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

Abstract

Bispecific modulators are disclosed. The bispecific modulators may bind to proteins of interest and internalizing receptors on the cell surface. Once bound, the protein of interest may internalize and / or degrade within the cell.

Description

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

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

[0003] This patent disclosure contains copyrighted material. The copyright owner does not object to the facsimile reproduction by anyone of the patent document or patent disclosure as it appears in the patent files or records of the United States Patent and Trademark Office, but reserves any and all copyright rights in all other respects. TECHNICAL FIELD

[0004] Aspects of the present invention relate to compositions and methods for modulating molecules on the cell surface, 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 growth. SEQUENCE LISTING

[0005] This application contains a sequence listing that has been electronically submitted in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on [], is named [], and is [] bytes in size. BACKGROUND OF THE INVENTION

[0006] Traditional small molecule inhibitor-based therapies have certain drawbacks, such as toxicity and limited efficacy. Examples of inhibitor characteristics contributing to their drawbacks include binding affinity as a determinant of inhibitor potency; treatment response that depends on continuous target binding; limited function of the target protein; and efficacy disrupted by target protein overexpression, natural ligand competition, and the generation of target protein mutations that limit binding or promote drug resistance. Thus, a desired therapeutic modality exhibits low toxicity and high efficacy. For example, a desired therapeutic modality includes multiple characteristics that contribute to overall potency; a treatment response that does not rely entirely or primarily on continuous target binding; the ability to completely or significantly limit the function of the target protein; and limited or no ability of target cells to disrupt efficacy, such as through target protein overexpression, natural ligand competition, and / or the generation of target protein mutations that limit binding or promote drug resistance.

[0007] Membrane proteins play a central role in numerous cellular functions and serve as targets for more than half of all drugs. Therefore, developing strategies to degrade membrane proteins is of particular interest for both basic research and therapeutic intervention purposes.

[0008] Targeted protein degradation (TPD) is a rapidly growing area in drug discovery and pharmacology. As a complement to traditional drug modalities, TPD molecules offer novel therapeutic mechanisms to tackle 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 use heterobifunctional biologics that recruit a specific "effector" protein such as a membrane E3 ligase (Cotton, A.D., Nguyen, D.P., Gramespacher, J.A., Seiple, I.B. & Wells, J.A. 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 a lysosomal shuttle receptor (Ahn, G. et al. LYTACs that engage the asialoglycoprotein receptor for targeted protein degradation. Nat Chem Biol 17, 937-946 (2021)) to the protein of interest (POI) to induce lysosome-mediated protein degradation. However, the effectiveness of these biological effectors is often limited by their tissue-specific expression patterns. For example, GalNAc-LYTAC targets the hepatocyte-specific receptor asialoglycoprotein receptor (ASGPR), making it only suitable for treating liver diseases or clearing circulating targets (Ahn, G. et al. Nat Chem Biol 17, 937-946 (2021); Zhou, Y., Teng, P., Montgomery, N.T., Li, X. & Tang, W. Development of Triantennary N-Acetylgalactosamine Conjugates as Degraders for Extracellular Proteins. ACS Cent Sci 7, 499-506 (2021)), while RNF43- or ZNRF3-based approaches are more effective for treating Wnt signaling upregulation disorders in which RNF43 and ZNRF3 are highly expressed. Thus, current techniques cannot cover all types of diseases, and the development of alternative effectors that are overexpressed in different diseases and tissues would greatly expand the range of cell surface targets that can be modulated and also increase targeting specificity.Accordingly, there is a need to improve targeted protein degradation, particularly the targeted degradation of membrane proteins, for the treatment of a range of diseases, most notably cancer. Summary of the Invention

[0010] Disclosed herein are new reagents and methods for modulating molecules on the cell surface (e.g., a molecule of interest, such as a protein of interest). 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 on the surface of cancer cells (e.g., epidermal growth factor receptor or EGFR, programmed death ligand 1 or PDL1) to inhibit cancer cell growth or modulate the immune response. In some embodiments, the reagents and methods can be used on cells that are not cancer cells.

[0011] In some embodiments, bispecific regulators as described herein are disclosed. In some embodiments, an antigen to which a cell surface molecule can bind or an antibody or antibody fragment that can bind a cell surface molecule is fused with a ligand of an internalizing receptor or an antibody or antibody fragment that can bind an internalizing receptor or an internalizing membrane protein. In embodiments, after binding, the bispecific regulator can cause the cell surface molecule (e.g., a molecule of interest, such as a protein of interest) to be internalized by the cell, and in some embodiments, the internalized cell surface molecule can be degraded. In various embodiments, the bispecific regulator can target single or multi-pass transmembrane proteins.

[0012] In some embodiments, reagents and methods for improving the degradation of internalized cell surface proteins using the bispecific regulators disclosed herein are disclosed. In some embodiments, a peptide linker sensitive to certain proteases is inserted into the bispecific regulator. In some embodiments, the peptide linker is sensitive to cathepsin.

[0013] Nucleic acids encoding these molecules, vectors containing these nucleic acids, and cells containing these vectors and / or expressing bispecific regulator molecules as disclosed herein are disclosed.

[0014] In some embodiments, methods of administering a bispecific regulator to a subject are disclosed. Brief Description of the Drawings

[0015] Certain illustrations, diagrams, or flowcharts are provided to allow for a better understanding of the present invention. However, it should be noted that the drawings only show selected embodiments of the present invention and should not be considered to limit the scope. There are additional and equally valid embodiments and applications of the present invention.

[0016] Figure 1It is a schematic diagram showing CAR-T cell therapy in patients.

[0017] Figure 2 Exemplary methods of treating toxicities observed after CAR-T cell therapy using immunosuppressive agents are shown.

[0018] Figure 3 Exemplary methods of treating toxicities observed after CAR-T cell therapy using suicide genes or ablation markers are shown.

[0019] Figure 4 Exemplary methods of treating toxicities observed after CAR-T cell therapy using reversible genetic switches are shown.

[0020] Figure 5 A reversible CAR-T regulatory mechanism as a strategy to enhance CAR-T cell efficacy is shown.

[0021] Figure 6 Exemplary methods for controlling CAR-T cell activation using targeting CAR internalization and / or degradation (bispecific modulators, including TransTAC molecules) (TransTAC is Trans ferrin receptor-mediated Targeted Chimera ( Trans ferrin receptor-mediated TA rgeting C himera)) are shown.

[0022] Figure 7 An exemplary schematic diagram of the TransTAC technology (e.g., a type of bispecific modulator) is shown. An extracellular protein (e.g., a membrane protein having an extracellular domain) can be selectively internalized and degraded by tethering an antibody (or a ligand of the target membrane protein) against the target membrane protein to the transferrin receptor using a bispecific modulator (shown as a membrane protein-specific antibody-transferrin fusion protein).

[0023] Figure 8 Another exemplary schematic diagram of the bispecific modulator / TransTAC technology is shown. This is not discussed in the text.

[0024] Figure 9 Another exemplary schematic diagram of the bispecific modulator / TransTAC technology is shown. This is not discussed in the text.

[0025] Figure 10The results are shown that display the expression of the anti-EGFR affibody Fc-Tr TransTAC molecule (left) and the effect on the EGFR level upon incubation of the TransTAC molecule with the MCF10A EGFR overexpressing cell line (right).

[0026] Figures 11A - 11B show the results that display the effect on the EGFR level (A) and the killing of MCF10A EGFR cells (B) upon incubation of the TransTAC molecule with A549 cells.

[0027] Figure 12 The results are shown that display the effective internalization of the receptor by TransTAC targeting.

[0028] Figure 13 Exemplary results are shown that display the expression of various TransTAC proteins in cultured cells.

[0029] Figure 14 The results are shown that display the effective reduction of the CAR level (e.g., internalization of the CAR) by TransTAC targeting for a CAR specific to CD19.

[0030] Figure 15 Exemplary results are shown that display the effective internalization of the CAR by TransTAC targeting for a CAR specific to CD19.

[0031] Figure 16 Exemplary results are shown that display the inhibition of Jurkat cell activation by TransTAC targeting for a CAR specific to CD19 on Jurkat cells in the presence of K562 cells. Figure 17 Exemplary results are shown that display the internalization of the CAR / inhibition of CAR-T activation by TransTAC targeting for a CAR specific to CD19.

[0032] Figure 18 The results are shown that display the blocking of CAR-T cell activation by a CD19-specific TransTAC molecule in the presence of K562 cells and also show the minimal effect of the TransTAC molecule on Jurkat cells in the absence of K562 cells.

[0033] Figures 19A - 19B show a schematic diagram of a CARTrap molecule (a domain to which the CAR can bind fused to an Fc region), a TransTAC molecule that can bind the CAR and internalize the receptor, and a dimer of the TransTAC molecule that can bind the CAR and internalize the receptor (A) and the results (B) of using these molecules at the CAR level on CAR-expressing cells.

[0034] Figure 19C Shows fluorescence microscopy of the target CAR on the cells in Figure 19B.

[0035] Figures 20A - 20B, Figure 20C , Figures 20D - 20E, Figures 20F - 20G, Figure 20H and Figure 20I Show examples where the internalized CAR is not degraded but engineering of the linker (here, incorporation of a cathepsin-sensitive linker) leads to CAR degradation. (A) Shows a schematic of the various molecules used in this study. GFLG indicates a Gly-Phe-Leu-Gly peptide linker sensitive to lysosomal cathepsins. (B) Shows a western blot of the target CAR (anti-CD3z) and an actin control (β-actin) when using the various molecules in (A). (C) Shows a graph of the data from (B) where the CAR level is normalized relative to the β-actin level. (D) Shows a western blot as described above using other molecules in (A). (E) Shows a graph of the normalized data from (D). (F) Shows a schematic of a dimer of a TransTAC molecule that can bind the CAR and the internalized receptor (this molecule also contains the GFLG linker). (G) Shows a western blot using the molecule shown in (F). (H) Shows a graph of the normalized data from (F). (I) Shows the results of screening additional cathepsin-sensitive TransTAC molecules with improved inhibitory potency.

[0036] Figures 21A - 21B show results demonstrating that the inhibition of T cell activity by TransTAC is more effective than the inhibition by CARTrap (a domain that can bind the CAR fused to the Fc region) in Jurkat cells (A) and in primary T cells (B).

[0037] Figure 22A and Figure 22B Show results demonstrating that CAR-TransTAC shuts off tumor cell killing by CAR-T cells and that CAR-T tumor cell killing is restored when the TransTAC molecule is removed.

[0038] Figures 23A - 23B show a schematic of the molecules used in the study (including an affibody-based EGFR TransTAC molecule) (A). (B) Shows the results of removing EGFR from the surface of A549 cells using the molecules shown in (A). The data show a ~10 - 50-fold improvement in IC 50 compared to that.

[0039] Figures 23C - 23D show the results of the inhibition of cell proliferation by the molecule shown in (A) as measured using the MTT cell proliferation assay. The data show that good results were obtained using the affibody-based TransTAC molecule (IC 50 of approximately 10 - 50-fold improvement).

[0040] Figures 24A - 24B show a schematic representation of the molecule (B) used in this study and the Western blot results (C) measuring molecule internalization and degradation.

[0041] Figure 24C A graph showing the normalized data from Figure 54(C) is shown.

[0042] Figures 25A - 25B present exemplary data demonstrating protein internalization by TransTAC and the reversibility of internalization.

[0043] Figure 26 Exemplary data demonstrating that TransTAC can interfere with IFNγ production are shown.

[0044] Figure 27A and Figure 27B Exemplary data showing transferrin receptor expression on various cells are shown.

[0045] Figure 28 Some examples of cell surface molecules that can be regulated by TransTAC are shown.

[0046] Figure 29A and Figure 29B Exemplary data demonstrating that TransTAC can degrade EGFR in cells and the potential cellular mechanisms mediating the degradation are shown.

[0047] Figure 30A 、 Figure 30B and Figures 30C - 30D show exemplary methods of treating lung cancer with TransTAC.

[0048] Figure 31 Exemplary data demonstrating that TransTAC with linker variants can degrade CAR in CAR-Jurkat cells are shown.

[0049] Figure 32A and Figure 32B Exemplary data demonstrating that TransTAC can degrade PD-L1 in breast cancer cells are shown.

[0050] Figure 33 Exemplary data demonstrating that TransTAC can degrade CD20 in lymphoma cells are shown.

[0051] Figures 34A - 34C andFigure 34D Exemplary TransTAC molecules containing protease-sensitive linkers and exemplary data obtained with these molecules are shown.

[0052] Figure 35 Exemplary data obtained with TransTAC molecules containing various protease-sensitive linkers are shown.

[0053] Figures 36A - 36C show exemplary TransTAC molecules containing antibody fragments specific for transferrin binding and exemplary data obtained with these molecules.

[0054] Figures 37A - 37B show examples of TransTAC molecules and exemplary data obtained with these molecules.

[0055] Figures 38A - 38E and 38F - 38H show an exemplary overview of the TransTAC technology and TfR expression analysis. (A) Schematic of an exemplary TransTAC technology. TransTAC induces close proximity of TfR and POI at the cell surface, resulting in co - internalization of the complex into early endosomes (EE), where cathepsin cleaves TransTAC and separates POI from TfR. Then the POI is transported to late endosomes (LE) / lysosomes for degradation, while TfR recycles back to the cell surface. (B) Illustration of an exemplary TransTAC protein. Some exemplary designs for preparing effective TransTAC degraders include: (1) two anti - TfR conjugates that contain binding and triggering of TfR dimerization for endocytosis, (2) a cathepsin B - sensitive linker between the anti - POI conjugate and Fc for endosomal cleavage to separate POI from recycling TfR, and (3) use of antibody conjugates instead of native TF ligands to reduce transport to recycling endosomes (RE). (C) Relative cell surface TfR expression levels in various non - oncogenic and cancer cell lines characterized by flow cytometry. Cancer cell lines express higher levels of TfR compared to non - oncogenic cell lines. Data represent 3 independent experiments. (D) Relative TFRC RNA expression levels in primary tumors compared to normal tissues based on the MERAV database. TFRC expression is significantly higher in most tumors compared to the corresponding normal tissues. (T - tests in Figures 38F and 38G show significance in the overall comparison of tumors to healthy tissues (p = 3.98e - 89), and in 14 out of 19 individual tumor / healthy tissue pairs. Female reproductive tissues are endometrium, cervix, fallopian tube, myometrium, ovary, placenta, and uterus. Central nervous system (CNS) tissues are basal ganglia, brainstem, cerebral cortex, hippocampus, spinal cord, and superior vestibular nucleus. Brain tissues are hypothalamus, pituitary gland, thalamus, ganglia, and nodose ganglion). (E) Relative TFRC RNA expression levels in natural T cells. Compared to unactivated T cells, TfR is upregulated approximately 6 - fold in activated CD4 and CD8 T cells with statistical significance (p = 1.25e - 68 for CD4 T cells and 4.81e - 68 for CD8 T cells, Figure 38H).

[0056] Figures 39A - 39L illustrate exemplary TransTAC degrader engineering. (A) Schematic diagrams of exemplary CAR-TransTACs and controls. TransTAC v0.1 has a single CD19NT.1 domain, a single TF, and a knob-in-hole (KIH) Fc, v0.2 has two CD19NT.1s, two TFs, and a homodimeric Fc linking the conjugates, v0.4 contains a cathepsin-sensitive linker between CD19NT.1 and Fc, v0.5 contains an H7 scFv for TfR binding, and v1.0 contains both H7 and a cathepsin-sensitive linker. (B) Schematic diagram of the myc-tagged anti-CD19 CAR receptor. (C) Flow cytometry measurement of cell surface CAR expression levels in CAR Jurkat treated with TransTAC v0.1, v0.2, and controls. TransTAC v0.2 led to higher CAR clearance from the cell surface compared to v0.1 and had no hook effect. Data represent 2 independent experiments. (D) Whole-cell CAR levels in CAR Jurkat treated with TransTAC were characterized by Western blot. TransTAC v1.0 degraded approximately 80% of the CAR; v0.2 did not result in significant CAR degradation. (E - H) Schematic diagrams showing the intracellular trafficking of the POI altered by different TransTACs. Cleavage of the cathepsin-sensitive linker in v0.4 and v1.0 results in the separation of the POI from the TfR, thus enhancing the LE / lysosome trafficking and degradation of the POI; when the cleavage linker is present, the H7 scFv in v0.5 and v1.0 reduces the trafficking of the complex to the RE, thus increasing the proportion of the POI in the EE and subsequent proteolytic processing. (I, J) Representative fluorescence images of Hela cells co-expressing CAR-GFP (green) and endosome / lysosome marker - mCherry (red) treated with various TransTAC molecules. The nuclei were stained with Hochest (blue). Untreated (UT) or control-treated cells had CAR-GFP located at the cell membrane. v0.5 and v1.0 led to efficient degradation of CAR-GFP, manifested as significantly lower GFP signals. Cells treated with v0.2 mainly transported CAR to the RE, showing co-localization of CAR-GFP with mCherry-Rab11 (white arrow). Cells treated with v0.5 transported CAR to the EE, showing co-localization of CAR-GFP with mCherry-Rab5 (white arrow). (K) Pearson correlation coefficient analysis of the co-localization of CAR-GFP with Rab5 (EE), EEA1 (EE), and Rab11 (RE) markers. T-test showed that the co-localization of Rab5, EEA1, Rab11 with CAR was statistically different for cells treated with v0.2 compared to cells treated with v0.5.(L) Pearson correlation coefficient analysis of co-localization of CAR-GFP with Rab7 (LE) and Lamp1 (lysosome) markers. T-test showed that co-localization of Rab7 and Lamp1 with CAR was statistically significant for v0.2 compared to v0.4 and v0.5 compared to v1.0. For k and l, the number of cells used for each analysis was as follows: for v0.2, for EEA1, Rab5, and Rab11 markers, N = 12, N = 12, and N = 13, respectively. For v0.5, for EEA1, Rab5, and Rab11 markers, N = 10, N = 22, and N = 15, respectively. For v0.2, v0.4, v0.5, and v1.0 with Lamp1 marker, N = 16, N = 21, N = 13, and N = 13, respectively.

[0057] Figures 40A - 40D show examples of developing TransTAC degrading agents for various membrane targets. (A) Schematic diagram of membrane proteins targeted by TransTAC in this study. These targets are synthetic or natural single-pass or multi-pass 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 form of atezolizumab was used as the PDL1-binding moiety. (C) Degradation of EGFR by TransTAC in A549 lung cancer cells. An affibody was used as the EGFR-binding moiety. (D) Degradation of CD20 by TransTAC. The Fab form of rituximab was used as the CD20-binding moiety.

[0058] Figures 41A - 41H and Figure 41IExemplary structure-activity relationship (SAR) studies of TransTAC, mechanism, and in vivo characterization are shown. (A) Time-course measurements of cell surface CAR levels in CAR-Jurkat treated with TransTAC revealed rapid kinetics of TransTAC-mediated CAR internalization. (B) Schematic of CAR-TransTAC variants consisting of one or two copies of anti-POI and anti-TfR conjugates with different protein geometries. (C) Measurement of cell surface CAR levels in CAR-Jurkat treated with the CAR-TransTAC variants outlined in (B). The results highlight the impact of having two versus one TfR conjugate (v0.5 versus v0.7) and geometry (v0.8 versus v0.9) on modulating protein internalization. Data represent 3 independent measurements. (D) Competition assay with H7-Fc fusion protein. A concentration-dependent decrease in CAR internalization was observed in the case of H7-Fc, demonstrating that internalization is mediated by TfR. Data represent 3 independent measurements. (E) Investigation of potential degradation pathways with TransTAC. Intact lysosomal function is crucial for degradation, as degradation was completely inhibited by bafilomycin in A549 cells treated with EGFR-TransTAC. (F) Measurement of total cellular TfR levels in the case of TransTAC treatment. In MDA-MB-231 cells treated with PDL1 TransTAC, the TfR levels remained unchanged while PD-L1 was degraded. (G) Schematic of a mouse experiment to evaluate TransTAC safety and serum half-life via IP injection. (H) Monitoring of mouse body weight over time after TransTAC or control IgG injection. The results revealed no observable effect on mouse body weight over time, indicating that the molecule is well tolerated. N = 2 per treatment group. (I) Western blot quantification of plasma levels of CD20-TransTAC and IgG control over time. N = 2 per treatment group.

[0059] Figures 42A-42G show examples of EGFR-TransTAC targeting TKI-resistant lung cancer cells. (A) Schematic representation of the development of resistant mutations in lung cancer cells and available treatment options. EGFR Del19 and L858R mutants can be targeted by first- and second-generation TKIs, T790M can be targeted by osimertinib, but cells with an additional C797S mutation have no available targeted therapy options. (B) Schematic of EGFR TransTAC designed with different cleavable linkers and TfR conjugates. (C) Cell viability assay of PC9 WT cells treated with the EGFR TransTAC variants shown in (B). v0.5 and v1.0 resulted in effective cell inhibition; the affibody-Fc control or v0.2 had no effect. Data represent 3 independent experiments. (D) Western blot showing efficient TransTAC1.0-mediated EGFR degradation in PC9WT cells and PC4 GR4 C797S cells. (E) Cell viability assay of lung cancer cells PC9 WT, PC9 GR4, PC9 GR4 C797S and normal fibroblast cell line HFF-1 treated with TransTAC and TKI. PC9 WT cells responded to all three TKIs; PC9-GR4 containing the T790M mutation was resistant to gefitinib; PC9 GR4 C797S was resistant to osimertinib in addition to gefitinib and afatinib; all three PC9 cell lines were inhibited by EGFR-TransTAC. Neither TKI nor TransTAC caused significant toxicity in the HFF-1 cell line. Data represent 3 independent experiments. (F) Efficacy and specificity of TransTAC were tested in co-culture assays of PC9 WT cancer cells and HFF-1 healthy cells compared to combinations of TKI and carboplatin / paclitaxel chemotherapy. PC9 WT cells and HFF-1 cells expressed GFP and mCherry, respectively. TransTAC and TKI specifically inhibited PC9 WT cancer cells without harming HFF-1 cells; chemotherapy inhibited both cell types. (G) Experiment of (F) using co-cultures of PC9 GR4 C797S cancer cells and HFF-1 healthy cells. TransTAC and chemotherapy but not TKI inhibited cancer cells; additionally, TransTAC and TKI did not cause cytotoxicity to HFF-1 cells, but chemotherapy treatment inhibited HFF-1.

[0060] Figures 43A - 43B show exemplary characterizations of wild-type (WT) CD19 extracellular domain and variant Fc fusions. CD19ecto-WT-Fc showed aggregation in SDS-PAGE gels, while the variants derived from yeast showed no aggregation. Among the four variants, CD19NT.1 was selected for CAR-TransTAC engineering given its high expression level.

[0061] Figures 44A - 44E show exemplary different CAR degradation efficiencies mediated by TransTAC variants. (A) Schematic diagrams of different generations of CAR-TransTAC and CD19NT.1-Fc controls. (B) Western blot showing that neither the control nor v0.2 led to CAR degradation. (C) Western blot showing that v0.4-GFLG, which contains a cathepsin-sensitive GFLG linker between CD19NT.1 and the Fc domain, led to approximately 40% - 50% CAR degradation. v0.3-GFLG, which contains a cleavable linker between the Fc and TF domains, did not lead to significant CAR degradation, probably due to Fc-mediated CAR recycling. (D) Western blot showing that different linker variants of v0.4 led to different CAR degradation efficiencies. (E) Western blot showing that different linker variants of v1.0 led to different CAR degradation efficiencies. Among all variants, the linkers GFLG-VR and VR showed the highest degradation.

[0062] Figures 45A - 45D and Figures 45E - 45F show exemplary co - localization analyses of internalized CAR with various endosomal / lysosomal markers. (A - C) Representative fluorescence images of Hela cells co - expressing CAR - GFP (green) and endosomal / lysosomal marker - mCherry (red) treated with various TransTACs or controls. EEA1: EE marker, Rab7: LE marker, Lamp1: lysosomal marker. The nuclei were stained with Hochest (blue). (D) Pearson correlation coefficient analysis of the co - localization of CAR - GFP with five endosomal / lysosomal markers. T - test showed that the co - localization of Rab5, EEA1, Rab11 with CAR was statistically different for cells treated with v0.2 compared to those treated with v0.5, and the co - localization of Rab7 and Lamp1 with CAR was statistically significant for v0.2 compared to v0.4 and v0.5 compared to v1.0. The number of cells used for analysis was as follows: for control, UT, v0.2, v0.4, v0.5, and v1.0, for the EEA1 marker, N = 5, N = 4, N = 12, N = 15, N = 10, and N = 11 respectively. For control, UT, v0.2, v0.4, v0.5, and v1.0, for the Rab5 marker, N = 12, N = 11, N = 12, N = 15, N = 22, and N = 17 respectively. For control, UT, v0.2, v0.4, v0.5, and v1.0, for the Rab7 marker, N = 9, N = 7, N = 8, N = 12, N = 26, and N = 11 respectively. For control, UT, v0.2, v0.4, v0.5, and v1.0, for the Rab11 marker, N = 9, N = 6, N = 13, N = 13, N = 15, and N = 22 respectively. For control, UT, v0.2, v0.4, v0.5, and v1.0, for the Lamp1 marker, N = 17, N = 12, N = 16, N = 21, N = 13, and N = 13 respectively. (E - F) Incorporating a cathepsin - sensitive linker into TransTAC enhances LE / lysosomal trafficking. Representative fluorescence images of Hela cells co - expressing CAR - GFP (green) and mCherry - Rab7 or Lamp1 - mCherry (red) treated with TransTAC v0.2 compared to v0.4. The nuclei were stained with Hochest (blue). The v0.4 GFP images were collected with 1000x more exposure than the v0.2 images to obtain sufficient GFP signal for Figure 2 the Pearson coefficient analysis in i. Cells treated with v0.4 showed co - localization of internalized CAR - GFP with mCherry - Rab7 and Lamp1 (white arrows).

[0063] Figures 46A - 46C show examples of TransTAC degrading agents that characterize various membrane proteins. Different linker and geometric structure designs result in different degradation efficiencies. (A) Western blot showing that the control or v0.2 and v0.4 PDL1 TransTAC variants do not cause much target degradation in MDA-MB-231 cells. (B) Western blot showing that EGFR TransTAC v0.2 does not cause much target degradation in A549 cells, while v0.4 or v1.0 with different linkers cause different degrees of degradation, where v1.0-EVR and GFLG-VR exhibit the highest degradation efficiency. (C) Western blot showing that the rituximab-scFv-Fc control does not cause significant CD20 degradation in Raji cells.

[0064] Figures 47A - 47E show examples of TransTAC modulating primary CAR-T cell activity. (A) Schematic diagram of reversibly controlling CAR-T cells using CAR-TransTAC. TransTAC-mediated removal of CAR from the cell surface prevents CAR-T cells from engaging CD19+ tumor cells, thus inhibiting cytokine release and cytotoxicity. (B) Schematic diagram of the setup of a primary CAR-T cell co-culture assay. The secreted IFN-γ level is measured to determine the level of CAR-T cell activation in the presence of CD19+ A375 cells and TransTAC; live cell fluorescence microscopy is used to determine the anti-tumor effect. (C) Measurement of IFN-γ release from human primary CAR-T cells in the co-culture assay described in (b) with the IFNγ split-luciferase assay (Promega). IFN-γ secretion is inhibited in a dose-dependent manner by TransTAC v0.4. TransTAC shows an IC50 of approximately 0.4 nM. Data represent 2 independent experiments. (D) Fluorescence microscopy of mCherry-labeled A375 cells showing reversibly controlling CAR-T cell-mediated killing of A375 by CAR-TransTAC v4. (E) Overlay of bright field and mCherry channel images showing the restoration of CAR-T cell-mediated A375 killing activity over time after TransTAC clearance.

[0065] Figures 48A - 48C show exemplary characterization of EGFR TransTAC. (A) Western blot showing that EGFR-TransTAC results in 40% - 50% target degradation in HEK293 cells overexpressing EGFR, a level significantly lower than in A549 and PC9 cells, likely due to lower TfR expression levels. (B) IC50 of TransTAC v1.0 and TKIs afatinib, gefitinib, and osimertinib in PC9 cells based on data presented elsewhere. (C) Flow cytometry analysis of PC9(GFP) / HFF-1(mCherry) cell ratios, which reflect differential sensitivities of tumor / healthy cells to various treatments. Data represent 3 independent experiments.

[0066] Figure 49A - Figure 49B Shows cleavage of the indicated linker on yeast by recombinant cathepsin B at pH 4.4 compared to cleavage of GFLGGVR (SEQ ID NO:144). (B) Cleavage of the indicated linker on yeast by recombinant cathepsin B at pH 6.4 compared to GFLGGVR (SEQ ID NO:144). Detailed Description

[0067] Targeted protein degradation (TPD) is a rapidly growing area in drug discovery and pharmacology. As a complement to traditional drug modalities, TPD molecules offer novel therapeutic mechanisms to tackle challenging targets, enhance the therapeutic potential of currently used drugs, etc. While many efforts in the field have focused on small molecules for intracellular targets, inducing the targeted degradation of extracellular proteins is a new opportunity. Developing strategies to degrade extracellular proteins is of particular interest for both basic research and therapeutic intervention purposes.

[0068] Iron is an essential element for cells, and the transport of iron is facilitated by the transferrin receptor (TfR). TfR undergoes rapid endocytosis as a recycling receptor with an average internalization rate of 500 molecules / cell / second, making it one of the fastest internalizing receptors known. Additionally, in cells with high iron demand, TfR is upregulated. This includes 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 at sufficient levels on non-cancerous cells such that the reagents and methods described herein can be used.

[0069] In this article, these features of TfR are exploited and protein engineering strategies are employed to develop new technologies for degrading membrane proteins. In this article, this technology is called receptor-mediated Targeted Chimera (receptor-mediated Ta rgeting C himeras , TransTAC). In some embodiments, TransTAC is a bispecific antibody that brings the protein of interest (POI) and TfR very close together on the cell surface and induces endocytosis of the POI / TfR complex and subsequent lysosome-mediated degradation of the POI. TransTAC effectively degrades various types of membrane proteins, including single-pass transmembrane receptors, multi-pass transmembrane receptors, native receptors, and synthetic receptors, showing a degradation efficiency of over 80% for all targets in various cell systems. A notable feature of TransTAC is its rapid kinetics of targeted internalization, which occurs on a time scale of minutes, making it a valuable molecular tool for rapid knockdown of cell surface expression and providing temporal specificity that is not achievable genetically for cell signaling studies. Additionally, the TransTAC molecule is fully recombinant, modular, and cancer-specific. These properties make TransTAC a general technology for manipulating cell surface targets in a disease-specific manner.

[0070] TransTAC can have broad applicability in basic research and translational applications. Non-limiting applications of TransTAC in the reversible control of CAR-T cells, targeting of drug-resistant EGFR-positive lung cancer, etc. are demonstrated herein. TransTAC represents a new molecular prototype for controlling cell surface proteins.

[0071] TransTAC is the first bispecific antibody technology that re-uses recycling ligand / receptor interactions for targeted protein internalization and degradation, which can significantly expand the range of cell surface effectors suitable for such purposes.

[0072] Chimeric antigen receptor (CAR) T cells have emerged as a promising therapy for patients with hematological malignancies ( Figure 1 ). However, in some cases, CAR-T therapy can cause side effects in patients receiving these cells, including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS).

[0073] Several regulatory mechanisms for controlling CAR-T cells in vivo have been developed to address these adverse events. However, these strategies have not satisfactorily met the current needs, as there have been continuous reports of toxicity and death in CAR-T clinical trials.

[0074] This disclosure describes new, modular, and reversible strategies for modulating CAR-T cell activity. Generally, these methods do not require additional genetic engineering of CAR-T cells. In some embodiments, these strategies can modulate CAR-T toxicity. In some embodiments, these strategies can increase the efficacy of CAR-T cell therapy. In some embodiments, these strategies are based on the reversible internalization of CAR receptors.

[0075] In some embodiments, bispecific modulators (including, for example, Trans ferritin receptor-mediated Targeted Chimera or TransTAC molecules) can co-localize CAR receptors to internalizing cell surface proteins. In embodiments, the bispecific modulator can downregulate the cell surface levels of CAR. In embodiments, the bispecific modulator can inhibit CAR-T cell activation and / or function.

[0076] In embodiments, the bispecific modulator can have a first portion (portion or moiety) that is an antibody, antibody fragment, or alternative antibody scaffold that specifically binds a target molecule on the cell (e.g., a molecule of interest, such as a protein of interest, like CAR, EGFR, CD20); and a second portion (e.g., transferrin or an antibody or antibody fragment) that can bind an internalizing molecule on the cell surface (e.g., the transferrin receptor). In embodiments, the bispecific modulator can have a first portion that is an antigen or ligand of a target protein on the cell (e.g., the CD19 antigen or a variant thereof for a CD19-specific CAR). In embodiments, the bispecific modulator can have a first portion that is an antibody or antibody fragment that can specifically bind a target molecule on the cell (e.g., a molecule of interest). The bispecific modulator can have a second portion that binds an internalizing protein on the cell surface (e.g., the transferrin receptor). Binding of the bispecific modulator to the target molecule and the internalizing protein results in the internalization of the target molecule.

[0077] In some embodiments, the bispecific modulator does not require engineering 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.

[0078] In other embodiments, the bispecific modulator can be reversible. Reversibility can provide fine-tuning of CAR-T cell activity, e.g., for toxicity management and / or for restoring cells for continued therapy.

[0079] In some embodiments, the bispecific modulator can be customized for CAR-T cells targeting different tumor antigens, for example, by replacing components used in the design (i.e., the capture agent and / or modulator can be modular).

[0080] Methods for enhancing CAR-T efficacy are also disclosed. The temporal "quiescence" of CAR-T cells can reverse CAR-T exhaustion. In some embodiments, the disclosed reversible CAR modulators can increase the efficacy of CAR-T cells by alternating the CAR-T cells between "active" and "quiescent" states.

[0081] Methods for targeting cancer cells, including cancer cells with drug-resistant mutations, are also disclosed. Cancers can rapidly evolve to evade therapy, often developing drug-resistant mutations that lead to treatment failure and disease recurrence. For example, the C797S mutation in EGFR poses challenges in the treatment of non-small cell lung cancer (NSCLC). The C797S mutation occurs in approximately 10%-26% of NSCLC patients after treatment with the third-generation EGFR tyrosine kinase inhibitor (TKI) osimertinib, and this mutation affects the key residue C797 that forms a covalent bond with irreversible TKIs. As a result, existing TKIs become ineffective against the disease. There is a need to develop drugs that target these drug-resistant oncogenes.

[0082] The development of EGFR TransTAC degraders that target EGFR-driven lung cancers, including patient populations with the C797S mutation, is disclosed. It is shown herein that (1) EGFR TransTAC can effectively degrade drug-resistant EGFR mutant proteins and thus inhibit cancer growth, and (2) EGFR TransTAC can specifically target cancer cells while sparing healthy cells due to the overexpression of TfR on cancer cells. The reagents and methods disclosed herein can be used for cells that are not cancer cells.

[0083] 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 those skilled in the art to employ the present invention in any suitable manner.

[0084] The singular forms "a / an" and "the" include plural referents unless the context clearly dictates otherwise. In the claims and / or the specification, when used in conjunction with the term "comprising," the use of the word "a / an" can refer to "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0085] Whenever any of the phrases "for example", "such as", "including", etc. are used herein, unless otherwise expressly specified, they shall be understood to be followed by the phrase "and without limitation". Similarly, "example", "exemplary", etc. shall be understood to be non-limiting.

[0086] The term "substantially" permits deviation from the descriptor that does not negatively affect the intended purpose. Descriptive terms shall be understood to be modified by the term "substantially" even if the word "substantially" is not explicitly recited.

[0087] The terms "comprising", "including", "having", and "involving" (and similarly "comprises", "includes", "has", and "involves") are used interchangeably and have the same meaning. In particular, the definition of each of these terms is consistent with the common definition of "comprising" under United States patent law and is thus construed as an open term meaning "at least the following" and is also construed as not excluding additional features, limitations, aspects, etc. Thus, for example, "a process involving steps a, b, and c" means that the process includes at least steps a, b, and c. Whenever the term "a" or "an" is used, it shall be understood to mean "one or more" unless such an interpretation is meaningless in the context.

[0088] As used herein, the term "about" may mean approximately, roughly, generally, or within a range of. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the upper and lower boundaries of the numerical values shown above. The term "about" is used herein to modify a change in a numerical value by plus or minus 20 percent (higher or lower) above and below the stated value. CAR - T Cells, Toxicity, and Controlling Toxicity

[0089] Chimeric antigen receptor (CAR) T cells have emerged as a promising treatment for patients with advanced B cell cancers ( Figure 1)。However, due to the lack of control over the infused CAR-T cells, the widespread use of the therapy may be limited by potentially life-threatening toxicities. Toxicity is an obstacle to the development of CAR-T therapies for blood cancers and solid tumors. Deaths reported as a result of CAR-T therapy have recently been discussed in the literature (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).

[0090] Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) are the two most common toxicities observed after CAR-T cell therapy.

[0091] CRS can be characterized by high fever, hypoxia, hypotension, or multi-organ toxicity; it develops in 37%-93% of lymphoma patients and 77%-93% of leukemia patients. ICANS is characterized by confusion, delirium, seizures, or cerebral edema; it develops in 23%-67% of lymphoma patients and 40%-62% of leukemia patients. Severe CRS and ICANS require monitoring and treatment in an intensive care setting, and multiple deaths have been reported due to unmanageable CRS or ICANS toxicities.

[0092] Currently, three types of treatments are being used for these toxicities.

[0093] In some cases, with immunosuppressants ( Figure 2) Treating patients, these immunosuppressive agents include systemic corticosteroids, IL-6 receptor antibodies (e.g., tocilizumab), lymphocytotoxic anti-CD52 antibodies (e.g., alemtuzumab), tyrosine kinase inhibitors (e.g., dasatinib) (LCK inhibitors do not inhibit already activated T cells), etc. However, these treatments have limitations. For example, treating with high-dose steroids can limit the time span during which CAR-T cells function and can induce hematological aplasia and toxicity. Anti-IL6 receptor antibodies have multiple biological activities and can non-specifically inhibit the immune system (Bonifant, Challice L. et al. “Toxicity and management in CAR T-cell therapy [Toxicity and management in CAR T-cell therapy].” Molecular Therapy-Oncolytics [Molecular Therapy-Oncology] 3 (2016): 16011).

[0094] In some cases, treating patients with suicide genes or ablation markers ( Figure 3 ), these markers include iCasp9, anti-CD20 (e.g., rituximab), anti-EGFR (e.g., cetuximab), etc. However, these treatments have limitations. For example, these treatments can irreversibly and / or permanently eliminate CAR-T cells from the body (Brandt, JB et al. “Emerging approaches for regulation and control of CAR T cells: a minireview [Emerging approaches for regulation and control of CAR T cells: a minireview].” Frontiers in Immunology [Frontiers in Immunology] 11 (2020): 326).

[0095] In some cases, CAR-T cells with switchable CAR receptors can be used for patients ( Figure 4 ), including split CAR, SMaSh-CAR, CAR PROTAC, etc. However, these treatments have limitations. For example, these treatments may impair CAR-T activity, the switch may be leaky, and the switch may be immunogenic (Labanieh, Louai et al. “Enhanced safety and efficacy of protease-regulated CAR-T cell receptors [Enhanced safety and efficacy of protease-regulated CAR-T cell receptors].” Cell [Cell] 185.10 (2022): 1745-1763).

[0096] However, reversible CAR-T regulatory mechanisms are known to be useful for enhancing CAR-T efficacy ( Figure 5 ). Constitutive CAR-T cells can exhibit increased levels of exhaustion-related proteins. However, in some embodiments, transient "rest" can reverse the exhausted phenotype. In some embodiments, the regulated CAR can be reversibly turned off and on to switch the CAR-T cell between "off" and "on" states (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).

[0097] 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. Bispecific Modulator

[0098] In some embodiments, the strategies disclosed herein for modulating a molecule on the cell surface (e.g., a molecule of interest, such as a protein of interest) and / or the regulatory activity of such a molecule can use a bispecific modulator approach. In some embodiments, the bispecific modulator molecule can have at least two parts. The first part can be a ligand to which one or more cell surface molecules can bind or an antibody or antibody fragment that can bind one or more cell surface molecules (e.g., a molecule of interest, such as a protein of interest). The second part can be a molecule that can bind an internalization receptor or membrane protein on the cell. In an embodiment, the second molecule can be an antibody or antibody fragment that binds an internalization receptor or membrane protein on the cell. In an embodiment, the bispecific modulator can be a bispecific antibody.

[0099] In some embodiments, the bispecific modulator method can modulate molecules other than those on the cell surface. In some embodiments, the bispecific modulator can bind and internalize (and optionally degrade) proteins present in the extracellular / external environment. In some embodiments, these can be soluble proteins. In some embodiments, by way of non-limiting example, these proteins can include autoantibodies, cytokines, enzymes, and the like.

[0100] In embodiments, the bispecific modulator having these two moieties can bind to or be bound by the cell surface or other molecules (e.g., a molecule of interest, such as a protein of interest), and can bind to an internalization receptor or a membrane protein. After such binding, the internalization receptor or membrane protein can cause the cell surface or other molecules to be internalized into the cell (e.g., endocytosis). In embodiments, the internalized cell surface or other molecules can be degraded. In embodiments, this reduces the amount of cell surface molecules on the cell surface. In embodiments, the internalized cell surface molecules are not functional. In some embodiments, the cell surface molecule targeted by the first moiety of the bispecific modulator is different from the molecule targeted by the second moiety.

[0101] In some embodiments, administering a bispecific modulator to a subject can be used for targeted internalization of a membrane or other protein. In some embodiments, administering a bispecific modulator to a subject can be used for targeted degradation of a membrane or other protein.

[0102] In some embodiments, adding a bispecific modulator to cells or administering it to a patient can cause targeted internalization and / or degradation of proteins on the cell surface or extracellularly. In some embodiments, this internalization / degradation is reversible. For example, when the cells are no longer exposed to the bispecific modulator, the membrane protein to which the bispecific modulator is specific is no longer internalized / degraded. Generally, the membrane protein is still synthesized and transported to the cell membrane. Thus, when the bispecific modulator is removed or no longer administered to the subject, there is no stimulant to internalize / degrade the protein. In some embodiments, a bispecific modulator that also contains a protease-sensitive linker can be used to both internalize and degrade cell membrane proteins that can be internalized by the bispecific modulator but not degraded. As discussed elsewhere, placing a protease-sensitive linker within the bispecific modulator can release the targeted cell protein of interest from the bispecific modulator inside the cell.

[0103] In some embodiments, the internalization and degradation of cell surface or other molecules (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 the cell surface molecule is EGFR). In some embodiments, the internalization and degradation of cell surface or other molecules 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 the cell surface or other molecule is a CAR).

[0104] In some embodiments, the internalization of a bispecific modulator or a portion thereof can involve receptor-mediated endocytosis, also known as clathrin-mediated endocytosis. In some embodiments, the internalization of a bispecific modulator can involve clathrin-independent endocytosis. In some embodiments, the internalization of a bispecific modulator can involve phagocytosis.

[0105] In embodiments, the cell surface molecule or the 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 on a CAR-T cell. In some embodiments, strategies for modulating CAR-T activity include internalizing the CAR receptor with a bispecific modulator. In some embodiments, the molecule of interest targeted by the first moiety can be a cell modulator, such as a protein that is part of an immune checkpoint pathway (e.g., PD-L1) or other signal transduction protein (e.g., EGFR). In some embodiments, the molecule of interest can be a marker of a particular cell type (e.g., CD20 for B cells).

[0106] 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, cytokine, enzyme, etc.

[0107] In some embodiments, the molecule of interest can bind a hormone, cytokine, growth factor, neurotransmitter, lipophilic signaling molecule (e.g., prostaglandin) or cell recognition molecule (e.g., integrin, selectin). 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).

[0108] 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 can be Na + 、K +, Ca 2+ or Cl - 2+

[0109] In some embodiments, the target molecule can be a transporter protein. In some embodiments, the target molecule can be an ion transporter protein.

[0110] In some embodiments, the molecule targeted by the first part of the bispecific modulator (i.e., the target molecule) can be a different molecule from the molecule targeted by the second part of the bispecific modulator (e.g., an internalizing receptor or a membrane protein).

[0111] 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 part and the second part of the bispecific modulator.

[0112] In some embodiments, the first part can be an antigen or epitope to which the target molecule can bind. In some embodiments, the antigen or epitope can bind to a receptor on the cell. In some embodiments, the antigen or epitope can be a ligand of the receptor. In embodiments, the receptor can be a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a B cell receptor (BCR).

[0113] In embodiments, the first part can be an antibody or antibody fragment that binds to an antigen or epitope specific to a tumor and / or cancer cell and / or tumor and / or cancer cell. In embodiments, the antigen or epitope that can be bound by a CAR and that can be bound by an antibody or antibody fragment can be CD19, B cell maturation antigen (BCMA), human epidermal growth factor 2 (HER2), etc.

[0114] In some embodiments, the antibody as the first part 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 section titled "Antibodies" in this application.

[0115] In embodiments, the second moiety binds to a receptor or membrane protein on the cell. In some embodiments, the receptor or membrane protein bound by the second moiety is an internalized receptor or membrane protein. In some embodiments, the internalized 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.

[0116] In some embodiments, the first and / or second moiety can be any type of moiety that can bind to a cell surface molecule or an extracellular molecular target. In some embodiments, the first and / or second moiety can be a polypeptide, ligand, aptamer, nanoparticle, small molecule, etc.

[0117] In embodiments, a non-limiting list of internalized receptors or membrane proteins that can be used for 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: implication of F-BAR protein Carom." Frontiers in bioscience (Landmark edition) 22 (2017): 1439). In some embodiments, GPCRs can include adrenergic receptors, chemokine receptors, thrombin 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, TMRs can include folate receptors, interleukin receptors (e.g., IL-2 receptor), low-density lipoprotein receptors, transferrin receptors, etc.

[0118] In embodiments, a non-limiting list of internalization receptors or membrane proteins that can be used for 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: implication 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, TMRs can include folate receptors, interleukin receptors (e.g., IL-2 receptor), low-density lipoprotein receptors, transferrin receptors, etc.)

[0119] In some embodiments, the internalization receptor or membrane protein can be a transferrin receptor. In embodiments, the ligand (e.g., the second part) that the transferrin receptor can bind to (the first part) can be transferrin or a fragment of transferrin. In some embodiments, the first part can be an antibody or an antibody fragment that can bind to the transferrin receptor.

[0120] In some embodiments, the internalization receptor or membrane protein can be a transferrin receptor (TfR). The transferrin receptor can be transferrin receptor 1 or transferrin receptor 2.

[0121] In some embodiments, the transferrin receptor can have a high endocytosis rate of approximately 500 molecules / cell / second, making it favorable for inducing protein endocytosis. In some embodiments, the expression of the transferrin receptor can be low in healthy tissues but higher in various tumors and some activated immune cells (such as brain cancer, liver cancer, breast cancer, lung cancer, colon cancer, and blood cancer).

[0122] In embodiments, the ligand (e.g., the second part) that the internalization receptor or membrane protein can bind to can be at least a part of a naturally occurring ligand. For example, the ligand can be at least a part of transferrin, cholesterol, low-density lipoprotein, and epidermal growth factor that can be bound by a homologous receptor.

[0123] In some embodiments, the ligand bound by the transferrin receptor can be transferrin or a fragment of transferrin. In some embodiments, the size of the ligand that the transferrin receptor can bind can be about 80 kDa and has glycosylation modification.

[0124] In some embodiments, the second part can be an antibody, antibody fragment or other molecule that can bind to an internalized receptor or a membrane protein. In some embodiments, the second part can be an scFv, Fab, single-domain antibody, nanobody, single antibody, DARPin or affibody.

[0125] In some embodiments, the antibody or antibody fragment that can bind to the transferrin receptor can be an anti-TfR1 antagonistic scFv antibody identified by phage display. This antibody can 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).

[0126] In one embodiment, the amino acid sequence of the H7 molecule can include the following molecule or can include 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% identical to the following amino acid sequence:

[0127] H7 scFV-LC (SEQ ID NO:1):

[0128] SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVMYGRNERPSGVPDRFSGSKSGTSASLAISGLQPEDEANYYCAGWDDSLTGPVFGGGTKLTVLG*

[0129] H7 scFV-HC (SEQ ID NO:2):

[0130] QVQLQESGGGVVQPGRSLRLSCAASRFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLSGYGDYPDYWGQGTLVTVSS

[0131] H7-scFv (SEQ ID NO:3)

[0132] SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVMYGRNERPSGVPDRFSGSKSGTSASLAISGLQPEDEANYYCAGWDDSLTGPVFGGGTKLTVLGGGGGSGGGGSGGGGSQVQLQESGGGVVQPGRSLRLSCAASRFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLSGYGDYPDYWGQGTLVTVSS

[0133] M16 (SEQ ID NO:4):

[0134] SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVMYGRNERPSGVPDRFSGSKSGTSASLAISGLQPEDEANYYCAGWDDSLTGPVFGGGTKLTVLGGGGGSGGGGSGGGGSQVQLQESGGGVVQPGRSLRLSCAASRYPFHHHDHHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLSGYGDYPDYWGQGTLVTVSS

[0135] 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 CAR-T cells (first part) and can bind to an internalized receptor or membrane protein on one or more identical or adjacent cells (second part: e.g., transferrin). The bispecific modulator can co-localize the CAR receptor to the internalized cell surface receptor or membrane protein. For example, internalized CAR cannot be activated or continue to function in the activated state. In some embodiments, the bispecific modulator can downregulate the cell surface level of CAR and inhibit CAR-T cell function( Figure 6 ).

[0136] Disclosed herein is a new mechanism for modulating proteins at the cell membrane. Endocytosis is a common mechanism for regulating membrane protein recycling and degradation. 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 / second). TfR imports iron by binding to the plasma protein transferrin (Tf) complexed with iron. It is highly expressed in various cancers and affects cancer cell proliferation, migration, invasion, apoptosis, and metastasis.

[0137] It has been shown that extracellular proteins, especially tumor-associated proteins of interest (POI), can be selectively degraded by tethering the POI to TfR with an antibody-Tf fusion protein( Figure 7 ). We named this technique Trans transferrin receptor-mediated Targeted Chimera (TransTAC). During TfR / TransTAC-mediated endocytosis, due to the different local environments of endosomes, the receptor dissociates from the complex( Figure 7 , red square) and undergoes lysosome-driven degradation.

[0138] This method is a new and general prototype for degrading proteins with fully recombinant biomolecules. The general method for degrading membrane / extracellular proteins opens up unlimited possibilities for manipulating cell behavior and thus serves as an important research tool as well as extending the attempts in the PROTAC field to target challenging extracellular targets. The fully recombinant nature of TransTAC allows it to be simply generalized to a wide range of targets and optimized for binding properties.

[0139] Improved tumor targeting specificity based on TfR degradation. TfR is used because: (1) TfR is a recycling receptor, so the level of TfR on the cell surface can remain constant, which is an important feature as a "vector" protein; (2) Tf has been studied for iron or small molecule drug delivery and thus has the necessary developability and stability as a therapeutic agent; and (3) TfR targeting provides additional tumor specificity; its expression is regulated by tumor-associated oxidative stress, inflammation, and hypoxia. In summary, the modular nature, genetic traceability, and tumor specificity of TransTAC are good methods for academic and translational applications.

[0140] In some embodiments, the bispecific modulators disclosed herein have an antigen to which a CAR can bind and a ligand for an internalizing receptor or membrane protein (e.g., transferrin receptor). In some embodiments, the bispecific modulators disclosed herein have an antibody that can bind a CAR or another molecule (such as EGFR, PD-L1, CD20) on the cell surface and a ligand for an internalizing receptor or membrane protein (e.g., a target molecule, such as a target protein).

[0141] In some embodiments, the bispecific modulator can be a fusion protein of the formula R1-R2-R3. In some embodiments, the 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, the bispecific modulator can be a dimer (homo-dimer) of R1-R2-R3 or R3-R2-R1.

[0142] In some embodiments, R1 can be a protein of interest binder (POIB) or a POIB element. In some embodiments, the POIB or element can be an antibody. In some embodiments, the POIB element can be part of a molecule to which the protein of interest normally binds (e.g., a peptide bound by a CAR). The POIB or element can bind a target molecule on the cell surface, such as a target protein. In some embodiments, the POIB or element can bind the extracellular domain of a transmembrane protein. In some embodiments, the POIB element can bind the extracellular domain of a chimeric antigen receptor (CAR), a receptor tyrosine kinase, a checkpoint inhibitor binding molecule, a cell lineage-specific marker, etc. In some embodiments, the POIB or element can bind the extracellular domain of epidermal growth factor receptor (EGFR), programmed death ligand (PD-L1), or CD20.

[0143] In some embodiments, the POIB element can bind the extracellular domain of a B cell receptor (BCR), human leukocyte antigen (HLA), fibroblast growth factor receptor (FGFR), Notch protein, or Claudin-18.2.

[0144] In some embodiments, R3 can be a transferrin receptor binding element (TRB). The TRB binds to the transferrin receptor on the cell surface. In some embodiments, the TRB can be an antibody that binds to the transferrin receptor (e.g., H7 or M16). In some embodiments, the TRB can be a polypeptide. In some embodiments, the TRB can be a ligand (e.g., transferrin) or a part of a ligand to which the transferrin receptor can bind. In some embodiments, a part of transferrin that can be used as the TRB is as follows (SEQ ID NO:5):

[0145] VPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGPSVACVKKASYLDCIRAIAANEADAVTLDAGLVYDAYLAPNNLKPVVAEFYGSKEDPQTFYYAVAVVKKDSGFQMNQLRGKKSCHTGLGRSAGWNIPIGLLYCDLPEPRKPLEKAVANFFSGSCAPCADGTDFPQLCQLCPGCGCSTLNQYFGYSGAFKCLKDGAGDVAFVKHSTIFENLANKADRDQYELLCLDNTRKPVDEYKDCHLAQVPSHTVVARSMGGKEDLIWELLNQAQEHFGKDKSKEFQLFSSPHGKDLLFKDSAHGFLKVPPRMDAKMYLGYEYVTAIRNLREGTCPEAPTDECKPVKWCALSHHERLKCDEWSVNSVGKIECVSAETTEDCIAKIMNGEADAMSLDGGFVYIAGKCGLVPVLAENYNKSDNCEDTPEAGYFAIAVVKKSASDLTWDNLKGKKSCHTAVGRTAGWNIPMGLLYNKINHCRFDEFFSEGCAPGSKKDSSLCKLCMGSGLNLCEPNNKEGYYGYTGAFRCLVEKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYELLCLDGTRKPVEEYANCHLARAPNHAVVTRKDKEACVHKILRQQQHLFGSNVTDCSGNFCLFRSETKDLLFRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLRKCSTSSLLEACTFRRP(SEQ ID NO:5).

[0146] In some embodiments, R2 can be a linker of the formula R4-R5 or R5-R4. In some embodiments, R4 can be from the Fc region of an antibody. In some embodiments, R4 can be from the Fc region of IgG, IgM, IgA, IgE, or IgD. In some embodiments, the Fc region can be (SEQ ID NO:6):

[0147] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0148] In some embodiments, the Fc region can dimerize to form a homodimeric or heterodimeric structure. In some embodiments, the Fc region can have or can be modified to have cysteine amino acids capable of forming disulfide bonds. In some embodiments, the dimer of the R1-R2-R3 fusion protein can be formed by disulfide bond(s) (one or more, such as two disulfide bonds) between cysteine residues in the R2 region of separate fusion protein molecules. In some embodiments, disulfide bonds are formed between R4s in separate fusion molecules (e.g., Fc with disulfide can be of the type of dimerization domain).

[0149] In certain embodiments, the Fc region can be a variant that contains amino acid substitutions that alter the effector functions independent of antigen, such as the circulatory half-life of the molecule linked thereto. Compared to Fc regions lacking these substitutions, molecules linked to these Fc regions can exhibit increased or decreased binding to FcRn and can have increased or decreased half-lives in serum, respectively. Fc variants with improved FcRn affinity are expected to have longer serum half-lives, and such molecules have useful applications in methods where a long half-life of the linked molecule is desired. In contrast, Fc variants with reduced FcRn binding affinity are expected to have shorter half-lives, and such molecules are also useful, for example, in situations where a shortened circulation time may be advantageous. Fc variants with reduced FcRn binding affinity are also less likely to cross the placenta. Additionally, other applications where reduced FcRn binding affinity may be desired include those that require targeting to the brain, kidney, and / or liver. In one embodiment, the molecule linked to the Fc variant can exhibit reduced transport across glomerular epithelium from the vascular system.

[0150] In another embodiment, a molecule conjugated to an Fc variant can exhibit reduced transport from the brain across the blood-brain barrier (BBB) into the vascular space. In one embodiment, an Fc region with altered FcRn binding comprises an Fc domain having one or more amino acid substitutions within the "FcRn-binding loop" of the Fc domain. The FcRn-binding loop is constituted by amino acid residues 280-299 (according to EU numbering). Exemplary amino acid substitutions with altered FcRn-binding activity are disclosed in PCT Publication No. WO05 / 047327, which is incorporated herein by reference. In certain exemplary embodiments, the bispecific modulators disclosed herein comprise an Fc domain having one or more of the following substitutions: V284E, H285E, N286D, K290E, and S304D (EU numbering).

[0151] In some embodiments, the molecules disclosed herein can be conjugated to an Fc variant comprising an amino acid substitution that alters glycosylation. For example, the Fc variant can have reduced glycosylation (e.g., N- or O-linked glycosylation). In some embodiments, the Fc variant comprises reduced glycosylation of the N-linked glycan typically found at amino acid position 297 (EU numbering). In another embodiment, these molecules can have amino acid substitutions near or within a glycosylation motif, such as an N-linked glycosylation motif containing the amino acid sequence NXT or NXS. In certain embodiments, the Fc variant can have an amino acid substitution at amino acid position 228 or 299 (EU numbering). Exemplary amino acid substitutions that confer reduced or altered glycosylation are described in PCT Publication No. WO 05 / 018572, which is incorporated herein by reference in its entirety.

[0152] In some embodiments, the molecules disclosed herein can be modified to eliminate glycosylation and can be referred to as "aglycosylated" molecules. Exemplary aglycosylated molecules can have an aglycosylated Fc region of an IgG4 antibody, which lacks Fc effector functions, thereby eliminating the possibility of Fc-mediated toxicity to normal vital tissues and cells. In still other embodiments, the molecules disclosed herein can have altered glycans. For example, there can be a reduced number of fucose residues on the N-glycan at Asn297 of the Fc region, i.e., be afucosylated. In some embodiments, there can be an altered number of sialic acid residues on the N-glycan at Asn297 of the Fc region.

[0153] In some embodiments, the CH2 or CH3 region of the Fc antibody domain can be truncated or modified to modulate the half-life of the molecule. In some embodiments, Fc truncation includes 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).

[0154] In some embodiments, R4 can be a dimerization domain. A dimerization domain can be any region that can associate with another dimerization domain, either covalently or non-covalently, to form a dimer (e.g., a bispecific modulator that is a homodimer or heterodimer). In some embodiments, R4 is not from the Fc region of an antibody.

[0155] There are many protein dimerization domains known in the art (e.g., see Dang, Dung Thanh. “Molecular Approaches to Protein Dimerization: Opportunities for Supramolecular Chemistry”. Frontiers in Chemistry 10 (2022): 829312). Exemplary dimerization domains can include zipper motifs, such as leucine zippers.

[0156] In some embodiments, dimerization can form between regions of the bispecific modulator that are not the R4 region.

[0157] In some embodiments, R5 can be a protease-sensitive linking element. In some embodiments, the protease-sensitive linking element 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 linking element can be a cathepsin-cleavable peptide. Some exemplary protease-sensitive linking elements are shown in Figure 35 . In some embodiments, the protease-sensitive linking element can be GGFLGGVRGVDG (SEQ ID NO:7) or GSGSGGEVRGVDG (SEQ ID NO:8).

[0158] In some embodiments, a linker (e.g., a linker) can be located between the respective segments of the R1-R2-R3 fusion protein. In some embodiments, this linker can be located between R2 and R3. In some embodiments, this linker can be located between R1 and R2. In some embodiments, the linker 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 4 amino acids that are glycine and / or serine. In some embodiments, other amino acids can be part of the GS linker as long as glycine and serine are in the majority.

[0159] In some embodiments, the bispecific modulators disclosed herein can include the following nucleotide and amino acid sequences, and molecules that are 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% identical to the following nucleotide and amino acid sequences. Specifically, the amino acid sequences of the bispecific modulators can be labeled as follows:

[0160] The underlined Times New Roman font is the signal peptide ;

[0161] Bold Times New Roman fonts are anti-target protein Fab-heavy chain, scFv or affibody;

[0162] Times New Roman italic fonts are linkers encoded by the creation of restriction enzyme sites;

[0163] The underlined and bold Times New Roman is the GS linker ;

[0164] The underlined, italic, and bold Times New Roman is the cleavable linker ;

[0165] Courier New font is H7-scFv;

[0166] Courier New underlined font is the Fc domain;

[0167] Courier New bold font is the TEV site;

[0168] Courier New italic fonts are fragments from transferrin;

[0169] Courier New underlined and bold font is the His tag;

[0170] Courier New bold and italic are light chains, and

[0171] Courier New underlined, italic, and bold font is the Avi - tag

[0172] pDP14-CD19 ETD_Fc(SEQ ID NO:17)

[0173]

[0174] (SEQ ID NO:18)

[0175]

[0176] pDP16-EGFR-Affibody-FC-Tf(SEQ ID NO:19)

[0177]

[0178] (SEQ ID NO:20)

[0179]

[0180] pDP18 - EGFR - Affibody (SEQ ID NO:21)

[0181]

[0182] (SEQ ID NO:22)

[0183]

[0184] pDP20-EGFR-Affibody-Fc (SEQ ID NO:23)

[0185] ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCGCTGCAGGTAGATAACAAATTCAACAAAGAAATGTGGGCGGCGTGGGAAGAAATTCGCAACCTGCCGAACCTGAACGGCTGGCAGATGACCGCGTTTATTGCGAGCCTGGTGGATGACCCAAGCCAAAGCGCTAACTTGCTAGCAGAAGCTAAAAAGCTAAATGATGCTCAGGCGCCGAAAGTAGACGGCAGCGGCAGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAA

[0186] (SEQ ID NO:24)

[0187]

[0188] pDP22-EGFR-Affibody (SEQ ID NO:25)

[0189] ATGCGAATGCAGCTGCTGCTGCTGATTGCGCTGAGCCTGGCGCTGGTGACCAACAGCACTAGTCTGCAGGTAGATAACAAATTCAACAAAGAAATGTGGGCGGCGTGGGAAGAAATTCGCAACCTGCCGAACCTGAACGGCTGGCAGATGACCGCGTTTATTGCGAGCCTGGTGGATGACCCAAGCCAAAGCGCTAACTTGCTAGCAGAAGCTAAAAAGCTAAATGATGCTCAGGCGCCGAAAGTAGACGGCAGCGGCAGCACTAGTTCTGGTGGTGGTGGTGAGAATCTGTACTTTCAGAGCTCGGGCGGAGGATCGGGTGGAGGCCACCACCATCATCACCACCATCACGGATCCGGCCTGAACGACATCTTCGAGGCTCAGAAAATCGAATGGCACGAAGGCTAA

[0190] (SEQ ID NO:26)

[0191]

[0192] pDP24-CD19-Fc-Tf (SEQ ID NO:27)

[0193]

[0194] (SEQ ID NO:28)

[0195]

[0196] pDP25-pFUSE-Tf-杵-Fc(SEQ ID NO:29)

[0197]

[0198] (SEQ ID NO:30)

[0199]

[0200] pDP32 - CD19 - FC - CD19(SEQ ID NO:31)

[0201]

[0202] (SEQ ID NO:32)

[0203]

[0204] pDP44-CD19 NT.1-FC-Tf(SEQ ID NO:33)

[0205]

[0206] (SEQ ID NO:34)

[0207]

[0208] pDP49-CD19 NT.1-FC-GFLG-Tf(SEQ ID NO:35)

[0209]

[0210] (SEQ ID NO:36)

[0211]

[0212] pDP50-His8-CD19 NT.1-GFLG-FC-Tf(SEQ ID NO:37)

[0213]

[0214] (SEQ ID NO:38)

[0215]

[0216] pDP85-cd20-scfv-GFLG-FC-Tf(SEQ ID NO:39)

[0217]

[0218] (SEQ ID NO:40)

[0219]

[0220] pDP95 - Herceptin_HC_Fc - N297G,S427C(SEQ ID NO:41)

[0221]

[0222] (SEQ ID NO:42)

[0223]

[0224] pDP69-His8-CD19 NT.1-2XGFLG-FC-Tf(SEQ ID NO:43)

[0225]

[0226] (SEQ ID NO:44)

[0227]

[0228] pDP70-His8-CD19 NT.1-3XGFLG-FC-Tf(SEQ ID NO:45)

[0229]

[0230] (SEQ ID NO:46)

[0231]

[0232] pDP71-His8-CD19 NT.1-GFLG-FK-FC-Tf(SEQ ID NO:47)

[0233]

[0234] (SEQ ID NO:48)

[0235]

[0236] pDP72-His8-CD19 NT.1-GFLG-VA-FC-Tf(SEQ ID NO:49)

[0237]

[0238] (SEQ ID NO:50)

[0239]

[0240] pDP73-His8-CD19 NT.1-GFLG-VK-FC-Tf(SEQ ID NO:51)

[0241]

[0242] (SEQ ID NO:52)

[0243]

[0244] pDP74-His8-CD19 NT.1-GFLG-VR-FC-Tf(SEQ ID NO:53)

[0245]

[0246] (SEQ ID NO:54)

[0247]

[0248] pDP75-His8-CD19 NT.1-GFLG-GGFG-FC-Tf(SEQ ID NO:55)

[0249]

[0250] (SEQ ID NO:56)

[0251]

[0252] pDP76-His8-CD19 NT.1-FK-FC-Tf(SEQ ID NO:57)

[0253]

[0254] (SEQ ID NO:58)

[0255]

[0256] pDP77-His8-CD19 NT.1-VA-FC-Tf(SEQ ID NO:59)

[0257]

[0258] (SEQ ID NO:60)

[0259]

[0260] pDP78-His8-CD19 NT.1-VK-FC-Tf(SEQ ID NO:61)

[0261]

[0262] (SEQ ID NO:62)

[0263]

[0264] pDP79-His8-CD19 NT.1-VR-FC-Tf(SEQ ID NO:63)

[0265]

[0266] (SEQ ID NO:64)

[0267]

[0268] pDP80-His8-CD19 NT.1-GGFG-FC-Tf(SEQ ID NO:65)

[0269]

[0270] (SEQ ID NO:66)

[0271]

[0272] pDP86 - PDL1 - scfv - GFLG - fc - Tf(SEQ ID NO:67)

[0273]

[0274] (SEQ ID NO:68)

[0275]

[0276] pDP96 - CD20_HC_Fc - N297G,S427C - Tf(SEQ ID NO:69)

[0277]

[0278] (SEQ ID NO:70)

[0279]

[0280] pDP97-cd20-scfv-GFLG-FC-N297G,S427C-Tf(SEQ ID NO:71)

[0281]

[0282] (SEQ ID NO:72)

[0283]

[0284] pDP98 - CD20_HC_Fc - N297G,S427C(SEQ ID NO:73)

[0285]

[0286] (SEQ ID NO:74)

[0287]

[0288] cd20 - scFV - FC - Tf(SEQ ID NO:75)

[0289]

[0290] (SEQ ID NO:76)

[0291]

[0292] PD-L1-scFV-FC-Tf(SEQ ID NO:77)

[0293]

[0294] (SEQ ID NO:78)

[0295]

[0296] pDP124-cd20-scfv-FC-TfR-H7(SEQ ID NO:79)

[0297]

[0298] pDP125-cd20-scfv-FC-TfR-M16(SEQ ID NO:80)

[0299]

[0300] pDP126-cd20-scfv-GFLG-FC-TfR-H7(SEQ ID NO:81)

[0301]

[0302] pDP127-cd20-scfv-GFLG-FC-TfR-M16(SEQ ID NO:82)

[0303]

[0304] pDP155-His8-CD19 NT.1-2XGFLG-FC-TfR-H7(SEQ ID NO:83)

[0305]

[0306] pDP156-His8-CD19 NT.1-3XGFLG-FC-TfR-H7(SEQ ID NO:84)

[0307]

[0308] pDP157-His8-CD19 NT.1-GFLG-FK-FC-TfR-H7(SEQ ID NO:85)

[0309]

[0310] pDP158-His8-CD19 NT.1-GFLG-VA-FC-TfR-H7(SEQ ID NO:86)

[0311]

[0312] pDP159-His8-CD19 NT.1-GFLG-VK-FC-TfR-H7(SEQ ID NO:87)

[0313]

[0314] pDP160-His8-CD19 NT.1-GFLG-VR-FC-TfR-H7(SEQ ID NO:88)

[0315]

[0316] pDP161-His8-CD19 NT.1-GFLG-GGFG-FC-TfR-H7(SEQ ID NO:89)

[0317]

[0318] pDP162-His8-CD19 NT.1-FK-FC-TfR-H7(SEQ ID NO:90)

[0319]

[0320] pDP163-His8-CD19 NT.1-VA-FC-TfR-H7(SEQ ID NO:91)

[0321]

[0322] pDP164-His8-CD19 NT.1-VK-FC-TfR-H7(SEQ ID NO:92)

[0323]

[0324] pDP165-His8-CD19 NT.1-VR-FC-TfR-H7(SEQ ID NO:93)

[0325]

[0326] pDP166-His8-CD19 NT.1-GGFG-FC-TfR-H7(SEQ ID NO:94)

[0327]

[0328] pDP167-CD20_HC_Fc-N297G,S427C-TfR-H7(SEQ ID NO:95)

[0329]

[0330] pDP168-CD20-scfv-GFLG-FC-N297G,S427C-TfR-H7(SEQ ID NO:96)

[0331]

[0332] pDP169-cd20-scfv-GFLG-FK-FC-(N297G,S427C)-TfR-H7(SEQ ID NO:97)

[0333]

[0334] pDP170-cd20-scfv-GFLG-VR-FC-(N297G,S427C)-TfR-H7 (SEQ ID NO:98)

[0335]

[0336] pDP171-His8-EGFR affi-FC-TfR-H7 (SEQ ID NO:99)

[0337]

[0338] pDP172-His8-EGFR affi-GFLG-FC-TfR-H7 (SEQ ID NO:100)

[0339]

[0340] pDP173-His8-EGFR affi-GFLG-FK-FC-TfR-H7 (SEQ ID NO:101)

[0341]

[0342] pDP174-His8-EGFR affi-GFLG-VR-FC-TfR-H7 (SEQ ID NO:102)

[0343]

[0344] pDP210-CD20-HC-(EVR)-FC(GRLR)-N297G-TfR-H7 (SEQ ID NO:103)

[0345]

[0346] pDP213-CD20-HC-(EVR)-FC(GRLR)-N297G(SEQ ID NO:104)

[0347]

[0348] pDP219-FC-TfR-H7(SEQ ID NO:105)

[0349]

[0350] pDP223-pFUSE-H7 scFV-杵-Fc-His(SEQ ID NO:106)

[0351]

[0352] pDP224-pFUSE-CD 19NT.1-杵-Fc-H7 scFV-His(SEQ ID NO:107)

[0353]

[0354] pDP225-2-臼Fc_H7 scFV(SEQ ID NO:108)

[0355]

[0356] pDP226-臼Fc_-avi标签(SEQ ID NO:109)

[0357]

[0358] pDP227-杵Fc_H7 scFV-His(SEQ ID NO:110)

[0359] PD-L1 TransTAC(pDP186)(SEQ ID NO:111)

[0360]

[0361] CD20 TransTAC:

[0362] Heavy chain (pDP210) (SEQ ID NO:112)

[0363]

[0364] Light chain (pDP118) (SEQ ID NO:113)

[0365]

[0366] CD20 TransTAC-M16 form (pDP127):

[0367] Heavy chain (SEQ ID NO:114) (light chain as described immediately above)

[0368]

[0369] EGFR TransTAC (pDP211) (SEQ ID NO:115)

[0370]

[0371] CD19 CAR TransTAC (pDP160) (SEQ ID NO:116)

[0372]

[0373] CD19 CAR TransTAC earlier form (pDP50) (SEQ ID NO:117)

[0374]

[0375] In some embodiments, the bispecific modulator can be a heterodimer between the fusion protein R1-R2-R3 (where R1 is a POIB; R2 is R4-R5 or R5-R4, where R4 is an antibody Fc region, and R5 is a protease-sensitive linker; R3 is a TRB, as described above) and the fusion protein R3-R4 or R4-R3, where R3 is a TRB and R4 is an antibody Fc region. In some embodiments, optionally, a protease-sensitive linking element may be present between R3 and R4 or R3-R4 or R4-R3.

[0376] In some embodiments, the bispecific modulators disclosed herein can have the formula R1-R6-R3, where R1 is a protein of interest binder (POIB), R6 is a dimerization element, and R3 is a transferrin receptor binding (TRB) element. In some embodiments, R6 can be a moiety that links R1 and R3 (e.g., a linking element that links R1 and R3). In some embodiments, R6 can be an amino acid linker. In some embodiments, the amino acid linker can be protease-sensitive. In some embodiments, R6 can be a dimerization domain that can form a dimer with another copy of R6 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 can be a combination of a linking molecule (e.g., one that can be protease-sensitive) and a dimerization domain as described above.

[0377] In some embodiments, the bispecific modulators disclosed herein can have the formula R1-R6-R3, where R1 is a protein of interest binder (POIB), R6 is a dimerization element, and R3 is a transferrin receptor binding (TRB) element. In some embodiments, R6 can be a moiety that links R1 and R3 and contains a dimerization domain. The dimerization domain can form a dimer with another copy of R6 via a covalent bond (e.g., a cysteine-containing Fc antibody region). In some embodiments, the dimerization domain can be an Fc antibody region having one or more cysteines. In some embodiments, or optionally, the linkage between R1 and R6 or between R6 and R3 can be a protease-sensitive linking element.

[0378] In some embodiments, the first component of the bispecific modulator can be R1-R6 or R6-R1, where R1 can be POIB, and R6 can be a dimerization element as above, and optionally can also be a protease-sensitive amino acid linker. The second component of the bispecific modulator can be R3-R7, where R3 can be a TRB element, and R7 can be a multimerization domain as above, and optionally can also be a protease-sensitive amino acid linker. When the multimerization domain (e.g., dimerization domain) of the first component forms a covalent or non-covalent bond with the multimerization domain of the second component, a bispecific modulator can be formed.

[0379] In some embodiments, the bispecific modulators disclosed herein are designed to target tumor cell surface molecules (e.g., target molecules such as target proteins) on tumor cells. In some embodiments, these cell surface molecules can regulate cell growth. 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 have an IC that is 10-50 times better than other treatments for tumors / cancer cells 50 .

[0380] In some embodiments, the bispecific modulators disclosed herein are used to internalize and degrade multi-transmembrane proteins (i.e., transmembrane proteins that span multiple times and produce multiple extracellular domains). In some embodiments, CD20 is the protein targeted using these bispecific modulators.

[0381] In some embodiments, the bispecific modulator can be a single polypeptide chain. In some embodiments, the bispecific modulator can be two polypeptide chains. In some embodiments of the double polypeptide configuration, the two conjugates are encoded in two polypeptide chains. In some embodiments, the two polypeptide chains can be linked or joined by a dimerization domain. In some embodiments, the two polypeptide chains can be linked or joined by a knob-into-hole structure Fc. Targeted Receptor Degradation via TransTAC

[0382] In some embodiments, the cell surface molecule (i.e., target molecule such as target protein) targeted by the bispecific modulator can be internalized by the bispecific modulator. In some embodiments, the internalized molecule may not be degraded or may be minimally degraded intracellularly. In some embodiments, the bispecific modulators disclosed herein are modified to more efficiently degrade the target protein. In some embodiments, the bispecific modulator is modified to contain a protease-sensitive amino acid sequence (e.g., see Figure 35) These proteases can be endosomal proteases or lysosomal proteases. In some embodiments, a peptide linker that is a target of a cathepsin can be used. When the linker is cleaved by a protease, the molecule of interest is released from the bispecific modulator.

[0383] In some embodiments, the linker is sensitive to cleavage by a cathepsin. In some embodiments, the cathepsin can be cathepsin A, B, C, D, E, F, G, H, K, L1, L2, O, S, W, or Z.

[0384] In some embodiments, the molecule of interest can be released from the bispecific modulator (e.g., by pH-dependent binding of the TRB) rather than by including a protease-sensitive linker.

[0385] Cleavage of the linker intracellularly (e.g., in an endosome) can release the molecule of interest from an internalized receptor or membrane protein and increase the likelihood that the molecule of interest is degraded. In some embodiments, the protease-sensitive peptide linker can 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.

[0386] In some embodiments, the molecule of interest can be released from the bispecific modulator (e.g., by pH-dependent binding of the TRB) rather than by including a protease-sensitive linker.

[0387] In some embodiments, the bispecific modulator is R1-R2-R3 as described earlier, and when R2 can be R4-R5 or R5-R4 (where R5 is a protease-sensitive linking element), the protease-sensitive linking element can be located between POIB (R1) and the Fc region (R4) from an antibody, as in R1-R5-R4-R3. In some embodiments, the protease-sensitive linking element can be located between the Fc region (R4) from an antibody and the TRB (R3), as in 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 inside an endosome, the low-pH environment can release / dissociate the target protein from the bispecific modulator, such that the target protein is degraded more efficiently.

[0388] In some embodiments, the linker can be sensitive to the low pH present in an endosome. In some embodiments, the low pH can cause cleavage of the linker.

[0389] In some embodiments, a transferrin receptor-binding element (TRB) can bind transferrin according to pH. For example, at a lower pH found in endosomes, the TRB may have a lower affinity for the transferrin receptor. The lower affinity can cause the TRB to release the transferrin receptor. This release can promote the degradation of the target protein bound to the POIB. Such a TRB can be, for example, Figure 35 the "M16" as shown.

[0390] In some embodiments, a protease-sensitive linker can be located between the first part (targeting the target molecule) and the second part (binding to an internalized receptor or membrane protein). In some embodiments, the linker can be positioned closer to the first part than to the second part.

[0391] In some embodiments, the protease-sensitive linking element can include Gly-Phe-Leu-Gly (GFLG; SEQ ID NO: 118). In some embodiments, the peptide linker can include valine-arginine (VR) and / or phenylalanine-lysine (FK) sequences. In some embodiments, the peptide linker can be 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).

[0392] In the experiment, ( Figure 49A - Figure 49B)Identify peptides that are not yet known to be sensitive to cathepsin cleavage using a yeast-displayed peptide library. These peptides can be from combinations of small motifs found in SEQ ID NO: 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).

[0393] Other sequences can be used.

[0394] In some embodiments, any combination of these peptide linkers and / or valine-citrulline (VC) linkers and / or glutamate-valine-arginine (EVR) linkers can be used. Antibody

[0395] Unique recombinant monoclonal antibodies are disclosed, which can be part of the bispecific modulators disclosed herein. In embodiments, the antibodies can be used in the first and / or second part of the bispecific modulators disclosed herein.

[0396] When related to a polypeptide (such as an antibody) or a polynucleotide, "recombinant" refers to a form of a polypeptide or polynucleotide that does not exist naturally, non-limiting examples of which can be created by combining polynucleotides or polypeptides that do not normally occur together. As used herein, "polypeptide" can encompass the singular "polypeptide" as well as the plural "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 any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, the terms peptide, dipeptide, tripeptide, oligopeptide, "protein", "amino acid chain", or any other term used to refer to any 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 modification of a polypeptide, these modifications including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant techniques, but is not necessarily translated from a designated nucleic acid sequence. It can be produced in any manner, including by chemical synthesis. For amino acid sequences, those skilled in the art will readily recognize that a single substitution, deletion, or addition to a nucleic acid, peptide, polypeptide, or protein sequence (which changes, adds, deletes, or substitutes a single amino acid or a small percentage of amino acids in the encoded sequence) is collectively referred to herein as a "conservative modified variant". In some embodiments, the change results in an amino acid being replaced by a chemically similar amino acid. Conservative substitution tables providing amino acids that are functionally similar are well known in the art. Such conservative modified variants of the antibodies disclosed herein can exhibit increased cross-reactivity compared to the unmodified antibody.

[0397] For example, a "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include the following side chains: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (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, an amino acid string can be replaced with a structurally similar string that differs in the order and / or composition of side chain family members.

[0398] Some embodiments are also characterized by antibodies having a specified percentage identity or similarity to the amino acid or nucleotide sequences of the antibodies described herein. For example, "homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions in each sequence that can be aligned for comparison purposes. When a position in the sequences being compared is occupied by the same base or amino acid, then 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 higher amino acid sequence identity when compared to a specified region or the full length of any 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 higher nucleic acid identity when compared to a specified region or the full length of any 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 by methods known in the art, such as using software programs known in the art, such as those described in the following: 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 percentage of sequence identity or similarity of the nucleic acids and proteins of the invention.

[0399] Aspects of the present invention provide isolated antibodies. As used herein, the term "isolated" with respect to a cell, nucleic acid (such as DNA or RNA) refers to a molecule that is separated from other DNA or RNA present in the natural source of the macromolecule. The term "isolated" can also refer to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or is substantially free of 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 can include both purified and recombinant polypeptides.

[0400] As used herein, the term "antibody" or "antigen-binding polypeptide" can refer to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. An antibody can be a whole antibody and any of its antigen-binding fragments or single chains. For example, an "antibody" can include any molecule containing a protein or peptide that comprises at least a portion of an immunoglobulin molecule having biological activity for binding an antigen. Non-limiting examples are complementarity determining regions (CDRs) of a heavy or light chain or ligand-binding portion thereof, variable regions of a heavy or light chain, constant regions of a heavy or light chain, framework (FR) regions, or any portion thereof, or at least a portion of a binding protein. As used herein, the term "antibody" can refer to an immunoglobulin molecule and the immunologically active portion of an immunoglobulin (Ig) molecule, i.e., a molecule containing an antigen-binding site that specifically binds an antigen (immunologically reacts with the antigen). "Specifically binds" or "immunologically reacts with" can mean that an antibody reacts with one or more antigenic determinants of a desired antigen and does not react with other polypeptides.

[0401] As used herein, the term "antibody fragment" or "antigen-binding fragment" is a part of an antibody, such as F (ab′)2 、F (ab)2 、F ab ′、F ab, Fv, scFv, etc. Regardless of the structure, the antibody fragment will bind to the same antigen recognized by the intact antibody. The term "antibody fragment" may include aptamers (such as mirror image aptamers), minibodies, and diabodies. The term "antibody fragment" may also include any synthetic or genetically engineered protein that functions like an antibody by binding to a specific antigen to form a complex. The 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′)2, Fd, Fv, single-chain Fv (scFv), single-chain antibodies, dAb (domain antibodies), minibodies, disulfide-linked Fv (sdFv), fragments containing VL or VH domains, fragments generated from a Fab expression library, and anti-idiotypic (anti-Id) antibodies.

[0402] "Single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy chain (V H ) and light chain (V L ) of an immunoglobulin. A single-chain Fv ("scFv") polypeptide molecule is a covalently linked VH:VL heterodimer that can be expressed from a gene fusion that includes a VH-encoding gene and a VL-encoding gene joined by a peptide-encoding linker. (See Huston et al. (1988) Proc Nat Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America] 85(16):5879-5883). In some aspects, the regions are joined by a short linker peptide of 10 to about 25 amino acids. The linker can be glycine-rich for flexibility or serine- or threonine-rich for solubility, and can join the N-terminus of V H to the C-terminus of V L , and vice versa. Although the constant regions are removed and a linker is introduced, the protein retains the specificity of the original immunoglobulin. Many methods have been described for resolving the chemical structure for converting the naturally aggregated but chemically separated light and heavy polypeptide chains from the antibody V regions into scFv molecules that will fold into a three-dimensional structure that is substantially similar to the structure of the antigen-binding site. See, for example, U.S. Patent Nos. 5,091,513; 5,892,019; 5,132,405; and U.S. Patent No. 4,946,778, each of which is incorporated herein by reference in its entirety.

[0403] Antibody molecules obtained from humans are divided into five classes of immunoglobulins: IgG, IgM, IgA, IgE, and IgD, which differ from each other due to the nature of the heavy chains present in the molecule. Those skilled in the art will understand that heavy chains are classified as gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε), and some of their subclasses (e.g., γ1-γ4). Certain classes also have subclasses, such as IgG1, IgG2, IgG3, and IgG4, etc. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., have been well characterized and are known to confer functional specialization. For IgG, a standard 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. These four chains are linked by disulfide bonds in a "Y" configuration, where the light chains are sandwiched between the heavy chains like brackets, starting from the mouth of the "Y" and continuing to the end of the variable region. The immunoglobulin or antibody molecules described herein can belong to 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 molecules.

[0404] Light chains are classified as kappa (κ) or lambda (λ). Each heavy chain class can bind to either a κ or λ light chain. For example, when an immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell, the light chain and heavy chain are covalently bonded to each other and the "tail" portions of the two heavy chains are bonded to each other either by covalent disulfide linkages or non-covalent linkages. In the heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the "Y" configuration to the C-terminus at the bottom of each chain.

[0405] Both light and heavy chains are divided into regions having structural and functional homology. The terms "constant" and "variable" are used functionally. The variable domains of the light chain (VL) and heavy chain (VH) portions determine antigen recognition and specificity. In contrast, the constant domains of the light chain (CL) and heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental movement, Fc receptor binding, complement binding, etc. 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 the amino acid residues of the N-terminal variable ("V") regions of the heavy chain ("H") and light chain ("L"). Three highly divergent segments (termed hypervariable regions) within the V regions of the heavy and light chains are interspersed between more conserved flanking segments called "framework regions" or "FRs". Thus, the term "FR" can refer to the amino acid sequences that naturally occur between and adjacent to the hypervariable regions of an immunoglobulin. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to one another in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementary determining regions" or "CDRs".

[0406] The six CDRs present in each antigen-binding domain are short discontinuous amino acid sequences that are specifically positioned to form the antigen-binding domain when the antibody assumes its three-dimensional conformation in an aqueous environment. The remaining amino acids in the antigen-binding domain (FR regions) exhibit less intermolecular variability. The framework regions predominantly adopt a β-sheet conformation, and the CDRs form loops that connect the β-sheet structures and, in some cases, form part of the β-sheet structure. The framework regions serve to form a scaffold that positions the CDRs in the correct orientation through interchain non-covalent interactions. The antigen-binding domain formed by the positioned CDRs provides a surface complementary to the epitope on the immunoreactive antigen, which facilitates the non-covalent binding of the antibody to its cognate epitope. One of ordinary skill in the art can readily identify the amino acids comprising the CDRs and framework regions for the heavy or light chain variable regions, as they have been previously defined (see, "Sequences of Proteins of Immunological Interest," Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0407] When two or more definitions exist for a term that is used and / or accepted within the art, the definition of the term as used herein is intended to include all such meanings, unless expressly stated to the contrary. A particular example is the use of the term "complementary determining region" ("CDR") to describe the non - contiguous antigen - combining sites found within the variable regions of heavy and light chain polypeptides. This region has been described by Kabat et al., U.S. Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al., J. Mol. Biol. 196:901 - 917 (1987) (which are hereby incorporated by reference in their entireties). The CDR definitions according to Kabat and Chothia include overlaps or subsets of amino acid residues when compared to each other. However, the application of either definition to refer to the CDRs of an antibody or its variants is intended to fall within the scope of the term as defined and used herein. The appropriate amino acid residues covering the CDRs defined by each of the above - cited references are listed in the table below for comparison. The exact number of residues covering a particular CDR will vary depending on the sequence and size of the CDR. Given the amino acid sequence of the variable region of an antibody, one of ordinary skill in the art can routinely determine which residues comprise a particular CDR. CDR Kabat Numbering Chothia Numbering VH CDR1 31-35 26-32 VH CDR2 50-65 52-58 VH CDR3 95-102 95-102 VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VL CDR3 89-97 91-96

[0408] Kabat et al. defined a numbering system for variable domain sequences that can be applied to any antibody. One of ordinary skill in the art can unambiguously assign this "Kabat numbering" system to any variable domain sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system proposed by Kabat et al., U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0409] In addition to the above table, the Kabat numbering system describes the CDR regions as follows: CDR-H1 starts 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 starts at the fifteenth residue after the end of CDR-H1, includes approximately 16 - 19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 starts at approximately the thirty-third amino acid residue after the end of CDR-H2; includes 3 - 25 amino acids; and ends at the sequence W - G - X - G, where X is any amino acid. CDR-L1 starts at approximately residue 24 (i.e., after the cysteine residue); includes approximately 10 - 17 residues; and ends at the next tryptophan residue. CDR-L2 starts at approximately the sixteenth residue after the end of CDR-L1 and includes approximately 7 residues. CDR-L3 starts at approximately the thirty-third residue (i.e., after the cysteine residue) after the end of CDR-L2; includes approximately 7 - 11 residues and ends at the sequence F or W - G - X - G, where X is any amino acid.

[0410] As used herein, the term "epitope" can include any protein determinant that can specifically bind an immunoglobulin, scFv, or T cell receptor. The variable regions allow an antibody to selectively recognize and specifically bind an epitope on an antigen. For example, the VL and VH domains of an antibody, or a subset combination of the complementarity determining regions (CDRs), form the variable region that defines the three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding sites present at the end of each arm of the Y. Epitope determinants can consist of groups of chemically reactive surface molecules (such as amino acids or sugar side chains) and can have specific three-dimensional structural features as well as specific charge features. For example, antibodies can be generated against the N-terminal or C-terminal peptides of a polypeptide. More particularly, 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.

[0411] As used herein, the terms "immunobinding" and "immunobinding properties" can refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific. The strength or affinity of an immunobinding interaction can be expressed by the dissociation constant (K d ) of the interaction, where a smaller K dIndicates greater affinity. Methods well known in the art can be used to quantify the immunobinding properties of the selected polypeptides. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, where these rates depend on the concentration of the complex partners, the affinity of the interaction, and geometric parameters that equally affect the rate in both directions. Thus, the "association rate constant" (K on ) and the "dissociation rate constant" (K off ) can be determined by calculating the concentrations and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). The ratio of K off / K on allows elimination of all parameters unrelated to affinity and is equal to the equilibrium binding constant K D . (See generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). As measured by kinetic assays such as radioligand binding assays or similar assays known to those skilled in the art such as BIAcore or Octet (BLI), the antibodies of the invention can specifically bind to an epitope when the equilibrium binding constant (K D ) ≤ 1 μM, ≤ 10 μM, ≤ 10 nM, ≤ 10 pM, or ≤ 100 pM to about 1 pM. For example, in some embodiments, K D is between about 1E-12 M and about 1E-11 M K D . In some embodiments, K D is between about 1E-11 M and about 1E-10 M K D . In some embodiments, K D is between about 1E-10 M and about 1E-9 M K D . In some embodiments, K D is between about 1E-9 M and about 1E-8 M K D . In some embodiments, K D is between about 1E-8 M and about 1E-7 M K D . In some embodiments, K D is between about 1E-7 M and about 1E-6 M K D . For example, in some embodiments, K D is about 1E-12 M, while in other embodiments, K D is about 1E-11 M. In some embodiments, K D is about 1E-10 M, while in other embodiments, K D is about 1E-9 M. In some embodiments, K D is about 1E-8 M, while in other embodiments, KD is about 1E-7 M. In some embodiments, K D is about 1E-6 M, and in other embodiments, K D is about 1E-5 M. For example, in some embodiments, K D is about 3E-11 M, and in other embodiments, K D is about 3E-12 M. In some embodiments, K D is about 6E-11 M. "Specifically binds" or "is specific for" can refer to an antibody that binds to an epitope via its antigen-binding domain, and this binding requires some complementarity between the antigen-binding domain and the epitope. For example, an antibody is said to "specifically bind" to an epitope when it binds to the epitope more readily via its antigen-binding domain than it binds to a random, unrelated epitope.

[0412] For example, an antibody can be monovalent or divalent and can comprise single-stranded or double-stranded. Functionally, the binding affinity of an antibody is in the range of 10 -5 M to 10 -12 M. For example, the binding affinity of an antibody is 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, 10 -7 M to 10 -10 M, 10 -8 M to 10 -10 M, 10 - 9 M to 10 -10 M, 10 -5 M to 10 -9 M, 10 -6M to 10 -9 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 -9 M, 10 -5 M to 10 -8 M, 10 -6 M to 10 -8 M, 10 -7 M to 10 -8 M, 10 -5 M to 10 -7 M, 10 -6 M to 10 -7 M, or 10 -5 M to 10 -6 M.

[0413] One of ordinary skill in the art will recognize that, without undue experimentation, it is possible to determine whether a human monoclonal antibody has the same specificity as a human monoclonal antibody of the invention by determining whether the human monoclonal antibody blocks the specific binding of the human monoclonal antibody of the invention. For example, if the human monoclonal antibody being tested competes with the human monoclonal antibody of the invention, as shown by a decrease in the binding of the human monoclonal antibody of the invention, then the two monoclonal antibodies bind to the same or closely related epitopes.

[0414] Another way to determine whether a human monoclonal antibody has the specificity of a human monoclonal antibody of the invention is to pre-incubate the human monoclonal antibody of the invention with an epitope that it normally reacts with, and then add the human monoclonal antibody being tested to determine whether the ability of the human monoclonal antibody being tested to bind the epitope is inhibited. If the human monoclonal antibody being tested is inhibited, then it has the same or functionally equivalent epitope specificity as the monoclonal antibody of the invention. The screening of the human monoclonal antibodies of the invention can also be carried out by utilizing an epitope and determining whether a test monoclonal antibody is able to neutralize a polypeptide containing the epitope.

[0415] A variety of procedures known in the art can be used to generate polyclonal or monoclonal antibodies against the proteins of the invention or their derivatives, fragments, analogs, homologs or orthologs. (See, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, which is incorporated herein by reference).

[0416] Antibodies can be purified by well-known techniques, such as affinity chromatography using Protein A or Protein G, which mainly provides the IgG fraction of immune sera. Subsequently or alternatively, the specific antigen or its epitope that is the target of the sought-after immunoglobulin can be immobilized on a column to purify the immunospecific antibody by immunoaffinity chromatography. The purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia, Pennsylvania, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0417] As used herein, the term "monoclonal antibody" or "mAb" or "Mab" or "monoclonal antibody composition" can refer to a population of antibody molecules that contains 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 the same in all molecules of the population. A MAb contains an antigen-binding site that can immunoreact with a specific epitope of an antigen, which is characterized by having a unique binding affinity for the antigen-binding site.

[0418] Monoclonal antibodies can be prepared using the hybridoma method, 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 elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro. Nucleic Acids, Vectors, and Cells Expressing Bispecific Modulators

[0419] 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.) comprising the nucleic acids. Also disclosed are various cells (e.g., prokaryotic cells, eukaryotic cells) containing the nucleic acids or vectors and capable of expressing the fusion proteins. Method

[0420] The present disclosure provides methods for administering the bispecific modulators described herein to a subject. In various embodiments, the bispecific modulators can internalize a target membrane protein (e.g., a cell receptor or other membrane protein) and selectively degrade and / or modulate these proteins. In some embodiments, the bispecific modulators can target the CAR receptor on CAR-T cells. In some embodiments, these methods are used to treat the 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, these methods are used to improve the efficacy of CAR-T cells that have been administered to a subject for the treatment of cancer.

[0421] In some embodiments, the membrane proteins on cancer cells can be targeted for degrading and / or modulating proteins (e.g., epidermal growth factor receptor or EGFR, programmed death ligand or PD-L1). In some instances, the bispecific modulators can be used to improve the anti-tumor response in this manner.

[0422] In some embodiments, the reagents and methods disclosed herein can be used with cells that are not cancer cells. Therapeutic Preparation

[0423] Aspects of the invention relate to therapeutic formulations. As used herein, the term "therapeutic formulation" can refer to any compound or composition (e.g., a bispecific modulator) that can be used or administered to achieve a therapeutic effect. As used herein, the term "therapeutic effect" can refer to an effect sufficient to result in an improvement in symptoms, such as the treatment, healing, prevention, or amelioration of a related medical condition, or an increase in the rate of treatment, healing, prevention, or amelioration of such a condition.

[0424] Embodiments as described herein may be administered to a subject in the form of a pharmaceutical composition or therapeutic formulation prepared for a desired route of administration. Such compositions and formulations may comprise, for example, one or more active ingredients and a pharmaceutically acceptable carrier. Such compositions and formulations may be in a form suitable for oral, subcutaneous, parenteral (such as intravenous, intraperitoneal), intramuscular, rectal, epidural, intratracheal, intranasal, dermal, vaginal, buccal, ophthalmic or pulmonary administration, such as in a form suitable for administration by a peripheral route, or suitable for oral administration or suitable for parenteral administration. Other routes of administration are subcutaneous, intraperitoneal and intravenous, and such compositions may be prepared in a manner well known to those skilled in the art, for example, as described in "Remington's Pharmaceutical Sciences", 17th edition, Alfonso R. Gennaro (editor), Mark Publishing Company, Easton, Pennsylvania, USA, 1985 and more recent editions, and monographs in the "Drugs and the Pharmaceutical Sciences" series, Marcel Dekker. These compositions and formulations may occur in conventional forms, such as solutions and suspensions for injection, capsules and tablets, in enteric-coated formulations (such as those disclosed in U.S. Patent No. 5,350,741) and for oral administration.

[0425] Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate or phosphate and agents for adjusting tonicity such as sodium chloride or dextrose. The pH may be adjusted with an acid or a base such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

[0426] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include saline, bacteriostatic water, Cremophor EM TM(BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition can be sterile and can be a fluid to the extent that it is easy to inject. In embodiments, it can be stable under manufacturing and storage conditions and can prevent the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing the following substances: for example, water, ethanol, pharmaceutically acceptable polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the dispersion, and by using surfactants. The action of preventing microorganisms can be achieved by various antibacterial and antifungal agents (for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal). In many cases, it can be useful to include isotonic agents such as sugars, polyols (such as mannitol, sorbitol), and sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption (for example, aluminum monostearate and gelatin) in the composition.

[0427] Sterile injectable solutions can be prepared by incorporating the compound in a suitable solvent, which optionally has one or a combination of the ingredients enumerated herein, in the required amounts, followed by filtration sterilization. Dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, examples of useful preparation methods are vacuum drying and freeze drying, which yield a powder of the active ingredient plus any additional required ingredients from its previously sterile filtered solution.

[0428] Oral compositions can include an inert diluent or an edible carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For purposes of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared using a fluid carrier as a mouthwash, where the compound in the fluid carrier is orally applied and swished and then either spat out or swallowed. For example, depending on the half-life of the drug, the oral formulation of the drug can be administered once, twice, three times, or four times a day.

[0429] It can include pharmaceutically compatible binders and / or adjuvant materials as part of the composition to be administered to a subject. Tablets, pills, capsules, lozenges, etc. can contain any one of the following ingredients or compounds with similar properties: binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, (Sodium starch glycolate) or corn starch; lubricants such as magnesium stearate or sterotes; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring.

[0430] Systemic administration can also be carried out by the transmucosal or transdermal route. For transmucosal or transdermal administration, permeants appropriate for the barrier to be penetrated are used in the formulation. Such permeants are known in the art and for transmucosal administration include, for example, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished by using nasal sprays or suppositories. For transdermal administration, these active compounds are formulated into ointments, salves, gels, or creams (as known in the art).

[0431] In an embodiment, administration can include placing the pharmaceutical composition in a subject by a method or route that causes the composition to be at least partially located at the desired site such that the desired effect is produced.

[0432] For example, the pharmaceutical composition can be administered by bolus or by infusion. Bolus can refer to the administration route where a syringe is connected to an IV access device and the drug is injected directly into the subject. The term "infusion" can refer to intravascular injection.

[0433] The embodiments described herein can be administered to a subject once (e.g., as a single injection, bolus, or deposition). Alternatively, it can be administered to the subject once or twice a day for a period of time, such as about 2 weeks to about 28 days. Administration can continue for up to one year. In an embodiment, administration can continue for the lifetime of the subject. It can also be administered to the subject once or twice a day for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times / year or a combination thereof.

[0434] In an embodiment, the compositions described herein can be administered to a subject for a long term. "Long-term administration" can refer to administration in a continuous manner so as to maintain the therapeutic effect (activity) over an extended period of time.

[0435] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody, its variants or derivatives used, the patient's age, weight, general health, gender and diet, as well as the time of administration, excretion rate, drug combination, and the severity of the particular disease being treated. The judgment of the medical caregiver regarding these factors is within the scope of those of ordinary skill in the art. The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0436] A therapeutically effective amount of the reagent or therapeutic composition of the present invention can be the amount required to achieve the therapeutic purpose. As described herein, this can be the binding interaction between the reagent or therapeutic composition and its target, which in some cases interferes with the target's ability to function. Additionally, the amount to be administered will depend on the binding affinity of the reagent or therapeutic composition for its specific target and will also depend on the rate at which the administered reagent or therapeutic composition is depleted from the free volume of the subject to which it is administered. The dose of the binding polypeptide described herein administered to a subject (e.g., a patient) is from about 0.1 mg / kg to 100 mg / kg of patient body weight, between 0.1 mg / kg and 20 mg / kg of patient body weight, or from 1 mg / kg to 10 mg / kg of patient body weight. Due to the immune response to foreign polypeptides, human antibodies have a longer half-life in the human body than antibodies from other species. Thus, it is generally possible to use lower doses of human antibodies and lower frequencies of administration. Further, the dose and frequency of administration of the reagents or therapeutic compositions disclosed herein can be reduced by enhancing the uptake and tissue permeability of these antibodies (e.g., into the brain) (by modification such as, for example, lipidation). By way of non-limiting example, a common range for the therapeutically effective dose of an antibody or antibody fragment of the present invention can be from about 0.1 mg / kg body weight to about 50 mg / kg body weight. A common range for the frequency of administration can be, for example, from twice daily to once weekly.

[0437] In the case of using fragments (e.g., antibody fragments), the minimal inhibitory fragment that specifically binds to the target protein binding domain is preferred. For example, based on the variable region sequence of an antibody, a peptide molecule can be designed to retain the ability to bind to the target protein sequence. Such a peptide can be produced synthetically and / or by recombinant DNA technology. (See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)). The formulation can also contain more than one active compound, for example those having complementary activities that do not adversely affect each other, depending on the needs of the specific indication being treated. Alternatively or in addition, the composition can contain agents that enhance its function, such as, for example, cytotoxic agents, cytokines (e.g., IL-15), chemotherapeutic agents, or growth inhibitors. Such molecules are suitably present in combinations effective for the intended purpose.

[0438] The active ingredient can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, e.g., in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions of hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules. Sustained-release preparations can be prepared.

[0439] The pharmaceutical or therapeutic carrier or diluent employed may be a conventional solid or liquid carrier. Non-limiting examples of solid carriers are lactose, talc powder, sucrose, cyclodextrin, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, stearic acid or lower alkyl ethers of cellulose. Non-limiting examples of liquid carriers are syrups, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyethylene oxides and water. Similarly, the carrier or diluent may include any sustained release substances known in the art, alone or mixed with waxes, such as glyceryl monostearate or glyceryl distearate.

[0440] When a solid carrier is used for oral administration, the preparation may be tableted, placed in a hard gelatin capsule in the form of powder or pellets or it may be in the form of a lozenge or troche. The amount of solid carrier will vary widely but may be from about 25 mg to about 1 g.

[0441] When a liquid carrier is used, the preparation may be in the form of a syrup, emulsion, soft gelatin capsule or a sterile injectable liquid (such as an aqueous or non-aqueous liquid suspension or solution).

[0442] The composition and / or preparation may also be in a form suitable for local or systemic injection or infusion and may thus be formulated with sterile water or isotonic saline or glucose solution. These compositions may be in a form suitable only for peripheral administration other than the form suitable for central administration. The composition and / or preparation may be in a form suitable for central administration.

[0443] The composition and / or preparation may be sterilized by conventional sterilization techniques well known in the art. The resulting aqueous solution may be packaged for use or filtered and lyophilized under aseptic conditions, and the lyophilized preparation is combined with a sterile aqueous solution before administration. The composition and / or preparation may contain pharmaceutically and / or therapeutically acceptable auxiliary substances such as buffering agents, tonicity regulators, etc., as required to approximate physiological conditions, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. Example

[0444] The following numbered paragraphs disclose exemplary embodiments disclosed herein.

[0445] 1. A bispecific modulator as disclosed herein.

[0446] 2. The bispecific modulator according to Example 1, the bispecific modulator comprising:

[0447] a. A first part, the first part comprising an antigen or epitope that can be bound by a chimeric antigen receptor (CAR); and

[0448] b. A second part, the second part being capable of binding to an internalization receptor or membrane protein on the cell.

[0449] 3. The bispecific regulator as described in Example 1, the bispecific regulator comprising:

[0450] a. A first part, the first part comprising an antibody capable of binding to a CAR; and

[0451] b. A second part, the second part capable of binding to an internalization receptor or membrane protein on the cell.

[0452] 4. The bispecific regulator as described in one of Examples 2 or 3, wherein the second part comprises an antibody.

[0453] 5. The bispecific regulator as described in one of Examples 2 or 3, wherein the binding of the first part to the CAR and the binding of the second part to the internalization receptor or membrane protein on the cell cause the CAR to internalize into the cell.

[0454] 6. The bispecific regulator as described in Example 5, wherein the binding of the first part to the CAR and the binding of the second part to the internalization receptor or membrane protein on the cell cause the CAR to internalize into the cell.

[0455] 7. The bispecific regulator as described in Example 5, wherein the binding of the first part to the CAR and the binding of the second part to the internalization receptor or membrane protein on the cell cause the CAR to internalize into the cell and the degradation of the CAR.

[0456] 8. A molecule having at least two parts, the at least two parts comprising:

[0457] a. A first part, the first part capable of binding to a target molecule on the cell; and

[0458] b. A second part, the second part capable of binding to an internalization molecule on the cell.

[0459] 9. The molecule as described in Example 29, wherein:

[0460] a. The first part comprises an antibody, antibody fragment, ligand, peptide, small molecule or aptamer; and

[0461] b. The second part comprises an antibody, antibody fragment, ligand, peptide, small molecule or aptamer.

[0462] 10. The molecule as described in Example 8, wherein the target molecule on the cell is different from the internalization molecule on the cell.

[0463] 11. The molecule as described in Example 8, wherein the first part and the second part comprise a polypeptide.

[0464] 12. The molecule as described in Example 9, wherein the internalization molecule on the cell comprises an internalization receptor.

[0465] 13. The molecule according to embodiment 12, wherein the internalizing molecule can be internalized by clathrin-mediated endocytosis.

[0466] 14. The molecule according to embodiment 12, wherein the internalizing molecule can be internalized by clathrin-independent endocytosis.

[0467] 15. The molecule according to embodiment 12, wherein the internalizing molecule comprises a transferrin receptor.

[0468] 16. The molecule according to embodiment 12, wherein the internalizing molecule comprises a G protein-coupled receptor (GPCR), a receptor tyrosine kinase (RTK), or a transmembrane receptor (TMR).

[0469] 17. The molecule according to embodiment 16, wherein the GPCR comprises an adrenergic receptor, a chemokine receptor, or a coagulation receptor.

[0470] 18. The molecule according to 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.

[0471] 19. The molecule according to embodiment 16, wherein the TMR comprises a folate receptor, an interleukin receptor (e.g., IL-2 receptor), a low-density lipoprotein receptor, or a transferrin receptor.

[0472] 20. The molecule according to embodiment 12, wherein the internalizing molecule comprises a transferrin receptor (TfR).

[0473] 21. The molecule according to embodiment 20, wherein the transferrin receptor comprises transferrin receptor 1 (TfR1) or transferrin receptor 2 (TfR2).

[0474] 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 the receptor.

[0475] 23. The molecule according to embodiment 22, wherein the ligand of the internalizing molecule comprises at least a portion of transferrin, cholesterol, low-density lipoprotein, and epidermal growth factor.

[0476] 24. The molecule according to embodiment 9, wherein the ligand of the internalizing molecule can be bound by a G protein-coupled receptor (GPCR), a receptor tyrosine kinase (RTK), or a transmembrane receptor (TMR).

[0477] 25. The molecule according to embodiment 22, wherein the ligand of the internalizing molecule can be bound by the transferrin receptor.

[0478] 26. The molecule according to embodiment 22, wherein the ligand of the internalizing molecule comprises transferrin or a part of transferrin.

[0479] 27. The molecule according to embodiment 9, wherein the second part comprises a Fab, scFv, single domain antibody, nanobody, single antibody, DARPin or affibody.

[0480] 28. The molecule according to embodiment 27, wherein the second part comprises an H7 scFv.

[0481] 29. The molecule according to embodiment 27, wherein the second part comprises an H7 Fab or an engineered H7 antibody variant.

[0482] 30. The molecule according to embodiment 9, wherein the second part can bind to a transferrin receptor, cholesterol receptor, low density lipoprotein receptor or epidermal growth factor receptor.

[0483] 31. The molecule according to embodiment 9, wherein the second part can bind to a G protein-coupled receptor (GPCR), receptor tyrosine kinase (RTK) or transmembrane receptor (TMR).

[0484] 32. The molecule according to embodiment 9, wherein the second part can bind to a transferrin receptor (TfR).

[0485] 33. The molecule according to embodiment 8, wherein the target molecule comprises a protein.

[0486] 34. The molecule according to embodiment 33, wherein the protein comprises a membrane protein.

[0487] 35. The molecule according to embodiment 33, wherein the protein comprises an integral membrane protein.

[0488] 36. The molecule according to embodiment 33, wherein the protein comprises an extracellular protein.

[0489] 37. The molecule according to embodiment 36, wherein the extracellular protein is found in the external environment.

[0490] 38. The molecule according to embodiment 36, wherein the extracellular protein is selected from the group consisting of autoantibodies, cytokines, enzymes and combinations thereof.

[0491] 39. The molecule according to embodiment 33, wherein the protein comprises a transmembrane protein.

[0492] 40. The molecule according to embodiment 39, wherein the transmembrane protein has one (1) or more transmembrane domains.

[0493] 41. The molecule according to embodiment 8, wherein the molecule of interest can bind a hormone, cytokine, growth factor, neurotransmitter, lipophilic signaling molecule (e.g., prostaglandin), or cell recognition molecule (e.g., integrin, selectin).

[0494] 42. The molecule according to embodiment 8, wherein the molecule of interest includes a receptor.

[0495] 43. The molecule according to embodiment 42, wherein the receptor includes a G protein-coupled receptor (GPCR).

[0496] 44. The molecule according to embodiment 42, wherein the receptor includes a receptor tyrosine kinase (RTK) or a transmembrane receptor (TMR).

[0497] 45. The molecule according to embodiment 39, wherein the receptor includes a ligand-gated ion channel-linked molecule, a transporter, an enzyme-linked molecule, or a G protein-linked receptor.

[0498] 46. The molecule according to 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.

[0499] 47. The molecule according to embodiment 45, wherein the enzyme-linked molecule includes a receptor tyrosine kinase, a tyrosine kinase-associated receptor (e.g., an enzyme associated with a cytokine), a receptor-like tyrosine phosphatase (e.g., a receptor-like tyrosine phosphatase that removes a phosphate group from tyrosine of an intracellular protein), a receptor serine / threonine kinase, a receptor guanylyl cyclase, or a histidine kinase-associated receptor.

[0500] 48. The molecule according to embodiment 8, wherein the molecule of interest includes 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).

[0501] 49. The molecule according to embodiment 8, wherein the molecule of interest includes a CAR, an EGFR, a CD20, or a PD-L1.

[0502] 50. The molecule according to embodiment 9, wherein the first portion contains an amino acid sequence to which a receptor can bind.

[0503] 51. The molecule according to embodiment 50, wherein the first portion includes a ligand of the receptor.

[0504] 52. The molecule according to embodiment 51, wherein the ligand comprises the extracellular domain of CD19, and the receptor comprises a CAR specific for CD19.

[0505] 53. The molecule according to embodiment 50, wherein the receptor comprises a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a B cell receptor (BCR).

[0506] 54. The molecule according to embodiment 9, wherein the first part comprises an scFv, a Fab, a single-domain antibody, a nanobody, a monobody, a DARPin, or an affibody.

[0507] 55. The molecule according to embodiment 8 or 9, wherein the molecule further comprises a peptide linker that can be cleaved by a protease.

[0508] 56. The molecule according to embodiment 55, wherein the protease comprises an endosomal / lysosomal protease.

[0509] 57. The molecule according to embodiment 56, wherein the protease comprises a cathepsin.

[0510] 58. The molecule according to embodiment 55, wherein the peptide linker is located between the first part and the second part on the polypeptide comprising the first part and the second part.

[0511] 59. The molecule according to embodiment 58, wherein the peptide linker is closer to the first part than to the second part.

[0512] 60. The molecule according to embodiment 55, wherein the peptide linker comprises Gly-Phe-Leu-Gly (GFLG).

[0513] 61. The molecule according to embodiment 55, wherein the peptide linker comprises valine-arginine (VR) and / or phenylalanine-lysine (FK).

[0514] 62. The molecule according to embodiment 55, wherein the peptide linker comprises GS, GFLG, 3xGFLG, GFLG-VA, GFLG-VK, GFLG-VR, GFLG-GFLG, FK, VA, EVR, VK linker, or a combination thereof ( Figure 35 ).

[0515] 63. The molecule according to embodiment 55, wherein the second part comprises an scFv, a Fab, a single-domain antibody, a nanobody, a monobody, a DARPin, or an affibody.

[0516] 64. The molecule according to embodiment 63, wherein the scFV comprises H7.

[0517] 65. A molecule as described in Example 55, wherein cleavage of the protease on the peptide linker can enable the target molecule to be captured and / or degraded inside the cell into which the molecule having at least two parts is internalized.

[0518] 66. A molecule as described in Example 8 or 9, the molecule comprising the amino acid sequence SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78 or 79 - 117, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0519] 67. A molecule as described in Example 8 or 9, wherein the nucleotide sequence encoding the molecule comprises SEQ ID NO: 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 or 77, or a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0520] 68. A bispecific modulator, the bispecific modulator comprising:

[0521] a. A first antibody or antibody fragment that binds to the transferrin receptor (TfR) on the cell surface; and

[0522] b. A second antibody or antibody fragment that binds to a transmembrane protein on the cell surface that is not TfR.

[0523] 69. A bispecific modulator, the bispecific modulator comprising:

[0524] a. Transferrin or a part of transferrin that binds to TfR on the cell surface; and

[0525] b. An antibody or antibody fragment that binds to a transmembrane protein on the cell surface that is not TfR.

[0526] 70. A bispecific modulator as described in Example 68 or 69, wherein the first antibody or antibody fragment and the second antibody or antibody fragment (Example 68), or transferrin / part of transferrin and the antibody or antibody fragment (Example 69) are part of a polypeptide.

[0527] 71. A bispecific modulator as described in Example 68 or 69, wherein the first antibody or antibody fragment and the second antibody or antibody fragment, or the transferrin / part of transferrin and the antibody or antibody fragment are more than one polypeptide.

[0528] 72. A bispecific modulator as described in Example 71, wherein the more than one polypeptide comprises two polypeptide chains linked by a dimerization domain.

[0529] 73. A bispecific modulator as described in Example 72, wherein the dimerization domain comprises a coiled-coil Fc.

[0530] 74. A bispecific modulator as described in Example 68 or 69, the bispecific modulator further comprising a peptide linker that can be cleaved by endosomal / lysosomal proteases.

[0531] 75. One or more nucleic acids encoding a molecule as described in any one of Examples 8-67 or a bispecific modulator as described in any one of Examples 68-74.

[0532] 76. A vector comprising the nucleic acid as described in Example 75.

[0533] 77. A cell comprising the vector as described in Example 76.

[0534] 78. A method for treating toxicity associated with CAR-T therapy or for increasing the efficacy of immune checkpoint and targeted cancer therapies, the method comprising administering to a subject a molecule as described in any one of Examples 8-67 or a bispecific modulator as described in any one of Examples 68-74. Example

[0535] Examples are provided below to facilitate a more comprehensive understanding of the present invention. 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 similar results can be obtained using alternative methods. Example 1 - Construction of a TransTAC (Bispecific Modulator) for Downregulating EGFR

[0536] Express anti-EGFR affibody-Fc-Tf TransTAC and verify by SDS-PAGE ( Figure 10 , left). Incubate TransTAC from zero to 60 nM with the MCF10A EGFR overexpressing cell line for 12, 36 or 68 h. A sharp dose-dependent decrease in EGFR levels was observed by flow cytometry, where at 68 h, the IC 50 <6 nM( Figure 10, right). We observed the same dose-dependent decrease in EGFR when using the adenocarcinoma lung cancer cell line A549 (Figure 11A). Then time-course experiments were performed on A549 cells with 30, 60, or 90 nM TransTAC, which showed that EGFR was effectively downregulated, with a half-life <30 min at 90 nM TransTAC. Additionally, we observed the killing of MCF10A-EGFR cells mediated by TransTAC (Figure 11B). These data demonstrate the effectiveness of the construct.

[0537] Notably, TransTAC downregulates more than 99% of cell surface EGFR at equilibrium, while the recently reported LYTAC only achieves a 70%-80% downregulation. Additionally, the kinetics are also different. TransTAC-driven EGFR internalization has a half-life of <30 min, while the half-life in the case of LYTAC is approximately 10-20 h. These differences are due to the different endocytosis kinetics of the carrier proteins used in the studies.

[0538] Additional data ( Figure 12 ) show that using A549 cells expressing the EGF receptor (EGFR) on the cell surface, TransTAC targeting EGFR internalizes the receptor to a greater extent than the EGFR-specific antibody alone. Example 2 - Construction of a TransTAC (Bispecific Modulator) for Downregulating Anti - CD19 CAR

[0539] Data show that transferrin fusion proteins with antibodies specific for CD19, EGFR, and HER2 are well-expressed in expi293 cells ( Figure 13 ).

[0540] Figure 14 Data showing that the mortar and pestle form of TransTAC targeting the anti-CD19 chimeric antigen receptor internalizes the receptor are presented.

[0541] Figure 15 Data showing the measurement of surface CAR levels in cells using anti-myc-biotin / streptavidin 647 are presented, similar to the studies shown in Figure 14 . Figure 15 The data in Figure 16 show that the homodimeric TransTAC effectively internalizes CAR for targeting. In the study shown in Figure 16 , cells expressing anti-CD19 CAR were incubated with K562 cells (expressing CD19) with the TransTAC bispecific modulator. Cell activation was measured. The data show that the TransTAC bispecific modulator begins to show inhibitory activity at concentrations below 10 nM. These data indicate that the TransTAC molecule effectively internalizes CAR and downregulates CAR-T cell activity.

[0542] Figure 17 The data in Figure 16 are similar to the data shown in Figure 12 The data in 50 show that TransTAC improves the IC that inhibits cell activation to approximately 10 - 20 nM.

[0543] Figure 18 The data in 50 show that the TransTAC molecule with the CD19NT.1 variant extracellular domain blocks CAR-T activation, where the IC Example 3 - TransTAC Molecules Containing Protease Sites to Increase Target Degradation

[0544] Figures 19A - 19B are schematic diagrams of the molecule (A) used in these studies and the results (B) obtained with the molecule. TransTAC1.0 in the accompanying drawings in this example is TransTAC0.4 as shown in Figure 44A.

[0545] Figure 19C Shows the fluorescence microscopy results of the target CAR on the cells in Figure 19B.

[0546] Figures 20A - 20B are schematic diagrams of the molecule (A) used in these studies and the protein blot results (B) of the target CAR and actin control.

[0547] Figure 20C A graph showing the data from Figure 20B is shown.

[0548] Figures 20D - 20E show the protein blot results (D) and data graph (E) of additional molecules used in these studies.

[0549] Figures 20F - 20G show the TransTAC molecule containing the GFLG linker (F) and the protein blot data (G) using the molecule.

[0550] Figure 20H A graph showing the data from Figure 20G is shown.

[0551] Figure 20I Results from other cathepsin-sensitive TransTAC molecules are shown.

[0552] Figures 21A - 21B show results that demonstrate that in experiments in which cells expressing an anti - CD19 CAR receptor were incubated with CD19 - positive A375 cells, the molecule shown in Figure 20A inhibits activation in Jukat cells (A) and inhibits the release of interferon - γ (IFN - γ) from primary T cells (B).

[0553] Figure 22A Results are shown that demonstrate that addition of the indicated molecule shown in Figure 20A blocks the killing of CD19 - positive A375 target cells by human primary anti - CD19 CAR - T cells. A375 cells express nuclear mCherry for fluorescence microscopy. Results show that removal of the molecule leads to re - activation of CAR - T cells and killing of CD19 - positive A375 target cells. The photographs show the red fluorescence channel of a fluorescence microscope.

[0554] Figure 22B Results are shown that demonstrate that removal of the indicated molecule leads to re - activation of CAR - T cells and killing of CD19 - positive A375 target cells. The photographs show an overlay of the red fluorescence channel and the white - light channel. Example 4 - TransTAC Molecules Targeting Epidermal Growth Factor Receptor (EGFR)

[0555] Figures 23A - 23B show a schematic of the molecules used in these studies (A). These molecules contain an antibody, an affibody, and a TransTAC molecule that are specific for EGFR. Results are also shown demonstrating the use of these molecules to reduce the level of EGFR on the surface of A549 cells (B).

[0556] Figures 23C - 23D show results of using the molecule of Figure 53A to inhibit cell proliferation. Example 5 - TransTAC Molecules Targeting CD20

[0557] These data show an exemplary method of using a TransTAC molecule that has an antibody specific for CD20 and a molecule that binds to the transferrin receptor (transferrin or an antibody).

[0558] Figure 24B - Figure 24C shows a schematic of the molecules used in these studies (B) and the results obtained from their use (C).

[0559] Figure 24D shows a graph of Figure 24C normalized data from

[0560] Data show that the TransTAC molecule internalizes / degrades faster than the anti - CD20 rituximab antibody that targets CD20 alone. Example 6 - Reversible Control of Receptor Function

[0561] Figure 25A shows cells expressing CAR. The cells in the left inset have been contacted with the CD19m-Fc antibody (CD19NT.1 variant). The cells in the right inset have been contacted with the TransTAC molecule that binds CAR. The cell nuclei in both insets have been stained with DAPI. The cells have also been stained with an anti-CD3z antibody that stains CAR. Immunofluorescence from the anti-CD3z antibody localizes to the cell surface in the left inset cells. Anti-CD3z antibody fluorescence localizes to the cytoplasm in the right inset cells. The data show that the TransTAC molecule causes internalization of CAR.

[0562] Figure 25B shows the reversibility of CAR internalization by TransTAC. In the first bar of the figure, the cell surface CAR-specific immunofluorescence of cells not contacted with TransTAC is relatively high (about 1.0). In the second bar of the figure, the cells have been contacted with TransTAC and the cell surface CAR-specific immunofluorescence is low (about 0.2). In the third bar of the figure, the cells have been contacted with TransTAC, but then the TransTAC is removed. After 24 hours, the cell surface CAR-specific immunofluorescence increases to a level similar to that of cells not exposed to TransTAC (about 1.0).

[0563] These data show that CAR receptor internalization achieved by TransTAC acts as a reversible CAR-T cell off-switch. This can be used to mitigate any toxicity associated with CAR-T therapy.

[0564] Figure 26 Shows that TransTAC inhibits / prevents interferon γ production.

[0565] As discussed in Example 8, Figure 22A Shows that TransTAC prevents primary human anti-CD19 CAR-T cells from killing CD19-positive A375 target cells. The data show that removal of TransTAC leads to reactivation of CAR-T cells and killing of CD19-positive A375 target cells. Figure 22B Shows that removal of the TransTAC molecule leads to reactivation of CAR-T cells and killing of the target. Example 7 - Targeted Membrane Protein Degradation

[0566] Figure 27A - Figure 27B Shows the expression of transferrin receptor on various cells as indicated by fluorescent antibody. The data indicate that transferrin receptor can be expressed at higher levels on the surface of tumor cells compared to non-tumor cells.

[0567] Figure 28 Shows examples of cell surface proteins that can be targeted by TransTAC.

[0568] Figure 29A It shows that the TransTAC molecules (DP81 and DP174) reduce the amount of EGFR in cells.

[0569] Figure 29B It shows that the TransTAC molecules can degrade EGFR. The data show that TransTAC-mediated EGFR degradation is sensitive to bafilomycin (an inhibitor of autophagosome-lysosome fusion) and MG132 (a proteasome inhibitor). These data show that TransTAC-induced EGFR degradation is mediated by the lysosomal pathway.

[0570] Figure 30A It shows a method for treating lung cancer using TransTAC. The high expression of TfR in cancer cells enables achieving target specificity.

[0571] Figure 30B It shows that the EGFR TransTAC molecule can inhibit PC9 cancer cells (lung adenocarcinoma).

[0572] Figures 30C - 30D show that the EGFR TransTAC molecule can inhibit PC9 cancer cells.

[0573] Figure 31 It shows that the anti-CAR TransTAC molecule can reduce the amount of CAR in these cells.

[0574] Figure 32A It shows that the anti-PD-L1 TransTAC molecules (DP186, DP187) can reduce the amount of PD-L1 in these cells.

[0575] Figure 32B It presents data demonstrating that the anti-PD-L1 TransTAC molecule can reduce the amount of PD-L1 in cells.

[0576] Figure 33 It shows that the anti-CD20 TransTAC molecules (DP209S, DP210, DP213) can reduce the amount of CD20 in cells.

[0577] Figures 34A - 34C and Figure 34D It shows exemplary TransTAC molecules containing protease-sensitive linkers and exemplary data obtained using these molecules.

[0578] Figure 35 It shows exemplary data obtained using TransTAC molecules containing various protease-sensitive linkers.

[0579] Figures 36A - 36C show exemplary TransTAC molecules that contain antibody fragments specific for transferrin binding and exemplary data obtained with these molecules.

[0580] Figures 37A - 37B show examples of TransTAC molecules and exemplary data obtained with these molecules. Example 8 - TransTAC for Cancer

[0581] Targeted therapy using tyrosine kinase inhibitors is the standard treatment for lung cancer with EGFR mutations. We reasoned that co - targeting EGFR and TfR receptors on lung cancer cells could lead to EGFR inhibition while maintaining high tumor specificity ( Figure 30A ). We generated the anti - EGFR affibody *H7*GFLG - VR TransTAC. Incubating the molecule with the human lung adenocarcinoma A549 cell line resulted in >90% EGFR degradation ( Figure 30B ). Notably, treating the non - tumorigenic HEK cell line engineered to overexpress EGFR with the TransTAC molecule led to much less EGFR degradation, highlighting the tumor - specificity of the technology. This specificity is due to higher TfR expression in tumor cells ( Figure 27A - Figure 2 7C). Example 9 - Transferrin Receptor Upregulation in Cancer Cell Lines, Primary Tumors, and Activated T Cells

[0582] Increased expression of TfR on cancer cells and some immune cells was demonstrated in Example 7.

[0583] In additional studies, we measured cell - surface TfR levels using flow cytometry on five non - tumorigenic cell lines (including HEK293T (embryonic kidney), MCF10A (mammary epithelial), HFF - 1 (foreskin fibroblast), MCR - 5 (lung fibroblast), and LF - 1 (fetal lung fibroblast)) and 10 cancer cell lines (including Hela (cervical cancer); Raji (lymphoma); Jurkat and K562 (leukemia); MDA - MB - 231 and MCF - 7 (breast cancer); PC9 and A549 (lung cancer), and PC9 cells with an EGFR - resistant mutant). We observed that cancer cell lines expressed 2 - 26 - fold more TfR on the cell surface compared to non - tumorigenic cell lines (Figure 38C). Among the cancer cell lines, the leukemia cell lines Jurkat and K562 exhibited the highest TfR expression. Our findings demonstrate the up - regulation of TfR in cancer cell lines.

[0584] Since cell lines have been modified to be immortalized, there is a possibility of protein expression variation. Therefore, further it is shown that by performing transcriptomic analysis of TFRC (i.e., the gene of TfR1), the expression of TfR in primary tumors is upregulated compared to primary healthy tissues. Microarray transcriptomic data of TFRC in primary healthy tissues and tumors are obtained from the MERAV database. A paired tissue analysis of healthy samples versus tumor samples is performed using a custom python script. TFRC expression is significantly increased statistically in cancers overall (p = 3.98e - 89) and in cancers of 14 out of 19 specific tissues, including cancers of breast, lung, pancreas, liver, bladder, skin, esophagus, thyroid, testis, stomach, salivary gland, kidney, central nervous system, and female reproductive system tumors (Figure 38D). These findings provide further evidence to support TfR as an upregulated target in cancer. To investigate whether TfR can also be a potential target for immunocyte regulation, the DICE dataset is analyzed, which contains gene expression profiles of human immune cells isolated from blood samples of healthy donors. Although most immune cells express low levels of TfR, approximately 6 - fold higher TfR expression is observed in activated CD4 and CD8 T cells compared to unactivated T cells, and this level is comparable to the TfR levels in some malignant tissues, indicating that TfR can be a target for regulating activated T cells (Figure 38E). These findings suggest that TfR is upregulated not only in tumors but also in activated T cells, highlighting its value as a cell - surface receptor for both cancer and immune regulation. Our transcriptomic analysis provides a detailed comparison of TfR expression in specific tissues, providing a roadmap for future selection of disease indications for our technology and other related technologies. Example 10 - Targeted Protein Endosomal Capture Using an Early Form of TransTAC Design

[0585] Experiments on TransTAC molecules specific to CD19 - specific CAR are also described in Example 2 but are more detailed in this example.

[0586] 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 extracellular domain of native CD19 as the CAR-binding component, but significant protein aggregation was observed in SDS-PAGE gels (Figure 43B, lanes 1-2). We then evaluated a small panel of CD19 extracellular domain variants developed earlier using yeast surface display (Klesmith, Justin R. et al. “Retargeting CD19 chimeric antigen receptor T cells via engineered CD19-fusion proteins.” Molecular pharmaceutics 16.8, 2019: 3544-3558) and showed that they expressed and performed well (Figure 43A-Figure 43B). Finally, we selected the mutant CD19NT.1, which exhibited better expression, for use in CAR-TransTAC.

[0587] We tested the following concept: that a molecule containing two TfR ligands is more effective than a molecule containing one ligand in driving targeted CAR internalization, as TfR is a homodimeric receptor and requires the binding of two transferrins (TFs) to fully initiate TfR dimerization to exert its physiological function. Accordingly, we created two forms of TransTAC: v0.1, which has a single TF for binding TfR and a knob-into-hole Fc construct of one CD19NT.1 against CAR; and v0.2, which has two TFs and an Fc fusion of two CD19NT.1s (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 hours, we measured cell surface CAR levels using an anti-myc antibody. We found that treatment with both v0.1 and v0.2 significantly reduced cell surface CAR levels, with v0.1 exhibiting 60% D max and v0.2 exhibiting 80% D max (Figure 39C). Interestingly, a hook effect was observed in the case of v0.1 but not v0.2. In contrast, treatment with the control CD19NT.1-Fc protein did not result in a decrease in cell surface CAR levels. These findings show that CAR-TransTAC can effectively internalize CAR from the cell surface via a TF-dependent mechanism, and in some embodiments, the dimeric TransTAC is more effective than the monomer.

[0588] However, despite the ability to efficiently remove CAR from the cell surface, TransTAC v0.2 did not result in CAR degradation, as shown by whole cell lysate western blotting (Figure 39D, Figures 44A - 44B), indicating that the internalized receptor was trapped intracellularly and not degraded.

[0589] To understand the subcellular destination of CAR internalized by v0.2, we stably expressed CAR - GFP and different endosomal and lysosomal markers tagged with mCherry in the Hela cell line, including Rab5+ or EEA+ (EE), Rab7+ (LE), Rab11+ (RE), and Lamp1+ (lysosome) (Figure 39I, Figure 39J, Figures 45A - 45D). Fluorescence microscopy imaging of v0.2 - treated cells showed co - localization of CAR - GFP with Rab11, indicating that internalized CAR is transported to the RE (Figure 39I, white arrow). Thus, TransTAC v0.2 effectively removes the POI from the cell membrane by trapping the POI in the recycling endosomal compartment in target cells. Example 11 - Engineering Degraders by Rational Rearrangement of the Intracellular Trafficking Pathway of Internalized Protein Complexes Engineering

[0590] Experiments related to intracellular trafficking are described herein and in more detail in this example.

[0591] Additional studies are to develop the next generation of TransTACs that not only trap the POI but also lead to its degradation. This may be particularly beneficial for cancer - related targets, as degradation can allow for more persistent inhibition of protein function.

[0592] To direct target protein degradation, we tested whether the POI (protein of interest) needs to be disengaged from the recycling Tf / TfR complex in the EE, where sorting to the degradation or recycling pathway occurs (Figure 38A, Figure 39E, Figure 39F). Proteases located in endosomes, such as the cysteine protease cathepsin, can be used to separate the POI from the Tf / TfR complex. Thus, 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) (Figure 39A, Figure 44A). Indeed, this linker modification altered the intracellular trafficking of CAR-GFP. In the case of TransTAC v0.4, a significant portion of the receptor now co-localizes with Rab7 (late endosome) and Lamp1 (lysosome) (Figure 39L, Figure 45D - Figure 45F). Additionally, using western blot, we observed that approximately 50% of the CAR was degraded (Figure 44C). Degradation was lower in the v0.3 case compared to v0.4 (Figure 44C), likely because CD19NT.1 remains linked to the Fc domain after separation from Tf / TfR, and the Fc domain can mediate recycling via the FcRn pathway. Overall, our study shows that incorporation of a protease-cleavable linker into TransTAC results in degradation of the POI.

[0593] Our next goal was to improve degradation efficiency and expand our understanding of proteolysis in endosomes. Traditionally, protein degradation was thought to occur mainly in acidic lysosomes or LEs, and little was known about proteolytic activity in EEs. To find the best protease substrates in EEs, we performed a small-scale linker screen, assuming that protease activity in EEs was related to degradation efficiency. We used Western blot assays to screen a set of 14 linkers containing single or combined cathepsin B cleavage motifs (Poreba, Marcin. “Protease-activated prodrugs: strategies, challenges, and future directions.” The FEBS Journal 287.10, 2020: 1936 - 1969) in both TransTAC v0.4 and v1.0, such as GFLG, Gly-Gly-Phe-Gly (GGFG), Phe-Lys (FK), Val-Ala (VA), Val-Lys (VK), and Val-Arg (VR) (Figure 44D - Figure 44E). In summary, we found that incorporation of the dipeptide motifs VK, VR, and FK contributed to improved cleavage activity compared to the GFLG sequence. Based on these results, we selected the GFLG-VR linker and the EVR linker to develop subsequent generations of TransTACs. We used the EVR linker to replace VR for in vivo studies because previous studies had found that including glutamate improved the stability of the linker in mouse serum.

[0594] Thus, another step in optimizing TransTAC was taken by replacing the TF ligand with an anti-TfR single-chain Fv (scFv) (designated H7, a TF-competing 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 replacement was aimed at reducing RE sorting (a step we felt was important in increasing degradation efficiency), as proteins sorted to the RE cannot be transported to the LE / lysosome for degradation (Figure 39G, Figure 39H). The logic is that certain molecular features of the TF / TfR complex are involved in RE sorting, and using a synthetic antibody conjugate such as H7 can alter this sorting decision and redirect the intracellular trafficking of the complex after iron release in the EE.

[0595] Based on this, we generated two forms of TransTAC: v0.5, which contains the H7 conjugate but no cleavable linker, and v1.0, which contains both H7 and the cleavable linker (Figure 39A). Cells treated with v0.5 showed that CAR-GFP mainly co-localized with the EE markers Rab5 and EEA (Figure 39G, Figure 39J, Figure 45A, Figure 45D). The Pearson co-localization coefficients of the corresponding markers were statistically different from those of cells treated with TransTAC v0.2 containing TF as the anti-TfR ligand (Figure 39K). This result confirmed that replacing TF with the anti-TfR antibody could reduce RE trafficking. In addition, recycling led to a significant improvement in the degradation efficiency. In the case of using TransTAC v1.0, we observed more than 80% CAR degradation in both western blot and fluorescence microscopy assays (Figure 39D, Figure 39I, Figure 39J; Figure 45A - Figure 45C). In addition to improving degradation, the H7 substitution also increased the protein yield by approximately seven-fold, making the expression level of TransTAC similar to that of conventional antibodies. In summary, our rational protein engineering efforts have successfully developed a novel protein design, namely TransTAC v1.0, as an effective molecular degrader of CARs, which is fully recombinant and robustly expressed. CAR-TransTAC v1.0 represents the first recombinant protein degrader prepared to target synthetic receptors. Example 12 - Reversible Control of Primary CAR - T Cell Function with CAR - TransTAC

[0596] Experiments related to the efficacy and reversibility of controlling CAR-T cells with TransTAC molecules are described in Example 2 and Example 6.

[0597] In additional studies, we investigated the use of TransTAC as an OFF switch to fine-tune CAR-T cell activity and manage related toxicities, such as those that can manifest as cytokine release syndrome (CRS) caused by overactivation of CAR-T cells (Figure 47A).

[0598] As a proof of concept, it was shown that CAR-TransTAC v0.4 could effectively inhibit primary human CAR-T cells.

[0599] We isolated primary CD8+ T cells from human PBMCs and generated anti-CD19 CAR-T cells by lentiviral transduction. For tumor cells, we used the adherent melanoma cell line A375, which had been engineered to express CD19 and nuclear mCherry to facilitate live cell imaging. We observed that CAR-TransTAC v0.4 potently inhibited IFN-γ secretion, with an IC50 of approximately 0.4 nM and D maxwas 88% (Figure 47B, Figure 47C). These molecules also effectively blocked tumor killing activity (Figure 47B, Figure 47D). In addition, the inhibition was reversible as removal of the TransTAC restored the tumor killing activity of the primary CAR-T cells (Figure 47B, Figure 47E).

[0600] To better understand the factors influencing the different properties of CAR-TransTAC and to determine the general structure-function activity (SAR) relationship of TransTAC, we generated and tested four TransTAC v0.5 variants v0.6 - v0.9, each containing one or two copies of CD19NT.1 or H7 with different geometries (Figure 41B). Our findings revealed that having two H7s was superior to having two CD19NT.1s in enhancing CAR internalization (Figure 41C, Figure 41D). This highlighted the importance of dual binding to the dimeric TfR rather than having two anti-POI conjugates in generating an effective TransTAC. It was also demonstrated that TransTAC-mediated CAR internalization was not the result of CAR crosslinking. Additionally, we observed significant differences in the internalization efficiency of molecules with different geometries (Figure 41C), indicating that the tertiary complex structure plays a role in influencing TransTAC efficiency. Furthermore, we generated Fc-H7 molecules as competitors for TfR binding and observed a dose-dependent decrease in CAR internalization in the presence of the competitor in solution when treated with TransTAC v0.5 (Figure 47D). This observation further indicated that TransTAC acts through a TfR-dependent mechanism.

[0601] Since CAR clustering may lead to low levels of spontaneous CAR-T cell activation, we hypothesized that our dimeric CAR "OFF" switch molecules might affect their inhibitory effect on CAR-T cells by inducing CAR clustering. Therefore, we developed CAR "OFF" switches containing only one CD19NT.1 domain and compared them with dimeric variants. The study showed that the potency of the CD19NT.1 monomer was significantly reduced 100-fold compared to the CD19NT.1-Fc dimer. This finding highlighted the importance of the avidity for CD19NT.1-Fc in achieving effective CAR-T cell inhibition. Since CAR-T / tumor interaction involves multiple CAR / antigen interactions at the immunological synapse, molecules with multiple copies appear to compete more effectively with tumor antigens for CAR binding. To further understand the role of multivalency, we also generated a tetrameric variant that showed similar potency in CAR-T cell inhibition compared to the dimer.

[0602] CAR-TransTAC does not rely on competition with tumor CD19 to exert its function and thus does not require a dimer form to be effective. We observed that monomeric CD19NT.1-based TransTAC (the domain to which the CAR can bind is fused to the Fc region) exhibited robust performance and was even superior to the dimer variant in blocking CAR-Jurkat cells. Example 13 - Expansion of TransTAC Addressable Targets

[0603] Experimental descriptions of TransTAC molecules specific for additional cell surface molecules are provided in Examples 1, 4, 5, and 7.

[0604] In additional studies, we explored the generality of TransTAC. To date, all biologic-based degraders have been developed to target single-pass transmembrane proteins. We sought to expand the range of targets by including single-pass transmembrane targets such as epidermal growth factor receptor (EGFR) and programmed death ligand 1 (PDL1), as well as the multi-pass 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 cancers and immune cells.

[0605] Our first target was programmed death ligand 1 (PD-L1) (the ligand for the immune checkpoint receptor), the downregulation of which can enhance antitumor T cell activity. Targeting of PD-L1 using monoclonal antibodies has seen modest success clinically, and thus a new mechanism for targeting this protein may be highly valuable. PD-L1-TransTAC was generated using the antigen-binding fragment (Fab) or single-chain variable fragment (scFv) of atezolizumab as the PDL1-binding domain. Up to 98% degradation of PD-L1 was observed in MDA-MB-231 breast cancer cells treated with PD-L1-TransTAC, while control groups lacking H7 or containing TransTAC v0.2 and v0.4 with TF ligand showed no or little PD-L1 degradation (Figure 40B; Figure 46A).

[0606] 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 types of cancer, such as lung cancer and brain cancer. An affibody (Friedman, Mikaela et al. "Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding affibody molecule." Journal of molecular biology 376.5, 2008: 1388-1402) was used to bind to EGFR and generate EGFR-TransTAC. Treatment of A549 lung cancer cells with EGFR-TransTAC v1.0 containing the GFLG-VR or EVR linker showed up to 80%-90% reduction in EGFR, while the control group showed little or no degradation (Figure 40C, Figure 46B). Different linkers in v1.0 led to different degrees of EGFR degradation, but all were lower than in the case of TransTAC with GFLG-VR or EVR, and v0.2 had no effect (Figure 46B). These results were consistent with the observations using CAR-TransTAC variants, thus validating the importance of those modifications made to improve TransTAC.

[0607] Cluster of differentiation 20 (CD20) is a B cell-specific surface marker with four transmembrane domains and unknown function. Knockdown of cell surface CD20 with a degrader might be valuable. The Fab form of rituximab (the first clinically approved CD20 antibody) was used to bind to CD20 to generate CD20-TransTAC. Treatment of Raji cells (a human B lymphoblastoid cell line) with the resulting CD20-TransTAC led to up to 97% reduction in CD20, while the control group did not cause degradation or caused significantly less degradation (Figure 40D, Figure 46C).

[0608] In summary, the successful generation of degrader against all four targets demonstrated the modularity and generality of TransTAC design. Importantly, high potency was observed for all four targets studied, reaching >80% in various cell systems, which demonstrated the efficiency of targeted degradation using TransTAC degrader design. Example 14 - Kinetics, Structure-Activity Relationship (SAR), Mechanism, and In Vivo Characterization of TransTAC

[0609] We further characterized the degrader to understand its basic mechanism and SAR.

[0610] First, we studied the kinetics of TransTAC-mediated protein internalization by measuring the time-course changes in the levels of CAR on the cell surface (Figure 41A). We observed a rapid elimination of CAR from the cell surface, with only 17% remaining after 10 minutes of treatment with TransTACv1.0-GFLG-VR and 13% remaining after 20 minutes. Additionally, this response was persistent, with 10% of CAR observed on the cell surface 3 hours after treatment with v1.0. This rapid and sustained protein downregulation highlights that TransTAC can be used as a promising research tool for knocking down cell surface proteins as an alternative to genetic methods, thereby providing time resolution for membrane protein regulation.

[0611] To further understand how the number of conjugates and the geometry of TransTAC affect its behavior, we generated and tested four CAR-TransTACv0.5 variants, v0.6-v0.9, each containing one or two copies of CD19NT.1 or H7 (Figure 41B). Our findings revealed that having two H7s was more critical than having two CD19NT.1s in enhancing CAR internalization (v0.6 compared to v0.7, Figure 41C). This highlights the importance of dual binding to the dimeric TfR rather than having two anti-POI conjugates in generating an effective TransTAC. It also shows that TransTAC-mediated CAR internalization is not the result of CAR crosslinking. Additionally, we observed significant differences in the internalization efficiency of molecules with different geometries, indicating that the tertiary complex structure plays a role in influencing TransTAC efficiency (v0.8 compared to v0.9, Figure 41C). Furthermore, we generated Fc-H7 molecules as competitors for TfR binding, and in the presence of the competitor in solution, we observed a dose-dependent decrease in CAR internalization when treated with TransTACv0.5 (Figure 41D). This observation further validates that TransTAC acts through a TfR-dependent mechanism. These SAR analyses provide valuable insights for guiding future TransTAC design.

[0612] We next investigated the potential cellular mechanisms of TransTAC-mediated protein degradation. Two major pathways involved in cellular protein degradation were tested: the lysosomal pathway and the proteasomal pathway. A549 cells were treated with bafilomycin (a vacuolar proton pump inhibitor that inhibits lysosomal acidification) or MG132 (a proteasome inhibitor). We observed that 1 μM bafilomycin prevented TransTAC-mediated EGFR degradation, while the effect of 1 μM MG132 was much less significant (Figure 41E). These results show that intact lysosomal function is required for TransTAC-mediated protein degradation.

[0613] To determine whether TfR levels remain unchanged or decrease upon treatment with TransTAC, we characterized whole-cell TfR expression using Western blot assays in the presence of 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 validated our hypothesis that the POI dissociates from the TfR before being directed to degradation while the TfR is recycled.

[0614] Finally, we investigated whether TransTAC is well tolerated in vivo and whether antibody clearance is similar to that of IgG. We intraperitoneally injected 5 or 7 mg / kg (body weight) of CD20 TransTAC or 5 mg / kg of IgG control into nude mice (Figure 41G). No significant change in body weight was observed in the case of TransTAC or the control (Figure 41H). Western blot analysis of plasma antibody levels revealed that TransTAC remained in the plasma for up to 10 days after injection, with a half-life of approximately 10 days, which is longer than that of the tested control IgG and comparable to the reported half-life of IgG in mice ( Figure 41I ). The scFv-H7 antibody is known to cross-react with murine TfR. Collectively, these results demonstrate that TransTAC is well tolerated and has favorable pharmacokinetics and is not rapidly cleared despite cross-reactivity with murine cells. Example 15 - Targeting Drug-Resistant Small Cell Lung Cancer with EGFR-TransTAC

[0615] Experiments related to TransTAC molecules for treating cancer are described in Example 8

[0616] In addition, cancers rapidly evolve to evade therapy, often developing drug-resistant mutations that lead to treatment failure and disease recurrence. The C797S mutation in EGFR poses a major challenge particularly in the treatment of non-small cell lung cancer (NSCLC), which accounts for 85% of all lung cancer cases. The C797S mutation occurs in approximately 10%-26% of NSCLC patients after treatment with the third-generation EGFR tyrosine kinase inhibitor (TKI) osimertinib, affecting the key residue C797 that forms a covalent bond with irreversible TKIs. As a result, existing TKI therapies become ineffective against the disease.

[0617] EGFR-TransTAC (which can induce targeted degradation of EGFR in cancer cells with upregulated TfR) can target EGFR-driven lung cancer patients, including the C797S-mutant population (Figure 42A). Three lung cancer cell lines were used: PC9-wild type (WT), PC9 GR4, and PC9 GR4 C797S. PC9-WT cells are a lung adenocarcinoma cell line with exon 19 deletion (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 carrying the T790M mutation (Del 19 / T790M) generated by a previously established drug selection protocol. Finally, PC9 GR4C797S (Del 19 / T790M / C797S) is a CRISPR-engineered cell line with an additional C797S mutation, making it further resistant to osimertinib.

[0618] We generated several EGFR affibody-based TransTAC variants (Figure 42B) and first evaluated the dose-dependent inhibitory efficiency of these variants on PC9-WT cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT cell viability assay. TransTAC v1.0 and v0.5 variants resulted in dose-dependent inhibition of the cells, with IC50s in the low nM range, while the control affibody-Fc fusion and v0.2 showed no or little response (Figure 42C). Consistently, more than 90% of maximal EGFR degradation was observed in PC9-WT cells and PC9 GR4 C797S cells treated with TransTAC v1.0, while the affibody-Fc control did not result in degradation (Figure 42D). The same TransTAC molecule induced only approximately 40%-50% of EGFR degradation in an engineered HEK293 cell line overexpressing EGFR, which expresses 3-10x less TfR compared to the three PC9 cell lines (Figure 48A). This result indicates that the efficiency of TransTAC-mediated protein degradation is positively correlated with the TfR expression level, highlighting the potential advantage of the cancer specificity of the TransTAC technology.

[0619] Next, we compared EGFR-TransTAC with the first-, second-, and third-generation EGFR TKIs gefitinib, afatinib, and osimertinib. The sensitivities of the three cell lines to these TKIs were consistent with previous reports (Figure 42E, Figure 48B). PC9-WT cells showed sensitivity to all three TKIs, with IC50s in the range of <0.1 to 33 nM. PC9-GR4 cells were less sensitive to afatinib and osimertinib, with IC50s of 168 nM and 207 nM, respectively, and showed complete resistance to gefitinib. PC9-GR4-C797S cells did not respond to any of the three inhibitors.

[0620] Unlike the TKIs, TransTAC v1.0 effectively inhibited all three cell lines, with IC50s in the low or sub-nM range (Figure 42E, Figure 48B). In particular, for PC9 GR4 C797S cells, the IC50 of TransTAC v1.0-GFLG-VR was 2 nM and that of TransTAC v1.0-EVR was 8 nM. To evaluate off-tumor toxicity, the healthy cell human fibroblast cell line (HFF-1) was included in the assay. Neither TransTAC nor the TKIs showed significant inhibition until molecular concentrations reached the high nM or μM range (Figure 42E, Figure 48B).

[0621] To further compare the efficacy and specificity of TransTAC with standard-of-care therapies, we performed co-culture assays of normal and cancer cells and monitored the action of the drugs using live-cell fluorescence imaging (Figure 42F - Figure 42G). PC9 and PC9GR4 C797S cells were engineered to express GFP, and HFF-1 cells expressed mCherry. The cells were mixed at a ratio of 1:10 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, the TKI inhibited PC9 WT cancer cells but not PC9 GR4 C797S cells. Neither the UT nor the affibody-Fc control molecule showed an effect on either cell type.

[0622] In addition, TransTAC was also compared with chemotherapeutic drugs. Unlike TransTAC, which did not kill HFF1 healthy cells, the combination of carboplatin and paclitaxel chemotherapy was cytotoxic to both cancer and normal cells (Figure 42F - Figure 42G). This is consistent with the previous notion that although chemotherapy is a first-line therapy for many cancer types, it generally has high off-tumor toxicity.

[0623] In addition, the results of live cell imaging were verified by performing flow cytometry analysis, which was used to determine the ratio of GFP / mCherry positive cells after treatment, and this ratio reflects the relative drug cytotoxicity to cancer cells compared to healthy cells (Figure 48C). Our analysis showed that TransTAC inhibited both PC9 WT and GR4 CS cells, showing a near-zero cancer cell / healthy cell ratio, indicating high cancer targeting potency and specificity. In contrast, the TKI was much less effective against GR4 CS cells, and cancer cells dominated the entire population of the cell mixture.

[0624] In summary, these findings demonstrate that the EGFR TransTAC molecule can target lung cancer cells carrying the EGFR C797S mutation. In addition, comparison with the current standard of care demonstrated the superior tumor efficacy and specificity of TransTAC. Example 16 - Schematic Diagram of the Design Principles We Have Discovered for Enhancing the Degradation Efficiency of TransTAC

[0625] Figure 38b is an illustration of an exemplary TransTAC degrader. Generally, TransTAC is a recombinant protein composed of an anti-POI conjugate and an anti-TfR conjugate, which is used to bridge POI and TfR closely on the cell surface. We found that, as outlined in Figures 39A and 44A, many forms of TransTAC can efficiently eliminate target proteins from the cell surface. Thus, all of these confer effectiveness as membrane protein regulators.

[0626] However, we found that at least three exemplary design principles can make TransTAC an efficient internalizer and degrader: (1) dimeric TransTAC drives more efficient protein internalization than monomeric heterobispecific TransTAC; (2) a cathepsin B-sensitive linker pair is important for lysosomal trafficking of POI; and (3) an antibody conjugate for targeting TfR (instead of the native transferrin (TF) ligand) can reduce the trafficking of POI to recycling endosomes (RE) and thus improve the degradation efficiency.

[0627] Using specific variants of the molecule, we can choose to induce endosomal capture or lysosomal degradation of the target, thus providing customizable possibilities for modular manipulation of membrane proteins. Example 17 - Identification of Peptides Not Yet Known to Be Sensitive to Cathepsin Cleavage

[0628] Peptides not previously known to be sensitive to cathepsin cleavage were identified using a yeast-displayed peptide library. These peptides can be from combinations of small motifs found in SEQ ID NO:144 and 145. At pH 4.4 ( Figure 49A ) and pH 6.4 ( Figure 49B) were studied. These peptides include GRLVGFD (SEQ ID NO:124), GRLVGFG (SEQ ID NO:125), RMLVGFV (SEQ ID NO:126), RRLYAFL (SEQ ID NO:127), VFRLLMF (SEQ ID NO:128), LVGVLLF (SEQ ID NO:129), VKLYGLG (SEQ ID NO:130), TWRVDLY (SEQ ID NO:131), EQLYLYA (SEQ ID NO:132), KLFLMIF (SEQ ID NO:133), NFVIILF (SEQ ID NO:134), MSLLIGV (SEQ ID NO:135), VRLLSLQ (SEQ ID NO:136), STLMWNV (SEQ ID NO:137), VRFLAAA (SEQ ID NO:138), HGWSFHE (SEQ ID NO:139), ENLYFQG (SEQ ID NO:140), VVMMFLH (SEQ ID NO:141), VFRLLMF (SEQ ID NO:142), or VGALVWL (SEQ ID NO:143). ***** Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention.

Claims

1. A fusion protein of formula I: R1-R2-R3 (I): Wherein: R1 is at least one protein of interest (POI) binder (POIB); R2 is a linker of formula R4-R5 or R5-R4, wherein: R4 is the IgG Fc region; and R5 is a protease-sensitive linking element; and R3 is a transferrin receptor-binding (TRB) element, and Optionally, the linkage between R2 and R3 is a glycine-rich linker.

2. The fusion protein according to claim 1, wherein the protease-sensitive linking element comprises a cathepsin-cleavable peptide.

3. The fusion protein according to claim 2, wherein the cathepsin-cleavable peptide linker is selected from the group consisting of: FK, VA, VK, SEQ ID NO:7, 8, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144 and 145.

4. The fusion protein according to claim 1, wherein the TRB-binding element is H7 or M16.

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

6. The fusion protein according to any one of claims 1-5, wherein the fusion protein of formula I is a heterodimer linked to a fusion protein of formula II: R4’-R3’ (II): Wherein: R4’ is the IgG Fc region; and R3’ is a transferrin receptor-binding (TRB) element; and Optionally, the linkage between R3’ and R4’ is a protease-sensitive linking element.

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

8. The fusion protein according to any one of claims 1-7, wherein the TRB is selected from the group consisting of SEQ ID NO:3, 4 and 5.

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

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

11. The fusion protein according to 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 according to any one of claims 1-11, 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 according to any one of claims 1-12, wherein the linkage between R2 and R3 is a glycine-rich linker selected from the group consisting of SEQ ID NO:9, 10, 11, 12, 13, 14, 15 and 16.

14. A nucleic acid sequence encoding a fusion protein as claimed in any one of claims 1-13.

15. A method for treating a subject suffering from cancer, the method comprising administering to the subject a fusion protein as claimed in any one of claims 1-13.

16. A fusion protein as claimed in any one of claims 1-13 for use in the treatment of cancer in a patient.

17. A homodimer of a fusion protein of formula I: R1-R2-R3 (III): Wherein: 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 linking element; and R3 is a transferrin receptor binding (TRB) element, and Optionally, the linkage between R2 and R3 is a glycine-rich linker.

18. A homodimer as claimed in claim 17, the homodimer further comprising a disulfide bond between cysteine amino acids in R4 of a separate fusion protein.

19. A homodimer of a fusion protein of formula I: R1-R6-R3 (I): Wherein: R1 is at least one protein of interest (POI) binder (POIB); R6 is a dimerization element; and And R3 is a transferrin receptor binding (TRB) element; Wherein the homodimer optionally has a protease-sensitive linking element between R1 and R6.

20. A pharmaceutical composition comprising a fusion protein or a homodimer of a fusion protein as claimed in any one of claims 1-13 or 17-19.

Images