provide greater IC against tumor / cancer cells than other therapies. 50 is 10 to 50 times better.

[0223] In some embodiments, the bispecific modulators disclosed herein are used to internalize and degrade multi-transmembrane proteins (i.e., transmembrane proteins that traverse the membrane multiple times and form multiple extracellular domains). In some embodiments, CD20 is the protein targeted using these bispecific modulators.

[0224] In some embodiments, the bispecific modulator can be one polypeptide chain. In some embodiments, the bispecific modulator can be two polypeptide chains. In some embodiments of a two-polypeptide configuration, the two binders are encoded on two polypeptide chains. In some embodiments, the two polypeptide chains can be linked or connected by a dimerization domain. In some embodiments, the two polypeptide chains can be linked or connected by a knob-in-hole Fc.

[0225] Receptor degradation targeted by TransTAC In some embodiments, the cell surface molecule targeted by the bispecific modulator (i.e., the molecule of interest, such as a protein of interest) can be internalized by the bispecific modulator. In some embodiments, the internalized molecule is not degraded or is minimally degraded intracellularly. In some embodiments, the bispecific modulators disclosed herein are modified to more efficiently degrade the targeted protein. In some embodiments, the bispecific modulators are modified to contain an amino acid sequence that is susceptible to a protease (see, e.g., Figure 35). The protease can be an endosomal or lysosomal protease. 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.

[0226] In some embodiments, the linker is susceptible to cleavage by a cathepsin, which can be cathepsin A, B, C, D, E, F, G, H, K, L1, L2, O, S, W, or Z.

[0227] In some embodiments, the molecule of interest can be released from the bispecific modulator other than the inclusion of a protease-sensitive linker (eg, pH-dependent binding of TRB).

[0228] Cleavage of the linker within the cell (e.g., endosome) liberates the molecule of interest from the internalized receptor or membrane protein, increasing the likelihood of degradation of the molecule of interest. In some embodiments, the protease-sensitive peptide linker may be positioned such that cleavage of the bispecific modulator by a protease releases or dissociates the target protein from the bispecific modulator, allowing for more complete degradation of the target protein.

[0229] In some embodiments, the molecule of interest can be released from the bispecific modulator other than the inclusion of a protease-sensitive linker (eg, pH-dependent binding of TRB).

[0230] In some embodiments, the bispecific modulator is R1-R2-R3 as described above, where R2 can be R4-R5 or R5-R4 (wherein R5 is a protease-sensitive linkage), and the protease-sensitive linkage can be located between the POIB (R1) and the Fc region from the antibody (R4), such as R1-R5-R4-R3. In some embodiments, the protease-sensitive linkage can be located between the Fc region from the antibody (R4) and the TRB (R3), such as R1-R4-R5-R3. In some embodiments, release of the target protein from the bispecific modulator can be achieved by incorporating a low pH-sensitive amino acid region into the bispecific modulator. In some embodiments, when the bispecific modulator is within an endosome, the low pH environment can release / dissociate the target protein from the bispecific modulator such that the target protein is more efficiently degraded.

[0231] In some embodiments, the linker may be sensitive to the low pH present in endosomes, hi some embodiments, the low pH may cause cleavage of the linker.

[0232] In some embodiments, the transferrin receptor binding means (TRB) can bind to transferrin in a pH-dependent manner. For example, TRB may have a lower affinity for the transferrin receptor at the low pH found in endosomes. This lower affinity may cause the TRB to release the transferrin receptor. This release may promote degradation of the target protein bound to the POIB. Such a TRB may be designated "M16," as shown in Figure 35.

[0233] In some embodiments, a protease-sensitive linker may be located between the first portion (which targets the molecule of interest) and the second portion (which binds to an internalizing receptor or membrane protein). In some embodiments, the linker may be located closer to the first portion than to the second portion.

[0234] In some embodiments, the protease-sensitive linking means can comprise Gly-Phe-Leu-Gly (GFLG; SEQ ID NO: 118). In some embodiments, the peptide linker can comprise a valine-arginine (VR) and / or a 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 (Figure 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).

[0235] In the experiments (Figures 49A-B), a yeast-display peptide library was used to identify peptides known to be insensitive to cathepsin cleavage. These peptides can be derived from combinations 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).

[0236] Other arrangements may also be used.

[0237] In some embodiments, any combination of these peptide linkers and / or valine-citrulline (VC) linkers and / or glutamic acid-valine-arginine (EVR) linkers may be used.

[0238] antibody Disclosed are specific recombinant monoclonal antibodies that can be part of the bispecific modulators disclosed herein. In embodiments, the antibodies can be used for the first and / or second portions of the bispecific modulators disclosed herein.

[0239] "Recombinant," with respect to a polypeptide (e.g., an antibody) or polynucleotide, refers to a form of a polypeptide or polynucleotide that does not occur in nature, including, but not limited to, those that can be produced by combining polynucleotides or polypeptides that do not normally occur together. As used herein, "polypeptide" encompasses the singular form "polypeptide" as well as the plural form "polypeptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also called peptide bonds). The term "polypeptide" refers to a chain of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or other terms 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 in place of or interchangeably with any of these terms. "Polypeptide" can also refer to the product of post-expression modifications of a polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. Polypeptides may be derived from natural biological sources or produced by recombinant technology, but are not necessarily translated from a designated nucleic acid sequence. They may be generated by any method, including chemical synthesis. With respect to amino acid sequences, those skilled in the art will readily recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter, add, delete, or substitute a single amino acid or a small percentage of amino acids in the encoded sequence are collectively referred to herein as "conservatively modified variants." 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 may exhibit increased cross-reactivity compared to the unmodified antibody.

[0240] For example, a "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain: a basic side chain (e.g., lysine, arginine, histidine), an acidic side chain (e.g., aspartic acid, glutamic acid), an uncharged polar side chain (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), a nonpolar side chain (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), a beta-branched side chain (e.g., threonine, valine, isoleucine), or an aromatic side chain (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential 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 the order and / or composition of the side chain family members.

[0241] Also featured in some embodiments are antibodies that share a specified percentage of amino acid or nucleotide sequence identity with the antibodies described herein. For example, "homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing a position in each sequence, which may be aligned for comparison. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. For example, an antibody can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more amino acid sequence identity when compared to a specific region or the entire length of any one of the antibodies described herein. For example, an antibody can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more nucleic acid sequence identity when compared to a specific region or the entire length of any one of the antibodies described herein. Sequence identity or similarity to the nucleic acids and proteins of the invention can be determined by sequence comparison and / or alignment using methods known in the art, for example, 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 used to determine the percent sequence identity or similarity of the nucleic acids and proteins of the invention.

[0242] An embodiment of the present invention provides an isolated antibody. As used herein, the term "isolated," with respect to a cell, a nucleic acid, such as DNA or RNA, refers to a molecule separated from other DNA or RNA, respectively, 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 technology, or chemical precursors or other chemicals when chemically synthesized. For example, an "isolated nucleic acid" can include a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in its natural state. "Isolated" can also refer to a cell or polypeptide that is separated from other cellular proteins or tissues. Isolated polypeptides include both purified and recombinant polypeptides.

[0243] As used herein, "antibody" or "antigen-binding polypeptide" can refer to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody, an antigen-binding fragment, or a single chain thereof. For example, "antibody" can include molecules containing proteins or peptides that constitute at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Non-limiting examples include the complementarity-determining regions (CDRs) of a heavy or light chain or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, or at least a portion of a binding protein. As used herein, the term "antibody" can refer to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts with" refers to an antibody that reacts with one or more antigenic determinants of a desired antigen and not with other polypeptides.

[0244] The term "antibody fragment" or "antigen-binding fragment" as used herein refers to F (ab’)2 , F (ab)2 , F ab ', Fab Antibody fragments are portions of antibodies, such as Fvs, scFvs, and the like. Regardless of structure, antibody fragments bind to the same antigen recognized by the intact antibody. The term "antibody fragment" also includes aptamers (such as spiegelmers), minibodies, and diabodies. The term "antibody fragment" may 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 antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab', and F(ab'), Fd, Fvs, single-chain Fvs (scFvs), single-chain antibodies, dAbs (domain antibodies), minibodies, disulfide-linked Fvs (sdFvs), fragments consisting of either the VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies.

[0245] "Single-chain variable fragment" or "scFv" refers to a fragment of an immunoglobulin heavy chain (V H ) and light chain (V L (scFv) refers to a fusion protein of the variable regions of a single-chain Fv ("scFv") polypeptide molecule, a covalently linked VH:VL heterodimer, can be expressed from a gene fusion containing 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 embodiments, the regions are linked by a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and the V H N-terminus and V LThe C-terminus of the antibody can be linked to the C-terminus of the antibody V region, or vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker. Various methods have been described for converting the naturally aggregated but chemically separated light and heavy polypeptide chains from antibody V regions into scFv molecules and identifying chemical structures that fold into a three-dimensional structure substantially similar to that of an antigen-binding site. See, for example, U.S. Pat. Nos. 5,091,513; 5,892,019; 5,132,405; and 4,946,778, which are incorporated herein by reference in their entireties.

[0246] Antibody molecules obtained from humans are classified into five immunoglobulin classes, including one or more of IgG, IgM, IgA, IgE, and IgD, which differ from one another in the nature of the heavy chains 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 several subclasses (e.g., γ1-γ4) within each. Particular classes also contain subclasses, such as IgG1, IgG2, IgG3, and IgG4. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, and IgG5, are well characterized and are known to confer functional specificity. With regard to IgG, a typical immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of approximately 53,000-70,000 daltons. The four chains are held together by disulfide bonds in a Y-shape, with the light chains continuing from the mouth of the "Y" through the variable regions and sandwiching the heavy chains. The 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 immunoglobulin molecule.

[0247] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can be associated with either kappa or lambda light chains. For example, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the light and heavy chains are covalently linked to each other, and the "tails" of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds. The amino acid sequence of the heavy chains runs from the N-terminus at the forked end of the Y-shape to the C-terminus at the end of each chain.

[0248] Both 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) chains determine antigen recognition and specificity. Conversely, the constant domains of the light (CL) and heavy (CH1, CH2, or CH3) chains confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, and complement fixation. The term "antigen-binding site" or "binding portion" can refer to the portion of an immunoglobulin molecule involved in antigen binding. The antigen-binding site is formed by amino acid residues in the N-terminal variable (V) regions of the heavy (H) and light (L) chains. Within the V regions of the heavy and light chains are three highly distinct regions called "hypervariable regions," which are sandwiched between highly conserved side chains called "framework regions" or "FR." Thus, the term "FR" can refer to the amino acid sequences naturally found between and adjacent to the 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 one another in three dimensional space to form an antigen-binding surface that 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."

[0249] The six CDRs present in each antigen-binding domain are short, noncontiguous amino acid sequences that are specifically arranged to form the antigen-binding domain when the antibody assumes a three-dimensional configuration in an aqueous environment. The remaining amino acids of the antigen-binding domain, the FR regions, show little inter-molecular variability. The framework regions largely adopt a beta-sheet structure, and the CDRs form loops that connect to, and in some cases form part of, the beta-sheet structure. The framework regions form a scaffold that orients the CDRs through interchain noncovalent interactions. The antigen-binding domain formed by the arranged CDRs provides a surface complementary to the epitope on the immunoreactive antigen, facilitating noncovalent binding of the antibody to its cognate epitope. The amino acids constituting the CDRs and framework regions, respectively, have already been defined and can be readily identified for heavy or light chain variable regions by those skilled in the art (see "Sequences of Proteins of Immunological Interest," Kabat, E., et al., USDapartment of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0250] Where there is more than one definition of a term used and / or accepted in the art, the definition of the term as used herein is intended to encompass all such meanings unless expressly stated to the contrary. As a specific example, the term "complementarity-determining region" ("CDR") is used to describe the non-contiguous antigen-binding sites found within the variable regions of heavy and light chain polypeptides. This region is described by Kabat et al., U.S. Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and Chothia et al., J. Mol. Biol. 196:901-917 (1987), both of which are incorporated herein by reference in their entireties. The Kabat and Chothia definitions of CDRs include overlapping amino acid residues and subsets when comparing amino acid residues. 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 that encompass the CDRs as defined by each of the above references are set forth in the table below for comparison. The exact residue numbers that 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 constitute a particular CDR from the amino acid sequence of the variable region of an antibody.

[0251] TIFF2025532646000109.tif53170

[0252] Kabat et al. defined a numbering system for variable domain sequences that is applicable to all antibodies. One of ordinary skill in the art can unambiguously assign this "Kabat numbering" system to any variable region sequence without reliance on experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system defined by Kabat et al., U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0253] In addition to the above table, the Kabat numbering 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 15th residue from 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 33rd amino acid residue from the end of CDR-H2, includes 3-25 amino acids, and ends with the sequence WGXG (where X is any amino acid). CDR-L1 begins at approximately residue 24 (i.e., following the cysteine ​​residue), includes approximately 10-17 residues, and ends at the next tryptophan residue. CDR-L2 begins at approximately the 16th residue from the end of CDR-L1 and includes approximately 7 residues. CDR-L3 begins at about the 33rd residue from the end of CDR-L2 (ie, following the cysteine ​​residue), contains about 7 to 11 residues, and ends with the sequence F or WGXG (X is any amino acid).

[0254] As used herein, the term "epitope" may include any protein determinant capable of specific binding to an immunoglobulin, scFv, or T-cell receptor. The variable region enables an antibody to selectively recognize and specifically bind to an epitope on an antigen. For example, the VL and VH domains of an antibody, or a subset of complementarity-determining regions (CDRs), combine to form the variable region, defining 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. Epitope determinants are composed of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and may have specific three-dimensional structural characteristics or specific charge characteristics. For example, antibodies can be raised against N- or C-terminal peptides of a polypeptide. More specifically, the antigen-binding site is defined by three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) on each of the VH and VL chains.

[0255] As used herein, the terms "immunological binding" and "immunological binding properties" can refer to the types of non-covalent interactions that occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity, of an immunological binding interaction is determined by the dissociation constant (K d ) and K d A smaller K represents a higher affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method is to measure the rates of formation and dissociation of the antigen-binding site / antigen complex; these rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Thus, the "on-rate constant" (K on ) and "off rate constant" (K off Both the K and K can be determined by calculating the concentration and the actual association and dissociation rates. (See Nature 361:186-87 (1993)). off / K onThe ratio of α to β allows all parameters unrelated to affinity to be cancelled out, resulting in the equilibrium binding constant K D (See generally Davies et al. (1990) Annual Rev Biochem 59:439-473). The antibodies of the present invention may have an equilibrium binding constant (K) as measured by a kinetic assay such as a radioligand binding assay or similar assay known to those skilled in the art, such as BIAcore or Octet (BLI). D ) is 1 μM or less, 10 μM or less, 10 nM or less, 10 pM or less, or 100 pM or less to about 1 pM. For example, in some embodiments, D Approximately 1E-12M~K D In some embodiments, K D Approximately 1E-11M~K D In some embodiments, K D Approximately 1E-10M~K D In some embodiments, K D Approximately 1E-9M~K D In some embodiments, K D Approximately 1E-8M~K D In some embodiments, K D Approximately 1E-7M~K D For example, in some embodiments, K D is about 1E-12M, but in other embodiments, K D is about 1E-11M. In some embodiments, K D is about 1E-10M, but in other embodiments, K D is about 1E-9M. In some embodiments, K D is about 1E-8M, but in other embodiments, K D is about 1E-7M. In some embodiments, K D is about 1E-6M, but in other embodiments, K D is about 1E-5M. In some embodiments, for example, K Dis about 3E-11M, but in other embodiments, K D is about 3E-12M. In some embodiments, K D is approximately 6E-11M. "Specifically binds" or "having specificity" can refer to an antibody that binds to an epitope via its antigen-binding domain and that requires some complementarity between the antigen-binding domain and the epitope for that binding. For example, an antibody is said to "specifically bind" to an epitope if it binds to that epitope via its antigen-binding domain more readily than it would bind to a random, unrelated epitope.

[0256] For example, antibodies can be monovalent or bivalent, and single-chain or double-chain. Functionally, antibodies have a binding affinity of 10 -5 M to 10 -12 For example, the binding affinity of an antibody is in the range of 10 -6 M to 10 -12 M, 10 -7 M to 10 -12 M, 10 -8 M to 10 -12 M, 10 -9 M to 10 -12 M, 10 -5 M to 10 -11 M, 10 -6 M to 10 -11 M, 10 -7 M to 10 -11 M, 10 -8 M to 10 -11 M, 10 -9 M to 10 -11 M, 10 -10 M to 10 -11 M, 10 -5 M to 10 -10 M, 10 - M to 10 -10 M to 10 -7 M to 10 -10 M to 10 -8 M to 10 -10 M to 10 -9 M to 10 -10 M to 10 -5 M to 10 -9 M to 10 -6M to 10 -9 M to 10 -7 M to 10 -9 M to 10 -8 M to 10 -9 M to 10 -5 M to 10 -8 M to 10 -6 M to 10 -8 M to 10 -7 M to 10 -8 M to 10 -5 M to 10 -7 M to 10 -6 M to 10 -7 Up to M or 10 -5 M to 10 -6 Up to M.

[0257] Those skilled in the art will recognize that whether a human monoclonal antibody has the same specificity as a human monoclonal antibody of the invention can be determined without undue experimentation by determining whether the former interferes with the specific binding of the latter. For example, if it is shown that the human monoclonal antibody under test competes with a human monoclonal antibody of the invention, resulting in reduced binding by the human monoclonal antibody of the invention, then the two monoclonal antibodies bind to the same or closely related epitopes.

[0258] Another method for determining whether a human monoclonal antibody has the specificity of a human monoclonal antibody of the present invention is to preincubate the human monoclonal antibody of the present invention with the epitope to which it normally reacts, then add the human monoclonal antibody to be tested and determine whether the ability of the human monoclonal antibody to be tested to bind to the epitope is inhibited. If the human monoclonal antibody to be tested is inhibited, then that antibody has the same or functionally equivalent epitope specificity as the monoclonal antibody of the present invention. Screening for human monoclonal antibodies of the present invention can also be performed by using an epitope to determine whether the test monoclonal antibody can neutralize a polypeptide containing the epitope.

[0259] Various procedures known in the art can be used to produce polyclonal or monoclonal antibodies to a protein of the invention, or to a derivative, fragment, analog, homolog, or ortholog thereof (see, e.g., Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).

[0260] 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, or epitope thereof, against which the immunoglobulin is sought, can be immobilized on a column, and immune-specific antibodies purified by immunoaffinity chromatography. Immunoglobulin purification 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).

[0261] As used herein, the term "monoclonal antibody" or "mAb" or "Mab" or "monoclonal antibody composition" 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 a monoclonal antibody are identical in all molecules of the population. mAbs have an antigen-binding site and are capable of immunoreacting with a specific epitope of an antigen.

[0262] Monoclonal antibodies can be prepared using hybridoma techniques, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal is immunized with an immunizing agent to induce lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0263] Nucleic acids, vectors and cells expressing bispecific modulators Also disclosed are nucleic acids encoding all or part of the bispecific modulators described herein. Also disclosed are various vectors (e.g., plasmids, viruses, etc.) that contain the nucleic acids. Also disclosed are various cells (e.g., prokaryotic, eukaryotic) that contain the nucleic acids or vectors and can express the fusion proteins.

[0264] method Disclosed herein are methods of administering the bispecific modulators 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 regulate these proteins. In some embodiments, the bispecific modulators can target CAR receptors on CAR-T cells. In some embodiments, the methods are used to treat toxicity (e.g., toxicity due to cytokine release) in a subject who has received a CAR-T cell infusion for the treatment of cancer. In some embodiments, the methods are used to improve the efficacy of CAR-T cells administered to a subject to treat cancer.

[0265] In some embodiments, membrane proteins on cancer cells can be targeted to degrade and / or modulate the protein (e.g., epidermal growth factor receptor or EGFR, programmed death ligand or PD-L1). In some examples, bispecific modulators can be used in this way to improve anti-tumor responses.

[0266] In some embodiments, the reagents and methods disclosed herein can be used with cells that are not cancer cells.

[0267] therapeutic preparations Aspects of the present invention relate to therapeutic preparations. As used herein, the term "therapeutic preparation" may refer to any compound or composition that can be used or administered for a therapeutic effect (e.g., a bispecific modulator). As used herein, the term "therapeutic effect" may refer to an effect sufficient to improve symptoms, e.g., treat, cure, prevent, or improve an associated pathology, or to increase the rate of treatment, cure, prevention, or improvement of such a pathology.

[0268]

[0013] The embodiments described herein may be administered to a subject in the form of a pharmaceutical composition or therapeutic formulation prepared for the intended route of administration. Such compositions and preparations may include, for example, an active ingredient and a pharmaceutically acceptable carrier. Such compositions and preparations may be in a form adapted for oral, subcutaneous, parenteral (e.g., intravenous, intraperitoneal), intramuscular, rectal, epidural, intratracheal, nasal, transdermal, vaginal, buccal, ocular, or pulmonary administration, e.g., a form adapted for administration by a peripheral route, suitable for oral administration, or suitable for parenteral administration. Other routes of administration are subcutaneous, intraperitoneal, and intravenous, and such compositions can be prepared by methods well known to those skilled in the art, for example, as described in "Remington's Pharmaceutical Sciences," 17th Ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, Pa., USA, 1985 and latest editions, and in Marcel Dekker's "Drugs and the Pharmaceutical Sciences" series of monographs. The compositions and formulations can be provided in conventional forms, such as solutions and suspensions for injection, capsules and tablets, enteric-coated formulations such as those disclosed in U.S. Pat. No. 5,350,741, and for oral administration.

[0269] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, and phosphates; and tonics such as sodium chloride or glucose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple-dose vials.

[0270] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Suitable carriers for intravenous administration include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, NJ), phosphate-buffered saline (PBS), and the like. In all cases, the composition will be sterile and fluid to the extent that easy syringability exists. In embodiments, it will be stable under the conditions of manufacture and storage and will 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, glycerol, propylene glycol, liquid polyethylene glycol, and suitable mixtures thereof, or a pharmaceutically acceptable polyol. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it may be useful to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[0271] Sterile injectable solutions can be prepared by incorporating the compound in the required amount in a suitable solvent with one or a combination of the ingredients listed herein, followed by filtration sterilization, if necessary. Dispersions are prepared by incorporating the active compound in a sterile solvent containing a basic dispersion medium and the required other ingredients from the ingredients listed herein. In the case of sterile powders for preparing sterile injectable solutions, useful methods include vacuum drying and freeze-drying, which can yield a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution.

[0272] Oral compositions may contain an inert diluent or an edible carrier. They may also be placed in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound may be incorporated with an excipient and used in the form of tablets, lozenges, or capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, in which case the compound in the fluid carrier is orally administered, gargled, and expectorated or swallowed. Oral formulations of drugs may be administered, for example, once a day, twice a day, three times a day, or four times a day, depending on the half-life of the drug.

[0273] Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition administered to a subject. Tablets, pills, capsules, troches, etc. can contain any of the following ingredients, or compounds of a similar nature: binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch, lactose, or disintegrating agents such as alginic acid, Primogel® (sodium starch glycolate), or corn starch; lubricants such as magnesium stearate or stearate; lubricants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; and flavoring agents such as peppermint, methyl salicylate, or orange flavor.

[0274] Systemic administration can also be via transmucosal or transdermal routes. For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such penetrants are known in the art, and include, for example, detergents, bile salts, fusidic acid derivatives, etc. for transmucosal administration. Transmucosal administration can be achieved through the use of nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams known in the art.

[0275] In embodiments, administration may include introducing a pharmaceutical composition into a subject by a method or route that results in at least partial localization of the composition at the desired site so that the desired effect occurs.

[0276] For example, the pharmaceutical composition may be administered by bolus injection or infusion. Bolus injection refers to a route of administration in which a syringe is connected to a venous access device and the agent is infused directly into the subject. The term "infusion" may refer to intravascular injection.

[0277] The embodiments described herein can be administered to a subject once (e.g., as a single injection, bolus, or deposition). Alternatively, they can be administered to a subject once or twice daily 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. They can also be administered to a subject once or twice daily, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or any combination thereof.

[0278] In embodiments, the compositions described herein can be administered chronically to a subject. "Chronic administration" can refer to administration in a continuous manner, such as maintaining a therapeutic effect (activity) over an extended period of time.

[0279] The specific dosage and treatment regimen for a particular patient will depend on a variety of factors, including the particular antibody, variant, or derivative thereof used, the patient's age, weight, general health, sex, and diet, as well as the time of administration, excretion rate, drug combination, and the severity of the particular disease being treated. Assessment of such factors by a medical professional is within the ordinary skill of those in the art. The amount will also vary depending on the individual patient being 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.

[0280] A therapeutically effective amount of a reagent or therapeutic composition of the present invention can be the amount necessary to achieve a therapeutic goal. As described herein, this is the binding interaction between the reagent or therapeutic composition and its target, which, in some cases, inhibits the function of the target. The amount required for administration further depends on the binding affinity of the reagent or therapeutic composition for its specific target and the rate at which the administered reagent or therapeutic composition depletes the free volume of the target to which it is administered. The binding polypeptides described herein can be administered to a subject (e.g., a patient) in the range of about 0.1 mg / kg to 100 mg / kg of the patient's body weight, 0.1 mg / kg to 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 in the human body than antibodies from other species due to immune responses to foreign polypeptides. Therefore, it is often possible to administer lower and less frequent doses of human antibodies. Furthermore, the dosage and frequency of administration of a reagent or therapeutic composition of the present disclosure can be reduced by enhancing antibody uptake and tissue penetration (e.g., into the brain) through modifications such as lipidation. A typical therapeutically effective dose range for an antibody or antibody fragment of the invention can be, by way of non-limiting example, from about 0.1 mg / kg body weight to about 50 mg / kg body weight. Typical dosing frequencies can range, for example, from twice daily to once weekly.

[0281] When a fragment (e.g., an antibody fragment) is used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, peptide molecules that retain the ability to bind to the target protein sequence can be designed based on the variable region sequence of an antibody. Such peptides can be synthesized chemically and / or by recombinant DNA technology. (See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)). The formulation may also contain two or more active compounds as needed for the particular indication being treated, e.g., those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may contain an agent that enhances its function, such as a cytotoxic agent, cytokine (e.g., IL-15), chemotherapeutic agent, or growth inhibitory agent. Such molecules are preferably present in combination in amounts effective for the intended purpose.

[0282] The active ingredient can also be encapsulated in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, prepared by coacervation techniques or interfacial polymerization, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, nanocapsules) or macroemulsions. Sustained-release formulations can also be prepared.

[0283] The pharmaceutical or therapeutic carrier or diluent used can be a conventional solid or liquid carrier.Non-limiting examples of solid carriers include lactose, terra alba, sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, or lower alkyl ether of cellulose.Non-limiting examples of liquid carriers include syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene, and water.Similarly, carriers or diluents include glyceryl monostearate or glyceryl distearate, and sustained-release materials known in the art can be used alone or mixed with wax.

[0284] If 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 in the form of a troche or lozenge. The amount of solid carrier varies widely but can be from about 25 mg to about 1 g.

[0285] If a liquid carrier is used, the preparation may 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.

[0286] The composition and / or preparation may also be in a form suitable for local or systemic injection or infusion, and may therefore be formulated with sterile water or isotonic saline or glucose solution. The composition may be in a form adapted for peripheral administration only, excluding forms that can be administered centrally. The composition and / or preparation may be in a form adapted for central administration.

[0287] The composition and / or preparation can be sterilized by conventional sterilization techniques well known in the art. The resulting aqueous solution can be packaged for use or filtered under aseptic conditions and lyophilized, and the lyophilized preparation can be combined with a sterile aqueous solution before administration. The composition and / or preparation can contain pharmaceutically and / or therapeutically acceptable auxiliary substances necessary to approximate physiological conditions, such as buffers, tonicity adjusters, etc., for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.

[0288] Embodiment Exemplary embodiments disclosed herein are disclosed below in numbered paragraphs.

[0289] 1. A bispecific modulator as disclosed herein.

[0290] 2. The bispecific modulator of embodiment 1, a. a first portion comprising an antigen or epitope capable of being bound by a chimeric antigen receptor (CAR); b. a second moiety capable of binding to an internalized receptor or membrane protein on a cell; A bispecific modulator comprising:

[0291] 3. The bispecific modulator of embodiment 1, a. a first portion comprising an antibody capable of binding to a CAR; b. a second moiety capable of binding to an internalized receptor or membrane protein on a cell; A bispecific modulator comprising:

[0292] 4. The bispecific modulator of one of embodiments 2 or 3, wherein the second moiety comprises an antibody.

[0293] 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 an internalizing receptor or membrane protein on the cell causes internalization of the CAR into the cell.

[0294] 6. The bispecific modulator of embodiment 5, wherein binding of the first moiety by the CAR and binding of the second moiety by an internalizing receptor or membrane protein on the cell causes internalization of the CAR into the cell.

[0295] 7. The bispecific modulator of embodiment 5, wherein binding of the first moiety by the CAR and binding of the second moiety by an internalizing receptor or membrane protein on the cell causes internalization of the CAR into the cell and degradation of the CAR.

[0296] 8. A molecule having at least two moieties, a. a first moiety capable of binding to a molecule of interest on a cell; b. a second moiety capable of binding to an internalization molecule on a cell; Contains, molecule.

[0297] 9. The molecule of embodiment 29, a. the first portion comprises an antibody, antibody fragment, ligand, peptide, small molecule, or apatamer; b. the second portion comprises an antibody, an antibody fragment, a ligand, a peptide, a small molecule, or an apatamer; molecule.

[0298] 10. The molecule of embodiment 8, wherein the molecule of interest on the cell is different from the internalized molecule on the cell.

[0299] 11. The molecule of embodiment 8, wherein the first portion and the second portion comprise one polypeptide.

[0300] 12. The molecule of embodiment 9, wherein the internalization molecule on the cell comprises an internalization receptor.

[0301] 13. The molecule of embodiment 12, wherein the internalized molecule can be internalized by clathrin-mediated endocytosis.

[0302] 14. The molecule of embodiment 12, wherein the internalized molecule can be internalized by clathrin-independent endocytosis.

[0303] 15. The molecule of embodiment 12, wherein the internalizing molecule comprises a transferrin receptor.

[0304] 16. The molecule of embodiment 12, wherein the internalizing molecule comprises a G protein-coupled receptor (GPCR), a receptor tyrosine kinase (RTK), or a transmembrane receptor (TMR).

[0305] 17. The molecule of embodiment 16, wherein the GPCR comprises an adrenergic receptor, a chemokine receptor, or a coagulation receptor.

[0306] 18. The molecule of embodiment 16, wherein the RTK comprises a colony-stimulating factor receptor, an epidermal growth factor receptor, a tyrosine kinase receptor, a fibroblast growth factor receptor, an insulin-like growth factor receptor, a platelet-derived growth factor receptor, or a transforming growth factor receptor.

[0307] 19. The molecule of embodiment 16, wherein the TMR comprises a folate receptor, an interleukin receptor (e.g., an IL-2 receptor), a low-density lipoprotein receptor, or a transferrin receptor.

[0308] 20. The molecule of embodiment 12, wherein the internalizing molecule comprises a transferrin receptor (TfR).

[0309] 21. The molecule of embodiment 20, wherein the transferrin receptor comprises transferrin receptor 1 (TfR1) or transferrin receptor 2 (TfR2).

[0310] 22. The molecule according to embodiment 9, wherein the ligand of the internalizing molecule comprises at least a portion of a naturally occurring ligand that can be bound by a receptor.

[0311] 23. The molecule according to embodiment 22, wherein the ligand of the internalization molecule comprises at least a portion of transferrin, cholesterol, low density lipoprotein, and epidermal growth factor.

[0312] 24. The molecule according to embodiment 9, wherein the ligand of the internalized molecule can be bound by a G protein-coupled receptor (GPCR), a receptor tyrosine kinase (RTK) or a transmembrane receptor (TMR).

[0313] 25. The molecule according to embodiment 22, wherein the ligand of the internalization molecule can be bound by the transferrin receptor.

[0314] 26. The molecule of embodiment 22, wherein the ligand of the internalization molecule comprises the transferrin protein or a portion of the transferrin protein.

[0315] 27. The molecule according to embodiment 9, wherein the second portion comprises a Fab, scFv, single domain antibody, nanobody, monobody, DARPin or affibody.

[0316] 28. The molecule of embodiment 27, wherein the second portion comprises an H7 scFv.

[0317] 29. The molecule of embodiment 27, wherein the second portion comprises an H7 Fab or an engineered H7 antibody variant.

[0318] 30. The molecule of embodiment 9, wherein the second moiety is capable of binding to a transferrin receptor, a cholesterol receptor, a low-density lipoprotein receptor, or an epidermal growth factor receptor.

[0319] 31. The molecule of embodiment 9, wherein the second moiety is capable of binding to a G protein-coupled receptor (GPCR), a receptor tyrosine kinase (RTK), or a transmembrane receptor (TMR).

[0320] 32. The molecule of embodiment 9, wherein the second moiety is capable of binding to the transferrin receptor (TfR).

[0321] 33. The molecule of embodiment 8, wherein the molecule of interest comprises a protein.

[0322] 34. The molecule of embodiment 33, wherein the protein comprises a membrane protein.

[0323] 35. The molecule of embodiment 33, wherein the protein comprises an integral membrane protein.

[0324] 36. The molecule of embodiment 33, wherein the protein comprises an extracellular protein.

[0325] 37. A molecule according to embodiment 36, wherein the extracellular protein is found in the external environment.

[0326] 38. The molecule according to embodiment 36, wherein the extracellular protein is selected from the group consisting of an autoantibody, a cytokine, an enzyme, and a combination thereof.

[0327] 39. The molecule of embodiment 33, wherein the protein comprises a transmembrane protein.

[0328] 40. The molecule according to embodiment 39, wherein the transmembrane protein has one or more transmembrane domains.

[0329] 41. The molecule of embodiment 8, wherein the molecule of interest is capable of binding to a hormone, cytokine, growth factor, neurotransmitter, lipophilic signaling molecule (e.g., prostaglandin), or cell recognition molecule (e.g., integrin, selectin).

[0330] 42. The molecule of embodiment 8, wherein the molecule of interest comprises a receptor.

[0331] 43. The molecule according to embodiment 42, wherein the receptor comprises a G protein-coupled receptor (GPCR).

[0332] 44. The molecule according to embodiment 42, wherein the receptor comprises a receptor tyrosine kinase (RTK) or a transmembrane receptor (TMR).

[0333] 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.

[0334] 46. ​​The molecule of embodiment 45, wherein the ligand-gated ion channel-linked molecule provides for the movement of Na+, K+, Ca2+, or Cl- across the plasma membrane of the cell.

[0335] 47. The molecule of embodiment 45, wherein the enzyme-linked molecule comprises a receptor tyrosine kinase, a tyrosine kinase-associated receptor (e.g., an enzyme that associates with a cytokine), a receptor-like tyrosine phosphatase (e.g., an enzyme that removes phosphate groups from tyrosines of intracellular proteins), a receptor serine / threonine kinase, a receptor guanylyl cyclase, or a histidine kinase-associated receptor.

[0336] 48. The molecule of embodiment 8, wherein the molecule of interest comprises a chimeric antigen receptor (CAR), a receptor tyrosine kinase (e.g., EGFR), a molecule to which a checkpoint inhibitor can bind (e.g., PD-L1), or a lineage-specific marker (e.g., CD20).

[0337] 49. The molecule of embodiment 8, wherein the molecule of interest comprises CAR, EGFR, CD20 or PD-L1.

[0338] 50. The molecule of embodiment 9, wherein the first portion comprises an amino acid sequence to which a receptor can bind.

[0339] 51. The molecule of embodiment 50, wherein the first portion comprises a ligand for a receptor.

[0340] 52. The molecule according to embodiment 51, wherein the ligand comprises the ectodomain of CD19 and the receptor comprises a CAR specific for CD19.

[0341] 53. The molecule of embodiment 50, wherein the receptor comprises a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a B cell receptor (BCR).

[0342] 54. The molecule according to embodiment 9, wherein the first portion comprises an scFv, a Fab, a single domain antibody, a nanobody, a monobody, a DARPin or an affibody.

[0343] 55. The molecule according to embodiment 8 or 9, further comprising a peptide linker that is cleavable by a protease.

[0344] 56. The molecule according to embodiment 55, wherein the protease comprises an endosomal / lysosomal protease.

[0345] 57. The molecule according to embodiment 56, wherein the protease comprises a cathepsin.

[0346] 58. The molecule according to embodiment 55, wherein the peptide linker is located between the first portion and the second portion on the polypeptide comprising the first portion and the second portion.

[0347] 59. The molecule of embodiment 58, wherein the peptide linker is closer to the first moiety than to the second moiety.

[0348] 60. The molecule according to embodiment 55, wherein the peptide linker comprises Gly-Phe-Leu-Gly (GFLG).

[0349] 61. The molecule according to embodiment 55, wherein the peptide linker comprises valine-arginine (VR) and / or phenylalanine-lysine (FK).

[0350] 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 a combination thereof (Figure 35).

[0351] 63. The molecule according to embodiment 55, wherein the second moiety comprises an scFv, a Fab, a single domain antibody, a nanobody, a monobody, a DARPin or an affibody.

[0352] 64. The molecule of embodiment 63, wherein the scFV comprises H7.

[0353] 65. A molecule according to embodiment 55, wherein cleavage of the peptide linker by a protease can provide for capture and / or degradation of the molecule of interest within a cell in which the molecule having at least two sites is internalized.

[0354] 66. The molecule of embodiment 8 or 9, comprising an amino acid sequence of 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.

[0355] 67. The molecule of embodiment 8 or 9, wherein the nucleotide sequence encoding the molecule consists of 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.

[0356] 68. A bispecific modulator, a. a first antibody or antibody fragment that binds to a cell surface transferrin receptor (TfR); b. A second antibody or antibody fragment that binds to a cell surface transmembrane protein different from TfR. A bispecific modulator comprising:

[0357] 69. A bispecific modulator, a. a transferrin protein or a portion of a transferrin protein that binds to TfR on the cell surface; b.An antibody or antibody fragment that binds to a cell surface transmembrane protein other than TfR. A bispecific modulator comprising:

[0358] 70. A bispecific modulator according to embodiment 68 or 69, wherein the first antibody or antibody fragment and the second antibody or antibody fragment (embodiment 68), or the transferrin / portion of transferrin and the antibody or antibody fragment (embodiment 69), are part of one polypeptide.

[0359] 71. The bispecific modulator of embodiment 68 or 69, wherein the first antibody or antibody fragment and the second antibody or antibody fragment, or the transferrin / portion of transferrin and the antibody or antibody fragment, are two or more polypeptides.

[0360] 72. A bispecific modulator according to embodiment 71, wherein the two or more polypeptides comprise two polypeptide chains linked by a dimerization domain.

[0361] 73. The bispecific modulator of embodiment 72, wherein the dimerization domain comprises a knob-in-hole Fc.

[0362] 74. The bispecific modulator of embodiment 68 or 69, further comprising a peptide linker that can be cleaved by an endosomal / lysosomal protease.

[0363] 75. A nucleic acid encoding a molecule according to any one of embodiments 8 to 67, or a bispecific modulator according to any one of embodiments 68 to 74.

[0364] 76. A vector comprising the nucleic acid of embodiment 75.

[0365] 77. A cell comprising the vector described in embodiment 76.

[0366] 78. A method for treating toxicity associated with CAR-T therapy, or a method for increasing the efficacy of immune checkpoint therapy and targeted cancer therapy, comprising administering to a subject a molecule described in any one of embodiments 8 to 67, or a bispecific modulator described in any one of embodiments 68 to 74. [Example]

[0367] In order that the present invention may be more fully understood, the following examples are set forth. The following examples illustrate exemplary modes of making and practicing the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples, which are for illustrative purposes only, as alternative methods may be utilized to achieve similar results.

[0368] Example 1 - Construction of TransTAC (bispecific regulator) to downregulate EGFR Anti-EGFR affibody-Fc-Tf TransTAC was expressed and verified by SDS-PAGE (Figure 10, left). Zero to 60 nM TransTAC was incubated with the MCF10A EGFR-overexpressing cell line for 12, 36, and 68 hours. Flow cytometry revealed a dramatic dose-dependent decrease in EGFR levels, with IC levels at 68 hours. 50 The dose-dependent downregulation of EGFR was less than 6 nM (Figure 10, right). We observed the same dose-dependent reduction of EGFR using the adenocarcinoma lung cancer cell line, A549 (Figure 11A). Next, we performed a time course experiment in which A549 cells were treated with 30, 60, or 90 nM TransTAC, demonstrating that 90 nM TransTAC effectively downregulated EGFR with a half-life of less than 30 minutes. Furthermore, we observed TransTAC-mediated killing of MCF10A-EGFR cells (Figure 11B). These data demonstrate the efficacy of this construct.

[0369] Notably, TransTAC down-regulates cell surface EGFR by over 99% at equilibrium, whereas recently reported LYTACs only achieved 70–80% down-regulation. Additionally, the kinetics are different: the half-life of EGFR internalization by TransTAC is less than 30 minutes, whereas that of LYTACs is approximately 10–20 hours. These differences are due to the different endocytic kinetics of the carrier proteins used in the study.

[0370] Additional data (Figure 12) show that using A549 cells expressing the EGF receptor (EGFR) on the cell surface, TransTAC targeting EGFR internalized the receptor to a greater extent than an EGFR-specific antibody alone.

[0371] Example 2 - Construction of TransTAC (bispecific regulator) to downregulate anti-CD19 CAR The data show that antibodies specific for CD19, EGFR, and HER2, as well as transferrin fusion proteins, are well expressed in expi293 cells (Fig. 13).

[0372] FIG. 14 presents data demonstrating that the knob-in-hole version of TransTAC targeted to the anti-CD19 chimeric antigen receptor internalized the receptor.

[0373] Figure 15 shows data from the same experiment shown in Figure 14, where intracellular surface CAR levels were measured using anti-myc-biotin / streptavidin 647. The data in Figure 15 demonstrate that CAR-targeting homodimeric TransTAC effectively internalized the CAR. In experiments similar to those shown in Figure 16, cells expressing an anti-CD19 CAR were incubated with K562 cells (expressing CD19) along with the TransTAC bispecific modulator. Cell activation was measured. The TransTAC bispecific modulator began to exhibit inhibitory activity at concentrations below 10 nM. These data demonstrate that the TransTAC molecule effectively internalized the CAR and downregulated CAR-T cell activity.

[0374] The data in Figure 17 are similar to those shown in Figure 16. The data in Figure 12 show that TransTAC inhibits cell activation at an IC 50 This shows that the concentration was improved to approximately 10-20 nM.

[0375] The data in Figure 18 demonstrate that TransTAC molecules with CD19NT.1 variant ectodomains exhibit an IC of approximately 800 pM. 50 This data indicates that TransTAC molecules have no effect on Jurkat cells in the absence of CD19-expressing K562 cells.

[0376] Example 3 - TransTAC molecules containing protease sites that increase target degradation Figures 19A-B are schematic diagrams of the molecules used in these studies (A) and the results obtained with those molecules (B). TransTAC1.0 for this example is TransTAC0.4 as shown in Figure 44A.

[0377] Figure 19C shows fluorescence microscopy of the targeted CAR in the cells of Figure 19B.

[0378] Figures 20A-B show a schematic diagram of the molecules used in these studies (A) and Western blot results of targeted CAR and actin controls (B).

[0379] FIG. 20C shows a graph of the data in FIG. 20B.

[0380] Figures 20D-E show Western blot results (D) and data graphs (E) for additional molecules used in these studies.

[0381] Figures 20F-G show a TransTAC molecule containing a GFLG linker (F) and Western blot data using that molecule (G).

[0382] Figure 20H shows a graph of the data in Figure 20G.

[0383] Figure 20I shows the results for other cathepsin-sensitive TransTAC molecules.

[0384] Figures 21A-B show that in an experiment in which cells expressing an anti-CD19 CAR receptor were incubated with CD19-positive A375 cells, the molecules shown in Figure 20A inhibited Jukat cell activation (A) and interferon gamma (IFN-γ) release from primary T cells (B).

[0385] Figure 22A shows that addition of the molecules shown in Figure 20A prevented human primary anti-CD19 CAR-T cells from killing CD19-positive A375 target cells. A375 cells express mCherry in the nucleus for fluorescence microscopy. Removal of the molecules resulted in reactivation of the CAR-T cells and killing of the CD19-positive A375 target cells. The photograph shows the red fluorescence channel of the fluorescence microscope.

[0386] Figure 22B shows that removal of the indicated molecules reactivated CAR-T cells and killed CD19-positive A375 target cells. The photograph shows an overlay of the red fluorescent channel and the white magnetic field channel.

[0387] Example 4 - TransTAC molecules targeting epidermal growth factor receptor (EGFR) Figures 23A-B show a schematic diagram of the molecules used in these studies (A), including EGFR-specific antibodies, affibodies, and TransTAC molecules, and also show results demonstrating the reduction of EGFR levels on the surface of A549 cells using these molecules (B).

[0388] Figures 23C-D show the results of cell growth inhibition using the molecules of Figure 53A.

[0389] Example 5 - TransTAC molecules targeting CD20 These data exemplify an approach using a TransTAC molecule with an antibody specific for CD20 and a molecule that binds to the transferrin receptor (transferrin or antibody).

[0390] Figures 24B-C are schematic diagrams of the molecules used in these studies (B) and the results obtained from their use (C).

[0391] FIG. 24D shows a graph of the normalized data of FIG. 24C.

[0392] This data indicates that the TransTAC molecule is internalized / degraded more rapidly than the anti-CD20 rituximab antibody, which targets only CD20.

[0393] Example 6 - Reversible control of receptor function Figure 25A shows cells expressing CAR. The cells in the left panel are contacted with a CD19m-Fc antibody (CD19NT.1 variant). The cells in the right panel are contacted with a TransTAC molecule that binds to CAR. In both panels, the cell nuclei are stained with DAPI. The cells are also stained with an anti-CD3z antibody, which stains CAR. In the left panel, the fluorescence from the anti-CD3z antibody is localized to the cell surface. In the right panel, the fluorescence from the anti-CD3z antibody is localized to the cytoplasm. This data indicates that the TransTAC molecule caused CAR internalization.

[0394] Figure 25B shows the reversibility of CAR internalization by TransTAC. In the first bar of the graph, cells not contacted with TransTAC have relatively high cell surface CAR-specific immunofluorescence (approximately 1.0). In the second bar of the graph, cells are contacted with TransTAC, and cell surface CAR-specific immunofluorescence is low (approximately 0.2). In the third bar of the graph, cells are contacted with TransTAC, but TransTAC is then removed. After 24 hours, cell surface CAR-specific immunofluorescence has increased to the same level (approximately 1.0) as cells not exposed to TransTAC.

[0395] These data demonstrate that TransTAC-mediated CAR receptor internalization functions as a reversible CAR-T cell off-switch, which can be used to mitigate toxicities associated with CAR-T therapy.

[0396] FIG. 26 shows that TransTAC inhibited / stopped interferon gamma production.

[0397] As discussed in Example 8, Figure 22A shows that TransTAC prevented primary human anti-CD19 CAR-T cells from killing CD19-positive A375 target cells. The data demonstrate that removal of TransTAC reactivates CAR-T cells and kills CD19-positive A375 target cells. Figure 22B shows that removal of the TransTAC molecule led to reactivation of CAR-T cells and target killing.

[0398] Example 7 - Targeted degradation of membrane proteins Figures 27A-B show the expression of transferrin receptor on various cells as revealed by fluorescent antibodies. The data indicate that transferrin receptor may be expressed at higher levels on the surface of tumor cells compared to non-tumor cells.

[0399] Figure 28 shows examples of cell surface proteins that can be targeted by TransTAC.

[0400] FIG. 29A shows that TransTAC molecules (DP81 and DP174) reduce the amount of EGFR in cells.

[0401] Figure 29B shows that TransTAC molecules can degrade EGFR. The data show that TransTAC-mediated degradation of EGFR is sensitive to bafilomycin (an autophagosome-lysosome fusion inhibitor) and MG132 (a proteosome inhibitor). These data suggest that TransTAC-mediated degradation of EGFR is mediated through the lysosomal pathway.

[0402] Figure 30A shows an approach to lung cancer treatment using TransTAC. High expression of TfR in cancer cells allows for target specificity.

[0403] FIG. 30B shows that EGFR TransTAC molecule can inhibit PC9 cancer cells (lung adenocarcinoma).

[0404] Figures 30C-D show that EGFR TransTAC molecules can inhibit PC9 cancer cells.

[0405] Figure 31 shows that the anti-CAR TransTAC molecule reduces the amount of CAR in these cells.

[0406] Figure 32A shows that anti-PD-L1 TransTAC molecules (DP186, DP187) can reduce the amount of PD-L1 in these cells.

[0407] Figure 32B presents data showing that anti-PD-L1 TransTAC molecules reduce the amount of PD-L1 in cells.

[0408] FIG. 33 shows that anti-CD20 TransTAC molecules (DP209S, DP210, DP213) reduce the amount of intracellular CD20.

[0409] Figures 34A-C and 34D show examples of TransTAC molecules containing protease-sensitive linkers and example data obtained with the molecules.

[0410] Figure 35 shows example data obtained with TransTAC molecules containing various protease-sensitive linkers.

[0411] 36A-C show an exemplary TransTAC molecule comprising an antibody fragment specific for transferrin binding and exemplary data obtained with this molecule.

[0412] 37A-B show an example of a TransTAC molecule and example data obtained with that molecule.

[0413] Example 8 - TransTAC for Cancer Targeted therapy with tyrosine kinase inhibitors is the standard of care for lung cancers with EGFR gene mutations. We hypothesized that co-targeting EGFR and TfR receptors on lung cancer cells would enable EGFR inhibition while maintaining high tumor specificity (Figure 30A). We engineered the anti-EGFR affibody *H7*GFLG-VR TransTAC. Incubation of this molecule with the human lung adenocarcinoma A549 cell line resulted in over 90% degradation of EGFR (Figure 30B). Notably, treatment of a non-tumorous HEK cell line engineered to overexpress EGFR with TransTAC resulted in much less EGFR degradation, demonstrating the tumor specificity of this technology. This specificity is due to the high expression of TfR in tumor cells (Figures 27A-C).

[0414] Example 9 - Upregulation of transferrin receptor in cancer cell lines, primary tumors and activated T cells Example 7 demonstrated increased expression of TfR in cancer cells and some immune cells.

[0415] In further studies, we used flow cytometry to measure cell surface TfR levels in five non-tumor cell lines, including HEK293T (embryonic kidney), MCF10A (mammary epithelial), HFF-1 (foreskin fibroblast), MCR-5 (lung fibroblast), and LF-1 (fetal lung fibroblast), as well as ten cancer cell lines, including HeLa (cervical cancer), Raji (lymphoma), Jurkat and K562 (leukemia), MDA-MB-231 and MCF-7 (breast cancer), PC9 and A549 (lung cancer), and PC9 cells harboring EGFR drug resistance mutants. We observed that cancer cell lines expressed 2- to 26-fold more TfR on the cell surface than non-tumor cell lines (Figure 38C). Among cancer cell lines, the leukemia cell lines Jurkat and K562 showed the highest TfR expression. Our findings demonstrate upregulation of TfR in cancer cell lines.

[0416] Because the cell lines were modified to be immortalized, changes in protein expression may have occurred. Therefore, we further demonstrated that TfR expression was upregulated in primary tumors compared to primary healthy tissues by performing transcriptomics analysis of TFRC, the gene encoding TfR1. Microarray transcriptomics data for TFRC in primary healthy tissues and tumors were obtained from the MERAV database. Paired tissue analysis of healthy versus tumor samples was performed using a custom Python script. TFRC expression was statistically significantly increased in overall cancer (p=3.98e-89) and in 14 of 19 specific tissues, including breast, lung, pancreatic, liver, bladder, skin, esophageal, thyroid, testicular, gastric, salivary gland, kidney, central nervous system, and female reproductive system cancers (Figure 38D). These findings provide further evidence supporting TfR as a target upregulated by cancer.

[0417] To investigate whether TfR may also be a potential target for immune cell modulation, we analyzed the DICE dataset, which contains gene expression profiles of human immune cells isolated from blood samples of healthy donors. While most immune cells express low levels of TfR, we observed approximately six-fold higher TfR expression in activated CD4 and CD8 T cells compared to inactivated T cells, a level comparable to TfR levels in some malignant tissues, indicating that TfR may be a target for regulating activated T cells (Figure 38E). These findings demonstrate that TfR is upregulated not only within tumors but also within activated T cells, highlighting its value as a cell surface receptor for both cancer and immune modulation. Our transcriptome analysis provides a detailed comparison of TfR expression in specific tissues, providing a roadmap for future selection of our technology and subsequent disease applications.

[0418] Example 10 - Endosomal capture of targeted proteins by an early version of the TransTAC design Experiments with TransTAC molecules specific for CD19-specific CARs are also described in Example 2, but are described in more detail in this Example.

[0419] In these experiments, we used Jurkat cells expressing an N-terminal myc epitope-tagged CAR to measure cell surface CAR levels (Figure 39B). Initially, we attempted to use the native CD19 ectodomain as the CAR-binding component, but observed significant protein aggregation in SDS-PAGE gels (Figure 43B, lanes 1-2). Next, we evaluated a small panel of previously developed CD19 ectodomain variants 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 that they expressed and behaved well (Figure 43A-B). Finally, we selected the variant CD19NT.1, which showed better expression, for use in CAR-TransTAC.

[0420] Because TfR is a homodimeric receptor and the full physiological function of the TfR dimer requires the binding of two transferrins (TFs), we hypothesized that molecules containing two TfR ligands would be more effective at promoting the internalization of targeted CARs than single ligands. Therefore, we generated two versions of TransTAC: v0.1, a knob-in-hole Fc construct with one TF for TfR binding and one CD19NT.1 for CAR, and v0.2, an Fc fusion with two TFs and two CD19NT.1 (Figure 39A). 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 h, we measured cell surface CAR levels using an anti-myc antibody. When processed with both v0.1 and v0.2, the D max is 60%, v0.2 D maxThe TF-dependent internalization of CAR from the cell surface was 80%, demonstrating a significant reduction in cell surface CAR levels (Figure 39C). Interestingly, a hook effect was observed with v0.1 but not with v0.2. In contrast, treatment with the control CD19NT.1-Fc protein did not reduce cell surface CAR levels. These findings demonstrate that CAR-TransTAC can effectively internalize CAR from the cell surface via a TF-dependent mechanism, and that in some embodiments, dimeric TransTAC is more effective than monomeric TransTAC.

[0421] However, despite its ability to efficiently remove CAR from the cell surface, TransTACv0.2 did not result in CAR degradation, as shown by whole cell lysate Western blots (Figures 39D, 44A-B), indicating that the internalized receptor was trapped intracellularly and not degraded.

[0422] To understand the intracellular destination of CAR internalized by v0.2, we stably expressed CAR-GFP and mCherry tagged with various endosomal and lysosomal markers, including Rab5+ or EEA+ (EE), Rab7+ (LE), Rab11+ (RE), and Lamp1+ (lysosomes), in the HeLa cell line (Figure 39I, J; Figure 45A-D). Fluorescence microscopy images of v0.2-treated cells showed colocalization of CAR-GFP and Rab11, indicating that internalized CAR was transported to the RE (Figure 39I, white arrow). Thus, TransTACv0.2 effectively removes POI from the plasma membrane by trapping it in the recycling endosomal compartment of target cells.

[0423] Example 11 - Rational rewiring of intracellular trafficking pathways of internalized protein complexes to engineer degradation-inducing compounds Experiments related to intracellular transport are described and explained in more detail in the present Examples.

[0424] Further research has been directed at developing next-generation TransTACs that not only capture POIs but also lead to their degradation, which may be beneficial for cancer-related targets in part because degradation can result in more persistent inhibition of protein function.

[0425] To target a target protein for degradation, we tested whether the POI (protein of interest) must be separated from the recycling TF / TFR complex in the EE, where it is sorted to the degradation or recycling pathway (Figures 38A, 39E, and 39F). Endosomal proteases, such as the cysteine ​​protease cathepsin, can be used to separate the POI from the Tf / TfR complex. Therefore, we incorporated a cathepsin B-sensitive Gly-Phe-Leu-Gly (GFLG) linker into TransTAC either between the Fc domain and the Tf ligand (v0.3) or between CD19NT.1 and the Fc domain (v0.4) (Figures 39A and 44A). Indeed, this linker modification altered the intracellular trafficking of CAR-GFP. With TransTAC v0.4, a significant portion of the receptor now colocalized with Rab7 (late endosomes) and Lamp1 (lysosomes) (Figures 39L, 45D-F). Furthermore, using Western blotting, we observed that approximately 50% of the CAR was degraded (Figure 44C). Degradation with v0.3 was lower compared to v0.4 (Figure 44C). This is likely because CD19NT.1 remained bound to the Fc domain after dissociation from Tf / TfR, which may mediate recycling via the FcRn pathway. Overall, our studies demonstrate that incorporating a protease-cleavable linker into TransTAC allows for degradation of the POI.

[0426] Our next goal was to improve degradation efficiency and deepen our understanding of proteolysis in endosomes. Traditionally, protein degradation has been thought to occur primarily in acidic lysosomes (LEs), and little is known about proteolytic activity in EEs. To identify optimal protease substrates in EEs, we performed small-scale linker screening, assuming that protease activity in EEs correlates with degradation efficiency. We screened a panel of 14 linkers containing cathepsin B cleavage motifs, such as GFLG, Gly-Gly-Phe-Gly (GGFG), Phe-Lys (FK), Val-Ala (VA), Val-Lys (VK), and Val-Arg (VR), either alone or in combination (Poreba, Marcin. "Protease-activated prodrugs: strategies, challenges, and future directions." The FEBS Journal 287.10, 2020:1936-1969), using Western assays with both TransTAC v0.4 and v1.0 (Figure 44D-E). Overall, we found that the incorporation of the dipeptide motifs VK, VR, and FK helped improve cleavage activity compared to the GFLG sequence. Based on these results, we selected the GFLG-VR and EVR linkers for developing subsequent generations of TransTAC. In preparation for in vivo studies, we used the EVR linker instead of VR, because previous studies have shown that the inclusion of glutamic acid improves the stability of the linker in mouse serum.

[0427] Next, we took a new step in optimizing TransTAC by replacing the TF ligand with an anti-TfR single-chain Fv (scFv) called H7, a TF-competitive antibody identified by phage display (Figure 39A) (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 was intended to reduce RE sorting, which we believe is an important step in improving degradation efficiency because proteins sorted to the RE are not transported to the LE / lysosome for degradation (Figure 39G,H). The logic was that certain molecular features of the TF / TfR complex were involved in RE sorting, and that synthetic antibody binders such as H7 could be used to alter this sorting decision and redirect the intracellular trafficking of the complex after iron release in the EE.

[0428] Based on this, we generated two versions of TransTAC: v0.5, which contains the H7 binder but no cleavable linker, and v1.0, which contains both the H7 binder and the cleavable linker (Figure 39A). Cells treated with v0.5 showed that CAR-GFP colocalized primarily with the EE markers Rab5 and EEA (Figures 39G, J, 45A, D). The Pearson colocalization coefficients of the corresponding markers were statistically different from those of cells treated with TransTACv0.2, which contains TF as the anti-TfR ligand (Figure 39K). This result demonstrated that replacing TF with an anti-TfR antibody reduced RE transport. Furthermore, retransport resulted in a significant improvement in degradation efficiency. Using TransTACv1.0, over 80% degradation of CAR was observed in both Western and fluorescent microscopy assays (Figures 39D, I, J; Figures 45A-C). In addition to improved degradation, the H7 substitution also increased protein yield by approximately 7-fold, bringing TransTAC expression levels comparable to those of conventional antibodies. Taken together, our rational protein engineering efforts have successfully developed a novel protein design, TransTACv1.0, as a potent degrader of CAR that is robustly expressed in a fully recombinant form. CAR-TransTACv1.0 is the first recombinant protein degrader engineered to target a synthetic receptor.

[0429] Example 12 - Reversible control of primary CAR-T cell function by CAR-TransTAC Experiments on the efficacy and reversibility of TransTAC molecules controlling CAR-T cells are described in Examples 2 and 6.

[0430] In further studies, we explored the use of TransTAC as an off-switch to fine-tune CAR-T cell activity and manage associated toxicities, such as cytokine release syndrome (CRS), caused by CAR-T cell overactivation (Figure 47A).

[0431] As a proof of concept, we showed that CAR-TransTACv0.4 can effectively inhibit human primary CAR-T cells.

[0432] We isolated primary CD8+ T cells from human PBMCs and generated anti-CD19 CAR-T cells by lentiviral transduction. For tumor cells, we used an adherent melanoma cell line, A375, engineered to express CD19 and nuclear mCherry to facilitate live-cell imaging. CAR-TransTACv0.4 potently inhibited IFN-γ secretion with an IC50 of approximately 0.4 nM and D max The tumoricidal activity of primary CAR-T cells was confirmed to be 88% (Figure 47B, C). This molecule also effectively inhibited tumoricidal activity (Figure 47B, D). Furthermore, this inhibition was reversible, and removal of TransTAC restored the tumoricidal activity of primary CAR-T cells (Figure 47B, E).

[0433] To better understand the factors influencing the various performances of CAR-TransTAC and to determine the general structure-function-activity (SAR) relationship of TransTAC, we constructed four TransTAC v0.5 variants, v0.6-v0.9, each containing one or two copies of CD19NT.1 or H7 in different configurations and tested them (Figure 41B). The results revealed that having two copies of H7 was superior to having two copies of CD19NT.1 in promoting CAR internalization (Figure 41C, D). This highlights the importance of dual binding to the dimeric TfR, rather than having two copies of the anti-POI binder, in generating potent TransTAC. We also demonstrated that TransTAC-mediated CAR internalization is not the result of CAR cross-linking. Furthermore, significant differences in internalization efficiency were observed between different molecular configurations (Figure 41C), indicating that tertiary complex structure plays a role in influencing TransTAC efficiency. Furthermore, we generated Fc-H7 molecules as a competitor for TfR binding and observed that CAR internalization was reduced in a dose-dependent manner by TransTACv0.5 treatment and in the presence of the competitor in solution (Figure 47D), further demonstrating that TransTAC functions through a TfR-dependent mechanism.

[0434] Because CAR clustering can lead to low levels of spontaneous CAR-T cell activation, we hypothesized that our dimeric CAR off-switch molecule might influence its inhibitory effect on CAR-T cells by inducing CAR clustering. Therefore, we developed a CAR off-switch containing only a single CD19NT.1 domain and compared it with a dimeric variant. In this study, CD19NT.1 monomers showed a significant 100-fold reduction in potency compared to CD19NT.1-Fc dimers. This finding emphasizes the importance of avidity for CD19NT.1-Fc to achieve effective CAR-T cell inhibition. Because CAR-T–tumor interactions involve multiple CAR / antigen interactions at the immunological synapse, molecules with multiple copies may be better able 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.

[0435] CAR-TransTAC did not require a dimeric format to be effective because it did not rely on competition with tumor CD19 for its efficacy. We observed that CD19NT.1-based monomeric TransTAC (fused to the Fc region and the CAR-binding domain) exhibited robust performance and even outperformed the dimeric variant in blocking CAR-Jurkat cells.

[0436] Example 13 - Expanding the targets that TransTAC can address Experiments with TransTAC molecules specific for additional cell surface molecules are described in Examples 1, 4, 5 and 7.

[0437] In further studies, we verified the generalizability of TransTAC. To date, all biologic-based degradation-inducing compounds have been developed targeting single-pass transmembrane proteins. We expanded our target pool to include both single-pass transmembrane targets, such as epidermal growth factor receptor (EGFR) and programmed death ligand 1 (PDL1), as well as the multipass transmembrane protein cluster of differentiation 20 (CD20) (Figure 40A). These targets have diverse functions and regulatory pathways and are found on a wide range of cancer and immune cells.

[0438] Our first target was the immune checkpoint receptor ligand, programmed death-ligand 1 (PD-L1), whose downregulation can enhance antitumor T cell activity. Targeting PD-L1 using monoclonal antibodies has met with moderate clinical success, and novel mechanisms for targeting this protein may be highly valuable. We generated PD-L1-TransTAC using the 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-TransTAC, whereas little or no PD-L1 degradation was observed in control groups containing TransTACv0.2 and v0.4 lacking H7 or carrying the TF ligand (Figure 40B; Figure 46A).

[0439] Next, we aimed to target the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase that plays an important role in the development and progression of various cancers, including lung and brain cancers. We used 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) to conjugate EGFR to generate EGFR-TransTAC. A549 lung cancer cells treated with EGFR-TransTACv1.0s containing either the GFLG-VR or EVR linker showed an 80–90% reduction in EGFR, whereas the control group showed almost no degradation (Figure 40C, Figure 46B). Although v1.0 induced varying degrees of EGFR degradation due to different linkers, all were lower than TransTAC with GFLG-VR or EVR, and v0.2 had no effect (Figure 46B). These results are consistent with observations with the CAR-TransTAC variants and demonstrate the importance of the modifications made to improve TransTAC.

[0440] Cluster of differentiation 20 (CD20) is a B cell-specific surface marker with four transmembrane domains and unknown function. Knocking down cell surface CD20 with a degrading compound is valuable. CD20-TransTAC was engineered to bind to CD20 using the Fab format of rituximab, the first clinically approved CD20 antibody. Treatment of Raji cells, a human B lymphoblastoid cell line, with the resulting CD20-TransTAC resulted in up to 97% reduction in CD20, whereas control cells showed no or significantly less degradation (Figure 40D, Figure 46C).

[0441] Collectively, the successful generation of degraders against these four targets demonstrates the modularity and versatility of the TransTAC design. Importantly, high activity was observed against all four targets investigated, reaching over 80% in various cell lines, demonstrating the high efficiency of target degradation using the TransTAC degrader design.

[0442] Example 14 - Kinetics, structure-activity relationship (SAR), mechanism, and in vivo characterization of TransTAC The present inventors further characterized the decomposition-inducing compounds to understand their underlying mechanisms and SAR.

[0443] We first investigated the kinetics of TransTAC-mediated protein internalization by measuring the time course of CAR levels on the cell surface (Figure 41A). We observed rapid removal of CAR from the cell surface, with only 17% remaining after 10 min and 13% remaining after 20 min of treatment with TransTACv1.0-GFLG-VR. Furthermore, this response persisted for a long time, with 10% CAR remaining on the cell surface after 3 h with v1.0. This rapid and sustained protein downregulation demonstrates that TransTAC offers a promising alternative to genetic approaches for studying cell surface protein knockdown, providing temporal resolution for membrane protein regulation.

[0444] To further understand how the number and shape of binders in TransTAC affect its behavior, we generated and tested four CAR-TransTAC v0.5 variants, v0.6-v0.9, containing one or two copies of CD19NT.1 or H7, respectively (Figure 41B). The results revealed that having two copies of H7 was more important for promoting CAR internalization than having two copies of CD19NT.1 (v0.6 vs. v0.7, Figure 41C). This highlights the importance of dual binding to the dimeric TfR, rather than having two anti-POI binders, in generating potent TransTAC. This also demonstrates that TransTAC-mediated CAR internalization is not the result of CAR cross-linking. Furthermore, significant differences were observed in the internalization efficiency of molecules with different shapes, indicating that tertiary complex structure plays a role in influencing TransTAC efficiency (v0.8 vs. v0.9, Figure 41C). Furthermore, we created an Fc-H7 molecule as a competitor for TfR binding and observed that CAR internalization was reduced in a dose-dependent manner by TransTACv0.5 treatment and in the presence of the competitor in solution (Figure 41D). This observation further supports the TfR-dependent mechanism of action of TransTAC. These SAR analyses provide valuable insights to guide future TransTAC design.

[0445] Next, we investigated the intracellular mechanisms underlying TransTAC-mediated protein degradation. We examined the two major pathways involved in cellular protein degradation: the lysosomal pathway and the proteosomal pathway. A549 cells were treated with either bafilomycin, which inhibits lysosomal acidification, a vacuolar proton pump inhibitor, or MG132, a proteasome inhibitor. Although 1 μM bafilomycin inhibited TransTAC-mediated EGFR degradation, 1 μM MG132 had a less significant effect (Figure 41E). These results indicate that intact lysosomal function is essential for TransTAC-mediated protein degradation.

[0446] To examine whether TfR levels remained constant or decreased upon TransTAC treatment, we characterized total cellular TfR expression by Western blotting assay using PD-L1-TransTAC. No change in TfR levels was observed, in stark contrast to the loss of PD-L1 in the same assay (Figure 41F). This result supports our hypothesis that TfR is recycled, while POI is separated from TfR and then targeted for degradation.

[0447] Finally, we investigated whether TransTAC exhibits antibody clearance similar to that of IgG in vivo and whether it is tolerable. Nude mice were intraperitoneally injected with 5 or 7 mg / kg body weight of CD20 TransTAC or 5 mg / kg of IgG control (Figure 41G). No significant weight changes were observed with either TransTAC or the control (Figure 41H). Western blot analysis of plasma antibody levels revealed that TransTAC persisted in plasma for up to 10 days after injection, with a half-life of approximately 10 days. This half-life is longer than the control IgG tested and comparable to the reported half-life of IgG in mice (Figure 41I). The scFv-H7 antibody is known to cross-react with the mouse TfR. Taken together, these results demonstrate that TransTAC is well tolerated, exhibits favorable pharmacokinetics, and is not rapidly eliminated despite cross-reactivity with mouse cells.

[0448] Example 15 - Targeting drug-resistant small cell lung cancer with EGFR-TransTAC Experiments regarding TransTAC molecules for cancer therapy are described in Example 8.

[0449] Furthermore, cancers rapidly progress to evade treatment, often developing drug-resistant mutations that lead to treatment failure and disease recurrence. The EGFR C797S mutation, in particular, poses a major challenge in the treatment of non-small cell lung cancer (NSCLC), which accounts for 85% of all lung cancer cases. The C797S mutation, which emerged in approximately 10–26% of NSCLC patients after treatment with the third-generation EGFR tyrosine kinase inhibitor (TKI) osimertinib, affects C797, a key residue that forms a covalent bond with the irreversible TKI. As a result, existing TKI therapeutics are ineffective against this disease.

[0450] EGFR-TransTAC can induce targeted degradation of EGFR in cancer cells with elevated TfR expression and can target EGFR-driven lung cancer patients, including the C797S mutant population (Figure 42A). Three lung cancer cell lines were used: PC9-wildtype (WT), PC9 GR4, and PC9 GR4 C797S. PC9-WT cells are a lung adenocarcinoma cell line with a deletion in exon 19 (Del 19) of the EGFR gene and are sensitive to all three generations of TKIs. PC9-GR4 is a gefitinib-resistant, osimertinib-sensitive cell line with a T790M mutation (Del 19 / T790M) that was generated according to a previously established drug selection protocol. Finally, PC9 GR4 C797S (Del19 / T790M / C797S) is a cell line in which the C797S mutation was newly introduced by CRISPR, further enhancing resistance to osimertinib.

[0451] Several EGFR-affibody-based TransTAC variants (Figure 42B) were generated and first evaluated for dose-dependent inhibitory efficacy in 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 dose-dependently inhibited cells with IC50 values ​​in the low nM range, whereas the control affibody-Fc fusion and v0.2 showed no or little response (Figure 42C). Consistently, >90% maximal EGFR degradation was observed in PC9-WT and PC9 GR4 C797S cells with TransTACv1.0 treatment, whereas no degradation was observed with the affibody-Fc control (Figure 42D). The same TransTAC molecule induced only approximately 40-50% EGFR degradation in the EGFR-overexpressing HEK293 cell line, which has 3-10-fold lower TfR expression compared to the three PC9 cell lines (Figure 48A). This result indicates that the efficiency of TransTAC-mediated proteolysis is positively correlated with TfR expression levels, highlighting the potential cancer-specific advantage of the TransTAC technology.

[0452] We next compared EGFR-TransTAC with the 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 (Figures 42E and 48B). PC9-WT cells were sensitive to all three TKIs, with IC50 values ​​ranging from <0.1 to 33 nM. PC9-GR4 cells were less sensitive to afatinib and osimertinib, with IC50 values ​​of 168 nM and 207 nM, respectively, and were completely resistant to gefitinib. PC9-GR4-C797S cells were insensitive to any of the three inhibitors.

[0453] Unlike TKIs, TransTACv1.0s effectively inhibited all three cell lines with IC50s in the low, sub-nM range (Figures 42E and 48B). Specifically, the IC50 of TransTACv1.0-GFLG-VR against PC9 GR4 C797S cells was 2 nM, and that of TransTACv1.0-EVR was 8 nM. To assess extratumor toxicity, a healthy human fibroblast cell line (HFF-1) was included in the assay. Neither TransTAC nor TKIs showed significant inhibition until the molecule concentrations reached the high nM or μM range (Figures 42E and 48B).

[0454] To further compare the efficacy and specificity of TransTAC with standard of care, we performed coculture assays of healthy and cancer cells and monitored the drug's effects using live-cell fluorescence imaging (Figure 42F-G). PC9 and PC9 GR4 C797S cells were engineered to express GFP, and HFF-1 cells expressed mCherry. The cells were mixed at a 1:10 ratio and treated with TransTAC or a TKI. Consistent with the MTT assay results, TransTAC demonstrated high efficacy against both PC9-WT and PC9 GR4 C797S cells. In contrast, TKIs inhibited PC9 WT cancer cells but not PC9 GR4 C797S cells. UT or affibody-Fc control molecules had no effect on either cell line.

[0455] Furthermore, TransTAC was compared with chemotherapy drugs. Unlike TransTAC, which did not kill healthy HFF1 cells, the combination chemotherapy of carboplatin and paclitaxel was cytotoxic to both cancer and normal cells (Figure 4F-G). This is consistent with the previous recognition that chemotherapy, although a first-line treatment for many types of cancer, often has high extratumoral toxicity.

[0456] Furthermore, we validated the live-cell imaging results by performing flow cytometry analysis to measure the ratio of GFP / mCherry-positive cells after treatment, which reflects the relative drug cytotoxicity toward cancer and healthy cells (Figure 48C). The analysis showed that TransTAC inhibited both PC9 WT and GR4 CS cells, with the ratio of cancer cells to healthy cells being nearly zero, demonstrating high cancer targeting activity and specificity. In contrast, the efficacy of TKIs against GR4 CS cells was much lower, with cancer cells dominating the overall mixed cell population.

[0457] Taken together, these findings demonstrate that EGFR TransTAC molecules can target lung cancer cells harboring the EGFR C797S mutation, and comparison with current standard of care demonstrated superior tumor efficacy and specificity of TransTAC.

[0458] Example 16 - Schematic of the design principles we discovered to enhance the degradation efficiency of TransTAC Figure 38b is an illustration of an example of a TransTAC degradation-inducing compound. Broadly speaking, TransTAC is a recombinant protein consisting of an anti-POI binder and an anti-TfR binder to bridge adjacent POI and TfR on the cell surface. As outlined in Figures 39A and 44A, we found that many forms of TransTAC can efficiently remove target proteins from the cell surface. Therefore, all of them are effective as membrane protein regulators.

[0459] However, we have found examples of at least three design principles that make TransTAC an efficient internalization and degradation-inducing compound: (1) dimeric TransTAC promotes more efficient protein internalization than monomeric heterobispecific TransTAC; (2) the cathepsin B-sensitive linker is important for enabling lysosomal transport of POI; and (3) antibody binders targeting TfR, but not the native transferrin (TF) ligand, may reduce POI transport to the recycling endosome (RE) and increase degradation efficiency.

[0460] Specific variants of the molecule can be used to induce targeted endosomal trapping or lysosomal degradation, offering customizable possibilities for modular engineering of membrane proteins.

[0461] Example 17 - Identification of peptides not known to be susceptible to cathepsin cleavage A yeast-display peptide library was used to identify peptides known to be insensitive to cathepsin cleavage. These peptides can be derived from combinations of small motifs found in SEQ ID NOs: 144 and 145. Tests were performed at pH 4.4 (Figure 49A) and pH 6.4 (Figure 49B). 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).

[0462] equivalent 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 which are considered to be within the scope of this invention.

Claims

1. Formula I: R1-R2-R3(I) a fusion protein of During the ceremony, R1 is at least one protein of interest (POI) binder (POIB); R2 is a linker of formula R4-R5 or R5-R4, wherein R4 is an IgG Fc region; and R5 is a protease-sensitive linker; and R3 is a transferrin receptor binding (TRB) means; and Optionally, the link between R2 and R3 is a glycine-rich linker; Fusion proteins.

2. The fusion protein of claim 1 , wherein the protease-sensitive linking means comprises a cathepsin-cleavable peptide.

3. 3. The fusion protein of claim 2, wherein the cathepsin-cleavable peptide linker is selected from the group consisting of FK, VA, VK, SEQ ID NOs: 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.

4. The fusion protein of claim 1 , wherein the TRB binding means is H7 or M16.

5. The fusion protein of any one of claims 1 to 4, wherein the fusion protein of formula I is a homodimer.

6. The fusion protein of formula I is of formula II: R4'-R3' (II) a heterodimer linked to a fusion protein of During the ceremony, R4' is an IgG Fc region; and R3' is a transferrin receptor binding (TRB) means; and Optionally, the bond between R3' and R4' is a protease-sensitive linkage; The fusion protein according to any one of claims 1 to 5.

7. The fusion protein of any one of claims 1 to 6, wherein the TRB comprises an antibody or a polypeptide.

8. The fusion protein of any one of claims 1 to 7, wherein the TRB is selected from the group consisting of SEQ ID NOs: 3, 4, and 5.

9. The fusion protein of any one of claims 1 to 8, wherein the POIB comprises an antibody.

10. The fusion protein of any one of claims 1 to 9, wherein the POIB binds to the extracellular domain of a transmembrane protein.

11. 11. The fusion protein of claim 10, wherein the extracellular region of the membrane protein comprises a chimeric antigen receptor (CAR), a receptor tyrosine kinase, a checkpoint inhibitor-binding molecule, or a cell lineage-specific marker.

12. The fusion protein of any one of claims 1 to 11, wherein the POIB binds to the extracellular domain of epidermal growth factor receptor (EGFR), programmed death-ligand 1 (PD-L1), or CD20.

13. The fusion protein of any one of claims 1 to 12, wherein the link between R2 and R3 is a glycine-rich linker selected from the group consisting of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15 and 16.

14. A nucleic acid sequence encoding the fusion protein of any one of claims 1 to 13.

15. 14. A method for treating a subject with cancer, comprising administering to said subject a fusion protein according to any one of claims 1 to 13.

16. A fusion protein according to any one of claims 1 to 13 for use in treating cancer in a patient.

17. Formula I: R1-R2-R3 (III) a homodimer of the fusion protein of During the ceremony, R1 is at least one protein of interest (POI) binder (POIB); R2 is a linker of formula R4-R5 or R5-R4, wherein R4 is an IgG Fc region; and R5 is a protease-sensitive linker; and R3 is a transferrin receptor binding (TRB) means; and Optionally, the link between R2 and R3 is a glycine-rich linker; Fusion protein homodimer.

18. 18. The homodimer of claim 17, further comprising a disulfide bond between the cysteine ​​amino acids in R4 of the separate fusion proteins.

19. Formula I: R1-R6-R3(I) a homodimer of the fusion protein of During the ceremony, R1 is at least one protein of interest (POI) binder (POIB); R6 is a dimerization means; and R3 is a transferrin receptor binding (TRB) means; the homodimer optionally has a protease-sensitive linkage between R1 and R6; Fusion protein homodimer.

20. A pharmaceutical composition comprising a fusion protein or a homodimer of a fusion protein according to any one of claims 1 to 13 or 17 to 19.

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