Meditope - Available T cells

Abstract

The present invention provides a composition that exhibits novel therapeutic ability and can reduce the off-target effects of therapeutic antibodies. The composition comprises a recombinant protein that, when expressed by T cells, can efficiently recruit therapeutic antibodies to their sites of action.

Description

[0001] Cross-reference regarding related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 611,924 filed December 29, 2017 and U.S. Provisional Application No. 62 / 680,442 filed June 4, 2018, the entirety of which is incorporated herein by reference for all purposes.

[0003] Reference to the appendix for the "sequence list," table, or computer program list submitted as an ASCII file

[0004] A sequence list recorded in file 048440-621001WO_Sequence_Listing_ST25, 29,672 bytes, machine format IBM-PC, MS Windows operating system, written on December 31, 2018, is incorporated herein by reference.

[0005] Summary of the Invention

[0006] In one aspect, a first recombinant protein is provided. The first recombinant protein comprises (i) a first non-CDR Fab-binding peptide domain; (ii) a first intracellular T cell signaling domain; and (iii) a first transmembrane domain connecting the first non-CDR Fab-binding peptide domain to the first intracellular T cell signaling domain. In an embodiment, the first recombinant protein comprises a first spacer region connecting the first non-CDR Fab-binding peptide domain to the first transmembrane domain. In an embodiment, the first spacer region is a first CH3 region.

[0007] In one aspect, an isolated nucleic acid encoding the first recombinant protein provided herein is provided, including embodiments thereof.

[0008] In one aspect, an expression vector comprising the nucleic acid provided herein is provided, including embodiments thereof. In an embodiment, the vector is a lentivirus or an onco-retrovirus.

[0009] In one aspect, a T lymphocyte comprising an expression vector provided herein, including an embodiment thereof, is provided.

[0010] In one aspect, a T lymphocyte comprising the first recombinant protein provided herein, including an embodiment thereof, is provided.

[0011] In one aspect, a T lymphocyte comprising the first recombinant protein provided herein, including embodiments thereof, is provided, wherein the transmembrane domain is located within the cell membrane of the T lymphocyte.

[0012] In one aspect, a method for treating cancer is provided. The method comprises the step of administering an effective amount of the T lymphocytes provided herein, including embodiments thereof, to a subject requiring treatment for cancer, wherein the first antigen-binding domain and the second antigen-binding domain are independently anti-cancer antigen-binding domains.

[0013] In one aspect, a recombinant protein is provided. The recombinant protein comprises (i) a non-CDR Fab-binding peptide domain; (ii) an intracellular T cell signaling domain; and (iii) a transmembrane domain connecting the non-CDR Fab-binding peptide domain to the intracellular T cell signaling domain.

[0014] In one aspect, isolated nucleic acids encoding a recombinant protein provided herein, including embodiments thereof, are provided.

[0015] In one aspect, an expression vector comprising nucleic acid provided herein is provided, including embodiments thereof.

[0016] In one aspect, a T lymphocyte comprising an expression vector provided herein, including an embodiment thereof, is provided.

[0017] In one aspect, a T lymphocyte comprising a recombinant protein provided herein is provided, including embodiments thereof.

[0018] In one aspect, a T lymphocyte comprising the recombinant protein provided herein, including embodiments thereof, is provided, wherein the transmembrane domain is located within the cell membrane of the T lymphocyte.

[0019] In one aspect, a method for treating cancer is provided. The method comprises the step of administering an effective amount of an antigen-binding domain capable of binding to a T lymphocyte and a non-CDR Fab-binding peptide domain provided herein, including embodiments thereof, to a subject requiring treatment for cancer, wherein the antigen-binding domain is a cancer antigen-binding domain. Brief explanation of the drawing

[0020] Figs. 1a-1c. Fig. 1a) Chimeric Antigen Receptor (CAR) T cells are typically generated by fusing antigen-specific single-strand Fab variable domains (e.g., heavy and light chain variable domains of mAbs) to the CD3 zeta chain. As such, each CAR T cell requires the generation of new genes to alter target specificity. Fig. 1b) To remove this limitation, the applicant used Meditope technology to 'snap' antibody fragments onto engineered T cells. Specifically, the applicant replaced scFv with cQFD Meditope. Fig. 1c) Cartoon illustration of current technology for single antigen-specific CAR T cells and how Meditope technology can be used to generate universal CARs. FIGS. 2a-2c. FIGS. 2a) CHO-S cells are transfected without a vector (Mock), with parental CAR (1), Meditope-CH3 (3), or Meditope-CD28 (2) to test the binding of Meditope-enabled I83E trastuzumab IgG (647^183E IgG) conjugated to the fluorescent dye Alexa Fluor 647, Meditope-enabled I83E trastuzumab Fab (647^183E Fab) conjugated to the fluorescent dye Alexa Fluor 647, and non-Meditope-enabled ipilimumab IgG (647^Ipi IgG) conjugated to the fluorescent dye Alexa Fluor 647. Live transfected cells undergo FSC / SSC → PI - → CD19 + This was confirmed using gating (see Fig. 3a). Cells were analyzed for the mean fluorescence intensity (MFI) of the APC signal. Fig. 2b) Cells without a vector (Mock), transfected with parental CAR (1) or Meditope-CD28 (2) did not show a shift in MFI when stained with 647^183E IgG, 647^183E Fab, or 647^183E IgG. Fig. 2c) Cells transfected with Meditope-CH3 (3) showed a significant shift in MFI when stained with 647^183E IgG or 647^183E Fab, but showed only a minimal shift when stained with 647^183E IgG. Figs. 3a-3b. Gating strategy for flow cytometry analysis. Fig. 3a) Cells were gated by FSC / SSC (left panel), PI staining (representing living cells; middle panel), and CD19 expression (transfection marker; right panel). Fig. 3b) Further analysis of the gated cells measured 647 (left panel) and VioBlue (right panel) signals indicating antibody and Her2 binding, respectively. Figs. 4a-4c. Meditope-available IgG and Fab bind to Meditope-CH3 CAR. Fig. 4a) Living transfected cells FSC / SSC → PI - → CD19 + It was confirmed using gating. Cells were analyzed for the mean fluorescence intensity (MFI) of the APC signal. APC using unstained cells + Gating was established for the cells. Cells stained with non-Meditope-available 647^Ipi IgG exhibited minimal migration in the APC MFI. Cells stained with Meditope-available 647^I83E IgG and Fab exhibited significant migration in the APC MFI, demonstrating the binding of 647-conjugated proteins to Meditope-CH3-expressing cells. (Fig. 4b) This trend was not observed in the Meditope-CD28 construct. (Fig. 4c) Live CD19 positive for 647^IgG or 647^Fab + Percentage of cells (Gated cells: FSC / SSC → PI - → CD19 + → APC + ; APC of the parent group + Cell frequency). Figs. 5a-5b. Meditope-CH3 expressing cells can bind to a Meditope-available antibody and subsequently bind to an antigen. Fig. 5a) APC + Histogram of Her2 mean fluorescence intensity (MFI) in Meditope-CH3 CAR cells (gated cells: FSC / SSC → PI - → CD19 + → APC + ). Fig. 5b) Live CD19 positive for Her2 + 647 + Percentage of cells (Her2 of the grandparent group) + Cell frequency, same gating strategy as above). FIG. 6a-6c. 647 + Her2 +Confirmation of double-positive cells. Live transfected cells are FSC / SSC → PI - → CD19 + This was confirmed using gating (see Fig. 3a). Subsequently, the cells were analyzed for 647 (x-axis) and Her2 levels (VioBlue). In particular, many 647 - Cells are on the y-axis. Fig. 6a) Analysis of cells stained with Meditope-available 647^I83E Fab. Fig. 6b) Analysis of cells stained with Meditope-available 647^I83E IgG. Fig. 6c) Analysis of cells stained with non-Meditope-available 647^Ipi IgG. Fig. 7. Her2 + Cell identification. Living transfected cells are FSC / SSC → PI - → CD19 + It was confirmed using gating. Subsequently, the gate is Her2 + To identify the cells, analysis was performed using samples without PacBlue^Her. Subsequently, the cells were analyzed for Her2(VioBlue) levels. Figs. 8a-8b. Meditope-available cetuximab and trastuzumab Fabs bind to Meditope-CH3 CAR. Fig. 8a) Live CD19 positive for 647^Fab + Cell Percentage Gated Cells: FSC / SSC → PI - → CD19 + → APC + ; APC of the parent group + Cell frequency). Fig. 8b) Living cells transfected with Meditope-CH3 FSC / SSC → PI - → CD19 + It was confirmed using gating. Cells were analyzed for the mean fluorescence intensity (MFI) of the APC signal. APC using unstained cells +Gating was established for the cells. Cells stained with non-meditope-available 647^Ipi Fab showed minimal migration in APC MFI. Cells stained with meditope-available 647^I83E cetuximab (cetux) Fab and 647^I83E trastuzumab (tras) Fab showed significant migration in APC MFI, demonstrating the binding of 647-conjugated proteins to meditope-CH3-expressing cells. Figs. 9a-9b. 647 + Her2 + Confirmation of double-positive cells. Live transfected cells are FSC / SSC → PI - → CD19 + Confirmed using gating. Subsequently, cells were analyzed for 647 (x-axis) and Her2 levels (Pacific Blue). Fig. 9a) Analysis of cells stained with Meditope-available 647^I83E cetuximab (cetux) Fab. Fig. 9b) Analysis of cells stained with Meditope-available 647^I83E trastuzumab (tras) Fab. Fig. 10. The figure shows the tumor death assay in ovarian cancer cell lines using different concentrations of Meditope-available Her2. Fig. 11. The figure shows the tumor death assay in breast cancer cell lines using different concentrations of Meditope-available Her2. Fig. 12. The figure shows a FabRack T cell. Fig. 13. The figure shows a convertible Fab CAR-T cell. Fig. 14. Jurkat cells were transduced with Meditope-CAR, and detachmented CD19 was co-expressed as a marker. After transduction, CD19-positive cells were sorted and expanded for the following experiments. Fig. 15. Meditope-CAR-expressing Jerkats cells were incubated with trastuzumab with or without the Meditope site, and then stained with secondary anti-kappa-647 (Abcam #202832) or anti-human IgG Fc-488 (ThermoFisher #H10120). The results indicated that only memAb trastuzumab could bind to Meditope-CAR-expressing Jerkats cells after the cells were analyzed by flow cytometry. Fig. 16. Left: Cancer cells, Jurkat-NFAT-Luc Meditope-CAR cells, and memAb trastuzumab were co-incubated in white-walled 96-well plates. The highest concentration in the figure was 15 nM, followed by 4-fold serial dilutions. After 6 hours of incubation, luciferase substrates were added to each well, and luminescence was immediately measured using a plate reader. In the presence of memAb trastuzumab, Jurkat cell activation increased dose-dependently at 15 nM, despite the hook effect setting. The EC50 for each cell line was 0.35 nM (SKOV3), 0.83 nM (SKBR3), 0.42 nM (MCF7), 0.27 nM (OVCAR3), and 0.26 nM (BT474). The level of Jerkat cell activation is quantitatively correlated with HER2 expression in cancer cells, excluding the BT474 cell line. Although BT474 exhibits high HER2 expression, it does not activate Jerkat cells to the same level as other high-HER2 expressing cells (SKOV3 and SKBR3). Right: 5x10 5Dog cells were treated with 100 nM memAb trastuzumab in 1% FBS in PBS for 30 minutes. After washing three times, the cells were labeled with a secondary anti-kappa-647 antibody for 30 minutes. The fluorescence intensity of the cells was analyzed using a BD Accuri C6 flow cytometer. The red peak represents cells treated with the secondary anti-kappa-647 antibody alone. The green peak represents cells treated with memAb trastuzumab and the secondary anti-kappa-647 antibody. HER2 expression levels were analyzed using flow cytometry by comparing cells stained with the secondary antibody alone or with memAb trastuzumab and the secondary antibody. The median fluorescence intensity (MFI) was 338 and 985 for MCF7 cells; 330 and 34405 for SKBR3 cells; and 392 and 31720 for BT474 cells. For SKOV3 cells, 370 and 43191; for OVCAR3 cells, 307 and 1436. Cells with HER2 expression ranging from high to low expression are SKOV3, SKBR3, BT474, OVCAR3, and MCF7. Fig. 17. Left: Cancer cells, FabRack Jurkat-NFAT-Luc cells, and cetuximab were co-incubated in white-walled 96-well plates. The highest concentration in the figure is 60 nM, followed by 4-fold serial dilutions. After 6 hours of incubation, luciferase substrate was added to each well, and luminescence was measured immediately using a plate reader. The EC50 for each cell line is 0.14 nM (SKOV3), 0.12 nM (SKBR3), 0.12 nM (MCF7), 0.11 nM (OVCAR3), and 1.1 nM (BT474). Right: 5*10 5Dog cells were treated with 100 nM cetuximab in 1% FBS PBS for 30 minutes. After washing three times, the cells were labeled with a secondary anti-kappa-647 antibody for 30 minutes. The fluorescence intensity of the cells was analyzed using a BD Accuri C6 flow cytometer. The red peak represents cells treated with the secondary anti-kappa-647 antibody alone. The green peak represents cells treated with cetuximab and the secondary anti-kappa-647 antibody. The median fluorescence intensity indicated that the cells with EGFR expression ranging from high to low expression were OVCAR3 (4819), SKOV3 (4479), SKBR3 (2865), BT474 (651), and MCF7 (480). Fig. 18. The figure shows Zerkat or cancer cells with memAb trastuzumab pre-mix and washing. Zerkat or cancer cells with memAb trastuzumab pre-binding followed by washing. Cancer cells (2.5 x 10⁻⁶ 4 100 µl / well of medium was seeded into 96-well white-walled plates. After cell attachment overnight, the medium was removed from the plates, and Jurkat-NFAT-Luc Meditope-CAR cells (1 x 10⁶ 560 µl of memAb trastuzumab was added to each well. MemAb trastuzumab was either continuously present or pre-bound to Jurkat-NFAT-Luc medi-CAR or cancer cells with washing. (The bars in the graph indicate moving from left to right: no treatment; Herceptin (4 nM) continuously present; memAb trastuzumab (4 nM) continuously present; FabRack Jurkat-NFAT-Luc cells with pre-bound and subsequently washed memAb trastuzumab (100 nM); cancer cells with pre-bound and subsequently washed memAb trastuzumab (100 nM).) Cells were incubated at 37°C for 6 hours, and then 50 µl of luciferase substrate (Invivogen #rep-qlc2) was added to each well. Luminescence was measured immediately using Biotek’s Synergy 4 multi-detector microplate reader. Fig. 19. The figure shows a T cell with pre-binding to memAb (left) and a cancer cell with pre-binding to memAb (right). Fig. 20. The figure indicates that Herceptin blocks I83E-mediated Jurkat-NFAT-Luc activation. MemAb trastuzumab and Herceptin were continuously present. Cancer cells (2.5 x 10⁶ 4 (100 µl) was seeded into 96-well white-walled plates. After cell attachment overnight, the medium was removed from the plates, and Jukat-NFAT-Luc me-CAR cells (1 x 10⁶ 560 µl was added to each well. MemAb trastuzumab was either continuously present, washed, or pre-bound to cancer cells. (The bars in the graph indicate moving from left to right: continuously present memAb trastuzumab (4 nM); continuously present memAb trastuzumab (4 nM) and Herceptin (40 nM); continuously present memAb trastuzumab (4 nM) and Herceptin (400 nM).) Cells were incubated at 37°C for 6 hours, after which 50 µl of luciferase substrate (Invivogen #rep-qlc2) was added to each well. Luminescence was immediately read by Biotech’s Synergy 4 multi-detector microplate reader. In the sustained presence of 4 nM memAb trastuzumab, Jurkat-NFAT-Luc medi-CAR cells were activated by binding to cancer cells via memAb trastuzumab. The sustained presence of Herceptin during incubation can block Jurkat cell activation, as Herceptin has the same epitope as our memAb trastuzumab and can compete for the same binding site on HER2. Herceptin (400 nM) at a 100-fold concentration of memAb trastuzumab (4 nM) almost completely blocks Jurkat cell activation, demonstrating that Jurkat cell activation is caused by the binding of memAb trastuzumab to the same HER2 epitope recognized by Herceptin. Fig. 21. The figure shows that Herceptin blocks memAb-mediated Jurkat-NFAT-Luc activation after pre-mixing with memAb trastuzumab and washing of Jurkat cells. MemAb trastuzumab is first pre-bound to Jurkat cells and washed. Herceptin was continuously present. Cancer cells (2.5 x 10⁶ 4 (100 µl) was seeded into 96-well white-walled plates. After cell attachment overnight, the medium was removed from the plates, and Jukat-NFAT-Luc me-CAR cells (1 x 10⁶5 60 µl was added to each well. MemAb trastuzumab was pre-bound to Jurkat-NFAT-Luc medi-CAR cells with continuous presence or washing. (The bars in the graph represent moving from left to right: memAb trastuzumab (4 nM) continuously present; Jurkat-NFAT-Luc medi-CAR cells with pre-conjugated and subsequently washed memAb trastuzumab (100 nM); Jurkat-NFAT-Luc medi-CAR cells with pre-conjugated and subsequently washed memAb trastuzumab (100 nM) + continuously present Herceptin (40 nM); Jurkat-NFAT-Luc medi-CAR cells with pre-conjugated and subsequently washed memAb trastuzumab (100 nM) + continuously present Herceptin (400 nM).) Cells were incubated at 37°C for 6 hours, and then 50 µl of luciferase substrate (Invivogen #rep-qlc2) was added to each well. Luminescence was measured immediately using Biotech’s Synergy 4 multi-detection microplate reader. Jurkat-NFAT-Luc medi-CAR cells washed after memAb trastuzumab pre-binding showed significantly reduced luminescent activity when co-incubated with high-HER2 expression cells (SKBR3, BT474, and SKOV3) compared to cells with persistent memAb, but not with low-HER2 expression cells (MCF7 and OVCAR3). The persistent presence of Herceptin during incubation can block Jurkat cell activation. The number of medi-CAR molecules on each Jurkat cell or target molecules on each cancer cell can determine how much activation occurs in each T cell or how many T cells are activated. Fig. 22. The figure indicates that Herceptin barely blocked memAb-trastuzumab-mediated Jurkat-NFAT-Luc activation after pre-mixing with memAb and washing in cancer cells. MemAb-trastuzumab is first pre-bound to cancer cells and washed. Herceptin was continuously present. Cancer cells that underwent washing following memAb-trastuzumab pre-binding did not significantly reduce luminescence activity compared to cells where memAb was continuously present. The continuous presence of Herceptin during incubation had no blocking effect on Jurkat cell activation or had a partial blocking effect. This data demonstrated that once memAb-trastuzumab binds to HER2 on cancer cells, it is difficult to compete with trastuzumab without the Meditope-binding site. Cancer cells (2.5 x 10⁶ 4 (100 µl) was seeded into 96-well white-walled plates. After cell attachment overnight, the medium was removed from the plates, and Jukat-NFAT-Luc me-CAR cells (1 x 10⁶ 5 ) was added to each well. memAb trastuzumab was either continuously present or pre-bound to cancer cells upon washing. (The bars in the graph represent moving from left to right: cancer cells pre-bound with memAb trastuzumab (4 nM) continuously present; cancer cells pre-bound with memAb trastuzumab (100 nM) followed by washing; cancer cells pre-bound with memAb trastuzumab (100 nM) followed by washing + continuously present Herceptin (40 nM); cancer cells pre-bound with memAb trastuzumab (100 nM) followed by washing + continuously present Herceptin (40 nM).) Cells were incubated at 37°C for 6 hours, after which 50 µl of luciferase substrate (Invivogen #rep-qlc2) was added to each well. Luminescence was measured immediately using Biotech’s Synergy 4 multi-detection microplate reader. Fig. 23. The figure shows that T cells kill cancer cells treated with memAb trastuzumab pre-mixing and washing. Survival rate (%) = [Luc (암세포 + T 세포 + 항체) -Luc (T 세포 + 항체) ] / [Luc (암세포 + T mock 세포) - Luc (T mock 세포) ]. In the sustained presence of 0.1 nM or 0.5 nM mem antibodies, the viability of cancer cells with FabRack T cell co-incubation was reduced compared to that of cancer cells with mock T cell co-incubation. Cell viability decreased in a dose-dependent manner at 0.1 and 0.5 nM in breast cancer cells, whereas ovarian cancer cells showed similar viability at these two concentrations. Cancer cells with antibody pre-binding and washing could still be killed by human FabRack T cells, despite a 5–18% reversal in viability compared to cancer cells with sustained antibody treatment. For tumor apoptosis assays, cancer cells (2.5 x 10⁶ 4 Human T cells (6,250 / 100 µl) and antibodies were seeded into 96-well round-bottom plates in the presence or absence of antibodies. After 72 hours of incubation, the cells were centrifuged at 250 xg for 5 minutes, and 100 µl of medium was removed from each well. To test cell viability, 100 µl of reagent from the Promega CellTiter kit was added to each well. After 2 minutes of incubation, 100 µl of the mixture was transferred to a white-walled 96-well plate and measured using Biotech’s Synergy 4 multi-detection microplate reader. Figure 24. The figure indicates that FabRack T cells effectively kill cancer cells at a low dose of memAb trastuzumab. Cancer cells were incubated with mock T cells or FabRack T cells in the presence of memAb trastuzumab for 3 days. This data indicated that FabRack T cells effectively killed HER2-positive cancer cells because they bind to cancer cells via memAb trastuzumab. The IC50s of cancer cells co-incubated with memAb trastuzumab and FabRack T cells were 0.33 nM (BT474), 0.11 nM (SKBR3), 0.069 nM (MCF7), 0.046 nM (SKOV3), and 0.027 nM (OVCAR3). The killing effect was not associated with the level of HER2 expression on the cancer cells. For tumor cell death testing, cancer cells (2.5 x 10 4 Human T cells (6,250 / 100 µl) and 6,250 / 100 µl were seeded into 96-well round-bottom plates in the presence or absence of antibodies. After 72 hours of incubation, the cells were centrifuged at 250 xg for 5 minutes, and 100 µl of medium was removed from each well. To test cell viability, 100 µl of reagent from the Promega Celltitor Kit was added to each well. After 2 minutes of incubation, 100 µl of the mixture was transferred to a white-walled 96-well plate and measured using Biotech’s Synergy 4 multi-detection microplate reader. FIG. 25. The figure shows tumor death of Meditope-CAR (T cells expressing the recombinant protein provided herein, including embodiments thereof) compared with scFv CAR and Fab CAR T cells. Figure 26. The figure shows the tumor death assay in ovarian cancer (FACS). Using flow cytometry, the number of cancer cells remaining alive after co-incubation with FabRack T cells and memAb trastuzumab was analyzed in comparison to cancer cells incubated with HER2 scFv CAR or HER2 Fab CAR T cells as positive controls. After 3 days of incubation, the number of viable cancer cells decreased sharply when co-incubated with FabRack T cells and memAb trastuzumab. The death effect of FabRack T cells was similar to or even superior to that of HER2 scFv CAR or HER2 Fab CAR T cells. Figure 27. The figure shows the tumor apoptosis assay in breast cancer (FACS). Using flow cytometry, the number of cancer cells remaining alive after co-incubation with FabRack T cells and memAb trastuzumab was analyzed in comparison to cancer cells incubated with HER2 scFv CAR or HER2 Fab CAR T cells as positive controls. After 3 days of incubation, the number of viable cancer cells decreased sharply when co-incubated with FabRack T cells and memAb trastuzumab. The apoptotic effect of FabRack T cells was similar to or even superior to that of HER2 scFv CAR or HER2 Fab CAR T cells. Fig. 28. The figure shows the histogram of CD107a and IFN-γ expression in T cells. Flow cytometry indicated that approximately 40% of human T cells were successfully transfected with Meditope-CAR. FabRack T cells increased the expression of CD107a and IFNγ when cancer cells were incubated with memAb trastuzumab pre-mixing and washing or in the continuous presence of memAb trastuzumab. To analyze CD107a and IFNγ expression, cancer cells (5 x 10⁶ 4Seed 100 µl of cells into a 96-well round-bottom plate and human T Mock or FabRack cells (5 x 10⁶ 4 100 µl of the effector was added to each well containing existing cancer cells. The ratio of effector to target was 1:1. The CD107a-FITC (BD #555800) antibody and the transporter inhibitor Golgistop (BD #554724) were added to each well during incubation. After 5 hours of incubation, cells were stained with a fixed viability dye (Thermo Fisher Scientific #L34965) for 30 minutes in the dark at 4°C. After two washes, cells were stained with CD4-PerCP (BD #347324), CD8-APCCy7 (BD #348793), and CD19-PECy7 (BD #557835) for 30 minutes in the dark at 4°C. After washing twice, the cells were fixed and permeated with the BD Cytofix / Cytoperm kit (BD 554714), and intracellular IFN was stained with IFN-APC (BD #554702) for 30 minutes at room temperature in the dark. After washing twice, the cells were resuspended to a final volume of 100 µl, and 40 µl of the sample was analyzed using a flow cytometer. Fig. 29. The figure shows CD107a and IFN-γ. Fig. 30. The figure indicates that I83E trastuzumab cannot activate FabRack T cells without target cells. Activation markers available for the study: upregulation CD69 (short lifespan), CD137 (4-1BB), CD44, CD27, CD45RO, CD154; downregulation CD62L, CCR7 (CD197) (CD25, CD69, CD137 (4-1BB), KLRG, CD62L, CD45RO, CD27, CD28). FabRack T cells did not show an increase in the expression of CD107a and IFNγ when incubated with 0.5 nM memAb trastuzumab for 5 hours. Fig. 31. Four constructs were generated. The top two constructs contain truncated CD19 genes to be used as markers for transformed cells. These two differ in the co-stimulation signal, C28 or 41BB. The bottom two constructs are identical without the CD19 readout marker. Fig. 32. Histogram of distal CD19 (CD19t) expression in Jurkat-NFAT-Luc cells before and after sorting. Cells were stained with CD19-PE-Cy7 and CD19t positivity was sorted using a BD Aria SORP flow cytometer. A homogeneous population of transformed cells was isolated by cell sorting using a distal CD19 marker. Fig. 33a-33c. Retrieve: Fig. 33a) Meditope-available IgG binds to only one of the four variants (in the presence or absence of CD19t and CD28 or 41BB co-stimulation signals). Fig. 33b) Non-transformed Jerkat cells do not bind to IgG. Fig. 33c) Likewise, parental antibodies (e.g., non-meditope-available) do not bind to FabRack Jerkat cells. In other words, different memAbs can be combined with different Fabrack variants. Fig. 34. Breast (MCF7, SKBR3, and BT474) or ovarian (SKOV3 and OVCAR3) cancer cell lines exhibited memAb binding to HER2 or EGFR. The median fluorescence intensity (MFI) of cells with or without memAb binding is shown below each graph. Her2, EGFR, and CD20 antigen densities were independently quantified for a series of cell lines using analytical cytometry, which we subsequently use to extensively test Fabrack's antigen specificity and ability to convert efficacy. Fig. 35. Breast or ovarian cancer cells (2.5 E4) were seeded into 96-well white-walled plates. After overnight cell adhesion, the medium was removed from the plates, and Jukat-NFAT-Luc Fabrack cells (CD28 version, 1E5) were added to each well along with various doses of memAb (anti-HER2 or anti-EGFR). Cells were incubated at 37°C for 6 hours, after which a luciferase substrate (Invivogen #rep-qlc2) was added to each well. Luminescence was measured immediately using Biotech’s Synergy 4 multi-detection microplate reader. EC50 was less than 1 nM in all cells, but the plateaus varied significantly. The increase correlated with antigen expression, except for BT474 cells. This exception requires further investigation (Her3 or other molecular interference?). A similar trend is observed for EGFR in the right panel. When using cetuximab, the MFI is 651 for BT474; 480 for MCF7; 4819 for OVCAR3; 2865 for SKBR3; and 4479 for SKOV3. BT474 expresses slightly more EGFR than MCF7, but the trend in the plateau tends to follow antigen density. Some of the characteristics of the BT474 cell line can be found on the website ncbi.nlm.nih.gov / pmc / articles / PMC3236329 / . Fig. 36. CD19-expressing cancer cells (5 E4) were seeded into 96-well white-walled plates and co-cultured with Jukat-NFAT-Luc Fabrack cells (41BB or CD28 versions, 1 E5) with varying doses of CD19 memAb. Cells were incubated at 37°C for 6 hours, after which a luciferase substrate (Invivogen #rep-qlc2) was added to each well. Luminescence was measured immediately using Biotech’s Synergy 4 multi-detector microplate reader. The left panel indicates that different CD19-expressing cells can be targeted. The plateau varies depending on antigen density. The MFI for Raji is 21566; for Daudi, 12263; and for Sup-B15, 12617. The right panel indicates that the CD28 activation signal is stronger than that of 41BB. Fig. 37. Activation of FabRack Jurkat cells in the presence of memAb and target cells. Different doses of memAb trastuzumab IgG or Fab were present in wells containing FabRack Jurkat cells and target cells for 6 hours. At the end of incubation, luciferase substrate was added to each well, and luminescence was immediately read using a plate reader. Breast or ovarian cancer cells (2.5 E4) were seeded into 96-well white-walled plates. After overnight cell attachment, the medium was removed from the plates, and Jukat-NFAT-Luc Fabrack cells (CD28 version, 1E5) were added to each well along with various doses of memAb trastuzumab or non-memAb pertuzumab. Cells were incubated at 37°C for 6 hours, after which luciferase substrate (Invivogen #rep-qlc2) was added to each well. Luminescence was immediately measured using Biotech’s Synergy 4 multi-detection microplate reader. Based on our FACS data, MCF7 exhibits the lowest amount of Her2 (MFI = 985). OvCAR3 expresses slightly more (MFI = 1435). SKBR3 (MFI = 34405) and SKOV3 (MFI = 43191) express significantly more than MCF7 and OvCAR3. Pertuzumab, which also binds to Her2, contains Fc but is not available at Meditope. The fact that it does not (as expected) indicates the need for Meditope-available Fab / Mab. The hook effect is present in all cell lines using the IgG format. This effect is established early in cells with 'low' antigen densities (the peak of the 'hook' is less than 10 nm for MCF7 and OvCAR3, and greater than 10 nM for SKBR3 and SKOV3). This finding is consistent with cell-derived antigens that saturate at low concentrations. Valency has a strong influence (e.g., the hook effect is established at lower concentrations when using IgG {divalent} compared to Fab {monovalent}).Interestingly, the 'flattop' is much higher when using Fab. The fact that there is a difference indicates that the effect can be optimized by adjusting the characteristics of memAb. Furthermore, it suggests that the hook effect can be used as a safety mechanism. The hook effect was not clear in Nb, Figures 8 and 9. This is partly because the concentration range used in their studies was lower (antibody concentrations were only up to 10 nM, whereas here they go to 1 uM). Information on the hook effect can be found on the website en.wikipedia.org / wiki / Hook_effect. Fig. 38. Activation of FabRack Jerkatt cells by co-culture with cancer cells in the presence of 4 nM memAb trastuzumab, memAb trastuzumab pre-conjugation to target cells, or memAb trastuzumab pre-conjugation to FabRack Jerkatt cells. Cancer cells or FabRack Jerkatt cells with 100 nM memAb trastuzumab pre-conjugation were washed in succession. The activation level of FabRack Jerkatt cells with 4 nM Herceptin treatment is similar to that of no treatment. Methods: Cancer cells (2.5 x 10⁶ 4 100 µl / well of medium was seeded into 96-well white-walled plates. After cell attachment overnight, the medium was removed from the plates, and Jurkat-NFAT-Luc Meditope-CAR cells (1 x 10⁶ 560 µl of memAb was added to each well. MemAb trastuzumab was pre-conjugated to Jurkat-NFAT-Luc medi-CAR or cancer cells, either continuously present or with washing. The bars in the graph represent the sequence from left to right: FabRack Jurkat-NFAT-Luc cells washed after pre-conjugation with memAb trastuzumab (100 nM), cancer cells washed after pre-conjugation with memAb trastuzumab (100 nM), continuously present with Herceptin (4 nM), and no treatment. Cells were incubated at 37°C for 6 hours, after which 50 µl of luciferase substrate (Invivogen #rep-qlc2) was added to each well. Luminescence was measured immediately using Biotech’s Synergy 4 multi-detection microplate reader. In all cases, the addition of 4 nM memAb trastuzumab to tumor cells mixed with Jurkat-NFAT-Luc, 'Meditope CAR' generated the largest signal. Two other samples were pre-incubated, washed, and then added to the third component. In one case, memAb trastuzumab was added to tumor cells, washed, and then exposed to Jurkat-NFAT-Luc, 'Meditope CAR'. In the other case, memAb trastuzumab was added to Jurkat-NFAT-Luc, 'Meditope CAR', washed, and then added to tumor cells. For cells with high antigen expression, pre-treatment of the tumor cells induced higher activation. For cells with low antigen expression (MCF7 and OVCAR3), pre-incubation of Jurkat-NFAT-Luc, 'Meditope CAR' cells induced higher levels of activation. It is important to note that while the concentration of memAb after 'washing' the cells is unknown, it is certainly less than 4 nM. These different treatments may explain the higher signal in the 4 nM treatment. Fig. 39. Herceptin blocked the activation of cancer cells and FabRack Zerkat cells co-incubated with 4 nM memAb trastuzumab. Herceptin and memAb trastuzumab were continuously present throughout the entire treatment period. Methods: Cancer cells (2.5 x 10⁶ 4 (100 µl) was seeded into 96-well white-walled plates. After cell attachment overnight, the medium was removed from the plates, and Jukat-NFAT-Luc me-CAR cells (1 x 10⁶ 5 60 µl was added to each well. The bars in the graph represent progress from left to right: persistent presence of memAb trastuzumab (4 nM), persistent presence of memAb trastuzumab (4 nM) and Herceptin (40 nM), and persistent presence of memAb trastuzumab (4 nM) and Herceptin (400 nM). Cells were incubated at 37°C for 6 hours, after which 50 µl of luciferase substrate (Invivogen #rep-qlc2) was added to each well. Luminescence was read immediately by Biotech’s Synergy 4 multi-detection microplate reader. Results: In the persistent presence of 4 nM memAb trastuzumab, Jurkat-NFAT-Luc medi-CAR cells were activated due to binding to cancer cells by memAb trastuzumab. The sustained presence of Herceptin during incubation can block Zykkat cell activation, as Herceptin possesses the same epitope as our memAb trastuzumab and can compete for the same binding site on HER2. Herceptin (400 nM) at a 100-fold concentration of memAb trastuzumab (4 nM) almost completely blocks Zykkat cell activation, demonstrating that Zykkat cell activation is caused by the binding of memAb trastuzumab to the same HER2 epitope recognized by Herceptin. Fabrack T cell activation requires memtope interactions, providing further evidence for the proposed mechanism of action. Fig. 40. Pre-binding of Herceptin to cancer cells blocked the activation of cancer cells and FabRack Zerkats co-incubated with 4 nM memAb trastuzumab. Cancer cells with 40 nM or 400 nM Herceptin pre-binding were washed in succession. The only difference between Herceptin and memAb trastuzumab is that Meditope became available later. Pre-treating cells with Herceptin blocks access to the antigen. Washing Herceptin-treated cells before memAb trastuzumab / Fabrack Zerkats removes unbound Herceptin. However, bound Herceptin will detach from the cells over time. Therefore, the reduction in activation is not dramatic in this experiment (compared to Fig. 41). Fig. 41. Herceptin barely blocks the activation of FabRack Zerkat cells in co-culture with cancer cells pre-bound to memAb. Cancer cells pre-bound to 100 nM memAb trastuzumab were washed in succession. Cancer cells washed after memAb trastuzumab pre-bound did not drastically reduce luminescence activity. The sustained presence of Herceptin during incubation had a minimal blocking effect on Zerkat cell activation. This data demonstrated that once memAb trastuzumab binds to HER2 on cancer cells, binding is minimally blocked by clinical trastuzumab. Methods: Cancer cells (2.5 x 10⁶ 4 (100 µl) was seeded into 96-well white-walled plates. After cell attachment overnight, the medium was removed from the plates, and Jukat-NFAT-Luc me-CAR cells (1 x 10⁶ 5) was added to each well. memAb trastuzumab was either continuously present or pre-bound to cancer cells upon washing. (The bars in the graph represent moving from left to right: cancer cells pre-bound with memAb trastuzumab (4 nM) continuously present; cancer cells pre-bound with memAb trastuzumab (100 nM) followed by washing; cancer cells pre-bound with memAb trastuzumab (100 nM) followed by washing + continuously present Herceptin (40 nM); cancer cells pre-bound with memAb trastuzumab (100 nM) followed by washing + continuously present Herceptin (40 nM).) Cells were incubated at 37°C for 6 hours, after which 50 µl of luciferase substrate (Invivogen #rep-qlc2) was added to each well. Luminescence was measured immediately using Biotech’s Synergy 4 multi-detection microplate reader. When memAb trastuzumab binds to a cell, it blocks Herceptin from binding to cell-derived antigens. Fig. 42. Expression of activation markers and cytokines in FabRack T cells. FabRack T cells were co-incubated with ovarian cancer cells, SKOV3 and OVCAR3, or breast cancer cells, SKBR3, BT474, and MCF7 for 5 hours in the presence of 0.5 nM memAb trastuzumab. The effector-to-target cell ratio was 1:1. HER2 scFv and Fab CAR were used as positive controls. After 5 hours of incubation, activation markers were analyzed by flow cytometry. Many controls were used in this experiment. The CDR loops of the scFv CAR and Fab CAR used here are identical to the memAb used in FabRack. Mocks are non-transformed T cells. The levels of CD69 and CD25, markers for T cell activation, were measured using cytometry. No activation of any variants was observed in the absence of tumor cells. Furthermore, in the presence of antigen-bearing tumor cells, little to no activation was observed for Fabrack T cells (without memAb), mock or mock, and I83E memAb trastuzumab. Finally, a significant increase in T cell activation was observed for Fabrack T cells + I83E memAb trastuzumab and scFV CAR T cells. Fab-CAR T cells were also activated, but exhibited greater variability. Fig. 43. FabRack T cells were co-cultured with ovarian cancer cells, SKOV3 and OVCAR3, or breast cancer cells, SKBR3, BT474, and MCF7 in the presence of 0.5 nM memAb trastuzumab for 5 hours. The effector-to-target cell ratio was 1:1. HER2 scFv CAR was used as a positive control. After 5 hours of incubation, degranulation markers CD107a and IFNγ were analyzed by flow cytometry. Fab T cells behave in a manner similar to conventional CAR T cells. Fig. 44. Cancer cells were incubated with mock T cells or FabRack T cells in the presence of various doses of memAb trastuzumab for 3 days. The ratio of effector to target cells was 1:4. At the end of incubation, cell viability was measured according to the instructions of the Promega Celltitor Kit. Results: Cancer cells were incubated with mock T cells or FabRack T cells in the presence of memAb trastuzumab for 3 days. This data indicates that FabRack T cells effectively killed HER2-positive cancer cells because they bind to cancer cells via memAb trastuzumab. The IC50s of cancer cells co-incubated with memAb trastuzumab and FabRack T cells were 0.33 nM (BT474), 0.11 nM (SKBR3), 0.069 nM (MCF7), 0.046 nM (SKOV3), and 0.027 nM (OVCAR3). The apoptotic effect was not associated with HER2 expression levels on cancer cells. Methods: For the tumor apoptosis assay, cancer cells (2.5 x 10⁶ 4 Human T cells (6,250 / 100 µl) and antibodies were seeded into 96-well round-bottom plates in the presence or absence of antibodies. After 72 hours of incubation, the cells were centrifuged at 250 xg for 5 minutes, and 100 µl of medium was removed from each well. To test cell viability, 100 µl of reagent from the Promega Celltitor Kit was added to each well. After 2 minutes of incubation, 100 µl of the mixture was transferred to a white-walled 96-well plate and measured using Biotech’s Synergy 4 multi-detection microplate reader. Fig. 45. The IC50 of cancer cells co-incubated with memAb trastuzumab and FabRack T cells is 0.33 nM (BT474), 0.11 nM (SKBR3), 0.069 nM (MCF7), 0.046 nM (SKOV3), and 0.027 nM (OVCAR3). Fig. 46. Results: In the continuous presence of 0.1 nM or 0.5 nM memAb antibodies, the viability of cancer cells with FabRack T cell co-incubation (center bar) was reduced compared to those with mock T cell co-incubation (left bar). In breast cancer cells, cell viability decreased in a dose-dependent manner at 0.1 and 0.5 nM, whereas ovarian cancer cells exhibited similar viability at both concentrations. Cancer cells with antibody pre-binding and washing (right bar) could still be killed by human FabRack T cells, despite a 5–18% reversal in viability compared to cancer cells with continuous antibody treatment. Methods: For the tumor apoptosis assay, cancer cells (2.5 x 10⁶ 4 6,250 / 100 µl of memAb antibody and human T cells (6,250 / 100 µl) were seeded into 96-well round-bottom plates in the presence or absence of memAb antibody. After 72 hours of incubation, the cells were centrifuged at 250 xg for 5 minutes, and 100 µl of medium was removed from each well. To test cell viability, 100 µl of reagent from the Promega Celltitor Kit was added to each well. After 2 minutes of incubation, 100 µl of the mixture was transferred to a white-walled 96-well plate and measured using Biotech’s Synergy 4 multi-detection microplate reader. Fig. 47. Cancer cell survival after DAPI staining was analyzed using flow cytometry. HER2 scFv CAR and HER2 Fab CAR were used as positive controls. Results: Flow cytometry was used to analyze how many cancer cells remained alive after co-incubation with FabRack T cells and memAb trastuzumab, compared to cancer cells incubated with HER2 scFv CAR or HER2 Fab CAR T cells as positive controls. After 3 days of incubation, the number of viable cancer cells decreased sharply in the case of co-incubation with FabRack T cells and memAb trastuzumab. The apoptotic effect of FabRack T cells was similar to or even superior to that of HER2 scFv CAR or HER2 Fab CAR T cells. Similar results are obtained when cell viability is read using alternative methods. Fig. 48. Cancer cell survival after DAPI staining was analyzed using flow cytometry. HER2 scFv CAR and HER2 Fab CAR were used as positive controls. Results: Flow cytometry was used to analyze how many cancer cells remained alive after co-incubation with FabRack T cells and memAb trastuzumab, compared to cancer cells incubated with HER2 scFv CAR or HER2 Fab CAR T cells as positive controls. After 3 days of incubation, the number of viable cancer cells decreased sharply when co-incubated with FabRack T cells and memAb trastuzumab. The apoptotic effect of FabRack T cells was similar to or even superior to that of HER2 scFv CAR or HER2 Fab CAR T cells. Fig. 49. Changes in T cell numbers in co-culture with target cells and different concentrations of memAb trastuzumab. During incubation, the ratio of effector to target cells is 1:4. These experiments demonstrate T cell activation and result in cell proliferation dependent on the presence of memAb. Fig. 50. Breast cancer cells (2.5 x 10⁶ 4 ) were seeded into 96-well white-walled plates. After overnight cell attachment, the medium was removed from the plates, and Jukat-NFAT-Luc Fabrack cells (41BB version, 1 x 10⁶) were seeded with various doses of 2N1 memAb. 5 ) was added to each well. Cells were incubated at 37°C for 6 hours, and then the luciferase substrate was added to each well. Luminescence was immediately read by Biotech’s Synergy 4 multi-detection microplate reader. Specific details for implementing the invention

[0021] definition

[0022] Although various embodiments and aspects of the present invention have been presented and described herein, it will be apparent to those skilled in the art that such embodiments and aspects are provided merely as examples. A number of modifications, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used to practice the invention.

[0023] Section titles used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or parts of documents cited in this application, including but not limited to patents, patent applications, articles, books, manuals, and papers, are expressly incorporated by reference in their entirety for any purpose.

[0024] The abbreviations used herein have their ordinary meanings in the fields of chemistry and biology. The chemical structures and formulas described herein are constructed according to standard rules for chemical atoms known in the field of chemistry.

[0025] When substituents are identified by conventional chemical formulas written from left to right, they contain the same chemically identical substituents resulting from writing the structure from right to left. For example, -CH2O- is identical to -OCH2-.

[0026] The term "alkyl" means a linear (i.e., unbranched) or branched acyclic carbon chain (or carbon) or a combination thereof, which, either itself or as part of other substituents, may be fully saturated, monounsaturated, or polyunsaturated, and may include divalent and polyvalent radicals having a specified number of carbon atoms (i.e., C1-C 10 (meaning 1 to 10 carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, and homologs and isomers such as n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. The unsaturated alkyl group is one having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethinyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers. The alkoxy is an alkyl group attached to the rest of the molecule via an oxygen linker (-O-). The alkyl moiety can be an alkenyl moiety. The alkyl moiety can be an alkenyl moiety. The alkyl portion can be completely saturated.

[0027] The term "alkylene" means a divalent radical derived from an alkyl, either in itself or as part of another substituent, exemplified but not limited to -CH2CH2CH2CH2- unless otherwise noted. Typically, the alkyl (or alkylene) group will have 1 to 24 carbon atoms, and a group having 10 or fewer carbon atoms is preferred in the present invention. "Lower alkyl" or "lower alkylene" is generally a short-chain alkyl or alkylene group having 8 or fewer carbon atoms. The term "alkenylene" means a divalent radical derived from an alken, either in itself or as part of another substituent, unless otherwise noted.

[0028] The term “heteroalkyl” means a stable acyclic straight or branched chain or combination thereof comprising, by itself or in combination with other terms, at least one carbon atom and at least one heteroatom (e.g., O, N, P, Si, or S), unless otherwise noted, wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, P, S, and Si may be placed at any internal position of the heteroalkyl group or at a position to which the alkyl group is attached to the rest of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, -O-CH-2-CH3, and -CN. For example, up to two or three heteroatoms may be consecutive, such as -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. The heteroalkyl moiety may include one heteroatom (e.g., O, N, S, Si, or P). A heteroalkyl moiety may comprise two optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may comprise three optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may comprise four optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may comprise five optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may comprise up to eight optionally different heteroatoms (e.g., O, N, S, Si, or P).

[0029] Likewise, the term "heteroalkylene" refers to -CH2-CH2-S-CH2-CH2, either as itself or as part of another substituent, unless otherwise noted. 2- and means a divalent radical derived from a heteroalkyl group, exemplified but not limited to -CH2-S-CH2-CH2-NH-CH2-. In the case of a heteroalkylene group, the heteroatom may also occupy one or both of the chain ends (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Additionally, in the case of alkylene and heteroalkylene linkers, the orientation of the linker is not implied by the direction in which the linker's chemical formula is written. For example, the chemical formula -C(O)2R'- represents both -C(O)2R'- and -R'C(O)2-. As previously stated, the heteroalkyl group used herein comprises a group attached to the rest of the molecule via a heteroatom, e.g., -C(O)R', -C(O)NR', ​​-NR'R'', -OR', -SR', and / or -SO2R'. Where "heteroalkyl" is mentioned and subsequently a specific heteroalkyl group such as NR'R'' is mentioned, the terms heteroalkyl and NR'R'' are to be understood as not overlapping or mutually exclusive. Rather, the specific heteroalkyl group is mentioned for the sake of clarity. Accordingly, the term "heteroalkyl" should not be interpreted herein as excluding specific heteroalkyl groups such as NR'R'''.

[0030] The terms “cycloalkyl” and “heterocycloalkyl” mean, either by themselves or in combination with other terms, non-aromatic cyclic versions of “alkyl” and “heteroalkyl”, respectively, which are not necessarily bonded to hydrogen due to all carbon atoms participating in bonding with non-hydrogen atoms. Additionally, in the case of heterocycloalkyl, the heteroatom may occupy a position where the heterocycle is attached to the rest of the molecule. Examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, 3-hydroxy-cyclobut-3-enyl-1,2,dione, 1H-1,2,4-triazolyl-5(4H)-one, 4H-1,2,4-triazolyl. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrotiene-2-yl, tetrahydrotiene-3-yl, 1-piperazinyl, 2-piperidinyl, etc. "Cycloalkylene" and "heterocycloalkylene" refer to divalent radicals derived from cycloalkyl and heterocycloalkyl, respectively, alone or as part of other substituents. The heterocycloalkyl moiety may include a single cyclic heteroatom (e.g., O, N, S, Si, or P). The heterocycloalkyl moiety may comprise two optionally different cyclic heteroatoms (e.g., O, N, S, Si, or P). The heterocycloalkyl moiety may comprise three optionally different cyclic heteroatoms (e.g., O, N, S, Si, or P). The heterocycloalkyl moiety may comprise four optionally different cyclic heteroatoms (e.g., O, N, S, Si, or P).The heterocycloalkyl moiety may include five optionally different cyclic heteroatoms (e.g., O, N, S, Si, or P). The heterocycloalkyl moiety may include up to eight optionally different cyclic heteroatoms (e.g., O, N, S, Si, or P).

[0031] The terms “halo” or “halogen” mean a fluorine, chlorine, bromine, or iodine atom, either as itself or as part of another substituent, unless otherwise noted. Additionally, terms such as “haloalkyl” mean including monohaloalkyl and polyhaloalkyl. For example, the term “halo(C1-C4)alkyl” includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, etc.

[0032] The term “acyl” means -C(O)R unless otherwise noted, where R is a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heteroalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl.

[0033] The term “aryl” means a polyunsaturated aromatic hydrocarbon substituent that may be a single ring or multiple rings (preferably 1 to 3 rings) that are fused together (i.e., fused ring aryl) or covalently linked, unless otherwise noted. A fused ring aryl refers to multiple rings fused together, wherein at least one of the fused rings is an aryl ring. The term “heteroaryl” refers to an aryl group (or ring) containing at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom(s) are optionally quaternized. Thus, the term “heteroaryl” comprises a fused ring heteroaryl group (i.e., multiple rings fused together in which at least one of the fused rings is a heteroaromatic ring). A 5,6-fused ring heteroarylene refers to two rings fused together, where one ring has 5 members and the other has 6 members, and at least one ring is a heteroaryl ring. Similarly, a 6,6-fused ring heteroarylene refers to two rings fused together, where one ring has 6 members and the other has 6 members, and at least one ring is a heteroaryl ring. A 6,5-fused ring heteroarylene refers to two rings fused together, where one ring has 6 members and the other has 5 members, and at least one ring is a heteroaryl ring. A heteroaryl group can be attached to the rest of the molecule via carbons or heteroatoms.Non-limiting examples of aryl and heteroaryl are phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolil, 2-pyrrolil, 3-pyrrolil, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazollyl, Includes furinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the aforementioned aryl and heteroaryl ring systems are selected from the group of acceptable substituents listed below. "Arylene" and "heteroarylene" refer to divalent radicals derived from aryl and heteroaryl, respectively, alone or as part of other substituents. Non-limiting examples of aryl and heteroaryl groups include pyridinyl, pyrimidinyl, thiophenyl, thienyl, furanyl, indolyl, benzoxadiazolyl, benzodioxolyl, benzodioxanyl, thianaphtanyl, pyrrolopyridinyl, indazolyl, quinolinyl, quinoxalinyl, pyridopyrazinyl, quinazolinonyl, benzisoxazolyl, imidazopyridinyl, benzofuranyl, benzothiophenyl, phenyl, naphthyl, biphenyl, pyrrolyl, pyrazolyl, imidazolyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl, furylthienyl, pyridyl, pyrimidyl, benzothiazolyl, furinyl, benzimidazolyl, isoquinolyl, thiadiazolyl, oxadiazolyl, pyrrolyl, Includes diazolyl, triazolyl, tetrazolyl, benzothiadiazolyl, isothiazolyl, pyrazolopyrimidinyl, pyrrolopyrimidinyl, benzotriazolyl, benzoxazolyl, or quinolyl. The above examples may be substituted or unsubstituted, and the divalent radical of each of the above heteroaryl examples is a non-limiting example of heteroarylene. The heteroaryl moiety may include one cyclic heteroatom (e.g., O, N, or S).A heteroaryl moiety may include two optionally different cyclic heteroatoms (e.g., O, N, or S). A heteroaryl moiety may include three optionally different cyclic heteroatoms (e.g., O, N, or S). A heteroaryl moiety may include four optionally different cyclic heteroatoms (e.g., O, N, or S). A heteroaryl moiety may include five optionally different cyclic heteroatoms (e.g., O, N, or S). An aryl moiety may have a single ring. An aryl moiety may have two optionally different rings. An aryl moiety may have three optionally different rings. An aryl moiety may have four optionally different rings. A heteroaryl moiety may have one ring. A heteroaryl moiety may have two optionally different rings. A heteroaryl moiety may have three optionally different rings. A heteroaryl residue may have four optionally different rings. A heteroaryl moiety may have five optionally different rings.

[0034] The fused ring heterocycloalkyl-aryl is an aryl fused to a heterocycloalkyl. The fused ring heterocycloalkyl-heteroaryl is a heteroaryl fused to a heterocycloalkyl. The fused ring heterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl. The fused ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkyl fused to another heterocycloalkyl. The fused ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkyl fused to another heterocycloalkyl. The fused ring heterocycloalkyl-aryl, the fused ring heterocycloalkyl-heteroaryl, the fused ring heterocycloalkyl-cycloalkyl, or the fused ring heterocycloalkyl-heterocycloalkyl may each be independently unsubstituted or substituted with one or more of the substituents described herein.

[0035] As used herein, the term "oxo" refers to oxygen double-bonded to a carbon atom.

[0036] As used herein, the term "alkylsulfonyl" means a moiety having the chemical formula -S(O2)-R', where R' is a substituted or unsubstituted alkyl group as defined above. R' may have a specific number of carbons (e.g., "C1-C4alkylsulfonyl").

[0037] Each of the above terms (e.g., "alkyl", "heteroalkyl", "cycloalkyl", "heterocycloalkyl", "aryl", and "heteroaryl") includes both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

[0038] Substituents for alkyl and heteroalkyl radicals (including groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) are, in numbers ranging from 0 to (2m'+1), non-limitingly -OR', =O, =NR', =N-OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -CO2R', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O)2R', -NR-C(NR'R'')=NR''', -S(O)R', It may be one or more of the various groups selected from -S(O)2R', -S(O)2N(R)('R''-NRSO2R'), -CN, and -NO2, where m' is the total number of carbon atoms in these radicals. R', R'', R''' and R'''' each preferably independently refer to hydrogen, a substituted or unsubstituted heteroalkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl (e.g., an aryl substituted with 1-3 halogens), a substituted or unsubstituted alkyl, alkoxy or thioalkoxy group, or an aryl alkyl group. If the compound of the present invention comprises more than one R group, for example, each R group is independently selected, such as each R', R'', R''' and R'''' group in the case where more than one R', R'', R''' and R'''' group are present. When R' and R'' are attached to the same nitrogen atom, they can combine with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example, -NR'R'' includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl.From the above discussion of substituents, those skilled in the art will understand that the term "alkyl" means a group comprising a carbon atom bonded to a group other than a hydrogen group, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, etc.).

[0039] Similar to the substituents described for alkyl radicals, the substituents for aryl and heteroaryl groups are diverse and range from 0 to the total number of open valencies of the aromatic ring system, such as -OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -CO2R', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', NR''C(O)2R', NRC(NR'R'')=NR''', S(O)R', -S(O)2R', -S(O)2N(R')(R'', -NRSO2R'), -CN, -NO2, -R', -N3, -CH(Ph)2, Selected from fluoro(C1-C4)alkoxy and fluoro(C1-C4)alkyl, wherein R', R'', R''' and R'''' are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. When the compound of the present invention comprises more than one R group, for example, each R group is independently selected, such as when more than one R', R'', R''' and R''''' are present, each R', R'', R''' and R''''''' is independently selected.

[0040] Where a moiety is substituted with an R substituent, the group may be referred to as "R-substituted." When a moiety is R-substituted, the moiety is substituted with at least one R substituent, and each R substituent is optionally different. For example, in this invention, the moiety is R 1A - In the case of a substituted or unsubstituted alkyl, multiple R 1A Substituents can be attached to alkyl residues, where each R 1A The substituents are optionally different. Where an R-substituted moiety is substituted with multiple R substituents, each R substituent may be distinguished herein by using a prime symbol ('), such as R', R'', etc. For example, if the moiety is R 3A - It is a substituted or unsubstituted alkyl, and the moiety is a plurality of R 3A In the case of substitution with a substituent, multiple R 3A The substituent is R 3A ', R 3A '', R 3A It can be distinguished by ''', etc. In some embodiments, the plurality of R substituents is 3. In some embodiments, the plurality of R substituents is 2.

[0041] Two or more substituents may be optionally linked to form an aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group. These so-called ring-forming substituents are not necessarily required but are typically found attached to the cyclic base structure. In one embodiment, the ring-forming substituents are attached to adjacent members of the base structure. For example, two ring-forming substituents attached to adjacent members of the cyclic base structure create a fused ring structure. In another embodiment, the ring-forming substituents are attached to a single member of the base structure. For example, two ring-forming substituents attached to a single member of the cyclic base structure create a spirocyclic structure. In yet another embodiment, the ring-forming substituents are attached to non-adjacent members of the base structure.

[0042] Two substituents on adjacent atoms of an aryl or heteroaryl ring are optionally of the chemical formula -TC(O)-(CRR') q It can form a -U- ring, where T and U are independently -NR-, -O-, -CRR'-, or a single bond, and q is an integer from 0 to 3. Alternatively, two substituents on adjacent atoms of an aryl or heteroaryl ring are optionally of the formula -A-(CH2) r It can be substituted with a -B- substituent, where A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'-, or single bonds, and r is an integer from 1 to 4. One of the single bonds of the new ring thus formed can optionally be replaced with a double bond. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring can optionally have the chemical formula -(CRR') s -X'-(C''R''R''') d It can be substituted with a substituent of -, where variables s and d are independently integers from 0 to 3, and X' is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-. Substituents R, R', R'' and R''' are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

[0043] The terms “heteroatom” or “ring heteroatom” as used herein mean that they include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0044] As used herein, "substituent" means a group selected from the following moiety:

[0045] (A) Oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC=(O)NHNH2, -NHC=(O) NH2, -NHSO2H, -NHC=(O)H, -NHC(O)-OH, -NHOH, -OCF3, -OCHF2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

[0046] (B) Substituted with at least one substituent selected from the following, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl:

[0047] (i) oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC=(O)NHNH2, -NHC=(O) NH2, -NHSO2H, -NHC=(O)H, -NHC(O)-OH, -NHOH, -OCF3, -OCHF2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

[0048] (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl substituted with at least one substituent selected from the following:

[0049] (a) oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC=(O)NHNH2, -NHC=(O) NH2, -NHSO2H, -NHC=(O)H, -NHC(O)-OH, -NHOH, -OCF3, -OCHF2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

[0050] (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl substituted with at least one substituent selected from the following: oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC=(O)NHNH2, -NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)-OH, -NHOH, -OCF3, -OCHF2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl.

[0051] As used herein, "size-limited substituent" or "size-limited substituent group" refers to a group selected from all substituents described above for the "substituent," wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C 20alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20-membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8-membered cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8-membered heterocycloalkyl, and each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C 10 It is an aryl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 10-membered heteroaryl.

[0052] As used herein, "lower substituent" or "lower substituent group" means a group selected from all substituents described above with respect to the "substituent," wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8-membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7-membered heterocycloalkyl, and the unsubstituted aryl is a substituted or unsubstituted C6-C 10 It is an aryl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 9-membered heteroaryl.

[0053] In some embodiments, each substituent described in the compounds of the present invention is substituted with one or more substituents. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene and / or substituted heteroarylene described in the compounds of the present invention is substituted with at least one substituent. In other embodiments, at least one or all of these groups are substituted with at least one size-limiting substituent. In other embodiments, at least one or all of these groups are substituted with at least one lower substituent.

[0054] In another embodiment of the compound of the present invention, each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C 20 They may be alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20-membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8-membered cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8-membered heterocycloalkyl, and each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C 10 It is an aryl, and / or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 10-membered heteroaryl. In some embodiments of the compounds of the present invention, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C 20 They are alkylenes, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 20-membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C8-membered cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 8-membered heterocycloalkylene, and each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C10 It is an arylene, and / or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 10 heteroarylene.

[0055] In some embodiments, each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8-membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7-membered heterocycloalkyl, and each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C 10 It is an aryl, and / or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 9-membered heteroaryl. In some embodiments, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C8 alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 8-membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C7 cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 7-membered heterocycloalkylene, and each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C 10 It is an arylene, and / or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 9-membered heteroarylene. In some embodiments, the compound is a chemical species presented in the following Examples section, drawings, or tables.

[0056] As used herein, the term “conjugate” refers to an association between atoms or molecules. The association may be direct or indirect. For example, a conjugate between a nucleic acid and a protein may be direct, for example, by a covalent bond, or indirect by a non-covalent bond (e.g., electrostatic interactions (e.g., ionic bonds, hydrogen bonds, halogen bonds), van der Waals interactions (e.g., dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effect), hydrophobic interactions, etc.). In the embodiments, the conjugate is formed using conjugate chemistry that includes, but is not limited to, nucleophilic substitution (e.g., reactions of amines and alcohols with acyl halides, reactions with active esters), electrophilic substitution (e.g., enamine reactions), and carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reactions, Diels-Alder addition). These and other useful reactions are discussed in the literature, for example (March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, DC, 1982). In an embodiment, the microparticles are non-covalently attached to a solid support through a non-covalent chemical reaction between a component of the microparticles and a component of the solid support. In another embodiment, the microparticles comprise one or more reactive moietyes, such as a covalent reactive moiety as described herein (e.g., an amine reactive moiety). In another embodiment, the microparticle comprises a linker having one or more reactive moiety as described herein, such as a covalent reactive moiety (e.g., an amine-reactive portion).

[0057] Useful reactive moiety or functional group used herein in conjugate chemistry (including "click chemistry" known in the art) includes, for example, the following:

[0058] (a) carboxyl groups and various derivatives thereof, including but not limited to N-hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid halides, acylimidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl, and aromatic esters;

[0059] (b) Hydroxyl group that can be converted into esters, ethers, aldehydes, etc.

[0060] (c) a haloalkyl group in which the halide may later be substituted with a nucleophilic group, such as an amine, carboxylate anion, thiol anion, carbocation, or alkoxide ion, to result in the covalent attachment of a new group at the site of the halogen atom;

[0061] (d) Chindient groups capable of participating in Diels-Alder reactions, such as maleimido groups, for example;

[0062] (e) an aldehyde or ketone group capable of subsequent derivatization through the formation of carbonyl derivatives, e.g., imines, hydrazones, semicarbazones, or oximes, or through mechanisms such as Grignard addition or alkyllithium addition;

[0063] (f) a sulfonyl halide group for a subsequent reaction with an amine to form a sulfonamide, for example;

[0064] (g) a thiol group that can be converted to a disulfide, react with an acyl halide, or bond to a metal such as gold;

[0065] (h) For example, an amine or sulfhydryl group that can be acylated, alkylated, or oxidized;

[0066] (i) alkenes that may undergo, for example, cycloaddition, acylation, Michael addition, etc.;

[0067] (j) Epoxide capable of reacting with, for example, amine and hydroxyl compounds;

[0068] (k) Other standard functional groups useful for phosphoramidite and nucleic acid synthesis;

[0069] (l) metal silicon oxide bond;

[0070] (m) a metal bonded to a reactive phosphorus group (e.g., phosphine) to form a phosphate diester bond, for example; and

[0071] (n) Sulfone, e.g. vinyl sulfone.

[0072] The chemical synthesis of compositions by combining small modular units using conjugate ("Click") chemistry is widely known and is described, for example, in the following literature: HC Kolb, MG Finn and KB Sharpless ((2001). "Click Chemistry: Diverse Chemical Function from a Few Good Reactions". Angewandte Chemie International Edition 40 (11): 2004-2021); RA Evans ((2007). "The Rise of Azide-Alkyne 1,3-Dipolar'Click' Cycloaddition and its Application to Polymer Science and Surface Modification". Australian Journal of Chemistry 60 (6): 384-395; WC Guida et al. Med. Res. Rev. p 3 1996; Spiteri, Christian and Moses, John E. ((2010). "Copper-Catalyzed Azide-Alkyne Cycloaddition: Regioselective Synthesis of 1,4,5-Trisubstituted 1,2,3-Triazoles". Angewandte Chemie International Edition 49 (1): 31-33); Hoyle, Charles E. and Bowman, Christopher N. ((2010). "Thiol-Ene Click Chemistry". Angewandte Chemie International Edition 49 (9): 1540-1573); Blackman, Melissa L. and Royzen, Maksim and Fox, Joseph M. ((2008).“Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity”. Journal of the American Chemical Society 130 (41): 13518-13519); Devaraj, Neal K. and Weissleder, Ralph and Hilderbrand, Scott A. ((2008). “Tetrazine Based Cycloadditions: Application to Pretargeted Live Cell Labeling”. Bioconjugate Chemistry 19 (12): 2297-2299); Stokmann, Henning; Neves, Andre; Stairs, Shaun; Brindle, Kevin; Leeper, Finian ((2011). “Exploring isonitrile-based click chemistry for ligation with biomolecules”. Organic & Biomolecular Chemistry) (all incorporated herein by reference in its entirety for all purposes).

[0073] Reactive functional groups may be selected so as not to contribute to or interfere with the chemical stability of the proteins or nucleic acids described herein. For example, the nucleic acid may comprise vinyl sulfone or other reactive moiety (e.g., maleimide). Optionally, the nucleic acid may comprise a reactive moiety having the chemical formula -SSR. R may be, for example, a protecting group. Optionally, R is hexanol. As used herein, the term hexanol refers to the chemical formula C6H 13Compounds having OH include 1-hexanol, 2-hexanol, 3-hexanol, 2-methyl-1-pentanol, 3-methyl-1-pentanol, 4-methyl-1-pentanol, 2-methyl-2-pentanol, 3-methyl-2-pentanol, 4-methyl-2-pentanol, 2-methyl-3-pentanol, 3-methyl-3-pentanol, 2,2-dimethyl-1-butanol, 2,3-dimethyl-1-butanol, 3,3-dimethyl-1-butanol, 2,3-dimethyl-2-butanol, 3,3-dimethyl-2-butanol, and 2-ethyl-1-butanol. Optionally, R is 1-hexanol.

[0074] As used herein, the term “approx.” means a range of values ​​including a specific value, which will be considered by those skilled in the art to be substantially similar to the specific value. In embodiments, the term “approx.” means within a standard deviation using measurements generally accepted in the art. In embodiments, “approx.” means a range extending up to + / -10% of a specific value. In embodiments, “approx.” means a specific value.

[0075] As used herein, terms in the singular form mean one or more. Additionally, as used herein, the phrase "substituted with" means that a specific group may be substituted with one or more of any or all named substituents. For example, a group such as an alkyl or heteroaryl group may be substituted with an "unsubstituted C1-C 20 In the case where "substituted with an alkyl, or an unsubstituted 2 to 20-membered heteroalkyl," the group comprises one or more unsubstituted C1-C 20 It may contain an alkyl group and / or one or more unsubstituted 2 to 20 heteroalkyl groups. Additionally, if a moiety is substituted with an R substituent, the group may be referred to as "R-substituted". If a moiety is R-substituted, the moiety is substituted with at least one R substituent, and each R substituent is optionally different.

[0076] Unless otherwise defined, technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art. For example, refer to the literature (Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989)). Any method, apparatus, and material similar or equivalent to those described herein may be used in the practice of the present invention. The following definitions are provided to aid in understanding specific terms frequently used herein and are not intended to limit the scope of the present disclosure.

[0077] "Biological sample" or "sample" refers to material obtained from or derived from a subject or patient. Biological samples include sections of tissue, such as biopsy and autopsy samples, and frozen sections taken for tissue examination. These samples include body fluids, such as blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, etc.), sputum, tissues, cultured cells (e.g., primary cultures, in vitro grafts, and transformed cells), feces, urine, synovial fluid, joint tissue, synovial tissue, synovial cells, fibroblast-like synovial cells, macrophage-like synovial cells, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, etc. Biological samples are typically obtained from eukaryotic organisms, such as primates, such as chimpanzees or mammals like humans; cattle; dogs; cats; rodents, such as guinea pigs, rats, mice; rabbits; or birds; reptiles; or fish.

[0078] As used herein, “cell” refers to a cell that performs metabolic or other functions sufficient to preserve or replicate its genomic DNA. Cells may be identified by methods widely known in the art, including, for example, the presence of an intact membrane, staining by a specific dye, the ability to produce offspring, or, in the case of germ cells, the ability to produce viable offspring in combination with a second germ cell. Cells may include prokaryotic and eukaryotic cells. Prokaryotic cells include, but are not limited to, bacteria. Eukaryotic cells include, but are not limited to, yeast cells and cells derived from plants and animals, such as mammals, insects (e.g., spodoptera), and human cells. Cells may be useful if they are naturally non-adherent or if they have been treated so as not to adhere to a surface, for example by trypsin treatment.

[0079] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues, wherein the polymer may optionally be conjugated to a moiety not composed of amino acids. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-natural amino acid polymers. “Fusion protein” refers to a chimeric protein encoding two or more individual protein sequences that are recombinantly expressed as a single moiety.

[0080] The terms “peptidyl” and “peptidyl moiety” refer to a peptide attached to the remainder of the molecule (e.g., the recombinant protein provided herein or the peptide domain-forming portion of the recombinant protein provided herein). The peptidyl moiety may be substituted with a chemical linker that serves to attach the peptidyl moiety to the remainder of the recombinant protein (e.g., a transmembrane domain, a spacer region, or a peptidyl linker). The peptidyl moiety may also be substituted with an additional chemical moiety (e.g., an additional R substituent). In an embodiment, the non-CDR Fab-binding peptide domain comprises a peptidyl moiety. In an embodiment, the non-CDR Fab-binding peptide domain is a peptidyl moiety. The term “meditope” as used herein refers to the peptidyl moiety contained in the peptide domain described herein. Thus, in an embodiment, the non-CDR Fab-binding peptide domain is a meditope. In an embodiment, the non-CDR Fab-binding peptide domain comprises a meditope.

[0081] The peptidyl moiety (e.g., Meditope) may be a linear or cyclic peptide moiety. Various methods for cyclizing the peptide moiety may be utilized, for example, to address in vivo stability and enable chemoselective control over subsequent conjugation chemistry. In some embodiments, the cyclization strategy is a lactam cyclization strategy involving head-to-tail (head-to-tail) lactam cyclization (between terminal residues of the acyclic peptide) and / or lactam linkage between other residues. Lactam formation may also be influenced by incorporating residues such as glycine, β-Ala, and / or 7-aminoheptanoic acid into the acyclic peptide cyclization precursor to produce different lactam ring sizes and linkage modes. Additional cyclization strategies, such as "click" chemistry and olefin metalysis, may also be utilized. These peptide and peptidomimetic cyclization methods are widely known in the art. In an embodiment, the peptidyl moiety (e.g., Meditope) is a linear peptidyl portion (e.g., linear Meditope). In an embodiment, the peptidyl moiety (e.g., Meditope) is a cyclic peptidyl moiety (e.g., cyclic Meditope).

[0082] "Label," "detectable domain," or "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, a useful label is 32 P, fluorescent dyes, electron-dense reagents, enzymes (e.g., those commonly used in ELISA), biotin, digoxigenin, or haptens and proteins, or other entities that may be detectable by incorporating, for example, a radiolabel into a peptide or antibody that is specifically reactive with the target peptide. Any suitable method known in the art to conjugate the antibody to the label may be used, such as the method described in the literature (Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego).

[0083] "Tagged protein or polypeptide" is one that is covalently bound to a label via a linker or chemical bond, or non-covalently bound via ionic, Wal der Waals, or hydrogen bonds, so that the presence of the labeled protein or polypeptide can be detected by detecting the presence of a label bound to the labeled protein or polypeptide. Alternatively, a method utilizing high-affinity interactions in which one of the pair of binding partners binds to the other, e.g., biotin or streptavidin, can achieve the same result.

[0084] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring cyclic amino acids are those encoded by the genetic code as well as amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoric acid. Amino acid analogs refer to compounds having the same basic chemical structure as natural amino acids—namely, hydrogen, a carboxyl group, an amino group, and an α-carbon bonded to an R group—such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. These analogs possess a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics refer to chemical compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids.

[0085] Amino acids may be referred to herein by commonly known three-character symbols or by one-character symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Likewise, nucleotides may be referred to by generally accepted single-character codes.

[0086] The amino acid or nucleotide base "position" is represented by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-terminus). Due to deletions, insertions, truncations, fusions, etc., that may be considered when determining optimal alignment, the amino acid residue number of the test sequence, generally determined by simply counting from the N-terminus, will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, if a variant has a deletion relative to the aligned reference sequence, there will be no amino acid in the variant corresponding to the position in the reference sequence at the deletion site. If there is an insertion in the aligned reference sequence, that insertion will not correspond to the numbered amino acid position in the reference sequence. In the case of truncations or fusions, there may be a stretch of amino acids in the reference or aligned sequence that does not correspond to any amino acid in the corresponding sequence.

[0087] The terms “numbered by reference” or “corresponding” used in relation to the numbering of a given amino acid or polynucleotide sequence refer to the numbering of residues of a specific reference sequence when the given amino acid or polynucleotide sequence is compared to a reference amino acid sequence. An amino acid residue of a protein is “corresponding” to a given residue when it occupies an essential structural position within the same protein as the given residue. For example, a selected residue in a selected antibody (or Fab domain) corresponds to light chain threonine at kavat position 40 when the selected residue occupies the same essential space or other structural relationship as light chain threonine at kavat position 40. In some embodiments, when the selected protein is aligned for maximum homology with the light chain of the antibody (or Fab domain), the position of the selected protein aligned with threonine 40 is referred to as corresponding to threonine 40. Instead of primary sequence alignment, a three-dimensional structural alignment may also be used, for example, in which the structure of the selected protein is aligned to correspond as much as possible to light chain threonine at kavat position 40 and the entire structure is compared. In this case, the amino acid occupying the same essential position as threonine 40 in the structural model is said to correspond to the threonine 40 residue.

[0088] The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences. With respect to a specific nucleic acid sequence, a conservatively modified variant refers to a nucleic acid encoding the same or essentially identical amino acid sequence, or, if the nucleic acid does not code for an amino acid sequence, an essentially identical sequence. Due to the degenerate nature of the genetic code, multiple functionally identical nucleic acid sequences code for any given amino acid residue. For example, codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Therefore, at any position where alanine is specified by a codon, the codon can be changed to any corresponding codon described without altering the encoded polypeptide. Such nucleic acid variations are "silencing variations," which are a type of conservatively modified variant. All nucleic acid sequences of the present invention encoding polypeptides also describe all possible silencing variations of the nucleic acid. Those skilled in the art will recognize that each codon of the nucleic acid (excluding AUG, the sole codon for methionine, and TGG, the sole codon for tryptophan) can be modified to produce a functionally identical molecule. Therefore, each silencing variant of the nucleic acid encoding the polypeptide is contained in the respective described sequence for the expression product, but not for the actual probe sequence.

[0089] With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter, add, or delete a single amino acid or a small percentage of amino acids in the coded sequence constitute "conservatively modified variants," and that such alterations result in the substitution of an amino acid with a chemically similar amino acid. Tables of conservative substitutions providing functionally similar amino acids are widely known in the art. Such conservatively modified variants are added to, but are not excluded from, the polymorphic variants, interspecies homologs, and alleles of the present invention.

[0090] The following eight groups each contain amino acids that are conservative substituents for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (e.g., see literature (Creighton, Proteins (1984))).

[0091] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded forms, their polymers, and their complements. The term "polynucleotide" refers to a linear sequence of nucleotides. The term "nucleotide" typically refers to a single unit of a polynucleotide, i.e., a monomer. A nucleotide may be a ribonucleotide, a deoxyribonucleotide, or a modified version thereof. Examples of polynucleotides considered herein include single- and double-stranded DNA, single- and double-stranded RNA (including siRNA), and hybrid molecules having a mixture of single- and double-stranded DNA and RNA. As used herein, "nucleic acid" also refers to a nucleic acid having the same basic chemical structure as naturally occurring nucleic acids. Such analogues have modified sugar and / or modified ring substituents but retain the same basic chemical structure as naturally occurring nucleic acids. "Nucleic acid mimics" refers to chemical compounds having a structure that functions in a manner similar to naturally occurring nucleic acids, although the general chemical structure of the nucleic acid differs. Examples of such analogs include, but are not limited to, phosphothioleates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNA).

[0092] The “sequence identity percentage” is determined by comparing two optimally aligned sequences across a comparison window, where a portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) for optimal alignment of the two sequences compared with a reference sequence (which does not include additions or deletions). The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in the two sequences to calculate the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying by 100 to calculate the sequence identity percentage.

[0093] With respect to two or more nucleic acid or polypeptide sequences, the term "identical" or "identical" percentage refers to two or more sequences or subsequences having a specific percentage of identical or identical amino acid residues or nucleotides (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity, measured by using one of the following sequence comparison algorithms or by manual alignment and visual inspection when compared and aligned by maximum correspondence across a comparison window or a designated region) with respect to the entire polypeptide sequence of the present invention or a specific region of an individual domain of the polypeptide of the present invention. In this case, such sequences are referred to as "substantially identical." This definition also refers to the complement of the test sequence. Optionally, the identity exists over a region of at least about 50 nucleotide lengths, or more preferably over a region of 100 to 500 or 1000 or more nucleotide lengths. The present invention comprises a polypeptide substantially identical to any of SEQ ID NOs 1 to 35.

[0094] For sequence comparison, typically one sequence serves as the reference sequence against which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Default program parameters may be used, or alternative parameters may be specified. Subsequently, the sequence comparison algorithm calculates the percentage of sequence identity for the test sequence against the reference sequence based on the program parameters.

[0095] The “comparison window” as used herein comprises a reference to any one segment of a whole sequence or a number of adjacent positions selected from a group consisting of 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides, wherein the sequence may be compared with a reference sequence of the same number of adjacent positions after optimally aligning two sequences. Optimal alignment of sequences for comparison is widely known in the art. Optimal alignment of sequences for comparison is, for example, described in the literature (Smith and Waterman (1970) Adv. Appl. Math. By the local homology algorithm of 2:482c), the literature (Needleman and Wunsch (1970) J. Mol. Biol. By the homology sorting algorithm of 48:443), the literature (Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA By similarity search methods of 85:2444), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA, Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (e.g., literature (Ausubel et al ., Current Protocols in Molecular Biology This can be done by (see 1995 supplement).

[0096] Examples of algorithms suitable for determining sequence identity percentage and sequence similarity are respectively from the literature (Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol These are the BLAST and BLAST 2.0 algorithms described in *. 215:403-410*. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in a query sequence that match or satisfy some positive threshold score T when aligned with words of the same length in the database sequence. T is referred to as the neighbor word score threshold (Altschul et al. , supraThese initial adjacent word hits initiate a search and act as seeds to find longer HSPs containing them. Word hits expand in both directions along each sequence as long as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for unsuitable residues; always < 0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. The expansion of word hits in each direction stops in the following cases: the cumulative alignment score drops from the maximum achieved value to quantity X; the cumulative score becomes zero or less due to the accumulation of one or more negative scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of alignment. (For nucleotide sequences) The BLASTN program uses by default a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and two-strand comparison. For amino acid sequences, the BLASTP program uses by default a word length of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915), alignment (B) of 50, an expectation (E) of 10, M=5, N=-4, and two-strand comparison.

[0097] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (e.g., literature (Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA(See 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences occurs by chance. For example, if the smallest sum probability in a comparison between a test nucleic acid and a reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001, the nucleic acid is considered similar to the reference sequence.

[0098] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the antibody produced against the polypeptide encoded by the second nucleic acid, as described below. Thus, the polypeptide is typically substantially identical to the second polypeptide, which differs, for example, only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize with each other under strict conditions, as described below. Another indication that two nucleic acid sequences are substantially identical is that the sequences can be amplified using the same primer.

[0099] With respect to genes, the expressions "expressed" or "expressed" as used herein refer to the transcription and / or decoding products of said gene. The expression level of a DNA molecule in a cell may be determined based on the amount of corresponding mRNA present within the cell or the amount of protein encoded by DNA produced by the cell. The expression level of a non-coding nucleic acid molecule (e.g., siRNA) may be detected by standard PCR or Northern blot methods widely known in the art. Literature (Sambrook et al ., 1989 Molecular Cloning: A Laboratory Manual Refer to , 18.1-18.88).

[0100] The expression of a transfected gene can occur transiently or stably in cells. During "transient expression," the transfected gene is not passed on to daughter cells during cell division. Because its expression is limited to the transfected cell, gene expression is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a screening advantage to the transfected cell. This screening advantage may be resistance to specific toxins provided to the cell. Expression of the transfected gene can be further achieved by transposon-mediated insertion into the host genome. During transposon-mediated insertion, the gene is positioned in a predictable manner between two transposon linker sequences that allow for insertion into the host genome as well as subsequent cleavage. Stable expression of the transfected gene can also be achieved by infecting cells with a lentiviral vector, which forms part of the cell genome after infection (integrating into the cell genome) and results in stable expression of the gene.

[0101] The terms "plasmid," "vector," or "expression vector" refer to a nucleic acid molecule encoding a gene and / or a regulatory element necessary for the expression of the gene. The expression of a gene from a plasmid can occur in a cis or trans form. When a gene is expressed in a cis form, the gene and the regulatory element are encoded by the same plasmid. Expression in a trans form refers to the case where the gene and the regulatory element are encoded by separate plasmids.

[0102] The terms “transfection,” “transduction,” “transfecting,” or “transduction” may be used interchangeably and are defined as the process of introducing nucleic acid molecules or proteins into a cell. Nucleic acids are introduced into cells using non-viral or virus-based methods. Nucleic acid molecules may be gene sequences encoding a complete protein or a functional part thereof. Non-viral methods of transfection include any suitable transfection method that does not use viral DNA or viral particles as a delivery system to introduce nucleic acid molecules into cells. Exemplary non-viral transfection methods include calcium phosphate transfection, liposome transfection, nucleofection, sonophoration, transfection via heat shock, magnetifection, and electroporation. In some embodiments, nucleic acid molecules are introduced into cells using electroporation according to standard procedures widely known in the art. For virus-based transfection methods, any useful viral vector may be used in the methods described herein. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, lentiviruses, and adeno-associated viral vectors. In some embodiments, nucleic acid molecules are introduced into cells using retroviral vectors according to standard procedures widely known in the art. The terms "transfection" or "transduction" refer to the introduction of a protein into a cell from the external environment. Typically, the transduction or transfection of a protein relies on the attachment of a peptide or protein capable of passing through the cell membrane to the protein of interest. For example, the literature (Ford et al. (2001) Gene Therapy 8:1-4 and Prochiantz (2007) Nat. Methods Refer to 4:119-20).

[0103] "Antibody" refers to a polypeptide comprising a framework region from an immunoglobulin gene or a fragment thereof that specifically binds to and recognizes an antigen. The recognized immunoglobulin gene includes kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. The light chain is classified as kappa or lambda. The heavy chain is classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. Typically, the antigen-binding region of an antibody plays a significant role in determining the specificity and affinity of the binding. In some embodiments, the antibody or a fragment of an antibody may be derived from different organisms, including humans, mice, rats, hamsters, camels, etc. The antibody of the present invention may include an antibody modified or mutated at one or more amino acid positions to improve or regulate the desired function of the antibody (e.g., glycosylation, expression, antigen recognition, effector function, antigen binding, specificity, etc.).

[0104] Antibodies are large, complex molecules with a complex internal structure (molecular weight of ~150,000 Da or about 1,320 amino acids). Natural antibody molecules contain two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain. Each light chain and heavy chain, in turn, consists of two regions: a variable ("V") region involved in binding to a target antigen and a constant ("C") region that interacts with other components of the immune system. The light and heavy chain variable regions assemble in three dimensions to form a variable region that binds to an antigen (e.g., a receptor on the cell surface). Within each light or heavy chain variable region, there are three short fragments (averaging 10 amino acids in length) called complementarity determining regions ("CDRs"). The six CDRs of the antibody variable domain (three from the light chain and three from the heavy chain) fold together in three dimensions to form the actual antibody binding site (paratope) that docks with the target antigen (epitope). The location and length of the CDR are from the literature (Kabat, E. et al. It was precisely defined by Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1983, 1987). The variable region not contained in the CDR refers to the framework ("FR") that forms the environment of the CDR.

[0105] "Antibody variants" provided herein refer to polypeptides capable of binding to an antigen and comprising one or more structural domains of an antibody or a fragment thereof (e.g., light chain variable domain, heavy chain variable domain). Non-limiting examples of antibody variants include single-domain antibodies or nanobodies, monospecific Fab2, bispecific Fab2, trispecific Fab3, monovalent IgG, scFv, bispecific diabodies, trispecific triabodidies, scFv-Fc, minibodies, IgNAR, V-NAR, hcIgG, VhH, or peptibodidies. "Peptibodidies" provided herein refer to peptide moieties attached to the Fc domain of an antibody (via covalent or non-covalent linkers). Further non-limiting examples of antibody variants known in the art include antibodies produced by cartilaginous fish or camels. A general description of antibodies from camels, their variable regions, and methods of their production, isolation, and use can be found in WO97 / 49805 and WO97 / 49805, which are incorporated herein by reference in their entirety for all purposes. Likewise, antibodies from cartilaginous fish, their variable regions, and methods of their production, isolation, and use can be found in WO2005 / 118629, which is incorporated herein by reference in its entirety for all purposes.

[0106] The terms "CDR L1," "CDR L2," and "CDR L3" provided herein refer to complementarity determining regions (CDRs) 1, 2, and 3 of the variable light chain (L) of the antibody. In the embodiments, the variable light chain provided herein comprises CDR L1, CDR L2, and CDR L3 in the direction from the N-terminus to the C-terminus. Likewise, the terms "CDR H1," "CDR H2," and "CDR H3" provided herein refer to complementarity determining regions (CDRs) 1, 2, and 3 of the variable heavy chain (H) of the antibody. In the embodiments, the variable heavy chain provided herein comprises CDR H1, CDR H2, and CDR H3 in the direction from the N-terminus to the C-terminus.

[0107] The terms "FR L1," "FR L2," "FR L3," and "FR L4" provided herein are used in accordance with their common meaning in the art and refer to framework regions (FR) 1, 2, 3, and 4 of the variable light chain (L) of the antibody. In the embodiments, the variable light chain provided herein comprises FR L1, FR L2, FR L3, and FR L4 in the direction from the N-terminus to the C-terminus. Likewise, the terms "FR H1," "FR H2," "FR H3," and "FR H4" provided herein are used in accordance with their common meaning in the art and refer to framework regions (FR) 1, 2, 3, and 4 of the variable heavy chain (H) of the antibody. In the embodiments, the variable heavy chain provided herein comprises FR H1, FR H2, FR H3, and FR H4 in the direction from the N-terminus to the C-terminus.

[0108] The term "antibody" is used according to the meaning generally known in the art. Antibodies exist as a number of well-characterized fragments produced by degradation, for example, into intact immunoglobulins or various peptidases. Thus, for example, pepsin degrades the antibody under the disulfide bond of the hinge region, and V by means of the disulfide bond H -C H1It generates F(ab)'2, a dimer of Fab, the light chain bound to it. F(ab)'2 can be reduced under mild conditions to convert the F(ab)'2 dimer into a Fab' monomer by breaking the disulfide bond at the hinge region. The Fab' monomer is essentially a Fab possessing part of the hinge region (see reference (Fundamental Immunology (Paul ed., 3rd ed. 1993)). While various antibody fragments are defined in terms of the degradation of an intact antibody, those skilled in the art will understand that such fragments can be de novo synthesized chemically or using recombinant DNA methodologies. Accordingly, the term antibody as used herein also includes antibody fragments produced by modification of a whole antibody, those synthesized using recombinant DNA methodologies (e.g., single-stranded Fv), or those identified using a phage display library (e.g., reference (McCafferty et al. , Nature See 348:552-554 (1990)).

[0109] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light chain" (about 25 kD) and one "heavy chain" (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The terms variable light chain (VL) or light chain variable region and variable heavy chain (VH) or heavy chain variable region refer to these light chain and heavy chain regions, respectively. The terms variable light chain (VL) and light chain variable region as mentioned herein may be used interchangeably. The terms variable heavy chain (VH) and heavy chain variable region as mentioned herein may be used interchangeably. The Fc (i.e., fragment crystallizable region) is the "base" or "tail" of the immunoglobulin and typically consists of two heavy chains that contribute to two or three constant domains, depending on the class of antibody. By binding to specific proteins, the Fc region ensures that each antibody generates an appropriate immune response to a given antigen. The Fc region also binds to various cellular receptors, such as Fc receptors, and other immune molecules, such as complement proteins.

[0110] The term “antigen” as provided herein refers to a molecule capable of binding to the antibody binding domain provided herein. The “antigen binding domain” as provided herein is a region of an antibody that binds to an antigen (epitope). As previously described, the antigen binding domain generally consists of one constant domain and one variable domain of each of the heavy chain and light chain (CH, CL, VH, and VL, respectively). A paratope or antigen binding site is formed at the N-terminus of the antigen binding domain. The two variable domains of the antigen binding domain typically bind to an epitope on the antigen.

[0111] Antibodies exist as numerous well-characterized fragments, for example, by being degraded into intact immunoglobulins or by various peptidases. Thus, for example, pepsin degrades the antibody under the disulfide bond of the hinge region to produce F(ab)'2, a dimer of Fab, a light chain bound to VH-CH1 by a disulfide bond. F(ab)'2 can be reduced under mild conditions to break the disulfide bond at the hinge region, converting the F(ab)'2 dimer into a Fab' monomer. The Fab' monomer is essentially an antigen-binding portion having part of the hinge region (see reference (Fundamental Immunology (Paul ed., 3rd ed. 1993)). While various antibody fragments are defined in terms of the degradation of intact antibodies, those skilled in the art will understand that these fragments can be de novo synthesized chemically or using recombinant DNA methodologies. Accordingly, as used herein, the term antibody also includes antibody fragments generated by modification of whole antibodies, or those synthesized using recombinant DNA methodologies (e.g., single-stranded Fv) or those identified using phage display libraries (e.g., literature (McCafferty et al. , Nature See 348:552-554 (1990)).

[0112] A single-strand variable fragment (scFv) is a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin, typically connected by a short linker peptide of 10 to about 25 amino acids. The linker is generally glycine-rich for flexibility and may be serine or threonine-rich for solubility. The linker may connect the N-terminus of the VH to the C-terminus of the VL or vice versa.

[0113] The epitope of an mAb is the region of the antigen to which the mAb binds. Two antibodies bind to the same or overlapping epitopes when each competitively inhibits (blocks) the binding of the other antibody to the antigen. That is, one antibody exceeding 1x, 5x, 10x, 20x, or 100x inhibits the binding of the other antibody by at least 30%, preferably 50%, 75%, 90%, or even 99%, as measured in a competitive binding assay (e.g., literature (Junghans) et al ., see Cancer Res. 50:1495, 1990). Alternatively, if essentially all amino acid mutations of an antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of another antibody, the two antibodies have the same epitope. If some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of another antibody, the two antibodies have overlapping epitopes.

[0114] For the preparation of the present invention and antibodies suitable for use according to the present invention, such as recombinant, monoclonal, or polyclonal antibodies, many techniques known in the art may be utilized (e.g., see Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986)). Genes encoding the heavy and light chains of the antibody of interest can be cloned from cells; for example, genes encoding monoclonal antibodies can be cloned from hybridomas and used to generate recombinant monoclonal antibodies. Gene libraries encoding the heavy and light chains of monoclonal antibodies can also be prepared from hybridomas or plasma cells. Random combinations of heavy and light chain gene products generate multiple antibody pools with different antigen specificities (see, for example, Kuby, Immunology (3rd ed. 1997)). The generation techniques of single-strand antibodies or recombinant antibodies (US Patent 4,946,778, US Patent No. 4,816,567) can be modified to generate antibodies against the polypeptide of the present invention. Additionally, other organisms, such as transgenic mice or other mammals, can be used to express humanized or human antibodies (see, for example, US Patent Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively, phage display technology can be used to identify antibodies and heterologous Fab fragments that specifically bind to selected antigens (e.g., see literature (McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992))). Antibodies can also be bispecific, meaning they can recognize two different antigens (e.g., see literature (WO 93 / 08829, Traunecker et al., EMBO J. 10:3655-3659 (1991); and Suresh et al., Methods in Enzymology 121:210 (1986))). The antibody may also be a heterozygous, for example, two covalently bonded antibodies or an immunotoxin (see, for example, US Patent No. 4,676,980, WO 91 / 00360; WO 92 / 200373; and EP 03089).

[0115] Methods for humanizing or primating non-human antibodies are widely known in the art (see, for example, literature (US Patent Nos. 4,816,567; 5,530,101; 5,859,205; 5,585,089; 5,693,761; 5,693,762; 5,777,085; 6,180,370; 6,210,671; and 6,329,511; WO 87 / 02671; EP Patent Application 0173494; Jones et al. (1986) Nature 321:522; and Verhoyen et al. (1988) Science 239:1534)). Humanized antibodies are further described, for example, in the literature (Winter and Milstein (1991) Nature 349: 293). Generally, humanized antibodies have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as import residues, which are typically taken from the import variable domain. Humanization is essentially achieved by substituting rodent CDRs or CDR sequences with the corresponding sequences of human antibodies, in accordance with the methods of Winter and his collaborators (e.g., literature (Morrison et al., PNAS USA, 81:6851-6855 (1984); Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyen et al., Science 239:1534-1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992), Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun., It can be performed according to 31(3):169-217 (1994)).Accordingly, such humanized antibodies are chimeric antibodies (US Patent No. 4,816,567), in which substantially less than the intact human variable domain is replaced with corresponding sequences from non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted with residues from similar sites of rodent antibodies. For example, a polynucleotide comprising a first sequence encoding a humanized immunoglobulin framework region and a second set of sequences encoding a desired immunoglobulin complementarity determining region can be generated synthetically or by combining appropriate cDNA and genomic DNA fragments. The human constant region DNA sequence can be isolated from various human cells according to known procedures.

[0116] A “chimeric antibody” is (a) an antibody molecule in which the constant region or a part thereof is modified, replaced, or exchanged so that the antigen-binding site (variable region) is linked to a different or modified class of constant region, effector function and / or species, or a completely different molecule, such as an enzyme, toxin, hormone, growth factor, drug, etc., which confers new characteristics to the chimeric antibody; or (b) an antibody molecule in which the variable region or a part thereof is modified, replaced, or exchanged with a variable region having different or modified antigen specificity. Antibodies preferred for use according to the present invention include humanized and / or chimeric monoclonal antibodies.

[0117] The term “therapeutic antibody” as provided herein refers to any antibody or functional fragment thereof used to treat cancer, autoimmune diseases, transplant rejection, cardiovascular diseases, or other diseases or conditions such as those described herein. Non-limiting examples of therapeutic antibodies include Erbitux (cetuximab), Leopro (absiximab), Simulect (basiliximab), Remicade (infliximab); Ortoclon OKT3 (muromonab-CD3); It includes murine antibodies, murineized or humanized chimeric antibodies, or human antibodies, including but not limited to Rituxan (rituximab), Bexar (tocitumomab), Humira (adalimumab), Campath (alemtuzumab), Simulect (baciliximab), Avastin (bevacizumab), Simzia (sertolizumab pegol), Zenapax (daclizumab), Soliris (eculizumab), Raptiva (epalizumab), Milotarg (gemtuzumab), Zavelin (ibritumomab tiuxetane), Tysabri (natalizumab), Xolair (omalizumab), Synagis (palivizumab), Vectibix (panitumomab), Lucentis (ranivizumab), and Herceptin (trastuzumab).

[0118] The technique of conjugating therapeutic agents to antibodies is widely known (e.g., literature (Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery" in Controlled Drug Delivery (2 ndSee also Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review" in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982)). As used herein, the terms "antibody-drug conjugate" or "ADC" refer to a therapeutic agent conjugated to or otherwise covalently bonded to an antibody. The "therapeutic agent" mentioned herein is a composition useful for treating or preventing diseases such as cancer.

[0119] When referring to proteins or peptides, the phrases "specifically (or selectively) binding" or "specifically (or selectively) immunoresponsive" to antibodies often refer to the binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other biopharmaceuticals. Thus, under specified immunoassay conditions, a specific antibody binds to a specific protein at least twice the background level, and more typically 10 to 100 times the background level. Under these conditions, specific binding to an antibody typically requires an antibody selected for its specificity to a specific protein. For example, polyclonal antibodies may be selected to yield only a subset of antibodies that specifically immunoresponsive to a selected antigen rather than other proteins. This selection can be achieved by excluding antibodies that cross-react with other molecules. Various immunoassay methods may be used to select antibodies that specifically immunoresponsive to a specific protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that specifically respond to proteins (e.g., for a description of immunoassay methods and conditions that can be used to determine specific immunoreactivity, see the literature (Harlow & Lane, Using Antibodies, A Laboratory Manual (1998))).

[0120] "Ligand" refers to an agent capable of binding to a receptor, such as a polypeptide or other molecule.

[0121] The term "recombinant," when used in relation to, for example, cells, nucleic acids, proteins, or vectors, indicates that the cells, nucleic acids, proteins, or vectors have been modified by laboratory methods or are the result thereof. Thus, for example, a recombinant protein comprises a protein produced by laboratory methods. A recombinant protein may contain amino acid residues not found in the natural (non-recombinant) form of the protein, or may contain modified, for example, labeled amino acid residues.

[0122] When used in relation to parts of nucleic acids, the term "heterogeneous" indicates that the nucleic acid contains two or more subsequences that are not naturally found in the same relationship. For example, a novel functional nucleic acid, such as a nucleic acid having two or more sequences from unrelated genes arranged to form a promoter from one source and a coding region from another source, is typically produced recombinantly. Similarly, a heterogeneous protein indicates that a protein contains two or more subsequences that are not naturally found in the same relationship (e.g., a fusion protein).

[0123] The term "isolated," when applied to nucleic acids or proteins, indicates that the nucleic acid or protein is essentially free of other cellular components associated with it in its natural state. This may exist, for example, in a homogeneous state and may exist in dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. Proteins, which are the dominant species present in the formulation, are substantially purified.

[0124] "Contact" is used in its obvious, general, and ordinary sense and refers to a process in which at least two distinct species (e.g., chemical compounds including biomolecules or cells) come close enough to react, interact, or physically come into contact. However, the resulting reaction product may be produced from a reaction between the added reagents or directly from an intermediate from one or more added reagents that may be produced in the reaction mixture.

[0125] The term “contact” may include causing two species to react, interact, or come into physical contact, wherein the two species may be, for example, the recombinant protein and the antigen-binding domain described herein. In an embodiment, contact includes, for example, causing the recombinant protein described herein to interact with the antigen-binding domain.

[0126] "Control" samples or values ​​refer to samples used as reference, generally known references, for comparison with test samples. For example, a test sample may be taken from test conditions, e.g., in the presence of a test compound, and may be compared with samples under known conditions, e.g., in the absence of a test compound (negative control) or in the presence of a known compound (positive control). Controls may also represent average values ​​collected from multiple tests or results. Those skilled in the art will recognize that controls may be designed for the evaluation of any number of parameters. For example, controls may be designed to compare therapeutic benefits based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of side effects). Those skilled in the art can understand which controls are useful in a given situation and analyze data based on comparisons with control values. Controls are also useful for determining the significance of data. For example, if the value of a given parameter varies widely in the control group, the variation in the test sample will not be considered significant.

[0127] “Patient” or “subject requiring treatment” refers to a living organism suffering from or susceptible to a disease or condition that can be treated by administration of the composition or pharmaceutical composition provided herein. Non-limiting examples include humans, other mammals, cattle, rats, mice, dogs, monkeys, goats, sheep, cattle, deer, and other non-mammalians. In some embodiments, the patient is a human.

[0128] The terms “disease” or “pathological condition” refer to the condition or health condition of a patient or subject that can be treated by the compounds, pharmaceutical compositions, or methods provided herein. In an embodiment, the disease is cancer (e.g., lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (e.g., Merkel cell carcinoma), testicular cancer, leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma).

[0129] The term “treating” or “treatment” refers to any indication of success in the treatment or improvement of an injury, disease, pathology, or condition, including any objective or subjective parameters such as alleviation; remission; reduction of symptoms or enabling the patient to better tolerate the injury, pathology, or condition; delaying the rate of regression or decline; making the final point of regression less debilitating; or promoting the patient’s physical or mental well-being. Treatment or improvement of symptoms may be based on objective or subjective parameters, including the results of a physical examination, neuropsychiatric examination, and / or psychiatric evaluation. The term “treating” and its application include the prevention of an injury, pathology, condition, or disease. In an embodiment, “treating” refers to the treatment of cancer.

[0130] "Effective dose" is an amount of compound sufficient to achieve the stated purpose in relation to the absence of the compound (e.g., achieving the effect of the administered compound, treating a disease, reducing or increasing enzyme activity, reducing signaling pathways, or reducing one or more symptoms of a disease or pathological condition). An example of a "therapeutic effective dose" is an amount sufficient to contribute to the treatment, prevention, or reduction of the symptoms or signs of a disease, and may also be referred to as a "therapeutic effective dose." "Reduction" of symptoms or signs (and grammatical equivalents of this phrase) means a reduction in the severity or frequency of the symptom(s) or the elimination of the symptom(s). The exact amount will depend on the purpose of treatment and may be determined by a person skilled in the art using known techniques (e.g., literature (Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy , 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0131] As used herein, the terms “Her2 protein” or “Her2” include any recombinant or naturally occurring form of receptor tyrosine-protein kinase erbB-2, also known as CD340 (differentiation cluster 340), the primary oncogene Neu, Erbb2 (rodent), or ERBB2 (human), or variants or homologs thereof that retain Her2 activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to Her2). In some aspects, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 consecutive amino acid portion) compared to the naturally occurring Her2 protein. In an embodiment, the Her2 protein is substantially identical to the protein identified by UniProt reference number P04626 or a variant or homolog having substantial identity therewith.

[0132] As used herein, the terms “EGFR protein” or “EGFR” include any recombinant or naturally occurring form of the epidermal growth factor receptor (EGFR), also known as ErbB-1 or HER1 in humans, or a variant or homolog thereof that retains EGFR activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity relative to EGFR). In some aspects, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 consecutive amino acid portion) relative to the naturally occurring EGFR protein. In an embodiment, the EGFR protein is substantially identical to the protein identified by UniProt reference number P00533 or a variant or homolog having substantial identity therewith.

[0133] As used herein, the terms “CD19 protein” or “CD19” include the CD19 molecule (differentiation cluster 19), any recombinant or naturally occurring form of B-lymphocyte antigen CD19 also known as B-lymphocyte surface antigen B4, T-cell surface antigen Leu-12, and CVID3, or variants or homologs thereof that retain CD19 activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity relative to CD19). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 consecutive amino acid portion) relative to the naturally occurring CD19 protein. In an embodiment, the CD20 protein is substantially identical to the protein identified by UniProt reference number P15391 or a variant or homolog having substantial identity therewith.

[0134] As used herein, the terms “CD20 protein” or “CD20” include any recombinant or naturally occurring form of B-lymphocyte antigen CD20 or differentiation cluster 20 (CD20), or variants or homologs thereof that retain CD20 activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity relative to CD20). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 consecutive amino acid portion) relative to the naturally occurring CD20 protein. In an embodiment, the CD20 protein is substantially identical to the protein identified by UniProt reference number P11836 or a variant or homolog having substantial identity therewith.

[0135] Recombinant protein composition

[0136] In particular, the present invention provides a recombinant protein expressed by T cells that binds to an antigen-binding domain (e.g., an antibody, a variant, or a fragment thereof) and can target T cells to cells (e.g., tumor cells) expressing an antigen bound by the antigen-binding domain. Through the binding of the recombinant protein expressed by T cells to the antigen-binding domain and the binding of the antigen-binding domain to the antigen expressed by the target cells, the T cells are activated and cytotoxic, thereby eliminating the target cells (e.g., cancer cells). The recombinant protein provided the present invention enables the rapid addition of functionality, particularly to adoptive immunotherapy, and is particularly useful for a wide range of therapeutic and diagnostic purposes. For example, the recombinant protein provided the present invention, including embodiments thereof, can be used as a means to reduce off-target effects by inducing effector T cells (e.g., autologous T cells) and therapeutic antibodies to their sites of action. The compositions provided the present invention enable rapid and efficient modification of target specificity without generating and optimizing individual CAR T cells.

[0137] The recombinant protein provided herein is a continuous single-strand polypeptide comprising, for example, a non-CDR Fab-binding peptide domain, an intracellular T cell signaling domain, and a transmembrane domain that links the non-CDR Fab-binding peptide domain to the intracellular T cell signaling domain. The recombinant protein provided herein may include additional elements, for example, a spacer region, a peptide linker, and an intracellular co-stimulatory signaling domain, all of which form part of the continuous single-strand polypeptide. The continuous single-strand polypeptide provided herein refers to a polypeptide chain comprising elements that are covalently attached to each other to form a continuous polypeptide chain.

[0138] Accordingly, in one aspect, a recombinant protein is provided. The recombinant protein comprises (i) a non-CDR Fab-binding peptide domain; (ii) an intracellular T cell signaling domain; and (iii) a transmembrane domain connecting the non-CDR Fab-binding peptide domain to the intracellular T cell signaling domain. In an embodiment, the intracellular T cell signaling domain is a CD3ζ intracellular T cell signaling domain.

[0139] The “non-CDR Fab binding peptide domain” provided herein refers to a peptide or peptide domain comprising a peptide capable of binding to a non-CDR binding site of an antibody, antibody variant, or fragment thereof. In an embodiment, the non-CDR Fab binding peptide domain is a peptide. In an embodiment, the non-CDR Fab binding peptide domain comprises a peptide. In an embodiment, the non-CDR Fab binding peptide domain binds to a non-CDR binding site. In an embodiment, the non-CDR Fab binding peptide domain is a peptidyl moiety. In an embodiment, the peptidyl moiety is a moiety described in published U.S. application US20120301400 A1, which is incorporated by reference in its entirety for all purposes.

[0140] In an embodiment, the non-CDR Fab-binding peptide domain comprises a peptide moiety of the following sequence:

[0141] X0-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12 (I).

[0142] In sequence formula (I), X0 is Ser or null. X1 is Ser, Cys, Gly, β-alanine, diaminopropionic acid, β-azidoalanine or null. X2 is Gln or null. X3 is Phe, Tyr, β,β'-diphenyl-Ala, His, Asp, 2-bromo-L-phenylalanine, 3-bromo-L-phenylalanine, 4-bromo-L-phenylalanine, Asn, Gln, modified Phe, a hydrated carbonyl-containing residue, or a boronic acid-containing residue. X4 is Asp or Asn. X5 is Leu, β,β'-diphenyl-Ala, Phe, Trp, Tyr; a non-natural analog of phenylalanine, tryptophan or tyrosine, a hydrated carbonyl-containing residue, or a boronic acid-containing residue. X6 is Cys or Ser. X7 is Cys, Thr, or Ser. X8 is protected Arg, Arg, or Ala. X9 is Cys, Arg, or Ala. X10 is Leu, Gln, Glu, β,β'-diphenyl-Ala, Phe, Trp, Tyr; a non-natural analog of phenylalanine, tryptophan, or tyrosine, a hydrateable carbonyl-containing residue, or a boronic acid-containing residue. X11 is Cys, Gln, Lys, or Arg. X12 is Ser, Cys, Gly, 7-aminoheptanoic acid, β-alanine, diaminopropionic acid, propargylglycine, isoaspartic acid, or null. X1 and X12 are optionally linked together to form a cyclic peptide moiety.

[0143] In an embodiment, the non-CDR Fab-binding peptide domain comprises a peptide moiety of the following sequence:

[0144] X0-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12 (I).

[0145] In sequence formula (I), X0 is Ser or null. X1 is Ser, Cys, Gly, β-alanine, or null. X2 is Gln or null. X3 is Phe, Tyr, His, Asp, Asn, or Gln. X4 is Asp or Asn. X5 is Leu, Phe, Trp, Tyr, tryptophan, or tyrosine. X6 is Cys or Ser. X7 is Cys, Thr, or Ser. X8 is Arg or Ala. X9 is Cys, Arg, or Ala. X10 is Leu, Gln, Glu, Phe, Trp, Tyr, tryptophan, or tyrosine. X11 is Cys, Gln, Lys, or Arg. X12 is Ser, Cys, Gly, or null. X1 and X12 are arbitrarily linked together to form a cyclic peptide moiety.

[0146] In an embodiment, the non-CDR Fab binding peptide domain comprises the sequence of SEQ ID NO. 32. In an embodiment, the non-CDR Fab binding peptide domain has the sequence of SEQ ID NO. 32. In an embodiment, the recombinant protein further comprises a signaling peptide having the sequence of SEQ ID NO. 37. In an embodiment, the signaling peptide is bound to the N-terminus of the non-CDR Fab binding peptide domain.

[0147] The term “non-CDR binding site” as provided herein refers to a binding site of an antigen-binding domain (e.g., a Fab domain of an antibody, an antibody variant, or a fragment thereof) that does not contain CDR residues of the heavy chain or light chain of an antibody. “Non-CDR peptide binding site” is a region of an antigen-binding domain capable of non-covalently binding to a non-CDR Fab-binding peptide domain of a recombinant protein provided herein. In an embodiment, the non-CDR binding site comprises a backbone region amino acid residue. In an embodiment, the non-CDR binding site comprises an FR residue of the heavy chain or light chain. In an embodiment, the non-CDR binding site comprises FR residues of both the heavy chain and the light chain. In an embodiment, the non-CDR binding site comprises a residue at a position corresponding to Kavat position 83, a residue at a position corresponding to Kavat position 30, or a residue at a position corresponding to Kavat position 52. In an embodiment, the non-CDR binding site includes a residue at a position corresponding to Kavat position 40, a residue at a position corresponding to Kavat position 41, a residue at a position corresponding to Kavat position 30, a residue at a position corresponding to Kavat position 52, a residue at a position corresponding to Kavat position 83, or a residue at a position corresponding to Kavat position 85. In an embodiment, the non-CDR binding site includes a residue at a position corresponding to Kavat position 40. In an embodiment, the non-CDR binding site includes a residue at a position corresponding to Kavat position 41. In an embodiment, the non-CDR binding site includes a residue at a position corresponding to Kavat position 30. In an embodiment, the non-CDR binding site includes a residue at a position corresponding to Kavat position 52. In an embodiment, the non-CDR binding site includes a residue at a position corresponding to Kavat position 83. In an embodiment, the non-CDR binding site includes a residue at a position corresponding to Kavat position 85.In an embodiment, the residue forming the non-CDR binding site is the residue described in the published U.S. application US20120301400 A1, which is incorporated by reference for all purposes.

[0148] The non-CDR binding site provided herein may also be referred to as the "meditope binding site." In the embodiments, the binding protein that binds to the non-CDR binding site via the non-CDR Fab binding peptide domain does not affect (e.g., does not affect, measurably) the binding of the antigen binding domain to the epitope. In other words, in the embodiments, the non-CDR binding site does not affect antigen binding. In the embodiments, the non-CDR binding site interacts non-covalently with the non-CDR Fab binding peptide domain (e.g., meditope) of the recombinant protein provided herein, including its embodiments. The amino acid residue capable of interacting with the non-CDR Fab binding peptide domain (e.g., meditope) may form part of an antibody, Fab, antibody cariant, or any fragment thereof. The non-CDR binding site may be manipulated with any suitable antibody, variant, or fragment thereof to form an antigen binding domain (antigen binding domain) having the non-CDR binding site. The antigen-binding domain containing a non-CDR binding site is also referred to herein as "meditope-available antibody," "meditope-available domain," or "meditope-available antibody region."

[0149] The “antigen-binding domain” provided herein is a region of an antibody, variant, or fragment thereof that binds to an antigen (epitope). As described herein, the antigen-binding domain generally consists of one constant and one variable domain of the heavy chain and light chain, respectively (VL, VH, CL, and CH1). A paratope or antigen-binding site is formed at the N-terminus of the antigen-binding domain. The two variable domains of the antigen-binding domain typically bind to an epitope on the antigen. In an embodiment, the antigen-binding domain forms part of an antibody. In an embodiment, the antigen-binding domain forms part of a therapeutic antibody. In an embodiment, the antigen-binding domain forms part of a Fab. In an embodiment, the antigen-binding domain is a Fab.

[0150] In an embodiment, the antigen binding domain comprises a heavy chain constant region (CH) and a light chain constant region (CL). In an embodiment, the heavy chain constant region (CH) is the constant region of the heavy chain of the antibody or its fragment. In an embodiment, the light chain constant region (CL) is the constant region of the light chain of the antibody or its fragment. In an embodiment, the heavy chain constant region (CH) is the constant region of Fab. In an embodiment, the light chain constant region (CL) is the light chain constant region of Fab. In an embodiment, the heavy chain constant region (CH) is the constant region of the F(ab)'2 dimer. In an embodiment, the light chain constant region (CL) is the light chain constant region of the F(ab)'2 dimer. In an embodiment, the antigen binding domain comprises an Fc domain. In an embodiment, the antigen binding domain is a humanized antigen binding domain. In an embodiment, the antigen binding domain is a humanized mouse antigen binding domain.

[0151] In an embodiment, the antigen binding domain is a trastuzumab Meditope-available domain, a pertuzumab Meditope-available domain, an M5A Meditope-available domain, or a rituximab Meditope-available domain. In an embodiment, the antigen binding domain is a humanized rituximab Meditope-available domain.

[0152] In an embodiment, the antigen-binding domain provided herein, including embodiments thereof, competes for antigen binding with one or more or all CDRs (or CDRs having at least about 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the CDR) including heavy chain CDRs 1, 2, and / or 3 and / or light chain CDRs 1, 2, and / or 3 of one or more known antibodies, such as any commercially available antibody as listed below, specifically binds to the same antigen or epitope, and / or contains: avagovomab, absiximab, adalimumab, adecatumumab, alemtuzumab, altumomab, altumomab pentetate, anatumomab, anatumomab maphenatox, arcitumomab, atilizumab, basiliximab, Vectumomab, Ectumomab, Belimumab, Benralizumab, Bevacizumab, Brentuximab, Canakinumab, Capromab, Capromab Pendette, Catumaxomab, Sertolizumab, Clibatuzumab Tetraxetane, Daclizumab, Denosumab, Eculizumab, Edrecolomab, Epalizumab, Etalacizumab, Ertumaxomab, Panolesomab, Fbta05, Pontolizumab, Gemtuzumab, Girentuximab, Rolimumab, Ibritumomab, Igovomab, Infliximab, Ipilimumab, Rabetuzumab, Mepolizumab, Muromonab, Muromonab-CD3, Natalizumab, Mesitumumab, Nimotuzumab, Ofatumumab, Omalizumab, Oregovomab, Palibizumab, panitumumab, ranibizumab, rituximab, satumomab, sulesomab, ibritumomab, ibritumomab tiuxetta, tocilizumab, tocitumomab, trastuzumab, Trbs07, ustekinumab, bicilizumab, botumumab, zalutumomab, and / or brodalumab; and / or anlukinumab, bapineuzumab, dalotuzumab, dempizumab, ganitumab, inotuzumab, mabrilimumab, moxetumomab pasodotox, rillotumumab, cipalimumab, tanezumab, tralokinumab, tremelimumab, urelumab, antibody produced by hybridoma 10B5 (Edelson & Unanue, Curr Opin Immunol, 2000 Aug;12(4):425-31), B6H12.2 (abcam) or other anti-CD47 antibodies (Chao et al.,; Cell , 142, 699-713, September 3, 2010).

[0153] In an embodiment, the antigen-binding domain specifically binds to an antigen selected from the group consisting of: CA-125, glycoprotein (GP) IIb / IIIa receptor, TNF-alpha, CD52, TAG-72, carcinogen-embryo antigen (CEA), interleukin-6 receptor (IL-6R), IL-2, interleukin-2 receptor α-chain (CD25), CD22, B cell activating factor, interleukin-5 receptor (CD125), VEGF, VEGF-A, CD30, IL-1 beta, prostate-specific membrane antigen (PSMA), CD3, EpCAM, EGF receptor (EGFR), MUC1, human interleukin-2 receptor, Tac, RANK ligand, complement protein, e.g., C5, EpCAM, CD11a, e.g., human CD11a, integrin, e.g., alpha-v beta-3 integrin, probenectin receptor alpha-v beta-3 integrin, HER2, neu, CD3, CD15, CD20 (small loop and / or large loop), interferon gamma, CD33, CA-IX, TNF alpha, CTLA-4, carcinoembryonic antigen, IL-5, CD3 epsilon, CAM, alpha-4-integrin, IgE, e.g., IgE Fc region, RSV antigen, e.g., F protein of respiratory syncytial virus (RSV), TAG-72, NCA-90 (granulocyte antigen), IL-6, GD2, GD3, IL-12, IL-23, IL-17, CTAA16.88, IL13, interleukin-1 beta, beta-amyloid, IGF-1 receptor (IGF-1R), delta-like ligand 4 (DLL4), alpha subunit of granulocyte macrophage colony-stimulating factor receptor, hepatocyte growth factor, IFN-alpha, nerve growth factor, IL-13, CD326, programmed cell death 1 ligand 1 (PD-L1, aka CD274, B7-H1), CD47, and CD137.

[0154] In an embodiment, the antigen binding domain is anti-CD19 protein, anti-CD20 protein, anti-CD22 protein, anti-CD30 protein, anti-CD33 protein, anti-CD44v6 / 7 / 8 protein, anti-CD123 protein, anti-CEA protein, anti-EGP-2 protein, anti-EGP-40 protein, anti-erb-B2 protein, anti-erb-B2,3,4 protein, anti-FBP protein, anti-fetal acetylcholine receptor protein, anti-GD2 protein, anti-GD3 protein, anti-Her2 / neu protein, anti-IL-13R-a2 protein, anti-KDR protein, anti-k-light chain protein, anti-LeY protein, anti-L1 cell adhesion molecule protein, anti-MAGE-A1 protein, anti-mesothelin protein, anti-murin CMV-infected cell protein, anti-MUC2 protein, anti-NKGD2 protein, anti-tumor-fetal antigen protein, anti-PCSA protein, It is an anti-PSMA protein, an anti-TAA (targeted by mAb IfE) protein, an anti-EGFR protein, an anti-TAG-72 protein, or an anti-VEGF-72 protein. In the embodiments, the antigen-binding domain is not cetuximab.

[0155] Additionally, in addition to non-covalent binding to the non-CDR Fab-binding peptide domain, the antigen-binding domain may be modified (e.g., genetically or chemically) to include a therapeutic moiety or an imaging or detectable moiety. Accordingly, in the embodiments, the antigen-binding domain includes a therapeutic moiety or a detectable moiety.

[0156] The term “therapeutic moiety” as provided herein is used in its obvious general sense and refers to a monovalent compound having a therapeutic benefit (e.g., prevention, eradication, or alleviation of the underlying disorder being treated) when provided to a subject requiring treatment. The therapeutic moiety provided herein may include, without limitation, peptides, proteins, nucleic acids, nucleic acid analogs, small molecules, antibodies, nanobodies, enzymes, prodrugs, cytotoxic agents (e.g., toxins), including but not limited to lysine, dioxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenofoside, vincristine, vinblastine, colchicine, dihydroxyanthracindione, actinomycin D, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrine, and glucocorticoids. In embodiments, the therapeutic moiety is an anticancer agent or chemotherapy agent as described herein. In an embodiment, the therapeutic moiety is a nucleic acid moiety, a peptide moiety, or a small molecule drug moiety. In an embodiment, the therapeutic moiety is a nucleic acid moiety. In an embodiment, the therapeutic moiety is an antibody moiety. In an embodiment, the therapeutic moiety is a peptide moiety. In an embodiment, the therapeutic moiety is a small molecule drug moiety. In an embodiment, the therapeutic moiety is a nuclease. In an embodiment, the therapeutic moiety is an immunostimulant. In an embodiment, the therapeutic moiety is a toxin. In an embodiment, the therapeutic moiety is a nuclease. In an embodiment, the therapeutic moiety is a cytokine (e.g., IL-2). In an embodiment, the therapeutic moiety comprises a non-natural amino acid. In an embodiment, the therapeutic moiety comprises siRNA. In an embodiment, the therapeutic moiety is siRNA. In an embodiment, the therapeutic moiety comprises an antisense nucleic acid. In an embodiment, the therapeutic moiety is an antisense nucleic acid.

[0157] The “imaging or detectable moiety” provided herein is a monovalent compound detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. In an embodiment, the imaging moiety is covalently attached to a peptide compound. Exemplary imaging moiety is, without limitation, 32 P, radionuclides, positron-emitting isotopes, fluorescent dyes, fluorophores, antibodies, bioluminescent molecules, chemiluminescent molecules, photoactive molecules, metals, electron density reagents, enzymes (e.g., those commonly used in ELISA), magnetic contrast agents, quantum dots, nanoparticles, biotin, digoxigenin, haptens, and proteins or other entities that may be made detectable by introducing a radiolabel to a peptide or antibody that is specifically reactive to a target label, for example. Any method known in the art may be used to conjugate an antibody to a moiety, for example, using the method described in the literature (Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego). Exemplary fluorophores include fluorescein, rhodamine, GFP, coumarin, FITC, alexafluoride, Cy3, Cy5, BODIPY, and cyanine dyes. Exemplary radionuclides include fluorine-18, gallium-68, and copper-64. Exemplary magnetic contrast agents include gadolinium, iron oxide and iron platinum, and manganese. In an embodiment, the imaging moiety is a bioluminescent molecule. In an embodiment, the imaging moiety is a photoactive molecule. In an embodiment, the imaging moiety is a metal. In an embodiment, the imaging moiety is a nanoparticle.

[0158] The recombinant protein provided herein comprises a plurality of domains (e.g., intracellular T cell signaling domain, transmembrane domain, spacer region, intracellular co-stimulatory signaling domain), all of which form part of a single continuous single-strand polypeptide.

[0159] The “intracellular T cell signaling domain” provided herein comprises an amino acid sequence capable of providing primary signaling in response to the binding of an antigen to an antibody region provided herein, including embodiments thereof. In an embodiment, signaling of the intracellular T cell signaling domain results in the activation of T cells expressing it. In an embodiment, signaling of the intracellular T cell signaling domain results in the proliferation (cell division) of T cells expressing it. In an embodiment, signaling of the intracellular T cell signaling domain results in the expression of proteins known in the art (e.g., CTLA-4, PD-1, CD28, CD69) by said T cells as characteristics of activated T cells. In an embodiment, the intracellular T cell signaling domain comprises the signaling domain of the zeta chain of the human CD3 complex. In an embodiment, the intracellular T cell signaling domain is a CD3ζ intracellular T cell signaling domain.

[0160] In an embodiment, the intracellular T cell signaling domain comprises the sequence of SEQ ID NO. 34. In an embodiment, the intracellular T cell signaling domain is the sequence of SEQ ID NO. 34. In an embodiment, the intracellular T cell signaling domain comprises the sequence of SEQ ID NO. 11. In an embodiment, the intracellular T cell signaling domain is the sequence of SEQ ID NO. 11.

[0161] In the case of the recombinant protein provided herein, the transmembrane domain connects the non-CDR Fab-binding peptide domain to the intracellular T cell signaling domain. In an embodiment, the transmembrane domain is located between the non-CDR Fab-binding peptide domain and the intracellular T cell signaling domain. That is, the transmembrane domain is connected directly or indirectly (e.g., via a spacer) to the C-terminus of the non-CDR Fab-binding peptide domain and directly or indirectly (e.g., via a co-stimulatory signaling domain) to the N-terminus of the intracellular T cell signaling domain.

[0162] The “transmembrane domain” provided herein refers to a polypeptide that forms part of a biological membrane. The transmembrane domain provided herein may extend across a biological membrane (e.g., a cell membrane) from one side of the membrane through another side of the membrane. In an embodiment, the transmembrane domain extends from the intracellular side of the cell membrane to the extracellular side. The transmembrane domain may include non-polar hydrophobic residues which immobilize the protein provided herein, including its embodiments, to a biological membrane (e.g., the cell membrane of a T cell). Any transmembrane domain capable of immobilizing the protein provided herein, including its embodiments, is considered. In an embodiment, the transmembrane domain is L-selectin. The term “L-selectin” as provided herein includes a recombinant or naturally occurring form of the L-selectin protein also known as CD62L, or a variant or homolog thereof that retains L-selectin activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity relative to L-selectin). In the embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 consecutive amino acid portion) relative to the naturally occurring L-selectin polypeptide. In an example, L-selectin is a protein identified by NCBI sequence reference GI:262206315, its homologs, or functional fragments. Non-limiting examples of transmembrane domains include transmembrane domains of CD8α, CD4, or CD3-zeta. In an example, the transmembrane domain is a CD8α transmembrane domain, a CD28 transmembrane domain, a CD4 transmembrane domain, or a CD3-zeta transmembrane domain. In an example, the transmembrane domain is a CD28 transmembrane domain.

[0163] The term “CD28 transmembrane domain” as provided herein comprises a transmembrane domain of CD28 in any recombinant or naturally occurring form, or a variant or homolog thereof that retains the activity of the CD28 transmembrane domain (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the CD28 transmembrane domain). In some aspects, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 consecutive amino acid portion) compared to the naturally occurring CD28 transmembrane domain polypeptide. In an embodiment, the CD28 transmembrane domain is the protein identified by SEQ ID NO. 18 or SEQ ID NO. 2, its variant, homolog, or functional fragment. In an embodiment, CD28 is the protein identified by NCBI sequence reference GI:340545506, its homolog, or its functional fragment.

[0164] In an embodiment, the transmembrane domain is a protein domain identified by SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 18, its homologs, or a functional fragment. In an embodiment, the transmembrane domain comprises a protein domain identified by SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 18, its homologs, or a functional fragment.

[0165] Likewise, the term “CD8α transmembrane domain” as provided herein comprises any recombinant or naturally occurring form of the CD8α transmembrane domain, or its variants or homologs that retain the CD8α transmembrane domain activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the CD8α transmembrane domain). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 consecutive amino acid portion) compared to the naturally occurring CD8α transmembrane domain polypeptide.

[0166] The term “CD4 transmembrane domain” as provided herein includes any recombinant or naturally occurring transmembrane domain of CD4, or a variant or homolog thereof that retains the activity of the CD4 transmembrane domain (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the CD4 transmembrane domain). In some aspects, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 consecutive amino acid portion) compared to the naturally occurring CD4 transmembrane domain polypeptide.

[0167] The term “CD3-zeta transmembrane domain” as provided herein comprises any recombinant or naturally occurring form of the CD3-zeta transmembrane domain, or a variant or homolog thereof that retains the CD3-zeta transmembrane domain activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the CD3-zeta transmembrane domain). In some aspects, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 consecutive amino acid portion) compared to the naturally occurring CD3-zeta transmembrane domain polypeptide.

[0168] In an embodiment, the recombinant protein comprises a spacer region that connects a non-CDR Fab-binding peptide domain to a transmembrane domain. In an embodiment, the spacer region is located between the transmembrane domain and the non-CDR Fab-binding peptide domain. That is, the spacer region is connected directly or indirectly (e.g., via a peptide linker) to the C-terminus of the non-CDR Fab-binding peptide domain and directly or indirectly (e.g., via another peptide linker) to the N-terminus of the transmembrane domain. Accordingly, the recombinant protein provided herein may comprise a first peptide linker and a second peptide linker, wherein the first peptide linker connects the C-terminus of the non-CDR Fab-binding peptide domain to the N-terminus of the spacer region and the second peptide linker connects the C-terminus of the spacer region to the N-terminus of the transmembrane domain.

[0169] The “spacer region” provided herein is a polypeptide that connects a non-CDR Fab-binding peptide domain to a transmembrane domain. In an embodiment, the binding affinity of the non-CDR Fab-binding peptide domain to an antigen-binding domain (e.g., Fab) is increased compared to the absence of the spacer region. In an embodiment, the steric hindrance between the non-CDR Fab-binding peptide domain and the antigen-binding domain (e.g., Fab) is reduced in the presence of the spacer region.

[0170] In an embodiment, the spacer region comprises an Fc region. Examples of spacer regions considered for the compositions and methods provided herein include, but are not limited to, immunoglobulin molecules or fragments thereof (e.g., IgG1, IgG2, IgG3, IgG4) and immunoglobulin molecules or fragments thereof (e.g., IgG1, IgG2, IgG3, IgG4) comprising mutations affecting Fc receptor binding. In an embodiment, the spacer region is a fragment of IgG (e.g., IgG4), said fragment comprising a deletion of the CH2 domain. In an embodiment, the spacer region is a fragment of IgG (e.g., IgG4), said fragment comprising a CH3 region. In an embodiment, the spacer region is a CH3 region. In an embodiment, the spacer region comprises a CH3 region. In an embodiment, the spacer region is a CH2 region. In an embodiment, the spacer region comprises a CH2 region. The spacer region may be a peptide linker. In an embodiment, the spacer region is a serine-glycine linker. In an embodiment, the spacer region has the sequence GGSG. The spacer region includes the sequence GGSG. In an embodiment, the spacer region has the sequence GSGSGSGS (Sequence No. 24). In an embodiment, the spacer region includes the sequence GSGSGSGS (Sequence No. 24). In an embodiment, the spacer region is at least 4 amino acids long. In an embodiment, the spacer region is about 4 amino acids long. In an embodiment, the spacer region is 4 to 250 amino acids long. The spacer region may include residues capable of extending the in vivo (e.g., plasma) half-life of the protein provided herein. In an embodiment, the spacer region is 10 amino acids long. In an embodiment, the spacer region is 229 amino acids long. In an embodiment, the spacer region is GGGSSGGGSG (Sequence No. 31). In an embodiment, the spacer region includes the sequence GGGSSGGGSG (Sequence No. 31).The spacer region may be "fasylated." The term "fasylated" or "fasylated" is used in its ordinary sense and refers to an amino acid sequence that forms a highly soluble biological polymer due to a high content of proline, alanine, and serine. Accordingly, in the embodiments, the spacer region comprises about 200 combined proline, alanine, and serine residues. In the embodiments, the spacer region comprises about 10 to about 200 combined proline, alanine, and serine residues. In the embodiments, the spacer region comprises hydrophilic residues. In the embodiments, the recombinant protein does not comprise a spacer region.

[0171] In an embodiment, the spacer region comprises the sequence of SEQ ID NO. 33. In an embodiment, the spacer region has the sequence of SEQ ID NO. 33.

[0172] In an embodiment, the recombinant protein comprises a peptide linker that links a non-CDR Fab-binding peptide domain to a spacer region. In an embodiment, the peptide linker is located between the non-CDR Fab-binding peptide domain and the spacer region. That is, the peptide linker is connected directly or indirectly (e.g., through the peptide linker) to the C-terminus of the non-CDR Fab-binding peptide domain and directly or indirectly (e.g., through another peptide linker) to the N-terminus of the spacer region. The peptide linker provided herein (e.g., the first or second peptide linker) may be 5 to 50 amino acids long. In an embodiment, the peptide linker (e.g., the first or second peptide linker) is 5 to 45 amino acids long. In an embodiment, the peptide linker (e.g., the first or second peptide linker) is 5 to 40 amino acids long. In an embodiment, the peptide linker (e.g., the first or second peptide linker) is 5 to 35 amino acids long. In an embodiment, the peptide linker (e.g., the first or second peptide linker) is 5 to 30 amino acids long. In an embodiment, the peptide linker (e.g., the first or second peptide linker) is 5 to 25 amino acids long. In an embodiment, the peptide linker (e.g., the first or second peptide linker) is 5 to 20 amino acids long. In an embodiment, the peptide linker (e.g., the first or second peptide linker) is 5 to 15 amino acids long. In an embodiment, the peptide linker (e.g., the first or second peptide linker) is 5 to 10 amino acids long. In an embodiment, the peptide linker (e.g., the first or second peptide linker) is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids long.In an embodiment, the peptide linker (e.g., the first or second peptide linker) is 18 amino acids long. In an embodiment, the peptide linker has the sequence of SEQ ID NO. 25. In an embodiment, the peptide linker includes the sequence of SEQ ID NO. 25. In an embodiment, the peptide linker is the sequence SAPASSASAPSAASAPAG (SEQ ID NO. 26).

[0173] In an embodiment, the peptide linker is a first peptide linker, and the recombinant protein comprises a second peptide linker, wherein the second peptide linker connects the spacer region to the transmembrane domain. Thus, in an embodiment, the second peptide linker is located between the spacer region and the transmembrane domain. In other words, the first peptide linker is connected to the C-terminus of the non-CDR Fab-binding peptide domain and to the N-terminus of the spacer region; and the second peptide linker is connected to the C-terminus of the spacer region and to the N-terminus of the transmembrane domain.

[0174] In an embodiment, the recombinant protein provided herein comprises an intracellular co-stimulatory signaling domain that links a transmembrane domain to an intracellular T cell signaling domain. In an embodiment, the intracellular co-stimulatory signaling domain is located between the transmembrane domain and the intracellular T cell signaling domain. That is, the intracellular co-stimulatory signaling domain is directly or indirectly linked (e.g., via a peptide linker) to the C-terminus of the transmembrane domain and directly or indirectly linked (e.g., via another peptide linker) to the N-terminus of the intracellular T cell signaling domain.

[0175] The “intracellular co-stimulatory signaling domain” provided herein comprises an amino acid sequence capable of providing co-stimulatory signaling in response to the binding of an antigen to an antibody region provided herein, including embodiments thereof. In an embodiment, signaling of the co-stimulatory signaling domain results in the production of cytokines and the proliferation of T cells expressing them. In an embodiment, the intracellular co-stimulatory signaling domain is a CD28 intracellular co-stimulatory signaling domain, a 4-1BB intracellular co-stimulatory signaling domain, an ICOS intracellular co-stimulatory signaling domain, or an OX-40 intracellular co-stimulatory signaling domain. In an embodiment, the intracellular co-stimulatory signaling domain comprises a CD28 intracellular co-stimulatory signaling domain. In an embodiment, the intracellular co-stimulatory signaling domain is a CD28 intracellular co-stimulatory signaling domain. In an embodiment, the intracellular co-stimulatory signaling domain comprises a 4-1BB intracellular co-stimulatory signaling domain. In an embodiment, the intracellular co-stimulatory signaling domain is a 4-1BB intracellular co-stimulatory signaling domain. In an embodiment, the intracellular co-stimulation signaling domain is the CD28 intracellular co-stimulation signaling domain, the 4-1BB intracellular co-stimulation signaling domain, the ICOS intracellular co-stimulation signaling domain, the OX-40 intracellular co-stimulation signaling domain, or any combination thereof.

[0176] Exemplary intracellular co-stimulation signaling domains, including sequences and accession numbers, are listed in Table 2. In an embodiment, the intracellular co-stimulation signaling domain is SEQ NO. 12, SEQ NO. 13, SEQ NO. 14, SEQ NO. 15, or SEQ NO. 16. In an embodiment, the intracellular co-stimulation signaling domain comprises a protein identified as SEQ NO. 12, SEQ NO. 13, SEQ NO. 14, SEQ NO. 15, or SEQ NO. 16. In an embodiment, the intracellular co-stimulation signaling domain is SEQ NO. 13. In an embodiment, the intracellular co-stimulation signaling domain is SEQ NO. 14.

[0177] In an embodiment, the recombinant protein comprises a detectable domain bound to the C-terminus of an intracellular T cell signaling domain. In an embodiment, the detectable domain is a truncated CD19 (CD19t) domain. The term "CD19t" refers to a truncated CD19 protein that lacks intracellular signaling ability. As used herein, truncated CD19 is an inactive molecule that functions as a detectable domain for identifying T cells containing the recombinant protein provided herein. In an embodiment, the detectable domain comprises the sequence of SEQ ID NO. 22. In an embodiment, the detectable domain is the sequence of SEQ ID NO. 22.

[0178] In an embodiment, the recombinant protein comprises a self-cleaving peptidyl sequence that links an intracellular T cell signaling domain to a detectable domain. In an embodiment, the self-cleaving peptidyl linker sequence is a T2A sequence or a 2A sequence. In an embodiment, the self-cleaving peptidyl sequence is located between the intracellular T cell signaling domain and the detectable domain. That is, the self-cleaving peptidyl sequence is linked directly or indirectly (e.g., via a peptide linker) to the C-terminus of the intracellular T cell signaling domain and directly or indirectly (e.g., via another peptide linker) to the N-terminus of the detectable domain.

[0179] In an embodiment, the self-cleaving peptidyl linker has the sequence PVKQLLNFDLLKLAGDVESNPGP (Sequence No. 27). In an embodiment, the self-cleaving peptidyl linker has the sequence of the equine rhinitis A virus. In an embodiment, the self-cleaving peptidyl linker has the sequence QCTNYALLKLAGDVESNPGP (Sequence No. 28). In an embodiment, the self-cleaving peptidyl has the sequence of the porcine Tescovirus 1. In an embodiment, the self-cleaving peptidyl has the sequence ATNFSLLKQAGDVEENPGP (Sequence No. 29). In an embodiment, the self-cleaving peptidyl linker has the sequence of the Thosea asigna virus. In an embodiment, the self-cleaving peptidyl linker has the sequence EGRGSLLTCGDVESNPGP (Sequence No. 30). In an embodiment, the self-cleaving peptidyl linker has the sequence of SEQ ID NO. 21. In an embodiment, the self-cleaving peptidyl linker is the sequence of SEQ ID NO. 21.

[0180] In an embodiment, the recombinant protein forms part of a cell. In an embodiment, the recombinant protein forms part of a T cell. In an embodiment, the transmembrane domain forms part of the cell membrane of a T cell.

[0181] As described above, the recombinant protein provided herein may bind to an antigen-binding domain. Including embodiments thereof, elements of the recombinant protein provided herein (e.g., a non-CDR Fab-binding peptide domain, a transmembrane domain, an intracellular T cell signaling domain, an intracellular co-stimulatory signaling domain) are covalently bound to each other to form a continuous single-strand polypeptide, but the recombinant protein binds non-covalently to the antigen-binding domain. In an embodiment, the non-CDR Fab-binding peptide domain is bound to the antigen-binding domain. In an embodiment, the non-CDR Fab-binding peptide domain is non-covalently bound to the antigen-binding domain.

[0182] As described above, the antigen-binding domain may be a Fab, IgG, or bispecific antibody. In an embodiment, the antigen-binding domain is a cetuximab medtop-available domain, a trastuzumab medtop-available domain, a pertuzumab medtop-available domain, an M5A medtop-available domain, or a rituximab medtop-available domain. In an embodiment, the antigen-binding domain may bind to a cancer antigen. In an embodiment, the antigen-binding domain may bind to a cancer antigen. In an embodiment, the antigen-binding domain may bind non-covalently to a cancer antigen. In an embodiment, the cancer antigen is Her2, EGFR, CD19, or CD20. In an embodiment, the cancer antigen forms part of the cell. In an embodiment, the cancer antigen is expressed on the surface of the cell. In an embodiment, the cell is a cancer cell. In an embodiment, the cancer is ovarian cancer, renal cell carcinoma, B-cell malignancy, leukemia, lymphoma, breast cancer, colorectal cancer, prostate cancer, neuroblastoma, melanoma, medulloblastoma, lung cancer, osteosarcoma, glioblastoma, or glioma.

[0183] The composition provided herein may include a plurality of (i.e., more than one, at least two) recombinant proteins provided herein, including embodiments thereof. Where the composition includes more than one recombinant protein provided herein, including embodiments thereof, the recombinant protein is referred to herein as the first, second, third, fourth, etc. recombinant protein. Accordingly, elements forming part of the first, second, third, fourth, etc. recombinant protein are referred to herein as the first, second, third, or fourth non-CDR Fab-binding peptide domain, the first, second, third, or fourth intracellular T cell signaling domain, the first, second, third, or fourth transmembrane domain, the first, second, third, or fourth spacer region, or the first, second, third, or fourth intracellular co-stimulation signaling domain. Where the composition includes a plurality of recombinant proteins provided herein, including embodiments thereof, the recombinant proteins may be different or identical. In other words, the recombinant protein may include the same domain or different domains (e.g., non-CDR Fab binding peptide domain, intracellular T cell signaling domain, transmembrane domain, spacer domain, intracellular co-stimulation signaling domain) or may bind to the same or different antigen binding domains.

[0184] When the composition provided herein comprises a plurality of recombinant proteins provided herein, the recombinant proteins may be dimerized with one another through non-covalent bonding of their respective spacer regions. For example, the first recombinant protein may comprise a first CH3 domain that non-covalently binds to the second CH3 domain of the second recombinant protein. Accordingly, in an embodiment, the recombinant protein is the first recombinant protein, the non-CDR Fab-binding peptide domain is the first non-CDR Fab-binding peptide domain, the intracellular T cell signaling domain is the first intracellular T cell signaling domain, the transmembrane domain is the first transmembrane domain, the spacer region is the first spacer region, and the intracellular co-stimulation signaling domain is the first intracellular co-stimulation signaling domain.

[0185] In an embodiment, the first recombinant protein is non-covalently bound to the second recombinant protein, and the second recombinant protein comprises (i) a second non-CDR Fab-binding peptide domain; (ii) a second intracellular T cell signaling domain; (iii) a second transmembrane domain connecting the second non-CDR Fab-binding peptide domain to the second intracellular T cell signaling domain; and (iv) a second spacer region, wherein the second spacer region connects the second non-CDR Fab-binding peptide domain to the second transmembrane domain, and wherein the first spacer region is non-covalently bound to the second spacer region. In an embodiment, the first spacer region and the second spacer region are a first constant heavy chain 3 (CH3) domain and a second constant heavy chain 3 (CH3) domain.

[0186] In an embodiment, the first non-CDR Fab-binding peptide domain and the second non-CDR Fab-binding peptide domain are chemically different. In an embodiment, the first non-CDR Fab-binding peptide domain and the second non-CDR Fab-binding peptide domain are chemically identical.

[0187] In an embodiment, the first non-CDR Fab binding peptide domain is non-covalently bound to the first antigen binding domain. In an embodiment, the second non-CDR Fab binding peptide domain is non-covalently bound to the second antigen binding domain. In an embodiment, the first antigen binding domain and the second antigen binding domain are chemically different or identical. In an embodiment, the first antigen binding domain and the second antigen binding domain are independently the cetuximab Meditope-available domain, the trastuzumab Meditope-available domain, the pertuzumab Meditope-available domain, the M5A Meditope-available domain, or the rituximab Meditope-available domain.

[0188] In one embodiment, the recombinant protein comprises a non-CDR Fab binding peptide domain of sequence 32, a spacer region of sequence 33, a CD28 transmembrane domain of sequence 18, a CD28 intracellular co-stimulator signaling domain of sequence no. 13, a CD3ζ intracellular T cell signaling domain of sequence no. 34, an autocleavage peptidyl linker sequence of sequence no. 21, and a detectable domain of sequence no. 22.

[0189] In one embodiment, the recombinant protein comprises a non-CDR Fab binding peptide domain of sequence 32, a spacer region of sequence 33, a CD28 transmembrane domain of sequence 18, a 4-1BB intracellular co-stimulation signaling domain of sequence no. 14, a CD3ζ intracellular T cell signaling domain of sequence no. 34, an autocleavage peptidyl linker sequence of sequence no. 21, and a detectable domain of sequence no. 22.

[0190] In one embodiment, the recombinant protein comprises a non-CDR Fab binding peptide domain of sequence 32, a spacer region of sequence 33, a CD28 transmembrane domain of sequence 18, an intracellular co-stimulator signaling domain of sequence no. 13, and an intracellular T cell signaling domain of sequence no. 34.

[0191] In one embodiment, the recombinant protein comprises a non-CDR Fab binding peptide domain of sequence 32, a spacer region of sequence 33, a CD28 transmembrane domain of sequence 18, a 4-1BB intracellular co-stimulation signaling domain of sequence no. 14, and a CD3ζ intracellular T cell signaling domain of sequence no. 34.

[0192] In one embodiment, the recombinant protein comprises a non-CDR Fab binding peptide domain of SEQ ID NO. 32, a peptide linker of SEQ ID NO. 25, a spacer region of SEQ ID NO. 33, a CD28 transmembrane domain of SEQ ID NO. 18, a CD28 intracellular co-stimulation signaling domain of SEQ ID NO. 13, a CD3ζ intracellular T cell signaling domain of SEQ ID NO. 34, an autocleavage peptidyl linker sequence of SEQ ID NO. 21, and a detectable domain of SEQ ID NO. 22.

[0193] In one embodiment, the recombinant protein comprises a non-CDR Fab binding peptide domain of SEQ ID NO. 32, a peptide linker of SEQ ID NO. 25, a spacer region of SEQ ID NO. 33, a CD28 transmembrane domain of SEQ ID NO. 18, a 4-1BB intracellular co-stimulation signaling domain of SEQ ID NO. 14, a CD3ζ intracellular T cell signaling domain of SEQ ID NO. 34, an autocleavage peptidyl linker sequence of SEQ ID NO. 21, and a detectable domain of SEQ ID NO. 22.

[0194] In an embodiment, the recombinant protein comprises the signal peptide of SEQ ID NO. 37. In an embodiment, the signal peptide is bound to the N-terminus of a non-CDR Fab-binding peptide domain.

[0195] In one embodiment, the recombinant protein comprises a non-CDR Fab binding peptide domain of sequence 32, a peptide linker of sequence 25, a spacer region of sequence 33, a CD28 transmembrane domain of sequence no. 18, a CD28 intracellular co-stimulation signaling domain of sequence no. 13, and a CD3ζ intracellular T cell signaling domain of sequence no. 34.

[0196] In one embodiment, the recombinant protein comprises the non-CDR Fab binding peptide domain of SEQ ID NO. 32, the peptide linker of SEQ ID NO. 25, the spacer region of SEQ ID NO. 33, the CD28 transmembrane domain of SEQ ID NO. 18, the 4-1BB intracellular co-stimulation signaling domain of SEQ ID NO. 14, and the CD3ζ intracellular T cell signaling domain of SEQ ID NO. 34.

[0197] In one embodiment, the recombinant protein comprises the sequence of SEQ ID NO. 35. In one embodiment, the recombinant protein is the sequence of SEQ ID NO. 35. In one embodiment, the recombinant protein comprises the sequence of SEQ ID NO. 36. In one embodiment, the recombinant protein is the sequence of SEQ ID NO. 36.

[0198] In an embodiment, the recombinant protein comprises a non-CDR Fab-binding peptide domain from the N-terminus to the C-terminus, a spacer region, a CD28 transmembrane domain, a CD28 intracellular co-stimulation signaling domain, a CD3ζ intracellular T cell signaling domain, a self-cleaving peptidyl linker sequence, and a detectable domain.

[0199] In an embodiment, the recombinant protein comprises a non-CDR Fab-binding peptide domain from the N-terminus to the C-terminus, a spacer region, a CD28 transmembrane domain, a 4-1BB intracellular co-stimulation signaling domain, a CD3ζ intracellular T cell signaling domain, a self-cleaving peptidyl linker sequence, and a detectable domain.

[0200] In an embodiment, the recombinant protein comprises a non-CDR Fab-binding peptide domain, a spacer region, a CD28 transmembrane domain, a CD28 intracellular co-stimulation signaling domain, and a CD3ζ intracellular T cell signaling domain from the N-terminus to the C-terminus.

[0201] In an embodiment, the recombinant protein comprises a non-CDR Fab binding peptide domain from the N-terminus to the C-terminus, a spacer domain, a CD28 transmembrane domain, a 4-1BB intracellular co-stimulation signaling domain, and a CD3ζ intracellular T cell signaling domain.

[0202] In an embodiment, the recombinant protein comprises a non-CDR Fab-binding peptide domain from the N-terminus to the C-terminus, a peptide linker, a spacer region, a CD28 transmembrane domain, a CD28 intracellular co-stimulation signaling domain, a CD3ζ intracellular T cell signaling domain, a self-cleavage peptidyl linker sequence, and a detectable domain.

[0203] In an embodiment, the recombinant protein comprises a non-CDR Fab-binding peptide domain from the N-terminus to the C-terminus, a peptide linker, a spacer region, a CD28 transmembrane domain, a 4-1BB intracellular co-stimulation signaling domain, a CD3ζ intracellular T cell signaling domain, an autocleavage peptidyl linker sequence, and a detectable domain.

[0204] In an embodiment, the recombinant protein comprises a non-CDR Fab-binding peptide domain from the N-terminus to the C-terminus, a peptide linker, a spacer region, a CD28 transmembrane domain, a CD28 intracellular co-stimulation signaling domain, and a CD3ζ intracellular T cell signaling domain.

[0205] In an embodiment, the recombinant protein comprises a non-CDR Fab binding peptide domain from the N-terminus to the C-terminus, a peptide linker, a spacer region, a CD28 transmembrane domain, a 4-1BB intracellular co-stimulation signaling domain, and a CD3ζ intracellular T cell signaling domain. nucleic acid composition

[0206] Nucleic acids encoding a recombinant protein provided herein, including embodiments thereof, are provided herein. Accordingly, in one aspect, nucleic acids encoding a recombinant protein provided herein, including embodiments thereof, are provided. In another aspect, an expression vector comprising the nucleic acid provided herein, including embodiments thereof, is provided. In an embodiment, the expression vector is a lentivirus or an onco-retrovirus. In an embodiment, the expression vector is a lentivirus. In an embodiment, the expression vector is an onco-retrovirus.

[0207] Cell composition

[0208] The recombinant proteins and nucleic acids provided herein may form part of a cell (i.e., are contained in and / or expressed by the cell). Accordingly, in one aspect, T lymphocytes comprising an expression vector provided herein are provided, including embodiments thereof.

[0209] In another aspect, a T lymphocyte comprising the recombinant protein provided herein, including embodiments thereof, is provided. A portion of the recombinant protein provided herein may form part of the cell membrane of the cell in which it is expressed. A transmembrane domain may extend across the cell membrane of the T cell, for example, from one side of the membrane through the other side of the membrane. In an embodiment, the transmembrane domain extends from the intracellular side of the cell membrane to the extracellular side. Thus, the non-CDR Fab-binding peptide domain and the spacer region are located on the extracellular side of the cell membrane, and the intracellular T cell signaling domain is located on the intracellular side of the cell. In an embodiment, the transmembrane domain is within the cell membrane of the T lymphocyte. In an embodiment, the T lymphocyte is an autologous T lymphocyte. In an embodiment, the T lymphocyte is a heterologous T lymphocyte.

[0210] In an embodiment, the binding peptide domain on the CDR is then bound to an antigen binding domain. In an embodiment, the antigen binding domain is a Fab, IgG, or bispecific antibody. In an embodiment, the antigen binding domain is bound to a cancer antigen.

[0211] The antigen binding domain may be an antibody comprising a first Fab domain and a second Fab domain, and the recombinant protein may bind to a non-CDR binding site of the first Fab domain and the second Fab domain may bind to a cancer antigen. In an embodiment, the recombinant protein binds to a non-CDR binding site of the first Fab domain and the first Fab domain binds to a cancer antigen. In an embodiment, the recombinant protein binds to a non-CDR binding site of the first Fab domain, the first Fab domain binds to a first cancer antigen, and the second Fab domain binds to a second cancer antigen. In an embodiment, the first recombinant protein binds to a non-CDR binding site of the first Fab domain and the second recombinant protein binds to a non-CDR binding site of the second Fab domain. In a further embodiment, the first Fab domain binds to a first cancer antigen and the second Fab domain binds to a second cancer antigen.

[0212] In an embodiment, the cancer antigen is Her2, EGFR, CD19, or CD20. In an embodiment, the antigen binding domain is a cancer antigen binding domain. In an embodiment, the antigen binding domain is a cetuximab Meditope-available domain, a trastuzumab Meditope-available domain, a pertuzumab Meditope-available domain, an M5A Meditope-available domain, or a rituximab Meditope-available domain.

[0213] Treatment methods

[0214] The compositions provided herein, including embodiments thereof, are particularly useful for providing effective treatment for diseases such as cancer. Accordingly, in one aspect, a method for treating cancer is provided. The method comprises the step of administering to a subject requiring cancer treatment an effective amount of T lymphocytes provided herein, including embodiments thereof, and an effective amount of an antigen-binding domain capable of binding to a non-CDR Fab-binding peptide domain, wherein the antigen-binding domain is a cancer antigen-binding domain.

[0215] In an embodiment, T lymphocytes and antigen-binding domains are administered simultaneously or sequentially. In an embodiment, T lymphocytes and antigen-binding domains are administered simultaneously.

[0216] T lymphocytes and antigen-binding domains may be administered in combination (e.g., as a mixture), individually but simultaneously (e.g., via separate intravenous lines), or sequentially (e.g., one agent is administered first and then a second agent). Thus, the term combination is used to refer to the joint, simultaneous, or sequential administration of T lymphocytes and antigen-binding domains. In an embodiment, when T lymphocytes and antigen-binding domains are administered sequentially, the T lymphocytes are administered at a first time point and the antigen-binding domains are administered at a second time point, wherein the first time point precedes the second time point. The course of treatment is best determined individually based on the specific characteristics of the subject and the selected type of treatment. Treatments such as those disclosed herein may be administered to the subject daily, twice daily, every other week, monthly, or at any applicable standard that is therapeutically effective. Treatment may be administered alone or in combination with any other treatment disclosed herein or known in the art. Additional treatment may be administered simultaneously with the first treatment, at a different time, or according to a completely different treatment schedule (e.g., the first treatment may be administered daily, but the additional treatment is administered weekly). Accordingly, in the embodiments, T lymphocytes and antigen-binding domains are administered simultaneously or sequentially.

[0217] In an embodiment, T lymphocytes are administered at a first time point and an antigen-binding domain is administered at a second time point, wherein the first time point precedes the second time point. In an embodiment, the second time point is within about 120, 90, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 10, 11, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1 day from the first time point. In an embodiment, the second time point is within about 120 days from the first time point. In an embodiment, the second time point is within about 90 days from the first time point. In an embodiment, the second time point is within about 60 days from the first time point. In an embodiment, the second time point is within about 50 days from the first time point. In an embodiment, the second time point is within about 40 days from the first time point. In an embodiment, the second time point is within about 30 days from the first time point. In an embodiment, the second time point is within about 20 days from the first time point.

[0218] In an embodiment, the antigen-binding domain is administered at a first time point and the T lymphocyte is administered at a second time point, wherein the first time point precedes the second time point. In an embodiment, the second time point is within about 120, 90, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 10, 11, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1 day from the first time point. In an embodiment, the second time point is within about 120 days from the first time point. In an embodiment, the second time point is within about 90 days from the first time point. In an embodiment, the second time point is within about 60 days from the first time point. In an embodiment, the second time point is within about 50 days from the first time point. In an embodiment, the second time point is within about 40 days from the first time point. In an embodiment, the second time point is within about 30 days from the first time point. In an embodiment, the second time point is within about 20 days from the first time point.

[0219] In an embodiment, the T lymphocytes and the antigen-binding domain are mixed prior to administration. In an embodiment, the method comprises: (i) forming a T lymphocyte-recombinant protein complex by binding a non-CDR Fab-binding peptide domain to an antigen-binding domain in vitro prior to administration; and (ii) administering the T lymphocyte-recombinant protein complex to a subject to treat cancer in the subject.

[0220] In an embodiment, T lymphocytes and an antigen-binding domain are administered sequentially. In an embodiment, T lymphocytes are administered at a first time point and the antigen-binding domain is administered at a second time point, wherein the first time point precedes the second time point. In an embodiment, the antigen-binding domain is administered at a first time point and T lymphocytes are administered at a second time point, wherein the first time point precedes the second time point.

[0221] In an embodiment, the cancer is ovarian cancer, renal cell carcinoma, B-cell malignancy, leukemia, lymphoma, breast cancer, colorectal cancer, prostate cancer, neuroblastoma, melanoma, medulloblastoma, lung cancer, osteosarcoma, glioblastoma, or glioma. In an embodiment, the antigen-binding domain is a cetuximab Meditope-available domain, a trastuzumab Meditope-available domain, a pertuzumab Meditope-available domain, an M5A Meditope-available domain, or a rituximab Meditope-available domain.

[0222] As used herein, the term "cancer" refers to all types of cancer, neoplasms, or malignant tumors found in mammals, including leukemia, lymphoma, melanoma, neuroendocrine tumors, carcinomas, and sarcomas. Exemplary cancers that can be treated by the compounds, pharmaceutical compositions, or methods provided herein include lymphoma, sarcoma, bladder cancer, bone cancer, brain tumor, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, renal cancer, multiple myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer (e.g., triple-negative, ER-positive, ER-negative, chemotherapy-resistant, Herceptin-resistant, HER2-positive, dioxorubicin-resistant, tamoxifen-resistant, ductal carcinoma, lobular carcinoma, primary, metastatic), ovarian cancer, pancreatic cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., non-small cell lung carcinoma, squamous cell lung carcinoma, adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma, carcinoid, sarcoma), glioblastoma multiforme, glioma, melanoma, prostate cancer, castration-resistant prostate cancer, breast cancer, triple-negative breast cancer, glioblastoma, Ovarian cancer, lung cancer, squamous cell carcinoma (e.g., head, neck, or esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B-cell lymphoma, or multiple myeloma.Additional examples include thyroid, endocrine system, brain, chest, neck, colon, head & neck, esophagus, liver, kidney, lung, cancer of non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterine or medulloblastoma, Hodgkin's disease, non-Hodgkin lymphoma, multiple myeloma, neuroblastoma, glioma, pleomorphic glioblastoma, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumor, cancer, malignant pancreatic insulinoma, malignant carcinoid, urinary bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, neoplasm of endocrine or exocrine pancreas, medullary thyroid cancer, medullary carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, Hepatocellular carcinoma, Paget's disease of the papilla, phyllodes tumor, lobular carcinoma, ductal carcinoma, cancer of pancreatic stellate cells, cancer of hepatic stellate cells, or. Includes prostate cancer.

[0223] The term "leukemia" broadly refers to a progressive malignant disease of the blood-forming organs, generally characterized by the abnormal proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified based on (1) the duration and characteristics of the disease—acute or chronic; (2) the type of cells involved, myeloid (myeloplastic), lymphoid (lymphoid), or mononuclear; and (3) an increase or non-increase in the number of abnormal cells in the blood—leukemic or nonleukemic (subleukemic). Exemplary leukemias that can be treated by the compounds, pharmaceutical compositions, or methods provided herein are, for example, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, mature T-cell leukemia, aleukemic leukemia, leukocytic leukemia, basophilic leukemia, blastocyst leukemia, bovine leukemia, chronic myeloid leukemia, leukemia cutis, embryonic leukemia, eosinophilic leukemia, growth leukemia, blastocyst leukemia, hemoblastic leukemia, hemocytic leukemia, histocytic leukemia, stem cell leukemia, acute mononucleosis, leukopenic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphoplastic leukemia, lymphocytic leukemia, lymphosarcomatous leukemia, mast cell leukemia. Includes megakaryotic leukemia, micromyeloid leukemia, monocytic leukemia, myeloid leukemia, myeloid leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Negeli leukemia, plasma cell leukemia, multiple myeloma, plasma cell leukemia, promyelocytic leukemia, Lider cell leukemia, Schilling leukemia, stem cell leukemia, subleukemic leukemia, or undifferentiated cell leukemia.

[0224] The term "sarcoma" generally refers to a tumor composed of dense cells organized in a substance such as embryonic connective tissue and typically embedded in fibrous or homogeneous material. Sarcomas that can be treated by the compounds, pharmaceutical compositions, or methods provided herein are chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, liposarcoma, liposarcoma, alveolar soft tissue sarcoma, enamelblastosarcoma, staphylocorticum, chloromolecular sarcoma, sarcoma carcinoma, embryonic sarcoma, Wilms tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing sarcoma, fasciosarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, B-cell immunoblastosarcoma, lymphoma, T-cell immunoblastosarcoma, Jensen sarcoma, Kaposi sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymal sarcoma, periosteal sarcoma, reticular sarcoma, Rhoe's sarcoma, serous cyst sarcoma, synovial sarcoma, or Includes dilated osteosarcoma.

[0225] The term "melanoma" is considered to mean a tumor arising from melanocytes of the skin and other organs. Exemplary melanomas that can be treated by the compounds, pharmaceutical compositions, or methods provided herein include, for example, acral melanoma, melanin-deficient melanoma, benign pediatric melanoma, Cloudman melanoma, S91 melanoma, Harding-Passey melanoma, pediatric melanoma, malignant melanoma, malignant melanoma, nodular melanoma, subungual melanoma, or superficial diffuse melanoma.

[0226] The term "carcinoma" refers to a new malignant growth composed of epithelial cells that tends to invade surrounding tissues and cause metastasis. Exemplary carcinomas that can be treated by the compounds, pharmaceutical compositions, or methods provided herein are, for example, medullary carcinoma of the thyroid, familial medullary carcinoma of the thyroid, acinar carcinoma, acinar-like carcinoma, adenocystic carcinoma, adenocarcinoma, adrenocortical carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basal-like carcinoma, basosquamous cell carcinoma, bronchioloalveolar carcinoma, bronchiole carcinoma, bronchosquamous carcinoma, cerebello-like carcinoma, cholangiocarcinoma, choriocarcinoma, colloid carcinoma, comedonal carcinoma, uterine body carcinoma, trichoid carcinoma, armored carcinoma, cutaneum carcinoma, columnar carcinoma, columnar cell carcinoma, duct carcinoma, ductal carcinoma, hard carcinoma, embryonic carcinoma, encephaloid carcinoma, epidermal carcinoma, epithelial adeno-like Carcinoma, ex ulcere carcinoma, fibrous carcinoma, gelatinoid carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, adenocarcinoma, granulosa cell carcinoma, pubic stromal carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hürtle cell carcinoma, hyaline carcinoma, adrenoid carcinoma, infantile embryonic carcinoma, carcinoma in situ, carcinoma in situ, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lobular carcinoma, lymphoepithelial carcinoma, medullary carcinoma medullary carcinoma, melanoma, soft carcinoma,Mucinous carcinoma, carcinoma muciparum, mucinous cell carcinoma, mucoepidermoid carcinoma, mucosal carcinoma, mucinous carcinoma, mucinomatous carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid tumor, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, acanthic cell carcinoma, dendritic carcinoma, renal cell carcinoma of the kidney, preparatory cell carcinoma, sarcomatous carcinoma, Schneiderian carcinoma, hard carcinoma, scrotal carcinoma (carcinoma scroti), signet ring cell carcinoma, simple carcinoma, small cell carcinoma, solanoid carcinoma, spherical carcinoma, spindle cell carcinoma, cavernous osteoma, squamous carcinoma, squamous cell carcinoma, adenocarcinoma (string carcinoma), telangiectatic carcinoma (carcinoma Includes telangiectaticum), carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tubular carcinoma, tuberous carcinoma, carcinoma villosum, or carcinoma villosum.

[0227] As used herein, the terms “metastasis,” “metastatic,” and “metastatic cancer” may be used interchangeably and refer to the spread of a proliferative disease or disorder, such as cancer, from one organ or another non-adjacent organ or part of the body. Cancer originates at a site of origin, such as the breast, which is referred to as the primary tumor, such as primary breast cancer. Some cancer cells from the primary tumor or the site of origin acquire the ability to infiltrate and invade surrounding normal tissues at the local site and / or the ability to infiltrate the walls of the lymphatic or vascular systems circulating to other parts and tissues of the body through the system. A second clinically detectable tumor formed from cancer cells of the primary tumor is referred to as metastasis or a secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are presumed to be similar to the cells of the original tumor. Thus, when lung cancer metastasizes to the breast, the secondary tumor in the breast area consists of abnormal lung cells and not abnormal breast cells. The secondary tumor in the breast is referred to as metastatic lung cancer. Therefore, the term metastatic cancer refers to a disease in which the subject has or had a primary tumor and has one or more secondary tumors. The term non-metastatic cancer refers to a disease in which the subject has a primary tumor but does not have one or more secondary tumors. For example, metastatic lung cancer refers to a disease in which the subject has a history of a primary lung tumor and has one or more secondary tumors in a second or multiple locations, such as the breast.

[0228] "Anticancer agent" is used in its obvious general sense and refers to a composition (e.g., compound, drug, antagonist, inhibitor, modifier) ​​having antineoplastic properties or the ability to inhibit cell growth or proliferation. In an embodiment, the anticancer agent is a chemotherapy agent. In an embodiment, the anticancer agent is an agent identified herein that has utility in a method of treating cancer. In an embodiment, the anticancer agent is an agent approved by the FDA or a similar regulatory agency in a country other than the United States for the treatment of cancer.

[0229] In relation to a substance or substance activity or function associated with a disease (e.g., cancer (e.g., prostate cancer, kidney cancer, metastatic cancer, melanoma, castration-resistant prostate cancer, breast cancer, triple-negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma (e.g., head, neck, or esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B-cell lymphoma, or multiple myeloma)), the term “associated” or “associated with” means that the symptoms of the disease (e.g., lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (e.g., Merkel cell carcinoma), testicular cancer, leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma) or the disease are caused (wholly or partially) by the substance or substance activity or function.

[0230] "Chemotherapeutic" or "chemotherapeutic agent" is used in its obvious general sense and refers to a chemical composition or compound having antineoplastic properties or the ability to inhibit cell growth or proliferation. As used herein, the term "aberrant" refers to something different from normal. When used to describe enzyme activity, aberrant refers to activity that is greater or less than the mean of a normal control or a normal non-disease control sample. Aberrant activity may refer to a disease-causing amount of activity, wherein returning the aberrant activity to a normal or non-disease-associated amount (e.g., using the methods described herein) reduces the disease or one or more disease symptoms.

[0231] Examples

[0232] CAR T cells have demonstrated remarkable success in treating liquid tumors and are rapidly expanding their use to treat solid tumors and other diseases. Current approaches involve generating CAR T cells that target a single antigen. While effective in eliminating tumor cells expressing a specific antigen, tumor cells that do not express the antigen escape, can proliferate, and often become more aggressive. Therefore, to target these cells, it is necessary to generate entirely new CAR T cells that express different tumor-targeting scFvs. Here, the applicant addresses this problem using Meditope technology. Specifically, the applicant replaces the tumor-targeting scFv with an ultra-high affinity Meditope (non-CDR Fab-binding peptide domain). Tumor specificity is subsequently added using an antigen-specific and Meditope-available Fab or mAb (antigen-binding domain).

[0233] The applicant generates universal CAR T cells using Meditope interactions. Specifically, the applicant has replaced the antigen targeting region with Meditope (a non-CDR Fab-binding peptide domain), and has demonstrated that Meditope-available Fab / mAb can be added and can bind to an antigen specific to Fab. The advantage of the present invention is that the applicant can generate Meditope-zeta chain T cells and replace the Meditope-available Fab with one that is specific to the disease or covers multiple antigens.

[0234] The field of CAR T cells is rapidly advancing, and as clinical results become available, the need to modify the specificity of CAR T cells has been demonstrated. The applicant provides a universal CAR T cell platform in which the applicant replaces scFv within the CAR T cell with Meditope (e.g., a non-CDR Fab-binding peptide domain) and adds antigen specificity using a Meditope-available mAb / Fab (antigen-binding domain). Through this platform technology, the applicant can rapidly and efficiently modify target specificity without generating and optimizing individual CAR T cells. The applicant demonstrates a proof of concept through FACs using two different Meditope-available mAbs. The applicant will demonstrate efficacy in animal models by optimizing linker design and docking and modifying the antigen specificity of Meditope-CAR in situ.

[0235] Single T cells expressing Meditope can be mixed and matched with Fabs / Mabs targeting different antigens / epitopes within the tumor, potentially producing superior products applicable to multiple forms of cancer. Fabs / mAbs are generally more stable and have a higher affinity for antigens than scFvs, and Fab panels binding to different epitopes on antigens can be easily generated, enabling rapid optimization (e.g., a current issue in this field is how the distance between the receptor epitope and the tumor membrane affects efficacy).

[0236] The applicant can generate FabRack (a recombinant protein provided herein) by packing Meditope into a lentivirus and characterize tumor eradication in vitro and in vivo. The applicant can modify and characterize specificity in situ. The applicant can generate a monovalent FabRack and generate, characterize, and modify specificity by modifying a linker (e.g., remover CH3 domain).

[0237] Conventional CARs and Meditope CARs: Basic Concepts. scFv variants are used in clinics. The 'simplest' FabRack binds to a Meditope-available IgG or Fab fragment. It is important to note that this is not limited to IgG or Fab; we can create dual-specific IgG, bionics, or single-arm Fabs. Essentially, anything we could fuse to a Fab was Meditope-available.

[0238] Fabrack-T cells can bind to tumor cells when mediated by Meditope-available IgG. There are numerous combinations and interactions that can lead to productive interactions. One Fab arm binds to the Fabrack T cell and the other arm binds to the tumor cell. The same Fab arm binds to both the Fabrack T cell and the tumor antigen. Both Fab arms bind to the tumor and one or the other Fab arm binds to the Fabrack T cell. Both Fab arms bind to the Fabrack T cell and the tumor. Etc.

[0239] The Meditope CAR construct can be packaged in a lentiviral vector. The extracellular domain comprises Meditope (non-CDR Fab-binding peptide domain), a linker (peptide linker), and a CH3 domain of IgG (spacer region), followed by a CD28 transmembrane domain (CD28tm), a CD28 co-stimulatory domain (CD28), and a CD3ζ cytolytic domain (also referred to herein as the intracellular T cell signaling domain) (CD3ζ). CD3ζ and CD19t are separated by the T2A sequence. The memAb (Meditope-available monoclonal antibody) can bind to target cells and FabRack-expressing Jerkats possessing NFAT-responsive luciferase. This is simple information for testing the concept before migrating to T cells.

[0240] memAb (meditope-available monoclonal antibody; antigen-binding domain) can be pre-bound to FabRack cells (T cells expressing the recombinant protein provided herein) or memAb (antigen-binding domain) can be pre-bound to target cells (e.g., cancer cells). The level of antigen expression on the target cells affects T cell activation. T cell activation was similar between T cells with memAb pre-binding and cancer cells with memAb pre-binding when antigen expression on the cancer cells was low. The highest T cell activation can be achieved when cancer cells are pre-bound to memAb and antigen expression on the cancer is high. Likewise, at high levels of antigen expression on the cancer, T cell activation is high when T cells are pre-bound to memAb (meditope-available monoclonal antibody, antigen-binding domain). Refer to Fig. 19.

[0241] Materials and Methods

[0242] Cloning . parents of Anti-Her2 scFv_IgG4op(HL-CH3)_CD28gg_op-Zeta_op-T2A-CD19t_epHIV7 The vector was kindly provided by Dr. C. Brown. GM-CSFr secretion signal_meditope_PASlinker17 Synthesized gene cassettes by DNA2.0 and using NheI and SbfI restriction sites IgG4op(HL-CH3) The plasmids were inserted into CAR vectors with or without the domains (Meditope-CH3 and Meditope-CD28, respectively). The cloned plasmids were purified using the MaxiPrep kit (Qiagen).

[0243] Fluorescent labeling of soluble proteins Alexafluoride dye (ThermoFisher) was attached to soluble proteins using amine conjugation according to the manufacturer's protocol. Briefly, Alexafluoride 647 NHS ester dye was conjugated to trastuzumab I83E IgG and Fab and ipilimumab IgG. A 280 and A maxLabeling degree (DOL) of 1 ≤ DOL ≤ 3 dyes per molecule was calculated using [method name]. Pacific Blue NHS ester dyes were conjugated to sHer2. Protein interactions were characterized by size exclusion chromatography (SEC) prior to FACS assay to evaluate binding activity.

[0244] Transfection On Day 0, CHO-S cells (Invitrogen) from passage 9 were transfected without a vector (Mock) or with the parental CAR, Meditope-CH3, or Meditope-CD28 vectors. Cells were transfected using the Freestyle MAX Transfection Kit (ThermoFisher) according to the manufacturer's protocol.

[0245] Flow cytometryOn day 5, cells were collected and counted. 3E6 cells were added to 5 mL FACS tubes (VWR) at a rate of 1E6 cells / mL with QS FACS staining solution (2% FCS, 0.5% NaN3 in Hanks equilibrium salt solution, batch #05092016). 0.1E6 cells were added to each well of a V-bottom 96-well plate (Corning Costar), with 3 wells per condition. Cells were washed twice with 100 μL of staining solution, rotated at 300 g for 3 minutes at 4°C, and the supernatant was decanted. Cells were resuspended in 100 μL of primary staining solution (PE-Cy7^CD-19 diluted 1:100 with 100 nM 647^IgG, 100 nM 647^Ipi, or 200 nM 647^Fab) for 30 minutes at 4°C in a light-protected environment. Cells were washed twice with 100 μL of staining solution and resuspended in secondary staining solution (200 nM PacBlue^Her2) for 30 minutes at 4°C in a light-protected environment. Cells were washed twice with 100 μL of staining solution and resuspended in 150 μL of PI solution (PI diluted 1:100 in the staining solution). Cell samples were analyzed using a MACSQuant instrument (#2, West side of Brown lab) with 40 μL per sample. Voltages were as follows: FSC = 358 V, SSC = 520 V. Analysis channels: PE(PI), PE-Cy7(CD19), APC(647), and VioBlue(Her2). Gating strategy:

[0246] FSC / SSC → PE - → PE-Cy7 + → APC + → VioBlue +

[0247] FSC / SSC → PI - → CD19 + → 647 + → Her2 +

[0248] Animal plan. Each group has 4 mice.

[0249] 1. Tumor Erythrones OVCAR3-luc or SKOV3-luc is superior.

[0250] 2. Tumors containing HER-2 CAR T cells Fab CAR or scFv CAR T cells are superior. 1E7 positive cells in each mouse.

[0251] 3. Tumors containing mock T cells : 1E7 mock T cells from each mouse.

[0252] 4. Tumors containing Meditope-CAR T cells : 1E7 positive CAR T cells in each mouse.

[0253] 5. Tumors with pre-mixed mock T cells + HER2 antibodies : 1E7 mock T cells in each mouse. HER2 antibody IP 1 day prior to T cell injection. 4 mg / kg T cells with 100 nM antibody and Pre-mixed It is washed. Antibodies are administered at 4 mg / kg IP twice a week for 2 weeks.

[0254] 6. Tumors with pre-mixed Meditope-CAR T cells + HER2 antibodies : 1E7 CAR T cells in each mouse. HER2 antibody IP 1 day prior to T cell injection. 4 mg / kg T cells with 100 nM antibody and Pre-mixed It is washed. Antibodies are administered at 4 mg / kg IP twice a week for 2 weeks.

[0255] 7. Tumors with Meditope-CAR T cells + HER2 antibodies without pre-mixing : 1E7 CAR T cells in each mouse. HER2 antibody IP 1 day prior to T cell injection. 4 mg / kg T cells and antibodies The dictionary is not mixed Antibodies are administered at 4 mg / kg IP twice a week for 2 weeks.

[0256]

[0257] FabRack animal data

[0258] Method (OVCAR3)

[0259] On day 1, 5,000,000 OVCAR3-gfp-luc cells were injected intraperitoneally (ip) into mice. In mice treated with Fabrack T cells (Groups 6 and 7), 4 mg / kg memAb trastuzumab (total of 5 doses) was administered every 3 days, with the first dose of the Ab administered on day 8. On day 9, 10,000,000 human T cells were injected into mice ip. In Group 6, Fabrack T cells were pre-mixed with memAb and washed. After injecting 150 μl of luciferin (28.57 mg / ml) into mice ip, the tumor burden of the mice was measured by luminescence. (Group 1: Tumor alone; Group 2: Mock T cells; Group 3: Fabrack T cells alone; Group 4: Mock T cells + Ab; Group 5: HER2 scFv CAR; Group 6: Fabrack T cells (pre-mixed) + Ab; Group 7: Fabrack T cells + Ab)

[0260] Result (OVCAR3)

[0261] Mice exhibited a substantial reduction in tumor size when provided with Fabrack T cells and memAb (Groups 6 and 7), regardless of whether the Fabrack T cells were pre-mixed with memAb. However, the tumors recurred around day 14. Based on flow cytometry results indicating HER2+ tumor cells in mouse abdominal fluid, the tumor recurrence was not attributed to the loss of HER2 antigens. Since very few Fabrack T cells remained in the mouse blood and abdominal fluid, the tumor recurrence may be due to the non-persistence of T cells. A dosing schedule to optimize tumor eradication is currently underway.

[0262] Method (MCF7)

[0263] On day 1, 5,000,000 MCF7-gfp-luc cells were injected intraperitoneally (ip) into mice. In the Fabrack group, mice were administered 4 mg / kg memAb trastuzumab approximately every 4 days, and the first dose of the antibody was injected together with T cells via ip. On day 8, the first dose of 2,000,000 human T cells was injected into mice via ip. An additional 2,000,000 Fabrack T cells were administered approximately every 6 days. After injecting 150 μl of luciferin (28.57 mg / ml) into mice via ip, the tumor burden of the mice was measured by luminescence.

[0264] Result (MCF7)

[0265] A reduction in tumor size was observed in mice provided with Fabrack T cells and memAb at d11 and d14. However, tumor recurrence occurred on day 16. Analysis of mouse blood on day 22 indicated the presence of Fabrack T cells and that memAb was bound to the Fabrack T cells. Analysis of mouse abdominal fluid on day 45 did not show antigen detachment in the tumor cells. Tumor recurrence may be attributed to a hook effect from the antibody, and its dose can saturate the antibody-binding sites on the tumor and T cells. Since the dose of Fabrack T cells was 2,000,000 in the MVC7 xenograft study compared to 10,000,000 in the OVCAR3 study, there were fewer Fabrack T cells for antibody binding. Additionally, because MVC7 and OVCAR3 exhibit low HER2 expression, HER2 is readily saturated by the antibody. Dosage schedules to optimize tumor eradication are currently underway.

[0266]

[0267]

[0268] P Implementation mode

[0269] Embodiment P1. A first recombinant protein comprising: (i) a first non-CDR Fab-binding peptide domain; (ii) a first intracellular T cell signaling domain; and (iii) a first transmembrane domain connecting the first non-CDR Fab-binding peptide domain to the first intracellular T cell signaling domain.

[0270] Embodiment P2. A first recombinant protein according to Embodiment P1, further comprising a first spacer region connecting the first non-CDR Fab-binding peptide domain to the first transmembrane domain.

[0271] Embodiment P3. A first recombinant protein in which the first spacer region of Embodiment P2 is a first CH3 region.

[0272] Embodiment P4. In Embodiment P3, the first recombinant protein is non-covalently bound to a second recombinant protein, wherein the second recombinant protein comprises (i) a second non-CDR Fab-binding peptide domain; (ii) a second intracellular T cell signaling domain; (iii) a second transmembrane domain connecting the second non-CDR Fab-binding peptide domain to the second intracellular T cell signaling domain; and (iv) a second spacer region, wherein the second spacer region connects the second non-CDR Fab-binding peptide domain to the second transmembrane domain, wherein the second spacer region comprises a second CH3 region, and wherein the first CH3 region is bound to the second CH3 region.

[0273] Embodiment P5. A first recombinant protein in any one of embodiments P1 to P5, wherein the first non-CDR Fab-binding peptide domain and the second non-CDR Fab-binding peptide domain are chemically different.

[0274] Embodiment P6. A first recombinant protein in any one of embodiments P1 to P5, wherein the first non-CDR Fab-binding peptide domain and the second non-CDR Fab-binding peptide domain are chemically identical.

[0275] Embodiment P7. In any one of Embodiments P1 to P6, a first recombinant protein in which the first and second intracellular T cell signaling domains are independently CD3ζ T cell signaling domains.

[0276] Embodiment P8. A first recombinant protein in any one of embodiments P1 to P7, wherein the first non-CDR Fab binding peptide domain is non-covalently bound to the first antigen binding domain.

[0277] Embodiment P9. A first recombinant protein in which, in any one of embodiments P1 to P8, the second non-CDR Fab binding peptide domain is non-covalently bound to the second antigen binding domain.

[0278] Embodiment P10. Isolated nucleic acid encoding a first recombinant protein of any one of Embodiments 1 to 3.

[0279] Embodiment P11. An expression vector comprising the nucleic acid of Embodiment P10.

[0280] Embodiment P12. An expression vector in which the virus of Embodiment P11 is a lentivirus or an onco-retrovirus.

[0281] Embodiment P13. T lymphocyte comprising an expression vector of Embodiment P11 or P12.

[0282] Embodiment P14. A T lymphocyte comprising a first recombinant protein of any one of embodiments P1 to P9.

[0283] Embodiment P15. A T lymphocyte comprising a first recombinant protein of any one of embodiments P1 to P9, wherein the transmembrane domain is located within the cell membrane of the T lymphocyte.

[0284] Embodiment P16. A method for treating cancer comprising the step of administering an effective amount of T lymphocytes of Embodiment P15 to a subject requiring treatment for cancer, wherein the first antigen-binding domain and the second antigen-binding domain are independently anti-cancer antigen-binding domains.

[0285] Mode of implementation

[0286] Embodiment 1. A recombinant protein comprising: (i) a non-CDR Fab-binding peptide domain; (ii) an intracellular T cell signaling domain; and (iii) a transmembrane domain connecting the non-CDR Fab-binding peptide domain to the intracellular T cell signaling domain.

[0287] Embodiment 2. The recombinant protein of Embodiment 1, wherein the intracellular T cell signaling domain is a CD3ζ intracellular T cell signaling domain.

[0288] Embodiment 3. A recombinant protein in which the transmembrane domain of Embodiment 1 or 2 is a CD8α transmembrane domain, a CD28 transmembrane domain, a CD4 transmembrane domain, or a CD3-zeta transmembrane domain.

[0289] Embodiment 4. A recombinant protein in any one of Embodiments 1 to 3, wherein the transmembrane domain is a CD28 transmembrane domain.

[0290] Embodiment 5. A recombinant protein in any one of Embodiments 1 to 4, further comprising a spacer region connecting the non-CDR Fab-binding peptide domain to the transmembrane domain.

[0291] Embodiment 6. The recombinant protein of Embodiment 5, wherein the spacer region is an invariant heavy chain 3 (CH3) domain.

[0292] Embodiment 7. A recombinant protein in any one of Embodiments 1 to 6, further comprising a peptide linker that connects the non-CDR Fab-binding peptide domain to the spacer region.

[0293] Embodiment 8. A recombinant protein in any one of Embodiments 1 to 7, further comprising an intracellular co-stimulation signaling domain that connects the transmembrane domain to the intracellular T cell signaling domain.

[0294] Embodiment 9. The recombinant protein of Embodiment 8, wherein the intracellular co-stimulation signaling domain is a CD28 intracellular co-stimulation signaling domain, a 4-1BB intracellular co-stimulation signaling domain, an ICOS intracellular co-stimulation signaling domain, or an OX-40 intracellular co-stimulation signaling domain.

[0295] Embodiment 10. The recombinant protein of Embodiment 8 or 9, wherein the intracellular co-stimulation signaling domain is a CD28 intracellular co-stimulation signaling domain.

[0296] Embodiment 11. A recombinant protein in any one of Embodiments 8 to 10, wherein the intracellular co-stimulation signaling domain is a 4-1BB intracellular co-stimulation signaling domain.

[0297] Embodiment 12. A recombinant protein comprising, in any one of Embodiments 1 to 11, an additional detectable domain bound to the C-terminus of the intracellular T cell signaling domain.

[0298] Embodiment 13. The recombinant protein of Embodiment 12, wherein the detectable domain is a terminally truncated CD19 protein.

[0299] Embodiment 14. A recombinant protein in any one of Embodiments 1 to 13, further comprising a self-cleaving peptidyl sequence connecting the intracellular T cell signaling domain to the detectable domain.

[0300] Embodiment 15. The recombinant protein of Embodiment 14, wherein the self-cleaving peptidyl linker sequence is a T2A sequence or a 2A sequence.

[0301] Embodiment 16. A recombinant protein in any one of Embodiments 1 to 15, wherein the recombinant protein forms part of a cell.

[0302] Embodiment 17. A recombinant protein in any one of Embodiments 1 to 16, wherein the recombinant protein forms part of a T cell.

[0303] Embodiment 18. A recombinant protein in which, in any one of Embodiments 1 to 17, the non-CDR Fab binding peptide domain is bound to an antigen binding domain.

[0304] Embodiment 19. The recombinant protein of Embodiment 18, wherein the antigen-binding domain is Fab, IgG, or a bispecific antibody.

[0305] Embodiment 20. A recombinant protein in which the antigen binding domain of Embodiment 18 or 19 is a cetuximab Meditope-available domain, a trastuzumab Meditope-available domain, a pertuzumab Meditope-available domain, an M5A Meditope-available domain, or a rituximab Meditope-available domain.

[0306] Embodiment 21. A recombinant protein in any one of embodiments 18 to 20, wherein the antigen binding domain is capable of binding to a cancer antigen.

[0307] Embodiment 22. A recombinant protein in any one of Embodiments 18 to 21, wherein the antigen-binding domain is capable of binding to a cancer antigen.

[0308] Embodiment 23. The recombinant protein in which the cancer antigen in Embodiment 21 or 22 is Her2, EGFR, CD19, or CD20.

[0309] Embodiment 24. A recombinant protein in which the cancer antigen forms part of a cell, in Embodiment 21 or 22.

[0310] Embodiment 25. In Embodiment 24, a protein in which the cell is a cancer cell.

[0311] Embodiment 26. The recombinant protein of Embodiment 25, wherein the cancer is ovarian cancer, renal cell carcinoma, B-cell malignancy, leukemia, lymphoma, breast cancer, colorectal cancer, prostate cancer, neuroblastoma, melanoma, medulloblastoma, lung cancer, osteosarcoma, glioblastoma, or glioma.

[0312] Embodiment 27. Isolated nucleic acid encoding any one of the recombinant proteins of Embodiments 1 to 26.

[0313] Embodiment 28. An expression vector comprising the nucleic acid of Embodiment 27.

[0314] Embodiment 29. An expression vector in which the virus of Embodiment 28 is a lentivirus or an onco-retrovirus.

[0315] Embodiment 30. T lymphocytes comprising the expression vector of Embodiment 28 or 29.

[0316] Embodiment 31. A T lymphocyte comprising a recombinant protein of any one of Embodiments 1 to 26.

[0317] Embodiment 32. A T lymphocyte comprising a recombinant protein of any one of Embodiments 1 to 26, wherein the transmembrane domain is located within the cell membrane of the T lymphocyte.

[0318] Embodiment 33. In any one of embodiments 30 to 32, the T lymphocyte is an autologous T lymphocyte.

[0319] Embodiment 34. In any one of embodiments 30 to 32, the lymphocyte in which the T lymphocyte is a heterologous T lymphocyte.

[0320] Embodiment 35. A T lymphocyte in any one of embodiments 30 to 34, wherein the non-CDR Fab binding peptide domain binds to an antigen binding domain.

[0321] Embodiment 36. The T lymphocyte of Embodiment 35, wherein the antigen-binding domain is Fab, IgG, or a bispecific antibody.

[0322] Embodiment 37. A T lymphocyte according to Embodiment 35 or 36, wherein the antigen binding domain binds to a cancer antigen.

[0323] Embodiment 38. The T lymphocyte of Embodiment 37, wherein the cancer antigen is Her2, EGFR, CD19, or CD20.

[0324] Embodiment 39. A T lymphocyte in any one of embodiments 35 to 38, wherein the antigen binding domain is a cancer antigen binding domain.

[0325] Embodiment 40. A T lymphocyte in any one of embodiments 35 to 39, wherein the antigen binding domain is a cetuximab Meditope-available domain, a trastuzumab Meditope-available domain, a pertuzumab Meditope-available domain, an M5A Meditope-available domain, or a rituximab Meditope-available domain.

[0326] Embodiment 41. A method for treating cancer comprising the step of administering an effective amount of any one of Embodiments 30 to 34 T lymphocytes and an antigen-binding domain capable of binding to the non-CDR Fab-binding peptide domain to a subject requiring cancer treatment, wherein the antigen-binding domain is a cancer antigen-binding domain.

[0327] Embodiment 42. A method according to Embodiment 41 in which the T lymphocyte and the antigen-binding domain are administered simultaneously or sequentially.

[0328] Embodiment 43. A method according to Embodiment 41 or 42, wherein the T lymphocyte is administered at a first time point and the antigen-binding domain is administered at a second time point, wherein the first time point precedes the second time point.

[0329] Embodiment 44. A method according to Embodiment 41 or 42, wherein the antigen binding domain is administered at a first time point and the T lymphocyte is administered at a second time point, wherein the first time point precedes the second time point.

[0330] Embodiment 45. The method of Embodiment 41, wherein the method comprises: (i) a step of forming a T lymphocyte-recombinant protein complex by binding the non-CDR Fab-binding peptide domain to the antigen-binding domain in vitro before administration; and (ii) a step of administering the T lymphocyte-recombinant protein complex to a subject to treat cancer in the subject.

[0331] Embodiment 46. A method in any one of embodiments 41 to 45, wherein the cancer is ovarian cancer, renal cell carcinoma, B-cell malignancy, leukemia, lymphoma, breast cancer, colorectal cancer, prostate cancer, neuroblastoma, melanoma, medulloblastoma, lung cancer, osteosarcoma, glioblastoma, or neuroglioma.

[0332] Embodiment 47. A method in any one of Embodiments 41 to 46, wherein the antigen binding domain is a cetuximab Meditope-available domain, a trastuzumab Meditope-available domain, a pertuzumab Meditope-available domain, an M5A Meditope-available domain, or a rituximab Meditope-available domain.

[0333] Embodiment 48. A recombinant protein in any one of Embodiments 1 to 26, wherein the recombinant protein is a first recombinant protein, the non-CDR Fab binding peptide domain is a first non-CDR Fab binding peptide domain, the intracellular T cell signaling domain is a first intracellular T cell signaling domain, the transmembrane domain is a first transmembrane domain, the spacer region is a first spacer region, and the intracellular co-stimulation signaling domain is a first intracellular co-stimulation signaling domain.

[0334] Embodiment 49. The recombinant protein according to Embodiment 48, wherein the first recombinant protein is non-covalently bound to a second recombinant protein, and the second recombinant protein comprises (i) a second non-CDR Fab-binding peptide domain; (ii) a second intracellular T cell signaling domain; (iii) a second transmembrane domain connecting the second non-CDR Fab-binding peptide domain to the second intracellular T cell signaling domain; and (iv) a second spacer region, wherein the second spacer region connects the second non-CDR Fab-binding peptide domain to the second transmembrane domain, and wherein the first spacer region is non-covalently bound to the second spacer region.

[0335] Embodiment 50. The recombinant protein of Embodiment 49, wherein the first spacer region and the second spacer region are a first constant heavy chain 3 (CH3) domain and a second constant heavy chain 3 (CH3) domain.

[0336] Embodiment 51. A recombinant protein in any one of embodiments 48 to 50, wherein the first non-CDR Fab-binding peptide domain and the second non-CDR Fab-binding peptide domain are chemically different.

[0337] Embodiment 52. A recombinant protein in any one of embodiments 48 to 50, wherein the first non-CDR Fab-binding peptide domain and the second non-CDR Fab-binding peptide domain are chemically identical.

[0338] Embodiment 53. A recombinant protein in which, in any one of embodiments 48 to 52, the first non-CDR Fab-binding peptide domain is non-covalently bound to the first antigen-binding domain.

[0339] Embodiment 54. A recombinant protein in which, in any one of embodiments 49 to 53, the second non-CDR Fab-binding peptide domain is non-covalently bound to the second antigen-binding domain.

[0340] Embodiment 55. A recombinant protein according to Embodiment 53 or 54, wherein the first antigen-binding domain and the second antigen-binding domain are chemically different or identical.

[0341] Embodiment 56. A recombinant protein in any one of embodiments 53 to 55, wherein the first antigen-binding domain and the second antigen-binding domain are independently a cetuximab medtop-available domain, a trastuzumab medtop-available domain, a pertuzumab medtop-available domain, an M5A medtop-available domain, or a rituximab medtop-available domain.

[0342] Informal ranking list

[0343] CD3z; GI:623041(Sequence No. 1)

[0344]

[0345] CD28; GI:340545506(Sequence No. 2)

[0346]

[0347] CD4; GI:179143(Sequence No. 3)

[0348]

[0349] CD8; GI:225007534(Sequence No. 4)

[0350]

[0351] CD8; GI:225007534(Sequence No. 5)

[0352]

[0353] CD8; GI:225007534(Sequence No. 6)

[0354]

[0355] 41BB; GI:315259099(Sequence No. 7)

[0356]

[0357] OX40; GI:315360637(Sequence No. 8)

[0358]

[0359] ICOS; GI:251823951(Sequence No. 9)

[0360]

[0361] CD62L; GI:262206314(Sequence No. 10)

[0362]

[0363] CD3ζ; GI:623041; Sequence No. 11:

[0364]

[0365] CD28; GI:340545506; Sequence No. 12:

[0366]

[0367] CD28gg*; GI:340545506; Sequence No. 13:

[0368]

[0369] 41BB; GI:315259099; Sequence No. 14:

[0370]

[0371] OX40; GI:315360637; Sequence No. 15:

[0372]

[0373] ICOS; GI:251823951; Sequence No. 16:

[0374]

[0375] Spacer (including IgG4-CH3) Sequence No. 17:

[0376]

[0377] CD28 Transversal Sequence Number 18:

[0378]

[0379] CD28cyto(LLmGG) Sequence No. 19:

[0380]

[0381] Intracellular T cell signaling domain (CD3-zeta) Sequence No. 20:

[0382]

[0383] Self-cleaving peptidyl linker (T2A) sequence number 21:

[0384]

[0385] Marker peptide (CD19t) sequence number 22:

[0386]

[0387] Self-cleaving peptidyl linker (2A) sequence number 23:

[0388]

[0389] Spacer region sequence number 24

[0390]

[0391] Peptide linker sequence number 25

[0392]

[0393] Sequence number 26

[0394]

[0395] Self-cleaving peptidyl linker sequence number 27

[0396]

[0397] Self-cleaving peptidyl linker Sequence No. 28

[0398]

[0399] Self-cleaving peptidyl linker sequence number 29

[0400]

[0401] Self-cleaving peptidyl linker sequence number 30

[0402]

[0403] Spacer region sequence number 31

[0404]

[0405] SEQ ID NO. 32; Meditope (non-CDR Fab-binding peptide domain)

[0406]

[0407] Sequence No. 33; CH3 (spacer region)

[0408]

[0409] SEQ ID NO. 34; CD3 zeta chain (intracellular T cell signaling domain)

[0410]

[0411] Sequence No. 35; Full 4-1BB FabRack sequence

[0412]

[0413]

[0414] Sequence No. 36; Full CD28 FabRack sequence

[0415]

[0416] SEQ ID NO. 37; signal peptide

[0417]

Claims

Claim 1 A recombinant protein comprising: (i) a Meditope capable of binding to an available Fab; (ii) an intracellular T cell signaling domain; and (iii) a transmembrane domain connecting the Meditope to the intracellular T cell signaling domain; wherein the recombinant protein is a continuous single-strand polypeptide and the Meditope comprises the sequence of SEQ ID NO.

32. Claim 2 A recombinant protein according to claim 1, further comprising a spacer region connecting the above-mentioned Meditope to the above-mentioned transmembrane domain. Claim 3 A recombinant protein according to paragraph 2, further comprising a peptide linker connecting the above Meditope to the above spacer region. Claim 4 A recombinant protein according to claim 3, further comprising an intracellular co-stimulation signaling domain that connects the transmembrane domain to the intracellular T cell signaling domain. Claim 5 A recombinant protein according to claim 4, further comprising a detectable domain bound to the C-terminus of the intracellular T cell signaling domain. Claim 6 A recombinant protein according to claim 5, further comprising a self-cleaving peptidyl sequence connecting the intracellular T cell signaling domain to the detectable domain. Claim 7 In claim 1, the recombinant protein is one that forms part of a cell. Claim 8 In claim 1, the recombinant protein is one that forms part of a T cell. Claim 9 A recombinant protein according to claim 1, wherein the above-mentioned Meditope is bound to an antigen-binding domain. Claim 10 In claim 9, the recombinant protein in which the antigen-binding domain is Fab, IgG, or a bispecific antibody. Claim 11 In claim 9, a recombinant protein in which the antigen-binding domain is capable of binding to a cancer antigen. Claim 12 In paragraph 11, the recombinant protein in which the cancer antigen forms part of a cell. Claim 13 In Clause 12, the recombinant protein in which the cell is a cancer cell. Claim 14 Isolated nucleic acid encoding the recombinant protein of claim 1. Claim 15 An expression vector containing the nucleic acid of claim 14. Claim 16 T lymphocytes containing the expression vector of claim 15. Claim 17 T lymphocytes containing the recombinant protein of claim 1. Claim 18 A T lymphocyte comprising the recombinant protein of claim 1, wherein the transmembrane domain is located within the cell membrane of the T lymphocyte. Claim 19 A pharmaceutical composition for use in a method for treating cancer comprising an effective amount of T lymphocytes of claim 16 and an antigen-binding domain capable of binding to Meditope, wherein the method comprises the step of administering an effective amount of T lymphocytes of claim 16 and an antigen-binding domain capable of binding to Meditope to a subject requiring treatment for cancer, wherein the antigen-binding domain is a cancer antigen-binding domain. Claim 20 A pharmaceutical composition according to claim 19, wherein the T lymphocytes and the antigen-binding domain are administered simultaneously or sequentially. Claim 21 A pharmaceutical composition according to claim 19, wherein the method comprises: (i) a step of binding the Meditope to the antigen-binding domain in vitro before administration to form a T lymphocyte-recombinant protein complex; and (ii) a step of administering the T lymphocyte-recombinant protein complex to a subject to treat cancer in the subject. Claim 22 In claim 4, the recombinant protein is the first recombinant protein, the Meditope is the first Meditope, the intracellular T cell signaling domain is the first intracellular T cell signaling domain, the transmembrane domain is the first transmembrane domain, the spacer region is the first spacer region, and the intracellular co-stimulation signaling domain is the first intracellular co-stimulation signaling domain. Claim 23 A recombinant protein according to claim 22, wherein the first recombinant protein is non-covalently bound to a second recombinant protein, and the second recombinant protein comprises: (i) a second Meditope; (ii) a second intracellular T cell signaling domain; (iii) a second transmembrane domain connecting the second Meditope to the second intracellular T cell signaling domain; and (iv) a second spacer region, wherein the second spacer region connects the second Meditope to the second transmembrane domain, and the first spacer region is non-covalently bound to the second spacer region. Claim 24 In paragraph 22, a recombinant protein in which the first Meditope is non-covalently bound to the first antigen-binding domain. Claim 25 A recombinant protein according to claim 23, wherein the second Meditope is non-covalently bound to the second antigen-binding domain. Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 delete Claim 30 delete Claim 31 delete Claim 32 delete Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 delete Claim 37 delete Claim 38 delete Claim 39 delete Claim 40 delete Claim 41 delete Claim 42 delete Claim 43 delete Claim 44 delete Claim 45 delete Claim 46 delete Claim 47 delete Claim 48 delete Claim 49 delete Claim 50 delete Claim 51 delete Claim 52 delete Claim 53 delete Claim 54 delete Claim 55 delete Claim 56 delete Claim 57 delete

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