Meditope-compatible T cells

Abstract

Presented herein are compositions that exhibit novel therapeutic capabilities and allow for the reduction of off-target effects of therapeutic antibodies. The compositions comprise recombinant proteins that, when expressed by T cells, efficiently recruit therapeutic antibodies to their site of action.

Description

[Technical Field]

[0001] Cross-reference of related applications 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, which are incorporated herein by reference in their entirety for all purposes.

[0002] References to "arrangement listings," tables, or appendices to computer program lists submitted as ASCII files. A sequence listing, written to the file 048440-621001WO_Sequence_Listing_ST25 and created on December 31, 2018, by the MS Windows operating system with a machine format of IBM-PC and 29,672 bytes, is incorporated herein by reference. [Overview of the project] [Means for solving the problem]

[0003] (Summary of the invention) In one embodiment, 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 ligating the first non-CDR Fab-binding peptide domain to the first intracellular T cell signaling domain. In several embodiments, the first recombinant protein comprises a first spacer region ligating the first non-CDR Fab-binding peptide domain to the first transmembrane domain. In several embodiments, the first spacer region is a first CH3 region.

[0004] In one embodiment, an isolated nucleic acid encoding a first recombinant protein provided herein, including embodiments thereof, is provided.

[0005] In one aspect, an expression vector comprising a nucleic acid provided herein including its embodiments is provided. In a plurality of embodiments, the vector is a lentivirus or an oncoretrovirus.

[0006] In one aspect, T lymphocytes comprising an expression vector provided herein including its embodiments are provided.

[0007] In one aspect, T lymphocytes comprising a first recombinant protein provided herein including its embodiments are provided.

[0008] In one aspect, T lymphocytes comprising a first recombinant protein provided herein including its embodiments, wherein the transmembrane domain is within the cell membrane of the T lymphocyte, are provided.

[0009] In one aspect, a method of treating cancer is provided. The method includes administering to a subject needing treatment of cancer an effective amount of T lymphocytes provided herein including its embodiments, wherein the first antigen-binding domain and the second antigen-binding domain are each independently an anti-cancer antigen-binding domain.

[0010] In certain aspects, a recombinant protein is provided. The recombinant protein includes (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.

[0011] In certain aspects, an isolated nucleic acid encoding a recombinant protein provided herein including its embodiments is provided.

[0012] In certain aspects, an expression vector comprising a nucleic acid provided herein including its embodiments is provided.

[0013] In one aspect, T lymphocytes comprising the expression vector provided herein, including that embodiment, are provided.

[0014] In one aspect, T lymphocytes comprising the recombinant protein provided herein, including that embodiment, are provided.

[0015] In one aspect, T lymphocytes comprising the recombinant protein provided herein, including that embodiment, and having a transmembrane domain within the cell membrane of the T lymphocyte are provided.

[0016] In one aspect, a method of treating cancer is provided. The method includes administering to a subject in need of treatment for cancer an effective amount of T lymphocytes provided herein, including that embodiment, and 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] <0000​​​​​​​​​This figure shows the results of transfecting CHO-S cells with no vector (mock), parental CAR(1), meditope-CH3(3), or meditope-CD28(2) to investigate the binding of meditope-compatible I83E trastuzumab IgG conjugated with the fluorescent dye Alexa Fluor 647 (647-I83E IgG), meditope-compatible I83E trastuzumab Fab conjugated with the fluorescent dye Alexa Fluor 647 (647-I83E Fab), and non-meditope-compatible ipilimumab IgG conjugated with the fluorescent dye Alexa Fluor 647 (647-Ipi IgG). Viable transfected cells were identified using FSC / SSC → PI- → CD19+ gating (see Figure 3A). Cells were analyzed for mean fluorescence intensity (MFI) of the APC signal. [Figure 2B] This figure shows that cells transfected with no vector (mock), parental CAR(1), or meditope-CD28(2) did not show a shift in MFI when stained with 647-I83E IgG, 647-I83E Fab, or 647-Ipi IgG. [Figure 2C] This figure shows that cells transfected with Meditope-CH3(3) showed a significant shift in MFI when stained with 647-I83E IgG or 647-I83E Fab, but showed only a minimal shift when stained with 647-Ipi IgG. [Figure 3A] This figure shows gating strategies for flow cytometry analysis. Cells were gated using FSC / SSC (left panel), PI staining (indicating viable cells; middle panel), and CD19 expression (transfection marker; right panel). [Figure 3B] This figure shows a gating strategy for flow cytometry analysis. Further analysis of the gated cells revealed 647 signals (left panel) and VioBlue signals (right panel), which indicate antibody and Her2 binding, respectively. [Figure 4A]This figure shows that meditope-compatible IgG and Fab bind to meditope-CH3 CAR. Viable transfected cells were identified using FSC / SSC → PI- → CD19+ gating. Cells were analyzed for mean fluorescence intensity (MFI) of the APC signal. APC+ cells were gated using cells without dye. Cells stained with non-meditope-compatible 647-Ipi IgG showed minimal shift in APC MFI. Cells stained with meditope-compatible 647-I83E IgG and 647-I83E Fab showed a marked shift in APC MFI, supporting the binding of 647 conjugate proteins to meditope-CH3 expressing cells. [Figure 4B] This figure shows that methyltope-compatible IgG and Fab bind to methyltope-CH3 CAR. This tendency was not observed in the methyltope-CD28 construct. [Figure 4C] This figure shows that methyltope-compatible IgG and Fab bind to the methyltope-CH3 CAR. The figure represents the survival percentage of CD19+ cells that are positive for 647-IgG or 647-Fab (gated cells: FSC / SSC → PI- → CD19+ → APC+; frequency of APC+ cells in the parent population). [Figure 5A] This figure shows that Meditope-CH3 expressing cells bind to Meditope-responsive antibodies, and subsequently, Meditope-responsive antibodies can bind to antigens. This is a histogram of the mean Her2 fluorescence intensity (MFI) on Meditope-CH3 CAR cells, which are APC+ (gated cells: FSC / SSC → PI- → CD19+ → APC+). [Figure 5B] This figure shows that Meditope-CH3 expressing cells bind to Meditope-responsive antibodies, and subsequently, Meditope-responsive antibodies bind to antigens. The figure also shows the survival percentage of CD19+647+ cells that are positive for Her2 (Her2+ cell frequency in the grandparent population using the same gating strategy as above). [Figure 6A]This figure shows the identification of 647+Her2+ double-positive cells. Viable transfected cells were identified using FSC / SSC→PI-→CD19+ gating (see Figure 3A). The cells were then analyzed at the 647 level (x axis) and the Her2 level (VioBlue). It is noteworthy that many 647- cells are located on the y axis. This is an analysis of cells stained with medotope-compatible 647-I83E Fab. [Figure 6B] This figure shows the identification of 647+Her2+ double-positive cells. Viable transfected cells were identified using FSC / SSC → PI- → CD19+ (see Figure 3A). The cells were then analyzed at the 647 level (x-axis) and Her2 level (VioBlue). It is noteworthy that many 647- cells are located on the y-axis. The analysis is of cells stained with medotope-compatible 647-I83E IgG. [Figure 6C] This figure shows the identification of 647+Her2+ double-positive cells. Viable transfected cells were identified using FSC / SSC → PI- → CD19+ (see Figure 3A). The cells were then analyzed at the 647 level (x-axis) and the Her2 level (VioBlue). It is noteworthy that many 647- cells are located on the y-axis. The analysis is for cells stained with non-meditope-compatible 647-Ipi IgG. [Figure 7] This figure shows the identification of Her2+ cells. Viable transfected cells were identified using FSC / SSC → PI- → CD19+ gating. Her2+ cells were identified using analysis of samples without PacBlue-Her2, followed by the following gate. The cells were then analyzed for Her2 (VioBlue) levels. [Figure 8]This figure shows that meditope-compatible cetuximab Fab and trastuzumab Fab bind to the meditope-CH3 CAR. (Figure 8A) This shows the survival percentage of CD19+ cells positive for 647-Fab (gated cells: FSC / SSC → PI- → CD19+ → APC+; frequency of APC+ cells in the parent population). (Figure 8B) Viable cells transfected with meditope-CH3 were identified using FSC / SSC → PI- → CD19+ gating. Cells were analyzed for mean fluorescence intensity (MFI) of the APC signal. APC+ cells were gated using cells without dye. Cells stained with non-meditope-compatible 647-Ipi Fab showed minimal shift in APC MFI. Cells stained with 647-I83E cetuximab (cetux)Fab and 647-I83E trastuzumab (tras)Fab, which are compatible with Meditope, showed a significant shift in APC MFI, confirming the binding of the 647 conjugate protein to Meditope-CH3 expressing cells. [Figure 9A] This figure shows the identification of 647+Her2+ double-positive cells. Viable transfected cells were identified using FSC / SSC→PI-→CD19+ gating. The cells were then analyzed at the 647 level (x axis) and the Her2 level (Pacific Blue). The analysis is for cells stained with medotope-compatible 647-I83E cetuximab (cetux) Fab. [Figure 9B] This figure shows the identification of 647+Her2+ double-positive cells. Viable transfected cells were identified using FSC / SSC → PI- → CD19+. The cells were then analyzed at the 647 level (x-axis) and the Her2 level (Pacific Blue). The analysis is for cells stained with medotope-compatible 647-I83E trastuzumab (tras)Fab. [Figure 10] This figure shows a tumor killing assay in ovarian cancer cell lines using different concentrations of medotope-compatible Her2. [Figure 11]This figure shows a tumor killing assay in breast cancer cell lines using different concentrations of medotope-compatible Her2. [Figure 12] This is a diagram showing FabRack T cells. [Figure 13] This is a diagram showing switchable Fab CAR-T cells. [Figure 14] This figure shows the results of transduction of Meditope-CAR into Jurkat cells and co-expression of cleaved CD19 as a marker. After transduction, CD19-positive cells were isolated and expanded for subsequent experiments. [Figure 15] This figure shows Jurkat cells expressing meditope-CAR incubated with trastuzumab with or without the meditope site, and then stained with secondary anti-kappa-647 (Abcam#202832) or secondary anti-human IgG Fc-488 (ThermoFisher#H10120). The results showed that, after cell analysis by flow cytometry, only memAb trastuzumab was able to bind to Jurkat cells expressing meditope-CAR. [Figure 16]Left: This figure shows the co-incubation of cancer cells, Jurkat-NFAT-Luc Meditope-CAR cells, and memAb trastuzumab in a 96-well plate with a white wall. The highest concentration in the figure is 15 nM, followed by a 4-fold dilution series. After 6 hours of incubation, luciferase substrate was added to each well, and luminescence was rapidly measured using a plate reader. In the presence of memAb trastuzumab, Jurkat cell activation increased in a dose-dependent manner, despite the hook effect at 15 nM. 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 activation levels of Jurkat cells were positively correlated with HER2 expression on cancer cells, except for the BT474 cell line. BT474 cells exhibit high HER2 expression but do not activate Jurkat cells to the same level as other high HER2-expressing cells (SKOV3 and SKBR3). Right: 5 × 10⁵ cells were treated with 100 nM memAb trastuzumab in 1% FBS in PBS for 30 minutes. After three washes, the cells were labeled with secondary anti-kappa-647 antibody for 30 minutes. Cell fluorophore intensity was analyzed using a BD Accuri C6 flow cytometer. Red peaks represent cells treated with secondary anti-kappa-647 antibody alone. Green peaks represent cells treated with memAb trastuzumab and secondary anti-kappa-647 antibody. HER2 expression levels were analyzed by comparing cells stained with secondary antibody alone with cells stained with memAb trastuzumab and secondary antibody using flow cytometry. The median fluorescence intensity (MFI) was 338 and 985 for MCF7 cells; 330 and 34405 for SKBR3 cells; 392 and 31720 for BT474 cells; 370 and 43191 for SKOV3 cells; and 307 and 1436 for OVCAR3 cells. Cells with HER2 expression, from high to low, were SKOV3, SKBR3, BT474, OVCAR3, and MCF7. [Figure 17]Left: This figure shows the co-incubation of cancer cells, FabRack Jurkat-NFAT-Luc cells, and cetuximab in a 96-well plate with white walls. The highest concentration shown in the figure is 60 nM, followed by a 4-fold dilution series. After 6 hours of incubation, luciferase substrate was added to each well, and luminescence was rapidly measured using a plate reader. The EC50 for each cell line was 0.14 nM (SKOV3), 0.12 nM (SKBR3), 0.12 nM (MCF7), 0.11 nM (OVCAR3), and 1.1 nM (BT474). Right: 5 × 10⁵ cells were treated with 100 nM cetuximab in 1% FBS in PBS for 30 minutes. After three washes, the cells were labeled with secondary anti-kappa-647 antibody for 30 minutes. Cellular fluorophore intensity was analyzed using a BD Accuri C6 flow cytometer. Red peaks represent cells treated with secondary anti-kappa-647 antibody alone. Green peaks represent cells treated with cetuximab and secondary anti-kappa-647 antibody. Median fluorescence intensity indicated that cells with EGFR expression, ranging from high to low, were OVCAR3 (4819), SKOV3 (4479), SKBR3 (2865), BT474 (651), and MCF7 (480). [Figure 18]This figure shows Jurkat cells or cancer cells that have been pre-mixed with memAb trastuzumab and washed. Jurkat cells or cancer cells are pre-bound with memAb trastuzumab and then washed. Cancer cells (2.5 × 10⁴ cells per 100 μl) were seeded in a 96-well plate with white walls. After cell adhesion overnight, the culture medium in the plate was removed, and Jurkat-NFAT-Luc meditope-CAR cells (1 × 10⁵ cells per 60 μl) were added to each well. memAb trastuzumab was either continuously present or pre-bound to Jurkat-NFAT-Luc medi-CAR cells or cancer cells, followed by washing (the bars in the graph, from left to right, represent: no treatment; continuously present herceptin (4nM); continuously present memAb trastuzumab (4nM); FabRack Jurkat-NFAT-Luc cells pre-bound with memAb trastuzumab (100nM) and then washed; and cancer cells pre-bound with memAb trastuzumab (100nM) and then washed). After incubation at 37°C for 6 hours, 50 μl of luciferase substrate (Invivogen#rep-qlc2) was added to each well. Luminescence was rapidly measured using Biotek's Synergy 4 multi-detection microplate reader. [Figure 19] This figure shows T cells (left) and cancer cells (right) that have been pre-bound with memAb. [Figure 20]This figure shows that Herceptin blocks I83E-mediated activation of Jurkat-NFAT-Luc. The memAb trastuzumab and Herceptin were continuously present. Cancer cells (2.5 × 10⁴ cells per 100 μl) were seeded in a 96-well plate with white walls. After cell adhesion overnight, the medium in the plate was removed, and Jurkat-NFAT-Luc me-CAR cells (1 × 10⁵ cells per 60 μl) were added to each well. memAb trastuzumab was either continuously present or pre-bound to Jurkat cells or cancer cells and then washed (the bars in the graph, from left to right, represent continuously present memAb trastuzumab (4nM); continuously present memAb trastuzumab (4nM) and herceptin (40nM); and continuously present memAb trastuzumab (4nM) and herceptin (400nM)). After incubating the cells at 37°C for 6 hours, 50 μl of luciferase substrate (Invivogen#rep-qlc2) was added to each well. Luminescence was rapidly read using Biotek's Synergy 4 multi-detection microplate reader. In the continuous presence of 4nM memAb trastuzumab, Jurkat-NFAT-Luc medi-CAR cells were activated due to memAb trastuzumab binding to cancer cells. Since Herceptin has the same epitope as our memAb trastuzumab and can compete for the same binding site on HER2, the sustained presence of Herceptin during incubation can block Jurkat cell activation. Herceptin (400nM), at a concentration 100 times higher than memAb trastuzumab (4nM), almost completely blocked Jurkat cell activation, confirming that Jurkat cell activation is caused by memAb trastuzumab, which binds to the same HER2 epitope recognized by Herceptin. [Figure 21]This figure shows that Herceptin blocks memAb-mediated activation of Jurkat-NFAT-Luc after Jurkat cells have been pre-bound with memAb trastuzumab and washed. First, memAb trastuzumab was pre-bound to Jurkat cells and washed. Herceptin was continuously present. Cancer cells (2.5 × 10⁴ cells per 100 μl) were seeded in a 96-well plate with a white wall. After cell adhesion overnight, the medium in the plate was removed and Jurkat-NFAT-Luc me-CAR cells (1 × 10⁵ cells per 60 μl) were added to each well. memAb trastuzumab was either continuously present or pre-bound to Jurkat-NFAT-Luc medi-CAR cells and then washed (the bars in the graph, from left to right, represent: continuously present memAb trastuzumab (4nM); Jurkat-NFAT-Luc medi-CAR cells pre-bound with memAb trastuzumab (100nM) and then washed; Jurkat-NFAT-Luc medi-CAR cells pre-bound with memAb trastuzumab (100nM) and then washed + continuously present herceptin (40nM); Jurkat-NFAT-Luc medi-CAR cells pre-bound with memAb trastuzumab (100nM) and then washed + continuously present herceptin (400nM)). 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 rapidly measured using Biotek's Synergy 4 multi-detection microplate reader. Pre-conjugation and washing of Jurkat-NFAT-Luc medi-CAR cells with memAb trastuzumab significantly reduced luminescence activity when co-incubated with high-HER2-expressing cells (SKBR3, BT474, and SKOV3) compared to cells with sustained memAb presence, but the reduction was less pronounced when co-incubated with low-HER2-expressing cells (MCF7 and OVCAR3).The sustained presence of Herceptin during incubation may block the activation of Jurkat cells. The number of medi-CARs on each Jurkat cell or target molecules on each cancer cell may determine how much activation is observed within each T cell, or how many T cells are activated. [Figure 22]This figure shows that Herceptin did little to block the activation of Jurkat-NFAT-Luc mediated by memAb trastuzumab after cancer cells were pre-mixed with memAb and washed. First, memAb trastuzumab was pre-bound to cancer cells and then washed. Herceptin was then continuously present. Pre-binding of cancer cells with memAb trastuzumab and washing did not dramatically reduce luminescence activity compared to cells with continuous memAb presence. Continuous presence of Herceptin during incubation had no or only a limited inhibitory effect on Jurkat cell activation. This data supports the idea that when memAb trastuzumab is bound to HER2 on cancer cells, it is hardly competed for by trastuzumab without a medotope-binding site. Cancer cells (2.5 × 10⁴ cells per 100 µl) were seeded in a 96-well plate with white walls. After cell adhesion overnight, the culture medium in the plate was removed, and Jurkat-NFAT-Luc me-CAR cells (1 × 10⁵) were added to each well. MemAb trastuzumab was either continuously present or pre-bound to the cancer cells and then washed (the bars in the graph, from left to right, represent: continuously present memAb trastuzumab (4 nM); cancer cells pre-bound with memAb trastuzumab (100 nM) and then washed; cancer cells pre-bound with memAb trastuzumab (100 nM) and then washed + continuously present herceptin (40 nM); cancer cells pre-bound with memAb trastuzumab (100 nM) and then washed + continuously present 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 rapidly measured using Biotek's Synergy 4 multi-detection microplate reader. [Figure 23]This figure shows that T cells kill cancer cells that have been pre-mixed with memAb and washed. The survival rate (%) is given by [Luc(cancer cells + T cells + antibody) - Luc(T cells + antibody)] / [Luc(cancer cells + Tmock cells) - Luc(Tmock cells)]. In the sustained presence of 0.1 nM or 0.5 nM mem antibody, the survival rate of cancer cells co-incubated with FabRack T cells was reduced compared to cancer cells co-incubated with mock T cells. In breast cancer cells, cell survival rates decreased in a dose-dependent manner at 0.1 and 0.5 nM concentrations, while ovarian cancer cells showed similar survival rates at both concentrations. Cancer cells pre-bound with antibody and washed away can still be killed by human FabRack T cells, despite a 5-18% reversal of survival rates compared to cancer cells treated with sustained antibody. For tumor killing assays, cancer cells (2.5 × 10⁴ cells per 100 μl) and human T cells (6,250 cells per 100 μl) were seeded in 96-well round-bottom plates, either in the presence or absence of the antibody. After incubation for 72 hours, the cells were centrifuged at 250 × g for 5 minutes, and 100 μl of medium was removed from each well. To examine cell viability, 100 μl of reagent from the Promega CellTiter kit was added to each well. After incubation for 2 minutes, the 100 μl mixture was transferred to a white-walled 96-well plate and measured using a Biotek Synergy 4 multi-detection microplate reader. [Figure 24]This figure shows that FabRack T cells effectively kill cancer cells with 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 shows 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 HER2 expression levels on the cancer cells. For tumor killing assays, cancer cells (2.5 × 10⁴ cells per 100 μl) and human T cells (6,250 cells per 100 μl) were seeded in 96-well round-bottom plates, either in the presence or absence of the antibody. After incubation for 72 hours, the cells were centrifuged at 250 × g for 5 minutes, and 100 μl of medium was removed from each well. To examine cell viability, 100 μl of reagent from the Promega CellTiter kit was added to each well. After incubation for 2 minutes, the 100 μl mixture was transferred to a white-walled 96-well plate and measured using a Biotek Synergy 4 multi-detection microplate reader. [Figure 25] This figure shows the tumor-killing ability of Meditope-CAR T cells (T cells expressing recombinant proteins provided herein, including embodiments thereof) compared to scFv CAR T cells and Fab CAR T cells. [Figure 26]This figure shows the results of a tumor killing assay (FACS) in ovarian cancer. Using flow cytometry, we analyzed how many cancer cells survived after co-incubation with FabRack T cells and memAb trastuzumab, compared to cancer cells incubated with HER2 scFv CAR or HER2Fab CAR T cells as a positive control. After 3 days of incubation, the number of surviving cancer cells dramatically decreased when co-incubated with FabRack T cells and memAb trastuzumab. The killing effect of FabRack T cells was similar to, or even better than, that of HER2 scFv CAR T cells or HER2Fab CAR T cells. [Figure 27] This figure shows the results of a tumor killing assay (FACS) in breast cancer. Using flow cytometry, we analyzed how many cancer cells survived after co-incubation with FabRack T cells and memAb trastuzumab, compared to cancer cells incubated with HER2 scFv CAR or HER2Fab CAR T cells as a positive control. After 3 days of incubation, the number of surviving cancer cells dramatically decreased when co-incubated with FabRack T cells and memAb trastuzumab. The killing effect of FabRack T cells was similar to, or even better than, that of HER2 scFv CAR T cells or HER2Fab CAR T cells. [Figure 28]This figure shows histograms of CD107a and IFN-γ expression within T cells. Flow cytometry showed that approximately 40% of human T cells successfully transduced the Meditope-CAR. When cancer cells were incubated with pre-mixed and washed memAb trastuzumab, or in the sustained presence of memAb trastuzumab, FabRack T cells increased the expression of CD107a and IFNγ. To analyze the expression of CD107a and IFNγ, cancer cells (5 × 10⁴ / 100 μl) were seeded in a 96-well round-bottom plate, and human T mock cells or FabRack cells (5 × 10⁴ cells per 100 μl) were added to each well containing cancer cells. The effector-to-target ratio was 1:1. CD107a-FITC (BD#555800) antibody and the transporter inhibitor Golgistop (BD#554724) were added to each well during incubation. After incubation for 5 hours, the cells were stained with Fixable Viability Dye (Thermo Fisher Scientific #L34965) in the dark at 4°C for 30 minutes. After washing twice, the cells were stained with CD4-PerCP (BD#347324), CD8-APCCy7 (BD#348793), and CD19-PECy7 (BD#557835) in the dark at 4°C for 30 minutes. After two washes, the cells were fixed and permeabilized using the BD Cytofix / Cytoperm kit (BD554714), followed by staining of intracellular IFN with IFN-APC (BD#554702) in the dark at room temperature for 30 minutes. After two washes, the cells were resuspended in a final volume of 100 μl, and 40 μl of the sample was analyzed by flow cytometry. [Figure 29] This is a diagram showing CD107a and IFN-γ. [Figure 30]This figure shows that I83E trastuzumab cannot activate FabRack T cells without target cells. Available activation markers for the study were: Upregulation: CD69 (short survival time), CD137 (4-1BB), CD44, CD27, CD45RO, CD154; Downregulation: CD62L, CCR7 (CD197) (CD25, CD69, CD137 (4-1BB), KLRG, CD62L, CD45RO, CD27, CD28). When incubated with 0.5 nM memAb trastuzumab for 5 hours, FabRack T cells did not show increased expression of CD107a and IFNγ. [Figure 31] This diagram shows the creation of four constructs. The top two schematic diagrams contain truncated CD19 genes, which are used as markers for transformed cells. These two differ by the co-stimulatory signal, C28 or 41BB. The bottom two schematic diagrams are identical except for the CD19 readout marker. [Figure 32] This is a histogram of the expression of cleaved CD19 (CD19t) in Jurkat-NFAT-Luc cells before and after cell sorting. Cells were stained with CD19-PE-Cy7, and CD19t-positive cells were sorted using a BD Aria SORP flow cytometer. This is a homogeneous population of transformed cells isolated by cell sorting using the cleaved CD19 marker. [Figure 33A] This diagram shows the challenges ahead. Meditope-compatible IgG binds to only one of four variants (with or without CD19t and CD28 costimulatory signals or 41BB costimulatory signals). [Figure 33B] This figure shows the challenges ahead. Untransformed Jurkat cells do not bind to IgG. [Figure 33C] This diagram shows the challenges ahead. Similarly, parental antibodies (e.g., non-meditope compatible) do not bind to FabRack Jurkat cells. In other words, different memAbs can be combined with different FabRack variants. [Figure 34]This figure shows the binding of memAbs to HER2 or EGFR in breast cancer cell lines (MCF7, SKBR3, and BT474) or ovarian cancer cell lines (SKOV3 and OVCAR3). The median fluorescence intensity (MFI) of cells with and without memAb binding is shown below each graph. Antigen densities of Her2, EGFR, and CD20 were independently quantified across a series of cell lines using analytical cytometry, which Fabrack subsequently uses extensively to investigate its ability and efficacy in switching antigen specificity. [Figure 35] This figure shows breast or ovarian cancer cells (2.5 × 10⁴ cells) seeded in a 96-well plate with white walls. After cell adhesion overnight, the medium in the plate was removed, and Jurkat-NFAT-Luc Fabrack cells (CD28 type, 1 × 10⁵ cells) with varying doses of memAb (anti-HER2 or anti-EGFR) were added to each well. The cells were incubated at 37°C for 6 hours, after which luciferase substrate (Invivogen#rep-qlc2) was added to each well. Luminescence was rapidly measured using Biotek's Synergy 4 multi-detection microplate reader. EC50 was less than 1 nM for all cells, but the plateaus varied considerably. The increases correlated with antigen expression, except for BT474 cells. This exception warrants further investigation (interference by Her3 or other molecules). A similar trend is observed for EGFR in the right panel. With cetuximab, MFIs were 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 within 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 / . [Figure 36]This figure shows the co-culture of CD19-expressing cancer cells (5 × 10⁴ cells) seeded in a white-walled 96-well plate with Jurkat-NFAT-Luc Fabrack cells (41BB or CD28 type, 1 × 10⁵ cells) accompanied by varying doses of CD19 memAb. The cells were incubated at 37°C for 6 hours, after which luciferase substrate (Invivogen#rep-qlc2) was added to each well. Luminescence was rapidly measured using Biotek's Synergy 4 multi-detection microplate reader. The left panel shows that different CD19-expressing cells can be targeted. The plateau fluctuates with antigen density. The MFI for Raji is 21566; for Daudi, it is 12263; and for Sup-B15, it is 12617. The right panel indicates that the CD28 activation signal is stronger than that of 41BB. [Figure 37]This figure shows the activation of FabRack Jurkat cells in the presence of memAb and target cells. Different doses of memAb trastuzumab IgG or Fab were added to 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 rapidly read using a plate reader. Breast cancer cells or ovarian cancer cells (2.5 × 10⁴ cells) were seeded in a 96-well plate with white walls. After cell adhesion overnight, the medium in the plate was removed, and Jurkat-NFAT-Luc Fabrack cells (CD28 type, 1 × 10⁵ cells) with varying doses of memAb trastuzumab or non-memAb pertuzumab were added to each well. After incubating the cells at 37°C for 6 hours, luciferase substrate (Invivogen#rep-qlc2) was added to each well. Luminescence was rapidly measured using Biotek's Synergy 4 multi-detection microplate reader. Based on our FACS data, MCF7 expresses the lowest amount of Her2 (MFI=985). OvCAR3 expresses a slightly higher amount (MFI=1435). SKBR3 (MFI=34405) and SKOV3 (MFI=43191) express significantly higher amounts than MCF7 and OvCAR3. Pertuzumab, which also binds to Her2, does not correspond to the meditope and contains Fc. The fact that pertuzumab does not bind indicates that a meditope-compatible Fab / Mab is required (as predicted). The hook effect is active in all cell lines using the IgG format. This effect begins early in cells with "low" antigen density (the "fitting" peak 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 being saturated at low concentrations. Valence has a significant impact (for example, the hook effect begins at lower concentrations when using IgG {bivalent} compared to when using Fab {monovalent}).Interestingly, the "plateau" is much higher when using Fab. The fact that a difference is observed suggests that the properties of the memAb can be fine-tuned to optimize the effect. This fact also suggests that the Hook effect can be used as a safety mechanism. Note: In Figures 8 and 9, the Hook effect was not evident. This is partly due to the low concentration range used in these studies (antibody concentrations reached only 10 nM, but 1 uM as used herein). Information on the "Hook effect" can be found on the website:en.wikipedia.org / wiki / Hook_effect. [Figure 38]This figure shows the activation of FabRack Jurkat cells co-cultured with cancer cells in the presence of 4 nM memAb trastuzumab, either pre-conjugated to target cells with memAb trastuzumab or pre-conjugated to FabRack Jurkat cells with memAb trastuzumab. Cancer cells or FabRack Jurkat cells were pre-conjugated with 100 nM memAb trastuzumab and then washed. The activation level of FabRack Jurkat cells with 4 nM Herceptin treatment was similar to that without treatment. Methods: Cancer cells (2.5 × 10⁴ cells per 100 μl) were seeded in a 96-well plate with white walls. After cell adhesion overnight, the medium in the plate was removed and Jurkat-NFAT-Luc medotope-CAR cells (1 × 10⁵ cells per 60 μl) were added to each well. memAb trastuzumab was either continuously present or pre-bound to Jurkat-NFAT-Luc medi-CAR cells or cancer cells, followed by washing. The bars in the graph, from left to right, represent continuously present memAb trastuzumab (4nM), FabRack Jurkat-NFAT-Luc cells pre-bound with memAb trastuzumab (100nM) and then washed, cancer cells pre-bound with memAb trastuzumab (100nM) and then washed, continuously present Herceptin (4nM), and no treatment. After incubating the cells at 37°C for 6 hours, 50 μl of luciferase substrate (Invivogen#rep-qlc2) was added to each well. Luminescence was rapidly measured using Biotek's Synergy 4 multi-detection microplate reader. In all cases, the addition of 4 nM memAB trastuzumab mixed with the “meditope CAR” Jurkat-NFAT-Luc to tumor cells yielded the greatest signal. The other two 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 the “meditope CAR” Jurkat-NFAT-Luc.In other cases, memAB trastuzumab was added to Jurkat-NFAT-Luc, a "meditope CAR," washed, and then added to tumor cells. For cells with high antigen expression, pretreatment of tumor cells resulted in high activation. For cells with low antigen expression (MCF7 and OVCAR3), pre-incubation of Jurkat-NFAT-Luc cells, a "meditope CAR" cell, resulted in high levels of activation. It is important to note that the concentration of memAb after "washing" the cells is unknown, but certainly less than 4 nM. This different treatment likely explains the high signal at 4 nM treatment. [Figure 39]This figure shows that Herceptin blocked the activation of FabRack Jurkat cells co-incubated with cancer cells and 4nM memAb trastuzumab. Herceptin and memAb trastuzumab were continuously present for the entire duration of the treatment. Method: Cancer cells (2.5 × 10⁴ cells per 100 μl) were seeded in a 96-well plate with white walls. After cell adhesion overnight, the medium in the plate was removed, and Jurkat-NFAT-Luc me-CAR cells (1 × 10⁵ cells per 60 μl) were added to each well. The bars in the graph, from left to right, represent continuously present memAb trastuzumab (4nM), continuously present memAb trastuzumab (4nM) and continuously present Herceptin (40nM), and continuously present memAb trastuzumab (4nM) and continuously present Herceptin (400nM). 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 rapidly read using Biotek's Synergy 4 multi-detection microplate reader. Results: In the sustained presence of 4 nM memAb trastuzumab, Jurkat-NFAT-Luc medi-CAR cells were activated due to binding of memAb trastuzumab to cancer cells. Since Herceptin has the same epitope as our memAb trastuzumab and can compete for the same binding site on HER2, the sustained presence of Herceptin during incubation may block the activation of Jurkat cells. Herceptin (400 nM) at a 100-fold increase in concentration compared to memAb trastuzumab (4 nM) nearly completely blocked Jurkat cell activation, supporting the idea that Jurkat cell activation is triggered by memAb trastuzumab, which binds to the same HER2 epitope recognized by Herceptin. Since fabrack T cell activation requires meditope interaction, this provides further evidence for the proposed mechanism of action. [Figure 40]This figure shows that Herceptin pre-bound to cancer cells blocked the activation of cancer cells and FabRack Jurkat cells co-incubated with 4 nM memAb trastuzumab. Cancer cells were pre-bound with 40 nM or 400 nM Herceptin and then washed. The only difference between Herceptin and memAb trastuzumab is that the latter corresponds to a medotope. Pre-treatment of cells with Herceptin blocks access to the antigen. Washing Herceptin-treated cells before memAb trastuzumab / FabRack Jurkat treatment removes unbound Herceptin. However, bound Herceptin dissociates from cells over time. Therefore, the reduction in activation is not as dramatic as in this experiment (compared to Figure 41). [Figure 41]This figure shows that Herceptin hardly blocks the activation of FabRack Jurkat cells co-cultured with cancer cells pre-bound to memAb. Cancer cells were pre-bound to 100 nM memAb trastuzumab and then washed. Pre-binding and washing of cancer cells with memAb trastuzumab did not dramatically reduce luminescence activity. The sustained presence of Herceptin during incubation had a small blocking effect on Jurkat cell activation. This data supports the idea that when memAb trastuzumab is bound to HER2 on cancer cells, the binding is hardly blocked by clinical trastuzumab. Methods: Cancer cells (2.5 × 10⁴ cells per 100 μl) were seeded in a 96-well plate with white walls. After cell adhesion overnight, the medium in the plate was removed and Jurkat-NFAT-Luc me-CAR cells (1 × 10⁵) were added to each well. Cells were either continuously treated with memAb trastuzumab or pre-bound to cancer cells and then washed (the bars in the graph, from left to right, represent: continuously treated memAb trastuzumab (4 nM); cancer cells pre-bound with memAb trastuzumab (100 nM) and then washed; cancer cells pre-bound with memAb trastuzumab (100 nM) and then washed + continuously treated herceptin (40 nM); cancer cells pre-bound with memAb trastuzumab (100 nM) and then washed + continuously treated herceptin (400 nM)). After incubation at 37°C for 6 hours, 50 μl of luciferase substrate (Invivogen#rep-qlc2) was added to each well. Luminescence was rapidly measured using Biotek's Synergy 4 multi-detection microplate reader. When the memAb trastuzumab is bound to cells, it blocks Herceptin from binding to cell-derived antigens. [Figure 42]This figure shows the expression of activation markers and cytokines in FabRack T cells. FabRack T cells were co-cultured for 5 hours in the presence of 0.5 nM memAb trastuzumab with ovarian cancer cells, SKOV3 and OVCAR3, or breast cancer cells, SKBR3, BT474, and MCF7. The ratio of effector cells to target cells was 1:1. HER2 scFv CAR and HER2 Fab CAR were used as positive controls. After a 5-hour incubation, activation markers were analyzed by flow cytometry; numerous controls were used in these experiments. The CDR loops of the scFv CAR and Fab CAR used here are the same as those used for FabRack. Mock cells are non-transformed T cells. Levels of CD69 and CD25, markers for T cell activation, were measured using cytometry. In the absence of tumor cells, no activation of the mutants was observed. Even in the presence of antigen-carrying tumor cells, activation was little to no observed in Fabrack T cells (without memAb), mock cells, or mock cells with I83E memAb trastuzumab. Finally, a marked increase in T cell activation was observed in Fabrack T cells + I83E memAb trastuzumab and scFVCAR T cells. Fab-CAR T cells were also activated, but with considerable variability. [Figure 43] This figure shows FabRack T cells 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 ratio of effector cells to target cells was 1:1. HER2 scFv CAR was used as a positive control. After 5 hours of incubation, the degranulation markers CD107a and IFNγ were analyzed by flow cytometry: Fab T cells behaved similarly to conventional CAR T cells. [Figure 44]This figure shows cancer cells incubated with mock T cells or FabRack T cells for 3 days in the presence of varying doses of memAb trastuzumab. The ratio of effector cells to target cells was 1:4. At the end of incubation, cell viability was measured according to the instructions on the Promega CellTiter kit. Results: Cancer cells were incubated with mock T cells or FabRack T cells for 3 days in the presence of memAb trastuzumab. 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 toxic effect was not associated with HER2 expression levels on cancer cells. Methods: For the tumor killing assay, cancer cells (2.5 × 10⁴ cells per 100 μl) and human T cells (6,250 cells per 100 μl) were seeded in 96-well round-bottom plates in or without the antibody. After incubation for 72 hours, the cells were centrifuged at 250 × g for 5 minutes, and 100 μl of medium was removed from each well. To examine cell viability, 100 μl of reagent from the Promega CellTiter kit was added to each well. After a 2-minute incubation, 100 µl of the mixture was transferred to a 96-well plate with white walls and measured using Biotek's Synergy 4 multi-detection microplate reader. [Figure 45] This figure shows that the IC50 values ​​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). [Figure 46]The results are shown in the figure: In the sustained presence of 0.1 nM or 0.5 nM memAb antibody, the survival rate of cancer cells co-incubated with FabRack T cells (center bar) was reduced compared to cancer cells co-incubated with mock T cells (left bar). In breast cancer cells, cell viability decreased in a dose-dependent manner at 0.1 and 0.5 nM concentrations, while ovarian cancer cells showed similar viability at both concentrations. Cancer cells pre-bound with the antibody and washed away (right bar) could still be killed by human FabRack T cells, despite a 5-18% reversal of survival rates compared to cancer cells treated with sustained antibody. Methods: For the tumor killing assay, cancer cells (2.5 × 10⁴ cells per 100 μl) and human T cells (6,250 cells per 100 μl) were seeded in 96-well round-bottom plates in or without memAb antibody. After a 72-hour incubation, the cells were centrifuged at 250 × g for 5 minutes, and 100 μl of medium was removed from each well. To examine cell viability, 100 μl of reagent from the Promega CellTiter kit was added to each well. After a 2-minute incubation, the 100 μl mixture was transferred to a 96-well plate with white walls and measured using Biotek's Synergy 4 multi-detection microplate reader. [Figure 47]This figure shows the analysis of cancer cell survival after DAPI staining 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 still survived after co-incubation with FabRack T cells and memAb trastuzumab, compared to cancer cells incubated with HER2 scFv CAR or HER2Fab CAR T cells as positive controls. After 3 days of incubation, the number of surviving cancer cells dramatically decreased when co-incubated with FabRack T cells and memAb trastuzumab. The killing effect of FabRack T cells was similar to, or even better than, that of HER2 scFv CAR T cells or HER2Fab CAR T cells. Similar results were obtained using alternative methods for reading cell viability. [Figure 48] This figure shows the analysis of cancer cell survival after DAPI staining using flow cytometry. HER2 scFv CAR and HER2 Fab CAR were used as positive controls. Results: Using flow cytometry, we analyzed how many cancer cells still survived after co-incubation with FabRack T cells and memAb trastuzumab, compared to cancer cells incubated with HER2 scFv CAR or HER2Fab CAR T cells as positive controls. After 3 days of incubation, the number of surviving cancer cells dramatically decreased when co-incubated with FabRack T cells and memAb trastuzumab. The killing effect of FabRack T cells was similar to, or even better than, that of HER2 scFv CAR T cells or HER2Fab CAR T cells. [Figure 49]This figure shows the multiplicative changes in T cell counts in target cells and co-cultures with different concentrations of the memAb trastuzumab. The ratio of effector cells to target cells is 1:4 at incubation. These experiments indicate T cell activation that leads to cell proliferation in response to the presence of memAb. [Figure 50] This figure shows breast cancer cells (2.5 × 10⁴ cells) seeded in a 96-well plate with white walls. After cell adhesion overnight, the culture medium in the plate was removed, and Jurkat-NFAT-Luc Fabrack cells (type 41BB, 1 × 10⁵ cells) with varying doses of 2N1 memAb were added to each well. The cells were incubated at 37°C for 6 hours, after which luciferase substrate was added to each well. Luminescence was rapidly read using a Biotek Synergy 4 multi-detection microplate reader. [Modes for carrying out the invention]

[0018] definition While various embodiments and aspects of the present invention have been described and illustrated herein, it will be apparent to those skilled in the art that such embodiments are presented for illustrative purposes only. Now, without departing from the present invention, those skilled in the art will conceive of numerous variations, alterations, and substitutions. It should be understood that various substitutes for the embodiments of the present invention described herein may be used in the practice of the present invention.

[0019] The section headings used herein are for organizational purposes only and should not be considered to limit the subject matter described. Without limitation, all documents or parts of documents cited herein, including patents, patent applications, articles, books, manuals and treatises, are expressly incorporated in whole by reference for any purpose.

[0020] The abbreviations used herein have their conventional meanings within the fields of chemistry and biotechnology. The chemical structures and formulas expressed herein are constructed in accordance with standard rules for chemical values ​​known in the fields of chemistry.

[0021] When substituents are specified by their conventional chemical formulas and written from left to right, they equally encompass chemically identical substituents that would result from writing the structure from right to left; for example, -CH2O- is equivalent to -OCH2-.

[0022] The term "alkyl" means, unless otherwise stated, a linear (i.e., unbranched) or branched acyclic carbon chain (or carbon) or combination thereof, which may be fully saturated, monounsaturated, or polyunsaturated, either by itself or as part of another substituent, and which has a number of carbon atoms as indicated (i.e., C1-C1). 10 A saturated hydrocarbon group may include divalent and polyvalent groups having 1 to 10 carbon atoms. Examples of saturated hydrocarbon groups 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 of n-pentyl, n-hexyl, n-heptyl, and n-octyl. An unsaturated alkyl group is an alkyl group having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, clotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-propynyl and 3-propynyl, 3-butynyl, and higher homologs and isomers. An alkoxy is an alkyl group bonded to the rest of the molecule via an oxygen linker (-O-). The alkyl portion can be an alkenyl portion. The alkyl portion can be an alkynyl portion. The alkyl portion can be a fully saturated alkyl portion.

[0023] The term "alkylene," unless otherwise stated, means a divalent group derived from an alkyl group, either by itself or as part of another substituent, exemplified by, but not limited to, -CH2CH2CH2CH2-. Typically, alkyl (or alkylene) groups have 1 to 24 carbon atoms, but in the present invention, groups having 10 or fewer carbon atoms are preferred. "Lower alkyl" or "lower alkylene" generally refers to a short-chain alkyl or short-chain alkylene group having 8 or fewer carbon atoms. The term "alkenylene," unless otherwise stated, means a divalent group derived from an alkene, either by itself or as part of another substituent.

[0024] The term "heteroalkyl," unless otherwise stated, means, by itself or in combination with another term, a stable acyclic linear or branched chain or combination thereof, comprising at least one carbon atom and at least one heteroatom (e.g., O, N, P, Si, or S), wherein the nitrogen and sulfur atoms may be optionally oxidized, and the heteroatom with nitrogen may be optionally quaternized. The heteroatoms O, N, P, S, and Si may be located at any internal position of the heteroalkyl group or at a position where the alkyl group is bonded 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, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3 can have up to two or three heteroatoms in a row. The heteroalkyl portion may contain one heteroatom (e.g., O, N, S, Si, or P). The heteroalkyl moiety may contain two arbitrarily distinct heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may contain three arbitrarily distinct heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may contain four arbitrarily distinct heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may contain five arbitrarily distinct heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may contain up to eight arbitrarily distinct heteroatoms (e.g., O, N, S, Si, or P).

[0025] Similarly, the term "heteroalkylene," unless otherwise stated, means a divalent group derived from a heteroalkyl group, either by itself or as part of another substituent, exemplified by, but not limited to, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, the heteroatom may also occupy one or both of the chain ends (e.g., alkylene oxy, alkylenedioxy, alkylene amino, alkylenediamino, etc.). Furthermore, for alkylene and heteroalkylene linkers, the orientation of the linker is not implied by the direction in which the formula of the linker is written. For example, the formula -C(O)2R'- represents both -C(O)2R'- and -R'C(O)2-. As stated above, the heteroalkyl groups used herein include groups that are bonded to the rest of the molecule via a heteroatom, such as -C(O)R', -C(O)NR', ​​-NR'R'', -OR', -SR', and / or -SO2R'. When "heteroalkyl" is enumerated followed by a list of specific heteroalkyl groups, such as -NR'R'', it should be understood that the terms heteroalkyl and -NR'R'' are neither redundant nor mutually exclusive. Rather, the specific heteroalkyl groups are enumerated for clarity. Therefore, the term "heteroalkyl" herein should not be interpreted as excluding specific heteroalkyl groups, such as -NR'R''.

[0026] The terms "cycloalkyl" and "heterocycloalkyl," unless otherwise stated, mean, either by themselves or in combination with other terms, the non-aromatic cyclic forms of "alkyl" and "heteroalkyl," respectively, in which case the carbon atoms constituting one or more rings do not necessarily have to be bonded to hydrogen, due to all carbon bond values ​​that participate in bonding with atoms other than hydrogen. In addition, for heterocycloalkyls, heteroatoms may occupy positions where the heterocycle is bonded 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-cyclobuta-3-enyl-1,2-dione, 1H-1,2,4-triazoyl-5(4H)-one, and 4H-1,2,4-triazoyl. Examples of heterocycloalkyls 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, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, etc. "Cycloalkylene" and "heterocycloalkylene" mean a divalent group derived from a cycloalkyl and a heterocycloalkyl, respectively, either alone or as part of another substituent. A heterocycloalkyl moiety may contain one ring heteroatom (e.g., O, N, S, Si, or P). A heterocycloalkyl moiety may contain two optionally distinct ring heteroatoms (e.g., O, N, S, Si, or P). A heterocycloalkyl moiety may contain three optionally distinct ring heteroatoms (e.g., O, N, S, Si, or P). The heterocycloalkyl moiety may contain four optionally distinct ring heteroatoms (e.g., O, N, S, Si, or P). The heterocycloalkyl moiety may contain five optionally distinct ring heteroatoms (e.g., O, N, S, Si, or P). The heterocycloalkyl moiety may contain up to eight optionally distinct ring heteroatoms (e.g., O, N, S, Si, or P).

[0027] The terms "halo" or "halogen" mean, unless otherwise stated, a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, either by themselves or as part of another substituent. In addition, terms such as "haloalkyl" are intended to include monovalent and polyvalent haloalkyls. For example, the term "halo(C1-C4)alkyl" includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, and 3-bromopropyl.

[0028] The term "acyl" means -C(O)R [wherein 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] unless otherwise stated.

[0029] The term "aryl" means a polyunsaturated aromatic hydrocarbon substituent that may be a single ring, fused to one another (i.e., a fused ring aryl), or a group of rings (preferably 1 to 3 rings) linked by covalent bonds, unless otherwise stated. A fused ring aryl refers to a group of rings fused to one another, in which case 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, in which case the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom(s) are optionally quaternized. Therefore, the term "heteroaryl" includes a fused ring heteroaryl group (i.e., a group of rings fused to one another, in which at least one of the fused rings is a heteroaromatic ring). A 5,6-fused ring heteroarylene refers to two rings fused to one another, one of which has 5 members and the other has 6 members, in which case at least one of the rings is a heteroaryl ring. Similarly, a 6,6-fused heteroarylene refers to two fused rings, one having six members and the other having six members, in which case at least one ring is a heteroaryl ring. Furthermore, a 6,5-fused heteroarylene refers to two fused rings, one having six members and the other having five members, in which case at least one ring is a heteroaryl ring. The heteroaryl group may be bonded to the rest of the molecule via a carbon atom or heteroatom.Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrzoyl, 2-imidazoyl, 4-imidazoyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2- This includes thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazoyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Each substituent of the aryl and heteroaryl ring systems mentioned above is selected from the group of acceptable substituents listed below. "Arylene" and "heteroarylene" mean divalent radicals derived from aryl and heteroaryl, respectively, either alone or as part of another substituent. Non-limiting examples of aryl and heteroaryl groups include pyridinyl, pyrimidinyl, thiophenyl, thienyl, furanyl, indolyl, benzoxadiazolyl, benzodioxolyl, benzodioxanyl, thianaphthalyl, pyrrolopyridinyl, indazolyl, quinolinyl, quinoxalinyl, pyridopyradinyl, quinazolinol, benzoisoxazolyl, imidazopyridinyl, benzofuranyl, benzothienyl, benzothiophenyl, phenyl, naphthyl, and biphenyl. This includes pyrrolyl, pyrazoyl, imidazoyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl, furylthienyl, pyridyl, pyrimidyl, benzothiazolyl, purinyl, benzimidazoyl, isoquinolyl, thiadiazolyl, oxadiazolyl, pyrrolyl, diazolyl, triazoyl, tetrazolyl, benzothiadiazolyl, isothiazolyl, pyrazolopyrimidinyl, pyrrolopyrimidinyl, benzotriazolyl, benzoxazolyl, or quinolyl. The above examples may be substituted heteroaryls or unsubstituted heteroaryls, and the divalent group in each of the above heteroaryl examples is an unrestricted example of a heteroarylene.The heteroaryl moiety may contain one ring heteroatom (e.g., O, N, or S). The heteroaryl moiety may contain two arbitrarily distinct ring heteroatoms (e.g., O, N, or S). The heteroaryl moiety may contain three arbitrarily distinct ring heteroatoms (e.g., O, N, or S). The heteroaryl moiety may contain four arbitrarily distinct ring heteroatoms (e.g., O, N, or S). The heteroaryl moiety may contain five arbitrarily distinct ring heteroatoms (e.g., O, N, or S). The aryl moiety may have a single ring. The aryl moiety may have two arbitrarily distinct rings. The aryl moiety may have three arbitrarily distinct rings. The aryl moiety may have four arbitrarily distinct rings. The heteroaryl moiety may have one ring. The heteroaryl moiety may have two arbitrarily distinct rings. The heteroaryl moiety may have three arbitrarily distinct rings. The heteroaryl moiety may have four arbitrarily distinct rings. The heteroaryl moiety may have five arbitrarily distinct rings.

[0030] A fused ring heterocycloalkyl-aryl is an aryl compound fused with a heterocycloalkyl group. A fused ring heterocycloalkyl-heteroaryl is a heteroaryl compound fused with a heterocycloalkyl group. A fused ring heterocycloalkyl-cycloalkyl is a heterocycloalkyl compound fused with a cycloalkyl group. A fused ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkyl compound fused with another heterocycloalkyl group. Each of a fused ring heterocycloalkyl-aryl, fused ring heterocycloalkyl-heteroaryl, fused ring heterocycloalkyl-cycloalkyl, or fused ring heterocycloalkyl-heterocycloalkyl may be unsubstituted or substituted with one or more substituents described herein.

[0031] As used herein, the term "oxo" refers to oxygen double-bonded to a carbon atom.

[0032] As used herein, the term "alkylsulfonyl" means a portion having the formula -S(O2)-R' [wherein R' is a substituted or unsubstituted alkyl group as defined above]. R' may have a specified number of carbon atoms (e.g., "C1-C4 alkylsulfonyl").

[0033] Each of the above terms (e.g., "alkyl," "heteroalkyl," "cycloalkyl," "heterocycloalkyl," "aryl," and "heteroaryl") includes both substituted and unsubstituted forms of the indicated group. Preferred substituents for each group are listed below.

[0034] Alkyl and heteroalkyl group substituents (including groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) are defined as -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', -S(O)2R', -S(O)2N(R)('R''-NRSO2R'), -CN, and -NO2 may be one or more of a variety of groups, selected from but not limited to these. R', R'', R''', and R'''' each preferably independently refers to hydrogen, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted heterocycloalkyl group, a substituted or unsubstituted aryl group (e.g., aryl substitution with one to three halogens), a substituted or unsubstituted alkyl group, an alkoxy group or a thioalkoxy group, or an arylalkyl group. If the compounds of the present invention contain, for example, more than one R group, each R group is selected independently, as are the groups selected when more than one of each R', R'', R''', and R'''' group is present. When R' and R'' are bonded to the same nitrogen atom, they can be combined with the nitrogen atom to form a four-membered, five-membered, six-membered, or seven-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" is intended to include groups containing carbon atoms bonded to groups other than hydrogen groups, such as haloalkyls (e.g., -CF3 and -CH2CF3) and acyls (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, etc.).

[0035] Similar to substituents described for alkyl groups, substituents for aryl and heteroaryl groups are diverse, ranging in number from zero to the total number of open bond values ​​on the aromatic ring system, for example, -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( The R groups are selected from O)2R', -S(O)2N(R')(R'', -NRSO2R'), -CN, -NO2, -R', -N3, -CH(Ph)2, fluoro(C1-C4)alkoxy, and fluoro(C1-C4)alkyl; 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. If the compounds of the present invention contain, for example, more than one R group, each R group is independently selected, as are the groups selected when more than one of each R', R'', R''', and R'''' is present.

[0036] If a portion is substituted with an R substituent, the group may be referred to as "R-substituted." When a portion is R-substituted, the portion is substituted with at least one R substituent, each R substituent being optionally different. For example, the portion in this specification is R 1A If it is an alkyl group that is substituted or unsubstituted by multiple R 1A The substituent may be bonded to the alkyl portion, in which case each R 1A The substituents are arbitrarily different. When an R-substituted moiety is substituted by multiple R substituents, each R substituent may be differentiated using a prime symbol ('), such as R', R'', etc. For example, if the moiety is R 3A A substituted or unsubstituted alkyl group, the portion being a plurality of R 3A If substituted by substituents, multiple R3A The substituent is R 3A ’, R 3A ’’, R 3A ’’’ etc. and can be differentiated. In some embodiments, the plurality of R substituents are three R substituents. In some embodiments, the plurality of R substituents are two R substituents.

[0037] Two or more substituents can be optionally connected to form an aryl group, a heteroaryl group, a cycloalkyl group or a heterocycloalkyl group. Such so-called ring-forming substituents are typically found to be joined to the cyclic base structure, although not necessarily. In one embodiment, the ring-forming substituent is joined to an adjacent member of the base structure. For example, two ring-forming substituents joined to adjacent members of the cyclic base structure create a fused ring structure. In another embodiment, the ring-forming substituent is joined to a single member of the base structure. For example, two ring-forming substituents joined to a single member of the cyclic base structure create a spiro ring structure. In yet another embodiment, the ring-forming substituent is joined to non-adjacent members of the base structure.

[0038] Two of the substituents on adjacent atoms of an aryl ring or heteroaryl ring can optionally form a ring of the formula -T-C(O)-(CRR’) q -U- [wherein, T and U are independently -NR-, -O-, -CRR’- or a single bond, and q is an integer from 0 to 3]. Alternatively, two of the substituents on adjacent atoms of an aryl ring or heteroaryl ring can optionally be replaced by a substituent of the formula -A-(CH2) r -B- [wherein, A and B are independently -CRR’-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR’- or a single bond, and r is an integer from 1 to 4]. One of the single bonds of the newly formed ring thus formed can optionally be replaced by a double bond. Alternatively, two of the substituents on adjacent atoms of an aryl ring or heteroaryl ring can optionally be of the formula -(CRR’) s -X’-(C’’R’’R’’’) d-[wherein the formula, the variables s and d are independently integers between 0 and 3, and X' is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-] may be replaced by substituents. The 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.

[0039] As used herein, the terms “heteroatom” or “ring heteroatom” are intended to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0040] As used herein, "substituent" refers to the following parts: (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 (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, (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 (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, (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 (b) Alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, which are substituted with at least one substituent selected from 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, and unsubstituted aryl. Alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from the above. Alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from the above. It means a base selected from.

[0041] As used herein, "size-limited substituent" means a group selected from all of the substituents described above for a substituent, in which case each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C 20Each alkyl group is a substituted or unsubstituted heteroalkyl group, each substituted or unsubstituted 2-20 member heteroalkyl group, each substituted or unsubstituted cycloalkyl group is a substituted or unsubstituted C3-C8 cycloalkyl group, each substituted or unsubstituted heterocycloalkyl group is a substituted or unsubstituted 3-8 member heterocycloalkyl group, and each substituted or unsubstituted aryl group 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.

[0042] As used herein, "lower substituent" means a group selected from all of the substituents described above for a substituent, in which case each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2-8 member heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3-7 member heterocycloalkyl, and each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C 10 It is an aryl group, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5- to 9-membered heteroaryl.

[0043] In some embodiments, each substituent described in the compounds herein is substituted with at least one substituent. More specifically, in some embodiments, each of the 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 herein 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.

[0044] In other embodiments of the compounds described herein, each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C 20 It can be alkyl, and each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2-20 member heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3-8 member heterocycloalkyl, and each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C 10 The aryl and / or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5- to 10-membered heteroaryl. In some embodiments of the compounds herein, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C 20 Each alkylene is a substituted or unsubstituted heteroalkylene, each substituted or unsubstituted 2-20 member heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C8 cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3-8 member 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 10-membered heteroarylene.

[0045] 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 2-8 member heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3-7 member heterocycloalkyl, and each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C 10Each 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 The compound 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 specified in the Examples section, figures, or tables below.

[0046] As used herein, the term “conjugate” refers to an association between atoms or molecules. Such associations may be direct or indirect. For example, a conjugate between nucleic acids and proteins may be a direct conjugate, for example, by covalent bonding, or an indirect conjugate, for example, by non-covalent bonding (e.g., electrostatic interactions (e.g., ionic, hydrogen, halogen bonds), van der Waals interactions (e.g., dipole-dipole interactions, dipole-induced dipole interactions, London dispersion forces), ring stacking (Pi effect), hydrophobic interactions, etc.). In several embodiments, conjugates are formed using conjugate chemical reactions, including but not limited to nucleophilic substitution (e.g., reactions of amines and alcohols with acyl halides and active esters), electrophilic substitution (e.g., enamine reactions), and addition to multiple bonds between carbon atoms and carbon-heteroatoms (e.g., Michael reactions, Diels-Alder additions). These useful reactions and other useful reactions are discussed, for example, in March, "Advanced Organic Chemistry," 3rd edition, 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 several embodiments, the microparticles are non-covalently bonded to the solid support via non-covalent chemical reactions between the components of the microparticles and the components of the solid support. In other embodiments, the microparticles comprise one or more reactive moieties, for example, covalently reactive moieties (e.g., amine-reactive moieties) as described herein. In other embodiments, the microparticles comprise a linker comprising one or more reactive moieties, for example, covalently reactive moieties (e.g., amine-reactive moieties) as described herein.

[0047] Useful reactive moieties or functional groups used in conjugate chemical reactions (including "click reactions" known in the art) as described herein include, for example, (a) Carboxyl groups and various derivatives thereof, including but not limited to N-hydroxysuccinimide esters, N-hydroxybenztriazole esters, halogen acids, acylimidazoles, thioesters, p-nitrophenyl esters, alkyls, alkenyls, alkynyls, and aromatic esters; (b) Hydroxyl groups that can be converted to esters, ethers, aldehydes, etc. (c) A haloalkyl group in which the halide is subsequently replaced by a nucleophile such as an amine, carboxylate anion, thiol anion, carbanion, or alkoxide ion, thereby resulting in a covalent bond at the halogen atom site of the new group; (d) Dienophile groups that can participate in the Diels-Alder reaction, such as maleimide groups; (e) an aldehyde or ketone group that allows subsequent derivatization, for example, through the formation of a carbonyl derivative such as an imine, hydrazone, semicarbazone, or oxime, or through a mechanism such as Grignard addition or alkyllithium addition; (f) For example, a sulfonyl halogenated group for reaction with a subsequent amine to form a sulfonamide; (g) Thiol groups that may be converted to disulfides, react with acyl halides, or bonded to metals such as gold; (h) For example, an amine group or sulfhydryl group that can be acylated, alkylated, or oxidized; (i) Alkenes, for example, that undergo cycloaddition, acylation, Michael addition, etc. (j) For example, epoxides that can react with amine compounds and hydroxyl compounds; (k) Phosphoramidites and other standard functional groups useful in nucleic acid synthesis; (l) Metal-silicon oxide bond; (m) For example, metallic bonding to a reactive phosphorus group (e.g., phosphine) that forms a phosphate diester bond, and (n) Sulfones, including vinyl sulfone, for example.

[0048] In the art, the chemical synthesis of compositions by linking low molecular weight modular units using conjugate ("click") chemical reactions is well known, for example, all of which are incorporated herein by reference in their entirety and for all purposes, including: HCOlb, MGFinn and KBSharpless ((2001), "Click Chemistry: Diverse Chemical Function from a Few Good Reactions," Angewandte Chemie International Edition, 40(11):2004~2021); RAEvans ((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; WCGuida 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.This is described in (2008), "Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity," Journal of American Chemical Society, 130(41):13518~13519; Devaraj, Neal K., Weissleder, Ralph, and Hilderbrand, Scott A. (2008), "Tetrazine Based Cycloadditions: Application to Pretargeted Live Cell Labeling," Bioconjugate Chemistry, 19(12):2297~2299); Stoeckmann, Henning; Neves, Andre; Stairs, Shaun; Brindle, Kevin; and Leeper, Finian (2011), "Exploring isonitrile-based click chemistry for ligation with biomolecules," Organic & Biomolecular Chemistry).

[0049] Reactive functional groups may be selected so as not to participate in or interfere with the chemical stability of the proteins or nucleic acids described herein. For example, nucleic acids may contain vinyl sulfone or other reactive moieties (e.g., maleimide). Optionally, nucleic acids may contain a reactive moiety having the formula -SSR, where R is, for example, a protecting group. Optionally, R is hexanol. As used herein, the term "hexanol" refers to a compound of the formula C6H 13The compounds include those with an OH group, and 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.

[0050] As used herein, the term “about” means a range of values ​​including the specified value, which a person skilled in the art would reasonably consider to be similar to the specified value. In some embodiments, the term “about” means within a standard deviation, using generally acceptable measurements in the art. In some embodiments, “about” means a range extending up to ±10% from the specified value. In some embodiments, “about” means the specified value.

[0051] As used herein, the terms “a” or “an” mean one or more. In addition, as used herein, the phrase “substituted with a[n]” means that the specified group may be substituted with one or more of the named substituents. For example, a group such as an alkyl group or heteroaryl group may be “unsubstituted C1-C 20 If the group is "substituted with an alkyl or unsubstituted 2-20 member heteroalkyl group", then the group is one or more unsubstituted C1-C 20 It may contain alkyl and / or one or more unsubstituted 2- to 20-membered heteroalkyl groups. Furthermore, if the portion is substituted with an R substituent, the group may be referred to as "R-substituted". If the portion is R-substituted, the portion is substituted with at least one R substituent, each R substituent being optionally different.

[0052] Unless otherwise specified, technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. See, for example, Singleton et al., "DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY," 2nd edition, 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 methods, devices, and materials similar to or equivalent to those described herein may be used in the practice of the present invention. The following definitions are provided to facilitate the understanding of certain terms that are frequently used herein and are not intended to limit the scope of this disclosure.

[0053] "Biological specimen" or "specimen" refers to material obtained from or derived from a subject or patient. Biological specimens include tissue sections and frozen sections taken for histological purposes, such as biopsy and autopsy specimens. Such specimens include bodily fluids such as blood and blood fractions or blood products (e.g., serum, plasma, platelets, red blood cells), sputum, tissues, cultured cells (e.g., primary cultures, explants, 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 specimens are typically obtained from primates, e.g., chimpanzees or humans; cattle; dogs; cats; rodents, e.g., guinea pigs, rats, mice; mammals or birds such as rabbits; and eukaryotes such as reptiles or fish.

[0054] As used herein, “cell” means a cell that performs metabolic or other functions sufficient to preserve or replicate its genomic DNA. Cells can be identified by methods well known in the art, including, for example, the presence of an intact membrane, staining with a specific dye, the ability to produce offspring, or, in the case of gametes, the ability to mamate with a second gamete to produce viable progeny cells. Cells may include prokaryotic cells 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 mammalian cells, insect (e.g., Spodoptera) cells, and human cells. Cells may be useful if they are non-adherent in nature or have been treated, for example, by trypsin treatment to prevent adhesion to a surface.

[0055] In this specification, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to polymers of amino acid residues, in which case the polymer may optionally be conjugated with a non-amino acid portion. The terms apply to amino acid polymers, which are artificial chemical mimics of corresponding natural amino acids, as well as natural and non-natural amino acid polymers, in addition to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding natural amino acids. A “fusion protein” refers to a chimeric protein that encodes two or more distinct protein sequences recombinantly expressed as a single portion.

[0056] The terms “peptidyl” and “peptidyl moiety” refer to a peptide conjugated to the remaining portion of a 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 by a chemical linker used to conjugate the peptidyl moiety to the remaining portion of the recombinant protein (e.g., a transmembrane domain, spacer region, or peptidyl linker). The peptidyl moiety may also be substituted by further chemical moieties (e.g., further R substituents). In several embodiments, a non-CDR Fab-binding peptide domain comprises a peptidyl moiety. In several embodiments, a non-CDR Fab-binding peptide domain is a peptidyl moiety. The term “meditope” as used herein refers to a peptidyl moiety contained within a peptide domain described herein. Therefore, in several embodiments, a non-CDR Fab-binding peptide domain is a meditope. In several embodiments, a non-CDR Fab-binding peptide domain comprises a meditope.

[0057] The peptidyl moiety (e.g., meditope) may be a linear peptide moiety or a cyclic peptide moiety. A variety of methods can be used for crystallizing the peptide moiety, for example, to address in vivo stability and to allow chemoselective control for subsequent conjugation chemical reactions. In some embodiments, the crystallization strategy is a lactam crystallization strategy, including head-to-tail (head-to-tail) lactam crystallization (between terminal residues of an acyclic peptide) and / or lactam linking between other residues. Lactam formation is also influenced by incorporating residues such as glycine, β-Ala, and / or 7-aminoheptanoic acid into the acyclic peptide crystal precursor to result in different lactam ring sizes and linkage patterns. Further crystallization strategies, such as "click" chemical reactions and olefin metathesis, may also be used. Methods for crystallizing such peptides and peptide mimetic compounds are well known in the art. In several embodiments, the peptidyl moiety (e.g., meditope) is a linear peptidyl moiety (e.g., linear meditope). In several embodiments, the peptidyl moiety (e.g., meditope) is a cyclic peptidyl moiety (e.g., cyclic meditope).

[0058] "Label," "detection domain," or "detection portion" refers to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, a useful label is: 32 The material includes P, a fluorescent dye, a high electron-density reagent, an enzyme (for example, commonly used in ELISA), biotin, digoxigenin or hapten, and a protein or other entity that can be detected 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 for conjugating the antibody with the label may be used, for example, the method described in Hermanson, "Bioconjugate Tecqueniques," 1996, Academic Press, Inc., San Diego.

[0059] A "labeled protein or labeled polypeptide" is a protein or polypeptide to which a label is bound, either covalently via a linker or chemical bond, or noncovalently via an ionic bond, van der Waals bond, electrostatic bond, or hydrogen bond, such that the presence of the labeled protein or labeled polypeptide can be detected by detecting the presence of the label bound to the labeled protein or labeled polypeptide. Alternatively, a method using high-affinity interactions, in which one of a pair of binding partners binds to the other, for example, biotin or streptavidin, can achieve the same result.

[0060] The term "amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimes that function similarly to natural amino acids. Natural amino acids include those encoded by the genetic code, as well as subsequently modified amino acids, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as natural amino acids, i.e., hydrogen, carboxyl group, amino group, and an α-carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as natural amino acids. Amino acid mimes refer to compounds that have a structure different from the general chemical structure of amino acids, but function similarly to natural amino acids.

[0061] In this specification, amino acids may be referred to by their commonly known three-letter or one-letter codes, as recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted one-letter codes.

[0062] The "position" of an amino acid or nucleotide base is represented by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position compared to the N-terminus (or 5'-terminus). Generally, the amino acid residue numbers in the test sequence, determined simply by counting from the N-terminus, may not necessarily be the same as the numbers of their corresponding positions in the reference sequence, due to deletions, insertions, cleavages, fusions, etc., which may be considered when determining optimal alignment. For example, if a mutant has a deletion compared to the aligned reference sequence, there is no amino acid in the mutant that corresponds to the position in the reference sequence at the deletion site. If an insertion exists in the aligned reference sequence, this insertion does not correspond to a numbered amino acid position in the reference sequence. In the case of cleavage or fusion, there may be sequences of amino acids in the reference sequence or the aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0063] When used in the context of numbering a given amino acid sequence or polynucleotide sequence, the terms “numbered in reference to” or “corresponding to” refer to the numbering of residues in a specified reference sequence when the given amino acid sequence or polynucleotide sequence is compared to a reference sequence. An amino acid residue in a protein “corresponds” to a given residue if it occupies the same essential structural position in the protein as the given residue. For example, a selected residue in a selected antibody (or Fab domain) corresponds to the light chain threonine at position 40 according to Kabat if the selected residue occupies the same essential spatial or other structural relationship as the light chain threonine at position 40 according to Kabat. In some embodiments, when a selected protein is aligned with the light chain of an antibody (or Fab domain) for maximum homology, the position in the selected protein that aligns with threonine 40 corresponds to threonine 40. For example, if the structure of a selected protein is aligned to the greatest correspondence between the light chain threonine at position 40 and the overall structure being compared, according to Kabat, then three-dimensional structural alignment may also be used instead of primary sequence alignment. In this case, the amino acid occupying the same essential position as threonine 40 in the structural model is considered to correspond to the threonine 40 residue.

[0064] The term "conservatively modified variant" applies to both amino acid sequences and nucleic acid sequences. For a given nucleic acid sequence, a conservatively modified variant refers to a nucleic acid encoding an identical or essentially identical amino acid sequence, or, if the nucleic acid does not encode an amino acid sequence, an essentially identical sequence. Due to the degeneracy of gene coding, numerous functionally identical nucleic acid sequences encode any given amino acid residue. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at any position where alanine is specified by the codon, the codon can be changed to one of the corresponding codons listed without altering the encoded polypeptide. Such nucleic acid mutations are a type of conservative modification mutation, known as "silent mutations." Every nucleic acid sequence encoding a polypeptide described herein also describes all possible silent mutations of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the codon for methionine only, and TGG, which is usually the codon for tryptophan only) can be modified to result in a functionally identical molecule. Therefore, each silent mutation in the nucleic acid encoding the polypeptide is implied within each described sequence with respect to the expression product, but not with respect to the probe sequence.

[0065] With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to nucleic acid sequences, peptide sequences, polypeptide sequences, or protein sequences, which alter, add, or delete a single amino acid or a small percentage of an amino acid within the encoded sequence, are "conservatively modified variants" if the alteration results in a substitution with an amino acid that is chemically similar to the original amino acid. Tables of conservative substitutions resulting in functionally similar amino acids are well known in the art. Such conservatively modified variants also include, but are not excluded, polymorphic variants, interspecific homologs, and alleles of the present invention.

[0066] The following eight groups each contain amino acids that are conserved substitutions for each other: 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) (see, for example, Creighton, "Proteins" (1984)).

[0067] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof, as well as their complements, in single-stranded or double-stranded form. The term "polynucleotide" refers to a linear sequence of nucleotides. The term "nucleotide" typically refers to a single polynucleotide unit, i.e., a monomer. A nucleotide may be a ribonucleotide, a deoxyribonucleotide, or a modified form thereof. Examples of polynucleotides envisioned herein include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA (including siRNA), and hybrid molecules having mixtures of single-stranded and double-stranded DNA and RNA. As used herein, nucleic acids also refer to nucleic acids that have the same basic chemical structure as natural nucleic acids. Such analogs have substituents of modified sugars and / or modified rings, but retain the same basic chemical structure as natural nucleic acids. A nucleic acid mimetic refers to a compound that has a different structure from the general chemical structure of nucleic acids, but functions similarly to natural nucleic acids. Examples of such analogues, without limitation, include phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs).

[0068] The "percentage of sequence identity" is determined by comparing two optimally aligned sequences across a comparison region, where portions of the polynucleotide or polypeptide sequence within the comparison region may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions in both sequences where identical nucleic acid bases or amino acid residues occur, dividing the number of matched positions by the total number of positions within the comparison region, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0069] In the context of two or more nucleic acid sequences or polypeptide sequences, the term “identical” or “identity” percentage means that two or more sequences or subsequences are the same, or that a specified percentage of amino acid residues or nucleotides are the same when compared and aligned for the greatest correspondence over a comparison or designated region measured by manual alignment and visual inspection using one of the following sequence comparison algorithms (i.e., for example, the entire polypeptide sequence of the present invention or individual domains of the polypeptide of the present invention, having 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity over a designated region). Thus, such sequences are considered “substantially identical.” This definition also refers to the complement of the sequence under test. Optionally, identity exists over a region of at least about 50 nucleotides in length, or more preferably over a region of 100 to 500 or 1000 nucleotides or more in length. The present invention includes a polypeptide that is substantially identical to any of SEQ ID NOs: 1 to 35.

[0070] For sequence comparison, typically one sequence acts as a reference sequence against which the sequence under comparison is compared. When using a sequence comparison algorithm, the sequence under comparison and the reference sequence are input into the computer, and if necessary, subsequence coordinates and parameters for the sequence algorithm program are specified. Default program parameters may be used, or alternative parameters may be specified. The sequence comparison algorithm then calculates the sequence identity percentage of the sequence under comparison compared to the reference sequence, based on the program parameters.

[0071] As used herein, the term “comparison region” includes references to a reference sequence having, for example, the full-length sequence or a segment having one of the following numbers of consecutive positions, selected from the group consisting of 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides, after the two sequences have been optimally aligned, so that the sequences can be compared to a reference sequence having the same number of consecutive positions. Sequence alignment methods for comparison are well known in the art. Optimal sequence alignment for comparison can be performed, for example, by the local homology algorithm by Smith and Waterman (1970), Adv. Appl. Math., 2:482c; by the homology alignment algorithm by Needleman and Wunsch (1970), J. Mol. Biol., 48:443; by the similarity search method by Pearson and Lipman (1988), Proc. Nat'l. Acad. Sci. USA, 85:2444; by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA at Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI); or by manual alignment and visual inspection (see, for example, Ausubel et al., "Current Protocols in Molecular Biology" (1995, augmented edition)).

[0072] Examples of algorithms suitable for determining sequence identity percentage and sequence similarity percentage are the BLAST algorithm and the BLAST 2.0 algorithm, respectively, described in Altschul et al. (1977), Nuc. Acids Res. 25:3389~3402 and Altschul et al. (1990), J. Mol. Biol. 215:403~410. Software for performing BLAST analysis is published by the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short word lengths W in the query sequence that match or satisfy a certain positive threshold score T when aligned with words of the same length in the database sequence. T is called the adjacent word score threshold (Altschul et al., previously mentioned). These initial adjacent word hits act as seeds to initiate a search for longer HSPs containing them. Word hits are extended in any direction along each sequence as long as the cumulative alignment score can increase. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for matching residue pairs; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of word hits in each direction stops if the cumulative alignment score falls by a quantity X from its maximum value achieved; if the cumulative score falls to zero or less due to the accumulation of alignments of one or more negative-scoring residues; or if it reaches the end of either sequence. The parameters W, T, and X of the BLAST algorithm determine the sensitivity and speed of alignment. The BLASTN program (for nucleotide sequences) uses 11 word length (W), 10 expected value (E), M=5, and N=-4 as defaults to compare both strands.The BLASTP program for amino acid sequences uses a word length of 3, and a BLOSUM62 scoring matrix of 10 (see Henikoff and Henikoff (1989), Proc. Natl. Acad. Sci. USA, 89:10915), alignment of 10, M=5, N=-4 as defaults to compare both strands.

[0073] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993), Proc. Natl. Acad. Sci. USA, 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indicator of the probability that a match between two nucleotide or amino acid sequences occurs by chance. For example, if the minimum sum probability when comparing the test nucleic acid against the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001, then the nucleic acid is considered similar to the reference sequence.

[0074] An indicator 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 an antibody induced against the polypeptide encoded by the second nucleic acid, as described below. Therefore, for example, if the two peptides differ only by conservative substitutions, the polypeptide is typically substantially identical to the second polypeptide. Another indicator 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. Yet another indicator that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.

[0075] In reference to a gene, the terms “expressed” or “expressed” as used herein mean the transcript and / or translation product of that gene. The intracellular expression level of a DNA molecule may be determined based on the amount of corresponding mRNA present in the cell or the amount of protein encoded by that DNA produced by the cell. The expression level of non-coding nucleic acid molecules (e.g., siRNA) may be detected by standard PCR or Northern blotting methods well known in the art. See Sambrook et al., 1989, “Molecular Cloning: A Laboratory Manual,” 18.1–18.88.

[0076] The expression of a transfected gene can occur transiently or stably within a cell. During "transient expression," the transfected gene is not transferred to daughter cells during cell division. Its expression is restricted to the transfected cell, and gene expression is lost over time. In contrast, stably expressed transfected genes can occur when the gene is co-transfected with another gene that confers a selective advantage to the transfected cell. Such a selective advantage may be resistance to a specific toxin provided to the cell. The expression of a transfected gene can also be further achieved through transposon-mediated insertion into the host genome. During transposon-mediated insertion, the gene is positioned predictably between two transposon linker sequences that enable insertion into the host genome and subsequent excision. Stable expression of transfected genes can be further achieved by infecting cells with lentiviral vectors that, after infection, form a portion of the cellular genome (integrate into the cellular genome), resulting in stable gene expression.

[0077] The terms "plasmid," "vector," or "expression vector" refer to nucleic acid molecules that encode genes and / or regulatory elements necessary for gene expression. Gene expression from a plasmid can occur in cis or trans configuration. When a gene is expressed in cis configuration, both the gene and its regulatory elements are encoded by the same plasmid. Trans expression refers to the case where the gene and its regulatory elements are encoded by separate plasmids.

[0078] The terms “transfection,” “transfer,” “transfect,” or “transfer” may be used interchangeably and are defined as the process of introducing a nucleic acid molecule or protein into a cell. Nucleic acids are introduced into cells using non-viral or viral methods. Nucleic acid molecules may be gene sequences encoding a complete protein or gene sequences encoding a functional portion. Non-viral transfection methods include any suitable transfection method that does not use viral DNA or viral particles as a delivery system for introducing nucleic acid molecules into cells. Exemplary non-viral transfection methods include calcium phosphate transfection, liposome transfection, nucleofection, sonoporation, heat shock transfection, magnetofection, and electroporation. In some embodiments, nucleic acid molecules are introduced into cells using electroporation following standard procedures well known in the art. For viral-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, retroviral vectors, adenoviral vectors, lentiviral vectors, and adeno-associated viral vectors. In some embodiments, nucleic acid molecules are introduced into cells using retroviral vectors following standard procedures well known in the art. The term “transfection” also refers to the introduction of proteins into cells from the external environment. Typically, protein transfection relies on the conjugation of a peptide or protein capable of crossing the cell membrane to the protein of interest. See, for example, Ford et al. (2001), Gene Therapy, 8:1-4 and Prochiantz (2007), Nat. Methods, 4:119-20.

[0079] An "antibody" refers to a polypeptide containing a framework region derived from an immunoglobulin gene or fragment thereof that specifically binds to and recognizes an antigen. Recognized immunoglobulin genes include the constant region genes of kappa, lambda, alpha, gamma, delta, epsilon, and mu, as well as numerous immunoglobulin variable region genes. The light chain is classified as a kappa chain or a lambda chain. The heavy chain is classified as a gamma chain, muon chain, alpha chain, delta chain, or epsilon chain, which define the classes of immunoglobulins: 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 binding. In some embodiments, the antibody or antibody fragment may originate from different organisms, including humans, mice, rats, hamsters, and camels. The antibodies of the present invention may include antibodies that have been modified or mutated at one or more amino acid positions to improve or modulate the desired function of an antibody (e.g., glycosylation, expression, antigen recognition, effector function, antigen binding, specificity, etc.).

[0080] Antibodies are large, complex molecules with a complex internal structure (molecular weight approximately 150,000 Da, or approximately 1320 amino acids). Natural antibody molecules contain two identical polypeptide chain pairs, each having one light chain and one heavy chain. Each light chain and each heavy chain consists of two regions: a variable ("V") region involved in binding to the target antigen and a constant ("C") region that interacts with other components of the immune system. The light chain variable region and the heavy chain variable region, together in three-dimensional space, form a variable region that binds to the antigen (e.g., a receptor on the surface of a cell). Within each light chain variable region or heavy chain variable region, there are three short segments (averaging 10 amino acids in length) called complementarity-determining regions ("CDRs"). The six CDR antibody variable domains (three derived from the light chain and three from the heavy chain) fold together in three-dimensional space to form the actual antibody-side binding site (paratope) that docks to the target antigen (epitope). The position and length of the CDR are precisely defined by Kabat, E. et al., "Sequences of Proteins of Immunological Interest," US Department of Health and Human Services, 1983, 1987. The portion of the variable region not contained within the CDR is called the framework ("FR"), which forms the environment for the CDR.

[0081] As provided herein, “antibody variant” refers to a polypeptide capable of binding to an antigen and comprising one or more structural domains (e.g., light chain variable domain, heavy chain variable domain) of an antibody or fragment thereof. Non-limiting examples of antibody variants include single-domain antibodies or nanobodies, monospecific Fab2, bispecific Fab2, trispecific Fab3, monovalent IgG, scFv, bispecific diabodies, trispecific triabodies, scFv-Fc, minibodies, IgNAR, V-NAR, hcIgG, VhH, or peptidebodies. As provided herein, “peptibody” refers to a peptide portion conjugated (via a covalent or non-covalent linker) to the Fc domain of an antibody. Further non-limiting examples of antibody variants known in the art include antibodies produced by cartilaginous fish or camelids. General descriptions of antibodies and their variable regions derived from camelid animals, as well as methods for their production, isolation, and use, can be found in WO97 / 49805 and WO97 / 49805, which are references incorporated herein by reference in their entirety for all purposes. Similarly, antibodies and their variable regions derived from cartilaginous fish, as well as methods for their production, isolation, and use, can be found in WO2005 / 118629, which are incorporated herein by reference in their entirety for all purposes.

[0082] The terms “CDR L1,” “CDR L2,” and “CDR L3” as provided herein refer to complementarity-determining regions (CDRs) 1, 2, and 3 of the variable light (L) chain of an antibody. In some embodiments, the variable light chain provided herein includes CDR L1, CDR L2, and CDR L3 in the direction from the N-terminus to the C-terminus. Similarly, the terms “CDR H1,” “CDR H2,” and “CDR H3” as provided herein refer to complementarity-determining regions (CDRs) 1, 2, and 3 of the variable heavy (H) chain of an antibody. In some embodiments, the variable heavy chain provided herein includes CDR H1, CDR H2, and CDR H3 in the direction from the N-terminus to the C-terminus.

[0083] The terms “FR L1,” “FR L2,” “FR L3,” and “FR L4” as provided herein are used according to their general meanings in the art and refer to framework regions (FRs) 1, 2, 3, and 4 of the variable light (L) chain of an antibody. In some embodiments, the variable light chain provided herein includes FR L1, FR L2, FR L3, and FR L4 in the direction from the N-terminus to the C-terminus. Similarly, the terms “FR H1,” “FR H2,” “FR H3,” and “FR H4” as provided herein are used according to their general meanings in the art and refer to framework regions (FRs) 1, 2, 3, and 4 of the variable heavy (H) chain of an antibody. In some embodiments, the variable heavy chain provided herein includes FR H1, FR H2, FR H3, and FR H4 in the direction from the N-terminus to the C-terminus.

[0084] The term "antibody" is used in accordance with its generally known meaning in the art. Antibodies exist, for example, as intact immunoglobulins or as numerous well-characterized fragments resulting from digestion with various peptidases. For example, pepsin digests the antibody below the disulfide linkage in the hinge region, yielding F(ab)'2, a dimer of Fab, which is a light chain linked to VH-CH1 by a disulfide bond. Under mild conditions, F(ab)'2 can be reduced to cleave the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into a Fab' monomer. The Fab' monomer is essentially Fab with a portion of the hinge region (see "Fundamental Immunology" (Paul, ed., 3rd edition, 1993)). In relation to the digestion of intact antibodies, a variety of antibody fragments are defined, and those skilled in the art will recognize that such fragments can be synthesized de novo, either chemically or by recombinant DNA methods. Therefore, the term antibody as used herein also includes antibody fragments produced by modification of whole antibodies, antibody fragments synthesized de novo using recombinant DNA methods (e.g., single-chain Fv), or antibody fragments identified using phage display libraries (see, for example, McCafferty et al., Nature, 348:552-554 (1990)).

[0085] The structural unit of an exemplary immunoglobulin (antibody) includes a tetramer. Each tetramer consists of two identical polypeptide chain pairs, each having one “light chain” (approximately 25 kD) and one “heavy chain” (approximately 50–70 kD). The N-terminus of each chain defines a variable region of approximately 100–110 or more amino acids, which 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 used herein may be used interchangeably. The terms variable heavy chain (VH) and heavy chain variable region as used herein may be used interchangeably. The Fc (i.e., the Fc (fragment crystallizable) region) is the "base" or "tail" of an immunoglobulin and typically consists of two heavy chains that contribute to two or three constant domains, depending on the antibody class. 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 the Fc receptor, and other immune molecules, such as complement proteins.

[0086] The term "antigen" as provided herein refers to a molecule capable of binding to an antibody-binding domain as provided herein. An "antigen-binding domain" as provided herein is a region of an antibody that binds to an antigen (epitope). As described above, an antigen-binding domain generally consists of one constant domain and one variable domain (CH, CL, VH, and VL, respectively) in both the heavy and light chains. The 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 epitopes on the antigen.

[0087] Antibodies exist, for example, as intact immunoglobulins, or as numerous well-characterized fragments resulting from digestion with various peptidases. For example, pepsin digests the antibody below the disulfide linkage within the hinge region, yielding F(ab)'2, a dimer of Fab, which is a light chain linked to VH-CH1 by a disulfide bond. Under mild conditions, F(ab)'2 can be reduced to a Fab' monomer by cleaving the disulfide linkage within the hinge region, thereby converting the F(ab)'2 dimer into a Fab' monomer. The Fab' monomer is essentially an antigen-binding moiety with a portion of the hinge region (see "Fundamental Immunology" (Paul, 3rd edition, 1993)). While a variety of antibody fragments are defined in relation to the digestion of intact antibodies, those skilled in the art will recognize that such fragments can be synthesized de novo, either chemically or by recombinant DNA methods. Therefore, the term antibody as used herein also includes antibody fragments produced by modification of the whole antibody, antibody fragments synthesized de novo using recombinant DNA methods (e.g., single-chain Fv), or antibody fragments identified using phage display libraries (see, for example, McCafferty et al., Nature, 348:552-554 (1990)).

[0088] Single-chain variable fragments (scFv) are typically fusion proteins of the heavy chain (VH) variable region and the light chain (VL) variable region of immunoglobulin, linked by a short linker peptide of approximately 10 to 25 amino acids. The linker is usually rich in glycine for flexibility, or in serine or threonine for solubility. The linker may connect the N-terminus of the VH to the C-terminus of the VL, or vice versa.

[0089] An mAb epitope is the region of an antigen to which the mAb binds. Two antibodies bind to the same or duplicate epitope if each competitively inhibits (blocks) the binding of the other antibody to the antigen. That is, a 1x, 5x, 10x, 20x, or 100x excess of one antibody 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 (see, for example, Junghans et al., Cancer Res., 50:1495, 1990). Alternatively, if essentially all amino acid mutations within the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody, then the two antibodies have the same epitope. If a certain amino acid mutation reduces or eliminates the binding of one antibody, and also reduces or eliminates the binding of the other antibody, then the two antibodies have a duplicate epitope.

[0090] Many techniques known in the art can be used for the preparation and use of suitable antibodies of the present invention, such as recombinant antibodies, monoclonal antibodies, or polyclonal antibodies (see, for example, Kohler and Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today, 4:72 (1983); Cole et al., pp. 77-96, "Monoclonal Antibodies and Cancer Therapy," Alan R. Liss, Inc. (1985); Coligan, "Current Protocols in Immunology" (1991); Harlow and Lane, "Antibodies, A Laboratory Manual" (1988); and Goding, "Monoclonal Antibodies: Principles and Practice" (2nd edition, 1986)). The genes encoding the heavy and light chains of the antibody of interest may be cloned from cells; for example, the gene encoding a monoclonal antibody may be cloned from a hybridoma and used to produce a recombinant monoclonal antibody. 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 create a large antibody pool with different antigen specificities (see, e.g., Kuby, "Immunology" (3rd edition, 1997)). Techniques for preparing single-chain or recombinant antibodies (U.S. Patent No. 4,946,778, U.S. Patent No. 4,816,567) can be adapted to prepare antibodies for the polypeptides of the present invention.Furthermore, other organisms, such as transgenic mice or other mammals, can also be used to express humanized antibodies or human antibodies (e.g., U.S. 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) See Biotechnology, 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol., 13:65-93 (1995). Alternatively, phage display technology can be used to identify Fab fragments of antibodies and heteromers that specifically bind to selected antigens (see, e.g., McCafferty et al., Nature, 348:552-554 (1990); Marks et al., Biotechnology, 10:779-783 (1992)). Antibodies can also be bispecific, i.e., capable of recognizing two different antigens (see, e.g., WO93 / 08829, Traunecker et al., EMBO J., 10:3655-3659 (1991) and Suresh et al., Methods in Enzymology, 121:210 (1986)). Antibodies can also be heteroconjugates, such as antibodies or immunotoxins linked by two covalent bonds. (See, for example, U.S. Patent No. 4,676,980, WO91 / 00360; WO92 / 200373 and EP03089.)

[0091] In the art, methods for humanizing or primate-like non-human antibodies are well known (e.g., U.S. 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; WO87 / 02671; European Patent Application No. 0173494; Jones et al. (1986), Nature, 321:522; and Verhoyen et al. (1988), Science, 239:1534). Humanized antibodies are further described, for example, in Winter and Milstein (1991), Nature, 349:293. Generally, humanized antibodies have one or more amino acid residues introduced into them from a non-human source. These non-human amino acid residues are typically often referred to as import residues, extracted from the import variable domain. Humanization is described by Winter and collaborators (e.g., 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 Prest This can essentially be carried out by substituting a rodent CDR or CDR sequence with the corresponding sequence of a human antibody, in accordance with (see a, Curr. Op. Struct. Biol., 2:593~596 (1992), Padlan, Molec. Immun., 28:489~498 (1991); Padlan, Molec. Immun., 31(3):169~217 (1994)). Thus, such a humanized antibody is essentially a chimeric antibody (U.S. Patent No. 4,816,567) in which a subintact human variable domain is substituted with the corresponding sequence derived from a non-human species.In reality, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted with residues derived from similar sites within rodent antibodies. For example, a polynucleotide containing a first sequence encoding the humanized immunoglobulin framework region and a second sequence set encoding the desired immunoglobulin complementarity-determining region may be synthesized or prepared by combining appropriate cDNA and genomic DNA segments. Human constant region DNA sequences can be isolated from various human cells according to well-known procedures.

[0092] A "chimeric antibody" is defined as (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 constant region of a different class, effector function, and / or species, or a constant region whose class, effector function, and / or species has been changed, or to a completely different molecule that confers new properties to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) an antibody molecule in which the variable region or a part thereof is modified, replaced, or exchanged by a variable region of a different antigen specificity, or a variable region whose antigen specificity has been changed. Preferred antibodies of the present invention and antibodies preferred for use according to the present invention include humanized monoclonal antibodies and / or chimeric monoclonal antibodies.

[0093] In this specification, “therapeutic antibodies” refers to any antibody or functional fragment used to treat cancer, autoimmune diseases, graft rejection, cardiovascular diseases, or other diseases or conditions described herein. Non-limited examples of therapeutic antibodies include Erbitux (cetuximab), ReoPro (absiximab), Simulect (basiliximab), Remicade (infliximab); Orthoclone OKT3 (muromonab-CD3); Rituxan (rituximab), Bexxar (tositumomab), Humira (adalimumab), Campath (aremtuzumab), Simulect (basiliximab), Avastin (bevacizumab), Cimzia (certolizumab pegol), Zenapax (daclizumab), Soliris (eculizumab), Raptiva (efalizumab), Mylot This includes, but is not limited to, arg (gemtuzumab), Zevalin (ibritumomab tiuxetan), Tysabri (natalizumab), Xolair (omalizumab), Synagis (palivizumab), Vectibix (panitumumab), Lucentis (ranibizumab), and Herceptin (trastuzumab), and includes mouse antibodies, murine chimeric antibodies, humanized chimeric antibodies, or human antibodies.

[0094] Techniques for conjugating therapeutic agents with antibodies are well known (for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," "Monoclonal Antibodies And Cancer Therapy," Reisfeld et al. (eds.), pp. 243-256 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," "Controlled Drug Delivery" (2nd edition), Robinson et al. (eds.), pp. 623-253 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," "Monoclonal Antibodies '84: Biological And Clinical Applications," Pinchera et al. (eds.), pp. 475-256 (1985); and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin See "Conjugates," Immunol. Rev. 62:119-58 (1982). As used herein, the terms “antibody-drug conjugate” or “ADC” refer to a therapeutic agent that is conjugated with an antibody or otherwise covalently bound to one. As used herein, “therapeutic agent” refers to a composition useful in the treatment or prevention of diseases such as cancer.

[0095] When referring to proteins or peptides, the phrases "specifically (or selectively) bind to" or "specifically (or selectively) immunoreactive with" an antibody refer to a binding reaction that determines the presence of a protein, and often proteins and other biological agents, within a heterogeneous population. Therefore, under specified immunoassay conditions, a specified antibody binds to a particular protein at least twice the background, more typically 10 to 100 times the background. Specific binding to an antibody under such conditions typically requires an antibody selected for its specificity to a particular protein. For example, polyclonal antibodies may be selected to obtain only a subset of antibodies that are specifically immunoreactive with a selected antigen but not specifically immunoreactive with other proteins. This selection can be achieved by excluding antibodies that cross-react with other molecules. Various immunoassay formats can be used to select antibodies that are specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select proteins and antibodies that are specifically immunoreactive (see, for example, Harlow and Lane, "Using Antibodies, A Laboratory Manual" (1998), for a description of immunoassay formats and conditions that may be used to determine specific immunoreactivity).

[0096] A "ligand" refers to a drug capable of binding to a receptor, such as a polypeptide or other molecule.

[0097] For example, when used in reference to cells, nucleic acids, proteins, or vectors, the term “recombinant” indicates that the cells, nucleic acids, proteins, or vectors have been modified by laboratory methods or are the result of laboratory methods. Therefore, for example, recombinant proteins include proteins produced by laboratory methods. Recombinant proteins may contain amino acid residues not found in the protein’s natural (non-recombinant) form, or they may contain modified, for example, labeled amino acid residues.

[0098] When used in reference to nucleic acids, the term “heterogeneous” refers to a nucleic acid that contains two or more subsequences that are not found to be related to each other in nature. For example, a novel functional nucleic acid, such as one containing two or more sequences derived from unrelated genes arranged to form a promoter from one source and a coding region from another source, is typically created by recombination. Similarly, heterogeneous proteins refer to proteins that contain two or more subsequences that are not found to be related to each other in nature (e.g., fusion proteins).

[0099] When applied to nucleic acids or proteins, the term "isolated" means that the nucleic acid or protein essentially contains no other cellular components to which it associates in its natural state. "Isolated" can be, for example, homogeneous, a dry solution, or an aqueous solution. Purity and homogeneity are typically determined using analytical chemistry methods such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The protein, which is the dominant species present in the preparation, is substantially purified.

[0100] "Bringing into contact" is used in its obvious and ordinary sense, referring to the process of bringing at least two distinctly different molecular species (e.g., compounds containing biomolecules or cells) close enough to react, interact, or physically come into contact. However, it should be noted that the resulting reaction products may be produced directly from the reaction between the added reagents, or from intermediates derived from one or more of the added reagents that may be formed in the reaction mixture.

[0101] The term "to bring into contact" can include reacting, interacting, or physically bringing two molecular species into contact, in which case the two molecular species may be, for example, a recombinant protein and an antigen-binding domain as described herein. In some embodiments, contact includes, for example, interacting a recombinant protein as described herein with an antigen-binding domain.

[0102] A "control" sample or "control" value refers to a sample used as a reference for comparison with the test sample, usually a known reference. For example, the test sample may be taken from test conditions in the presence of the test compound and compared to a sample under known conditions, such as in the absence of the test compound (negative control) or in the presence of a known compound (positive control). A control may also represent the average value collected from a number of tests or results. Those skilled in the art will recognize that controls can be designed for 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 measurements (e.g., comparison of side effects). Those skilled in the art will understand which controls are valuable in a given situation and which allow for data analysis based on comparison with the control value. Controls are also valuable for determining the significance of data. For example, if the value for a given parameter varies widely in the control, the variation in the test sample is not considered significant.

[0103] "Patient" or "subject requiring it" means an organism suffering from or predisposed to a disease or condition that can be treated by administration of the compositions or pharmaceutical compositions provided herein. Non-limited examples include humans, other mammals, bovids, rats, mice, dogs, monkeys, goats, sheep, cattle, deer, and other non-mammals. In some embodiments, the patient is human.

[0104] The terms “disease” or “condition” refer to the current condition or health state of a patient or subject that can be treated by the compounds, pharmaceutical compositions or methods provided herein. In some embodiments, 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).

[0105] The terms “treatment” or “treatment” refer to any sign of successful treatment or improvement of injury, disease, pathology or condition, including any objective or subjective parameters, such as reduction of symptoms; remission; disappearance or making the injury, pathology or condition more tolerable to the patient; slowing the rate of degeneration or decline; making the final degenerative point less debilitating; or improving 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 general examination, neuropsychiatric examination and / or psychiatric assessment. The terms “treatment” and their conjugations include the prevention of injury, pathology, condition or disease. In some embodiments, “treatment” refers to the treatment of cancer.

[0106] An “effective dose” is the amount of a compound sufficient to achieve a stated purpose (for example, to achieve the effect it is administered for, to treat a disease, to reduce enzyme activity, to increase enzyme activity, to reduce a signaling pathway, or to reduce one or more symptoms of a disease or condition) compared to the absence of the compound. An example of an “effective dose” is an amount sufficient to contribute to the treatment, prevention, or reduction of one or more symptoms of a disease, which may also be called a “therapeutic effective dose.” “Reduction” of one or more symptoms (and grammatical equivalents thereof) means a reduction in the severity or frequency of the symptoms or the disappearance of the symptoms. The exact amount depends on the purpose of the treatment and can be determined by those skilled in the art using known techniques (see, for example, Lieberman, "Pharmaceutical Dosage Forms" (vols. 1-3, 1992); Lloyd, "The Art, Science and Technology of Pharmaceutical Compounding" (1999); Pickar, "Dosage Calculations" (1999); and Remington, "Science and Practice of Pharmacy," 20th edition, 2003, edited by Gennaro, Lippincott, Williams & Wilkins).

[0107] As used herein, the terms “Her2 protein” or “Her2” include any recombinant or native form of a variant or homolog of CD340 (cluster of differentiation 340), erbB-2, a receptor tyrosine protein kinase also known as the oncogene Neu, Erbb2 (rodent), or ERBB2 (human), or Her2, that maintains the activity of Her2 (e.g., activity within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to Her2). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identity across the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 consecutive amino acids) compared to the native Her2 protein. In several embodiments, the Her2 protein is substantially identical to the protein identified by UniProt reference number P04626, or a variant or homolog thereof that is substantially identical to it.

[0108] As used herein, the terms “EGFR protein” or “EGFR” include, in humans, either the epidermal growth factor receptor (EGFR), also known as ErbB-1 or HER1, or any recombinant or native form of its variants or homologs that maintain EGFR activity (e.g., activity within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to EGFR). In some embodiments, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 consecutive amino acids) compared to the native EGFR protein. In several embodiments, the EGFR protein is substantially identical to the protein identified by UniProt reference number: P00533 or a variant or homolog having substantial identity therewith.

[0109] As used herein, the terms “CD19 protein” or “CD19” include the CD19 molecule (cluster of differentiation 19), the B lymphocyte antigen CD19, or any recombinant or native form of its variants or homologs that maintain the activity of CD19 (e.g., activity within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to CD19), also known as the B lymphocyte antigen CD19 molecule (cluster of differentiation 19), the B lymphocyte surface antigen B4, the T cell surface antigen Leu-12, and CVID3. In some embodiments, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 consecutive amino acids) compared to the native CD19 protein. In several embodiments, the CD19 protein is substantially identical to the protein identified by UniProt reference number P15391, or a variant or homolog thereof that is substantially identical to it.

[0110] As used herein, the terms “CD20 protein” or “CD20” include either the B lymphocyte antigen CD20 or CD20 (cluster of differentiation 20) or any recombinant or native form of its variants or homologs that maintain the activity of CD20 (e.g., activity within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to CD20). In some embodiments, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 consecutive amino acids) compared to the native CD20 protein. In several embodiments, the CD20 protein is substantially identical to the protein identified by UniProt reference number: P11836 or a variant or homolog having substantial identity therewith.

[0111] Recombinant protein composition This specification presents recombinant proteins that are expressed by T cells and bind to antigen-binding domains (e.g., antibodies, mutants, or fragments thereof), thereby enabling T cells to target cells (e.g., tumor cells) that express the antigen to which the antigen-binding domain is bound. 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 cell, the T cell is activated and becomes cytotoxic, thereby eliminating the target cell (e.g., cancer cell). The recombinant proteins provided herein enable the rapid addition of functionality to adoptive immunotherapy and are particularly useful for a wide variety of therapeutic and diagnostic purposes. For example, the recombinant proteins provided herein, including their embodiments, can be used as a means of directing effector T cells (e.g., autologous T cells) and therapeutic antibodies to their sites of action, thereby reducing off-target effects. The compositions provided herein enable rapid and efficient modification of target specificity without creating and optimizing individual CAR T cells.

[0112] The recombinant proteins provided herein are, for example, continuous single-chain polypeptides comprising a non-CDR Fab-binding peptide domain, an intracellular T cell signaling domain, and a transmembrane domain connecting the non-CDR Fab-binding peptide domain to the intracellular T cell signaling domain. All recombinant proteins provided herein may include further elements that form the portion of the continuous single-chain polypeptide, such as spacer regions, peptide linkers, and intracellular costimulatory signaling domains. The continuous single-chain polypeptides provided herein refer to polypeptide chains comprising elements covalently linked to each other, thereby forming a continuous polypeptide chain.

[0113] Accordingly, in one embodiment, 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 ligating the non-CDR Fab-binding peptide domain to the intracellular T cell signaling domain. In several embodiments, the intracellular T cell signaling domain is a CD3ζ intracellular T cell signaling domain.

[0114] In this specification, “non-CDR Fab-binding peptide domain” means a peptide or a peptide-containing peptide domain capable of binding to a non-CDR binding site of an antibody, antibody variant, or fragment thereof. In several embodiments, the non-CDR Fab-binding peptide domain is a peptide. In several embodiments, the non-CDR Fab-binding peptide domain contains a peptide. In several embodiments, the non-CDR Fab-binding peptide domain binds to a non-CDR binding site. In several embodiments, the non-CDR Fab-binding peptide domain is a peptidyl moiety. In several embodiments, the peptidyl moiety is the portion described in U.S. Patent Publication No. US20120301400A1, which is incorporated herein by reference in its entirety for all purposes.

[0115] In several embodiments, the non-CDR Fab-binding peptide domain is given by formula: X0-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12(I) It contains the peptide portion. In 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, phenylalanine, a non-natural analog of 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 hydrated 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 to each other to form a cyclic peptidyl moiety.

[0116] In several embodiments, the non-CDR Fab-binding peptide domain is given by formula: X0-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12(I) It contains the peptide portion. In 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 optionally connected to each other to form a cyclic peptidyl moiety.

[0117] In several embodiments, the non-CDR Fab-binding peptide domain contains the sequence of SEQ ID NO: 32. In several embodiments, the non-CDR Fab-binding peptide domain has the sequence of SEQ ID NO: 32. In several embodiments, the recombinant protein further contains a signaling peptide having the sequence of SEQ ID NO: 37. In several embodiments, the signaling peptide is bound to the N-terminus of the non-CDR Fab-binding peptide domain.

[0118] The term "non-CDR binding site" as provided herein refers to a binding site of an antigen-binding domain (e.g., the Fab domain of an antibody, an antibody variant, or a fragment thereof) that does not include CDR residues of the heavy chain or light chain of the antibody. "Non-CDR peptide binding site" refers to a region of an antigen-binding domain of a recombinant protein provided herein that can be bound non-covalently to a non-CDR Fab-binding peptide domain. In several embodiments, the non-CDR binding site includes amino acid residues of the framework region. In several embodiments, the non-CDR binding site includes FR residues of the heavy chain or light chain. In several embodiments, the non-CDR binding site includes FR residues of both the heavy and light chains. In several embodiments, the non-CDR binding site includes a residue at the position corresponding to Kabat's 83rd position, a residue at the position corresponding to Kabat's 30th position, or a residue at the position corresponding to Kabat's 52nd position. In multiple embodiments, the non-CDR binding site includes a residue at the position corresponding to Kabat's 40, a residue at the position corresponding to Kabat's 41, a residue at the position corresponding to Kabat's 30, a residue at the position corresponding to Kabat's 52, a residue at the position corresponding to Kabat's 83, or a residue at the position corresponding to Kabat's 85. In multiple embodiments, the non-CDR binding site includes a residue at the position corresponding to Kabat's 40. In multiple embodiments, the non-CDR binding site includes a residue at the position corresponding to Kabat's 41. In multiple embodiments, the non-CDR binding site includes a residue at the position corresponding to Kabat's 30. In multiple embodiments, the non-CDR binding site includes a residue at the position corresponding to Kabat's 52. In multiple embodiments, the non-CDR binding site includes a residue at the position corresponding to Kabat's 83. In multiple embodiments, the non-CDR binding site includes a residue at the position corresponding to Kabat's 85.In several embodiments, the residues forming non-CDR binding sites are the residues described in U.S. Patent Publication No. US20120301400A1, which are incorporated herein by reference in their entirety for all purposes.

[0119] The non-CDR binding sites provided herein may also be referred to as “meditope binding sites.” In several embodiments, recombinant proteins that bind to the non-CDR binding site via the non-CDR Fab-binding peptide domain do not affect (e.g., have a measurable effect on) the binding of the antigen-binding domain to the epitope. In other words, in several embodiments, the occupancy of the non-CDR binding site does not affect binding to the antigen. In several embodiments, the non-CDR binding site interacts non-covalently with the non-CDR Fab-binding peptide domain (e.g., meditope) of recombinant proteins provided herein, including those embodiments. Amino acid residues capable of interacting with the non-CDR Fab-binding peptide domain (e.g., meditope) may form a portion of an antibody, a Fab, a variant of an antibody, or any fragment thereof. The non-CDR binding site may be manipulated into any suitable antibody, variant, or fragment thereof to form an antigen-binding domain with the non-CDR binding site. In this specification, antigen-binding domains including non-CDR binding sites are also referred to as "meditope-compatible antibody," "meditope-compatible domain," or "meditope-compatible antibody region."

[0120] As provided herein, an "antigen-binding domain" is a region of an antibody, variant, or fragment thereof that binds to an antigen (epitope). As described herein, an antigen-binding domain generally consists of one constant domain and one variable domain (VL, VH, CL, and CH1, respectively) in the heavy chain and light chain. The paratope or antigen-binding site is formed on the N-terminus of the antigen-binding domain. Two variable domains of the antigen-binding domain typically bind to an epitope on the antigen. In several embodiments, the antigen-binding domain forms part of an antibody. In several embodiments, the antigen-binding domain forms part of a therapeutic antibody. In several embodiments, the antigen-binding domain forms part of a Fab. In several embodiments, the antigen-binding domain is a Fab.

[0121] In several embodiments, the antigen-binding domain includes a heavy chain constant region (CH) and a light chain constant region (CL). In several embodiments, the heavy chain constant region (CH) is the constant region of the antibody heavy chain or a fragment thereof. In several embodiments, the light chain constant region (CL) is the constant region of the antibody light chain or a fragment thereof. In several embodiments, the heavy chain constant region (CH) is the constant region of Fab. In several embodiments, the light chain constant region (CL) is the constant region of the Fab light chain. In several embodiments, the heavy chain constant region (CH) is the constant region of the F(ab)'2 dimer. In several embodiments, the light chain constant region (CL) is the constant region of the F(ab)'2 dimer light chain. In several embodiments, the antigen-binding domain includes an Fc domain. In several embodiments, the antigen-binding domain is a humanized antigen-binding domain. In several embodiments, the antigen-binding domain is a humanized mouse antigen-binding domain.

[0122] In several embodiments, the antigen-binding domain is a trastuzumab medotope-compatible domain, a pertuzumab medotope-compatible domain, an M5A medotope-compatible domain, or a rituximab medotope-compatible domain. In several embodiments, the antigen-binding domain is a humanized rituximab medotope-compatible domain.

[0123] In several embodiments, the antigen-binding domains provided herein, including those embodiments, are antigen-binding domains such as avagovomab, absiximab, adalimumab, adecatumumab, alemtuzumab, artumomab, artumomab penteate, anatumomab, anatumomab mafenatox, artitumomab, atilizumab, basiliximab, vectumomab, ectumomab, belimumab, and benralizumab. Bevacizumab, Brentuximab, Canakinumab, Capromab, Capromab Pendetide, Katsumakisomab, Cartolizumab, Cribatuzumab Tetraxetan, Daclizumab, Denosumab, Eculizumab, Edrecolomab, Efalizumab, Etalacizumab, Erzumakisomab, Fanoresomab, Fbta05, Fontrizumab, Gemtuzumab, Gilentuximab, Golimumab, Ibritumomab, Igobomab, In Fliximab, Ipilimumab, Labetuzumab, Mepolizumab, Muromonab, Muromonab-CD3, Natalizumab, Necitumumab, Nimotuzumab, Ofatumumab, Omalizumab, Olegobomab, Palivizumab, Panitumumab, Ranibizumab, Rituximab, Saturomab, Thresomab, Ibritumomab, Ibritumomab Chiuxetan, Tocilizumab, Tocitumomab, Trastuzumab, Trbs07, Ustekinumab, Bi Zilizumab, botumumab, zaltumumab and / or brodalumab and / or anlukinzumab, bapineuzumab, dalotuzumab, demcizumab, ganitumab, inotuzumab, mabrilimumab, moxetumomab pasdotox, rilotumumab, cifalimumab, tanezumab, tralokinumab, tremelimumab, urelumab, and antibodies produced by hybridoma 10B5 (see Edelson and Unanue, Curr Opin Immunol, August 2000, 12(4):425~31).), B6H12.2(abcam) or other anti-CD47 antibodies (see Chao et al., Cell, 142, 699-713, September 3, 2010), including one or more known antibodies that specifically bind to and / or compete with the same antigen or epitope as one or more CDRs (or CDRs containing at least, or about 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to a CDR), including heavy chain CDR1, 2, and / or 3, and / or light chain CDR1, 2, and / or 3.

[0124] In several embodiments, the antigen-binding domain is CA-125, glycoprotein (GP) IIb / IIIa receptor, TNF-alpha, CD52, TAG-72, carcinoembryonic antigen (CEA), interleukin-6 receptor (IL-6R), IL-2, interleukin-2 receptor a chain (CD25), CD22, B cell activator, interleukin-5 receptor (CD125), VEGF, VEGF-A, CD30, IL-1 beta, prostate-specific membrane antigen (PSMA), CD3, EpCAM, EGF receptor (EGFR), MUC1, human interleukin-1 Kin2 receptor, Tac, RANK ligand, complement proteins, e.g., C5, EpCAM, CD11a, e.g., human CD11a, integrins, e.g., alpha-v-beta-3 integrin, vitronectin 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 It specifically binds to antigens selected from the group consisting of the Fc region, RSV antigen (e.g., the F protein of RSV (respiratory syncytial virus)), TAG-72, NCA-90 (granulocyte antigen), IL-6, GD2, GD3, IL-12, IL-23, IL-17, CTAA16, 88, IL-13, 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 ligand 1 (PD-L1, also known as CD274, B7-H1), CD47, and CD137.

[0125] In several embodiments, 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-mouse CMV-infected cell protein, anti-MUC2 protein, anti-NKGD2 protein, anti-carcinoembryonic antigen protein, anti-PCSA protein, anti-PSMA protein, anti-TAA(mAb) The protein is an IgE-targeted protein, an anti-EGFR protein, an anti-TAG-72 protein, or an anti-VEGF-72 protein. In several embodiments, the antigen-binding domain is not cetuximab.

[0126] In addition to non-covalent binding to the non-CDR Fab-binding peptide domain, the antigen-binding domain may be modified (e.g., genetically modified or chemically modified) to include a therapeutic, imaging, or detection portion. Therefore, in several embodiments, the antigen-binding domain includes a therapeutic or detection portion.

[0127] The term “therapeutic portion” as provided herein is used in its obvious and ordinary sense and refers to a monovalent compound that, when applied to a target requiring it, provides a therapeutic benefit (e.g., prevention, eradication, or improvement of the underlying disorder being treated). The therapeutic portions provided herein may include, but are not limited to, peptides, proteins, nucleic acids, nucleic acid analogs, small molecules, antibodies, nanobodies, enzymes, prodrugs, lysine, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracinedione, actinomycin D, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, and glucocorticoids, as cytotoxic agents (e.g., toxins). In some embodiments, the therapeutic portion is an anticancer agent or chemotherapeutic agent as described herein. In some embodiments, the therapeutic portion is a nucleic acid portion, a peptide portion, or a small molecule drug portion. In multiple embodiments, the therapeutic portion is a nucleic acid portion. In multiple embodiments, the therapeutic portion is an antibody portion. In multiple embodiments, the therapeutic portion is a peptide portion. In multiple embodiments, the therapeutic portion is a small molecule drug portion. In multiple embodiments, the therapeutic portion is a nuclease. In multiple embodiments, the therapeutic portion is an immunostimulant. In multiple embodiments, the therapeutic portion is a toxin. In multiple embodiments, the therapeutic portion is a nuclease. In multiple embodiments, the therapeutic portion is a cytokine (e.g., IL-2). In multiple embodiments, the therapeutic portion contains a non-natural amino acid. In multiple embodiments, the therapeutic portion contains siRNA. In multiple embodiments, the therapeutic portion is siRNA. In multiple embodiments, the therapeutic portion contains an antisense nucleic acid. In multiple embodiments, the therapeutic portion is an antisense nucleic acid.

[0128] The “imaging portion or detection portion” provided herein is a monovalent compound detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. In several embodiments, the imaging portion is covalently bonded to the peptide compound. Exemplary imaging portions, without limitation, are described below. 32 P, radionuclides, positron-emitting isotopes, fluorescent dyes, fluorophores, antibodies, bioluminescent molecules, chemiluminescent molecules, photoreactive molecules, metals, high electron-density reagents, enzymes (e.g., commonly used in ELISA), magnetic contrast agents, quantum dots, nanoparticles, biotin, digoxigenin, haptens, and proteins, or other entities that can be detected by incorporating, for example, a radiolabel into a peptide or antibody that is specifically reactive with the target peptide. Any method known in the art for conjugating an antibody with a portion, such as the method described in Hermanson, "Bioconjugate Tecqueniques," 1996, Academic Press, Inc., San Diego, may be used. Exemplary fluorophores include fluorescein, rhodamine, GFP, coumarin, FITC, ALEXA fluor, 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 alloys, and manganese. In several embodiments, the imaging portion is a bioluminescent molecule. In several embodiments, the imaging portion is a photoreactive molecule. In several embodiments, the imaging portion is a metal. In several embodiments, the imaging portion is a nanoparticle.

[0129] All recombinant proteins provided herein contain multiple domains (e.g., an intracellular T cell signaling domain, a transmembrane domain, a spacer region, and an intracellular costimulatory signaling domain) that form a single continuous single-chain polypeptide segment.

[0130] The “intracellular T cell signaling domain” provided herein includes an amino acid sequence capable of inducing primary signaling in response to the binding of an antigen to an antibody region provided herein, including its embodiments. In several embodiments, signaling of the intracellular T cell signaling domain results in the activation of T cells expressing the intracellular T cell signaling domain. In several embodiments, signaling of the intracellular T cell signaling domain results in the proliferation (cell division) of T cells expressing the intracellular T cell signaling domain. In several embodiments, signaling of the intracellular T cell signaling domain results in the expression of proteins by the T cells that are known in the art to be characteristic of activated T cells (e.g., CTLA-4, PD-1, CD28, CD69). In several embodiments, the intracellular T cell signaling domain includes a signaling domain of the zeta chain of the human CD3 complex. In several embodiments, the intracellular T cell signaling domain is a CD3ζ intracellular T cell signaling domain.

[0131] In several embodiments, the intracellular T cell signaling domain includes the sequence of SEQ ID NO: 34. In several embodiments, the intracellular T cell signaling domain is the sequence of SEQ ID NO: 34. In several embodiments, the intracellular T cell signaling domain includes the sequence of SEQ ID NO: 11. In several embodiments, the intracellular T cell signaling domain is the sequence of SEQ ID NO: 11.

[0132] In the recombinant proteins provided herein, the transmembrane domain annexes the non-CDR Fab-binding peptide domain to the intracellular T cell signaling domain. In several embodiments, the transmembrane domain lies between the non-CDR Fab-binding peptide domain and the intracellular T cell signaling domain. In other words, the transmembrane domain is directly or indirectly (e.g., via a spacer) attached to the C-terminus of the non-CDR Fab-binding peptide domain and directly or indirectly (e.g., via a co-stimulatory signaling domain) attached to the N-terminus of the intracellular T cell signaling domain.

[0133] As provided herein, “transmembrane domain” refers to a polypeptide-forming portion of a biological membrane. A transmembrane domain as provided herein can extend from one side of the membrane to the other side of the membrane, within a biological membrane (e.g., a cell membrane). In several embodiments, the transmembrane domain extends from the intracellular to the extracellular side of the cell membrane. The transmembrane domain may include a nonpolar hydrophobic residue that anchors the protein provided herein, including its embodiments, within a biological membrane (e.g., the cell membrane of a T cell). Any transmembrane domain capable of anchoring the protein provided herein, including its embodiments, is envisioned. In several embodiments, the transmembrane domain is L-selectin. As provided herein, the term “L-selectin” includes the L-selectin protein also known as CD62L, or any of its variants or homologous recombinant or native forms that maintain L-selectin activity (e.g., activity within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of L-selectin activity). In several embodiments, the variant or homologue 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 portion of 50, 100, 150, or 200 consecutive amino acids) compared to the native L-selectin polypeptide. In several embodiments, L-selectin is a protein, its homologue, or a functional fragment identified by NCBI sequence reference number GI:262206315. Non-limiting examples of transmembrane domains include CD8α, CD4, or CD3 zeta transmembrane domains. In several embodiments, the transmembrane domain is a CD8α transmembrane domain, a CD28 transmembrane domain, a CD4 transmembrane domain, or a CD3 zeta transmembrane domain. In several embodiments, the transmembrane domain is a CD28 transmembrane domain.

[0134] The term “CD28 transmembrane domain” as provided herein includes the transmembrane domain of CD28, or any recombinant or native form of its variant or homologue that maintains the transmembrane domain activity of CD28 (e.g., activity within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to the CD28 transmembrane domain). In some embodiments, the variant or homologue has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 consecutive amino acids) compared to the native CD28 transmembrane domain polypeptide. In several embodiments, the CD28 transmembrane domain is a protein identified by SEQ ID NO: 18 or SEQ ID NO: 2, its variant, homologue, or functional fragment. In several embodiments, CD28 is a protein, its homolog, or a functional fragment identified by NCBI sequence reference number GI:340545506.

[0135] In several embodiments, the transmembrane domain is a protein domain identified by SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 18, its homologue, or a functional fragment thereof. In several embodiments, the transmembrane domain includes a protein domain identified by SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 18, its homologue, or a functional fragment thereof.

[0136] Similarly, the term “CD8α transmembrane domain” as provided herein includes either a recombinant or native form of a variant or homologue of CD8α that maintains the transmembrane domain of CD8α or the transmembrane domain activity of CD8α (e.g., activity within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to the CD8α transmembrane domain). In some embodiments, the variant or homologue has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 consecutive amino acids) compared to the native CD8α transmembrane domain polypeptide.

[0137] The term “CD4 transmembrane domain” as provided herein includes any recombinant or native form of a variant or homologue thereof that maintains the CD4 transmembrane domain or CD4 transmembrane domain activity (e.g., activity within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to the CD4 transmembrane domain). In some embodiments, the variant or homologue has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 consecutive amino acids) compared to the native CD4 transmembrane domain polypeptide.

[0138] The term “CD3 zeta transmembrane domain” as provided herein includes any recombinant or native form of a variant or homologue of the CD3 zeta transmembrane domain or the CD3 zeta transmembrane domain that maintains CD3 zeta transmembrane domain activity (e.g., activity within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to the CD3 zeta transmembrane domain). In some embodiments, the variant or homologue has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 consecutive amino acids) compared to the native CD3 zeta transmembrane domain polypeptide.

[0139] In several embodiments, the recombinant protein includes a spacer region connecting a non-CDR Fab-binding peptide domain to a transmembrane domain. In several embodiments, the spacer region is located between the transmembrane domain and the non-CDR Fab-binding peptide domain. In other words, the spacer region is directly or indirectly (e.g., via a peptide linker) connected to the C-terminus of the non-CDR Fab-binding peptide domain and directly or indirectly (e.g., via another peptide linker) connected to the N-terminus of the transmembrane domain. Thus, the recombinant protein provided herein may include a first peptide linker and a second peptide linker, the first peptide linker connecting the C-terminus of the non-CDR Fab-binding peptide domain to the N-terminus of the spacer region, and the second peptide linker connecting the C-terminus of the spacer region to the N-terminus of the transmembrane domain.

[0140] The “spacer region” provided herein is a polypeptide that connects a non-CDR Fab-binding peptide domain to a transmembrane domain. In several embodiments, the binding affinity of the non-CDR Fab-binding peptide domain to the antigen-binding domain (e.g., Fab) is increased compared to the absence of the spacer region. In several embodiments, 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.

[0141] In several embodiments, the spacer region includes an Fc region. Examples of spacer regions envisioned 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) including mutations that affect binding to the Fc receptor. In several embodiments, the spacer region is a fragment of IgG (e.g., IgG4), in which case the fragment includes a deletion of the CH2 domain. In several embodiments, the spacer region is a fragment of IgG (e.g., IgG4), in which case the fragment includes a CH3 region. In several embodiments, the spacer region is a CH3 region. In several embodiments, the spacer region includes a CH3 region. In several embodiments, the spacer region is a CH2 region. In several embodiments, the spacer region includes a CH2 region. The spacer region may be a peptide linker. In several embodiments, the spacer region is a serine-glycine linker. In several embodiments, the spacer region has the sequence GGSG. In several embodiments, the spacer region includes the sequence GGSG. In several embodiments, the spacer region has the sequence GSGSGSGS (SEQ ID NO: 24). In several embodiments, the spacer region includes the sequence GSGSGSGS (SEQ ID NO: 24). In several embodiments, the spacer region is at least 4 amino acids long. In several embodiments, the spacer region is about 4 amino acids long. In several embodiments, the spacer region is between 4 and 250 amino acids long. The spacer region may contain residues that can extend the in vivo (e.g., in plasma) half-life of the protein provided herein. In several embodiments, the spacer region is 10 amino acids long. In several embodiments, the spacer region is 229 amino acids long. In several embodiments, the spacer region is GGGSSGGGSG (SEQ ID NO: 31). In several embodiments, the spacer region includes the sequence GGGSSGGGSG (SEQ ID NO: 31).Spacer regions can be “PAS-ified.” The term “PAS-ified” is used in its general sense to refer to an amino acid sequence that forms a highly soluble biological polymer due to its high proline, alanine, and serine content. Thus, in several embodiments, the spacer region contains a combined approximately 200 proline, alanine, and serine residues. In several embodiments, the spacer region contains a combined approximately 10 to approximately 200 proline, alanine, and serine residues. In several embodiments, the spacer region contains hydrophilic residues. In several embodiments, the recombinant protein does not contain a spacer region.

[0142] In several embodiments, the spacer region includes the sequence of sequence number 33.

[0143] In several embodiments, the recombinant protein includes a peptide linker connecting a non-CDR Fab-binding peptide domain to a spacer region. In several embodiments, the peptide linker is located between the non-CDR Fab-binding peptide domain and the spacer region. In other words, the peptide linker is directly or indirectly (e.g., via another peptide linker) connected to the C-terminus of the non-CDR Fab-binding peptide domain and directly or indirectly (e.g., via another peptide linker) connected to the N-terminus of the spacer region. The peptide linkers provided herein (e.g., a first peptide linker or a second peptide linker) may be 5 to 50 amino acids long. In several embodiments, the peptide linker (e.g., a first peptide linker or a second peptide linker) is 5 to 45 amino acids long. In several embodiments, the peptide linker (e.g., a first peptide linker or a second peptide linker) is 5 to 40 amino acids long. In several embodiments, the peptide linker (e.g., the first peptide linker or the second peptide linker) is 5 to 35 amino acids long. In several embodiments, the peptide linker (e.g., the first peptide linker or the second peptide linker) is 5 to 30 amino acids long. In several embodiments, the peptide linker (e.g., the first peptide linker or the second peptide linker) is 5 to 25 amino acids long. In several embodiments, the peptide linker (e.g., the first peptide linker or the second peptide linker) is 5 to 20 amino acids long. In several embodiments, the peptide linker (e.g., the first peptide linker or the second peptide linker) is 5 to 15 amino acids long. In several embodiments, the peptide linker (e.g., the first peptide linker or the second peptide linker) is 5 to 10 amino acids long.In several embodiments, the peptide linker (e.g., the first peptide linker or the 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 several embodiments, the peptide linker (e.g., the first peptide linker or the second peptide linker) is 18 amino acids long. In several embodiments, the peptide linker has the sequence of SEQ ID NO: 25. In several embodiments, the peptide linker contains the sequence of SEQ ID NO: 25. In several embodiments, the peptide linker is sequence SAPASSASAPSAASAPAG (SEQ ID NO: 26).

[0144] In several embodiments, the peptide linker is a first peptide linker, and the recombinant protein includes a second peptide linker, the second peptide linker connecting the spacer region to the transmembrane domain. Thus, in several embodiments, 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; the second peptide linker is connected to the C-terminus of the spacer region and to the N-terminus of the transmembrane domain.

[0145] In several embodiments, the recombinant proteins provided herein include an intracellular costimulatory signaling domain that connects a transmembrane domain to an intracellular T cell signaling domain. In several embodiments, the intracellular costimulatory signaling domain is located between the transmembrane domain and the intracellular T cell signaling domain. In other words, the intracellular costimulatory signaling domain is directly or indirectly (e.g., via a peptide linker) connected to the C-terminus of the transmembrane domain and directly or indirectly (e.g., via another peptide linker) connected to the N-terminus of the intracellular T cell signaling domain.

[0146] The “intracellular co-stimulatory signaling domain” provided herein includes an amino acid sequence capable of inducing a co-stimulatory signal in response to the binding of an antigen to an antibody region provided herein, including its embodiments. In several embodiments, the signaling of the co-stimulatory signaling domain results in cytokine production and proliferation of T cells expressing cytokines. In several embodiments, 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 several embodiments, the intracellular co-stimulatory signaling domain includes a CD28 intracellular co-stimulatory signaling domain. In several embodiments, the intracellular co-stimulatory signaling domain is a CD28 intracellular co-stimulatory signaling domain. In several embodiments, the intracellular co-stimulatory signaling domain includes a 4-1BB intracellular co-stimulatory signaling domain. In several embodiments, the intracellular co-stimulatory signaling domain is a 4-1BB intracellular co-stimulatory signaling domain. In several embodiments, the intracellular co-stimulus signaling domain is the CD28 intracellular co-stimulus signaling domain, the 4-1BB intracellular co-stimulus signaling domain, the ICOS intracellular co-stimulus signaling domain, the OX-40 intracellular co-stimulus signaling domain, or any combination thereof.

[0147] Exemplary intracellular costimulatory signaling domains, including sequences and accession numbers, are listed in Table 2. In several embodiments, the intracellular costimulatory signaling domain comprises a protein identified by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In several embodiments, the intracellular costimulatory signaling domain is3. In several embodiments, the intracellular costimulatory signaling domain is SEQ ID NO: 14.

[0148] In several embodiments, the recombinant protein includes a detection domain bound to the C-terminus of the intracellular T cell signaling domain. In several embodiments, the detection domain is a cleaved CD19 (CD19t) domain. The term "CD19t" refers to a cleaved CD19 protein that lacks intracellular signaling ability. As used herein, cleaved CD19 is an inactive molecule that functions as a detection domain for identifying T cells containing the recombinant protein provided herein. In several embodiments, the detection domain includes the sequence of SEQ ID NO: 22.

[0149] In several embodiments, the recombinant protein includes a self-cleaving peptidyl sequence that connects the intracellular T cell signaling domain to the detection domain. In several embodiments, the self-cleaving peptidyl linker sequence is a T2A sequence or a 2A sequence. In several embodiments, the self-cleaving peptidyl sequence is located between the intracellular T cell signaling domain and the detection domain. In other words, the self-cleaving peptidyl sequence is directly or indirectly (e.g., via a peptide linker) connected to the C-terminus of the intracellular T cell signaling domain and directly or indirectly (e.g., via another peptide linker) connected to the N-terminus of the detection domain.

[0150] In several embodiments, the self-cleaving peptidyl linker has the sequence PVKQLLNFDLLKLAGDVESNPGP (SEQ ID NO: 27). In several embodiments, the self-cleaving peptidyl linker has the sequence of equine rhinitis virus type A. In several embodiments, the self-cleaving peptidyl linker has the sequence QCTNYALLKLAGDVESNPGP (SEQ ID NO: 28). In several embodiments, the self-cleaving peptidyl has the sequence of porcine rhinitis virus type 1. In several embodiments, the self-cleaving peptidyl has the sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 29). In several embodiments, the self-cleaving peptidyl linker has the sequence of Tosea asigna virus. In several embodiments, the self-cleaving peptidyl linker has the sequence EGRGSLLTCGDVESNPGP (SEQ ID NO: 30). In several embodiments, the self-cleaving peptidyl linker has the sequence of SEQ ID NO: 21. In several embodiments, the self-cleaving peptidyl linker is the sequence of Sequence ID No. 21.

[0151] In several embodiments, the recombinant protein forms part of a cell. In several embodiments, the recombinant protein forms part of a T cell. In several embodiments, the transmembrane domain forms part of the T cell membrane.

[0152] As described above, the recombinant proteins provided herein can bind to antigen-binding domains. While the elements of the recombinant proteins provided herein, including those embodiments (e.g., non-CDR Fab-binding peptide domain, transmembrane domain, intracellular T cell signaling domain, intracellular costimulatory signaling domain), are covalently bound to each other to form a continuous single-chain polypeptide, the recombinant proteins bind to the antigen-binding domain noncovalently. In several embodiments, the non-CDR Fab-binding peptide domain is bound to the antigen-binding domain. In several embodiments, the non-CDR Fab-binding peptide domain is bound to the antigen-binding domain noncovalently.

[0153] As described above, the antigen-binding domain may be Fab, IgG, or a bispecific antibody. In several embodiments, the antigen-binding domain is a cetuximab medotope-compatible domain, a trastuzumab medotope-compatible domain, a pertuzumab medotope-compatible domain, an M5A medotope-compatible domain, or a rituximab medotope-compatible domain. In several embodiments, the antigen-binding domain is capable of binding to a cancer antigen. In several embodiments, the antigen-binding domain is capable of binding to a cancer antigen. In several embodiments, the antigen-binding domain is capable of binding to a cancer antigen by non-covalent bond. In several embodiments, the cancer antigen is Her2, EGFR, CD19, or CD20. In several embodiments, the cancer antigen forms part of a cell. In several embodiments, the cancer antigen is expressed on the surface of a cell. In several embodiments, the cell is a cancer cell. In several embodiments, 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.

[0154] The compositions provided herein may comprise a plurality (i.e., at least two, more than one) of the recombinant proteins provided herein, including embodiments thereof. If a composition comprises a plurality of recombinant proteins provided herein, including embodiments thereof, the recombinant proteins are referred herein to as the first, second, third, fourth, etc. recombinant proteins. Accordingly, the elements forming a portion of the first, second, third, fourth, etc. recombinant proteins are referred herein to, respectively, 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 costimulatory signaling domain. If a composition comprises a plurality of recombinant proteins provided herein, including embodiments thereof, the recombinant proteins may be different or the same. In other words, recombinant proteins may contain the same or different domains (e.g., non-CDR Fab-binding peptide domain, intracellular T cell signaling domain, transmembrane domain, spacer region, intracellular costimulatory signaling domain), and may be capable of binding to the same or different antigen-binding domains.

[0155] If a composition provided herein comprises a plurality of recombinant proteins provided herein, the recombinant proteins may dimerize with each other via non-covalent bonding of their respective spacer regions. For example, the first recombinant protein may include a first CH3 domain that non-covalently binds to a second CH3 domain of the second recombinant protein. Thus, in some embodiments, recombinant protein is the first recombinant protein, non-CDR Fab-binding peptide domain is the first non-CDR Fab-binding peptide domain, intracellular T cell signaling domain is the first intracellular T cell signaling domain, transmembrane domain is the first transmembrane domain, spacer region is the first spacer region, and intracellular costimulatory signaling domain is the first intracellular costimulatory signaling domain.

[0156] In several embodiments, the first recombinant protein is non-covalently bound to the second recombinant protein, the second recombinant protein comprising (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 connecting the second non-CDR Fab-binding peptide domain to the second transmembrane domain, to which the first spacer region is non-covalently bound. In several embodiments, the first and second spacer regions are a first heavy chain constant 3(CH3) domain and a second heavy chain constant 3(CH3) domain.

[0157] In several embodiments, the first non-CDR Fab-binding peptide domain and the second non-CDR Fab-binding peptide domain are chemically different. In several embodiments, the first non-CDR Fab-binding peptide domain and the second non-CDR Fab-binding peptide domain are chemically identical.

[0158] In several embodiments, the first non-CDR Fab-binding peptide domain is non-covalently bound to the first antigen-binding domain. In several embodiments, the second non-CDR Fab-binding peptide domain is non-covalently bound to the second antigen-binding domain. In several embodiments, the first antigen-binding domain and the second antigen-binding domain are chemically different or chemically identical. In several embodiments, the first antigen-binding domain and the second antigen-binding domain are independently a cetuximab medotope-compatible domain, a trastuzumab medotope-compatible domain, a pertuzumab medotope-compatible domain, an M5A medotope-compatible domain, or a rituximab medotope-compatible domain.

[0159] In one embodiment, the recombinant protein includes the non-CDR Fab-binding peptide domain of SEQ ID NO: 32, the spacer region of SEQ ID NO: 33, the CD28 transmembrane domain of SEQ ID NO: 18, the CD28 intracellular costimulatory signaling domain of SEQ ID NO: 13, the CD3ζ intracellular T cell signaling domain of SEQ ID NO: 34, the autocleavable peptidyl linker sequence of SEQ ID NO: 21, and the detection domain of SEQ ID NO: 22.

[0160] In one embodiment, the recombinant protein includes the non-CDR Fab-binding peptide domain of SEQ ID NO: 32, the spacer region of SEQ ID NO: 33, the CD28 transmembrane domain of SEQ ID NO: 18, the 4-1BB intracellular costimulatory signaling domain of SEQ ID NO: 14, the CD3ζ intracellular T cell signaling domain of SEQ ID NO: 34, the autocleavable peptidyl linker sequence of SEQ ID NO: 21, and the detection domain of SEQ ID NO: 22.

[0161] In one embodiment, the recombinant protein comprises the non-CDR Fab-binding peptide domain of SEQ ID NO: 32, the spacer region of SEQ ID NO: 33, the CD28 transmembrane domain of SEQ ID NO: 18, the CD28 intracellular costimulatory signaling domain of SEQ ID NO: 13, and the CD3ζ intracellular T cell signaling domain of SEQ ID NO: 34.

[0162] In one embodiment, the recombinant protein includes the non-CDR Fab-binding peptide domain of SEQ ID NO: 32, the spacer region of SEQ ID NO: 33, the CD28 transmembrane domain of SEQ ID NO: 18, the 4-1BB intracellular costimulatory signaling domain of SEQ ID NO: 14, and the CD3ζ intracellular T cell signaling domain of SEQ ID NO: 34.

[0163] In one embodiment, the recombinant protein includes 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 CD28 intracellular costimulatory signaling domain of SEQ ID NO: 13, the CD3ζ intracellular T cell signaling domain of SEQ ID NO: 34, the self-cleaving peptidyl linker sequence of SEQ ID NO: 21, and the detection domain of SEQ ID NO: 22.

[0164] In one embodiment, the recombinant protein includes 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 costimulatory signaling domain of SEQ ID NO: 14, the CD3ζ intracellular T cell signaling domain of SEQ ID NO: 34, the self-cleaving peptidyl linker sequence of SEQ ID NO: 21, and the detection domain of SEQ ID NO: 22.

[0165] In several embodiments, the recombinant protein contains the signal peptide of SEQ ID NO: 37. In several embodiments, the signal peptide is bound to the N-terminus of a non-CDR Fab-binding peptide domain.

[0166] 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 CD28 intracellular costimulatory signaling domain of SEQ ID NO: 13, and the CD3ζ intracellular T cell signaling domain of SEQ ID NO: 34.

[0167] In one embodiment, the recombinant protein includes 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 costimulatory signaling domain of SEQ ID NO: 14, and the CD3ζ intracellular T cell signaling domain of SEQ ID NO: 34.

[0168] In one embodiment, the recombinant protein contains 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 contains the sequence of SEQ ID NO: 36. In one embodiment, the recombinant protein is the sequence of SEQ ID NO: 36.

[0169] In several embodiments, the recombinant protein comprises, from the N-terminus to the C-terminus, a non-CDR Fab-binding peptide domain, a spacer region, a CD28 transmembrane domain, a CD28 intracellular costimulatory signaling domain, a CD3ζ intracellular T cell signaling domain, a self-cleaving peptidyl linker sequence, and a detection domain.

[0170] In several embodiments, the recombinant protein comprises, from the N-terminus to the C-terminus, a non-CDR Fab-binding peptide domain, a spacer region, a CD28 transmembrane domain, a 4-1BB intracellular costimulatory signaling domain, a CD3ζ intracellular T cell signaling domain, a self-cleaving peptidyl linker sequence, and a detection domain.

[0171] In several embodiments, the recombinant protein comprises, from the N-terminus to the C-terminus, a non-CDR Fab-binding peptide domain, a spacer region, a CD28 transmembrane domain, a CD28 intracellular costimulatory signaling domain, and a CD3ζ intracellular T cell signaling domain.

[0172] In several embodiments, the recombinant protein comprises, from the N-terminus to the C-terminus, a non-CDR Fab-binding peptide domain, a spacer region, a CD28 transmembrane domain, a 4-1BB intracellular costimulatory signaling domain, and a CD3ζ intracellular T cell signaling domain.

[0173] In several embodiments, the recombinant protein comprises, from the N-terminus to the C-terminus, a non-CDR Fab-binding peptide domain, a peptide linker, a spacer region, a CD28 transmembrane domain, a CD28 intracellular costimulatory signaling domain, a CD3ζ intracellular T cell signaling domain, a self-cleaving peptidyl linker sequence, and a detection domain.

[0174] In several embodiments, the recombinant protein comprises, from the N-terminus to the C-terminus, a non-CDR Fab-binding peptide domain, a peptide linker, a spacer region, a CD28 transmembrane domain, a 4-1BB intracellular costimulatory signaling domain, a CD3ζ intracellular T cell signaling domain, a self-cleaving peptidyl linker sequence, and a detection domain.

[0175] In several embodiments, the recombinant protein comprises, from the N-terminus to the C-terminus, a non-CDR Fab-binding peptide domain, a peptide linker, a spacer region, a CD28 transmembrane domain, a CD28 intracellular costimulatory signaling domain, and a CD3ζ intracellular T cell signaling domain.

[0176] In several embodiments, the recombinant protein comprises, from the N-terminus to the C-terminus, a non-CDR Fab-binding peptide domain, a peptide linker, a spacer region, a CD28 transmembrane domain, a 4-1BB intracellular costimulatory signaling domain, and a CD3ζ intracellular T cell signaling domain.

[0177] Nucleic acid composition This specification presents nucleic acids encoding recombinant proteins provided herein, including embodiments thereof. Thus, in one embodiment, nucleic acids encoding recombinant proteins provided herein, including embodiments thereof, are presented. In another embodiment, an expression vector comprising nucleic acids provided herein, including embodiments thereof, is presented. In several embodiments, the expression vector is a lentivirus or an oncholoretrovirus. In several embodiments, the expression vector is a lentivirus. In several embodiments, the expression vector is an oncholoretrovirus.

[0178] cell composition The recombinant proteins and nucleic acids provided herein can form part of a cell (i.e., be contained in and / or expressed in a cell). Accordingly, in one embodiment, a T lymphocyte comprising an expression vector provided herein, including embodiments thereof, is provided.

[0179] In another embodiment, T lymphocytes comprising recombinant proteins provided herein, including embodiments thereof, are provided. Some of the recombinant proteins provided herein may form part of the cell membrane of the cell on which they are expressed. The transmembrane domain can span the cell membrane, for example, from one side of the membrane to the other side of the membrane of a T cell. In some embodiments, the transmembrane domain spans the cell membrane from the intracellular to the extracellular side. Thus, the non-CDR Fab-binding peptide domain and spacer region are located on the extracellular side of the cell membrane, while the intracellular T cell signaling domain is located on the intracellular side of the cell. In some embodiments, the transmembrane domain is located within the cell membrane of the T lymphocyte. In some embodiments, the T lymphocytes are autologous T lymphocytes. In some embodiments, the T lymphocytes are heterologous T lymphocytes.

[0180] Therefore, in several embodiments, the non-CDR Fab-binding peptide domain is bound to the antigen-binding domain. In several embodiments, the antigen-binding domain is Fab, IgG, or a bispecific antibody. In several embodiments, the antigen-binding domain is bound to a cancer antigen.

[0181] The antigen-binding domain can be an antibody containing a first Fab domain and a second Fab domain, the recombinant protein can bind to a non-CDR binding site of the first Fab domain, and the second Fab domain can bind to a cancer antigen. In several embodiments, 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 several embodiments, 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 several embodiments, 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 further embodiments, the first Fab domain binds to a first cancer antigen, and the second Fab domain binds to a second cancer antigen.

[0182] In several embodiments, the cancer antigen is Her2, EGFR, CD19, or CD20. In several embodiments, the antigen-binding domain is a cancer antigen-binding domain. In several embodiments, the antigen-binding domain is a cetuximab medotope-compatible domain, a trastuzumab medotope-compatible domain, a pertuzumab medotope-compatible domain, an M5A medotope-compatible domain, or a rituximab medotope-compatible domain.

[0183] Treatment method The compositions provided herein, including those embodiments, are particularly useful for providing effective treatment for diseases such as cancer. Accordingly, in one embodiment, a method for treating cancer is provided. The method comprises administering to a subject in need an effective amount of T lymphocytes provided herein, including those embodiments, 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.

[0184] In several embodiments, T lymphocytes and antigen-binding domains are administered simultaneously or sequentially.

[0185] T lymphocytes and antigen-binding domains may be administered synchronically (e.g., as a mixture), individually but simultaneously (e.g., via separate intravenous lines), or sequentially (e.g., one drug is administered first, followed by the administration of a second drug), or in combination. Therefore, the term “combination” is used to refer to the synchronous, simultaneous, or sequential administration of T lymphocytes and antigen-binding domains. In embodiments where 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, with the first time point preceding the second. The course of treatment is best determined on an individual basis, depending on the specific characteristics of the subject and the type of treatment selected. Treatments such as those disclosed herein may be administered to the subject daily, twice daily, every other week, monthly, or on any applicable basis that is therapeutically effective. The treatment may be administered alone or in combination with any other treatment disclosed herein or known in the art. Further treatments may be administered simultaneously with the first treatment, at different times, or according to entirely different treatment schedules (for example, the first treatment may be a daily treatment while the further treatment is a weekly treatment). Thus, in several embodiments, T lymphocytes and antigen-binding domains may be administered simultaneously or sequentially.

[0186] In several embodiments, T lymphocytes are administered at time point 1, and antigen-binding domains are administered at time point 2, with time point 1 preceding time point 2. In several embodiments, time point 2 is less than approximately 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 1 day from time point 1. In several embodiments, time point 2 is less than approximately 120 days from time point 1. In several embodiments, time point 2 is less than approximately 90 days from time point 1. In several embodiments, time point 2 is less than approximately 60 days from time point 1. In several embodiments, time point 2 is less than approximately 50 days from time point 1. In several embodiments, the second time point is within approximately 40 days from the first time point. In several embodiments, the second time point is within approximately 30 days from the first time point. In several embodiments, the second time point is within approximately 20 days from the first time point.

[0187] In several embodiments, the antigen-binding domain is administered at time point 1, and T lymphocytes are administered at time point 2, with time point 1 preceding time point 2. In several embodiments, time point 2 is less than approximately 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 1 day from time point 1. In several embodiments, time point 2 is less than approximately 120 days from time point 1. In several embodiments, time point 2 is less than approximately 90 days from time point 1. In several embodiments, time point 2 is less than approximately 60 days from time point 1. In several embodiments, time point 2 is less than approximately 50 days from time point 1. In several embodiments, the second time point is within approximately 40 days from the first time point. In several embodiments, the second time point is within approximately 30 days from the first time point. In several embodiments, the second time point is within approximately 20 days from the first time point.

[0188] In several embodiments, the T lymphocytes and antigen-binding domains are mixed before administration. In several embodiments, the method includes (i) conjugating a non-CDR Fab-binding peptide domain to an antigen-binding domain in vitro before administration, thereby forming a T lymphocyte-recombinant protein complex; and (ii) administering the T lymphocyte-recombinant protein complex to a subject, thereby treating cancer in the subject.

[0189] In several embodiments, T lymphocytes and antigen-binding domains are administered sequentially. In several embodiments, T lymphocytes are administered at a first time point, and the antigen-binding domains are administered at a second time point, with the first time point preceding the second. In several embodiments, the antigen-binding domains are administered at a first time point, and the T lymphocytes are administered at a second time point, with the first time point preceding the second.

[0190] In several embodiments, 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 several embodiments, the antigen-binding domain is a cetuximab medotope-compatible domain, a trastuzumab medotope-compatible domain, a pertuzumab medotope-compatible domain, an M5A medotope-compatible domain, or a rituximab medotope-compatible domain.

[0191] 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 may 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, kidney cancer, myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer (e.g., triple-negative breast cancer, ER-positive breast cancer, ER-negative breast cancer, chemotherapy-resistant breast cancer, Herceptin-resistant breast cancer, HER2-positive breast cancer, doxorubicin-resistant breast cancer, tamoxifen-resistant breast cancer, fallopian tube cancer, This includes lobular carcinoma, primary breast cancer, metastatic breast cancer, ovarian cancer, pancreatic cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer, squamous cell lung cancer, adenocarcinoma, large cell lung cancer, small cell lung cancer, carcinoid, sarcoma), glioblastoma pleomorphonosum, 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. Further examples include thyroid cancer, endocrine cancer, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, non-small cell lung cancer, melanoma, mesothelioma, ovarian cancer, sarcoma, stomach cancer, uterine cancer or medulloblastoma, Hodgkin's disease, non-Hodgkin lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytopenia, primary macroglobulinemia, and primary brain tumors. This includes cancer, malignant islet cell tumor, malignant carcinoid, bladder cancer, pre-malignant skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, urogenital tract cancer, malignant hypercalcemia, endometrial cancer, adrenocortical cancer, neoplasms of the pancreatic endocrine or exocrine glands, medullary thyroid carcinoma, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid carcinoma, hepatocellular carcinoma, Paget's disease of the nipple, phyllodes tumor, lobular carcinoma, ductal carcinoma, astrocellular carcinoma of the pancreas, hepatic astrocellular carcinoma, or prostate cancer.

[0192] The term "leukemia" broadly refers to a progressive malignant disease of the hematopoietic system, generally characterized by the abnormal proliferation and development of white blood cells 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 (myelogenous), lymphoid (lymphogenous), or monocytes); and (3) the increase or non-increase in the number of abnormal cells in the blood (leukemic or non-leukemic (subleukemic)). Exemplary leukemias that can be treated by the compounds, pharmaceutical compositions, or methods provided herein include, for example, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, non-leukemic leukemia, leukemia, and basophilic leukemia. , blastic leukemia, bovine leukemia, chronic myeloid leukemia, cutaneous leukemia, fetal leukemia, eosinophilic leukemia, Gross leukemia, hair cell leukemia, hemoblastic leukemia, hemocyte progenitor leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcomacytic leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myeloid leukemia, myelogranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia This includes cell-mediated leukemia, multiple myeloma, plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemia, or anaplastic cell leukemia.

[0193] The term "sarcoma" generally refers to a tumor composed of embryonic connective tissue-like material, and generally consists of tightly packed cells embedded in small fibrous material or homogeneous material. Sarcomas that can be treated by the compounds, pharmaceutical compositions or methods provided herein include chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abernesi's sarcoma, and liposarcoma (adipose). This includes sarcoma, liposarcoma, alveolar soft part sarcoma, amelophyte fibrosarcoma, staphyloid sarcoma, chloroplastoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, B-cell immunoblastic sarcoma, lymphoma, T-cell immunoblastic sarcoma, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukemosarcoma, malignant mesenchymal sarcoma, paraosteal sarcoma, reticular sarcoma, Rous sarcoma, serous cystic sarcoma, synovial sarcoma, or tubulodilator sarcoma.

[0194] The term "melanoma" is understood to mean tumors arising from the pigment cell system of the skin and other organs. Melanomas that can be treated by the compounds, pharmaceutical compositions or methods provided herein include, for example, acral lentiginous melanoma, achromatic melanoma, benign juvenile melanoma, Cloudmann melanoma, S91 melanoma, Harding-Passé melanoma, juvenile melanoma, lentigo malignant melanoma, malignant melanoma, nodular melanoma, subungual melanoma, or superficial spreading melanoma.

[0195] The term "carcinoma" refers to a malignant new growth composed of epithelial cells that tends to invade surrounding tissues and metastasize.Exemplary carcinomas that can be treated with the compounds, pharmaceutical compositions, or methods provided herein include, for example, medullary thyroid carcinoma, familial medullary thyroid carcinoma, acinar carcinoma, acinar cell carcinoma, adenoid carcinoma, adenoid cystic carcinoma, carcinoma in adenoma, adrenocortical carcinoma, alveolar epithelial carcinoma, alveolar epithelial cell carcinoma, basal cell carcinoma (carcinoma basocellulare), basal cell carcinoma, basal cell type squamous cell carcinoma, bronchioloalveolar carcinoma, bronchogenic carcinoma, cerebral carcinoma, cholangiocarcinoma, choriocarcinoma, gliocarcinoma, comedo carcinoma, endometrial carcinoma, cribriform carcinoma, armory carcinoma, skin carcinoma, columnar epithelial carcinoma, columnar epithelial cell carcinoma, adenoid carcinoma, ductal carcinoma, compact carcinoma, fetal carcinoma, cerebral carcinoma, epidermoid carcinoma, adenoid epithelial carcinoma, exotropic carcinoma, ulcerative carcinoma, fibrous carcinoma, gelatinous carcinoma, gliocarcinoma, and giant cell carcinoma. cell carcinoma, carcinoma gigantocellulare), adenocarcinoma, granulosa cell carcinoma, pilocomatous carcinoma, hepatoid carcinoma, hepatocellular carcinoma, Hürthle cell carcinoma, glassy carcinoma, renal clear cell carcinoma, pediatric embryonal carcinoma, carcinoma in situ, intraepithelial carcinoma, intraepithelial carcinoma, Crompecher carcinoma, Kruczyski cell carcinoma, large cell carcinoma, lenticular carcinoma, carcinoma lenticulare), fatty carcinoma, lobular carcinoma, lymphoepithelial carcinoma, medullary carcinoma (carcinoma medullare, medullary carcinoma), pigmented carcinoma, soft carcinoma, mucinous carcinoma (carcinoma muciparum), mucinous cell carcinoma, mucoepidermoid carcinoma, mucosal carcinoma (carcinoma mucosus, mucous) This includes carcinoma, myxomatous carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, pre-invasive carcinoma, squamous cell carcinoma, atherosclerotic carcinoma, renal cell carcinoma, preliminary cell carcinoma, sarcomatoid carcinoma, Schneiderian carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, simple carcinoma, small cell carcinoma, eggplant-shaped carcinoma, spheroid cell carcinoma, spindle cell carcinoma, cavernous carcinoma, squamous cell carcinoma, squamous cell carcinoma, string-like carcinoma, telangiectaticum (carcinoma telangiectaticum, carcinoma telangiectodes), transitional cell carcinoma, nodular carcinoma (carcinoma tuberosum), tubular carcinoma, tuberous carcinoma, verrucous carcinoma, or choriocarcinoma.

[0196] As used herein, the terms “metastasis,” “metastatic,” and “metastatic cancer” may be used interchangeably and refer to the spread of a proliferative disorder or disorder, such as cancer, from one organ or another non-adjacent organ or part of the body. Cancer that arises in the site of origin, such as the breast, is called a primary tumor, such as primary breast cancer. Some cancer cells in a primary tumor or site of origin acquire the ability to penetrate and invade surrounding normal tissue within a local area, and / or to penetrate the inner walls of the lymphatic or vascular system and circulate to other parts and tissues of the body via the lymphatic and vascular systems. A clinically detectable secondary tumor formed from cancer cells of a primary tumor is called a metastatic tumor or secondary tumor. When cancer cells metastasize, it is assumed that the metastatic tumor and its cells are similar to the cells of the original tumor. Therefore, if lung cancer metastasizes to the breast, the secondary tumor in the breast site will consist of abnormal lung cells and not abnormal mammary gland cells. Secondary tumors within the breast are referred to as metastatic lung cancer. Therefore, the term metastatic cancer refers to a disease in which the subject has, or has had, a primary tumor and has one or more secondary tumors. The term non-metastatic cancer, or a subject with 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 a subject that has, or has a history of having, a primary lung tumor and has one or more secondary tumors in a second or more locations, such as within the breast.

[0197] "Anticancer" is used in its obvious and ordinary sense and refers to a composition (e.g., compound, drug, antagonist, inhibitor, modulator) having antineoplastic properties or the ability to inhibit cell growth or proliferation. In some embodiments, an anticancer agent is a chemotherapeutic agent. In some embodiments, an anticancer agent is a drug identified herein as having utility in a method of treating cancer. In some embodiments, an anticancer agent is a drug approved by the FDA or a similar regulatory agency in a country other than the United States for the treatment of cancer.

[0198] In the context of a substance or the activity or function of a substance 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 terms “associated” or “associated with ~” mean that 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) is caused (whole or partially) by the substance or the activity or function of the substance, or that the symptoms of the disease are caused (whole or partially).

[0199] "Chemotherapy" or "chemotherapeutic agent" is used in its obvious and ordinary sense to mean a chemical composition or compound having antineoplastic properties or the ability to inhibit cell growth or proliferation.

[0200] As used herein, the term “abnormal” means different from normal. When used to describe enzyme activity, abnormal means activity that is above or below the mean of a normal control or normal non-affected control sample. Abnormal activity may mean activity at a disease-causing level, in which case restoring the abnormal activity to a normal or non-disease-related level (e.g., by using the methods described herein) results in the disease or reduction of one or more disease symptoms. [Examples]

[0201] CAR T cells have demonstrated remarkable success in treating humoral tumors and are rapidly being expanded to treat solid tumors and other diseases. Current approaches involve creating CAR T cells that target a single antigen. While effective in eliminating tumor cells expressing the specified antigen, tumor cells that do not express the antigen can escape and proliferate, often becoming more invasive. Therefore, to target these cells, there is a need to create entirely new CAR T cells that express scFvs that target different tumors. In this specification, the applicants address this problem using meditope technology. Specifically, the applicants replace tumor-targeting scFvs with ultra-high affinity meditopes (non-CDR Fab-binding peptide domains). Tumor specificity is then added using antigen-specific meditope-compatible Fab or mAb (antigen-binding domains).

[0202] The applicants have created universal CAR T cells using meditope interactions. Specifically, the applicants have demonstrated that the antigen targeting region can be replaced with a meditope (non-CDR Fab-binding peptide domain), a meditope-compatible Fab / mAb can be added, and the Fab can be bound to an antigen specific to that Fab. The advantage described herein is that the applicants can create meditope-zeta chain T cells and replace them with disease-specific meditope-compatible Fabs or target multiple antigens.

[0203] The field of CAR T cells is rapidly developing, and as clinical results become available, the need to modify the specificity of CAR T cells is becoming increasingly apparent. We present a universal CAR T cell platform in which we replace the scFv within CAR T cells with a meditope (e.g., a non-CDR Fab-binding peptide domain) and add antigen specificity using a meditope-compatible mAb / Fab (antigen-binding domain). This platform technology allows us to rapidly and efficiently modify target specificity without creating and optimizing individual CAR T cells. We demonstrate proof of concept via FACS using two different meditope-compatible mAbs. We can optimize linker design and docking to support efficacy in animal models by modifying the antigen specificity of meditope-CAR cells in situ.

[0204] By mixing and matching single T cells expressing meditopes with Fab / mAbs that target different antigens / epitopes within tumors, it is possible to potentially create superior products applicable to multiple forms of cancer. Fab / mAbs are generally more stable than scFv, generally have a higher affinity for antigens than scFv, and panels of Fabs that bind to different epitopes on antigens can be easily fabricated and rapidly optimized (for example, a current question in the field is how much distance between the receptor epitope and the tumor membrane affects efficacy).

[0205] The applicants can package meditopes within lentiviruses to produce FabRack (recombinant proteins provided herein) and characterize tumor eradication in vitro and in vivo. The applicants can modify and characterize specificity in situ. The applicants can create, produce, characterize, and modify the specificity of monovalent FabRack by modifying linkers (e.g., the CH3 domain, which is a remover).

[0206] Conventional CARs and medotope CARs: Basic concept: scFv variants are used in clinical settings. The "simplest" FabRack is bound to a medotope-compatible IgG or Fab fragment. It is noteworthy that this is not limited to IgG or Fab, and bispecific IgG, bionics, or single-arm Fabs can also be created. Basically, anything can be fused to a medotope-compatible Fab.

[0207] Fabrack-T cells may bind to tumor cells via meditope-responsive IgG. Multiple combinations / interactions exist that can result in productive interactions. These include: one Fab arm binding to a Fabrack T cell and the other arm binding to a tumor cell; the same Fab arm binding to both the Fabrack T cell and the tumor antigen; both Fab arms binding to the tumor and one or the other Fab arm binding to the Fabrack T cell; and both Fab arms binding to both the Fabrack T cell and the tumor.

[0208] The Meditope CAR construct can be packed into a lentiviral vector. The extracellular domain includes the Meditope (non-CDR Fab-binding peptide domain), a linker (peptide linker), and the IgG CH3 domain (spacer region), followed by the CD28 transmembrane domain (CD28tm), the CD28 costimulatory domain (CD28), and the CD3ζ cell-lytic domain (also referred herein as the intracellular T cell signaling domain) (CD3ζ). CD3ζ and CD19t are separated by a T2A sequence. A memAb (Meditope-compatible monoclonal antibody) is conjugated to target cells and FabRack-expressing Jurkat cells possessing NFAT-responsive luciferase. This is a simple readout to test the concept before transfer to T cells.

[0209] memAb (meditope-compatible monoclonal antibody; antigen-binding domain) can be pre-bound to FabRack cells (T cells expressing recombinant proteins provided herein), and memAb (antigen-binding domain) can also be pre-bound to target cells (e.g., cancer cells). The level of antigen expression on target cells affects T cell activation. T cell activation is equivalent for T cells pre-bound with memAb and for cancer cells pre-bound with memAb, where antigen expression on the cancer cells is low. The best T cell activation can be achieved when cancer cells are pre-bound with memAb and antigen expression in the cancer is high. Similarly, if the level of antigen expression in the cancer is high, T cell activation is high if T cells are pre-bound with memAb (meditope-compatible monoclonal antibody, antigen-binding domain). See Figure 19.

[0210] Materials and methods Cloning: The parental anti-Her2 scFv_IgG4op(HL-CH3)_CD28gg_op-Zeta_op-T2A-CD19t_epHIV7 vector was donated by Dr. C. Brown. The GM-CSFr secretion signal_meditope_PASlinker17 gene cassette was synthesized using DNA2.0 and inserted into CAR vectors with or without the IgG4op(HL-CH3) domain (meditope-CH3 and meditope-CD28, respectively) using the NheI and SbfI restriction sites. The cloned plasmids were purified using the MaxiPrep kit (Qiagen).

[0211] Labeling of soluble proteins with fluorophores: Alexa Fluor dye (ThermoFisher) was conjugated to soluble proteins using amine conjugation according to the manufacturer's protocol. Specifically, Alexa Fluor 647 NHS Ester dye was conjugated with trastuzumab I83E IgG and trastuzumab I83E Fab, and ipilimumab IgG. The degree of labeling (DOL) was A. 280 and A max Using [a specific method], the amount of dye per molecule was calculated to be between 1 ≤ DOL ≤ 3. Pacific Blue NHS Ester dye was conjugated with sHer2. Protein interactions were characterized by size exclusion chromatography (SEC), followed by a FACS assay to evaluate binding activity.

[0212] Transfection: On day 0, 9th passage CHO-S cells (Invitrogen) were transfected with either no vector (mock), parental CAR vector, meditope-CH3 vector, or meditope-CD28 vector. The cells were transfected using the FreeStyle MAX transfection kit (ThermoFisher) according to the manufacturer's protocol.

[0213] Flow cytometry: On day 5, cells were collected and counted. 3 x 10⁶ cells 6 The cells were added to a 5 mL FACS tube (VWR), and stained with FACS staining solution (Hanks' Balanced Salt Solution with 2% FCS and 0.5% NaN3; Batch #05092016), resulting in 1 × 10 cells per 1 mL. 6 The amount was adjusted to suit each individual cell. 0.1 × 10 6The cells were added to each well of a V-bottom 96-well plate (Corning Costar), with three cells per condition. The cells were washed twice with 100 μL of staining solution, spun at 300 g at 4°C for 3 minutes, and the supernatant was decanted. The cells were resuspended in 100 μL of primary staining solution (PE-Cy7-CD-19 diluted 1:100 and 100 nM 647-IgG, 100 nM 647-Ipi, or 200 nM 647-Fab) protected from light, at 4°C for 30 minutes. The cells were washed twice with 100 μL of staining solution and resuspended in secondary staining solution (200 nM PacBlue-Her2) protected from light, at 4°C for 30 minutes. 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 analyzer (#2, West side of Brown lab), using 40 μL per sample. Voltages were as follows: FSC = 358V, SSC = 520V. The analysis channels were PE (PI), PE-Cy7 (CD19), APC (647), and VioBlue (Her2). The gating strategy was: FSC / SSC → PE- → PE-Cy7 + →APC + →VioBlue + FSC / SSC → PI - →CD19 + →647 + →Her2 + That was the case.

[0214] Animal allocation: Each group will have 4 mice.

[0215] 1. Tumor only: Either OVCAR3-luc or SKOV3-luc is acceptable.

[0216] 2. Tumors with HER-2 CAR T cells: Fab CAR or scFv CAR T cells are acceptable. 1 × 10⁶ positive cells per mouse. 7 Let's consider them as one.

[0217] 3. Tumors with mock T cells: 1 × 10⁶ mock T cells per mouse 7 Let's consider them as one.

[0218] 4. Meditope-CAR T cell-associated tumors: 1 × 10⁶ positive CAR T cells per mouse 7 Let's consider them as one.

[0219] 5. Tumors with pre-mixed mock T cells + HER2 antibody: 1 × 10⁶ mock T cells per mouse 7 The T cells are divided into individual cells. One day before T cell injection, 4 mg / kg of HER2 antibody is administered intraperitoneally. The T cells are pre-mixed with 100 nM antibody and washed. The antibody is administered intraperitoneally at a dose of 4 mg / kg twice a week for two weeks.

[0220] 6. Tumors with pre-mixed Meditope-CAR T cells + HER2 antibody: 1 × 10⁶ CAR T cells per mouse 7 The T cells are divided into individual cells. One day before T cell injection, 4 mg / kg of HER2 antibody is administered intraperitoneally. The T cells are pre-mixed with 100 nM antibody and washed. The antibody is administered intraperitoneally at a dose of 4 mg / kg twice a week for two weeks.

[0221] 7. Tumors with pre-mixed Meditope-CAR T cells + HER2 antibody: 1 × 10⁶ CAR T cells per mouse 7 The T cells are administered intraperitoneally at a dose of 4 mg / kg of HER2 antibody one day before T cell injection. The T cells are not mixed with the antibody beforehand. The antibody is administered intraperitoneally at a dose of 4 mg / kg twice a week for two weeks.

[0222] [Table 1]

[0223] Animal data about Fabrack Method (OVCAR3) Five million OVCAR3-gfp-luc cells were intraperitoneally (ip) injected into mice on day 1. In mice treated with Fabrack T cells (groups 6 and 7), mice were administered 4 mg / kg of the memAb trastuzumab every 3 days (total of 5 administrations), and the first Ab administration was on day 8. Ten million human T cells were ip injected into mice on day 9. In group 6, Fabrack T cells were pre-mixed with and washed with the memAb. After 150 μl of luciferin (28.57 mg / ml) was ip injected into mice, the tumor burden of the mice was measured by luminescence (group 1: only tumors; group 2: mock T cells; group 3: only Fabrack T cells; 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).

[0224] Results (OVCAR3) Mice show a substantial decrease in tumor size when treated with Fabrack T cells and memAb (groups 6 and 7), regardless of whether the Fabrack T cells were pre-mixed with the memAb or not. However, the tumors recurred around day 14. The recurrence of the tumors did not coincide with the loss of the HER2 antigen based on flow cytometry results showing HER2+ tumor cells in mouse ascites. The recurrence of the tumors may be due to the non-survival of T cells as only a very small number of Fabrack T cells remained in the blood and ascites of the mice. A dosing schedule is being developed to optimize tumor eradication.

[0225] Method (MCF7) Five million MCF7-gfp-luc cells were intraperitoneally (ip) injected into mice on day 1. In the Fabrack group, mice were administered 4 mg / kg of memAb trastuzumab approximately every 4 days, and the first Ab administration was ip injected together with T cells. The first administration of 2 million human T cells was ip injected into mice on day 8. An additional 2 million Fabrack T cells were administered approximately every 6 days. After 150 μl of luciferin (28.57 mg / ml) was ip injected into mice, the tumor burden of the mice was measured by luminescence.

[0226] Results (MCF7) A decrease in tumor size was observed on days 11 and 14 in mice administered Fabrack T cells and memAb. However, tumor recurrence was observed on day 16. Analysis of mouse blood on day 22 showed the presence of Fabrack T cells and that the memAb was bound to the Fabrack T cells. Analysis of mouse ascites on day 45 did not show antigen escape of tumor cells. Tumor recurrence may be due to the hook effect derived from the Ab, whose administration can saturate the Ab-binding sites on the tumor and on T cells. Since the dose of Fabrack T cells was 2 million in the MCF7 xenograft study compared to 10 million in the OVCAR3 study, there were fewer Fabrack T cells for binding to the Ab. In addition, since MCF7 and OVCAR3 have low HER2 expression, their HER2 is easily saturated by the Ab. A dosing schedule is being developed to optimize tumor eradication.

[0227] Table

[0228] [Table 2]

[0229] [Table 3]

[0230] P embodiment 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 ligated to the first non-CDR Fab-binding peptide domain.

[0231] Embodiment P2. The 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.

[0232] Embodiment P3. The first recombinant protein according to Embodiment P2, wherein the first spacer region is a first CH3 region.

[0233] Embodiment P4. The first recombinant protein according to Embodiment P3, wherein 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 CH3 region connecting the second non-CDR Fab-binding peptide domain to the second transmembrane domain, the first CH3 region being bound to the second CH3 region, and the first recombinant protein comprising a second spacer region.

[0234] Embodiment P5. The first recombinant protein according to 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.

[0235] Embodiment P6. The first recombinant protein according to 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.

[0236] Embodiment P7. The first intracellular T cell signaling domain and the second intracellular T cell signaling domain are independently the CD3ζ intracellular T cell signaling domain, and the first recombinant protein according to any one of Embodiments P1 to P6.

[0237] Embodiment P8. The first non-CDR Fab-binding peptide domain is non-covalently bound to the first antigen-binding domain, and the first recombinant protein according to any one of Embodiments P1 to P7.

[0238] Embodiment P9. The second non-CDR Fab-binding peptide domain is non-covalently bound to the second antigen-binding domain, and the first recombinant protein according to any one of Embodiments P1 to P8.

[0239] Embodiment P10. An isolated nucleic acid encoding the first recombinant protein according to any one of Embodiments 1 to 3.

[0240] Embodiment P11. An expression vector containing the nucleic acid according to Embodiment P10.

[0241] Embodiment P12. The virus is a lentivirus or an oncoretrovirus, and the expression vector according to Embodiment P11.

[0242] Embodiment P13. A T lymphocyte containing the expression vector according to any one of Embodiments P11 to P12.

[0243] Embodiment P14. A T lymphocyte containing the first recombinant protein according to any one of Embodiments P1 to P9.

[0244] Embodiment P15. A T lymphocyte containing the first recombinant protein according to any one of Embodiments P1 to P9, and the transmembrane domain is within the cell membrane of the T lymphocyte.

[0245] 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.

[0246] Embodiment 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.

[0247] Embodiment 2. The recombinant protein according to Embodiment 1, wherein the intracellular T cell signaling domain is a CD3ζ intracellular T cell signaling domain.

[0248] Embodiment 3. The recombinant protein according to Embodiment 1 or 2, wherein the transmembrane domain is a CD8α transmembrane domain, a CD28 transmembrane domain, a CD4 transmembrane domain, or a CD3 zeta transmembrane domain.

[0249] Embodiment 4. The recombinant protein according to any one of Embodiments 1 to 3, wherein the transmembrane domain is a CD28 transmembrane domain.

[0250] Embodiment 5. The recombinant protein according to any one of Embodiments 1 to 4, further comprising a spacer region connecting the non-CDR Fab-binding peptide domain to the transmembrane domain.

[0251] Embodiment 6. The recombinant protein according to Embodiment 5, wherein the spacer region is a heavy chain constant 3(CH3) domain.

[0252] Embodiment 7. The recombinant protein according to any one of Embodiments 1 to 6, further comprising a peptide linker connecting the non-CDR Fab-binding peptide domain to the spacer region.

[0253] Embodiment 8. The recombinant protein according to any one of Embodiments 1 to 7, further comprising an intracellular costimulatory signaling domain that connects the transmembrane domain to the intracellular T cell signaling domain.

[0254] Embodiment 9. The recombinant protein according to Embodiment 8, wherein the intracellular co-stimulus signaling domain is a CD28 intracellular co-stimulus signaling domain, a 4-1BB intracellular co-stimulus signaling domain, an ICOS intracellular co-stimulus signaling domain, or an OX-40 intracellular co-stimulus signaling domain.

[0255] Embodiment 10. The recombinant protein according to Embodiment 8 or 9, wherein the intracellular co-stimulatory signaling domain is a CD28 intracellular co-stimulatory signaling domain.

[0256] Embodiment 11. The recombinant protein according to any one of Embodiments 8 to 10, wherein the intracellular co-stimulus signaling domain is the 4-1BB intracellular co-stimulus signaling domain.

[0257] Embodiment 12. The recombinant protein according to any one of Embodiments 1 to 11, further comprising a detection domain bound to the C-terminus of the intracellular T cell signaling domain.

[0258] Embodiment 13. The recombinant protein according to Embodiment 12, wherein the detection domain is a cleaved CD19 protein.

[0259] Embodiment 14. The recombinant protein according to any one of Embodiments 1 to 13, further comprising a self-cleaving peptidyl sequence connecting the intracellular T cell signaling domain to the detection domain.

[0260] Embodiment 15. The recombinant protein according to Embodiment 14, wherein the self-cleaving peptidyl linker sequence is a T2A sequence or a 2A sequence.

[0261] Embodiment 16. The recombinant protein according to any one of Embodiments 1 to 15, wherein the recombinant protein forms part of a cell.

[0262] Embodiment 17. The recombinant protein according to any one of Embodiments 1 to 16, wherein the recombinant protein forms part of a T cell.

[0263] Embodiment 18. The recombinant protein according to any one of Embodiments 1 to 17, wherein the non-CDR Fab-binding peptide domain is bound to the antigen-binding domain.

[0264] Embodiment 19. The recombinant protein according to Embodiment 18, wherein the antigen-binding domain is Fab, IgG, or a bispecific antibody.

[0265] Embodiment 20. The recombinant protein according to any one of Embodiments 18 to 19, wherein the antigen-binding domain is a cetuximab medotope-compatible domain, a trastuzumab medotope-compatible domain, a pertuzumab medotope-compatible domain, an M5A medotope-compatible domain, or a rituximab medotope-compatible domain.

[0266] Embodiment 21. The recombinant protein according to any one of Embodiments 18 to 20, wherein the antigen-binding domain is capable of binding to a cancer antigen.

[0267] Embodiment 22. The recombinant protein according to any one of Embodiments 18 to 21, wherein the antigen-binding domain is capable of binding to a cancer antigen.

[0268] Embodiment 23. The recombinant protein according to Embodiment 21 or 22, wherein the cancer antigen is Her2, EGFR, CD19, or CD20.

[0269] Embodiment 24. The recombinant protein according to Embodiment 21 or 22, wherein the cancer antigen forms part of a cell.

[0270] Embodiment 25. The recombinant protein according to Embodiment 24, wherein the cells are cancer cells.

[0271] Embodiment 26. The recombinant protein according to 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.

[0272] Embodiment 27. An isolated nucleic acid encoding a recombinant protein according to any one of Embodiments 1 to 26.

[0273] Embodiment 28. An expression vector containing the nucleic acid described in Embodiment 27.

[0274] Embodiment 29. The expression vector according to Embodiment 28, wherein the virus is a lentivirus or an onchoretrovirus.

[0275] Embodiment 30. T lymphocytes containing the expression vector described in any one of Embodiments 28 to 29.

[0276] Embodiment 31. A T lymphocyte containing the recombinant protein described in any one of Embodiments 1 to 26.

[0277] Embodiment 32. A T lymphocyte comprising the recombinant protein described in any one of Embodiments 1 to 26, wherein the transmembrane domain is located within the cell membrane of the T lymphocyte.

[0278] Embodiment 33. The T lymphocyte according to any one of Embodiments 30 to 32, wherein the T lymphocyte is an autologous T lymphocyte.

[0279] Embodiment 34. The T lymphocyte according to any one of Embodiments 30 to 32, wherein the T lymphocyte is a heterologous T lymphocyte.

[0280] Embodiment 35. A T lymphocyte according to any one of Embodiments 30 to 34, wherein the non-CDR Fab-binding peptide domain is bound to an antigen-binding domain.

[0281] Embodiment 36. The T lymphocyte according to Embodiment 35, wherein the antigen-binding domain is Fab, IgG, or a bispecific antibody.

[0282] Embodiment 37. A T lymphocyte according to Embodiment 35 or 36, wherein the antigen-binding domain is bound to a cancer antigen.

[0283] Embodiment 38. The T lymphocyte according to Embodiment 37, wherein the cancer antigen is Her2, EGFR, CD19, or CD20.

[0284] Embodiment 39. A T lymphocyte according to any one of Embodiments 35 to 38, wherein the antigen-binding domain is a cancer antigen-binding domain.

[0285] Embodiment 40. A T lymphocyte according to any one of Embodiments 35 to 39, wherein the antigen-binding domain is a cetuximab medotope-compatible domain, a trastuzumab medotope-compatible domain, a pertuzumab medotope-compatible domain, an M5A medotope-compatible domain, or a rituximab medotope-compatible domain.

[0286] Embodiment 41. A method for treating cancer, comprising the step of administering to a subject in need of cancer treatment an effective amount of T lymphocytes and an antigen-binding domain capable of binding to the non-CDR Fab-binding peptide domain described in any one of Embodiments 30 to 34, wherein the antigen-binding domain is a cancer antigen-binding domain.

[0287] Embodiment 42. The method according to Embodiment 41, wherein the T lymphocytes and the antigen-binding domain are administered simultaneously or sequentially.

[0288] Embodiment 43. The method according to Embodiment 41 or 42, wherein the T lymphocytes are administered at a first time point, the antigen-binding domain is administered at a second time point, and the first time point precedes the second time point.

[0289] Embodiment 44. The method according to Embodiment 41 or 42, wherein the antigen-binding domain is administered at a first time point, the T lymphocytes are administered at a second time point, and the first time point precedes the second time point.

[0290] Embodiment 45. The method according to Embodiment 41, comprising the steps of (i) in vitro conjugating the non-CDR Fab-binding peptide domain to the antigen-binding domain before administration, thereby forming a T lymphocyte-recombinant protein complex; and (ii) administering the T lymphocyte-recombinant protein complex to the subject, thereby treating the cancer in the subject.

[0291] Embodiment 46. The method according to 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 glioma.

[0292] Embodiment 47. The method according to any one of Embodiments 41 to 46, wherein the antigen-binding domain is a cetuximab medotope-compatible domain, a trastuzumab medotope-compatible domain, a pertuzumab medotope-compatible domain, an M5A medotope-compatible domain, or a rituximab medotope-compatible domain.

[0293] Embodiment 48. The recombinant protein according to any one of Embodiments 1 to 26, wherein 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 costimulatory signaling domain is the first intracellular costimulatory signaling domain.

[0294] Embodiment 49. The recombinant protein according to Embodiment 48, wherein the first recombinant protein is non-covalently bound to the second recombinant protein, and the second recombinant protein includes (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 connecting the second non-CDR Fab-binding peptide domain to the second transmembrane domain and to which the first spacer region is non-covalently bound.

[0295] Embodiment 50. The recombinant protein according to Embodiment 49, wherein the first spacer region and the second spacer region are a first heavy chain constant 3(CH3) domain and a second heavy chain constant 3(CH3) domain.

[0296] Embodiment 51. The recombinant protein according to 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.

[0297] Embodiment 52. The recombinant protein according to 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.

[0298] Embodiment 53. The recombinant protein according to any one of Embodiments 48 to 52, wherein the first non-CDR Fab-binding peptide domain is non-covalently bound to the first antigen-binding domain.

[0299] Embodiment 54. The recombinant protein according to any one of Embodiments 49 to 53, wherein the second non-CDR Fab-binding peptide domain is non-covalently bound to the second antigen-binding domain.

[0300] Embodiment 55. The recombinant protein according to Embodiment 53 or 54, wherein the first antigen-binding domain and the second antigen-binding domain are chemically different or chemically the same.

[0301] Embodiment 56. The recombinant protein according to any one of Embodiments 53 to 55, wherein the first antigen-binding domain and the second antigen-binding domain are independently a cetuximab medotope-compatible domain, a trastuzumab medotope-compatible domain, a pertuzumab medotope-compatible domain, an M5A medotope-compatible domain, or a rituximab medotope-compatible domain.

[0302] Unofficial sequence list CD3z;GI:623041(Sequence ID 1) LCYLLDGILFIYGVILTALFL

[0303] CD28;GI:340545506(Sequence ID 2) FWVLVVVGGVLACYSLLVTVAFIIFWV

[0304] CD4;GI:179143(Sequence ID 3) MALIVLGGVAGLLLFIGLGIFF

[0305] CD8;GI:225007534(Sequence ID 4) IYIWAPLAGTCGVLLLSLVIT

[0306] CD8;GI:225007534(Sequence ID 5) IYIWAPLAGTCGVLLLSLVITLY

[0307] CD8;GI:225007534(Sequence ID 6) IYIWAPLAGTCGVLLLSLVITLYC

[0308] 41BB;GI:315259099(Sequence ID 7) IISFFLALTSTALLFLLFF LTLRFSVV

[0309] OX40;GI:315360637(Sequence ID 8) VAAILGLGLVLGLLGPLAILL

[0310] ICOS;GI:251823951(Sequence ID 9) FWLPIGCAAFVVVCILGCILI

[0311] CD62L;GI:262206314(Sequence ID 10) PLFIPVAVMVTAFSGLAFIIWLA

[0312] CD3ζ;GI:623041;Sequence ID 11:

[0313] [ka]

[0314] CD28;GI:340545506;Sequence ID 12: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0315] CD28gg * ;GI:340545506;Sequence ID 13: RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(ref)

[0316] 41BB;GI:315259099;Sequence ID 14: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0317] OX40;GI:315360637;Sequence ID 15: ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

[0318] ICOS;GI:251823951;Sequence ID 16: CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL

[0319] Spacer (containing IgG4-CH3); Sequence ID 17:

[0320] [ka]

[0321] CD28 transmembrane; Sequence ID No. 18: MFWVLVVVGGVLACYSLLVTVAFIIFWV

[0322] CD28cyto(LLmGG); Sequence ID 19: RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0323] Intracellular T cell signaling domain (CD3 zeta): Sequence ID No. 20:

[0324] [ka]

[0325] Self-cleaving peptidyl linker (T2A): Sequence ID No. 21: LEGGGEGRGSLLTCGDVEENPGPTR

[0326] Marker peptide (CD19t): SEQ ID NO: 22

[0327] [ka]

[0328] Self-cleaving peptidyl linker (2A): Sequence ID No. 23: GGSTSEGRGSLLTCGDVEENPGP

[0329] Spacer area: Sequence ID 24 GSGSGSGS

[0330] Peptide linker: Sequence ID No. 25 SAPASSASAPSAASAPAG

[0331] Sequence ID 26 SAPASSASAPSAASAPAG

[0332] Self-cleaving peptidyl linker: Sequence ID No. 27 PVKQLLNFDLLKLAGDVESNPGP

[0333] Self-cleaving peptidyl linker: Sequence ID No. 28 QCTNYALLKLAGDVESNPGP

[0334] Self-cleaving peptidyl linker: Sequence ID No. 29 ATNFSLLKQAGDVEENPGP

[0335] Self-cleaving peptidyl linker: SEQ ID NO: 30 EGRGSLLTCGDVESNPGP

[0336] Spacer area: Sequence ID 31 GGGSSGGGSG

[0337] Sequence ID 32: Meditope (non-CDR Fab-binding peptide domain) CQFDLSTRRLQC

[0338] Sequence ID 33: CH3 (Spacer region)

[0339] [ka]

[0340] Sequence ID 34: CD3 zeta chain (intracellular T cell signaling domain)

[0341] [ka]

[0342] Sequence ID 35: Total 4-1BB Fabrack sequence

[0343] [ka]

[0344] Sequence ID 36: All CD28 Fabrack array

[0345] [ka]

[0346] SEQ ID NO: 37: Signal Peptide MLLLVTSLLLCELPHPAFLLIP

Claims

1. (i) Meditopes that can be bound to a medotope-compatible Fab; (ii) intracellular T cell signaling domains; and (iii) Transmembrane domain connecting the medoplast to the intracellular T cell signaling domain A recombinant protein comprising a recombinant protein which does not contain an antigen-binding domain.

2. The recombinant protein according to claim 1, further comprising a spacer region connecting the medopole to the transmembrane domain.

3. The recombinant protein according to claim 2, wherein the spacer region is a heavy chain constant 3 (CH3) domain.

4. The recombinant protein according to claim 3, further comprising a peptide linker connecting the medop to a spacer region.

5. The recombinant protein according to claim 1, further comprising a detectable domain bound to the C-terminus of an intracellular T cell signaling domain.

6. The recombinant protein according to claim 5, further comprising a self-cleaving peptidyl linker connected to a detectable domain of an intracellular T cell signaling domain.

7. An isolated nucleic acid encoding a recombinant protein as described in claim 1.

8. An expression vector comprising the nucleic acid described in claim 7.

9. A T lymphocyte comprising the expression vector described in claim 8.

10. A T lymphocyte containing the recombinant protein described in claim 1.

11. A composition for use in a method of treating cancer, comprising an antigen-binding domain capable of binding to the T lymphocytes and the medotope described in Claim 9, wherein the method comprises the step of administering an effective amount of the antigen-binding domain capable of binding to the T lymphocytes and the medotope described in Claim 9 to a subject in need of cancer treatment, wherein the antigen-binding domain is a cetuximab medotope-compatible domain, a trastuzumab medotope-compatible domain, a pertuzumab medotope-compatible domain, an M5A medotope-compatible domain, or a rituximab medotope-compatible domain.

12. The composition according to claim 11, wherein T lymphocytes and antigen-binding domains are administered simultaneously or sequentially.

13. The method described above, (i) Before administration, the step of binding the medop to the antigen-binding domain in vitro, thereby forming a T lymphocyte-recombinant protein complex; and (ii) A step of administering T lymphocyte-recombinant protein complexes to a target to treat cancer in the target. The composition according to claim 11, comprising:

14. The recombinant protein according to claim 2, wherein 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, and the spacer region is the first spacer region.

15. The first recombinant protein is bound to the second recombinant protein by a non-covalent bond, and the second recombinant protein, (i) The second medop; (ii) Second intracellular T cell signaling domain; (iii) A second transmembrane domain connecting a second medoploc to a second intracellular T cell signaling domain; and (iv) A second spacer region connecting the second medopope to the second transmembrane domain, The recombinant protein according to claim 14, wherein the first spacer region is non-covalently bound to the second spacer region, and the second recombinant protein does not contain an antigen-binding domain.

16. The recombinant protein according to claim 15, wherein the first medotope and the second medotope are chemically different or chemically the same.

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