For folate receptor 1-specific chimeric antigen receptor

Abstract

This invention provides proteins having an antigen-binding domain specific to FolR1. Target-specific binding has been demonstrated in several different applications. Depending on the application, the proteins of this invention may include additional structural features and / or domains. Depending on the structural features, proteins having an antigen-binding domain specific to FolR1 may be, for example, chimeric antigen receptors, protein-drug conjugates, bispecific T-cell adaptors (BITEs), or detection reagents.

Description

Technical Field

[0001] This invention relates to antigen-binding proteins (e.g., antibodies or binding fragments) specific to folate receptor α (FolR1). Antigen-binding proteins can be used in several applications, such as for the detection and / or treatment of cancer. Background Technology

[0002] FolR1 is a membrane protein that binds to folic acid with high affinity and mediates the cellular uptake of this vitamin via receptor-mediated endocytosis. 2 Folic acid is a fundamental component of cell metabolism, DNA synthesis, and repair. Rapidly dividing cancer cells have an increased need for folic acid to maintain DNA synthesis. Compared to normal cells, FolR1 levels are higher in certain epithelial-origin malignancies and are positively correlated with tumor stage and grade, prompting investigation into its role in tumor etiology and progression. It has been suggested that FolR1 may confer a growth advantage on tumors by regulating folic acid uptake from serum or by generating regulatory signals. 3 .

[0003] FolR1 expression levels are particularly high in cancers such as triple-negative breast cancer, gastric cancer, lung cancer, kidney cancer, pancreatic cancer, or ovarian cancer. FolR1 expression plays a crucial role, especially in ovarian cancer.

[0004] Ovarian cancer is defined as a group of tumors originating in the ovary. It is the fourth most common cancer in women and a leading cause of gynecological cancer-related death in women. The majority of clinical cases are epithelial ovarian cancer, representing approximately 90% of diagnosed patients. Epithelial ovarian cancer has four main histological subtypes: serous, endometrioid, mucinous, and clear cell. Approximately 70% of patients present with serous histology, and clinical trials have demonstrated the prognostic importance of the patient's subtype. Other less common types of ovarian tumors include primary peritoneal tumors, fallopian tube tumors, and malignant germ cell tumors. 1 .

[0005] Ovarian cancer patients have an approximate overall five-year survival rate of 49.1%. As with most cancers, the five-year survival rate can vary depending on the stage at diagnosis. If the cancer is detected early, at a limited stage (stage I-II), the five-year survival rate is 92.6%. Ovarian cancer that has spread to different parts of the body, also known as regional ovarian cancer (stage III), has a five-year survival rate of 74.8%. The five-year survival rate further decreases to 30.3% in metastatic (stage IV) ovarian cancer. Most patients present with advanced disease at diagnosis, and despite a high response to initial treatment, most eventually relapse, resulting in an incurable disease. 1 .

[0006] Immunotherapy, such as the use of protein (especially antibody) drug conjugates and chimeric antigen receptors, has shown effectiveness in cancer treatment. Chimeric antigen receptors (CARs) offer a promising approach for adoptive cellular immunotherapy in cancer.

[0007] Surgery in combination with chemotherapy remains the standard treatment. However, most patients relapse and eventually develop resistance to chemotherapy. Therefore, ovarian cancer presents a significant unmet medical need, and there is an urgent need for alternative treatments such as immunotherapy using cell or protein drug conjugates expressing CARs. In particular, high expression of tumor-associated FolR1 has been identified in triple-negative breast cancer, gastric cancer, lung cancer, kidney cancer, and pancreatic cancer, and it therefore represents a promising target for CAR T-cell therapy. There is a need in the art for improved or alternative immunotherapies, such as FolR1-specific CARs, for the treatment of FolR1-expressing cancers.

[0008] Detection of cancer cells in patient samples plays a crucial role in determining disease stage and predicting the effectiveness of immunotherapy. For this purpose, proteins containing antigen-binding domains of the protein used in immunotherapy (e.g., CARs or protein-drug conjugates) should preferably be used. However, proteins containing antigen-binding domains commonly used in immunotherapy exhibit weak binding properties for other applications such as imaging or flow cytometry. In most cases, such proteins do not bind to patient tissue (which has been treated with a fixative) at all. Therefore, they cannot be used for the detection and prediction of outcomes for such immunotherapies.

[0009] Therefore, there is a need in the art for improved or alternative proteins that include an antigen-binding domain, which can be used to detect FolR1-expressing cancer cells, stage the disease, predict treatment success, and for treatment using immunotherapies such as chimeric antigen receptors or protein drug conjugates. Summary of the Invention

[0010] The cell surface antigen FolR1 is expressed on cancer cells, such as triple-negative breast cancer, gastric cancer, lung cancer, kidney cancer, pancreatic cancer, or ovarian cancer, and can be used for targeted immunotherapy. The inventors have discovered that proteins containing a specific antigen-binding domain specific to FolR1 can be used to detect FolR1-positive cells (especially cancer cells; Figure 6) and simultaneously for use in immunotherapy such as CAR-T cell therapy (Examples 4-5).

[0011] Therefore, a first aspect of the present invention is a protein comprising an antigen-binding domain, said antigen-binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. - The heavy chain variable region (VH) of the antibody includes a first complementarity-determining region (HCDR1) containing the amino acid sequence SEQ ID No. 20 (GYSFTSYW), a second complementarity-determining region (HCDR2) containing the amino acid sequence IYPGDSDT (SEQ ID No. 21), and a third complementarity-determining region (HCDR3) containing the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22). - The light chain variable region (VL) of the antibody includes a first complementarity-determining region (LCDR1) containing the amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity-determining region (LCDR2) containing the amino acid sequence DDS (SEQ ID No. 37), and a third complementarity-determining region (LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38).

[0012] In addition, the inventors have discovered that proteins containing antigen-binding domains as disclosed herein can be used in immunotherapies such as CAR T-cell therapy and protein (e.g., antibody) drug conjugates. The inventors have demonstrated that the CAR effectively kills cancer cells in vivo and in vitro (Figures 4 and 5). Therefore, one aspect of the invention is a protein that is a chimeric antigen receptor comprising an antigen-binding domain, a spacer domain, a transmembrane domain, and an intracellular signal transduction domain as defined by the first aspect of the invention.

[0013] In another aspect of the invention, the protein may be a protein-pharmaceutical conjugate (e.g., an antibody-pharmaceutical conjugate), wherein the protein comprises an antigen-binding domain and a cytotoxic payload portion as described in the first aspect of the invention. Such constructs can be used for immunotherapeutic applications.

[0014] In another aspect of the invention, the protein may include an antigen-binding domain and a detection portion as defined in the first aspect. Such proteins can be used to detect FolR1-positive cells (especially cancer cells) in a sample (preferably a patient sample). They can be used for flow cytometry and / or imaging applications. Such methods can be used for disease staging. Attached Figure Description

[0015] Figure 1CAR T-cell binding domain candidates. 189 cross-reactive anti-FOLR1 candidate binders were identified from a human antibody library via phage display. Nineteen candidates were further selected by flow cytometry screening of cells expressing the FOLR variant to identify FOLR1-specific candidates. CAR T-cell screening was then used to evaluate functionality, specificity, and cell expansion. Four candidates were selected for further in vitro analysis, identifying a lead anti-FOLR1 CAR candidate.

[0016] Figure 2: Unique binding agent sequences with cross-reactivity between hFOLR1 and mFOLR1 were identified via phage display. A) Five different panning strategies were used to identify binding agent candidates, with hFOLR1 and mFOLR1 antigens used for positive selection, respectively. hFOLR2, hFOLR3, and hIgG1-Fc antigens were used for competition in all strategies. Strategy 2 used a lower amount of antigen in the third round of panning compared to Strategy 1. B) The phage display antibody library used revealed 2266 first hits for hFOLR1 and / or mFOLR1 in ELISA. C) The five different selection strategies led to the discovery of scFvs that preferentially bind to the targets used in the panning strategies; for example, panning strategies 4 and 5 selected scFvs that preferentially bind to both hFOLR1 and mFOLR1 antigens. D) Sequencing results confirmed 349 unique sequences derived from 723 processable sequences.

[0017] Figure 3: Flow cytometry screening revealed potential CAR T cell candidates in scFv-Fc form with high specificity for FOLR1 compared to other FOLR variants. Candidates were generated in scFv-Fc form via transfection of HEK293 cells. Jurkat cells expressing human or mouse FOLR variants, respectively, were used to test the specificity of the candidates for FOLR1. 1x10 5Jurkat cells expressing FOLR were plated and stained with 1 µg scFv-Fc at 4°C for 30 min. Anti-IgG (Fc)-PE secondary antibody staining was performed at 4°C for 10 min. Flow cytometry measurements were performed in triplicate and displayed as a percentage of stained cells. Candidates that stained for FOLR2 or FOLR4 but showed low staining for FOLR1 were excluded. Selected candidates were marked in black, and unselected candidates were marked in gray. A) After excluding debris, duplexes, and dead cells, determine the gating strategy for the specificity of candidates in scFv-Fc form for hFOLR1 and mFOLR1. B) After excluding debris, duplexes, and dead cells, determine the gating strategy for the specificity of candidates in scFv-Fc form for hFOLR2 and mFOLR2. C) After excluding debris, duplexes, and dead cells, determine the gating strategy for the specificity of candidates in scFv-Fc form for hFOLR4 and mFOLR4. D) E) Flow cytometry results of candidates in scFv-Fc form combined with Jurkat cells overexpressing hFOLR1. F) G) Flow cytometry results of candidates in scFv-Fc form combined with Jurkat cells overexpressing mFOLR1. H) I) Flow cytometry results of candidates in scFv-Fc form combined with Jurkat cells overexpressing hFOLR2. J) K) Flow cytometry results of candidates in scFv-Fc form combined with Jurkat cells overexpressing mFOLR2. L) M) Flow cytometry results of candidates in scFv-Fc form combined with Jurkat cells overexpressing hFOLR4. N) O) Flow cytometry results of candidates in scFv-Fc form combined with Jurkat cells overexpressing mFOLR4.

[0018] Figure 4: CAR T cell screening revealed functional and specific anti-FOLR1 CAR T cell candidates. Seeding 2x10 4 5 x 10 target cells expressing GFP and FOLR1 (OV-90) and with 5 x 10 4 Anti-FOLR1 CAR T cells were co-cultured to measure antigen-dependent ovarian cancer cell lysis. After 92 hours of co-culture, an additional 2 x 10⁶ CAR T cells were added. 4 One ovarian cancer cell (OV-90), followed by 5x10 cells after 164 hours. 4 Ovarian cancer cells (OV-90) were co-cultured with FOLR1-deficient ovarian cancer cells (OV-90 FOLR1 KO) in parallel. Measurements were taken using Green Calibrated Units (µm). 2The images, spanning 11 days, analyzed target cell lysis induced by CAR T cell candidates by the decrease in GFP signal over time. As a negative control, target cells (OV-90) were cultured without the addition of CAR T cells or co-cultured with untransduced T cells. Two positive control CAR T cell constructs known to successfully induce FOLR1-specific target cell lysis were included. IFN-γ secretion was measured 24 hours after each addition of ovarian cancer cells. CAR T cell expansion was quantitatively evaluated by flow cytometry at 92, 164, and 260 hours of co-culture, i.e., before each addition of target cells and at the endpoint. A), B), and C) show the lysis of FOLR1-expressing ovarian cancer cells (OV-90) by 19 CAR T candidates identified as FOLR1-specific from three different donors in a previously performed flow cytometry screening. D), E), F) Lysis of FOLR1-deficient ovarian cancer cells (OV-90 FOLR1 KO) by 19 CAR candidates from three different donors identified as FOLR1-specific in a previously performed flow cytometry screening. G), H), I) Expansion of CAR T cells from all candidates from three different donors as CD3+ LNGFR+ cells / well after 92, 164, and 260 hours of co-culture. J), K), L) IFN-γ secretion in repeated co-cultures 24 hours after each addition of candidate CAR T cells to FOLR1-expressing ovarian cancer cells (OV-90). M), N), O) IFN-γ secretion in repeated co-cultures 24 hours after each addition of candidate CAR T cells to FOLR1-deficient ovarian cancer cells (OV-90 FOLR1 KO). P) Workflow protocols for identifying functional and specific CAR T cells in a series of in vitro co-culture assays.

[0019] Figure 5: Advanced in vitro CAR T cell assay identified the lead anti-FOLR1 CAR T cell candidate. Seeded 2x10 4 Cells expressing GFP, and with 1x10 4 Anti-FOLR1 CAR T cells were co-cultured for five days to measure antigen-dependent lysis of ovarian cancer cells. After 48 hours of co-culture, 2x10-1 CAR-1 cells were... 4Ovarian cancer cells were added to a co-culture. Cell lysis induced by CAR T cell candidates was tracked by measuring the confluence of green regions. As a negative control, ovarian cancer cells were cultured without the addition of CAR T cells or with untransduced T cells. Positive control CAR T cell constructs were included, which are known to successfully induce FOLR1-specific target cell lysis. In this assay containing three donors each, the top four candidates identified through CAR T cell screening were included. Flow cytometry measurements of activation marker expression CD137 and CD69 were performed at 48 and 120 hours. Additionally, activation markers were measured on a fourth donor after co-culturing with FOLR1-low (OVCAR-3) expression cells. A) CAR structures of the four FOLR1-targeting CAR T cell candidates, consisting of a FOLR1-targeting scFv, a CD8 hinge and transmembrane region, a 4-1BB co-stimulatory region, and a CD3ζ domain. B), C), D) Lysis of FOLR1-expressing ovarian cancer cells (OV-90 wt) by four CAR T cell candidates from three different donors, identified as functional in a previously performed CAR T cell screening. E), F), G) Lysis of FOLR1-deficient ovarian cancer cells (OV-90 FOLR1 KO) by four CAR T cell candidates from three different donors, identified as functional in a previously performed CAR T cell screening. H), I), J) Expression of CD69 activation markers on CAR T cell candidates from the three donors after 48 and 120 hours of co-culture with ovarian cancer cells. K), L), M) Expression of CD137 activation markers on CAR T cell candidates from the three donors after 48 and 120 hours of co-culture with ovarian cancer cells. The expression of CD69 and CD137 activation markers on CART candidates from three donors after co-culturing with FOLR1 high (OV-90 wt), FOLR1 low (OVCAR-3), and FOLR1- (OV-90 FOLR1 KO) cells for 48 hours and 120 hours, respectively.

[0020] Figure 6: Immunofluorescence analysis of human cell lines and tissues reveals potential CAR T cell candidates in the form of scFv-Fc. Cells were seeded at a concentration of 1E6 (Jurkat) or 2E5 (OV90) cells / 24 wells and fixed with 4% PFA prior to DAPI pre-staining. Staining of each conjugate was performed automatically, with signal removal and sample washing. A monoclonal antibody against the target of interest was used as a positive reference. Cyclic imaging of the scFv-Fc-PE conjugate on cells was performed at a concentration of 5 µg / mL. The scale bar indicates 200 µm. A 20x objective lens was used and the exposure time was set to 80 ms. A) Staining of the target-specific scFv-Fc-PE conjugate and the positive reference on target cells (Jurkat wt) that do not express the antigen of interest, and B) Quantification of the corresponding signal intensity and the signal overlapping with the positive reference. C) Staining of the target scFv-Fc-PE conjugate and positive reference on transgene knock-in cells (Jurkat KI) with the antigen of interest, and D) quantification of the corresponding signal intensity and the signal overlapping with the positive reference. E) Staining of the target scFv-Fc-PE conjugate and positive reference on cells expressing the physiological target (Ov-90 wt), and F) quantification of the corresponding signal intensity and the signal overlapping with the positive reference. G) Staining of the target scFv-Fc-PE conjugate and positive reference on cells with CRISPR / Cas9-mediated antigen knockout (Ov-90 KO). I) Staining of the target scFv-Fc-PE conjugate and positive reference on diseased tissue (high-grade serous ovarian cancer) expressing the target, and J) quantification of the corresponding signal intensity and the signal overlapping with the positive reference. Detailed Implementation

[0021] This invention provides proteins having an antigen-binding domain specific to FolR1. Target-specific binding has been demonstrated in several different applications. Depending on the application, the proteins of this invention may include additional structural features and / or domains. Depending on the structural features, proteins having an antigen-binding domain specific to FolR1 may be, for example, chimeric antigen receptors, protein-drug conjugates (e.g., antibody-drug conjugates), bispecific T-cell adaptors (BITEs), or detection reagents. The following paragraphs define the structural features of the protein required for each application / construct.

[0022] Proteins containing an antigen-binding domain specific to FolR1 In a first aspect, the present invention provides a protein comprising an antigen-binding domain (specific to FolR1), said antigen-binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. - The heavy chain variable region (VH) of the antibody includes a first complementarity-determining region (HCDR1) containing the amino acid sequence GYSFTSYW (SEQ ID No. 20), a second complementarity-determining region (HCDR2) containing the amino acid sequence IYPGDSDT (SEQ ID No. 21), and a third complementarity-determining region (HCDR3) containing the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22). - The light chain variable region (VL) of the antibody includes a first complementarity-determining region (LCDR1) containing the amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity-determining region (LCDR2) containing the amino acid sequence DDS (SEQ ID No. 37), and a third complementarity-determining region (LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38).

[0023] The protein may contain: The heavy chain variable region (VH) of the antibody may include a first framework region (HFR1) containing the amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) containing the amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) containing the amino acid sequence of SEQ ID No. 25, and a fourth framework region (HFR4) containing the amino acid sequence of SEQ ID No. 26. The light chain variable region (VL) of the antibody may include a first framework region (LFR1) containing the amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) containing the amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) containing the amino acid sequence of SEQ ID No. 41, and a fourth framework region (LFR4) containing the amino acid sequence of SEQ ID No. 42.

[0024] In one embodiment of the invention, the protein comprising the antigen-binding domain may include a heavy chain variable region (VH) comprising an antibody containing the amino acid sequence SEQ ID No:19, and a light chain variable region (VL) comprising an antibody containing the amino acid sequence SEQ ID No:35.

[0025] In one embodiment of the invention, the protein containing the antigen-binding domain may contain the amino acid sequence SEQ ID No:80.

[0026] According to this aspect of the invention, the protein comprises an antigen-binding domain. Any molecule that specifically binds to the target antigen (FolR1) can serve as the antigen-binding domain. The antigen-binding domain can be, or may comprise, for example, an antibody or its antigen-binding fragment (e.g., a Fab fragment or a single-chain Fv (scFv) fragment), a VHH fragment, a bivalent single-chain antibody, or a dual antibody. In a preferred embodiment of the invention, the antigen-binding domain can be an antibody or its antigen-binding fragment. In a preferred embodiment of the invention, the antigen-binding domain can be scFv.

[0027] In one embodiment of the invention, the protein containing the antigen-binding domain (preferably scFv) may include at least one additional domain selected from the following: a cytotoxic portion, a detection portion, and an Fc terminus. In one embodiment of the invention, the protein containing the antigen-binding domain (preferably scFv) may include a crystallizable fragment region (Fc domain) of an antibody.

[0028] In another embodiment of the invention, the protein containing the antigen-binding domain (preferably scFv) may be a chimeric antigen receptor (CAR) comprising the antigen-binding domain, spacer domain, transmembrane domain, and intracellular signal transduction domain that are specific to FolR1.

[0029] For FolR1-specific chimeric antigen receptor In a second aspect, the present invention provides a protein that is a chimeric antigen receptor (CAR), the chimeric antigen receptor (CAR) comprising an antigen-binding domain, a spacer domain, a transmembrane domain and an intracellular signal transduction domain that are specific to FolR1.

[0030] Any molecule that specifically binds to a given antigen can be used as an antigen-binding domain. Exemplary antigen-binding domains can be, or may contain, for example, a Fab fragment, a single-chain Fv (scFv) fragment, a VHH fragment, a bivalent single-chain antibody, or a biantibody. Preferably, the antigen-binding domain is an scFv. Typically, in an scFv, variable regions of the immunoglobulin heavy and light chains are fused together via a flexible linker to form the scFv. Such a linker can be, for example, a “(G4 / S)3-linker” or a “whitlow linker,” preferably a “(G4 / S)3-linker.”

[0031] The CAR includes an antigen-binding domain (e.g., scFv) as defined by the first aspect of the invention. In other words, the protein of this aspect of the invention is a chimeric antigen receptor (CAR) comprising an antigen-binding domain (e.g., scFv), a spacer domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody, wherein the heavy chain variable region (VH) of the antibody comprises a first complementarity-determining region (HCDR1) containing the amino acid sequence GYSFTSYW (SEQ ID No. 20), a second complementarity-determining region (HCDR2) containing the amino acid sequence IYPGDSDT (SEQ ID No. 21), and a third complementarity-determining region (HCDR3) containing the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22), and wherein the light chain variable region (VL) of the antibody comprises a first complementarity-determining region (LCDR1) containing the amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity-determining region (LCDR2) containing the amino acid sequence DDS (SEQ ID No. 37), and a third complementarity-determining region (LCDR3) containing the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 20). The third complementary determinant region (LCDR3) of ID No. 38.

[0032] The protein (which is a CAR) includes the antigen-binding domain (e.g., scFv), wherein the antigen-binding domain may include a heavy chain variable region (VH) of the antibody, which may include a first framework region (HFR1) containing the amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) containing the amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) containing the amino acid sequence of SEQ ID No. 25, and a fourth framework region (HFR4) containing the amino acid sequence of SEQ ID No. 26, and wherein the light chain variable region (VL) of the antibody may include a first framework region (LFR1) containing the amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) containing the amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) containing the amino acid sequence of SEQ ID No. 41, and a fourth framework region (LFR4) containing the amino acid sequence of SEQ ID No. 42.

[0033] The protein (which is a CAR) includes the antigen-binding domain (e.g., scFv), wherein the antigen-binding domain may include: a heavy chain variable region (VH) of an antibody that may contain the amino acid sequence SEQ ID No: 19, and a light chain variable region (VL) of an antibody that may contain the amino acid sequence SEQ ID No: 35.

[0034] In one embodiment of this aspect of the invention, the protein (which is a CAR) includes the antigen-binding domain (e.g., scFv), wherein the antigen-binding domain may contain the amino acid sequence SEQ ID No: 80.

[0035] As defined in this aspect of the invention, the protein (which is a CAR) may include a hinged domain serving as a spacer region, wherein the hinged domain may include a hinge sequence of, for example, CD8α (SEQ ID No:2) or IGG4 (SEQ ID No:72). In a preferred embodiment, the CAR may include a hinged domain of CD8α, wherein the hinged domain of CD8α may include SEQ ID NO:2.

[0036] In other words, the protein described in this aspect of the invention is a chimeric antigen receptor (CAR) comprising: The antibody comprises an antigen-binding domain (preferably scFv), a spacer domain, a transmembrane domain, and an intracellular signal transduction domain. The antigen-binding domain includes a heavy chain variable region (VH) and a light chain variable region (VL) of the antibody. The heavy chain variable region (VH) includes a first complementarity-determining region (HCDR1) containing the amino acid sequence GYSFTSYW (SEQ ID No. 20), a second complementarity-determining region (HCDR2) containing the amino acid sequence IYPGDSDT (SEQ ID No. 21), and a third complementarity-determining region (HCDR3) containing the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22). The light chain variable region (VL) includes a first complementarity-determining region (LCDR1) containing the amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity-determining region (LCDR2) containing the amino acid sequence DDS (SEQ ID No. 37), and a region containing the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 36). The third complementary determinant region (LCDR3) of 38), and The hinge structure domain of CD8α, preferably, may include SEQ ID NO:2.

[0037] The protein (which is a CAR) may comprise: The antigen-binding domain (preferably scFv), spacer domain, transmembrane domain, and intracellular signal transduction domain, wherein the antigen-binding domain comprises the heavy chain variable region (VH) of the antibody, which may include a first framework region (HFR1) containing the amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) containing the amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) containing the amino acid sequence of SEQ ID No. 25, and a fourth framework region (HFR4) containing the amino acid sequence of SEQ ID No. 26, and wherein the light chain variable region (VL) of the antibody may include a first framework region (LFR1) containing the amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) containing the amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) containing the amino acid sequence of SEQ ID No. 41, and a fourth framework region (LFR4) containing the amino acid sequence of SEQ ID No. 42, and The hinge structure domain of CD8α, preferably, may include SEQ ID NO:2.

[0038] The protein (which is a CAR) may comprise: The antigen-binding domain (preferably scFv), spacer domain, transmembrane domain, and intracellular signal transduction domain, wherein the antigen-binding domain includes a heavy chain variable region (VH) of an antibody that may contain the amino acid sequence SEQ ID No: 19, and a light chain variable region (VL) of an antibody that may contain the amino acid sequence SEQ ID No: 35. The hinge structure domain of CD8α, preferably, may include SEQ ID NO:2.

[0039] In one embodiment of this aspect of the invention, the protein is a CAR and comprises: The antigen-binding domain (preferably scFv), spacer domain, transmembrane domain, and intracellular signal transduction domain, wherein the antigen-binding domain may contain the amino acid sequence SEQ ID No: 80, and The hinge structure domain of CD8α, preferably, may include SEQ ID NO:2.

[0040] As defined in this aspect of the invention, the protein (which is a CAR) may include a transmembrane domain containing a sequence of a transmembrane domain from CD8α (SEQ ID No:4) or CD28 (SEQ ID No:76), preferably CD8α (SEQ ID No:4).

[0041] In other words, the protein (which is a CAR) of this aspect of the invention may comprise: The antibody comprises an antigen-binding domain (preferably scFv), a spacer domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the antigen-binding domain includes a heavy chain variable region (VH) and a light chain variable region (VL) of the antibody. The heavy chain variable region (VH) includes a first complementarity-determining region (HCDR1) containing the amino acid sequence GYSFTSYW (SEQ ID No. 20), a second complementarity-determining region (HCDR2) containing the amino acid sequence IYPGDSDT (SEQ ID No. 21), and a third complementarity-determining region (HCDR3) containing the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22). The light chain variable region (VL) includes a first complementarity-determining region (LCDR1) containing the amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity-determining region (LCDR2) containing the amino acid sequence SEQ ID No. 37 (DDS), and a region containing the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 20). The third complementary determinant region (LCDR3) of 38) The hinge structure domain of CD8α, preferably, may include SEQ ID NO:2, and The transmembrane domain of CD8α, preferably the transmembrane domain of CD8α may include SEQ ID NO:4.

[0042] The protein (which is a CAR) may comprise: The antigen-binding domain (preferably scFv), spacer domain, transmembrane domain, and intracellular signal transduction domain, wherein the antigen-binding domain comprises the heavy chain variable region (VH) of the antibody, which may include a first framework region (HFR1) containing the amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) containing the amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) containing the amino acid sequence of SEQ ID No. 25, and a fourth framework region (HFR4) containing the amino acid sequence of SEQ ID No. 26, and wherein the light chain variable region (VL) of the antibody may include a first framework region (LFR1) containing the amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) containing the amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) containing the amino acid sequence of SEQ ID No. 41, and a fourth framework region (LFR4) containing the amino acid sequence of SEQ ID No. 42. The hinge structure domain of CD8α, preferably, may include SEQ ID NO:2, and The transmembrane domain of CD8α, preferably the transmembrane domain of CD8α may include SEQ ID NO:4.

[0043] The protein (which is a CAR) may comprise: The antigen-binding domain (preferably scFv), spacer domain, transmembrane domain, and intracellular signal transduction domain, wherein the antigen-binding domain includes a heavy chain variable region (VH) of an antibody that may contain the amino acid sequence SEQ ID No: 19, and a light chain variable region (VL) of an antibody that may contain the amino acid sequence SEQ ID No: 35. The hinge structure domain of CD8α, preferably, may include SEQ ID NO:2, and The transmembrane domain of CD8α, preferably the transmembrane domain of CD8α may include SEQ ID NO:4.

[0044] In one embodiment of the invention, the protein (which is a CAR) comprises an intracellular signal transduction domain, such as the sequence of the intracellular signal transduction domain of CD3ζ (SEQ ID No: 8), and / or one or more of CD28 (SEQ ID NO: 78), CD137 (41BB; SEQ ID NO: 6), and OX40 (SEQ ID NO: 74). In a preferred embodiment, the CAR may comprise or consist of the following: a transmembrane domain of CD8α, an intracellular primary (stimulatory) signal transduction domain of CD3ζ, and an intracellular costimulatory signal transduction domain of CD137 (41BB).

[0045] In other words, the protein of this aspect of the invention may comprise or consist of the following: The antibody comprises an antigen-binding domain (preferably scFv), a spacer domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the antigen-binding domain includes a heavy chain variable region (VH) and a light chain variable region (VL) of the antibody. The heavy chain variable region (VH) includes a first complementarity-determining region (HCDR1) containing the amino acid sequence GYSFTSYW (SEQ ID No. 20), a second complementarity-determining region (HCDR2) containing the amino acid sequence IYPGDSDT (SEQ ID No. 21), and a third complementarity-determining region (HCDR3) containing the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22). The light chain variable region (VL) includes a first complementarity-determining region (LCDR1) containing the amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity-determining region (LCDR2) containing the amino acid sequence DDS (SEQ ID No. 37), and a region containing the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 36). The third complementary determinant region (LCDR3) of 38) The hinge structure domain of CD8α (preferably, the hinge structure domain of CD8α may include SEQ ID NO:2), The transmembrane domain of CD8α (preferably the transmembrane domain of CD8α may include SEQ ID NO:4), and Intracellular signal transduction domains containing the co-stimulatory domain of CD137 and the stimulatory domain of CD3ζ.

[0046] The protein (which is a CAR) may comprise or consist of the following: The antigen-binding domain (preferably scFv), spacer domain, transmembrane domain, and intracellular signal transduction domain, wherein the antigen-binding domain comprises a heavy chain variable region (VH) of the antibody, which may include a first framework region (HFR1) containing the amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) containing the amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) containing the amino acid sequence of SEQ ID No. 25, and a fourth framework region (HFR4) containing the amino acid sequence of SEQ ID No. 26, and wherein the light chain variable region (VL) of the antibody may include a first framework region (LFR1) containing the amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) containing the amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) containing the amino acid sequence of SEQ ID No. 41, and a fourth framework region (LFR4) containing the amino acid sequence of SEQ ID No. 42; The hinge structure domain of CD8α (preferably, the hinge structure domain of CD8α may include SEQ ID NO:2), The transmembrane domain of CD8α (preferably the transmembrane domain of CD8α may include SEQ ID NO:4), and Intracellular signal transduction domains containing the co-stimulatory domain of CD137 and the stimulatory domain of CD3ζ.

[0047] The protein (which is a CAR) may comprise or consist of the following: The antigen-binding domain (preferably scFv), spacer domain, transmembrane domain, and intracellular signal transduction domain, wherein the antigen-binding domain includes a heavy chain variable region (VH) of an antibody that may contain the amino acid sequence SEQ ID No: 19, and a light chain variable region (VL) of an antibody that may contain the amino acid sequence SEQ ID No: 35. The hinge structure domain of CD8α (preferably, the hinge structure domain of CD8α may include SEQ ID NO:2), The transmembrane domain of CD8α (preferably the transmembrane domain of CD8α may include SEQ ID NO:4), and Intracellular signal transduction domains containing the co-stimulatory domain of CD137 and the stimulatory domain of CD3ζ.

[0048] In one embodiment of this aspect of the invention, the protein (which is a CAR) may comprise or consist of the following: The antigen-binding domain (preferably scFv), spacer domain, transmembrane domain, and intracellular signal transduction domain, wherein the antigen-binding domain may contain the amino acid sequence SEQ ID No: 80; The hinge structure domain of CD8α (preferably, the hinge structure domain of CD8α may include SEQ ID NO:2), The transmembrane domain of CD8α (preferably the transmembrane domain of CD8α may include SEQ ID NO:4), and Intracellular signal transduction domains containing the co-stimulatory domain of CD137 and the stimulatory domain of CD3ζ.

[0049] In one embodiment of the invention, the protein (which is a CAR) may comprise or consist of the amino acid sequence SEQ ID No:44.

[0050] This aspect of the invention further provides isolated nucleic acid sequences that encode FolR1-CAR as disclosed herein (especially with respect to this aspect of the invention).

[0051] The nucleic acid sequence encoding the CAR as disclosed herein can be contained in a vector, such as a viral vector. The vector can be a DNA vector, RNA vector, plasmid vector, granular vector, herpesvirus vector, measlesvirus vector, lentiviral vector, adenovirus vector, or retroviral vector, or a combination thereof. In a preferred embodiment, the nucleic acid sequence encoding the CAR as disclosed herein can be contained in a lentiviral vector.

[0052] In some embodiments of the present invention, the vector further comprises a promoter, wherein the promoter is an inducible promoter, a tissue-specific promoter, a constitutive promoter, a suicide promoter, or any combination thereof.

[0053] In another embodiment of the invention, the vector encoding CAR may be further modified to include one or more operational elements to control the expression of CAR T cells or to eliminate CAR-T cells by means of a suicide switch. The suicide switch may include, for example, apoptosis-inducing signaling cascades or drugs that induce cell death. In a preferred embodiment, the vector expressing CAR may be further modified to express enzymes such as thymidine kinase (TK) or cytosine deaminase (CD).

[0054] This aspect of the invention further provides a composition comprising a lentiviral vector containing a nucleic acid sequence encoding a FolR1-CAR as disclosed herein (particularly with respect to this aspect of the invention). The composition can be used in immunotherapy. The composition can be used to treat diseases such as cancer, autoimmune diseases, or infectious diseases.

[0055] This aspect of the invention further provides a composition for a method of treating a subject in need, the method comprising administering a lentiviral vector comprising a nucleic acid sequence encoding a FolR1-CAR as disclosed herein (particularly with respect to this aspect of the invention).

[0056] This aspect of the invention further provides engineered cells expressing the protein, which is a FolR1-specific CAR as disclosed herein (especially this aspect of the invention).

[0057] The cells may be immune cells. In a preferred embodiment of the invention, the cells may be T cells, tumor-infiltrating lymphocytes (TILs), or NK cells. In a more preferred embodiment of the invention, the cells are T cells.

[0058] This aspect of the invention further provides engineered cells expressing the protein for immunotherapy, the protein being the FolR1 CAR as disclosed herein (especially in this aspect of the invention). Immunotherapy can be used to treat cancer in a subject suffering from cancer, wherein the cancer cells of said cancer express FolR1. Immunotherapy can be used to treat cancer in a subject suffering from cancer, wherein at least one subpopulation of the cancer cells of said cancer expresses FolR1.

[0059] In all cancer cells of a subject suffering from said cancer, the FolR1-expressing cancer cell subpopulation may contain at least one cell expressing FolR1. Preferably, the FolR1-expressing cancer cell subpopulation may account for at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of all cancer cells in the subject suffering from said cancer.

[0060] This aspect of the invention further provides a cell population comprising cells expressing the protein, which is FolR1-CAR as disclosed herein (especially this aspect of the invention).

[0061] The modified cell population or isolated population can be expanded to a therapeutically effective amount of cells before being used in the immunotherapy. The cancer may be selected from triple-negative breast cancer, gastric cancer, lung cancer, kidney cancer, pancreatic cancer, or ovarian cancer. The cancer may be ovarian cancer. The cells may be immune cells or subsets of immune cells, preferably T cells, tumor-infiltrating lymphocytes (TILs), or NK cells, more preferably T cells.

[0062] This aspect of the invention further provides a method for treating cancer, comprising administering to a subject in need of modified cells expressing a protein, said protein being FolR1-CAR as disclosed herein (especially in this aspect of the invention). Cancer treatment may be performed in a subject suffering from cancer, wherein at least a subset of cancer cells in said cancer express FolR1.

[0063] This aspect of the invention further provides compositions for treating diseases comprising cells expressing genetically modified CARs specific to the antigen FolR1 as disclosed herein (especially this aspect of the invention). Such diseases may be selected from cancer, autoimmune diseases, and infectious diseases. In one embodiment, the composition can be used to treat cancer (cancer expressing FolR1). In another embodiment, the composition can be used to treat cancer in a subject with this need.

[0064] This aspect of the invention further provides pharmaceutical compositions comprising cells expressing genetically modified CARs specific to antigen FolR1 as disclosed herein (especially this aspect of the invention), and optionally a pharmaceutically acceptable carrier.

[0065] Pharmaceutically acceptable carriers, diluents, or excipients may include buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0066] This aspect of the invention further provides a composition for a method of treating a subject in need, the method comprising administering cells expressing a genetically modified CAR as disclosed herein (especially this aspect of the invention) that is specific to the antigen FolR1.

[0067] CAR connector A third aspect of the invention provides a composition comprising a protein as disclosed in the first aspect of the invention, wherein the composition comprises the protein (which is a tagged protein) and an anti-tag CAR comprising an antigen-binding domain, a transmembrane domain and an intracellular signal transduction domain specific to the tag of the tagged protein.

[0068] In other words, this aspect of the invention provides a composition comprising a protein as disclosed in the first aspect of the invention and an anti-tag CAR, wherein the protein is a tagged protein, and the anti-tag CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain specific to the tag of the tagged protein. The tagged protein comprises an antigen-binding domain (specific to FolR1) that includes a heavy chain variable region (VH) and a light chain variable region (VL) of the antibody. - The heavy chain variable region (VH) of the antibody includes a first complementarity-determining region (HCDR1) containing the amino acid sequence GYSFTSYW (SEQ ID No. 20), a second complementarity-determining region (HCDR2) containing the amino acid sequence IYPGDSDT (SEQ ID No. 21), and a third complementarity-determining region (HCDR3) containing the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22), and - The light chain variable region (VL) of the antibody includes a first complementarity-determining region (LCDR1) containing the amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity-determining region (LCDR2) containing the amino acid sequence DDS (SEQ ID No. 37), and a third complementarity-determining region (LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38).

[0069] The antigen-binding domain of the tagged protein may include the heavy chain variable region (VH) of the antibody, which may include a first framework region (HFR1) containing the amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) containing the amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) containing the amino acid sequence of SEQ ID No. 25, and a fourth framework region (HFR4) containing the amino acid sequence of SEQ ID No. 26. The light chain variable region (VL) of the antibody may include a first framework region (LFR1) containing the amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) containing the amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) containing the amino acid sequence of SEQ ID No. 41, and a fourth framework region (LFR4) containing the amino acid sequence of SEQ ID No. 42.

[0070] In one embodiment of this aspect of the invention, the tagged protein may include the antigen-binding domain, which comprises: The antibody may contain the heavy chain variable region (VH) of the amino acid sequence SEQ ID No: 19, and The light chain variable region (VL) of the antibody containing the amino acid sequence SEQ ID No: 35.

[0071] In one embodiment of the invention, the tagged protein may include the antigen-binding domain, and the antigen-binding domain may include the amino acid sequence SEQ ID No: 80.

[0072] The tagged protein can be any protein or protein fragment that specifically binds to a given antigen. Exemplary such proteins can be: antibodies, antigen-binding fragments of antibodies, scFv, heterologous antibody (fusion scFv-IgG) VHH fragments, bivalent single-chain antibodies, or biantibodies or analogs. In a preferred embodiment, the tagged protein can be an antibody or an antigen-binding fragment. In another embodiment, the antigen-binding domain is fused to the crystallizable fragment region (Fc domain) of an antibody.

[0073] As described above, tagged proteins may contain a tag. The tag can be selected from a variety of known tags, proteins, and molecules. However, it is essential that the tag can be bound to / recognized by the antigen-binding domain of an anti-tag CAR. The tag can be or may contain a portion of the protein. It can be selected from standard tags commonly known in the art. Exemplary tags are, for example, c-Myc tags, Strep-tag II, Flag tags, multihistidine tags, Avi tags, calmodulin-binding protein tags, Yol tags (derived from α-tubulin), E tags, HA tags, S tags, SBP tags, or V5 tags. Additionally, other tags such as biotin can be selected.

[0074] The composition of this aspect of the invention comprises two parts: a tagged protein and an anti-tagged CAR. The anti-tagged CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain specific to the tag of the tagged protein.

[0075] An antigen-binding domain needs to be specific to the tag on the tagged protein. An antigen-binding domain can contain, for example, a Fab fragment, a single-chain Fv (scFv) fragment, a VHH fragment, a bivalent single-chain antibody, or a biantibody. Any molecule that specifically binds to a given antigen, such as an affibody or a ligand-binding domain from a naturally occurring receptor, can be used as an antigen-binding domain. Often, the antigen-binding domain is the scFv. Typically, in a scFv, variable regions of the immunoglobulin heavy and light chains are fused together by a flexible linker to form the scFv. Such a linker can be, for example, a “(G4 / S)3-linker” or a “whitlow linker.”

[0076] The tag-resistant CAR disclosed herein may include a spacer domain (also known as a hinge domain). Exemplary spacer domains may be selected from CD8α and IGG4.

[0077] In addition, the tag-resistant CAR may also include a transmembrane domain, which may be selected from CD28 or CD8α. In a preferred embodiment, the transmembrane domain may be CD8α.

[0078] Furthermore, the anti-tag CAR may comprise an intracellular signal transduction domain, such as the sequence of the intracellular signal transduction domain of CD3ζ (SEQ ID NO:8), and / or one or more of CD28 (SEQ ID NO:78), CD137 (41BB; SEQ ID NO:6), and OX40 (SEQ ID NO:74). In a preferred embodiment, the CAR may comprise or consist of the following: a transmembrane domain of CD8α, an intracellular primary (stimulatory) signal transduction domain of CD3ζ, and intracellular co-stimulatory signal transduction domains of CD137 (41BB) and CD28.

[0079] Protein drug conjugates One aspect of the present invention provides a protein that is a protein drug conjugate (e.g., an antibody drug conjugate) comprising an antigen-binding domain and a cytotoxic payload portion as described in the first aspect of the present invention.

[0080] In other words, the protein in the protein-drug conjugate comprises an antigen-binding domain (specific to FolR1) and a cytotoxic payload portion, wherein the antigen-binding domain comprises the heavy chain variable region (VH) and the light chain variable region (VL) of the antibody. -The heavy chain variable region (VH) of the antibody includes a first complementarity-determining region (HCDR1) containing the amino acid sequence GYSFTSYW (SEQ ID No. 20), a second complementarity-determining region (HCDR2) containing the amino acid sequence IYPGDSDT (SEQ ID No. 21), and a third complementarity-determining region (HCDR3) containing the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22), and -The light chain variable region (VL) of the antibody includes a first complementarity-determining region (LCDR1) containing the amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity-determining region (LCDR2) containing the amino acid sequence DDS (SEQ ID No. 37), and a third complementarity-determining region (LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38).

[0081] The protein (protein-drug conjugate) comprises an antigen-binding domain and a cytotoxic payload portion, wherein the antigen-binding domain comprises, wherein the heavy chain variable region (VH) of the antibody may include a first framework region (HFR1) comprising the amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) comprising the amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) comprising the amino acid sequence of SEQ ID No. 25, and a fourth framework region (HFR4) comprising the amino acid sequence of SEQ ID No. 26, and wherein the light chain variable region (VL) of the antibody comprises a first framework region (LFR1) comprising the amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) comprising the amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) comprising the amino acid sequence of SEQ ID No. 41, and a fourth framework region (LFR4) comprising the amino acid sequence of SEQ ID No. 42.

[0082] In one embodiment of the present invention, the protein (which is a protein drug conjugate) may comprise the antigen-binding domain and the cytotoxic payload portion, wherein the antigen-binding domain comprises: The antibody may contain the heavy chain variable region (VH) of the amino acid sequence SEQ ID No: 19, and The light chain variable region (VL) of the antibody containing the amino acid sequence SEQ ID No: 35.

[0083] In one embodiment of the present invention, the protein (which is a protein-drug conjugate) may include the antigen-binding domain and the cytotoxic payload portion, wherein the antigen-binding domain may include the amino acid sequence SEQ ID No: 80.

[0084] Any molecule that specifically binds to FolR1 can be used as an antigen-binding domain. The antigen can be, or may contain, for example, a Fab fragment, a single-chain Fv (scFv) fragment, a VHH fragment, a bivalent single-chain antibody, or a dual antibody. In a preferred embodiment, the antigen-binding domain can be an antibody or an antigen-binding fragment.

[0085] In another embodiment, the antigen-binding domain (e.g., an antigen-binding fragment of the antibody or scFv) may be fused to a crystallizable fragment region (Fc domain) of the antibody. The fusion protein can be generated by methods known in the art. Exemplary fusion proteins can be generated by enzymatic or chemical fusion. In another embodiment, the fusion protein can be generated by recombinant protein expression.

[0086] In one embodiment of the invention, the cytotoxic payload portion may be a chemotherapeutic agent (e.g., as described by Fu et al.). 4 The cytotoxic payload portion (or chemotherapeutic agent) is selected from DNA alkylating agents, topoisomerase 1 inhibitors, topoisomerase 2 inhibitors, transcription inhibitors, Bcl-xL inhibitors, tyrosine kinase inhibitors, DHFR inhibitors, microtubule inhibitors, or DNA targets. The cytotoxic domain can be fused to the protein using methods known in the art, such as click chemistry. Exemplarily, one of the methods / exemplary methods disclosed in Fu et al. can be used. 4 middle.

[0087] The protein drug conjugates disclosed in this aspect of the invention can be used in immunotherapy.

[0088] This aspect of the invention further provides a method for treating cancer, comprising administering the protein pharmaceutical conjugate disclosed herein (this aspect of the invention) to a subject in need of treatment. Cancer treatment may be performed in a subject suffering from cancer, wherein at least a subset of cancer cells in said cancer express FolR1.

[0089] This aspect of the invention further provides pharmaceutical compositions comprising a protein pharmaceutical conjugate for antigen FolR1 as disclosed herein (especially this aspect of the invention), and optionally a pharmaceutically acceptable carrier.

[0090] Pharmaceutically acceptable carriers, diluents, or excipients may include buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0091] Bispecific T-cell adaptor (BiTE) One aspect of the invention provides a protein that is a bispecific T-cell adaptor (BITE), the bispecific T-cell adaptor comprising an antigen-binding domain (first antigen-binding domain) as disclosed in the first aspect of the invention and at least one additional antigen-binding domain (second antigen-binding domain), wherein the target of the additional antigen-binding domain is specific for antigens other than FolR1.

[0092] In other words, this aspect of the invention provides a protein comprising a first antigen-binding domain (specific to FolR1) and at least one additional antigen-binding domain (a second antigen-binding domain), wherein the first antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. -The heavy chain variable region (VH) of the antibody includes a first complementarity-determining region (HCDR1) containing the amino acid sequence GYSFTSYW (SEQ ID No. 20), a second complementarity-determining region (HCDR2) containing the amino acid sequence IYPGDSDT (SEQ ID No. 21), and a third complementarity-determining region (HCDR3) containing the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22), and - The light chain variable region (VL) of the antibody includes a first complementarity-determining region (LCDR1) containing the amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity-determining region (LCDR2) containing the amino acid sequence DDS (SEQ ID No. 37), and a third complementarity-determining region (LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38), and the second antigen-binding domain is specific for antigens other than FolR1.

[0093] The protein comprises a first antigen-binding domain (specific to FolR1) and at least one additional antigen-binding domain (a second antigen-binding domain), wherein the antigen-binding domain comprises, wherein the heavy chain variable region (VH) of the antibody may comprise a first framework region (HFR1) comprising the amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) comprising the amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) comprising the amino acid sequence of SEQ ID No. 25, and a fourth framework region (HFR4) comprising the amino acid sequence of SEQ ID No. 26, and wherein the light chain variable region (VL) of the antibody comprises a first framework region (LFR1) comprising the amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) comprising the amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) comprising the amino acid sequence of SEQ ID No. 41, and a fourth framework region comprising SEQ ID No. 49. The fourth framework region (LFR4) of the amino acid sequence of 42, wherein the second antigen-binding domain is specific for antigens other than FolR1.

[0094] In one embodiment of the invention, the protein may comprise a first antigen-binding domain (specific to FolR1) and at least one additional antigen-binding domain (a second antigen-binding domain), wherein the antigen-binding domain comprises: a heavy chain variable region (VH) of an antibody that may comprise the amino acid sequence SEQ ID No: 19, and a light chain variable region (VL) of an antibody that comprises the amino acid sequence SEQ ID No: 35, and wherein the second antigen-binding domain is specific for antigens other than FolR1.

[0095] In one embodiment of the invention, the protein may comprise a first antigen-binding domain (specific to FolR1) and at least one additional antigen-binding domain (a second antigen-binding domain), the antigen-binding domain comprising the amino acid sequence SEQ ID No: 80, and wherein the second antigen-binding domain is specific to antigens other than FolR1.

[0096] Any molecule or any variant thereof that specifically binds to a target antigen such as FolR1 or an antigen other than FolR1 can be used as an antigen-binding domain. The antigen-binding domain can be or may contain, for example, a Fab fragment, a single-chain Fv (scFv) fragment, a VHH fragment, a bivalent single-chain antibody, or a biantibody. In a preferred embodiment, the antigen-binding domain can be an antibody or an antigen-binding fragment. In another embodiment, the antigen-binding domain (e.g., an antigen-binding fragment of an antibody or scFv) can be fused to a crystallizable fragment region (Fc domain) of the antibody.

[0097] The additional antigen-binding domain (second antigen-binding domain) is specific for antigens / targets other than FolR1, which may be CD19, CD20, CD33, CD123, FcRH5, FLT3, BCMA, GPRC5D, PSMA, EGFRvIII, DLL3, MUC17, or CLDN18.2.

[0098] The two antigen-binding domains can be fused using methods known in the art. Exemplary methods are disclosed in Kujawski et al. 5 middle.

[0099] The protein drug conjugate can be used in immunotherapy.

[0100] This aspect of the invention further provides a method for treating cancer, comprising administering to a subject in need of the protein drug conjugate disclosed in this aspect of the invention. Cancer treatment may be performed in a subject suffering from cancer, wherein at least one subpopulation of cancer cells in said cancer expresses FolR1.

[0101] This aspect of the invention further provides pharmaceutical compositions comprising a protein pharmaceutical conjugate for antigen FolR1 as disclosed herein, and optionally a pharmaceutically acceptable carrier.

[0102] Pharmaceutically acceptable carriers, diluents, or excipients may include buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0103] Detection of FolR1 positive cells In another aspect of the invention, a protein containing an antigen-binding domain, as described in the first aspect of the invention, may include a detection portion.

[0104] In other words, the protein comprises an antigen-binding domain (specific to FolR1) and a detection moiety, wherein the antigen-binding domain comprises the heavy chain variable region (VH) and the light chain variable region (VL) of the antibody. - The heavy chain variable region (VH) of the antibody includes a first complementarity-determining region (HCDR1) containing the amino acid sequence GYSFTSYW (SEQ ID No. 20) SEQ, a second complementarity-determining region (HCDR2) containing the amino acid sequence IYPGDSDT (SEQ ID No. 21), and a third complementarity-determining region (HCDR3) containing the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22), and - The light chain variable region (VL) of the antibody includes a first complementarity-determining region (LCDR1) containing the amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity-determining region (LCDR2) containing the amino acid sequence DDS (SEQ ID No. 37), and a third complementarity-determining region (LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38).

[0105] The protein comprising an antigen-binding domain and a detection portion may include an antigen-binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. The heavy chain variable region (VH) may include a first framework region (HFR1) comprising the amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) comprising the amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) comprising the amino acid sequence of SEQ ID No. 25, and a fourth framework region (HFR4) comprising the amino acid sequence of SEQ ID No. 26. The light chain variable region (VL) of the antibody includes a first framework region (LFR1) comprising the amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) comprising the amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) comprising the amino acid sequence of SEQ ID No. 41, and a fourth framework region (LFR4) comprising the amino acid sequence of SEQ ID No. 42.

[0106] In one embodiment of the present invention, the protein may include the antigen-binding domain and the detection portion, wherein the antigen-binding domain includes: The antibody may contain the heavy chain variable region (VH) of the amino acid sequence SEQ ID No: 19, and The light chain variable region (VL) of the antibody containing the amino acid sequence SEQ ID No: 35.

[0107] In one embodiment of the present invention, the protein comprises the antigen-binding domain and a detection portion, wherein the antigen-binding domain may comprise the amino acid sequence SEQ ID No: 80.

[0108] As described above, a protein comprises an antigen-binding domain and a detection portion. The detection portion may be a fluorescent portion. The detection portion may be selected from fluorescent dyes, fluorescent proteins, or biotin.

[0109] In one embodiment, a protein containing an antigen-binding domain can be fused to an Fc terminus using standard methods known in the art. This Fc terminus can then serve as a detection portion.

[0110] Examples of fluorescent dyes are small organic molecule dyes, such as xanthones like fluorescein, or rhodamine dyes, coumarin dyes, cyanine dyes, pyrene dyes, oxazine dyes, pyridyloxazole dyes, pyrrolemethylene dyes, acridine dyes, oxadiazole dyes, carbopyronine dyes, benzopyranium dyes, fluorene dyes, or organometallic complexes, such as Ru, Eu, and Pt complexes. In addition to monomolecules, this also includes small organic molecule dyes, fluorescent oligomers, or fluorescent polymers such as clusters of polyfluorene.

[0111] Additionally, the fluorescent component can be based on proteins such as phycobiliproteins, nanoparticles such as quantum dots, upconversion nanoparticles, gold nanoparticles, or stained polymer nanoparticles. Commonly known fluorescent proteins such as GFP, RFP, YFP, and their variants can be used.

[0112] The detection section can be directly covalently coupled or coupled via a connector unit.

[0113] Any molecule that specifically binds to a target antigen (specifically for FolR1) can be used as an antigen-binding domain. The antigen can be, or may contain, for example, a Fab fragment, a single-chain Fv (scFv) fragment, a VHH fragment, a bivalent single-chain antibody, or a biantibody. In a preferred embodiment, the antigen-binding domain can be an antibody or an antigen-binding fragment. In another embodiment, the antigen-binding domain (e.g., an antigen-binding fragment of an antibody or scFv) can be fused to a crystallizable fragment region (Fc domain) of the antibody.

[0114] The protein comprising the antigen-binding domain and the detection portion, as disclosed in this aspect of the invention, can be used in a method for detecting cells expressing FolR1 (preferably cancer cells) (Figure 6). The method for detecting cells expressing FolR1 (especially cancer cells) in a biological sample may include the following steps: - A) Contact a sample containing cells expressing FolR1 (especially cancer cells) with a protein according to this aspect of the invention, wherein the protein contains a detection portion. - B) Detect the protein that binds to the cells expressing FolR1.

[0115] In other words, this aspect of the invention provides a method for detecting cells (especially cancer cells) expressing FolR1 in a biological sample, comprising the steps of: A) contacting a sample containing cells (especially cancer cells) expressing FolR1 with a protein comprising an antigen-binding domain (specific to FolR1) and a detection portion, wherein the antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. - The heavy chain variable region (VH) of the antibody includes a first complementarity-determining region (HCDR1) containing the amino acid sequence GYSFTSYW (SEQ ID No. 20), a second complementarity-determining region (HCDR2) containing the amino acid sequence IYPGDSDT (SEQ ID No. 21), and a third complementarity-determining region (HCDR3) containing the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22). - The light chain variable region (VL) of the antibody includes a first complementarity-determining region (LCDR1) containing the amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity-determining region (LCDR2) containing the amino acid sequence DDS (SEQ ID No. 37), and a third complementarity-determining region (LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38).

[0116] - B) Detect the protein that binds to the cells expressing FolR1.

[0117] The method for detecting cells (especially cancer cells) expressing FolR1 in a biological sample may include the following steps: A) contacting a sample containing cells (especially cancer cells) expressing FolR1 with a protein comprising an antigen-binding domain (specific to FolR1) and a detection portion, wherein the antigen-binding domain comprises, wherein the heavy chain variable region (VH) of the antibody may comprise a first framework region (HFR1) comprising the amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) comprising the amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) comprising the amino acid sequence of SEQ ID No. 25, and a fourth framework region (HFR4) comprising the amino acid sequence of SEQ ID No. 26, and wherein the light chain variable region (VL) of the antibody comprises a first framework region (LFR1) comprising the amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) comprising the amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) comprising the amino acid sequence of SEQ ID No. 41, and a fourth framework region comprising the amino acid sequence of SEQ ID No. 26. A) The fourth framework region (LFR4) of the amino acid sequence of 42, and the detection portion; B) Detection of the protein that binds to the cells expressing FolR1.

[0118] In one embodiment, the present invention provides a method for detecting cells (especially cancer cells) expressing FolR1 in a biological sample, comprising the steps of: a) contacting a sample containing cells (especially cancer cells) expressing FolR1 with a protein comprising an antigen-binding domain (specific to FolR1) and a detection portion, wherein the antigen-binding domain comprises: a heavy chain variable region (VH) of an antibody that may contain the amino acid sequence SEQ ID No: 19, and a light chain variable region (VL) of an antibody that contains the amino acid sequence SEQ ID No: 35; b) detecting the protein bound to the cells expressing FolR1.

[0119] In one embodiment, the present invention provides a method for detecting cells (especially cancer cells) expressing FolR1 in a biological sample, comprising the steps of: a) contacting a sample containing cells (especially cancer cells) expressing FolR1 with a protein comprising an antigen-binding domain (specific to FolR1) and a detection portion, wherein the antigen-binding domain may comprise an amino acid sequence SEQ ID No: 80; b) detecting the protein bound to the cells expressing FolR1.

[0120] In a preferred embodiment, the antigen-binding domain is fused to the crystallizable fragment region (Fc domain) of the antibody, and the detection portion is a fluorescent portion or biotin, more preferably a fluorescent portion.

[0121] The method can be used for immunofluorescence or flow cytometry analysis. Specific protocols and methods are known in the art, such as those described by Kinkhabwala et al. 6 and McKinnon 7 It was made public in China.

[0122] In the first step, a sample containing cells expressing FolR1 (especially cancer cells) is contacted with the protein comprising the antigen-binding domain and the detection portion. If the detection portion is, for example, a fluorescent portion, detection can be performed directly, or, for example, if the detection portion is a biotinylate portion, detection can be performed indirectly. In this case, an additional protein can be used, such as an antibody containing a fluorescent detection portion and specific to biotin.

[0123] The sample that can be used in the methods disclosed in this aspect of the invention can be a tissue sample. This tissue sample can be human-derived. The sample can be, or may be derived from, a cancer patient. Therefore, the sample can contain cancer cells expressing folate receptor 1. Furthermore, the sample can be a sample of triple-negative breast cancer, gastric cancer, lung cancer, kidney cancer, pancreatic cancer, or ovarian cancer. The sample can be an ovarian cancer sample.

[0124] Depending on the detection method used, the sample can be a tissue section (e.g. for imaging applications), or the sample can exist in a dissociated form, such as a single-cell solution (e.g. for flow cytometry-based detection methods).

[0125] For optimal detection, tissue sections can be 5-20 µm, preferably 8 µm. Tissue can be fixed according to methods known in the art. Standard fixation methods are based on formaldehyde, formalin, or acetone.

[0126] In immunofluorescence applications, step b) can be performed using a standard fluorescence microscope. For single-cell detection by flow cytometry, standard instruments such as the MACS Quant analyzer can be used.

[0127] After detection, cells can be sorted using fluorescence-activated cell sorting (FACS).

[0128] This aspect of the invention further provides a method for detecting FolR1-expressing cancer cells and subsequent tumor treatment. The method may include the following steps: A) contacting a sample containing cells (especially cancer cells) expressing FolR1 with a protein according to this aspect of the invention, wherein the protein contains a detection portion; B) detecting the protein binding to the FolR1-expressing cells; and D) administering a therapeutically effective amount of the protein, such as CAR, BITE, or protein-drug conjugates as disclosed herein, to a subject.

[0129] This aspect of the invention further provides a method for detecting cancer cells expressing FolR1 and predicting the probability of a subject responding to a protein as disclosed herein. The method may include the following steps: A) contacting a sample containing cells (especially cancer cells) expressing FolR1 with a protein according to this aspect of the invention, wherein the protein contains a detection portion; B) detecting the protein binding to the cells expressing FolR1; C) assigning a score to the detection step (B), wherein the score is assigned based on a comparison with one or more reference samples; D) comparing the score in step (C) with the scores of one or more reference samples, wherein a score indicating a cancer FolR1 level greater than the score of a reference sample with normal or low FolR1 expression, or a score indicating a cancer FolR1 level equal to or greater than the score of a reference sample with high FolR1 expression, identifies the cancer as likely to respond to immunotherapeutic treatment based on CAR, BITE, or protein drug conjugates as disclosed herein.

[0130] The data obtained from step (b) can be used for scoring and to determine whether immunotherapy using this particular conjugate will be successful for treatment. Such scoring can be based on the amount and frequency of cells detected, as well as expression profile and staining intensity.

[0131] For scoring, values ​​are assigned based on comparisons with reference samples.

[0132] If the score is equal to or greater than (preferably greater) compared to the score of a reference sample, the cancer sample contains cancer cells. The individual from whom the sample was obtained is likely to respond to immunotherapy based on CAR, BITE, or protein drug conjugates as disclosed herein.

[0133] Reference samples can be standard tissues, such as non-neoplastic tissues, such as ovaries, kidneys, lungs, and cerebellum.

[0134] All definitions, features, and embodiments defined herein with respect to the first aspect of the invention disclosed herein are applicable, with necessary modifications, to other aspects of the invention disclosed herein.

[0135] definition Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0136] As used herein, the terms “comprising” or “comprises” are used when referring to compositions, methods, and their respective components that are essential to the method or composition, but may still include unspecified elements (whether or not they are essential).

[0137] The terms folate receptor 1, FolR1, FOLR, FOLR1, and FR1 are used interchangeably. It should be understood that this also includes other prior art synonyms such as folate receptor α, FRalpha or FRα, folate-binding protein, FBP, or neurodegenerative disease due to folate transport defects in the brain, NCFTD.

[0138] An antigen-binding domain is a region of a protein that specifically binds to an antigen (usually a variable region of an antibody or its fragment). It consists of a constant domain and a variable domain, one for the heavy chain and one for the light chain. The key and most important elements for antigen-specific binding are the complementarity-determining region (CDR) and the framework region (FR).

[0139] Complementarity-determining regions (CDRs) are portions of the variable chain in an antibody where molecules bind to their specific antigens. CDRs are crucial for the specific binding of antigen-binding proteins.

[0140] The framework region (FR) is a subregion of the antibody's variable region. The framework region is responsible for acting as a scaffold for the complementarity-determining region (CDR).

[0141] Chimeric antigen receptors (CARs) are recombinant antigen receptors that redirect the specificity and function of T lymphocytes and / or other immune cells within a single molecule. The concept behind using CARs in immunotherapy lies in the fact that CARs programmed to target tumor-associated antigens or disease-associated antigens can replicate rapidly and uniformly.

[0142] Generally, a CAR can consist of: an extracellular domain containing an antigen-binding domain (extracellular portion), a transmembrane domain, and a cytoplasmic signal transduction domain (intracellular signal transduction domain). The extracellular domain can connect to the transmembrane domain via a linker or spacer. The extracellular domain may also contain a signal peptide.

[0143] "Signal peptide" refers to a peptide sequence that guides the transport and localization of proteins within the cell, such as to specific organelles (e.g., endoplasmic reticulum) and / or the cell surface.

[0144] Generally, the antigen-binding domain of a CAR refers to the region that specifically binds to an antigen, such as a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), or a disease-associated antigen. The CAR of the present invention may comprise one or more antigen-binding domains (e.g., a tandem CAR). Generally, the target region of the CAR is located extracellularly. The antigen-binding domain may comprise an antibody, a single-domain antibody, or an antigen-binding fragment thereof. The antigen-binding domain may comprise, for example, a Fab fragment, a single-chain Fv (scFv) fragment, a VHH fragment, a bivalent single-chain antibody, or a biantibody. Often, the antigen-binding domain is an scFv. Typically, in an scFv, the variable regions of the immunoglobulin heavy and light chains are fused together by a flexible linker to form the scFv. Such a linker may be, for example, a "(G4 / S)3-linker" or a "whitlow linker."

[0145] As used herein, the term "spacer region" or "hinge" refers to the hydrophilic region located between the antigen-binding domain and the transmembrane domain of a CAR. Spacer regions may include, for example, an Fc fragment or a fragment thereof of an antibody, a hinge region or a fragment thereof of an antibody, the CH2 or CH3 region of an antibody, accessory proteins, artificial spacer sequences, or combinations thereof. Typical examples of spacer regions are the CD8α hinge or the IgG4 hinge.

[0146] The transmembrane domain of a CAR can originate from any desired natural or synthetic source of such a domain. When the source is natural, the domain can originate from any membrane-binding or transmembrane protein. The transmembrane domain can, for example, originate from CD8α or CD28.

[0147] If the corresponding CAR is an activating CAR (generally, as discussed herein, CAR refers to an activating CAR), then the cytoplasmic signaling domain of the CAR (intracellular signaling domain or activating intracellular domain) is responsible for activating at least one normal effector function of the immune cell in which the CAR is expressed. "Effector function" refers to a specialized function of the cell; for example, in T cells, an effector function could be cytolytic activity or co-activation including cytokine secretion. An intracellular signaling domain refers to a portion of a protein that transduces effector function signals and directs the CAR-expressing cell to perform its specialized function. An intracellular signaling domain can include any complete, mutated, or truncated portion of the intracellular signaling domain of a given protein sufficient to transduce signals that initiate or block immune cell effector functions. Typical examples of intracellular signaling domains used in CARs include cytoplasmic signaling sequences of the T cell receptor (TCR) and co-receptors that initiate signal transduction after antigen-receptor binding.

[0148] Generally, T cell activation can be mediated by two different classes of cytoplasmic signaling sequences: a first class (primary cytoplasmic signaling sequences, primary cytoplasmic signaling domains) that initiates antigen-dependent primary activation via the TCR, and a second class (secondary cytoplasmic signaling sequences, co-stimulatory signaling domains) that acts in an antigen-independent manner to provide secondary or co-stimulatory signals. Therefore, the intracellular signaling domains of a CAR can contain one or more primary cytoplasmic signaling domains and / or one or more secondary cytoplasmic signaling domains.

[0149] Primary cytoplasmic signaling domains that function via stimulation can contain ITAMs (immune receptor tyrosine activation motifs). Examples of ITAM-containing primary cytoplasmic signaling domains frequently used in CARs are those derived from TCRζ (CD3ζ), FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. The most typical are sequences derived from CD3ζ.

[0150] The cytoplasmic domain of a CAR can be designed to contain a single CD3ζ signaling domain, or in combination with any other desired cytoplasmic domain. The cytoplasmic domain of a CAR can contain a portion of the CD3ζ chain and a co-stimulatory signaling region (domain). The co-stimulatory signaling region refers to the portion of the intracellular domain of the CAR containing the co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules, other than antigen receptors or their ligands, that are essential for an effective lymphocyte response to antigens. Examples of co-stimulatory molecules are CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0151] Cytoplasmic signaling sequences within the cytoplasmic signaling region of the CAR can be linked together in random or designated order, with or without linkers. Short oligopeptides or polypeptide linkers, preferably 2 to 10 amino acids in length, can form bonds. A typical linker is a glycine-serine duplex.

[0152] In some embodiments of the invention, the antigen-binding domain of the CAR binds to a tag or hapten (“haptened” or “tagged” peptide / protein) conjugated to a protein or peptide, wherein the peptide may bind to a disease-associated antigen, such as a tumor-associated antigen (TAA) that may be expressed on the surface of a cancer cell, FolR1 herein.

[0153] Such CARs may be referred to as “tag-resistant” CARs, “connector CARs”, or “generic CARs”, as disclosed, for example, in US9233125B2.

[0154] A hapten or tag can be directly or indirectly coupled to a polypeptide (a tagged polypeptide) that can bind to the disease-associated antigen expressed on the surface of a target (cell), such as FolR1. The tag can be, for example, a dextran or a hapten such as biotin or fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or thiamine; however, the tag can also be, for example, a peptide sequence chemically or recombinantly coupled to a polypeptide moiety in a tagged polypeptide. The tag can also be streptavidin. The tag moiety of the tagged polypeptide is limited to molecules that can be recognized and specifically bound by a tag-specific antigen-binding domain for a CAR. For example, when the tag is FITC (fluorescein isothiocyanate), the tag-binding domain can constitute an anti-FITC scFv. Alternatively, when the tag is biotin or PE (phycoerythrin), the tag-binding domain can constitute an anti-biotin scFv or an anti-PE scFv, respectively.

[0155] As used herein, the terms "tagged protein" or "tagged polypeptide" refer to a polypeptide having at least one additional component, i.e., a tag, which binds directly or indirectly to it. As used herein, a tagged polypeptide is capable of binding an antigen expressed on a target cell. The polypeptide can be an antibody or its antigen-binding fragment that binds to an antigen expressed on the surface of a target cell, such as a tumor-associated antigen (TAA) on cancer cells. Alternatively, the polypeptide in a tagged polypeptide can be a cytokine or growth factor or another soluble polypeptide capable of binding an antigen to a target cell. As used herein, the terms "adaptor" or "adaptor molecule" or "tagged polypeptide" are used interchangeably. The tag can be, for example, a hapten or a dextran, and the hapten or dextran can be bound by an antigen-binding domain of a CAR containing an antigen-binding domain specific to the tag. Haptens, such as FITC, biotin, PE, thiamine, streptavidin, or dextran, are small molecules that elicit an immune response only when attached to a large carrier, such as a protein; the carrier can also be a carrier that cannot elicit an immune response on its own.

[0156] However, the tag can also be, for example, a peptide sequence chemically or recombinantly coupled to the polypeptide moiety in the tagged polypeptide. The peptide can be selected from c-Myc tags, Strep tags, Flag tags, and multihistidine tags. The tag can also be streptavidin. The tag moiety of the tagged polypeptide is limited to molecules that can be recognized and specifically bound to a tag-specific antigen-binding domain for CAR. For example, when the tag is FITC (fluorescein isothiocyanate), the tag-binding domain can constitute an anti-FITC scFv. Alternatively, when the tag is biotin or PE (phycoerythrin), the tag-binding domain can constitute an anti-biotin scFv or an anti-PE scFv.

[0157] As used herein, the term "modified" can refer to one or more artificially designed alterations to nucleic acids (e.g., nucleic acids within the genome of an organism). "Modified cell" or "genetically modified cell" can refer to a cell with added, deleted, and / or altered genes. In particular, the term refers to the fact that a cell (preferably a T cell) can be manipulated by recombination methods well known in the art to stably or transiently express peptides or proteins that are not expressed in these cells under natural conditions. For example, T cells (preferably human T cells) are modified to express artificial constructs such as chimeric antigen receptors on their cell surface.

[0158] The CAR-expressing cells can be further modified using genetic engineering methods well-known in the art, such as macronucleases, TALENs, CRISPRCases, zinc finger nucleases, shRNAs, and / or miRNAs. The cells can be modified to reduce or eliminate the expression of specific genes commonly expressed in cells, such as T-cell receptors (TCRs), MHC, and co-repressive molecules like PD-1, CTLA-4, BTLA, TIGIT, Tim-3, CD244, LAIR, Lag-3, CD160, and HVEM. The cells can also be modified to express additional transgenes such as therapeutic controls, cytokines and / or fragments, cytokine receptors and / or fragments, cytokine receptor fusion proteins, co-stimulatory receptors, or armoring molecules.

[0159] The terms "immune cell" and "immune effector cell" are used interchangeably and refer to cells that can be part of the immune system and perform specific effector functions, such as T cells, αβT cells, NK cells, NKT cells, B cells, innate lymphocytes (ILCs), cytokine-induced killer (CIK) cells, lymphokine-activated killer (LAK) cells, γδT cells, monocytes, or macrophages. Preferably, these immune cells are human immune cells. Preferred immune cells are cells with cytotoxic effector functions, such as T cells, αβT cells, NK cells, NKT cells, ILCs, CIK cells, LAK cells, macrophages, or γδT cells. The most preferred immune effector cells are T cells and NK cells. "Effector function" refers to a cell's specialized function; for example, in T cells, an effector function can be cell lytic activity or co-operational activity including cytokine secretion.

[0160] As used herein, the term “antibody” is used in the broadest sense to encompass a wide range of antibody structures, including but not limited to monoclonal and polyclonal antibodies (including full-length antibodies), single-domain antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments (i.e., antigen-binding fragments of antibodies), immunoadhesins, and antibody-immunoadhesin chimeras that specifically recognize (i.e. bind) antigens.

[0161] An "antigen-binding fragment" comprises a portion of a full-length antibody, preferably its variable domain, or at least its antigen-binding site ("antigen-binding fragment of the antibody"). Examples of antigen-binding fragments include Fab (antigen-binding fragment), scFv (single-chain variable fragment), VHH fragment, biantibody, dsFv, Fab', biantibody, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0162] The terms "specific to," "specifically binding," or "specific to" refer to antigen-binding domains of antibodies, their fragments, or CARs that recognize and bind to a specific antigen but substantially do not recognize or bind to other molecules in the sample. An antigen-binding domain that specifically binds to an antigen from one species can also bind to the same antigen from another species. This cross-species reactivity does not contradict the definition of the antigen-binding domain as specific. Similarly, an antigen-binding domain that specifically binds to an antigen can also bind to different allelic forms of the antigen (allelic variants, splice variants, isotypes, etc.). This cross-reactivity does not contradict the definition of the antigen-binding domain as specific.

[0163] As used herein, the term "antigen" is intended to include substances that bind to or induce the production of one or more antibodies, and may include, but is not limited to, proteins, peptides, polypeptides, oligopeptides, lipids, carbohydrates such as dextran, haptens, and combinations thereof, such as glycosylated proteins or glycolipids. As used herein, the term "antigen" refers to a molecular entity that can be expressed on the surface of a target cell and can be recognized by means of: an adaptive immune system including, but not limited to, antibodies or TCRs, or engineered molecules including, but not limited to, endogenous or transgenic TCRs, CARs, scFvs or multimers thereof, Fab fragments or multimers thereof, antibodies or multimers thereof, single-chain antibodies or multimers thereof, or any other molecule that can perform binding to a structure with high affinity.

[0164] The term "epitope" refers to a portion of an antigen, such as a soluble antigen, that can be recognized and specifically bound by an antibody or its antigen-binding fragment (antigen-binding domain).

[0165] As used herein, tumor-associated antigens (TAAs) refer to antigenic substances produced by tumor cells. TAAs are useful tumor or cancer markers for identifying tumors / cancer cells using diagnostic tests and are potential candidates for cancer treatment. Preferably, TAAs are expressed on the cell surface of tumors / cancer cells, allowing them to be recognized by antigen-binding receptors as disclosed herein.

[0166] As used herein, the term "target cell" refers to a cell on which an antigen is expressed on its cell surface, said antigen being to be recognized (bound) by a protein according to the invention. The target cell may be, for example, a cancer cell, a cell associated with an autoimmune disease, or a cell associated with an infectious disease.

[0167] Immunotherapy is a medical term defined as "the treatment of disease by inducing, enhancing, or suppressing an immune response." Immunotherapy designed to elicit or amplify an immune response is classified as activating immunotherapy, while immunotherapy that reduces or suppresses an immune response is classified as suppressive immunotherapy.

[0168] As used in this article, the term "treatment" means reducing the frequency or severity of at least one sign or symptom of a disease.

[0169] The terms “therapeutic effective amount” or “therapeutic effective population” refer to the amount of cell population that provides therapeutic benefit in a subject.

[0170] As used herein, the term "subject" refers to an animal. Preferably, the subject is a mammal such as a mouse, rat, cow, pig, goat, chicken, dog, monkey, or human. More preferably, the individual is a human. The subject may be a subject suffering from a disease such as cancer.

[0171] As used herein, the term “expression” is defined as the transcription and / or translation of a specific nucleotide sequence in a cell, driven by its promoter.

[0172] The term "isolated" is used herein to indicate that a polypeptide, nucleic acid, or host cell exists in a different physical environment than in which it occurs in nature. For example, an isolated polypeptide may be substantially isolated (e.g., enriched or purified) relative to the complex cellular environment in which it naturally occurs.

[0173] The term "cancer" is medically termed a malignant growth. Cancer is a broad class of diseases involving uncontrolled cell growth and includes all types of leukemia. In cancer, cells (cancer cells) divide and grow uncontrollably, forming a malignant tumor and invading neighboring parts of the body. Cancer can also spread to more distant parts of the body via the lymphatic system or bloodstream. More than 200 different types of cancer affecting humans are known.

[0174] The term antibody-drug conjugate (ADC) is a molecule comprising an antibody (or an antigen-binding fragment of an antibody) conjugated to a drug, such as a cytotoxic agent. ADCs can be used to specifically target drugs to cancer cells by the specific binding of the antibody to tumor antigens expressed on the cell surface. Exemplary drugs used in conjunction with ADCs include DNA alkylating agents, topoisomerase 1 inhibitors, topoisomerase 2 inhibitors, transcription inhibitors, Bcl-xL inhibitors, tyrosine kinase inhibitors, DHFR inhibitors, microtubule inhibitors, or DNA targeting agents.

[0175] A bispecific T-cell adaptor, or BiTE, is an antigen-binding molecule. It consists of a single-chain polypeptide and can bind to two different antigens.

[0176] Autoimmune diseases are conditions caused by an imbalance in the autoimmune system or immune homeostasis, leading to pathological conditions that can affect multiple different organ systems. Examples include Behcet's disease, juvenile idiopathic arthritis, type 1 diabetes, rheumatoid arthritis, Wegener's granulomatosis, systemic lupus erythematosus, systemic sclerosis, Crohn's disease, Graves' disease, Hashimoto's thyroiditis, Goodpassuia syndrome, primary biliary cholangitis, myasthenia gravis, dermatomyositis / polymyositis, vasculitis, mixed connective tissue disease, scleroderma, multiple sclerosis, psoriasis, ulcerative colitis, and uveitis.

[0177] An infection (infectious disease) is the invasion of an organism's tissues by a pathogenic agent, its proliferation, and the host tissue's response to the infectious agent and its toxins. Infections are caused by infectious agents (pathogens), including viruses, bacteria, fungi, and parasites. The infection can be acute or chronic.

[0178] The term "administration" refers to both local and systemic administration, including intravenous, parenteral, pulmonary, and local / percutaneous administration. Administration can be direct intratumoral administration. Routes of administration of the drug component include, for example, oral, nasal, or inhalation administration to the subject, administration as a suppository, local contact, percutaneous delivery, intrathecal administration, intravenous administration, intraperitoneal administration, intramuscular administration, intralesional administration, or subcutaneous administration. Administration can be carried out via any route, including parenteral and transmucosal (e.g., oral, nasal, vaginal, rectal, or percutaneous) routes. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intrarenal, intraurethral, ​​intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, ionophoretic, and intracranial administration. Example

[0179] The following embodiments are intended to provide a more detailed explanation of the invention, but are not intended to limit the invention to these embodiments.

[0180] The selection process for anti-FOLR1 CAR T candidates consists of the following: initial phage display selection, followed by flow cytometry screening to ensure the specificity of candidates for FOLR1 compared to other FOLR variants. Subsequently, CAR T screening methods and advanced in vitro co-culture assays allow for the identification of novel, functional, and specific anti-FOLR1 CAR candidates. Figure 1 ).

[0181] Example 1: Identification of antibody fragments Two native scFv phage display libraries constructed at Miltenyi Biotec were used to identify novel binding agents against FOLR1. These libraries differed in the variable domains of the light chain and contained either the κ or λ isotype. For panning, mouse FOLR1 (mFOLR1), human FOLR1-hIgG1Fc (hFOLR1-Fc), and human FOLR2 (hFOLR2) were purchased from AcroBiosystems. Human FOLR3 was purchased from R&D Systems, and human IgG1-Fc (hIgG1Fc) was purchased from amsbio. Five different panning strategies were used to identify binding agent candidates, with hFOLR1 and mFOLR1 antigens used for positive selection, respectively. In all strategies, hFOLR2, hFOLR3, and hIgG1-Fc antigens were used for competition (Figure 2A). For panning, each library was incubated on antigen immobilized on a MaxiSorb ELISA plate (Nunc). After washing, scFv phage particles were eluted with trypsin. Eluted phage particles were used to infect *E. coli* TG1, and phage production was promoted using M13K07 helper phage. After a third round of panning, eluted phage particles were used to infect *E. coli* TG1, and single-cell colonies were inoculated the following day for scFv expression.

[0182] The ability of expressed scFvs to bind to hFOLR1-Fc, mFOLR1, hFOLR2, or hIgG1Fc was analyzed by ELISA. This resulted in the identification of 2266 first hits, which bound hFOLR1 and / or mFOLR1, but not hFOLR2 or hIgG1-Fc (Figure 2B). Separating the ELISA hits using a panning strategy became apparent: the binders selected in the strategy without mFOLR1 during the selection process did indeed preferentially recognize hFOLR1 over mFOLR1. On the other hand, as expected, panning strategy 3, which selected using mFOLR1, revealed preferential binding to mFOLR1 over hFOLR1. A panning strategy using both hFOLR1 and mFOLR1 resulted in the selection of scFvs with cross-reactivity against both targets (Figure 2C).

[0183] Of the 2266 initial hits, the scFv gene was sequenced in 817 clones. Alignment of the scFv gene revealed that 349 of the 817 clones contained unique scFv sequences. From these unique sequences, 189 clones were cloned as scFv-Fc fusion proteins into mammalian expression vectors (Figure 2D). After expression and purification, 94 of these scFv-Fc proteins were analyzed by flow cytometry.

[0184] Example 2: Expression and purification of anti-FOLR1 scFv-Fc candidate Potential anti-FOLR1 scFv-Fc candidates were manufactured via transient transfection of HEK293 cells. On the day of transfection, HEK293 cells were introduced at a rate of 2 x 10⁻⁶ cells / cells. 6 Cells were seeded at a density of 10 cells / well into a total volume of 800 µl of expression medium in 96-well deep-well plates. Next, the transfection reagent was mixed with diluted plasmid DNA encoding the corresponding anti-FOLR1 scFv-Fc candidate and added to the cells after a 10-minute incubation period. Cells were incubated at 37°C on a track-shaker at 8% CO2. On day 4 post-transfection, the supernatant was collected, and the scFv-Fc candidate was purified using a Protein A PhyTip® column (Biotage) in conjunction with an automated workstation (Beckman Coulter). Purity was confirmed via SDS-PAGE, and protein concentration was determined using a microplate reader by measuring UV absorbance at 280 nm. The purified protein was stored at -20°C until further processing.

[0185] Example 3: Flow cytometry screening of anti-FOLR1 scFv-Fc candidates The anti-FOLR1 scFv-Fc candidate was prepared as described in Example 2, and the protein concentration was normalized. For flow cytometry validation, non-target cells were labeled with CellTrace™ Violet or CellTrace™ FarRed (ThermoFisher), and 1x10 5Cells / cell lines were seeded into 96-well plates. Cells were pelleted at 1200 g for 2 min at 4°C and stained with a 50 µL staining mixture containing 1 µg scFv-Fc. Staining was performed at 4°C for 30 min, followed by two consecutive washes with 150 μL PBS / 2 mM EDTA / 0.5% BSA (referred to as PEB, 1200 g, 2 min). Next, a 50 µL secondary staining mixture containing AffinitPure F(ab')2Fragment Goat Anti-HumanIgG-PE (Jackson ImmunoResearch) and 7-AAD (Miltenyi Biotec) as a dead cell marker was added to the cells. Staining was performed at 4°C for 10 min, followed by two consecutive washes with 150 μL PEB. Cells were resuspended in 50 μL PEB for subsequent flow cytometry analysis on a MACSQuant® Analyzer 10 (Miltenyi Biotec). Cells were gated according to size, unicellularity, and viability before being distinguished by CellTrace from cell lines expressing the corresponding mouse or human FOLR variants (hFOLR1 SEQ ID 45, 51, 52; hFOLR2 SEQ ID 46, 53, 54; hFOLR3 SEQ ID 47, 48, 55, 56; hFOLR4 SEQ ID 49, 50, 57, 58; mFOLR1 SEQ ID 59, 60, 65, 66; mFOLR2 SEQ ID 61, 62, 67, 68; mFOLR4 SEQ ID 63, 64, 69, 70). (Fig. 3A-C) The corresponding FOLR-positive target cell lines were stained with the anti-FOLR1 scFv-Fc candidate, and the frequency (%) of stained cells is shown. Of the 94 anti-FOLR1 scFv-Fc candidates that have been successfully generated and purified, 65 candidates showed activity against hFOLR1 in flow cytometry analysis. + Positive results were observed in Jurkat cells (frequency >15%) (Figures 3D, E). Of these candidates, 58 additional candidates showed activity against mFOLR1. + Positive results in Jurkat cells (frequency >15%) (Figure 3F, G). Nineteen of these 58 candidates showed efficacy against mouse FOLR2. + Non-specific staining of Jurkat cells (frequency >5%), while for hFOLR2 + mFOLR4 + and hFOLR4 +No staining was observed in the cells (Fig. 3H-O). The remaining 39 candidates were then excluded based on their binding to Jurkat wt cells and for hFOLR1. + and mFOLR1 + Candidates with a signal-to-noise ratio of <10 were further selected for use against FOLR1 in humans and mice. + Jurkat cells showed specific binding to both, but not to h / m FOLR2. + and h / m FOLR4 + For Jurkat cells, no signal-free anti-FOLR1 scFv-Fc candidates were selected, and a total of 19 candidates (black bars) were chosen for further analysis.

[0186] Example 4: Identification of chimeric antigen receptors by using CAR transgenic T cells to recognize target cells expressing FOLR1. (CAR).

[0187] A human-derived natural single-stranded variable fragment (scFv) was cloned into a lentiviral vector construct having an scFv – human CD8α hinge (SEQ ID 1, 2) – CD8α transmembrane domain (SEQ ID 3, 4) – human 41BB co-stimulatory domain (SEQ ID 5, 6) – human CD3ζ domain configuration (SEQ ID 7, 8). The scFv contains a (G4S)3 connector domain (SEQ ID 9, 10). The lentiviral vector contains a truncated human low-affinity nerve growth factor receptor (ΔLNGFR) expression cassette separated by 2A elements for co-expression as a transduction marker. The lentiviral particles were used to transduce primary human T cells from three independent donors for CAR expression. CD3 was quantified using flow cytometry. + LNGFR + The event determined the frequency and absolute number of CAR T cells. CAR T cells were cultured at an effector:target ratio of 2.5:1 with FOLR1- or FOLR1-KO OV-90 cells (which were transduced to express GFP). As controls, CAR T cells were cultured in the absence of target cells, and OV-90 or OV-90 FOLR1 KO cells were cultured in the absence of CAR T cells. Additionally, OV-90 and OV-90 FOLR1 KO cells were cultured with untransduced T cells or T cells expressing one of the two positive control CARs, respectively. At 24 hours, the cell culture supernatant was analyzed for IFN-γ secretion. At 92 hours, CAR T cells (CD3+) showed increased IFN-γ secretion. + LNGFR +Quantification was performed by flow cytometry. Additionally, fresh OV-90 and OV-90 FOLR1 KO cells were added according to the experimental setup to initiate a second round of co-culture (Figure 4P). After 24 hours, the supernatant was analyzed again for IFN-γ secretion. At 164 hours, CAR T cells (CD3+)... + LNGFR + Quantification was performed by flow cytometry. According to the experimental setup, the third round of co-culture was initiated by adding fresh OV-90 and OV-90FOLR1 KO cells. After 24 hours, the supernatant was analyzed again for IFN-γ secretion. After 260 hours, CAR T cells (CD3+)... + LNGFR + Quantification was performed by flow cytometry. The total duration of the three rounds of co-culture was 11 days, and quantification was performed by measuring green calibration units / µm. 2 The image shows GFP signals acquired over time, with cytotoxicity against OV-90 and OV-90 FOLR1 KO cells quantified every 24 hours.

[0188] Three consecutive rounds of co-culture of CAR T cells with OV-90 target cells allowed for the identification of CAR T candidates capable of mediating the killing of FOLR1-expressing cells (Fig. 4A). Candidates FOLR1 CAR 8 and 19 (SEQ ID NO: 88 and 98) failed to express and were excluded from further analysis. FOLR1 CAR 5, 7, 12, 16, and 18 (SEQ ID NO: 85, 87, 80, 95, and 97) effectively lysed tumor cells during the three rounds of co-culture. These functional CAR T candidates were confirmed in co-cultures of OV-90 cells with CAR T cells from two other donors (Fig. 4B, 4C). In contrast, candidates FOLR1 CAR 6, 9, 10, 11, 13, 15, 17, 20, and 21 (SEQ ID NO: 86, 89, 90, 91, 92, 94, 96, 99, and 100) showed very poor or no function after three rounds of co-culture from at least one donor. Co-culture of CAR T cells with OV-90 FOLR1 KO cells allowed for the identification of CAR T candidates that mediated cytotoxicity (Fig. 4D-F), i.e., antigen-independent cell lysis, in the first and up to the third rounds of co-culture. Candidate FOLR1 CAR 16 nonspecifically lysed tumor cells in the absence of FOLR1.

[0189] At the end of the first and third rounds, CAR T cell expansion was observed in all three donors in the co-culture with OV-90 cells for four CAR constructs (i.e., FOLR1 CAR 5, 7, 12, and 18), exceeding the initial number of CD3+LNGFR+ T cells plated on day 0 (Fig. 4 GI). The same was true for T cells expressing the CAR (i.e., FOLR1 CAR16), which mediated cytotoxicity against OV-90 FOLR1 KO cells. For candidates FOLR1 CAR 1, 2, 3, 4, 6, 9, 10, 11, 13, 14, 15, 17, 20, and 21 (SEQ ID NO: 81, 82, 83, 84, 86, 89, 90, 91, 92, 93, 94, 96, 99, and 100), proliferation was unmeasurable in at least one donor.

[0190] These four functional CARs (FOLR1 CARs 5, 7, 12, and 18) were associated with IFN-γ secretion in each round of co-culture with OV-90 cells via T cell expression (Fig. 4 JL). In contrast, IFN-γ was unmeasurable in at least one donor for candidate FOLR1 CARs 1, 2, 3, 4, 6, 9, 10, 11, 13, 14, 15, 17, 20, and 21. CAR candidates that induced T cell-mediated cytotoxicity in OV-90 FOLR1 KO co-cultures also induced IFN-γ secretion in each round of co-culture with OV-90 and OV-90 FOLR1 KO cells, respectively (Fig. 4 MO). In summary, in vitro co-culture allows for the selection of CARs that equip T cells with the ability to mediate repeated killing, cytokine production, and expansion upon encountering target cells, without killing target antigen KO cells, namely FOLR1 CARs 5, 7, 12, and 18.

[0191] Example 5: Generation and identification of a lead chimeric antigen receptor (CAR) for target cell recognition of FOLR1 expression.

[0192] Four lead CAR T candidates (FOLR1 CAR5, FOLR1 CAR7, FOLR1 CAR12, and FOLR1 CAR18) identified in previous CAR T screening methods were further tested in more advanced in vitro assays. CAR constructs lacking the LNGFR reporter gene were generated by cloning an scFv targeting native human FOLR1 along with the CD8α hinge (SEQ ID 1, 2), CD8α transmembrane domain (SEQ ID 3, 4), human 41BB co-stimulatory domain (SEQ ID 5, 6), and human CD3ζ domain linker (SEQ ID 7, 8) into a lentiviral vector construct (Figure 5A). The heavy and light chains of the scFv were linked via (G4S)3 linkers (SEQ ID 9, 10). Lentiviral particles were produced via transfection of HEK293T cells and collected from the cell culture supernatant. To determine the functional titers of different lentiviral particles, SUP-T1 cells were transduced with varying amounts of lentiviral vector, and transduction efficiency was determined by flow cytometry using CAR assays of the biotinylated FOLR1-Fc protein. All four candidate CARs were expressed at high and comparable levels.

[0193] To validate the functionality and specificity of the CAR T candidates, CAR T cells from three different donors were co-cultured with repeatedly added GFP-expressing target cells (OV-90 wt) or non-target cells (OV-90 FOLR1KO) at an initial effector:target ratio of 0.5:1. As controls, OV-90 cells were cultured without T cells and with untransduced T cells. Additionally, positive control CAR T cells were co-cultured with OV-90 cells, which are known to specifically and effectively induce lysis of FOLR1-expressing cells. Fresh OV-90 cells were added to the co-culture after 48 hours. OV-90 cell lysis resulted in a decrease in GFP signal, while ineffective or no lysis increased GFP signal over time. OV-90 cell lysis was measured at 120 hours and is shown as confluence [%] in green areas. After two additions of OV-90 cells, all CAR T candidates from all three donors effectively eliminated FOLR1-expressing OV-90 cells, slightly better than the positive control (Fig. 5B-D). In addition, none of the CAR T candidates from all three donors eliminated OV-90 FOLR1 KO cells (Fig. 5E-G).

[0194] At 48 and 120 hours after co-culture setup, the expression of activation markers CD137 and CD69 in the CAR T candidates was measured by flow cytometry. CAR T cells from all three donors were efficiently activated, as evidenced by increased CD69 and CD137 expression compared to untransduced T cells. Although the activation profiles were similar among the candidates after 48 hours of co-culture, the 120-hour co-culture results showed more significant differences between the candidates. After co-culture with FOLR1-expressing OV-90 cells, FOLR1 CAR 18 and FOLR1 CAR 7 showed lower expression of activation markers compared to FOLR1 CAR 5 and FOLR1 CAR 12. After co-culture with OV-90 FOLR1 KO cells, CD137 activation marker expression in all candidates was comparable to that in untransduced T cells (Fig. 5K-M). However, after co-culturing with OV-90 FOLR1 KO cells, CD69 expression in FOLR1 CAR 18 and FOLR1 CAR 7 was increased compared with FOLR1CAR 5, FOLR1 CAR 12 and positive control CAR T cells (Fig. 5 HJ).

[0195] In another co-culture experiment, the anti-FOLR1 CAR T candidate was compared with FOLR1. 低 (OVCAR-3) expressing cells were co-cultured with OV-90 wt (FOLR1) 高 The expression of CD69 and CD137 activation markers of CAR T candidates was increased after co-culturing with OV-90 cells expressing FOLR1 for 48 hours, while minimal activation of all CAR T candidates was observed after co-culturing with OV-90 FOLR1 KO cells. Although OVCAR-3 cells showed only 15% FOLR1 expression, the positive control CAR T cells and FOLR1 CAR 12 showed higher expression of activation markers CD69 and CD137 compared with untransduced T cells and other candidates (Fig. 5N, O).

[0196] In summary, all four tested CAR T candidates were functional and specific in vitro. This is because FOLR1CAR 12 is controlled by FOLR1. 低 Expression of target cell activation, and in conjunction with FOLR1 高 The expression cells showed superior activation compared to other candidates after co-culture, therefore FOLR1 CAR 12 was selected as the lead candidate (SEQ ID 11-44).

[0197] Example 6: Immunization of FOLR1 expression in human cell lines and tissues using anti-FOLR1 scFv-Fc candidate Fluorescence detection.

[0198] Four lead CAR T candidates (FOLR1 CAR5, FOLR1 CAR7, FOLR1 CAR12, and FOLR1 CAR18) identified in previous CAR T screening methods were further characterized by immunofluorescence assays to assess their ability to detect FOLR1-expressing cells. The scFv-Fc fusion protein as described in Example 3 was used. Furthermore, the candidates were directly labeled with a fluorescent dye, PE.

[0199] In the first step, Jurkat cells that do not express FOLR1 were analyzed using PE-labeled scFv-Fc fusion protein (Figure 6A). Here, the specificity of the anti-FOLR1 scFv-Fc fusion protein was evaluated, and no significant signal of the candidate was detected relative to the positive and negative control antibodies (Figure 6B).

[0200] Next, Jurkat cells were transduced to express FOLR1, and the scFv-Fc fusion protein was analyzed using PE-labeled protein (Fig. 6C). Here, the specificity of the anti-FOLR1 scFv-Fc fusion protein was evaluated, and an increased signal was detected in the candidate compared to the negative control antibody (Fig. 6D) and compared to Jurkat cells not expressing FOLR1 (Fig. 6B). Furthermore, the signal of the scFv-Fc candidate co-localized with the positive control.

[0201] Third, OV-90 cells expressing FOLR1 were analyzed using PE-labeled scFv-Fc fusion protein (Figure 6E). Here, the specificity of the anti-FOLR1 scFv-Fc fusion protein was evaluated, and an increased signal was detected in the candidate compared to the negative control antibody (Figure 6F).

[0202] Next, OV-90 cells were modified via CRISPR / Cas9 to suppress endogenous FOLR1 expression. These OV-90 FOLR1 KO cells were then analyzed using a PE-labeled scFv-Fc fusion protein (Fig. 6G). The specificity of the anti-FOLR1 scFv-Fc fusion protein was evaluated, and no significant signal was detected relative to the positive and negative control antibodies (Fig. 6H). Furthermore, the scFv-Fc candidate signal co-localized with the positive control.

[0203] Finally, the PE-labeled anti-FOLR1 scFv-Fc fusion protein was used to analyze primary human ovarian cancer (Figure 6I). Here, the specificity of the anti-FOLR1 scFv-Fc fusion protein was evaluated, and a significant signal of the candidate was detected relative to the positive and negative control antibodies (Figure 6J).

[0204] Sequence description of sequence scheme SEQ ID NO: 1: Human CD8α hinge nucleotide sequence SEQ ID NO: 2: Human CD8α hinge amino acid sequence SEQ ID NO: 3: Human CD8α transmembrane domain nucleotide sequence SEQ ID NO: 4: Amino acid sequence of human CD8α transmembrane domain SEQ ID NO: 5: Nucleotide sequence of human 41BB co-stimulatory domain SEQ ID NO: 6: Amino acid sequence of human 41BB co-stimulatory domain SEQ ID NO: 7: Human CD3ζ domain nucleotide sequence SEQ ID NO: 8: Human CD3ζ domain amino acid sequence SEQ ID NO: 9: (G4S)3 linker nucleotide sequence SEQ ID NO: 10: (G4S)3 linker amino acid sequence SEQ ID NO: 11: FOLR1 CAR 12 scFv Vh nucleotide sequence SEQ ID NO: 12: FOLR1 CAR 12 scFv Vh CDR1 nucleotide sequence SEQ ID NO: 13: FOLR1 CAR 12 scFv Vh CDR2 nucleotide sequence SEQ ID NO: 14: FOLR1 CAR 12 scFv Vh CDR3 nucleotide sequence SEQ ID NO: 15: FOLR1 CAR 12 scFv Vh FR1 nucleotide sequence SEQ ID NO: 16: FOLR1 CAR 12 scFv Vh FR2 nucleotide sequence SEQ ID NO: 17: FOLR1 CAR 12 scFv Vh FR3 nucleotide sequence SEQ ID NO: 18: FOLR1 CAR 12 scFv Vh FR4 nucleotide sequence SEQ ID NO: 19: FOLR1 CAR 12 scFv Vh amino acid sequence SEQ ID NO: 20: FOLR1 CAR 12 scFv Vh CDR1 amino acid sequence SEQ ID NO: 21: FOLR1 CAR 12 scFv Vh CDR2 amino acid sequence SEQ ID NO: 22: FOLR1 CAR 12 scFv Vh CDR3 amino acid sequence SEQ ID NO: 23: FOLR1 CAR 12 scFv Vh FR1 amino acid sequence SEQ ID NO: 24: FOLR1 CAR 12 scFv Vh FR2 amino acid sequence SEQ ID NO: 25: FOLR1 CAR 12 scFv Vh FR3 amino acid sequence SEQ ID NO: 26: FOLR1 CAR 12 scFv Vh FR4 amino acid sequence SEQ ID NO: 27: FOLR1 CAR 12 scFv Vl nucleotide sequence SEQ ID NO: 28: FOLR1 CAR 12 scFv Vl CDR1 nucleotide sequence SEQ ID NO: 29: FOLR1 CAR 12 scFv Vl CDR2 nucleotide sequence SEQ ID NO: 30: FOLR1 CAR 12 scFv Vl CDR3 nucleotide sequence SEQ ID NO: 31: FOLR1 CAR 12 scFv Vl FR1 nucleotide sequence SEQ ID NO: 32: FOLR1 CAR 12 scFv Vl FR2 nucleotide sequence SEQ ID NO: 33: FOLR1 CAR 12 scFv Vl FR3 nucleotide sequence SEQ ID NO: 34: FOLR1 CAR 12 scFv Vl FR4 nucleotide sequence SEQ ID NO: 35: FOLR1 CAR 12 scFv Vl amino acid sequence SEQ ID NO: 36: FOLR1 CAR 12 scFv Vl CDR1 amino acid sequence SEQ ID NO: 37: FOLR1 CAR 12 scFv Vl CDR2 amino acid sequence SEQ ID NO: 38: FOLR1 CAR 12 scFv Vl CDR3 amino acid sequence SEQ ID NO: 39: FOLR1 CAR 12 scFv Vl FR1 amino acid sequence SEQ ID NO: 40: FOLR1 CAR 12 scFv Vl FR2 amino acid sequence SEQ ID NO: 41: FOLR1 CAR 12 scFv Vl FR3 amino acid sequence SEQ ID NO: 42: FOLR1 CAR 12 scFv Vl FR4 amino acid sequence SEQ ID NO: 43: FOLR1 CAR 12 nucleotide sequence SEQ ID NO: 44: FOLR1 CAR 12 amino acid sequence SEQ ID NO: 45: Human FORL1, P15328, aa25-235 SEQ ID NO: 46: Human FORR2, P14207, aa17-230 SEQ ID NO: 47: Human FORR3 isotype 1, P41439, aa23-245 SEQ ID NO: 48: Human FORR3 isotype 2, P41439-4, aa23-172 SEQ ID NO: 49: Human FORR4 isotype 1, A6ND01, aa20-228 SEQ ID NO: 50: Human FORR4 isotype 2, A6ND01-2, aa20-243 SEQ ID NO: 51: Recombinant hFOLR1 nucleotide sequence including 3x HA tags SEQ ID NO: 52: Recombinant hFOLR1 amino acid sequence including 3x HA tags SEQ ID NO: 53: Recombinant hFOLR2 nucleotide sequence including 3x HA tags SEQ ID NO: 54: Recombinant hFOLR2 amino acid sequence including 3x HA tags SEQ ID NO: 55: Recombinant hFOLR3 nucleotide sequence including 3x HA tags and hFOLR1 GPI anchoring domain SEQ ID NO: 56: Recombinant hFOLR3 amino acid sequence including 3x HA tags and hFOLR1 GPI anchoring domain SEQ ID NO: 57: Recombinant hFOLR4 nucleotide sequence including 3x HA tags SEQ ID NO: 58: Recombinant hFOLR4 amino acid sequence including 3x HA tags SEQ ID NO: 59: Recombinant mFOLR1 nucleotide sequence including 3x HA tags SEQ ID NO: 60: Recombinant mFOLR1 amino acid sequence including 3x HA tags SEQ ID NO: 61: Recombinant mFOLR2 nucleotide sequence including 3x HA tags SEQ ID NO: 62: Recombinant mFOLR2 amino acid sequence including 3x HA tags SEQ ID NO: 63: Recombinant mFOLR4 nucleotide sequence including 3x HA tags SEQ ID NO: 64: Recombinant mFOLR4 amino acid sequence including 3x HA tags SEQ ID NO: 65: mFOLR1 nucleotide sequence, 14275 SEQ ID NO: 66: mFOLR1 amino acid sequence, P35846 SEQ ID NO: 67: mFOLR2 nucleotide sequence, 14276 SEQ ID NO: 68: mFOLR2 amino acid sequence, Q05685 SEQ ID NO: 69: mFOLR4 nucleotide sequence, 64931 SEQ ID NO: 70: mFOLR4 amino acid sequence, Q9EQF4 SEQ ID NO: 71: Human IgG4 hinge nucleotide sequence SEQ ID NO: 72: Human IgG4 hinge amino acid sequence SEQ ID NO: 73: Nucleotide sequence of the intracellular domain of OX40 SEQ ID NO: 74: Amino acid sequence of the intracellular domain of OX40 SEQ ID NO: 75: CD28 transmembrane nucleotide sequence SEQ ID NO: 76: CD28 transmembrane amino acid sequence SEQ ID NO: 77: CD28 intracellular domain nucleotide sequence SEQ ID NO: 78: Amino acid sequence of the CD28 intracellular domain SEQ ID NO: 79: FOLR1 CAR 12scFv nucleotide sequence SEQ ID NO: 80: FOLR1 CAR 12scFv amino acid sequence SEQ ID NO: 81: FOLR1 CAR 1 scFv sequence SEQ ID NO: 82: FOLR1 CAR 2 scFv sequence SEQ ID NO: 83: FOLR1 CAR 3 scFv sequence SEQ ID NO: 84: FOLR1 CAR 4 scFv sequence SEQ ID NO: 85: FOLR1 CAR 5 scFv sequence SEQ ID NO: 86: FOLR1 CAR 6 scFv sequence SEQ ID NO: 87: FOLR1 CAR 7 scFv sequence SEQ ID NO: 88: FOLR1 CAR 8 scFv sequence SEQ ID NO: 89: FOLR1 CAR 9 scFv sequence SEQ ID NO: 90: FOLR1 CAR 10 scFv sequence SEQ ID NO: 91: FOLR1 CAR 11 scFv sequence SEQ ID NO: 92: FOLR1 CAR 13 scFv sequence SEQ ID NO: 93: FOLR1 CAR 14 scFv sequence SEQ ID NO: 94: FOLR1 CAR 15 scFv sequence SEQ ID NO: 95: FOLR1 CAR 16 scFv sequence SEQ ID NO: 96: FOLR1 CAR 17 scFv sequence SEQ ID NO: 97: FOLR1 CAR 18 scFv sequence SEQ ID NO: 98: FOLR1 CAR 19 scFv sequence SEQ ID NO: 99: FOLR1 CAR 20 scFv sequence SEQ ID NO: 100: FORL1 CAR 21 scFv sequence.

[0205] References 1. Hansen T., Datamonitor Healthcare, Informa Pharma Intelligence, Ovarian cancer disease analysis, Ref Code: DMKC0215129, 2021 2. Leamon CP, Low PS, 1991, PNAS; Kandalaft et al., 2012, J Transl Med 3. Kelemen, 2006, Int. J. Cancer 4. Fu, Z., Li, S., Han, S. et al. Antibody drug conjugate: the “biological missile” for targeted cancer therapy. Sig Transduct Target Ther 7, 93 (2022). https: / / doi.org / 10.1038 / s41392-022-00947-7 5. Kujawski M, Li L, Bhattacharya S, Wong P, Lee WH, Williams L, Li H, Chea J, Poku K, Bowles N, Vaidehi N, Yazaki P, Shively JE. Generation of dual specific bivalent BiTEs (dbBIspecific T-cell engaging antibodies) for cellular immunotherapy. BMC Cancer. 2019 Sep 5;19(1):882. doi: 10.1186 / s12885-019-6056-8. PMID: 31488104; PMCID: PMC6727398. 6. Kinkhabwala A, Herbel C, Pankratz J, Yushchenko DA, Rüberg S,Praveen P, Reiß S, Rodriguez FC, Schäfer D, Kollet J, Dittmer V, Martinez-Osuna M, Minnerup L, Reinhard C, Dzionek A, Rockel TD, Borbe S, Büscher M,Krieg J, Nederlof M, Jungblut M, Eckardt D, Hardt O, Dose C, Schumann E,Peters RP, Miltenyi S, Schmitz J, Müller W, Bosio A. MACSima imaging cyclicstaining (MICS) technology reveals combinatorial target pairs for CAR T celltreatment of solid tumors. Sci Rep. 2022 Feb 3;12 (1):1911. doi: 10.1038 / s41598-022-05841-4. PMID: 35115587; PMCID: PMC8813936. 7. McKinnon KM. Flow Cytometry: An Overview. Curr Protoc Immunol.2018 Feb 21;120:5.1.1-5.1.11. doi: 10.1002 / cpim.40. PMID: 29512141; PMCID:PMC5939936。

Claims

1. A protein comprising an antigen-binding domain specific to FolR1, said antigen-binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. - The heavy chain variable region (VH) of the antibody includes a first complementarity-determining region (HCDR1) containing the amino acid sequence GYSFTSYW (SEQ ID No. 20), a second complementarity-determining region (HCDR2) containing the amino acid sequence IYPGDSDT (SEQ ID No. 21), and a third complementarity-determining region (HCDR3) containing the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22). - The light chain variable region (VL) of the antibody includes a first complementarity-determining region (LCDR1) containing the amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity-determining region (LCDR2) containing the amino acid sequence DDS (SEQ ID No. 37), and a third complementarity-determining region (LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38).

2. The protein according to claim 1, wherein the heavy chain variable region (VH) of the antibody comprises a first framework region (HFR1) containing the amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) containing the amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) containing the amino acid sequence of SEQ ID No. 25, and a fourth framework region (HFR4) containing the amino acid sequence of SEQ ID No. 26, and wherein the light chain variable region (VL) of the antibody comprises a first framework region (LFR1) containing the amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) containing the amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) containing the amino acid sequence of SEQ ID No. 41, and a fourth framework region (LFR4) containing the amino acid sequence of SEQ ID No.

42.

3. The protein according to any one of claims 2-3, wherein the antigen-binding domain comprises a heavy chain variable region (VH) of an antibody comprising the amino acid sequence SEQ ID No: 19, and a light chain variable region (VL) of an antibody comprising the amino acid sequence SEQ ID No:

35.

4. The protein according to any one of claims 1-3, wherein the antigen-binding domain comprises the amino acid sequence SEQ ID No:

80.

5. The protein according to any one of claims 1-4, wherein the protein is a chimeric antigen receptor (CAR) comprising the antigen-binding domain, spacer domain, transmembrane domain and intracellular signal transduction domain.

6. The protein of claim 5, wherein the spacer domain comprises a hinge domain of CD8α.

7. The protein according to any one of claims 5-6, wherein the CAR comprises a transmembrane domain of CD8α, and wherein the intracellular signal transduction domain comprises a co-stimulatory domain of CD137 and a stimulatory domain of CD3ζ.

8. The protein according to any one of claims 5-7, wherein the CAR comprises the amino acid sequence SEQ ID No:

44.

9. The protein according to any one of claims 1-4, wherein the protein comprises the antigen-binding domain and the detection portion.

10. The protein according to any one of claims 9, wherein the detection portion is selected from the group consisting of fluorescent dyes, fluorescent proteins, or biotin.

11. A method for detecting cells expressing FolR1 in a biological sample, comprising the following steps: a. Contact a sample containing cells expressing FolR1 with the protein according to claim 9 or 10. b. Detect the protein that binds to the cells expressing FolR1.

Images