Methods for diagnosing and treating ovarian cancer

Abstract

Provided are methods for the prognosis and / or diagnosis of cancer, particularly ovarian cancer. Also provided are methods for the treatment of ovarian cancer, as well as chimeric antigen receptors (CARs) and CAR-expressing cells for the treatment of cancer, particularly ovarian cancer.

Description

[Technical Field]

[0001]

[0002] The present invention relates to methods for diagnosing, treating, and determining the prognosis of ovarian cancer. In particular, the present invention relates to the identification of glypican-1 expression in ovarian cancer, such as high-grade serous ovarian cancer, and the use of immunotherapy in the treatment of glypican-1 positive cancers. [Background technology]

[0002]

[0003] The following discussion of the background of the invention is intended to facilitate an understanding of the present invention. However, it should be understood that this discussion is not an admission or acknowledgement that any of the material referred to was previously published, publicly known, or part of the common general knowledge as of the priority date of any one of the claims herein.

[0003]

[0004] Ovarian cancer is the most lethal gynecological malignancy. High-grade serous ovarian cancer (HGSOC) accounts for nearly 70% of ovarian cancers, with 90% of patients presenting with advanced-stage disease. Current treatment for HGSOC consists of debulking surgery followed by platinum-taxane combination-based chemotherapy. Although initial response to first-line treatment is high, more than 75% of patients ultimately relapse and develop chemotherapy resistance, which is the leading cause of death from ovarian cancer and a major limitation to its successful treatment.

[0004]

[0005] Currently, there are no treatment options for ovarian cancer (especially recurrent ovarian cancer). Furthermore, there is a lack of biomarkers that allow for the diagnosis or prognosis of ovarian cancer. Therefore, there is a need to provide alternatives to current therapies (e.g., surgery and chemotherapy) for the treatment of ovarian cancer. There is also a need for improved diagnosis or prognosis of ovarian cancer. Summary of the Invention

[0005]

[0006] The present invention is based on the surprising discovery that the heparan sulfate proteoglycan glypican-1 (GPC1) is associated with ovarian cancer, including high-grade serous ovarian cancer.

[0006]

[0007] Thus, in one aspect, a chimeric antigen receptor (CAR) is provided that includes an antigen recognition domain, a transmembrane domain, and a signaling domain, where the antigen recognition domain recognizes glypican-1 (GPC1). When the CAR is expressed by an appropriate cell, it can be used to target cancer cells (particularly ovarian cancer cells) that express glypican-1.

[0007]

[0008] The antigen recognition domain can be any suitable binding molecule that recognizes glypican-1, but in preferred embodiments, the antigen recognition domain comprises a binding portion of an antibody that recognizes glypican-1. In some embodiments, the antibody portion is selected from the group consisting of an antigen-binding fragment (Fab), an antibody variable heavy chain, or an antibody variable light chain.

[0008]

[0009] The antigen recognition domain can also be a fusion protein (eg, a single chain variable fragment (scFv)) that has sequence identity to the variable heavy and variable light chains of an antibody that binds to glypican-1.

[0009]

[0010] Preferably, the CAR comprises a linker between the antigen recognition domain and the transmembrane domain, hi some embodiments, the linker comprises an IgG4 hinge region, and / or an IgG4 CH3 region, and / or an IgG4 CH2 region (which may include mutations L235D or N297Q).

[0010]

[0011] The present disclosure also provides cells comprising the CAR of the present invention. The chimeric antigen receptor constructs can be transduced into a variety of cell types, with particularly contemplated embodiments being immune cells such as lymphocytes, CD3+ lymphocytes, CD8+ lymphocytes (e.g., CD8+ T cells), CD4+ lymphocytes (e.g., CD4+ T cells), natural killer (NK) cells, or NKT cells.

[0011]

[0012] Also provided is the use of a CAR or a cell expressing a CAR for treating or preventing ovarian cancer in a subject, wherein the ovarian cancer has elevated expression of glypican-1.

[0012]

[0013] Also provided are methods for diagnosing a subject with ovarian cancer or assessing the prognosis of the subject, comprising determining the amount of glypican-1 in ovarian cells or suspected cancer cells from the subject, wherein an elevated amount of glypican-1 indicates the presence of ovarian cancer and / or indicates a poor prognosis for the subject. In some embodiments, a poor prognosis indicates lower overall survival or shorter progression-free survival.

[0013]

[0014] In some embodiments, increased glypican-1 gene expression is indicative of lower overall survival, hi some embodiments, increased glypican-1 gene expression and / or increased glypican-1 protein expression is indicative of shorter progression-free survival.

[0014]

[0015] In some embodiments, the ovarian cancer is recurrent ovarian cancer.

[0015]

[0016] In some embodiments, the ovarian cancer is high-grade serous ovarian cancer.

[0016]

[0017] In some embodiments, the diagnostic or prognostic evaluation method is performed on a subject who has already been treated for ovarian cancer with one or more of chemotherapy, surgical resection or debulking, or radiation therapy.

[0017]

[0018] In some embodiments, the ovarian cancer is recurrent ovarian cancer and the elevated amount of glypican-1 is compared to cancer tissue before the recurrence.

[0018]

[0019] In some embodiments, the elevated amount of glypican-1 is compared to non-cancerous ovarian tissue.

[0019]

[0020] In some embodiments, determining the amount of glypican-1 comprises quantifying glypican-1 protein expression and / or mRNA expression. Glypican-1 protein expression may be surface expression of glypican-1 protein, and / or intracellular expression of glypican-1 protein, and / or secreted amount of glypican-1.

[0020]

[0021] In some embodiments, protein expression is determined by an agent that preferentially or selectively binds to glypican 1. Such an agent may be an antibody or a binding fragment of an antibody.

[0021]

[0022] In some embodiments, the agent that binds to glypican 1 is a fusion protein, such as a single chain variable fragment comprising the sequences of the variable light and variable heavy chains of an antibody.

[0022]

[0023] In some embodiments, the antibody is MIL-38.

[0023]

[0024] The present invention also provides methods for treating a subject with ovarian cancer or preventing ovarian cancer in a subject, the methods comprising killing cells that express glypican-1. In some embodiments of the methods, the cells that express glypican-1 are determined to express elevated amounts of glypican-1 protein.

[0024]

[0025] In some embodiments, increasing the amount of glypican-1 protein expression comprises increasing surface expression of glypican-1.

[0025]

[0026] In some embodiments of this method of treatment, the ovarian cancer is recurrent ovarian cancer.

[0026]

[0027] In some embodiments of this method of treatment, the ovarian cancer is high-grade serous ovarian cancer (HSOC).

[0027]

[0028] In some embodiments of this method of treatment, the subject has already been treated for ovarian cancer with one or more of chemotherapy, surgical resection or debulking, or radiation therapy.

[0028]

[0029] In some embodiments of this method of treatment, the ovarian cancer is recurrent ovarian cancer and the elevated amount of glypican-1 is compared to cancerous tissue and / or non-cancerous ovarian tissue before the recurrence.

[0029]

[0030] In some embodiments of this method of treatment, the cells are killed by administering or introducing into the subject an agent that preferentially or selectively binds to glypican-1 expressed by the cells. Such an agent may, in some embodiments, be an antibody or a binding fragment of an antibody. In some embodiments, the agent that binds to glypican-1 may be a fusion protein, such as a single-chain variable fragment comprising the variable light and variable heavy chain sequences of an antibody. In some embodiments, the antibody is a humanized antibody. One particularly contemplated antibody is an antibody that is MIL-38.

[0030]

[0031] In some embodiments of this method of treatment, the agent that binds to glypican-1 is a chimeric antigen receptor (CAR)-expressing cell. In some embodiments, the CAR is a CAR disclosed herein.

[0031]

[0032] In some embodiments of treatment, a diagnostic or prognostic method (disclosed herein) is performed before killing the cells expressing glypican-1.

[0032]

[0033] Also provided is the use of a CAR or agent against glypican-1 in the manufacture of a medicament for preventing or treating ovarian cancer in a subject, preferably the ovarian cancer has high expression of glypican-1. Also provided is a method for preventing or treating ovarian cancer in a subject, preferably the ovarian cancer has high expression of glypican-1.

[0033]

[0034] For a fuller understanding of the aspects and advantages of the present invention, reference should be made to the following detailed description taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0034] [Figure 1A] GPC1 expression is increased in HGSOC tissues compared with non-cancerous tissues. (A) GPC1 mRNA expression data obtained from the GENT2 database, including ovarian surface epithelium (OSE) (n = 66), fallopian tube (FT) (n = 40), and high-grade serous ovarian cancer HGSOC (n = 807). Higher GPC1 expression was observed in HGSOC compared with FT (****P < 0.0001, Kruskal-Wallis with Dunn's multiple comparison test). (B) GPC1 H-index scores of OSE, benign serous cystadenoma, and HGSOC tissues assessed by GPC1 immunohistochemistry (IHC) staining and measured with Qupath. Data are presented as mean ± SEM. (*P < 0.05, one-way ANOVA with Tukey's multiple comparison test). Representative images of GPC1 protein expression by IHC in (C) OSE, (D) FT, (E) benign serous cystadenoma, and (F) HGSOC (scale bar = 50 μm). All images are at the same magnification. [Figure 1B]GPC1 expression is increased in HGSOC tissues compared with non-cancerous tissues. (A) GPC1 mRNA expression data obtained from the GENT2 database, including ovarian surface epithelium (OSE) (n = 66), fallopian tube (FT) (n = 40), and high-grade serous ovarian cancer HGSOC (n = 807). Higher GPC1 expression was observed in HGSOC compared with FT (****P < 0.0001, Kruskal-Wallis with Dunn's multiple comparison test). (B) GPC1 H-index scores of OSE, benign serous cystadenoma, and HGSOC tissues assessed by GPC1 immunohistochemistry (IHC) staining and measured with Qupath. Data are presented as mean ± SEM. (*P < 0.05, one-way ANOVA with Tukey's multiple comparison test). Representative images of GPC1 protein expression by IHC in (C) OSE, (D) FT, (E) benign serous cystadenoma, and (F) HGSOC (scale bar = 50 μm). All images are at the same magnification. [Figure 1C] GPC1 expression is increased in HGSOC tissues compared with non-cancerous tissues. (A) GPC1 mRNA expression data obtained from the GENT2 database, including ovarian surface epithelium (OSE) (n = 66), fallopian tube (FT) (n = 40), and high-grade serous ovarian cancer HGSOC (n = 807). Higher GPC1 expression was observed in HGSOC compared with FT (****P < 0.0001, Kruskal-Wallis with Dunn's multiple comparison test). (B) GPC1 H-index scores of OSE, benign serous cystadenoma, and HGSOC tissues assessed by GPC1 immunohistochemistry (IHC) staining and measured with Qupath. Data are presented as mean ± SEM. (*P < 0.05, one-way ANOVA with Tukey's multiple comparison test). Representative images of GPC1 protein expression by IHC in (C) OSE, (D) FT, (E) benign serous cystadenoma, and (F) HGSOC (scale bar = 50 μm). All images are at the same magnification. [Figure 1D]GPC1 expression is increased in HGSOC tissues compared with non-cancerous tissues. (A) GPC1 mRNA expression data obtained from the GENT2 database, including ovarian surface epithelium (OSE) (n = 66), fallopian tube (FT) (n = 40), and high-grade serous ovarian cancer HGSOC (n = 807). Higher GPC1 expression was observed in HGSOC compared with FT (****P < 0.0001, Kruskal-Wallis with Dunn's multiple comparison test). (B) GPC1 H-index scores of OSE, benign serous cystadenoma, and HGSOC tissues assessed by GPC1 immunohistochemistry (IHC) staining and measured with Qupath. Data are presented as mean ± SEM. (*P < 0.05, one-way ANOVA with Tukey's multiple comparison test). Representative images of GPC1 protein expression by IHC in (C) OSE, (D) FT, (E) benign serous cystadenoma, and (F) HGSOC (scale bar = 50 μm). All images are at the same magnification. [Figure 1E] GPC1 expression is increased in HGSOC tissues compared with non-cancerous tissues. (A) GPC1 mRNA expression data obtained from the GENT2 database, including ovarian surface epithelium (OSE) (n = 66), fallopian tube (FT) (n = 40), and high-grade serous ovarian cancer HGSOC (n = 807). Higher GPC1 expression was observed in HGSOC compared with FT (****P < 0.0001, Kruskal-Wallis with Dunn's multiple comparison test). (B) GPC1 H-index scores of OSE, benign serous cystadenoma, and HGSOC tissues assessed by GPC1 immunohistochemistry (IHC) staining and measured with Qupath. Data are presented as mean ± SEM. (*P < 0.05, one-way ANOVA with Tukey's multiple comparison test). Representative images of GPC1 protein expression by IHC in (C) OSE, (D) FT, (E) benign serous cystadenoma, and (F) HGSOC (scale bar = 50 μm). All images are at the same magnification. [Figure 1F]GPC1 expression is increased in HGSOC tissues compared with non-cancerous tissues. (A) GPC1 mRNA expression data obtained from the GENT2 database, including ovarian surface epithelium (OSE) (n = 66), fallopian tube (FT) (n = 40), and high-grade serous ovarian cancer HGSOC (n = 807). Higher GPC1 expression was observed in HGSOC compared with FT (****P < 0.0001, Kruskal-Wallis with Dunn's multiple comparison test). (B) GPC1 H-index scores of OSE, benign serous cystadenoma, and HGSOC tissues assessed by GPC1 immunohistochemistry (IHC) staining and measured with Qupath. Data are presented as mean ± SEM. (*P < 0.05, one-way ANOVA with Tukey's multiple comparison test). Representative images of GPC1 protein expression by IHC in (C) OSE, (D) FT, (E) benign serous cystadenoma, and (F) HGSOC (scale bar = 50 μm). All images are at the same magnification. [Figure 2A] High GPC1 expression is associated with poor prognosis. Kaplan Meier survival analysis of the relationship between GPC1 mRNA expression and progression-free survival (PFS) and overall survival (OS) in patients with HGSOC. (A) PFS HR=1.3, 95% CI, 1.1-1.53, p=0.0015, n=1029) and (B) OS (HR=1.3, (95% CI, 1.15-1.58, p=0.00026, n=1144). Data were evaluated using the online Kaplan Meier tool. (C) Representative images by IHC of HGSOC with low GPC1 protein expression and (D) HGSOC with high GPC1 expression (scale bar = 50 μm). All images are at the same magnification. Kaplan Meier survival analysis demonstrated a significant correlation between GPC1 protein expression and HGSOC. The relationship between PFS and OS is shown. Using the maximum H-index score as a cutoff point, samples were divided into those with high (H-index > 70) or low (H-index ≤ 70) GPC1 protein expression. (E) PFS, log-rank test, P = 0.031 (n = 96). (F) OS, log-rank test, p = 0.536 (n = 100). [Figure 2B]High GPC1 expression is associated with poor prognosis. Kaplan Meier survival analysis of the relationship between GPC1 mRNA expression and progression-free survival (PFS) and overall survival (OS) in patients with HGSOC. (A) PFS HR=1.3, 95% CI, 1.1-1.53, p=0.0015, n=1029) and (B) OS (HR=1.3, (95% CI, 1.15-1.58, p=0.00026, n=1144). Data were evaluated using the online Kaplan Meier tool. (C) Representative images by IHC of HGSOC with low GPC1 protein expression and (D) HGSOC with high GPC1 expression (scale bar = 50 μm). All images are at the same magnification. Kaplan Meier survival analysis demonstrated a significant correlation between GPC1 protein expression and HGSOC. The relationship between PFS and OS is shown. Using the maximum H-index score as a cutoff point, samples were divided into those with high (H-index > 70) or low (H-index ≤ 70) GPC1 protein expression. (E) PFS, log-rank test, P = 0.031 (n = 96). (F) OS, log-rank test, p = 0.536 (n = 100). [Figure 2C]High GPC1 expression is associated with poor prognosis. Kaplan Meier survival analysis of the relationship between GPC1 mRNA expression and progression-free survival (PFS) and overall survival (OS) in patients with HGSOC. (A) PFS HR=1.3, 95% CI, 1.1-1.53, p=0.0015, n=1029) and (B) OS (HR=1.3, (95% CI, 1.15-1.58, p=0.00026, n=1144). Data were evaluated using the online Kaplan Meier tool. (C) Representative images by IHC of HGSOC with low GPC1 protein expression and (D) HGSOC with high GPC1 expression (scale bar = 50 μm). All images are at the same magnification. Kaplan Meier survival analysis demonstrated a significant correlation between GPC1 protein expression and HGSOC. The relationship between PFS and OS is shown. Using the maximum H-index score as a cutoff point, samples were divided into those with high (H-index > 70) or low (H-index ≤ 70) GPC1 protein expression. (E) PFS, log-rank test, P = 0.031 (n = 96). (F) OS, log-rank test, p = 0.536 (n = 100). [Figure 2D]High GPC1 expression is associated with poor prognosis. Kaplan Meier survival analysis of the relationship between GPC1 mRNA expression and progression-free survival (PFS) and overall survival (OS) in patients with HGSOC. (A) PFS HR=1.3, 95% CI, 1.1-1.53, p=0.0015, n=1029) and (B) OS (HR=1.3, (95% CI, 1.15-1.58, p=0.00026, n=1144). Data were evaluated using the online Kaplan Meier tool. (C) Representative images by IHC of HGSOC with low GPC1 protein expression and (D) HGSOC with high GPC1 expression (scale bar = 50 μm). All images are at the same magnification. Kaplan Meier survival analysis demonstrated a significant correlation between GPC1 protein expression and HGSOC. The relationship between PFS and OS is shown. Using the maximum H-index score as a cutoff point, samples were divided into those with high (H-index > 70) or low (H-index ≤ 70) GPC1 protein expression. (E) PFS, log-rank test, P = 0.031 (n = 96). (F) OS, log-rank test, p = 0.536 (n = 100). [Figure 2E]High GPC1 expression is associated with poor prognosis. Kaplan Meier survival analysis of the relationship between GPC1 mRNA expression and progression-free survival (PFS) and overall survival (OS) in patients with HGSOC. (A) PFS HR=1.3, 95% CI, 1.1-1.53, p=0.0015, n=1029) and (B) OS (HR=1.3, (95% CI, 1.15-1.58, p=0.00026, n=1144). Data were evaluated using the online Kaplan Meier tool. (C) Representative images by IHC of HGSOC with low GPC1 protein expression and (D) HGSOC with high GPC1 expression (scale bar = 50 μm). All images are at the same magnification. Kaplan Meier survival analysis demonstrated a significant correlation between GPC1 protein expression and HGSOC. The relationship between PFS and OS is shown. Using the maximum H-index score as a cutoff point, samples were divided into those with high (H-index > 70) or low (H-index ≤ 70) GPC1 protein expression. (E) PFS, log-rank test, P = 0.031 (n = 96). (F) OS, log-rank test, p = 0.536 (n = 100). [Figure 2F]High GPC1 expression is associated with poor prognosis. Kaplan Meier survival analysis of the relationship between GPC1 mRNA expression and progression-free survival (PFS) and overall survival (OS) in patients with HGSOC. (A) PFS HR=1.3, 95% CI, 1.1-1.53, p=0.0015, n=1029) and (B) OS (HR=1.3, (95% CI, 1.15-1.58, p=0.00026, n=1144). Data were evaluated using the online Kaplan Meier tool. (C) Representative images by IHC of HGSOC with low GPC1 protein expression and (D) HGSOC with high GPC1 expression (scale bar = 50 μm). All images are at the same magnification. Kaplan Meier survival analysis demonstrated a significant correlation between GPC1 protein expression and HGSOC. The relationship between PFS and OS is shown. Using the maximum H-index score as a cutoff point, samples were divided into those with high (H-index > 70) or low (H-index ≤ 70) GPC1 protein expression. (E) PFS, log-rank test, P = 0.031 (n = 96). (F) OS, log-rank test, p = 0.536 (n = 100). [Figure 3A] GPC1 is highly expressed in recurrent tissues compared with tissues at diagnosis from matched HGSOC patients. Representative images of GPC1 protein expression in HGSOC tissues at (A-B) diagnosis and (C-D) recurrence by IHC (scale bar 50 μm). All images are at the same magnification. (E) GPC1 staining in HGSOC tissues at diagnosis (n=4) and recurrence (n=4) was measured using QuPath (*P<0.05, paired t-test). [Figure 3B] GPC1 is highly expressed in recurrent tissues compared with tissues at diagnosis from matched HGSOC patients. Representative images of GPC1 protein expression in HGSOC tissues at (A-B) diagnosis and (C-D) recurrence by IHC (scale bar 50 μm). All images are at the same magnification. (E) GPC1 staining in HGSOC tissues at diagnosis (n=4) and recurrence (n=4) was measured using QuPath (*P<0.05, paired t-test). [Figure 3C]GPC1 is highly expressed in recurrent tissues compared with tissues at diagnosis from matched HGSOC patients. Representative images of GPC1 protein expression in HGSOC tissues at (A-B) diagnosis and (C-D) recurrence by IHC (scale bar 50 μm). All images are at the same magnification. (E) GPC1 staining in HGSOC tissues at diagnosis (n=4) and recurrence (n=4) was measured using QuPath (*P<0.05, paired t-test). [Figure 3D] GPC1 is highly expressed in recurrent tissues compared with tissues at diagnosis from matched HGSOC patients. Representative images of GPC1 protein expression in HGSOC tissues at (A-B) diagnosis and (C-D) recurrence by IHC (scale bar 50 μm). All images are at the same magnification. (E) GPC1 staining in HGSOC tissues at diagnosis (n=4) and recurrence (n=4) was measured using QuPath (*P<0.05, paired t-test). [Figure 3E] GPC1 is highly expressed in recurrent tissues compared with tissues at diagnosis from matched HGSOC patients. Representative images of GPC1 protein expression in HGSOC tissues at (A-B) diagnosis and (C-D) recurrence by IHC (scale bar 50 μm). All images are at the same magnification. (E) GPC1 staining in HGSOC tissues at diagnosis (n=4) and recurrence (n=4) was measured using QuPath (*P<0.05, paired t-test). [Figure 4A]GPC1 expression in ovarian cancer cells. Quantification of GPC1 mRNA expression in (A) ovarian cancer cell lines and (B) primary HGSOC cells from independent experiments (n = 4) performed in triplicate by qRT-PCR. Data were analyzed using the 2-Δct method and normalized to the housekeeper gene β-actin. GPC1 protein abundance was assessed by Western blotting of GPC1 (65 kDa) and β-actin (48 kDa). 20 μg of protein from each (C) ovarian cancer cell line and (D) primary ovarian cancer cells was loaded and electrophoresed on a 4-20% TGX gel. Quantification of Western blots of (E) ovarian cancer cell lines (n = 9) and (F) primary cells (n = 7) from two to three independent gels. (Data are presented as mean ± SD, normalized to β-actin.) [Figure 4B] GPC1 expression in ovarian cancer cells. Quantification of GPC1 mRNA expression in (A) ovarian cancer cell lines and (B) primary HGSOC cells from independent experiments (n = 4) performed in triplicate by qRT-PCR. Data were analyzed using the 2-Δct method and normalized to the housekeeper gene β-actin. GPC1 protein abundance was assessed by Western blotting of GPC1 (65 kDa) and β-actin (48 kDa). 20 μg of protein from each (C) ovarian cancer cell line and (D) primary ovarian cancer cells was loaded and electrophoresed on a 4-20% TGX gel. Quantification of Western blots of (E) ovarian cancer cell lines (n = 9) and (F) primary cells (n = 7) from two to three independent gels. (Data are presented as mean ± SD, normalized to β-actin.) [Figure 4C]GPC1 expression in ovarian cancer cells. Quantification of GPC1 mRNA expression in (A) ovarian cancer cell lines and (B) primary HGSOC cells from independent experiments (n = 4) performed in triplicate by qRT-PCR. Data were analyzed using the 2-Δct method and normalized to the housekeeper gene β-actin. GPC1 protein abundance was assessed by Western blotting of GPC1 (65 kDa) and β-actin (48 kDa). 20 μg of protein from each (C) ovarian cancer cell line and (D) primary ovarian cancer cells was loaded and electrophoresed on a 4-20% TGX gel. Quantification of Western blots of (E) ovarian cancer cell lines (n = 9) and (F) primary cells (n = 7) from two to three independent gels. (Data are presented as mean ± SD, normalized to β-actin.) [Figure 4D] GPC1 expression in ovarian cancer cells. Quantification of GPC1 mRNA expression in (A) ovarian cancer cell lines and (B) primary HGSOC cells from independent experiments (n = 4) performed in triplicate by qRT-PCR. Data were analyzed using the 2-Δct method and normalized to the housekeeper gene β-actin. GPC1 protein abundance was assessed by Western blotting of GPC1 (65 kDa) and β-actin (48 kDa). 20 μg of protein from each (C) ovarian cancer cell line and (D) primary ovarian cancer cells was loaded and electrophoresed on a 4-20% TGX gel. Quantification of Western blots of (E) ovarian cancer cell lines (n = 9) and (F) primary cells (n = 7) from two to three independent gels. (Data are presented as mean ± SD, normalized to β-actin.) [Figure 4E]GPC1 expression in ovarian cancer cells. Quantification of GPC1 mRNA expression in (A) ovarian cancer cell lines and (B) primary HGSOC cells from independent experiments (n = 4) performed in triplicate by qRT-PCR. Data were analyzed using the 2-Δct method and normalized to the housekeeper gene β-actin. GPC1 protein abundance was assessed by Western blotting of GPC1 (65 kDa) and β-actin (48 kDa). 20 μg of protein from each (C) ovarian cancer cell line and (D) primary ovarian cancer cells was loaded and electrophoresed on a 4-20% TGX gel. Quantification of Western blots of (E) ovarian cancer cell lines (n = 9) and (F) primary cells (n = 7) from two to three independent gels. (Data are presented as mean ± SD, normalized to β-actin.) [Figure 4F] GPC1 expression in ovarian cancer cells. Quantification of GPC1 mRNA expression in (A) ovarian cancer cell lines and (B) primary HGSOC cells from independent experiments (n = 4) performed in triplicate by qRT-PCR. Data were analyzed using the 2-Δct method and normalized to the housekeeper gene β-actin. GPC1 protein abundance was assessed by Western blotting of GPC1 (65 kDa) and β-actin (48 kDa). 20 μg of protein from each (C) ovarian cancer cell line and (D) primary ovarian cancer cells was loaded and electrophoresed on a 4-20% TGX gel. Quantification of Western blots of (E) ovarian cancer cell lines (n = 9) and (F) primary cells (n = 7) from two to three independent gels. (Data are presented as mean ± SD, normalized to β-actin.) [Figure 5A]Effect of GPC1 CAR-T cell treatment on ovarian cancer cell viability in a 2D monolayer assay. Ovarian cancer cells were plated at 10,000 cells / well in a 96-well plate and treated with medium alone, untransduced (UT) CD3 T cells (blue), or GPC1 CAR-T cells (pink) at 2:1, 5:1, and 10:1 E:T ratios for 48 hours. Cell viability was calculated using an MTT assay. (A) OVCAR3, (B) COV362, (C) OV90, (D) SKOV-3, (E) Patient 1, and (F) Patient 3. Data are presented as mean ± SD (*P<0.05, unpaired t-test from three independent experiments). GPC1 CAR-T cells demonstrated potent killing effects at all concentrations in SKOV3 and COV362 cell lines compared with UT T-CD3 T cells. OV90 and OVCAR3 cells treated with GPC1 CAR T cells at 5:1 and 2:1 resulted in significantly reduced cell viability compared to UT CD3 T cells. Cells from both patients 1 and 3 showed significantly reduced cell viability at 10:1, but not at 5:1, and only patient 3 showed reduced cell viability at 2:1. [Figure 5B]Effect of GPC1 CAR-T cell treatment on ovarian cancer cell viability in a 2D monolayer assay. Ovarian cancer cells were plated at 10,000 cells / well in a 96-well plate and treated with medium alone, untransduced (UT) CD3 T cells (blue), or GPC1 CAR-T cells (pink) at 2:1, 5:1, and 10:1 E:T ratios for 48 hours. Cell viability was calculated using an MTT assay. (A) OVCAR3, (B) COV362, (C) OV90, (D) SKOV-3, (E) Patient 1, and (F) Patient 3. Data are presented as mean ± SD (*P<0.05, unpaired t-test from three independent experiments). GPC1 CAR-T cells demonstrated potent killing effects at all concentrations in SKOV3 and COV362 cell lines compared with UT T-CD3 T cells. OV90 and OVCAR3 cells treated with GPC1 CAR T cells at 5:1 and 2:1 resulted in significantly reduced cell viability compared to UT CD3 T cells. Cells from both patients 1 and 3 showed significantly reduced cell viability at 10:1, but not at 5:1, and only patient 3 showed reduced cell viability at 2:1. [Figure 5C]Effect of GPC1 CAR-T cell treatment on ovarian cancer cell viability in a 2D monolayer assay. Ovarian cancer cells were plated at 10,000 cells / well in a 96-well plate and treated with medium alone, untransduced (UT) CD3 T cells (blue), or GPC1 CAR-T cells (pink) at 2:1, 5:1, and 10:1 E:T ratios for 48 hours. Cell viability was calculated using an MTT assay. (A) OVCAR3, (B) COV362, (C) OV90, (D) SKOV-3, (E) Patient 1, and (F) Patient 3. Data are presented as mean ± SD (*P<0.05, unpaired t-test from three independent experiments). GPC1 CAR-T cells demonstrated potent killing effects at all concentrations in SKOV3 and COV362 cell lines compared with UT T-CD3 T cells. OV90 and OVCAR3 cells treated with GPC1 CAR T cells at 5:1 and 2:1 resulted in significantly reduced cell viability compared to UT CD3 T cells. Cells from both patients 1 and 3 showed significantly reduced cell viability at 10:1, but not at 5:1, and only patient 3 showed reduced cell viability at 2:1. [Figure 5D]Effect of GPC1 CAR-T cell treatment on ovarian cancer cell viability in a 2D monolayer assay. Ovarian cancer cells were plated at 10,000 cells / well in a 96-well plate and treated with medium alone, untransduced (UT) CD3 T cells (blue), or GPC1 CAR-T cells (pink) at 2:1, 5:1, and 10:1 E:T ratios for 48 hours. Cell viability was calculated using an MTT assay. (A) OVCAR3, (B) COV362, (C) OV90, (D) SKOV-3, (E) Patient 1, and (F) Patient 3. Data are presented as mean ± SD (*P<0.05, unpaired t-test from three independent experiments). GPC1 CAR-T cells demonstrated potent killing effects at all concentrations in SKOV3 and COV362 cell lines compared with UT T-CD3 T cells. OV90 and OVCAR3 cells treated with GPC1 CAR T cells at 5:1 and 2:1 resulted in significantly reduced cell viability compared to UT CD3 T cells. Cells from both patients 1 and 3 showed significantly reduced cell viability at 10:1, but not at 5:1, and only patient 3 showed reduced cell viability at 2:1. [Figure 5E]Effect of GPC1 CAR-T cell treatment on ovarian cancer cell viability in a 2D monolayer assay. Ovarian cancer cells were plated at 10,000 cells / well in a 96-well plate and treated with medium alone, untransduced (UT) CD3 T cells (blue), or GPC1 CAR-T cells (pink) at 2:1, 5:1, and 10:1 E:T ratios for 48 hours. Cell viability was calculated using an MTT assay. (A) OVCAR3, (B) COV362, (C) OV90, (D) SKOV-3, (E) Patient 1, and (F) Patient 3. Data are presented as mean ± SD (*P<0.05, unpaired t-test from three independent experiments). GPC1 CAR-T cells demonstrated potent killing effects at all concentrations in SKOV3 and COV362 cell lines compared with UT T-CD3 T cells. OV90 and OVCAR3 cells treated with GPC1 CAR T cells at 5:1 and 2:1 resulted in significantly reduced cell viability compared to UT CD3 T cells. Cells from both patients 1 and 3 showed significantly reduced cell viability at 10:1, but not at 5:1, and only patient 3 showed reduced cell viability at 2:1. [Figure 5F]Effect of GPC1 CAR-T cell treatment on ovarian cancer cell viability in a 2D monolayer assay. Ovarian cancer cells were plated at 10,000 cells / well in a 96-well plate and treated with medium alone, untransduced (UT) CD3 T cells (blue), or GPC1 CAR-T cells (pink) at 2:1, 5:1, and 10:1 E:T ratios for 48 hours. Cell viability was calculated using an MTT assay. (A) OVCAR3, (B) COV362, (C) OV90, (D) SKOV-3, (E) Patient 1, and (F) Patient 3. Data are presented as mean ± SD (*P<0.05, unpaired t-test from three independent experiments). GPC1 CAR-T cells demonstrated potent killing effects at all concentrations in SKOV3 and COV362 cell lines compared with UT T-CD3 T cells. OV90 and OVCAR3 cells treated with GPC1 CAR T cells at 5:1 and 2:1 resulted in significantly reduced cell viability compared to UT CD3 T cells. Cells from both patients 1 and 3 showed significantly reduced cell viability at 10:1, but not at 5:1, and only patient 3 showed reduced cell viability at 2:1. [Figure 6A] Effect of GPC1 CAR-T cells on ovarian cancer 3D spheroid cultures. Representative images of (A) COV362, (B) SKOV3, and (C) OVCAR-3 ovarian cancer spheroids 48 hours after treatment with medium alone, untransduced (UT) CD3 T cells (5:1), or GPC1 CAR-T cells (5:1). Spheroid images were collected in duplicate from three separate experiments and measured from five randomly selected areas within each well using ImageJ software. Data were expressed as a percentage of the control spheroid area. A significant decrease in spheroid size was observed between the UT CD3 T cell and GPC1 CAR-T cell groups for all cell lines. Statistical significance was assessed by one-way ANOVA with Tukey's multiple comparison test. *P<0.05, **P<0.01, ***P<0.001. Scale bars represent 1000 μm. All images are at the same magnification. [Figure 6B]Effect of GPC1 CAR-T cells on ovarian cancer 3D spheroid cultures. Representative images of (A) COV362, (B) SKOV3, and (C) OVCAR-3 ovarian cancer spheroids 48 hours after treatment with medium alone, untransduced (UT) CD3 T cells (5:1), or GPC1 CAR-T cells (5:1). Spheroid images were collected in duplicate from three separate experiments and measured from five randomly selected areas within each well using ImageJ software. Data were expressed as a percentage of the control spheroid area. A significant decrease in spheroid size was observed between the UT CD3 T cell and GPC1 CAR-T cell groups for all cell lines. Statistical significance was assessed by one-way ANOVA with Tukey's multiple comparison test. *P<0.05, **P<0.01, ***P<0.001. Scale bars represent 1000 μm. All images are at the same magnification. [Figure 6C] Effect of GPC1 CAR-T cells on ovarian cancer 3D spheroid cultures. Representative images of (A) COV362, (B) SKOV3, and (C) OVCAR-3 ovarian cancer spheroids 48 hours after treatment with medium alone, untransduced (UT) CD3 T cells (5:1), or GPC1 CAR-T cells (5:1). Spheroid images were collected in duplicate from three separate experiments and measured from five randomly selected areas within each well using ImageJ software. Data were expressed as a percentage of the control spheroid area. A significant decrease in spheroid size was observed between the UT CD3 T cell and GPC1 CAR-T cell groups for all cell lines. Statistical significance was assessed by one-way ANOVA with Tukey's multiple comparison test. *P<0.05, **P<0.01, ***P<0.001. Scale bars represent 1000 μm. All images are at the same magnification. [Figure 7A]Effect of GPC1 CAR-T cells on primary ovarian cancer cell 3D spheroid cultures. Representative images of spheroids from (A) Patient 1 and (B) Patient 3, cultured 48 hours after treatment with medium alone, untransduced (UT) CD3 T cells (5:1), or GPC1 CAR-T cells (5:1). Spheroid images were collected in duplicate from three separate experiments and measured using ImageJ from five randomly selected areas within each well. Data are expressed as a percentage of the spheroid area. A significant decrease in spheroid size was observed between the control and GPC1 CAR-T cell-treated groups for both primary cell types. Statistical significance was assessed by one-way ANOVA with Tukey's multiple comparison test. *P<0.05, **P<0.01. Scale bar represents 1000 μm, and all images are at the same magnification. [Figure 7B] Effect of GPC1 CAR-T cells on primary ovarian cancer cell 3D spheroid cultures. Representative images of spheroids from (A) Patient 1 and (B) Patient 3, cultured 48 hours after treatment with medium alone, untransduced (UT) CD3 T cells (5:1), or GPC1 CAR-T cells (5:1). Spheroid images were collected in duplicate from three separate experiments and measured using ImageJ from five randomly selected areas within each well. Data are expressed as a percentage of the spheroid area. A significant decrease in spheroid size was observed between the control and GPC1 CAR-T cell-treated groups for both primary cell types. Statistical significance was assessed by one-way ANOVA with Tukey's multiple comparison test. *P<0.05, **P<0.01. Scale bar represents 1000 μm, and all images are at the same magnification. [Figure 8A]Effect of GPC1 CAR-T cells in patient-derived explant assays. Treatment with GPC1 CAR-T cells induces apoptosis in patient-derived explants with high GPC1 expression. A–F: Quantification of cleaved caspase 3. Data are expressed as % of positively stained cells / mm², and bar graphs show mean ± SD. Cleaved caspase 3 expression was significantly increased in the CAR-T-treated group compared to the untransduced group (unpaired t-test) for patients 1–4 (A–D), but not for patients 5 and 6 (E–F). G. Representative images of GPC1 expression by immunohistochemistry in explant tissue that did not respond to treatment with GPC1 CAR-T cells and responded to treatment with GPC1 CAR-T cells. Scale bar = 50 μm. All images are at the same magnification. GPC1 expression, measured by H-score using QuPath, was significantly increased in responders compared to non-responders. [Figure 8B] Effect of GPC1 CAR-T cells in patient-derived explant assays. Treatment with GPC1 CAR-T cells induces apoptosis in patient-derived explants with high GPC1 expression. A–F: Quantification of cleaved caspase 3. Data are expressed as % of positively stained cells / mm², and bar graphs show mean ± SD. Cleaved caspase 3 expression was significantly increased in the CAR-T-treated group compared to the untransduced group (unpaired t-test) for patients 1–4 (A–D), but not for patients 5 and 6 (E–F). G. Representative images of GPC1 expression by immunohistochemistry in explant tissue that did not respond to treatment with GPC1 CAR-T cells and responded to treatment with GPC1 CAR-T cells. Scale bar = 50 μm. All images are at the same magnification. GPC1 expression, measured by H-score using QuPath, was significantly increased in responders compared to non-responders. [Figure 8C]Effect of GPC1 CAR-T cells in patient-derived explant assays. Treatment with GPC1 CAR-T cells induces apoptosis in patient-derived explants with high GPC1 expression. A–F: Quantification of cleaved caspase 3. Data are expressed as % of positively stained cells / mm², and bar graphs show mean ± SD. Cleaved caspase 3 expression was significantly increased in the CAR-T-treated group compared to the untransduced group (unpaired t-test) for patients 1–4 (A–D), but not for patients 5 and 6 (E–F). G. Representative images of GPC1 expression by immunohistochemistry in explant tissue that did not respond to treatment with GPC1 CAR-T cells and responded to treatment with GPC1 CAR-T cells. Scale bar = 50 μm. All images are at the same magnification. GPC1 expression, measured by H-score using QuPath, was significantly increased in responders compared to non-responders. [Figure 8D] Effect of GPC1 CAR-T cells in patient-derived explant assays. Treatment with GPC1 CAR-T cells induces apoptosis in patient-derived explants with high GPC1 expression. A–F: Quantification of cleaved caspase 3. Data are expressed as % of positively stained cells / mm², and bar graphs show mean ± SD. Cleaved caspase 3 expression was significantly increased in the CAR-T-treated group compared to the untransduced group (unpaired t-test) for patients 1–4 (A–D), but not for patients 5 and 6 (E–F). G. Representative images of GPC1 expression by immunohistochemistry in explant tissue that did not respond to treatment with GPC1 CAR-T cells and responded to treatment with GPC1 CAR-T cells. Scale bar = 50 μm. All images are at the same magnification. GPC1 expression, measured by H-score using QuPath, was significantly increased in responders compared to non-responders. [Figure 8E]Effect of GPC1 CAR-T cells in patient-derived explant assays. Treatment with GPC1 CAR-T cells induces apoptosis in patient-derived explants with high GPC1 expression. A–F: Quantification of cleaved caspase 3. Data are expressed as % of positively stained cells / mm², and bar graphs show mean ± SD. Cleaved caspase 3 expression was significantly increased in the CAR-T-treated group compared to the untransduced group (unpaired t-test) for patients 1–4 (A–D), but not for patients 5 and 6 (E–F). G. Representative images of GPC1 expression by immunohistochemistry in explant tissue that did not respond to treatment with GPC1 CAR-T cells and responded to treatment with GPC1 CAR-T cells. Scale bar = 50 μm. All images are at the same magnification. GPC1 expression, measured by H-score using QuPath, was significantly increased in responders compared to non-responders. [Figure 8F] Effect of GPC1 CAR-T cells in patient-derived explant assays. Treatment with GPC1 CAR-T cells induces apoptosis in patient-derived explants with high GPC1 expression. A–F: Quantification of cleaved caspase 3. Data are expressed as % of positively stained cells / mm², and bar graphs show mean ± SD. Cleaved caspase 3 expression was significantly increased in the CAR-T-treated group compared to the untransduced group (unpaired t-test) for patients 1–4 (A–D), but not for patients 5 and 6 (E–F). G. Representative images of GPC1 expression by immunohistochemistry in explant tissue that did not respond to treatment with GPC1 CAR-T cells and responded to treatment with GPC1 CAR-T cells. Scale bar = 50 μm. All images are at the same magnification. GPC1 expression, measured by H-score using QuPath, was significantly increased in responders compared to non-responders. [Figure 8G]Effect of GPC1 CAR-T cells in patient-derived explant assays. Treatment with GPC1 CAR-T cells induces apoptosis in patient-derived explants with high GPC1 expression. A–F: Quantification of cleaved caspase 3. Data are expressed as % of positively stained cells / mm², and bar graphs show mean ± SD. Cleaved caspase 3 expression was significantly increased in the CAR-T-treated group compared to the untransduced group (unpaired t-test) for patients 1–4 (A–D), but not for patients 5 and 6 (E–F). G. Representative images of GPC1 expression by immunohistochemistry in explant tissue that did not respond to treatment with GPC1 CAR-T cells and responded to treatment with GPC1 CAR-T cells. Scale bar = 50 μm. All images are at the same magnification. GPC1 expression, measured by H-score using QuPath, was significantly increased in responders compared to non-responders. [Figure 9] Development of GPC1 CD3 CAR-T cells. Flowchart of the established protocol for preparation of CAR-T cells in Professor Simon Barry's laboratory. [Figure 10] Flow cytometry analysis (FACs) of batch 98 cells. EGFR expression in CD3 untransduced (UT) T cells and GPC1 CD3 CART cells. [Figure 11] Flow cytometry analysis (FACs) of Batch 98 cells - cell maturation panel. CD45RA and CD62L markers in CD4 and CD8 T cell populations. Q9: TEMRA (effector memory cells re-expressing CD45RA, CD45RA+ CD62L-), Q10: naive phenotype (CD45RA+ CD62L+), Q11: central memory (CD45RA- CD62L+), Q12: effector memory (CD45RA- CD62L-). [Figure 12A] Flow cytometry analysis (FACs) of batch 2 cells - Exhaustion panel. (A) Programmed cell death-1 (PD1), LAG3, and TIM expression in CD4 T cell populations. (B) Programmed cell death-1 (PD1), LAG3, and TIM expression in CD8 T cell populations. [Figure 12B]Flow cytometry analysis (FACs) of batch 2 cells - Exhaustion panel. (A) Programmed cell death-1 (PD1), LAG3, and TIM expression in CD4 T cell populations. (B) Programmed cell death-1 (PD1), LAG3, and TIM expression in CD8 T cell populations. [Figure 13A] GPC1 expression in controls. Representative images of GPC1 protein expression in mouse kidneys when used for IHC analysis. (A) Positive control, and (B) Negative control. [Figure 13B] GPC1 expression in controls. Representative images of GPC1 protein expression in mouse kidneys when used for IHC analysis. (A) Positive control, and (B) Negative control. [Figure 14A] Relationship between GPC1 and progression-free survival and overall survival using quartile measurements of the H-index. Quartile color legend: 1 (blue), 2 (red), 3 (green), 4 (orange). A. Relationship between GPC1 tumor measurements and progression-free survival (n=93). P=0.363, H-index quartile 1 (15 / 21), H-index quartile 2 (19 / 25), H-index quartile 3 (19 / 23), H-index quartile 4 (17 / 24). B. Relationship between GPC1 tumor measurements and overall survival (n=100), p=0.973, H-index quartile 1 (17 / 25), H-index quartile 2 (16 / 25), H-index quartile 3 (17 / 25), H-index quartile 4 (16 / 25). [Figure 14B] Relationship between GPC1 and progression-free survival and overall survival using quartile measurements of the H-index. Quartile color legend: 1 (blue), 2 (red), 3 (green), 4 (orange). A. Relationship between GPC1 tumor measurements and progression-free survival (n=93). P=0.363, H-index quartile 1 (15 / 21), H-index quartile 2 (19 / 25), H-index quartile 3 (19 / 23), H-index quartile 4 (17 / 24). B. Relationship between GPC1 tumor measurements and overall survival (n=100), p=0.973, H-index quartile 1 (17 / 25), H-index quartile 2 (16 / 25), H-index quartile 3 (17 / 25), H-index quartile 4 (16 / 25). [Figure 15A]Construction of CNA500200, CNA510200, CNA500300, CNA510300, CNA500400, and CNA510400. Six different CAR constructs were generated. These consisted of two different scFv fusion proteins providing the binding domains and three different linker domains. The two scFvs contained (i) the MIL-38 leader sequence (1) linked to the variable light (VL) chain of the MIL-38 antibody (2) fused via a Whitlow linker (3) to the variable heavy (VH) chain of the MIL-38 antibody (4); and (ii) the MIL-38 leader sequence (1) linked to the variable heavy (VH) chain of the MIL-38 antibody (4) fused via a Whitlow linker (3) to the variable light (VL) chain of the MIL-38 antibody (2). The three linked domains included (i) an IgG4 hinge (5), (ii) an IgG4 hinge + IgG4 CH3 (11), and (iii) an IgG4 hinge + IgG4 CH2 L235D and N297Q mutations + IgG4 CH3 (12). The CAR further included a CD28 transmembrane domain (6), a costimulatory domain with a portion of 4-1BB (7), an activation domain with a portion of CD3 zeta (8), a T2A autocleavage site (9), and a truncated EGFR (10). [Figure 15B]Construction of CNA500200, CNA510200, CNA500300, CNA510300, CNA500400, and CNA510400. Six different CAR constructs were generated. These consisted of two different scFv fusion proteins providing the binding domains and three different linker domains. The two scFvs contained (i) the MIL-38 leader sequence (1) linked to the variable light (VL) chain of the MIL-38 antibody (2) fused via a Whitlow linker (3) to the variable heavy (VH) chain of the MIL-38 antibody (4); and (ii) the MIL-38 leader sequence (1) linked to the variable heavy (VH) chain of the MIL-38 antibody (4) fused via a Whitlow linker (3) to the variable light (VL) chain of the MIL-38 antibody (2). The three linked domains included (i) an IgG4 hinge (5), (ii) an IgG4 hinge + IgG4 CH3 (11), and (iii) an IgG4 hinge + IgG4 CH2 L235D and N297Q mutations + IgG4 CH3 (12). The CAR further included a CD28 transmembrane domain (6), a costimulatory domain with a portion of 4-1BB (7), an activation domain with a portion of CD3 zeta (8), a T2A autocleavage site (9), and a truncated EGFR (10). [Figure 15C]Construction of CNA500200, CNA510200, CNA500300, CNA510300, CNA500400, and CNA510400. Six different CAR constructs were generated. These consisted of two different scFv fusion proteins providing the binding domains and three different linker domains. The two scFvs contained (i) the MIL-38 leader sequence (1) linked to the variable light (VL) chain of the MIL-38 antibody (2) fused via a Whitlow linker (3) to the variable heavy (VH) chain of the MIL-38 antibody (4); and (ii) the MIL-38 leader sequence (1) linked to the variable heavy (VH) chain of the MIL-38 antibody (4) fused via a Whitlow linker (3) to the variable light (VL) chain of the MIL-38 antibody (2). The three linked domains included (i) an IgG4 hinge (5), (ii) an IgG4 hinge + IgG4 CH3 (11), and (iii) an IgG4 hinge + IgG4 CH2 L235D and N297Q mutations + IgG4 CH3 (12). The CAR further included a CD28 transmembrane domain (6), a costimulatory domain with a portion of 4-1BB (7), an activation domain with a portion of CD3 zeta (8), a T2A autocleavage site (9), and a truncated EGFR (10). [Figure 15D]Construction of CNA500200, CNA510200, CNA500300, CNA510300, CNA500400, and CNA510400. Six different CAR constructs were generated. These consisted of two different scFv fusion proteins providing the binding domains and three different linker domains. The two scFvs contained (i) the MIL-38 leader sequence (1) linked to the variable light (VL) chain of the MIL-38 antibody (2) fused via a Whitlow linker (3) to the variable heavy (VH) chain of the MIL-38 antibody (4); and (ii) the MIL-38 leader sequence (1) linked to the variable heavy (VH) chain of the MIL-38 antibody (4) fused via a Whitlow linker (3) to the variable light (VL) chain of the MIL-38 antibody (2). The three linked domains included (i) an IgG4 hinge (5), (ii) an IgG4 hinge + IgG4 CH3 (11), and (iii) an IgG4 hinge + IgG4 CH2 L235D and N297Q mutations + IgG4 CH3 (12). The CAR further included a CD28 transmembrane domain (6), a costimulatory domain with a portion of 4-1BB (7), an activation domain with a portion of CD3 zeta (8), a T2A autocleavage site (9), and a truncated EGFR (10). [Figure 15E]Construction of CNA500200, CNA510200, CNA500300, CNA510300, CNA500400, and CNA510400. Six different CAR constructs were generated. These consisted of two different scFv fusion proteins providing the binding domains and three different linker domains. The two scFvs contained (i) the MIL-38 leader sequence (1) linked to the variable light (VL) chain of the MIL-38 antibody (2) fused via a Whitlow linker (3) to the variable heavy (VH) chain of the MIL-38 antibody (4); and (ii) the MIL-38 leader sequence (1) linked to the variable heavy (VH) chain of the MIL-38 antibody (4) fused via a Whitlow linker (3) to the variable light (VL) chain of the MIL-38 antibody (2). The three linked domains included (i) an IgG4 hinge (5), (ii) an IgG4 hinge + IgG4 CH3 (11), and (iii) an IgG4 hinge + IgG4 CH2 L235D and N297Q mutations + IgG4 CH3 (12). The CAR further included a CD28 transmembrane domain (6), a costimulatory domain with a portion of 4-1BB (7), an activation domain with a portion of CD3 zeta (8), a T2A autocleavage site (9), and a truncated EGFR (10). [Figure 15F]Construction of CNA500200, CNA510200, CNA500300, CNA510300, CNA500400, and CNA510400. Six different CAR constructs were generated. These consisted of two different scFv fusion proteins providing the binding domains and three different linker domains. The two scFvs contained (i) the MIL-38 leader sequence (1) linked to the variable light (VL) chain of the MIL-38 antibody (2) fused via a Whitlow linker (3) to the variable heavy (VH) chain of the MIL-38 antibody (4); and (ii) the MIL-38 leader sequence (1) linked to the variable heavy (VH) chain of the MIL-38 antibody (4) fused via a Whitlow linker (3) to the variable light (VL) chain of the MIL-38 antibody (2). The three linked domains included (i) an IgG4 hinge (5), (ii) an IgG4 hinge + IgG4 CH3 (11), and (iii) an IgG4 hinge + IgG4 CH2 L235D and N297Q mutations + IgG4 CH3 (12). The CAR further included a CD28 transmembrane domain (6), a costimulatory domain with a portion of 4-1BB (7), an activation domain with a portion of CD3 zeta (8), a T2A autocleavage site (9), and a truncated EGFR (10). DETAILED DESCRIPTION OF THE INVENTION

[0035]

[0065] The present invention is based in part on the inventors' recognition that glypican-1 (GPC1) is expressed by ovarian cancer cells.Therefore, GPC1 expression can provide information about the presence of disease in individuals.Therefore, the inventors have demonstrated that GPC1 can also serve as a prognostic marker for patients with ovarian cancer, and elevated expression indicates poor patient outcomes.

[0036]

[0066] Furthermore, the present inventors demonstrate that GPC1 can be targeted to kill cancer cells, such as ovarian cancer cells, and therefore may provide a target for cancer therapy.

[0037]

[0067] Glypican-1

[0068] Glypican proteins belong to the heparan sulfate proteoglycan (HSPC) family, numbered from 1 (GPC-1) to 6 (GPC-6).

[0038]

[0069] Glypican-1 (GPC1) is a glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan. The GPC1 cDNA sequence is listed in NCBI Reference Sequence: NM_002081.3, and the GPC1 protein sequence is listed in NCBI Reference Sequence: NP_002072.2. GPC1 consists of a 558-amino acid core protein and three predicted heparan sulfate chains attached at S486, S488, and S490. It exists in both a membrane-anchored form (via a GPI at S530) and a secreted, soluble form.

[0039]

[0070] During embryonic development, GPC1 is expressed primarily in the nervous and skeletal systems and at lower levels in adult tissues such as the heart and testis, where it is involved in organ development by regulating extracellular growth signals and morphogen gradient formation.

[0040]

[0071] Cancer treatment

[0072] As shown above and exemplified herein, GPC1 represents a target for cancer cell therapy.

[0041]

[0073] In some aspects, the present invention provides methods of treating a subject with cancer, the method comprising killing cells that express glypican-1.

[0042]

[0074] Also provided is a method of treating or preventing cancer in a subject, comprising administering to or introducing into the subject an agent that targets glypican-1.

[0043]

[0075] The present inventors have demonstrated that glypican-1 is associated with ovarian cancer. Thus, in some embodiments of this method, the cancer is ovarian cancer. In some embodiments of this method, the ovarian cancer is recurrent ovarian cancer.

[0044]

[0076] Most epithelial ovarian / fallopian tube cancers are of the serous type and are divided into low-grade serous carcinoma (LGSC or LSOC) and high-grade serous carcinoma (HGSC or HSOC). These tumors differ in their genetic alterations and biological characteristics.

[0045]

[0077] The present inventors have shown that glypican-1 is particularly associated with high-grade serous ovarian cancer. Thus, in some embodiments of the present methods of treatment or prevention, the ovarian cancer is epithelial cell ovarian cancer, particularly serous ovarian cancer, and most particularly high-grade serous ovarian cancer. In some embodiments, the ovarian cancer is germ cell ovarian cancer. In some embodiments, the ovarian cancer is stromal cell ovarian cancer.

[0046]

[0078] In some embodiments, the ovarian cancer is recurrent ovarian cancer.

[0047]

[0079] Suitable agents for targeting or killing cells expressing glypican-1 include, but are not limited to, antibodies and binding fragments thereof, antibody-drug conjugates (ADCs), radionuclide-labeled antigen-binding molecules, fusion proteins, chimeric antigen receptor (CAR)-expressing cells, bispecific binding molecules such as bispecific T cell engagers and bispecific antibodies, and vaccines designed to mount an immune response against GPC1.

[0048]

[0080] Thus, in some embodiments of the present methods of treatment or prevention, the subject is administered an agent or cells are exposed to an agent that preferentially or selectively binds to glypican 1. Such an agent may, in some embodiments, be an antibody or a binding fragment of an antibody.

[0049]

[0081] Antibody-binding fragments can be derived from antibodies or can be recombinantly produced using sequences identical to the CDRs of the antibody or antibody fragment. Indeed, the CDRs may be derived from an affinity-matured antibody and therefore may not be identical to the in vivo derived antibody.

[0050]

[0082] Antibodies are composed of four chains (two heavy and two light) that can be divided into an Fc domain (crystallizable region) and an Fab domain (antigen-binding region). The Fc portion of an antibody interacts with Fc receptors and the complement system. Therefore, the Fc portion is important for the immune function of the antibody. However, the Fab portion contains the binding region of the antibody and is crucial for the specificity of the antibody for the desired epitope.

[0051]

[0083] Thus, in some embodiments, the antibody fragment is a Fab fragment of an antibody. The Fab fragment may be an individual Fab fragment (i.e., the antibody fragment is generated without being linked by disulfide bridges) or a F(ab')2 fragment containing two Fab fragments of an antibody linked by disulfide bridges. These fragments are typically generated by fragmenting antibodies using digestive enzymes such as pepsin. Methods for preparing such Fabs are known in the art (see, for example, Sjoegren, J. et al., Methods Mol Biol. 2017;1535:pp.319-329).

[0052]

[0084] An antibody consists of a total of six CDRs, with the VH and VL chains each containing three CDRs (within a framework of four framework regions). Individual VH and VL chains (each containing only three CDRs) have been shown to bind specifically with high affinity. Typically, individual binding regions are known as single antibody domains (sdAbs). Alternatively, VH and VL chains can be linked by a linker to form a fusion protein known as a single-chain variable fragment (scFv, also known as a diabody). Unlike Fabs, scFvs are not fragmented from antibodies but rather are typically recombinantly formed based on the CDRs and framework regions of antibodies. Furthermore, sdAbs and scFvs can also be recombinantly produced to form the binding components of larger fusion proteins that may also contain additional moieties. Thus, in some embodiments, the agent is or may include an scFv or sdAb containing CDRs from an antibody that binds to GPC1. An scFv can comprise multiple VH and VL chains linked together to form a multivalent scFv, such as a di-scFv or tri-scFv.

[0053]

[0085] In some embodiments, the antibody that binds to GPC1 is MIL-38 (Miltuximab™).

[0054]

[0086] In some embodiments, the agent that binds to glypican-1 can be a fusion protein, such as a single-chain variable fragment comprising the variable light and variable heavy chain sequences of an antibody, such as the antibody MIL-38. In some embodiments, the agent that binds to glypican-1 comprises the VH or VL chain of an antibody (e.g., MIL-38) that binds to glypican-1.

[0055]

[0087] Antibodies to specific analytes can be obtained commercially or generated by methods known in the art. For example, antibodies to specific analytes can be prepared using methods generally disclosed by Howard and Kaser (Making and Using Antibodies: a Practical Handbook, CRC Press, 2007).

[0056]

[0088] In some embodiments, the variable heavy region has the amino acid sequence of SEQ ID NO: 2, or a variant of this amino acid sequence having sequence identity to this sequence. In some embodiments, the variable light region has the amino acid sequence of SEQ ID NO: 1, or a variant of this amino acid sequence having sequence identity to this sequence. In some embodiments, the variable heavy or variable light chain variant is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.2%, at least 98.4%, at least 98.6%, at least 98.8%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% identical to the variable heavy and / or variable light chain of SEQ ID NO:2 and / or SEQ ID NO:1.

[0057]

[0089] In some embodiments, the variable heavy region comprises a heavy chain CDR1 having the amino acid sequence DYSMN or having the amino acid sequence DYSMN with up to one, two, or three amino acid modifications; a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:4 or having the amino acid sequence set forth in SEQ ID NO:4 with up to one, two, or three amino acid modifications; and a heavy chain CDR3 having the amino acid sequence HYDYGGFPY or having the amino acid sequence HYDYGGFPY with up to one, two, or three amino acid modifications.

[0058]

[0090] In some embodiments, the variable light chain comprises a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:3, or having the amino acid sequence set forth in SEQ ID NO:3 with up to one, two, or three amino acid modifications; a light chain CDR2 having the amino acid sequence TAKTLAD, or having the amino acid sequence TAKTLAD with up to one, two, or three amino acid modifications; and a light chain CDR3 having the amino acid sequence QHFWSNPWT, or having the amino acid sequence QHFWSNPWT with up to one, two, or three amino acid modifications.

[0059]

[0091] In some embodiments, the antibody or antigen-binding fragment comprises heavy chain CDR1, CDR2, and CDR3 having the amino acid sequences of DYSMN, SEQ ID NO: 4, and HYDYGGFPY, wherein up to 1, 2, or 3 amino acids are modified.

[0060]

[0092] In some embodiments, the antibody or antigen-binding fragment comprises a light chain CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NO:3, TAKTLAD, and QHFWSNPWT, in which up to 1, 2, or 3 amino acids are altered.

[0061]

[0093] The specificity, avidity, and affinity of antibodies generated in a subject can be altered by in vitro processes such as affinity maturation (see, e.g., Fujino Y. et al. Biochem Biophys Res Comm., 2012; 428(3):395-400; Li, B. et al. MAbs. 2014; 6(2):pp. 437-45, and Ho M and Pastan I, "In vitro Antibody Affinity Maturation Targeting Germline Hotspots", Method Mol Biol., 2009; 525:293-xiv). These techniques include, but are not limited to, site-directed mutagenesis and PCR-driven mutagenesis, phage library development, and affinity screening. For example, mutations flanking hotspot sites defined by A / GGC / TA / T (RGYW) and AG-C / T (AGY) sequences (based on the encoding immunoglobulin DNA) are likely to alter the affinity of the produced antibodies. Alternatively, a process such as in vitro scanning saturation mutagenesis (Chen, G et al. Protein Eng Des Sel., 1999; (12) 4:346-356) can be used to replace every possible mutation in the CDR region with every other possible mutation. Each variant is then evaluated for antigen affinity and specificity. Thus, in vitro-derived antibodies or binding fragments thereof can be further modified to generate distinct, yet still linearly related, antibodies. Thus, the term "antibody" (and fragments thereof) encompasses in vivo-derived antibodies and in vitro-derived molecules that have undergone a mutational process to alter the CDR-binding site and have unique sequences compared to antibodies generated in vivo. Furthermore, the binding portions of antibodies (particularly the CDRs) can be affinity-matured and mutated using techniques known in the art.

[0062]

[0094] The term "antibody" also includes non-conventional antibodies generated from species such as camelids, sharks, and jawfish. Thus, the term antibody includes heavy chain antibodies such as camelid antibodies, IgNARs, and variable lymphocyte receptors (VLRs). Furthermore, these may be fragmented into binding moieties (e.g., VNARs, which are single-binding moieties of IgNARs) or recombinantly incorporated into fusion proteins. Methods for generating and adapting such non-conventional antibodies are known in the art; see, for example, Nuttall, S., Methods Mol. Biol. 2012; 911: pp. 27-36 and Vincke C. et al., Methods Mol. Biol. 2012; 907: pp. 145-76.

[0063]

[0095] Antibodies that bind to GPC1 can be generated. Furthermore, such antibodies can be affinity matured to optimize sustained affinity and avidity. Thus, in some embodiments, the binding domain contains a sequence identical to the binding region of an antibody that binds to GPC1, or a sequence corresponding to an affinity-matured form of the binding region that binds to GPC1. While the affinity-matured binding region can be significantly different from the original antibody binding region, in preferred forms, the affinity-matured form of the binding region has at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the antibody that binds to GPC1.

[0064]

[0096] In some embodiments, the agent that binds to glypican-1 is an antibody drug conjugate (ADC).

[0065]

[0097] Antibody-drug conjugates use immunoconjugates in which a cytotoxic agent is chemically or enzymatically linked to an antibody that selectively binds to an internalizing tumor-associated antigen, thereby selectively delivering the cytotoxic agent to specific cells. Most ADCs contain an IgG1 antibody conjugated to a microtubule inhibitor, such as maytansine or auristatin.

[0066]

[0098] Other known conjugates used in ADCs include monomethyl auristatin E (MMAE - used in brentuximab vedotin (Adcetris™) and enfortumab vedotin (Padcev™)); monomethyl auristatin F (MMAF) - used in belantamab mafodotin (Blenrep™); calicheamicin-gemtuzumab ozogamicin (Mylotarg™); maytansinoid DM1 - used in trastuzumab emtansine (Kadcyla™); maytansinoid DM4 - used in used in betuximab soravtansine; pyrrolobenzodiazepine (PBD) dimer - used in Rova-T; camptothecin analog - sacituzumab govitecan (Trodelvy™); duocarmycin analog - used in trastuzumab duocarmazine; camptothecin derivative SN-38-sacituzumab govitecan (Trodelvy™); irinotecan metabolite SN-38-trastuzumab deruxtecan (Enhertu™); and topoisomerase I inhibitor - used in trastuzumab deruxtecan (Enhertu™). Any of the listed conjugates may be used in the present invention. Further conjugates and information regarding the preparation of such ADCs are described in Riccardi F, et al. (2023), A comprehensive overview on antibody-drug conjugates: from the conceptualization to cancer therapy. Front Pharmacol, 14:1274088, the contents of which are incorporated herein.

[0067]

[0099] Antibody drug conjugates for glypican-1 are known in the art. Examples of such conjugates include those disclosed in the following publications: Matsuzaki S, et al. (2017). Anti-glypican-1 antibody-drug conjugate exhibits potent preclinical antitumor activity against glypican-1 positive uterine cervical cancer. Int J Cancer, 1; 142(5), 1056-1066; Yokota K, et al. (2021). Anti-Glypican-1 Antibody-Drug Conjugate as Potential Therapy Against Tumor Cells and Tumor Vasculature for Glypican-1-Positive Cholangiocarcinoma. Mol Cancer Ther, 20(9), 1713-1722; Munekage E, et al. (2021). A glypican-1-targeted antibody-drug conjugate exhibits potent tumor growth inhibition in glypican-1-positive pancreatic cancer and esophageal squamous cell carcinoma. carcinoma. Neoplasia, 23(9), 939-950; and Tsujii S et al.(2021). Glypican-1 Is a Novel Target for Stroma and Tumor Cell Dual-Targeting Antibody-Drug Conjugates in Pancreatic Cancer. Mol Cancer Ther, 20(12), 2495-2505 (the contents of the above-listed references are incorporated herein).

[0068]

[0100] The methods of treatment or prevention provided herein may be used in patients diagnosed with cancer, particularly ovarian cancer. In some embodiments, the methods of treatment or prevention are carried out after analyzing the expression of glypican-1 in a subject (particularly in the subject's cancer cells). In some embodiments of the methods of prevention or treatment, the cells expressing glypican-1 have been determined to express elevated amounts of glypican-1 (particularly glypican-1 protein or glypican-1 mRNA). Factors that cause elevated amounts of glypican-1 are known in the art and defined herein. Methods for assessing protein and mRNA levels are known in the art and provided herein.

[0069]

[0101] In some embodiments, increasing the amount of glypican-1 protein expression comprises increasing surface expression of glypican-1 protein and / or increasing intracellular expression of glypican-1 protein.

[0070]

[0102] In some embodiments of the treatment method, the ovarian cancer is recurrent ovarian cancer, and the elevated amount of glypican-1 is compared to cancerous tissue and / or non-cancerous ovarian tissue before the recurrence.

[0071]

[0103] In some embodiments of the present prophylactic or therapeutic methods, a diagnostic or prognostic method described herein is performed before or after treatment.

[0072]

[0104] The therapeutic and preventative methods of the present invention can be performed alone or in combination with other treatments for cancer, including, but not limited to, chemotherapy, surgical resection or debulking, or radiation therapy.

[0073]

[0105] In some embodiments, the methods of treatment or prevention are performed as an adjuvant therapy in combination with another treatment, such as immunotherapy. In some embodiments, the agent that binds to glypican 1 is administered together with an immune checkpoint inhibitor, such as a PD1 inhibitor, a PD-L1 inhibitor, a CTLA4 inhibitor, a LAG3 inhibitor, a TIM3 inhibitor, or a TIGIT inhibitor (including antibodies or binding agents that bind to these targets).

[0074]

[0106] In some embodiments of the present methods of treatment, the agent that binds to glypican-1 is a chimeric antigen receptor (CAR) (expressed on the cell). In some embodiments, the CAR is an anti-GPC1 CAR disclosed herein.

[0075]

[0107] Chimeric Antigen Receptor

[0108] Chimeric antigen receptors (CARs) are artificially constructed proteins that can induce antigen-specific cellular responses upon expression on the cell surface. In their most basic form, CARs contain at least three domains. The first domain is an extracellular antigen-recognition domain that specifically recognizes an antigen (more specifically, the epitope portion of an antigen) or a portion of an antigen. The second domain is an intracellular signaling domain that can induce or be involved in the induction of an intracellular signaling pathway. The third domain is a transmembrane domain that spans the cell membrane and bridges the extracellular antigen-recognition domain and the intracellular signaling domain.

[0076]

[0109] The combination of the first two domains determines the antigen specificity of the CAR and its ability to induce the desired cellular response, the latter of which also depends on the host cell of the CAR. For example, activation of a CAR expressed on helper T cells and having a signaling domain containing a CD3 activation domain can induce the secretion of various cytokines by CD4+ helper T cells when activated by its cognate antigen. In a further example, the same CAR, when expressed on CD8+ cytotoxic T cells, can induce the release of cytotoxins when activated by cells expressing the cognate antigen, ultimately leading to the induction of apoptosis of the antigen-expressing cells.

[0077]

[0110] The third domain (transmembrane domain) may comprise a portion of the signaling domain of the CAR or may be associated with the signaling domain of the CAR. This transmembrane domain is typically one or more hydrophobic helices that penetrate the lipid bilayer of the cell, embedding the CAR within the cell membrane. The transmembrane domain of the CAR may be a determinant of the expression pattern of the CAR when associated with a cell. For example, the use of a transmembrane domain associated with the CD3 co-receptor may, among other things, enable expression of the CAR in naive T cells, while the use of a transmembrane domain from the CD4 co-receptor may induce expression of the CAR in helper T cells. The use of a CD8 co-receptor transmembrane domain may induce expression in cytotoxic T lymphocytes (CTLs), and the CD28 transmembrane domain may enable expression in both CTLs and helper T cells and may help stabilize the CAR.

[0078]

[0111] An additional component or portion of a chimeric antigen receptor may be a linker domain. The linker domain extends from the extracellular side of the transmembrane domain to the antigen-recognition domain, thereby connecting the antigen-recognition domain and the transmembrane domain. Typically, in the art, the linker domain is considered an optional domain because some CARs function without the linker domain.

[0079]

[0112] Thus, in one aspect, the present invention provides a chimeric antigen receptor (CAR) comprising an antigen recognition domain, a transmembrane domain, and a signaling domain, wherein the antigen recognition domain recognizes glypican-1 (GPC1).

[0080]

[0113] As used throughout this specification, the term "recognize" (with respect to glypican-1) refers to the ability of the binding domain to associate with a desired epitope of GPC1 or any portion of the GPC1 molecule. Preferably, this recognition is selective, in that the binding domain binds exclusively or preferentially to GPC1. In some embodiments, the binding domain may directly bind to GPC1 or an epitope thereof. In some embodiments, the binding domain may indirectly bind to GPC1 or an epitope thereof, for example, via an intermediate or bispecific molecule (e.g., fifth-generation CDR). In some embodiments, the antigen recognition domain may bind to a processed form of GPC1. When used in this context, the term "processed form" typically refers to a form of GPC1 that is truncated or digested as a result of intracellular processing, including the form of GPC1 that is presented on the major histocompatibility complex (e.g., human leukocyte antigen) and the epitope.

[0081]

[0114] The CAR binding domain can be any suitable domain capable of recognizing GPC1 or an epitope thereof. As used throughout this specification, the term "binding domain" refers to the portion of a CAR that confers specificity to GPC1. In the context of the present invention, the binding domain comprises only a portion of the extracellular region (or ectodomain) of the CAR.

[0082]

[0115] The binding domain of the present CAR can comprise a variety of binding molecules. These molecules include antibodies (including non-traditional antibodies such as heavy chain antibodies), antibody binding fragments (including scFvs, Fabs, and sdAbs described herein), and protein binding scaffolds. In some embodiments, the binding domain comprises the variable heavy chain of an antibody that binds to GPC1, and / or the binding domain comprises the variable light chain of an antibody that binds to GPC1 (including the antibodies and binding fragments disclosed herein). In some embodiments, the binding domain comprises a Fab. The antigen recognition domain can also be a fusion protein (e.g., a single-chain variable fragment (scFv)) having sequence identity to an antibody that binds to glypican-1. In some embodiments, the antigen binding domain comprises SEQ ID NO:28 or SEQ ID NO:29 (with or without the MIL-38 leader of SEQ ID NO:27), or a functional variant of SEQ ID NO:28 or SEQ ID NO:29 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.5% sequence identity.

[0083]

[0116] For the avoidance of doubt, the binding domain of the present CAR may comprise any antibody or antibody fragment sequence (e.g., CDRs) disclosed herein in relation to an anti-GPC-1 antibody (e.g., one described as a potential "agent"), including any possible modifications disclosed herein.

[0084]

[0117] Antibodies that can bind to GPC1 are discussed herein and include MIL-38.

[0085]

[0118] Linker Domain

[0119] The linker domain connects the transmembrane domain and the antigen recognition domain of the CAR. Functional CAR T cells have been generated without including a linker domain, and therefore, in this context, the linker domain is not generally considered essential for the function of all CARs.

[0086]

[0120] Without wishing to be bound by theory, the linker domain provides the CAR ectodomain (extracellular domain) with an appropriate molecular length to enable epitope recognition by the antigen recognition domain, while at the same time forming an appropriate immune synapse distance between the effector cell expressing the CAR and the target cell. Furthermore, the linker domain may provide appropriate flexibility so that the antigen recognition domain can be oriented in an appropriate direction to recognize its epitope.

[0087]

[0121] Thus, in some embodiments, the extracellular domain comprises a linker domain connecting the binding domain to the transmembrane domain. In some embodiments, the linked domain is at least 12 amino acids in length. In some embodiments, the linked domain is at least about 12 amino acids in length. In some embodiments, the linked domain is more than 12 amino acids in length. In some embodiments, the linked domain is at least 119 amino acids in length. In some embodiments, the linked domain is at least about 119 amino acids in length. In some embodiments, the linked domain is more than 119 amino acids in length. In some embodiments, the linked domain is at least 229 amino acids in length. In some embodiments, the linked domain is at least about 229 amino acids in length. In some embodiments, the linked domain is more than 229 amino acids in length.

[0088]

[0122] In some embodiments, the linked domains are up to 119 amino acids in length. In some embodiments, the linked domains are up to about 119 amino acids in length. In some embodiments, the linked domains are up to 229 amino acids in length. In some embodiments, the linked domains are up to about 229 amino acids in length.

[0089]

[0123] Selection of a suitable linker domain may be based on (i) reducing binding affinity to Fc receptors (such as Fcγ and FcRn receptors), thereby minimizing "off-target" activation of CAR-expressing cells, and (ii) optimizing the efficacy of the CAR construct by increasing the flexibility of the antigen-binding region, reducing spatial constraints on the formation of the immune synapse (e.g., reducing steric hindrance and optimizing synaptic distance).

[0090]

[0124] In some embodiments, the linker domain comprises a sequence identical to an immunoglobulin hinge region or a hinge region or extracellular region from a membrane-bound molecule involved in T-cell synapse formation. For example, the linker domain can comprise a region having amino acid sequence homology to a hinge region from CD4, CD8, CD3, CD7, or CD28.

[0091]

[0125] In some embodiments, the linker domain comprises a sequence identical to a portion of an immunoglobulin. In some embodiments, the portion is one or more of a hinge region (e.g., an IgG4 hinge region or a modified version thereof), a constant heavy (CH)1 region, a CH2 region, a CH3 region, or a CH4 region. In some embodiments, the portion is the CH2 region, CH3 region, or hinge region of an immunoglobulin, or has at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity to said CH region. In some embodiments, the portion is the CH2 region or CH3 region and hinge region of an immunoglobulin. In some embodiments, the immunoglobulin is selected from an IgG subtype.

[0092]

[0126] In some embodiments, the linker domain comprises a sequence having similarity to one or more portions of an IgG1, IgG2, IgG3, or IgG4 Fc region, for example, an IgG1 hinge region and an IgG4 CH2 or CH3 region, or a functional variant thereof having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.5% sequence identity.

[0093]

[0127] In some embodiments, the linker domain comprises a sequence identical to an immunoglobulin CH3 domain, an immunoglobulin CH2 domain, or both a CH2 domain and a CH3 domain. In some embodiments, the linker domain comprises a sequence identical to an immunoglobulin hinge region and one or more of a CH3 domain or a CH2 domain. In some embodiments, the CH2 and / or CH3 region is derived from an IgG4 subclass of IgG antibody.

[0094]

[0128] In some embodiments, the linker domain comprises all or a portion of an immunoglobulin hinge region. As is understood in the art, the specific regions that form the hinge region of an immunoglobulin vary depending on the isotype. For example, immunoglobulins of the IgA, IgD, and IgG isotypes have a hinge region between the CH1 and CH2 regions, while in immunoglobulins of the IgE and IgM isotypes, the function of the hinge region is provided by the CH2 region.

[0095]

[0129] In some embodiments, the linker comprises an IgG4 hinge region, and / or an IgG4 CH3 region, and / or an IgG4 CH2 region (which may contain the mutations L235D, N297Q).

[0096]

[0130] A non-exhaustive list of sequences that may be incorporated into the linker domain is shown in Table 1 below. In some embodiments, the linker domain of the present invention may comprise any one or more of the components shown in Table 1. In some embodiments, the linker domain may consist of any one or more of the linkers shown in Table 1. Additionally, the linker domain may be an artificially synthesized sequence, such as a polyglycine sequence or a repeat of the GGGGS(Gly4Ser) sequence (e.g., (Gly4Ser)3).

[0097]

[0131] [Table 1]

[0098]

[0132] The hinge, CH2, and CH3 regions of immunoglobulins (particularly IgG isotype antibodies) can bind to Fc receptors, such as Fc gamma receptors and Fc neonatal receptors. Binding of the linker domain of a chimeric antigen receptor can reduce the effectiveness of the receptor and cause off-target killing. Thus, in some embodiments, the linker domain is designed to have reduced or no Fc receptor binding ability. In some embodiments, the linker domain is identical to an immunoglobulin that has reduced Fc receptor binding ability compared to other immunoglobulin isotypes. In some embodiments, the linker domain of a chimeric antigen receptor does not contain an amino acid sequence that substantially binds to an Fc receptor.

[0099]

[0133] The ability of Fc receptors to bind various IgG isotypes is shown in Table 2 below.

[0100]

[0134] [Table 2]

[0101]

[0135] In some embodiments, when the linker domain contains a portion identical to the Fc region of an immunoglobulin, this portion can be modified to reduce binding to Fc receptors. Methods for modifying proteins to reduce binding to Fc receptors are known in the art. Fc gamma receptors primarily bind to the lower hinge region and the n-terminus of the CH2 region of immunoglobulin domains, whereas neonatal Fc receptors primarily bind to amino acids at the C-terminus of the CH2 region and the N-terminus of the CH3 region. Guidance on Fc receptor binding to IgG antibodies can be found in Chapter 7 of "Antibody Fc: Linking Adaptive and Innate Immunity," Ackerman and Nimmerjahn, Elsevier Science & Technology 2014. Thus, modifications in these regions can alter Fc receptor binding to linker domains that share homology with the Fc portion of immunoglobulins. A non-exhaustive, exemplary list of mutations into human IgG1 that have been shown to reduce Fc-gamma receptor and FcRn binding includes: E116P, L117V, L118A, G119 deletion, P121A, S122A, I136A, S137A, R138A, T139A, E141A, D148A, S150A, S150A, E152A, D153A, E155A, N159A, D163A, H168A, N169A, K171A, K173A, R175A, E176A, Q178A, Y179F, N180A, S181A, R184A, V 188A, T190A, L192A, Q194A, D195A, N198A, K200A, K205A, K209A, A210Q, A210S, A210G, P212A, P214A, E216A, K217A, S220A, K221A, A222T, K243A, Q245A, H251A, D259A, A261Q, E263A, E265A, V286A, S288A, K297A, S307A, E313A, H316A, N317A, H318A, Y319A (numbering corresponds to the sequence listed in Uniprot reference number P01857-1)

[0102]

[0136] In some embodiments, the linked domain has a sequence selected from the group consisting of SEQ ID NO: 15, 16, or 17, or is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15, 16, or 17.

[0103]

[0137] Transmembrane and intracellular domains

[0138] The transmembrane domain of CAR bridges the extracellular part (ectodomain) and the intracellular part (endodomain), and mainly plays a structural role.Therefore, the transmembrane domain can be composed of any sequence that can anchor and penetrate the lipid bilayer of the cell.However, the nature of the transmembrane domain can affect its localization and expression.

[0104]

[0139] In preferred embodiments, the transmembrane domain has sequence identity to the sequence of a molecule involved in T cell synapse formation or T cell signaling. In some embodiments, a chimeric antigen receptor of the invention comprises a transmembrane domain comprising a sequence identical to all or a portion of the transmembrane domain of CD3, CD4, CD8, or CD28. In some embodiments, the transmembrane domain comprises a sequence having identity to all or a portion of the transmembrane domain of CD8 or CD28. In some embodiments, the transmembrane domain has sequence identity to all or a portion of the transmembrane domain of CD28. In some embodiments, the transmembrane domain has amino acid sequence identity to SEQ ID NO: 18, or a functional variant of SEQ ID NO: 18 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.5% sequence identity.

[0105]

[0140] In addition to the antigen recognition domain, linker domain, and transmembrane domain, the chimeric antigen receptor of the present invention comprises an intracellular (endo) domain that contains a signal transduction moiety (signaling domain).

[0106]

[0141] In addition to the antigen recognition domain, linker domain, and transmembrane domain, the chimeric antigen receptor of the present invention comprises an intracellular (endo) domain that contains a signal transduction moiety (signaling domain).

[0107]

[0142] The intracellular signaling domain of the present chimeric antigen receptor can be any suitable domain capable of inducing or participating in the induction of an intracellular signaling cascade upon activation of the CAR as a result of antigen recognition by the antigen recognition domain. The signaling domain of the CAR will be specifically selected depending on the intended cellular outcome after CAR activation. While many signaling domains are possible, for use in immunotherapy and cancer therapy, signaling domains can be classified into two general categories based on the receptor from which they are derived: activating receptors and costimulatory receptors (see further details below). Thus, in some embodiments, the signaling domain comprises a portion having an amino acid sequence identical to the signaling portion of an activating receptor, or a functional variant thereof. In some embodiments, the signaling domain comprises a portion having an amino acid sequence identical to the signaling portion of a costimulatory receptor, or a functional variant thereof.

[0108]

[0143] As used throughout this specification, the term "portion," when used in reference to an activating or costimulatory receptor, relates to any segment of the receptor that contains sequences that are responsible for or involved in the initiation / induction of an intracellular signaling cascade following interaction of the receptor with its cognate antigen or ligand. Examples of initiation / induction of intracellular signaling cascades of the T cell receptor (TCR) via CD3 are outlined below.

[0109]

[0144] Without wishing to be bound by theory, the extracellular portion of the TCR primarily comprises heterodimers of either the clonotypic TCRα and TCRβ chains (TCRα / β receptors) or the TCRγ and TCRδ chains (TCRγδ receptors). These TCR heterodimers generally lack intrinsic signaling capabilities and therefore noncovalently associate with multiple signaling subunits of CD3 (primarily CD3 zeta, gamma, delta, and epsilon). The CD3 gamma, delta, and epsilon chains each have an intracellular (cytoplasmic) portion containing a single immunoreceptor tyrosine-based activation motif (ITAM), whereas the CD3 zeta chain contains three tandem ITAMs. When the TCR binds its cognate antigen in the presence of MHC and engages with the required coreceptors, such as CD4 or CD8, signaling is initiated, resulting in tyrosine kinase (i.e., Lck) phosphorylating two tyrosine residues within the intracellular ITAMs of the CD3 chains. Subsequently, another tyrosine kinase (ZAP-70, also activated by Lck phosphorylation) is recruited and diphosphorylates the ITAMs, resulting in the activation of several downstream target proteins, ultimately leading to intracellular structural changes, calcium mobilization, and rearrangements of the actin cytoskeleton, which ultimately lead to the activation of transcription factors and the induction of T cell immune responses.

[0110]

[0145] As used throughout this specification, the term "activating receptor" relates to a receptor or co-receptor that forms a component of or is involved in the formation of the T cell receptor (TCR) complex, or to a receptor that is involved in the specific activation of an immune cell as a result of recognition of an antigenic or other immunogenic stimulus.

[0111]

[0146] Non-limiting examples of such activating receptors include components of the T cell receptor-CD3 complex (CD3 zeta, gamma, delta, and epsilon), CD4 co-receptors, CD8 co-receptors, Fc receptors, or natural killer (NK) cell-associated activating receptors, such as LY-49 (KLRA1), natural cytotoxicity receptors (NCRs, preferably NKp46, NKp44, NKp30, or NKG2, or CD94 / NKG2 heterodimers). Thus, in some embodiments of the CARs of the invention, the signaling domain comprises a portion derived from any one or more of a member of the CD3 co-receptor complex (preferably at least the signaling portion of the CD3 zeta (ζ) chain), a CD4 co-receptor, a CD8 co-receptor, a signaling portion of an Fc receptor (FcR) (preferably the signaling portion of FcεRI or FcγRI), or an NK-associated receptor, such as LY-49.

[0112]

[0147] The specific intracellular signaling portions of each of the CD3 chains are known in the art, see, e.g., WO 2022 / 104424 (the entire contents of which are incorporated herein by reference, particularly with respect to the linker, transmembrane domain, and intracellular domain of the CAR).

[0113]

[0148] In some embodiments of the invention, the signaling domain comprises a portion derived from or having sequence homology to CD3 (preferably, the CD3-zeta chain or a portion thereof). In some embodiments, the signaling domain comprises a sequence identical to all or a portion of the intracellular domain of CD3 zeta (CD3-zeta). In some embodiments, the portion of the CD3-zeta co-receptor complex comprises the amino acid sequence set forth in SEQ ID NO: 19, or a functional variant of this amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.5% sequence identity.

[0114]

[0149] Alternative signaling domains include the intracellular portions of Fc receptors known in the art, such as the intracellular portions of the FcεR1 receptor or the FcγRI receptor (see WO 2022 / 104424 for specific sequences). Various combinations of portions of activating receptors can be used to form the transmembrane (TM) and intracellular (IC) portions of CARs, e.g., CD3ζ TM and CD3ζ IC (Landmeier S, et al. (2007). Gene-Engineered Varicella-Zoster Virus-Reactive CD4+ Cytotoxic T Cells Exert Tumor-Specific Effector Function, Cancer Res, 67, 8335-43; Guest RD, et al. (2005). The role of extracellular spacer regions in the optimal design of chimeric immune receptors: evaluation of four different scFvs and antigens, J Immunother, 28(3), 203-211; Hombach AA, et al. (2007). T cell activation by antibody-like immunoreceptors: the position of the binding epitope within the target molecule determines the efficiency of activation of redirected T cells, J Immunol, 178, 4650-7; James SE, et al. (2008). Antigen sensitivity of CD22-specific chimeric TCR is modulated by target epitope distance from the cell membrane, J Immunol, 180(10), 7028-38; Patel SD, et al. (1999).Impact of chimeric immune receptor extracellular protein domains on T cell function,Gene Ther,6,412-419;Haynes NM,et al.(2001).Redirecting Mouse CTL Against Colon Carcinoma:Superior Signaling Efficacy of Single-Chain Variable Domain Chimeras Containing TCR-ζ vs FcεRI-γ,J Immunol,166,182-1877;Annenkov AE,et al.(1998).Loss of Original Antigenic Specificity in T Cell Hybridomas Transduced with a Chimeric Receptor Containing Single-Chain Fv of an Anti-Collagen Antibody and FcεRI-Signaling γ Subunit,J Immunol,161,6604-6613). .

[0115]

[0150] As discussed above, in some embodiments of the chimeric antigen receptor of the present invention, the signaling domain comprises a portion having an amino acid sequence identical to the signaling portion of a costimulatory receptor.

[0116]

[0151] As used throughout this specification, the term "costimulatory receptor" refers to a receptor or coreceptor that assists in the activation of immune cells upon antigen-specific induction of an activating receptor. As will be understood, costimulatory receptors do not require the presence of an antigen and are not antigen-specific, but are typically one of two signals (the other being an activation signal) required for the induction of an immune cell response. In the context of an immune response, costimulatory receptors are typically activated by the presence of a ligand expressed on the surface of antigen-presenting cells (APCs), such as dendritic cells or macrophages. With regard to T cells in particular, costimulation is necessary to trigger cell activation, proliferation, differentiation, and survival (all of which are commonly referred to as the umbrella of T cell activation), and antigen presentation to T cells in the absence of costimulation can lead to anergy, clonal deletion, and / or the development of antigen-specific tolerance. Importantly, costimulatory molecules can influence T cell responses to simultaneously encountered antigens. Generally, antigens encountered in the context of "positive" costimulatory molecules activate T cells and initiate a cellular immune response aimed at eliminating cells expressing this antigen, whereas antigens encountered in the context of "negative" co-receptors induce tolerance to the simultaneously encountered antigen.

[0117]

[0152] Non-limiting examples of T cell costimulatory receptors include CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS. Specifically, CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS all represent "positive" costimulatory molecules that enhance activation of T cell responses. Accordingly, in some embodiments of the first aspect of the invention, the signaling domain comprises a portion derived from any one or more of CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS.

[0118]

[0153] In some embodiments of the invention, the signaling domain comprises a portion derived from CD28, OX40, or 4-1BB costimulatory receptor, hi some embodiments, the signaling domain comprises a portion of 4-1BB set forth in SEQ ID NO:20, or a functional variant of this portion having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.5% sequence identity.

[0119]

[0154] Various portions of costimulatory receptors can be used alone or in combination to form the transmembrane (TM) and intracellular (IC) portions of the CAR. Exemplary combinations include CD8 TM and DAP10 IC, or CD8 TM and 4-1BB IC (Marin V. et al. Exp Hematol., 2007; 35: 1388-97), CD28 TM and CD28 IC (Wilkie S. et al. J Immunol., 2008; 180: 4901-9; Maher J. et al. Nat Biotechnol., 2002; 20: 70-5), and CD8 TM and CD28 IC (Marin V. et al. Exp Hematol., 2007; 35: 1388-97).

[0120]

[0155] Sequence information for the above activating and costimulatory receptors is readily accessible in various databases. For example, examples of the human amino acid, gene, and mRNA sequences for these receptors are shown in Table 3.

[0121]

[0156] [Table 3]

[0122]

[0157] While Table 3 is presented with respect to human activating and costimulatory receptors, one of skill in the art will understand that homologous and orthologous versions of each receptor exist in most mammalian and vertebrate species. Thus, the above sequences are provided only as non-limiting examples of receptor sequences that may be included in a CAR of the first aspect of the invention, and homologous and orthologous sequences from any desired species may be used to generate a CAR suitable for a given species.

[0123]

[0158] In some embodiments of the present invention, the transmembrane domain and a portion of the signaling domain share homology with the same molecule. For example, a portion containing the transmembrane domain and signaling domain of CD3 may be utilized. In some embodiments, the transmembrane domain comprises or consists of a sequence identical to all or a portion of the transmembrane domain of CD28, and the signaling domain comprises or consists of a sequence identical to all or a portion of the intracellular domain of CD28.

[0124]

[0159] In some embodiments of the present invention, the signaling domain comprises a portion derived from an activating receptor and a portion derived from a costimulatory receptor. Without wishing to be bound by theory, in this context, antigen recognition by the antigen recognition domain of the CAR simultaneously induces both an intracellular activation signal and an intracellular costimulatory signal. Consequently, this mimics antigen presentation by an APC expressing a costimulatory ligand. Alternatively, the CAR may have a signaling domain comprising a portion derived from either an activating receptor or a costimulatory receptor. In this alternative form, the CAR induces only either the activation intracellular signaling cascade or the costimulatory intracellular signaling cascade.

[0125]

[0160] In some embodiments of the invention, the signaling domain comprises or consists of a sequence identical to all or part of the intracellular domain of 4-1BB and CD3-ζ chain.

[0126]

[0161] In some embodiments, the CAR has a signaling domain comprising a portion of a single activating receptor and a portion of multiple costimulatory receptors. In some embodiments, the CAR has a signaling domain comprising a sequence identical to a portion of multiple activating receptors and a portion from a single costimulatory receptor. In some embodiments, the CAR has a signaling domain comprising a sequence identical to a portion of multiple activating receptors and a portion from multiple costimulatory receptors. In some embodiments, the CAR has a signaling domain comprising a sequence identical to a portion of a single activating receptor and a portion from two costimulatory receptors. In some embodiments, the CAR has a signaling domain comprising a sequence identical to a portion of a single activating receptor and a portion from three costimulatory receptors. In some embodiments, the CAR has a signaling domain comprising a sequence identical to a portion of two activating receptors and a portion from one costimulatory receptor. In some embodiments, the CAR has a signaling domain comprising a sequence identical to a portion of two activating receptors and a portion from two costimulatory receptors. As will be appreciated, there are further variations in the number of activating receptors and costimulatory receptors, and the above examples are not intended to limit the possible combinations included herein.

[0127]

[0162] In some embodiments of the invention, the sequence of the transmembrane domain and at least a portion of the signaling domain have sequence similarity to portions of different molecules, hi some embodiments, the transmembrane domain comprises or consists of a sequence identical to all or a portion of the transmembrane domain of CD28, and the signaling domain comprises or consists of a sequence identical to all or a portion of the intracellular domain of 4-1BB and the CD3-zeta chain.

[0128]

[0163] CARs are now referred to as first through fifth generation (see Labanieh L and Mackall CL. (2023), CAR immune cells: design principles, resistance, and the next generation. Nature, 614(7949): pg635-648; and Zheng Z, et al. (2023). Fine-Tuning through Generations: Advances in Structure and Production of CAR-T Therapy. Cancers (Basel). 3;15(13):3476, the entire disclosures of which are incorporated herein). In some embodiments, the CAR of the present invention is a third generation or later CAR (i.e., comprises an activation domain and two or more costimulatory domains). In some embodiments, the CAR is a fourth generation or later CAR (i.e., T cells redirected to TRUCK—universal cytokine-mediated killing). In some embodiments, the CAR T cells comprise a termination receptor to allow for elimination of the CAR T cells after administration.

[0129]

[0164] Chimeric Antigen Receptor

[0165] An exemplary chimeric antigen receptor (CAR) of the present invention was prepared using two scFv fusion proteins with two orientations: the variable light domain and the variable heavy domain of the MIL-38 antibody (WO 2016 / 168885 A1; and Truong Q, et al. (2016). Glypican-1 as a Biomarker for Prostate Cancer: Isolation and Characterization. J Cancer. May 21;7(8):1002-9.).

[0130]

[0166] As shown in Figures 15A-15F, two scFv domains were prepared in the following orientation:

[0167] 1. Light chain variable region (2)-feline linker (3) (PMID: 8309948)-heavy chain variable region (4) (designated by the code CNA500xxx); and

[0168] 2. Heavy chain variable region (4)-fetal linker (3)-light chain variable region (2) (designated by the code CNA510xxx).

[0169] In addition, three different linker domains were utilized: Linker1 - IgG4 hinge (5) (SEQ ID NO: 15) - (shown with code CNA5x02xx); Linker2-IgG4 hinge+IgG4 CH3(11) (SEQ ID NO: 16)-(designated by code CNA5x03xx); and Linker 3 - IgG hinge + IgG4 CH2 L235D and N297Q mutations + IgG4 CH3(12) (SEQ ID NO: 17) - (denoted by code CNA5x04xx).

[0131]

[0170] The sequences (SEQ ID NOs) of the chimeric antigen receptors and their component parts are shown in Table 4.

[0132]

[0171] [Table 4] TIFF2025540193000006.tif103149

[0133]

[0172] 15A-15F, as a specific embodiment, the CAR exemplified in the present invention comprises the following components: a MIL-38 leader sequence (1), a transmembrane region with sequence identity to a portion of CD28 (6), a costimulatory domain with sequence identity to a portion of 4-1BB (7), and an activation domain with sequence identity to a portion of CD3 zeta (8). The exemplified CAR also contained a truncated ECF receptor (EGFRt) (10), which allows for transduction and expression analysis in cells. The EGFRt was linked by a self-cleavage site, T2a (9), which allows for separation of the EGFRt from the CAR. The sequences of EGFRt and T2a are known in the art and are published in WO 2022 / 104424.

[0134]

[0173] In some embodiments, the CAR comprises an antigen recognition domain specific for GPC1, a linker domain with sequence identity to an IgG4 hinge region, a transmembrane region with sequence identity to a CD28 transmembrane sequence, an intracellular portion with sequence identity to the signaling region of 4-1BB, and / or an intracellular portion with sequence identity to the signaling region of CD3 zeta, or a functional variant of the described portions, domains, or regions.

[0135]

[0174] In some embodiments, the CAR comprises an antigen recognition domain specific for GPC1, a linker domain with sequence identity to an IgG4 hinge region combined with an IgG4 CH3 region, a transmembrane region with sequence identity to a CD28 transmembrane sequence, an intracellular portion with sequence identity to the signaling region of 4-1BB, and / or an intracellular portion with sequence identity to the signaling region of CD3 zeta, or a functional variant of the described portions, domains, or regions.

[0136]

[0175] In some embodiments, the CAR comprises an antigen recognition domain specific for GPC1, an IgG4 CH2 region (which may include the L235D and N297Q mutations), and a linker domain with sequence identity to an IgG4 hinge region combined with an IgG4 CH3 region, a transmembrane region with sequence identity to the CD28 transmembrane sequence, an intracellular portion with sequence identity to the signaling region of 4-1BB, and / or an intracellular portion with sequence identity to the signaling region of CD3 zeta, or a functional variant of the described portions, domains, or regions.

[0137]

[0176] In some embodiments of the invention, the chimeric antigen receptor comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 23, 24, 25, and 26 (CNA500200, CNA500300, CNA500400, CNA510200, CNA510300, and CNA510400), or a functional variant of this amino acid sequence.

[0138]

[0177] As should be understood by those skilled in the art, modifications of the CAR receptors described herein can be made without departing from the scope of the present invention. For example, with respect to SEQ ID NOs: 21, 22, 23, 24, 25, and 26, the preferred function of the CAR is to recognize GPC1 and induce an intracellular signal that leads to the activation of T cells expressing the CAR. Therefore, modifications to a portion of the amino acid sequence of the chimeric antigen receptor can be made without significantly altering the specificity of the CAR and / or the activation of cells (e.g., T cells) expressing the CAR. Such modifications include, but are not limited to, modifications in the hinge region of the chimeric antigen receptor, modifications in the transmembrane domain, and modifications in portions of the activating receptor and / or costimulatory receptor, including the intracellular domain of the chimeric antigen receptor. When making such modifications, those skilled in the art will utilize their knowledge and techniques with the intention of arriving at an effective CAR. Therefore, the scope of modifications does not include modifications that would be readily recognized by those skilled in the art as resulting in the abrogation of CAR function.

[0139]

[0178] In some embodiments of the invention, the chimeric antigen receptor has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 129% or consisting of a variant of SEQ ID NO: 21, 22, 23, 24, 25, or 26 having an amino acid sequence with at least 98%, at least 98.2%, at least 98.4%, at least 98.6%, at least 98.8%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity thereto.

[0140]

[0179] Nucleic acid constructs and genetic modification of cells

[0180] CAR described herein can be produced by any means known in the art, but preferably by recombinant DNA technology.If necessary, the nucleic acid encoding some regions of chimeric receptor can be prepared by standard molecular cloning techniques known in the art (such as genome library screening, PCR, primer-assisted ligation, site-directed mutagenesis, etc.) to assemble into complete coding sequence.The obtained coding region can preferably be inserted into expression vector and used to transform suitable expression host cell line (preferably T lymphocyte cell line, most preferably autologous T lymphocyte cell line).

[0141]

[0181] Therefore, the present invention further provides a nucleic acid molecule having a nucleic acid sequence encoding the above-mentioned chimeric antigen receptor, or a nucleic acid construct comprising the nucleic acid molecule.

[0142]

[0182] Furthermore, the nucleic acid construct may be an expression vector comprising a nucleic acid sequence encoding the chimeric antigen receptor.

[0143]

[0183] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 23, 24, 25, and 26, or a variant of these sequences as previously defined.

[0144]

[0184] The nucleic acid molecule can contain any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified or modified RNA or DNA. For example, the nucleic acid molecule can include single-stranded and / or double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, and hybrid molecules containing DNA and RNA that can be single-stranded, more typically double-stranded, or a mixture of single- and double-stranded regions. In addition, the nucleic acid molecule can contain triple-stranded regions containing RNA, DNA, or both RNA and DNA. The nucleic acid molecule can also contain one or more modified bases or modified DNA or RNA backbones for stability or other reasons. Various modifications can be made to DNA and RNA, and therefore the term "nucleic acid molecule" encompasses chemically, enzymatically, or metabolically modified forms.

[0145]

[0185] In some embodiments of the invention, the nucleic acid molecule comprises a portion of the nucleotide sequence set forth in SEQ ID NO: 7, 8, 9, 10, 11, or 12, or a functional variant thereof, that encodes the amino acids set forth in SEQ ID NO: 21, 22, 23, 24, 25, and 26.

[0146]

[0186] For the avoidance of doubt, functional variants of relevant portions of SEQ ID NO: 7, 8, 9, 10, 11, or 12 include sequence variants with one or more different nucleic acids, but still encode the same amino acid sequence. Due to the degeneracy of the genetic code, a large number of nucleic acids can encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Those skilled in the art will recognize that each codon in a nucleic acid sequence (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to obtain a functionally identical molecule. Thus, each silent variation of a nucleotide sequence encoding a polypeptide is implicit in each described sequence.

[0147]

[0187] It will be understood that the nucleic acid construct, according to the present invention, may further comprise one or more of: an origin of replication in one or more hosts; a selectable marker gene active in one or more hosts; and / or one or more transcription control sequences, wherein expression of the nucleic acid molecule is under the control of the transcription control sequences.

[0148]

[0188] As used herein, the term "selectable marker gene" includes any gene that confers a phenotype on a cell in which it is expressed, facilitating the identification and / or selection of cells that have been transfected or transduced with a construct.

[0149]

[0189] A "selection marker gene" includes any nucleotide sequence that, when expressed by cells transduced with a construct, confers a phenotype on the cells that facilitates identification and / or selection of those transduced cells. A range of nucleotide sequences encoding suitable selection markers are known in the art (e.g., Mortesen, R. M. and Kingston, R. E. Curr Protoc Mol Biol, 2009; Unit 9.5). Exemplary nucleotide sequences encoding selectable markers include, among others, an adenosine deaminase (ADA) gene; a cytosine deaminase (CDA) gene; a dihydrofolate reductase (DHFR) gene; a histidinol dehydrogenase (hisD) gene; a puromycin-N-acetyltransferase (PAC) gene; a thymidine kinase (TK) gene; a xanthine-guanine phosphoribosyltransferase (XGPRT) gene, or antibiotic resistance genes, such as the ampicillin resistance gene, the puromycin resistance gene, the bleomycin resistance gene, the hygromycin resistance gene, the kanamycin resistance gene, and the ampicillin resistance gene; fluorescent reporter genes, such as genes encoding green, red, yellow, or blue fluorescent proteins; and luminescence-based reporter genes, such as the luciferase gene, which allow for optical selection of cells using techniques such as fluorescence-activated cell sorting (FACS). Furthermore, cell selection markers for T cells are specifically discussed in Barese, CN and Dunubar CE, Hum. Gene Ther., 2011;22(6):pp.659-68. These markers include the neomycin (NEO) resistance gene, ΔNGFR (non-signaling NGFR), truncated CD34, and truncated non-signaling CD19 (ΔCD19). In embodiments of the present invention (described further herein), a truncated form of the epidermal growth factor receptor (EGFRt) is utilized. Additional techniques for tracking CAR T cells in vivo, such as modified eDHFD, have been developed (see Sellmyer, MA et al. Mol. Ther., 2020;28(1):pp.42-51).

[0150]

[0190] Furthermore, it should be noted that the selectable marker gene can be a separate open reading frame in the construct or can be expressed as a fusion protein with another polypeptide (e.g., CAR).

[0151]

[0191] As described above, the nucleic acid construct may also include one or more transcription control sequences. The term "transcription control sequence" should be understood to include any nucleic acid sequence that affects the transcription of an operably linked nucleic acid. Transcription control sequences may include, for example, a leader, a polyadenylation sequence, a promoter, an enhancer sequence or upstream activation sequence, and a transcription terminator. Typically, a transcription control sequence includes at least a promoter. The term "promoter," as used herein, describes any nucleic acid that confers, activates, or enhances the expression of a nucleic acid in a cell.

[0152]

[0192] In some embodiments, the nucleic acid molecule of the second aspect of the invention is operably linked to at least one transcription control sequence. For purposes herein, a transcription control sequence is considered to be "operably linked" to a given nucleic acid molecule if it is capable of promoting, inhibiting, or otherwise regulating transcription of the nucleic acid molecule. Thus, in some embodiments, the nucleic acid molecule is under the control of a transcription control sequence, such as a constitutive promoter or an inducible promoter.

[0153]

[0193] A promoter can constitutively or differentially control the expression of an operably linked nucleic acid molecule with respect to the cell, tissue, or organ in which expression occurs. Thus, a promoter can include, for example, a constitutive promoter or an inducible promoter. A "constitutive promoter" is a promoter that is active under most environmental and physiological conditions. An "inducible promoter" is a promoter that is active under specific environmental or physiological conditions. The present invention contemplates the use of any promoter that is active in the cell of interest. Thus, a variety of promoters can be readily identified by one of skill in the art.

[0154]

[0194] Constitutive mammalian promoters may include, but are not limited to, Simian Virus 40 (SV40), cytomegalovirus (CMV), P-actin, ubiquitin C (UBC), elongation factor-1 alpha (E3A), phosphoglycerate kinase (PGK), and CMV early enhancer / chicken beta actin (CAGG).

[0155]

[0195] Inducible promoters may include, but are not limited to, chemically inducible promoters and physically inducible promoters. Chemically inducible promoters include promoters whose activity is controlled by compounds such as alcohol, antibiotics, steroids, metal ions, or other compounds. Examples of chemically inducible promoters include, among others, tetracycline-regulated promoters (see, e.g., U.S. Pat. Nos. 5,851,796 and 5,464,758); steroid-responsive promoters, such as glucocorticoid receptor promoters (see, e.g., U.S. Pat. No. 5,512,483), ecdysone receptor promoters (see, e.g., U.S. Pat. No. 6,379,945), and the like; and metal-responsive promoters, such as metallothionein promoters (see, e.g., U.S. Pat. Nos. 4,940,661, 4,579,821, and 4,601,978).

[0156]

[0196] As mentioned above, the control sequence may also include a terminator. The term "terminator" refers to a DNA sequence at the end of a transcription unit, which indicates the termination of transcription. A terminator is a 3'-terminal untranslated DNA sequence that generally contains a polyadenylation signal, which promotes the addition of a polyadenylation sequence to the 3' end of a primary transcript. Similar to a promoter sequence, a terminator may be any terminator sequence that is operable in the cells, tissues, or organs intended for use. Suitable terminators will be known to those skilled in the art.

[0157]

[0197] As will be appreciated, the nucleic acid constructs of the present invention may further comprise additional sequences, such as sequences enabling enhanced expression, cytoplasmic or membrane transport, and location signals. Non-limiting examples include, among others, an internal ribosome entry site (IRES), an N-terminal interleukin-2 signal peptide (Moot R. et al., Mol Ther Oncolytics, 2016;3:16026), CSF2RA, an IgE leader sequence (WO 2017147458), and an influenza hemagglutinin signal sequence (Quitterer, U. et al., Biochem. Biophys. Res., 2011:409(3):pp.544-579). A review of signal peptides is provided in Owki, H. et al., Eur. J. Cell Biol., 2018;97(6):pp.422-441, which is incorporated herein by reference.

[0158]

[0198] The present invention extends to all genetic constructs essentially as described herein, which constructs may further comprise nucleotide sequences intended for the maintenance and / or replication of the genetic construct in eukaryotes and / or for the integration of the genetic construct, or parts thereof, into the genome of eukaryotic cells.

[0159]

[0199] The nucleic acid construct may be in any suitable form (e.g., in the form of a plasmid, phage, transposon, cosmid, chromosome, vector, etc.) that is replicable when associated with appropriate control elements and capable of transferring the genetic sequences contained within the construct between cells.

[0160]

[0200] Thus, the term vector includes not only viral vectors, but also cloning vehicles and expression vehicles. In some embodiments, the nucleic acid construct is a vector. In some embodiments, the vector is a viral vector, and thus the present invention provides a viral vector comprising a nucleic acid molecule or nucleic acid construct encoding a CAR as described above. In some embodiments, the vector is a DNA vector or an mRNA vector.

[0161]

[0201] In at least some embodiments, the present invention provides nucleic acid molecules or nucleic acid constructs encoding the above-described CARs for use in preparing genetically modified cells. Additionally, in at least some embodiments, the present invention provides use of the nucleic acid molecules in preparing vectors for transforming, transfecting, or transducing cells, such as those described herein. Cells suitable for genetic modification can be xenogeneic or autologous.

[0162]

[0202] In some embodiments, the cells are used in a method or in the preparation of a medicament for the prevention or treatment of cancer. Thus, in some embodiments, the invention provides for the use of a vector in the preparation of a medicament for the prevention or treatment of cancer, particularly ovarian cancer, which expresses glypican-1.

[0163]

[0203] Methods for the deliberate introduction (transfection / transduction) of exogenous genetic material, such as the present nucleic acid constructs, into eukaryotic cells are known in the art. It will be understood that the optimal method for introducing a nucleic acid construct into a desired host cell will depend on many factors, including the size of the nucleic acid construct, the type of host cell, the desired efficiency of transfection / transduction, and the ultimate desired or required viability of the transfected / transduced cells. Non-limiting examples of such methods include: chemical transfection using chemicals such as cationic polymers, calcium phosphate, or structures such as liposomes and dendrimers; non-chemical methods such as electroporation (see Potter and Heller. "Transfection by Electroporation." Curr. Prot. Mol. Bio., ed. Frederick M. Ausubel et al. 2003:Unit-9.3), sonoporation (Wang, M et al. Sci. Reps., 2018;8:3885), heat shock or optical transfection; particle-based methods such as "gene gun" delivery, magnetofection, or imparefection, lipid nanoparticles, or viral transduction.

[0164]

[0204] Various viral transduction techniques for mammalian cells are known in the art. Common viral vectors include lentivirus and retrovirus. Exemplary protocols are shown in Wang L et al., Proc. Natl. Acad. Sci., 2011; 108: E803-12. Alternative viral vectors include HSV, adenovirus, and AAV (Howarth J et al., Cell. Bio. & Toxic., 2010, vol. 26, issue 1, pp 1-20).

[0165]

[0205] In some embodiments, the present invention provides a lentivirus comprising a nucleic acid encoding a chimeric antigen receptor described herein. Additionally, the present invention provides the use of a viral vector (preferably a lentivirus or a retrovirus such as a gammaretrovirus) in the preparation of a genetically modified cell or medicament for the prevention or treatment of cancer, or for the killing of cells that express glypican-1 or aberrantly express glypican-1.

[0166]

[0206] By transducing cells, the DNA encoding the CAR can be integrated into the genome. Alternatively, this DNA can be transiently expressed in the transduced cells. Each of these has its advantages and disadvantages. The DNA integrated into the genome is stably expressed and replicated in progeny cells during cell replication. This ensures a robust immune response and ensures a significant increase in CAR-expressing T cells in vivo.

[0167]

[0207] Alternatively, transient transduction (often achieved by transduction with mRNA) allows temporary expression of the CAR in cells, which usually results in significantly reduced responsiveness but allows the physician additional control to increase or decrease the "dose" as needed.

[0168]

[0208] As explained above, in some embodiments, the present invention provides the use of a DNA vector or recombinant DNA in the preparation of a viral vector for gene transfer into cells. The cells may be any cells, but the following are preferred examples.

[0169]

[0209] The nucleic acid construct will be selected depending on the desired transfection / transduction method. In some embodiments, the nucleic acid construct is a viral vector, and the method for introducing the nucleic acid construct into host cells is viral transduction. Methods using viral transduction to induce CAR expression in PBMCs, such as T cells, are known in the art (Parker, LL. et al. Hum Gene Ther. 2000; 11:2377-87), and more generally, methods using retroviral systems to transduce mammalian cells are known (Cepko, C. and Pear, W. Curr Protoc Mol Biol. 2001, unit 9.9). In some embodiments, the nucleic acid construct is a plasmid, cosmid, artificial chromosome, etc., and can be transfected into cells by any suitable method known in the art.

[0170]

[0210] Techniques for selecting / isolating cell subsets are known in the art. These techniques include fluorescence-activated cell sorting (Basu S. et al. J. Vis. Exp. 2010; 41:1546), techniques that utilize antibodies immobilized on a substrate, such as the magnetic cell sorting (MACS®) device (Zola H. et al. Blood, 2005; 106(9):3123-6), which immunomagnetically selects cells expressing a desired marker, or the use of microfluidic chips.A range of cell markers may be used to isolate cells of the immune system, including, but not limited to, BCR, CCR10, CD1a, CD1b, CD1c, CD1d, CD3, CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD13, CD16, CD19, CD21, CD23, CD25, CD27, CD31, CD32, CD33, CD34, CD38, CD39, CD40, CD43, CD45, CD45RA, CD45RO, CD D48, CD49d, CD49f, CD51, CD56, CD57, CD62, CD62L, CD68, CD69, CD62, CD62L, CD66b, CD68, CD69, CD73, CD78, CD79a, CD79b, CD80, CD 81, CD83, CD84, CD85g, CD86, CD94, CD103, CD106, CD115, CD117, CD122, CD123, CD126, CD127, CD130, CD138, CD140a, CD140b, CD141, CD152, CD159a, CD160, CD161, CD163, CD165, CD169, CD177, CD178, CD183, CD185, CD192, CD193, CD194, CD195, CD196, CD198, CD200, CD200R, CD203c, CD205, CD206, CD207, CD209, CD212, CD217, CD218alpha, CD229, CD244, CD268, CD278, CD279, CD282, CD284, CD289, CD294, CD303, CD304, CD314, CD319, CD324, CD335, CD336, CXCR3, Dectin-1, Tc epsilon R1 alpha, Flt3, Granzyme A, Granzyme B, IL-9, IL-13apha1, IL-21R, iNOS, KLRG1, MARCO, MHC class II, RAG, ROR gamma T, Singlec-8, ST2, TCR alpha / beta, TCR gamma / delta, TLR4, TLR7, VEGF, ZAP70.

[0171]

[0211] Of particular note are the T cell markers CCR10, CD1a, CD1c, CD1d, CD2, CD3, CD4, CD5, CD7, CD8, CD9, CD10, CD11b, CD11c, CD13, CD16, CD23, CD25, CD27, CD31, CD34, CD38, CD39, CD43, CD45, CD45RA, CD45RO, CD48, CD49d, CD56, CD62, CD62L, CD68, CD69, CD73, CD79a, CD80, CD81, CD83, CD84, CD86, CD94, CD103, CD122, CD126, CD127, CD130, CD140a, and CD140. b, CD152, CD159a, CD160, CD161, CD165, CD178, CD183, CD185, CD192, CD193, CD194, CD195, CD196, CD198, CD200, CD200R, CD212, CD217, CD218alpha, CD229, CD244, CD278, CD279, CD294, CD304, CD314, CXCR3, Flt3, Granzyme A, Granzyme B, IL-9, IL-13alpha1, IL-21R, KLRG1, MHC class II, RAG, ROR gamma T, ST2, TCR alpha / beta, TCR gamma / delta, ZAP70. Particularly preferred cell markers for T cell selection include TCR gamma, TCR delta, CD3, CD4, and CD8.

[0172]

[0212] The isolated cells can then be cultured to alter, expand, or activate cell activity. Techniques for expanding and activating cells are known in the art (Wang X. and Riviere I. Mol. Thera. Oncolytics. 2016;3:16015). These techniques include the use of anti-CD3 / CD28 microbeads (Miltenyi Biotec or Thermofisher Scientific—as per manufacturer's instructions) or other forms of immobilized CD3 / CD28 activating antibodies. The activated / genetically modified cells can then be expanded in vitro in the presence of cytokines (e.g., IL-2, IL-12, IL-15, or IL-17) and then cryopreserved. A summary of methods for expanding CAR T cells is provided in Wang and Riviera (ibid.).

[0173]

[0213] The present invention further provides genetically modified cells comprising the chimeric antigen receptor, nucleic acid molecule, or nucleic acid construct described above. In some embodiments, the genetically modified cell comprises a genomically integrated version of the nucleic acid molecule or construct. In some embodiments, the genetically modified cell is a leukocyte. In some embodiments, the genetically modified cell is a peripheral blood mononuclear cell (PBMC). In some embodiments, the genetically modified cell is a bone marrow cell. In some embodiments, the genetically modified cell is a monocyte. In some embodiments, the genetically modified cell is a macrophage. In some embodiments, the genetically modified cell is a lymphocyte. In some embodiments, the genetically modified cell is a T cell. In some embodiments, the genetically modified cell is an alpha beta (αβ) T cell. In some embodiments, the genetically modified cell is a gamma delta (γδ) T cell. In some embodiments, the genetically modified cell is a CD3+ T cell (e.g., a naive CD3+ T cell or a memory CD3+ T cell). In some embodiments, the T cells are CD4+ T cells (e.g., naive CD4+ T cells or memory CD4+ T cells). In some embodiments, the T cells are CD8+ T cells (e.g., naive CD8+ T cells or memory CD8+ T cells). In some embodiments, the genetically modified cells are natural killer cells. In some embodiments, the genetically modified cells are natural killer T (NKT) cells.

[0174]

[0214] Use of CARs to treat or prevent cancer

[0215] In addition to our confirmation of high expression of glypican-1 in ovarian cancer, high expression of glypican-1 has also been reported in pancreatic ductal adenocarcinoma, breast cancer, cervical cancer, lung cancer, malignant pleural mesothelioma, and glioblastoma (Nishigaki T, et al. (2020). Anti-glypican-1 antibody-drug conjugate is a potential therapy against pancreatic cancer. Br J Cancer, 122, 1333-41; Duan L, et al. (2013). GPC-1 may serve as a predictor of perineural invasion and a prognosticator of survival in pancreatic cancer. Asian J Surg, 36, 7-12; Matsuda K, et al. (2001). Glypican-1 is overexpressed in human breast cancer and modulates the mitogenic effects of multiple heparin-binding growth factors in breast cancer cells. Cancer Res, 61, 5562-9; Matsuzaki S,et al.(2018).Anti-glypican-1 antibody-drug conjugate exhibits potent preclinical antitumor activity against glypican-1-positive uterine cervical cancer.Int J Cancer,142,1056-66;Chiu K,et al.(2018).Glypican-1 immunohistochemistry does not separate mesothelioma from pulmonary adenocarcinoma.Mod Pathol, 31, 1400-3; and Saito T, et al. (2017).High expression of glypican-1 predicts dissemination and poor prognosis in glioblastomas. (See World Neurosurg, 105, 282-8.)

[0175]

[0216] Thus, the CARs of the present invention may be used to treat or prevent any cancer associated with glypican-1 expression (for example, but not limited to, pancreatic ductal adenocarcinoma, breast cancer, cervical cancer, lung cancer, malignant pleural mesothelioma, glioblastoma, and ovarian cancer). A particularly contemplated embodiment of a method for treating or preventing cancer is a method for treating or preventing ovarian cancer in a subject, comprising administering anti-GPC1 CAR cells to the subject.

[0176]

[0217] The present invention also provides a pharmaceutical composition comprising a genetically modified cell containing the above-mentioned chimeric antigen receptor, nucleic acid molecule, or nucleic acid construct and one or more pharmaceutically acceptable carriers, excipients, or diluents, for use in the prevention or treatment of cancer (for example, but not limited to, pancreatic ductal adenocarcinoma, breast cancer, cervical cancer, lung cancer, malignant pleural mesothelioma, glioblastoma, and ovarian cancer). In a preferred embodiment, the cancer is ovarian cancer.

[0177]

[0218] Diagnostic and prognostic methods

[0219] Also provided is a method for diagnosing a subject with ovarian cancer or assessing the prognosis of said subject, comprising determining the amount of glypican-1 in ovarian cells from the subject or cells suspected of being cancerous, wherein an elevated amount of glypican-1 indicates the presence of ovarian cancer and / or a poor prognosis.

[0178]

[0220] In some embodiments, ovarian cancer is classified according to the FIGO system and includes ovarian cancer, fallopian tube cancer, or peritoneal cancer (see Kehoe, S and Bhatla, N, FIGO cancer report 2021, International Journal of Gynecology & Obstetrics).

[0179]

[0221] "Elevated expression of glypican-1," as used herein, refers to an increase in glypican-1 mRNA or protein compared to a control value. In some embodiments, the control value is a normal expression level. In some embodiments, the control value is a threshold value. In some embodiments, the control value is a pre-cancerous value. In some embodiments, if the ovarian cancer is recurrent ovarian cancer, the control value is the value before recurrence.

[0180]

[0222] In some embodiments, normal expression is determined from non-cancerous ovarian cells, or in the case of some ovarian cancers, fallopian tube cells, which in some embodiments are the same type of cells (e.g., epithelial cells) as the cancer cells.

[0181]

[0223] In some embodiments, the threshold is a predetermined threshold, which may be based on previous analyses of the subject or may be based on a population value, such as a median or mean value determined from multiple samples of non-cancerous or healthy cells from comparable members of the population.

[0182]

[0224] In some embodiments, expression of glypican-1 is increased by at least or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120%, 140%, 160%, 180%, 200%, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold.

[0183]

[0225] In some embodiments, poor prognosis refers to a lower overall survival rate or lower progression-free survival rate for a subject. In some embodiments, elevated gene expression indicates lower overall survival rate. In some embodiments, elevated protein expression indicates lower overall survival rate. In some embodiments, elevated gene expression and elevated protein indicate lower overall survival. In some embodiments, elevated gene expression indicates lower progression-free survival. In some embodiments, elevated protein expression indicates lower progression-free survival. In some embodiments, elevated gene expression and elevated protein indicate lower progression-free survival.

[0184]

[0226] In some embodiments, a poor prognosis refers to a fast progression of the cancer or fast growth of the tumor. In some embodiments, a poor prognosis refers to a high likelihood that the cancer will progress to a higher stage. In some embodiments, a poor prognosis refers to a high likelihood of metastasis of the primary cancer.

[0185]

[0227] The stages of ovarian cancer (including fallopian tube cancer and peritoneal cancer) are shown in Table 5.

[0186]

[0228] [Table 5] JPEG2025540193000008.jpg161149

[0187]

[0229] In some embodiments, the ovarian cancer is recurrent ovarian cancer.

[0188]

[0230] In some embodiments, the ovarian cancer is high-grade serous ovarian cancer.

[0189]

[0231] In some embodiments, the diagnostic or prognostic evaluation method is performed on a subject who has previously been treated for ovarian cancer, where such treatment included one or more of chemotherapy, surgical resection or debulking, radiation therapy, hormone therapy, or immunotherapy.

[0190]

[0232] In some embodiments of the method of diagnosis or prognosis evaluation, the ovarian cancer is recurrent ovarian cancer and the elevated amount of glypican-1 is compared to cancer tissue before the recurrence.

[0191]

[0233] In some embodiments of the diagnostic or prognostic evaluation methods, elevated levels of glypican-1 are compared to non-cancerous ovarian tissue or comparison tissue not suspected of cancer. Such non-cancerous tissue may be obtained from the same individual or a different individual. In some embodiments, the comparison tissue is from the same sample as the suspected or confirmed cancer cells. In some embodiments, the comparison tissue is from a different ovary of the same individual.

[0192]

[0234] In some embodiments of the diagnostic or prognostic assessment methods, protein expression is determined by a binding agent that preferentially or selectively binds to glypican 1. Such binding agents include antibodies or binding fragments of antibodies, or other such binding agents described herein as binding agents that can be used to treat cancer. Also included are fusion proteins, such as single-chain variable fragments comprising the variable light and variable heavy chain sequences of an antibody.

[0193]

[0235] How to analyze RNA

[0236] RNA isolation

[0237] Various methods for RNA isolation are known in the art, and an appropriate method will be selected by the skilled artisan, taking into account particular requirements and constraints.

[0194]

[0238] There are at least three major techniques widely used in the art for RNA extraction: organic extractions such as phenol-guanidine isothiocyanate (GITC)-based solutions, silica membrane-based spin column techniques, and paramagnetic particle techniques.

[0195]

[0239] Various commercially available kits are known in the art for isolating RNA, including, for example, AxyPrep Multisource Total RNA Miniprep (Axygen), RNeasy® Mini (Qiagen), EasySpin (Citomed), Ilustra RNAspin Mini RNA Isolation Kit (GE), TRIzol® and TRIzol plus RNA Purification System (Invitrogen), and EZNA™ Total RNA Kit II (omega bio-tek). A comparison of the advantages, disadvantages, and performance of these kits can be found in Tavares, L., et al. (2011), Comparison of different methods for DNA-free RNA isolation from SK-N-MC neuroblastoma, BMC Res Notes; 4, 3. Alternatively, protocols for RNA isolation are provided in Liu and Harada (2013), RNA Isolation from Mammalian Samples, Current Protocols in Molecular Biology; 103: 4.16.1-4.16.16.

[0196]

[0240] Reverse transcription polymerase chain reaction (RT-PCR)

[0241] RT-PCR is one of the most sensitive techniques for quantifying a specific nucleic acid sample.

[0197]

[0242] To perform RT-PCR, RNA is extracted and purified from tissue samples. This RNA is then reverse transcribed using retroviral reverse transcriptase to convert it into complementary DNA (cDNA). This cDNA is then combined with a thermostable DNA polymerase, deoxynucleotides, and forward and reverse primers in a buffer solution, and then thermally cycled to denature (separate) the double-stranded DNA, allow primers to anneal to the separated DNA strands, and allow DNA polymerase to extend new DNA copies. This process is repeated to amplify the sequence of the strand between the forward and reverse primers, resulting in a short DNA sequence known as an amplicon. This amplicon can then be visualized and / or quantified. An example protocol for performing RT-PCR is described in Mitchel, J. (2002) RT-PCR Protocols. Methods in Molecular Biology, Vol. 193.

[0198]

[0243] In situ hybridization

[0244] In situ hybridization allows for the identification and localization of nucleic acids (e.g., RNA) within a biological sample, and therefore, unlike some other techniques, in situ hybridization does not simply confirm the presence of a nucleic acid or quantify the expression of this nucleic acid, but can indicate the tissue distribution of the nucleic acid within the sample.

[0199]

[0245] In situ hybridization utilizes hybridization of a target nucleic acid (e.g., mRNA) with an oligonucleotide (e.g., cDNA) or RNA probe (riboprobe). Each probe is conjugated with a detection moiety, such as a radioactive label, enzyme, or fluorophore. Hybridization of the complementary probe nucleic acid sequence with the target sequence can then be detected or visualized to identify the location and amount of the target nucleotide.

[0200]

[0246] Techniques for performing in situ hybridization are known in the art, e.g., Henley SR et al. (2021), RNA in situ hybridization for human papillomavirus testing in oropharyngeal squamous cell carcinoma on a routine clinical diagnostic platform. Journal of Oral Pathology & Medicine; 50, 1, pp. 68-75.

[0201]

[0247] Nuclease protection assay

[0248] Techniques for performing ribonuclease protection assays are known in the art, and include those described in Henttu P. (2001), Quantification of mRNA levels using ribonuclease protection assay. Methods in Molecular Biology; 169, pp. 65-79.

[0202]

[0249] Northern analysis

[0250] After purification of RNA samples from tissue or cell samples, they are separated by size by gel (e.g., agarose) electrophoresis under denaturing conditions (e.g., in the presence of formaldehyde or glyoxal / DMSO). The size-separated RNA is then transferred to a membrane (e.g., a nitrocellulose or nylon membrane). This transfer can be performed by techniques such as capillary transfer, vacuum transfer, salt gradient transfer, or electrophoretic transfer. The RNA is then crosslinked or fixed to the membrane and hybridized with a specifically labeled probe.

[0203]

[0251] Northern blotting allows for the size-based analysis and quantification of transcripts, which allows for the analysis of different expression variants of genes.

[0204]

[0252] An example of Northern blot techniques is described in Brown, T et al. (2004), Analysis of RNA by Northern and Slot Blot Hybridization, Current Protocols in Molecular Biology; 4.9.1-4.9.19.

[0205]

[0253] RNA microarray

[0254] Microarrays utilize a series of specific oligonucleotide probes immobilized in an array on a solid support, each location-specific probe having a known sequence that hybridizes specifically to a complementary nucleic acid.

[0206]

[0255] Nucleic acid samples for microarray analysis are typically prepared by reverse transcribing mRNA isolated from the sample to produce cDNA. A fluorescent label may be added to the generated cDNA during reverse transcription or may be added upon completion.

[0207]

[0256] Labeled cDNA from the sample to be analyzed is then incubated with the immobilized probes on the microarray under high stringency conditions, after which unhybridized cDNA is removed. Fluorescence at each location is then quantified to indicate the amount of hybridized sample nucleic acid complementary to each immobilized probe.

[0208]

[0257] Various commercially available microarray chips are known in the art, including products from Affymetrix, Illumina, Agilent, Applied Microarrays, Eppendorf, and Arrayit. Additionally, microarray protocols are known in the art, including those provided by the National Human Genome Research Institute (https: / / research.nhgri.nih.gov / microarray / protocols.shtml) and Grant, GR, et al. (2007), Analysis and Management of Microarray Gene Expression Data. Current Protocols in Molecular Biology, 77:19.6.1-19.6.30. https: / / doi.org / 10.1002 / 0471142727.mb1906s77.

[0209]

[0258] RNA sequencing (RNA-Seq)

[0259] RNA-Seq utilizes next-generation sequencing platforms to analyze the sequence and expression of RNA in cells at any given time point. RNA-Seq can be used to analyze total RNA, microRNA, transfer RNA, and mRNA. Messenger RNA is reverse transcribed into cDNA, and then adapters are ligated to each end of the cDNA. Sequencing can be performed in either one direction (single-end sequencing) or both directions (paired-end sequencing), and sequences are aligned in silico to a reference genome database or assembled to obtain de novo transcripts. RNA quantification is performed by counting the number of reads that map to each locus in the reference genome. Counts can be quantified using various tools, such as HTSeq, FeatureCounts, Rcount, Maxcounts, FIXSEQ, Cuffquant, Sailfist, and Kallisto.

[0210]

[0260] Differential expression between two tissues (eg, cancer and non-cancer) can be calculated by known tools such as DESeq, edgeR, and Voom+limma.

[0211]

[0261] Protocols for performing RNA-Seq and analyzing the data are known in the art, including Kukurba KRand Montgomery SB (2015), RNA Sequencing and Analysis. Cold Spring Harbor Protocols, 11:951-969; and Costa-Silva J, et al. (2017), RNA-Seq differential expression analysis: An extended review and a software tool. PLoS ONE 12(12):e0190152. https: / / doi.org / 10.1371 / journal.pone.0190152.

[0212]

[0262] How to analyze proteins

[0263] Immunohistochemistry / immunostaining

[0264] One of the most common techniques for protein quantification and localization is immunohistochemistry. This technique involves fixing and embedding tissue, followed by sectioning and incubation with a primary antibody against the protein of interest. This primary antibody can be directly labeled or detected by a labeled secondary antibody. Common labels include enzymes (e.g., horseradish peroxidase), fluorescent tags, radioactive labels, or conjugates such as biotin. This label can then be detected and used to identify the location and / or quantity of the protein of interest.

[0213]

[0265] Methods for performing IHC are known in the art and include Schlederer M, et al. (2014) Reliable Quantification of Protein Expression and Cellular Localization in Histological Sections. PLoS ONE 9(7); Goldstein, M. and Watkins, S. (2008), Immunohistochemistry. Current Protocols in Molecular Biology, 81:14.6.1-14.6.23; and Goldstein, M. and Watkins, S. (2008), Immunohistochemistry. Current Protocols in Molecular Biology, 81:14.6.1-14.6.23.

[0214]

[0266] Alternative methods for protein quantification include: high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC / MS), enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunoelectrophoresis, SDS-page, and Western blot. Protocols for carrying out these techniques are known in the art and include, for example, Mitulovic G. and Mechtler K (2006), HPLC techniques for proteomics analysis—a short overview of the latest developments, Briefings in Functional Genomics, 5, 4, pp. 249-260; Gao Z, et al. (2009), Identification and Verification of the Main Differentially Expressed Proteins in Gastric Cancer via iTRAQ Combined with Liquid Chromatography-Mass Spectrometry, Analytical Cellular Pathology (Amsterdam), 2019: 5310684; Lorne F et al. (2001), Whole cell ELISA for detection of tumor antigen expression in tumor samples, Journal of Immunological Methods, 258, 1-2, pp. 47-53; Kim, SM, et al. (2017). Two different protein expression profiles of oral squamous cell carcinoma analyzed by immunoprecipitation. high-performance liquid chromatography.World journal of surgical oncology,15(1),151;Osborne C, Brooks SA.(2006)SDS-PAGE and Western blotting to detect proteins and glycoproteins of interest in breast cancer research.Methods in Molecular Medicine,120:p.217-29;Ni,D.,Xu,P.,and Gallagher,S.2016.Immunoblotting and immunodetection.Curr.Protoc.Mol.Biol.114:10.8.1-10.8.37; and Adams, LDand Gallagher, SR (2004), Two-Dimensional Gel Electrophoresis.Current Protocols in Molecular Biology,67:10.4.1-10.4.23.doi:10.1002 / 0471142727.mb1004s67. .

[0215]

[0267] Another method for quantifying protein expression on and in cells is flow cytometry. Briefly, a tissue sample is collected and dissected into tissues of interest, which are then dissociated, digested, and filtered into a single-cell suspension. The cell suspension is then stained with an antibody against the protein of interest, such as glypican-1 (i.e., using a fluorescently labeled primary antibody or a two-step labeling method including a primary antibody followed by a fluorescently labeled secondary antibody against the primary antibody). To analyze intracellular staining, the cells may be permeabilized before staining (often after fixation). The cells may then be run through a flow cytometer to identify cells expressing the protein of interest (e.g., glypican-1), and the protein expression in each cell may be quantified.

[0216]

[0268] Methods for performing flow cytometry are known in the art and include El-Hajjar, L. et al., (2023) Guide to Flow Cytometry: Components, Basic Principles, Experimental Design, and Cancer Research Applications. Curr Protoc; 3(3): e721; and Nolan, JP and Condello, D. (2013), Spectral Flow Cytometry. Current Protocols in Cytometry, 63: 1.27.1-1.27.13.

[0217]

[0269] Techniques for simultaneous quantification of mRNA and protein expression are also known, including REAP-seq and CITE-seq. Techniques known in the art include: Peterson V. et al. (2017) Multiplexed quantification of proteins and transcripts in single cells. Nature Biotechnology, 35, 936-939 https: / / doi.org / 10.1038 / nbt.3973; and Stoeckius M, et al. (2017), Simultaneous epitope and transcriptome measurement in single cells. Nature Methods, 14(9): p.865-868. doi:10.1038 / nmeth.4380. Epub 2017 Jul 31. PMID: 28759029; PMCID: PMC5669064.

[0218]

[0270] Analysis System

[0271] The present invention also provides a method for diagnosing or prognosticating a subject with ovarian cancer, comprising: obtaining a sample of suspected or confirmed ovarian cancer cells from the subject; quantitating the expression of glypican-1 in the sample or quantifying the expression of glypican-1 in a control sample; comparing the quantified expression in the sample from the subject with a control sample or control value (as defined herein); and performing an analysis of glypican-1 expression in the sample compared to a control sample or control value, wherein elevated expression in the sample from the subject is indicative of ovarian cancer or a poor prognosis for the subject. The present invention provides a method comprising:

[0219]

[0272] In some embodiments of the above methods, the control value is stored in a computer database or computer system.

[0220]

[0273] The method of the present invention can be carried out by any suitable method known in the art. However, in some embodiments, the expression of glypican-1 in a sample from a subject is compared to a control value (as defined herein) by a computer system. Preferably, the predetermined amount of the control value is stored in a database. This allows the database to serve as a standard for comparison to multiple cancer or suspected cancer samples.

[0221]

[0274] In such embodiments, after quantifying the expression level of glypican-1, this data is input (e.g., uploaded or entered) into a computer system which compares the expression level in the sample with control values ​​stored in a database. The computer system and associated computer-readable media can then perform any necessary statistical analyses which may provide a diagnosis of the likelihood that the sample is cancerous and / or an indication of the subject's prognosis.

[0222]

[0275] Thus, in some embodiments of the present invention, a computer system is provided comprising a computer processor and a computer readable medium encoded with programming instructions executable by the computer processor to compare the quantified expression of one or more markers and perform a comparison with a reference standard, preferably stored in a database.

[0223]

[0276] In some aspects, the present invention includes a detection system comprising a sample receiving portion configured to receive an RNA sample from an ovarian cancer sample or a suspected ovarian cancer sample, and a detection portion comprising one or more nucleic acids configured to hybridize to a glypican-1 nucleic acid.

[0224]

[0277] In some embodiments, the detection system may also include a computer system as described herein. In such embodiments, the computer-readable medium may have programming instructions encoded thereon, or the computer-readable medium may be executed by a computer processor to process data associated with the detection unit to determine the expression of glypican-1 in the received sample. Furthermore, the programming instructions may include a control value for glypican-1, or may process data associated with the control sample to determine the control value. The programming instructions for processing the data to compare the expression of glypican-1 in the sample received from the subject to the control value may enable evaluation of the sample to determine or predict the likelihood of the sample being ovarian cancer or to assess the subject's prognosis, where an elevated amount of glypican-1 indicates the presence of ovarian cancer and / or indicates a poor prognosis for the subject.

[0225]

[0278] treatment

[0279] If a patient is determined to have or is likely to have cancer, such as ovarian cancer, by any of the diagnostic or prognostic methods described herein, appropriate treatment may be administered. What constitutes appropriate treatment will be determined by those skilled in the art based on available and approved treatments. Currently available treatments include, but are not limited to, surgical resection or debulking of the cancer, systemic or localized chemotherapy, systemic or localized immunotherapy (as described herein), radiation, or CAR T-cell therapy (e.g., CARs described herein). Accordingly, any of the diagnostic or prognostic methods may include or form part of a therapeutic method. For the avoidance of doubt, provided herein are methods of treating a subject confirmed to have or suspected of having cancer by performing a diagnostic or prognostic method described herein.

[0226]

[0001] This application claims priority from Australian Provisional Patent Application No. 2022903762, filed December 9, 2022, the entire disclosure of which is incorporated herein by reference.

[0227]

[0351] Definitions and Qualifications

[0352] This specification includes the discussion of documents, acts, materials, devices, articles and the like for the sole purpose of providing a context for the present invention. No intention is made or represented that any or all of these items form part of the prior art existing before the priority date of each claim in this application or common general knowledge in any field relevant to the present invention.

[0228]

[0353] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of the specified element or integer or group of elements or integers, but not the exclusion of any other element or integer or group of elements or integers.

[0229]

[0354] It should be further understood that terminology such as "comprise," or variations such as "comprises" or "comprising," inherently include within their scope (but are not limited to) versions of the invention that exclude other elements directly related to the invention. Thus, terminology such as "consisting of" or "consisting essentially of" may be substituted with terminology such as "comprise," "comprises," or "comprising," which has the effect of limiting the scope of the invention to the elements specifically recited. In particular, where it is expressly intended that the invention be considered comprehensively, such limitations should be considered to relate only to the inventive concept disclosed herein, and other features outside the scope of the inventive concept may be added. Such features or elements may include, but are not limited to, excipients, formulations, additives, diluents, packaging, adjuvants, and coexisting features not excluded by terminology such as "consisting of" or "essentially consisting of."

[0230]

[0355] The referenced documents, publications, and patents are incorporated by reference in their entirety, and the teachings and disclosures in such documents, publications, and patents are therefore deemed to form part of the disclosure herein.

[0356] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any examples or exemplary language (e.g., "such as," "i.e.") provided herein is intended merely to more clearly describe exemplary embodiments and does not limit the scope of the claimed invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential.

[0357] The description provided herein is of several embodiments sharing common properties and characteristics. It should be understood that one or more features of one embodiment may be combined with one or more features of other embodiments. In addition, single features or combinations of features of embodiments may constitute additional embodiments.

[0231]

[0358] The subject headings used herein are included for ease of reference only and should not be used to limit the subject matter found throughout this disclosure or the claims. The subject headings should not be used in interpreting the claims or claim limitations.

[0232]

[0359] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention is to be understood to include all such variations and modifications. The invention also includes all steps, features, compositions, and compounds individually or collectively referred to or indicated herein, and any combination of any two or more of these steps or features.

[0233]

[0360] Also, it should be noted that as used herein, the singular forms "a," "an," and "the" include plural references unless the context already dictates otherwise.

[0234]

[0361] Future patent applications may be filed based on a claim of priority to this application or as continuations or divisions of this application. It is understood that the claims below are not intended to limit the scope of what may be claimed in any such future applications. Features may be added to, or deleted from, the claims at a later date in order to further define or redefine the claimed invention.

[0235]

[0362] Although the invention has been described in detail herein for purposes of clarity and understanding, it will be apparent to those skilled in the art that various modifications and variations can be made to the embodiments and methods described herein without departing from the scope of the inventive concepts disclosed herein. [Example]

[0236]

[0281] The present invention is further described and illustrated in the following examples, which are intended only to illustrate particular embodiments of the invention and are not intended to be limiting with respect to the scope of the invention as described above and as claimed in this application or any future application claiming priority from this application.

[0237]

[0282] Example 1 - Analysis of Glypican-1 (GPC1) Expression in Ovarian Cancer

[0283] Analysis of microarray expression data and immunohistochemistry (IHC) demonstrated that GPC1 is increased in ovarian cancer cells and is associated with poor patient prognosis (e.g., lower overall survival and lower progression-free survival).

[0238]

[0284] Materials and Methods

[0285] Microarray analysis

[0286] Using the GENT2 database (Park SJ, et al. (2019). GENT2: an updated gene expression database for normal and tumor tissues. BMC Medical Genomics 12(Suppl 5), 101. DOI:10.1186 / s12920-019-0514-7), GPC1 mRNA abundance was assessed in normal tissues (ovarian surface epithelium (n=66), fallopian tube (n=40), and HGSOC tissues (n=807)) based on annotated Gene Expression Omnibus (U133Plus2) data. The Kaplan-Meier plotter tool was used to evaluate the relationship between GPC1 mRNA expression (GPC1:202755_s_at and 202756_s_at) and progression-free survival (PFS) and overall survival (OS) in patients with HGSOC (Fekete JT et al. (2020). Predictive biomarkers of platinum and taxane resistance using the transcriptomic data of 1816 ovarian cancer patients. Gynecol Oncol 156, 654-661).

[0239]

[0287] Immunohistochemistry (IHC)

[0288] IHC was performed on tissue sections from high-grade serous ovarian cancer (HGSOC) (n = 37), benign (n = 7), normal ovaries (n = 14), fallopian tubes (FT, n = 10), and matched HGSOC tissues at diagnosis and recurrence (n = 4). A tissue microarray (TMA) cohort of HGSOC patients (n = 101) was also evaluated (Ricciardelli C et al. (2017). Keratin 5 overexpression is associated with serous ovarian cancer recurrence and chemotherapy resistance. Oncotarget 8, 17819-17832). Clinical and pathological parameters of these tissues are listed in Tables 6 and 7, respectively.

[0240]

[0289] [Table 6]

[0241]

[0290] [Table 7]

[0242]

[0291] This methodology was adapted from a previously described method (Lokman NA et al. (2013). Annexin A2 is regulated by ovarian cancer-peritoneal cell interactions and promotes metastasis. Oncotarget 4, 1199-1211). After citrate antigen retrieval, tissue sections were incubated overnight at 4°C with a primary antibody: Rb polyclonal GPC1 (1 / 75, 16700-1-AP, Proteintech™). Sections were then incubated with a secondary antibody (biotinylated goat anti-rabbit, 1 / 400, Dako™, Australia), followed by incubation with streptavidin-HRP (1 / 500, Dako™, Australia) for 1 hour at room temperature. Peroxidase activity was detected using diaminobenzidine and H2O2 (Sigma-Aldrich™). Using the Human Protein Atlas online database, kidney and liver tissues were selected as positive and negative controls, respectively. High and low GPC1 immunostaining was observed in mouse kidney and liver, respectively (Figures 13A and 13B, respectively).

[0243]

[0292] Immunohistochemical evaluation

[0293] IHC slides were scanned using a Nanozoomer™ digital pathology system (Hamamatsu Photonics™, Japan). The level of GPC1 staining intensity in tumor cells was assessed using Qupath™ software (Bankhead P, et al. (2017). QuPath: Open source software for digital pathology image analysis. Scientific Reports 7, 16878. DOI: 10.1038 / s41598-017-17204-5). The H-index was measured using the percentage and intensity of positively stained cancer cells on a scale of 0 to 300 and scored using three thresholds: weak (1+), moderate (2+), and strong (3+).

[0244]

[0294] cell culture

[0295] OVCAR3, OV90, and SKOV3 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). COV362, COV318, A2780, and OAW28 cell lines were purchased from the European Collection of Authenticated Cell Cultures (ECACC). OVCAR-5 cells were obtained from Dr. Thomas Hamilton (Fox Chase Cancer Center, PA, USA). Cell lines were cultured in DMEM (Thermo Fisher Scientific™) or RPMI (Thermo Fisher Scientific™) medium supplemented with 10% fetal bovine serum (FBS, Scientifix Pty Ltd) and antibiotics (100 U penicillin G, 100 μg / ml streptomycin sulfate, and 0.25 μg / ml amphotericin B (Sigma-Aldrich™)). All cells were maintained at 37°C in a 5% CO2 environment.

[0245]

[0296] Primary HGSOC cells (n=7) were obtained from ascites collected from patients with advanced HGSOC from the Royal Adelaide Hospital and cultured as previously reported (Ricciardelli C et al. (2015). Transketolase is upregulated in metastatic peritoneal implants and promotes ovarian cancer cell proliferation. Clin Exp Metastasis 32, 441-455). Table 8 shows the clinical and pathological parameters of these patients. Primary HGSOC cells were maintained in advanced RPMI (Life Technologies), 10% FBS, 2 mM Glutamax™ (Life Technologies), and antibiotics.

[0246]

[0297] [Table 8]

[0247]

[0298] Quantitative real-time reverse transcription PCR

[0299] Ovarian cancer cells (COV362, COV318, OAW28, OV90, OVCAR3, A2780, and OVCAR5) were plated at 30,000 cells per well for 24 hours. RNA was isolated from the cells and reverse transcribed using TaqMan Gene expression Cells to CT™ (Life Technologies) according to the manufacturer's guidelines, as previously described (Lokman NA et al. (2019). 4-Methylumbelliferone Inhibits Cancer Stem Cell Activation and Overcomes Chemoresistance in Ovarian Cancer. Cancers (Basel) 11, 1187. DOI: 10.3390 / cancers11081187). Complementary DNA (cDNA) was stored and used for subsequent PCR analysis using a Quantstudio 12K Flex Real Time PCR System™ (Applied Biosystems). PCR solution was prepared to 10 μL using TaqMan™ Gene Expression Master Mix (2X), GPC1 primer (Hs00892476_m1), nuclease-free water, and cDNA sample. Negative controls included samples without RNA or cDNA. PCR cycling conditions were used as previously described (Lokman NA et al. (2019). 4-Methylumbelliferone Inhibits Cancer Stem Cell Activation and Overcomes Chemoresistance in Ovarian Cancer. Cancers (Basel) 11, 1187. DOI: 10.3390 / cancers11081187). Cycle threshold (CT) values ​​were normalized to β-actin (Applied Biosystems™, Life Technologies) and 2 μg / mL. -ΔCT was calibrated using the method.

[0248]

[0300] Western blot

[0301] Ovarian cancer cell lines and primary ovarian cancer cells (n = 7) were cultured to confluence, and protein extracts were collected as previously described (Lokman NA et al. (2013). Annexin A2 is regulated by ovarian cancer-peritoneal cell interactions and promotes metastasis. Oncotarget 4, 1199-1211). Twenty micrograms of each sample was then loaded onto a 4-20% TGX gel (Bio-Rad) at 50 V for 30 minutes and 110 V for 90 minutes. The gel was transferred to a PVDF membrane (GE Healthcare™) at 33 V overnight at 4°C. The membrane was then incubated with Rb polyclonal GPC1 (1 / 500, 16700-1-AP, Proteintech™) for 2 hours and peroxidase-conjugated anti-rabbit IgG (1 / 4000, Millipore™) for 1 hour. Protein expression was visualized using chemiluminescence (ECL Hyperfilm™, GE Healthcare), scanned using a Chemidoc™ MP Imaging System (Bio-Rad Laboratories™, Inc.), and analyzed using Imagelab™. Beta-actin anti-rabbit antibody (1 / 5000, ab8226, Abcam™) was used as a loading control.

[0249]

[0302] statistical analysis

[0303] For the GENT2 database, the Kruskal-Wallis test and Dunn's multiple comparison test were used. One-way ANOVA with Tukey's multiple comparison test was used to evaluate the GPC1 H-index score measured by Qupath. Survival curves were generated using the Kaplan Meier plotter database to determine the relationship between GPC1 mRNA and patient outcomes. Kaplan Meier analysis with the log-rank test was performed to evaluate the relationship between GPC1 protein and progression-free survival (PFS) and overall survival (OS) (SPSS software, version 28.0, SPSS Inc, USA). Paired Student's t-test was used to evaluate the statistical significance of H-index scores for matched HGSOC patient tissues at diagnosis and recurrence.

[0250]

[0304] result

[0305] GPC1 is elevated in high-grade serous ovarian cancer

[0306] Analysis of the GENT2 database revealed significantly elevated GPC1 mRNA levels in HGSOC compared with FT (Figure 1A, P < 0.0001). However, there was no significant difference in GPC1 expression between ovarian surface epithelium (OSE) and HGSOC (Figure 1A). Immunohistochemical (IHC) evaluation of GPC1 protein levels, measured as the H-index, showed a significant increase in HGSOC compared with benign serous cystadenomas (Figure 1B). However, no differences were observed in HGSOC GPC1 H-index compared with either OSE or FT. Representative images show cytoplasmic and membranous GPC1 staining in OSE (Figure 1C) and FT (Figure 1D), as well as low GPC1 staining in benign serous cystadenoma tissue (Figure 1E). High GPC1 cytoplasmic and membranous staining was present in HGSOC tissue (Figure 1F).

[0251]

[0307] High GPC1 expression is associated with poor patient outcomes

[0308] Survival curves were generated using the Kaplan Meier online plotter tool to examine the relationship between GPC1 mRNA levels and patient outcomes (Figures 2A-F). High GPC1 mRNA expression was significantly associated with decreased PFS (Figure 2A, hazard ratio (HR) = 1.3, p = 0.0015) and OS (Figure 2B, HR = 1.35, p = 0.00026).

[0252]

[0309] GPC1 protein abundance was assessed in a TMA cohort of HGSOC. The median H-index observed in this cohort was 73.7. Examples of HGSOC tissues with low and high GPC1 protein expression are shown in Figures 2C and 2D, respectively. The GPC1 H-index was divided into quartiles for initial Kaplan-Meier survival analysis (Figure 14). A significant difference was observed between the higher quartiles (Q3 and Q4) and the lower quartiles (Q1 and Q2) in the PFS analysis, but not in the OS analysis. Using an H-index score cutpoint of 70 (close to the median), a GPC1 abundance greater than 70 was associated with decreased PFS (Figure 2E, P = 0.031) but not decreased OS (Figure 2F, p = 0.536).

[0253]

[0310] GPC1 expression is elevated after recurrence

[0311] GPC1 protein abundance was assessed in matched HGSOC tissues at diagnosis and recurrence. Examples of GPC1 immunostaining in HGSOC tissues at diagnosis are shown in Figures 3A and 3B, and matched tissues at recurrence are shown in Figures 3C and 3D. Quantitation of IHC staining using QuPath demonstrated elevated GPC1 abundance in matched tumor tissues at recurrence compared with tissues at diagnosis (Figure 3E, P = 0.0014, paired t-test).

[0254]

[0312] GPC1 expression in ovarian cancer cells

[0313] Quantitative PCR (qRT-PCR) showed that GPC1 mRNA was expressed in all ovarian cancer cell lines (Figure 4A) and primary HGSOC cells (Figure 4B). The highest GPC1 expression was observed in OVCAR5 and primary cells from patient 4. Western blot detected a band with the predicted molecular weight of 65 kDa, confirming GPC1 expression in ovarian cancer cell lines (Figure 4C) and primary HGSOC cells (Figure 4D). Western blot quantification showed the highest GPC1 protein abundance in OAW28 and OV90 (Figure 4E). Primary cells from patient 1 and patient 3, isolated from HGSOC patients with recurrent disease, showed the highest GPC1 protein abundance (Figure 4F). Ovarian cancer cell lines (OVCAR3, OV90, COV362, and SKOV3) and primary cells (patient 1 and patient 3) with varying GPC1 abundance were selected for further in vitro studies.

[0255]

[0314] Discussion of results

[0315] The above results showed that: i) GPC1 mRNA and protein expression is increased in HGSOC compared with non-cancerous tissues, but this amount does not necessarily correlate within patients or cell lines; ii) elevated GPC1 mRNA amount is associated with PFS and OS; and iii) high GPC1 protein expression in tumor cells is associated with decreased PFS.

[0256]

[0316] Examination of GPC1 expression showed that GPC1 mRNA expression was significantly increased in HGSOC compared with the FT, the site of origin of HGSOC.Furthermore, GPC1 protein expression was significantly increased in HGSOC compared with benign tissue.

[0257]

[0317] Kaplan Meier plotter analysis and TMA cohort results demonstrated a significant association between high GPC1 expression and poor prognosis in patients with HGSOC. We observed only faint GPC1 staining in the interstitium, and GPC1 was mainly localized in the cell membrane and cytosol.

[0258]

[0318] The results also demonstrate that GPC1 was expressed to various degrees in ovarian cancer cell lines and primary cells.

[0259]

[0319] Example 2 - Anti-glypican-1 chimeric antigen receptor T cells efficiently kill ovarian cancer cells.

[0260]

[0320] Based on the demonstration of GPC1 expression in ovarian cancer cells, particularly HGSOC, we determined whether ovarian cancer cells could be targeted with drugs that induce cell death in GPC1-expressing cells. To target GPC1-expressing cells, we developed anti-GPC1 CAR-T cells and used them in cancer cell lysis assays. This experiment demonstrated that ovarian cancer cells could be killed by anti-GPC1 drugs, including CAR-T cells.

[0261]

[0321] CAR-T cell production and characterization.

[0322] CNA500200 CAR-T cells were generated in Professor Simon Barr's laboratory using established protocols (Jensen MC & Riddell SR (2015). Designing chimeric antigen receptors to effectively and safely target tumors. Curr Opin Immunol 33, 9-15; Wang X, et al. (2012). Phenotypic and functional attributes of lentivirus-modified CD19-specific human CD8+ central memory T cells manufactured at clinical scale. J Immunother 35, 689-701; and WO 2022 / 104424A). The GPC1-binding domain was cloned into a second-generation CAR scaffold encoding linker and intracellular domains of CD3, CD28, and epidermal growth factor (EGFR) receptors (Jensen MC & Riddle SR (2015), supra, and Wang X et al. (2012), supra).

[0262]

[0323] Lentivirus was generated by transfecting 293T cells with third-generation self-inactivating lentiviral plasmids and packaging plasmids encoding REV, VSV-G, and gag-pol using established methods (Barry SC et al. (2000). Lentiviral and murine retroviral transduction of T cells for expression of human CD40 ligand. Hum Gene Ther 111, 323-332). An outline of the transduction protocol is shown in Figure 9.

[0263]

[0324] The CAR-T cells used in the assay were fully characterized using fluorescence-activated cell sorting (FACs) by Batjargal Gundsambuu (Molecular Immunology, University of Adelaide). Transduction efficiency was measured by EGFR expression. In the CD4 T cell population, 80.4% of cells were EGFR-positive, in the CD8 T cell population, 66.3% of cells were positive, and in the total lymphocyte population, 78.3% were positive (Figure 10).

[0264]

[0325] Markers of cell maturation, CD45RA and CD62L, were also assessed. In the CD4 cell population, 51.7% of the cells were effector memory T cells (T EM ) phenotype, and 32.7% were central memory T cells (T CM ) phenotype, and 8.29% were effector memory cells (T EMRA ) phenotype, with 7.3% of cells exhibiting a naive T cell phenotype (Figure 11). In contrast, the CD8 cell population was slightly more prevalent (19.3%) with 20.7% exhibiting a central memory T cell phenotype, 29.8% exhibiting an effector memory T cell phenotype, and 20.2% exhibiting a T EMRA The phenotype was shown (Figure 11).

[0265]

[0326] An exhaustion staining panel was also evaluated using PD1, LAG3, and TIM3 antibodies. FAC analysis showed that in the CD4 cell population, 1.38% of cells expressed very low levels of PD1, and of these cells, only 2.31% of cells expressed LAG3 and TIM3 (Figure 12). In the CD8 cell population, 3.92% of cells expressed PD1, and of these cells, only 1.56% expressed LAG3 and TIM3. These results indicate low levels of T cell exhaustion.

[0266]

[0327] Materials and Methods

[0328] MTT cell viability assay

[0329] SKOV3, COV362, OVCAR3, OV90, and primary HGSOC cells (n=2) were plated in their respective growth media at 10,000 cells / well in 96-well plates. After 24 hours, cells were treated with either (i) control media, (ii) untransduced (UT) CD3 T cells, or (iii) anti-GPC1 CAR T cells (effector T cell:target cancer cell (E:T) ratios of 2:1, 5:1, and 10:1) for 48 hours. Cell monolayers were washed twice with RPMI media to remove T cells. MTT assays were performed as previously described (Ricciardelli C, et al. (2013). Chemotherapy-induced hyaluronan production: a novel chemoresistance mechanism in ovarian cancer. BMC Cancer 13, 476. DOI: 10.1186 / 1471-2407-13-476).

[0267]

[0330] Spheroid assay

[0331] SKOV3, COV362, OVCAR3, and primary ovarian cancer cells (n=2) were plated at 20,000 cells / well in their respective growth media onto polyHEMA (30 mg / mL in 95% ethanol, Sigma-Aldrich™)-coated 24-well plates. After 24 hours, cells were treated with X-VIVO 15 medium (Lonza™, 5% human serum, Sigma-Aldrich™, 2 mM L-glutamine, Sigma™, 20 mM HEPES) or UT CD3 T cells or anti-GPC1 CAR-T cells (E:T ratio 5:1). Spheroid formation was monitored over 6 days, and brightfield images were acquired using an EVOS® Light Microscope FL Imaging System (Life Technologies™). Spheroid area (μm) of spheroids greater than 50 μm in diameter was measured for each of the treatment groups (n = 5 images / well in duplicate wells from three independent experiments) using Image J 32 software (Image J I.50i, National Institute Health, Bethesda, MD, USA) as previously described. 2 ) was measured (Lokman NA, et al. (2019). 4-Methylumbelliferone Inhibits Cancer Stem Cell Activation and Overcomes Chemoresistance in Ovarian Cancer. Cancers (Basel) 11, 1187).

[0268]

[0332] Patient-derived explant (PED) assay

[0333] Tissues were collected at the time of surgery and cryopreserved in liquid nitrogen containing 15% DMSO and 25% FBS. Cryopreserved patient-derived tissues (Figure 11) were cut into 1 mm 3The tissue was dissected into pieces and explanted onto gelatin dental sponges (Spongostan™, Johnson & Johnson™) in CD3 T cell X-VIVO medium (containing the cytokines IL-2 (50 U / mL), IL-7 (5 ng / mL), and IL-15 (0.5 ng / mL)) and incubated at 37°C in a humidified atmosphere containing 5% CO2 with either: (i) control medium; (ii) anti-GPC1 CAR-T cells (2 × 10 6 cells / mL), or (iii) UT CD3 T cells (2 × 10 6 Cells were treated with either 1000mg / mL of 1000mg ...

[0269]

[0334] result

[0335] Effect of GPC1 CAR-T cells on ovarian cancer monolayers in vitro

[0336] All ovarian cancer cells and primary cells responded to treatment with GPC1 CAR-T cells. Significant decreases in cell viability were observed in OVCAR3 (Figure 5A), COV362 (Figure 5B), OV90 (Figure 5C), and SKOV3 (Figure 5D) cell lines at E:T ratios of 5:1 and 2:1 compared with UT CD3 T cells. Anti-GPC1 CAR-T cells reduced the viability of COV362 (Figure 5B) and SKOV3 cells (Figure 5D) at E:T ratios of 10:1 compared with UT CD3 T cells. Primary cells from patient 1 showed a statistically significant decrease in cell viability when incubated with anti-GPC1 CAR-T cells at an E:T ratio of 10:1 (Figure 5E), but not at 5:1 or 2:1. Anti-GPC1 CAR-T cells also showed a statistically significant effect on primary cell viability in patient 3 at E:T ratios of 10:1 and 2:1, but not at 5:1 (Figure 5F).

[0270]

[0337] Effect of GPC1 CAR-T cells on cancer cells in 3D spheroid formation

[0338] Spheroids containing COV362 (Figure 6A), SKOV3 (Figure 6B), and OVCAR3 (Figure 6C) cell lines responded to anti-GPC1 CAR-T cell treatment at a 5:1 E:T ratio, resulting in a statistically significant decrease in spheroid area compared to UT CD3 T cells. Significant differences between control and anti-GPC1 CAR-T cells were also observed in spheroids containing COV362 (Figure 6A) and SKOV3 (Figure 6B) cells, but not in OVCAR3 cells (Figure 6C). A statistically significant decrease in spheroid size was observed between UT CD3 T cells and anti-GPC1 CAR-T cells in spheroids composed of cells from patient 3 (Figure 7B), but not in spheroids from patient 1 (Figure 7A), although a decrease was observed in this patient. Compared to control media, anti-GPC1 CAR-T cell treatment resulted in a statistically significant decrease in spheroid size for both patient 1 (Figure 7A) and patient 3 (Figure 7B). No cells showed a statistically significant difference between UT CD3 T cell treatment and control treatment.

[0271]

[0339] Effect of GPC1 CAR-T cells on patient-derived explants

[0340] Ovarian cancer tissues from six patients were selected for the PDE assay (Figures 8A-8F). The PDE assay was performed by treating explanted patient tissues with UT CD3 T cells or anti-GPC1 CAR-T cells, followed by cleaved caspase 3 immunostaining to assess apoptosis in the explanted tissues.

[0272]

[0341] Patients 1-4 had a statistically significant increase in cleaved caspase 3 staining (Figures 8A-8D), indicating increased cell death in explant tumor tissue after treatment with anti-GPC1 CAR-T cells compared with UT CD3 T cells. Comparison of GPC1 expression between tissues that responded to CAR T-cell treatment and those that did not (e.g., patients 5 and 6) showed low levels of GPC1 expression in explants that did not respond to CAR T-cell treatment (Figure 8G), indicating that GPC1 expression correlates with treatment outcome.

[0273]

[0342] Discussion of results

[0343] The efficacy of anti-GPC1 CAR-T cells was evaluated in ovarian cancer cell lines with different GPC1 expression and in two primary cell lines from patients with relapsed ovarian cancer.

[0274]

[0344] The results of the 2D monolayer assay showed that anti-GPC1 CAR-T cells exhibited a dose-dependent killing effect.

[0275]

[0345] A further significant decrease in cancer cell viability was observed at the lowest concentration, a 2:1 ratio, for all ovarian cancer cells except for primary cells from patient 1. For OVCAR3 and OV90, no statistically significant effect was observed at a 10:1 ratio, most likely due to an increased cytotoxic effect of the greater number of UT CD3 T cells, although the data still showed increased killing at these ratios.

[0276]

[0346] Evaluation of CAR-T cell efficacy in 3D spheroid assays allowed for an accurate physiological representation of the tumor microenvironment, particularly when such structures formed in the malignant ascites of ovarian cancer patients.

[0277]

[0347] This study demonstrated significant antitumor activity of anti-GPC1 CAR-T cells against primary ovarian cancer 3D spheroids in vitro.

[0278]

[0348] The efficacy of anti-GPC1 CAR-T cells was also investigated in an ex vivo model, the PDE assay, which importantly preserves tissue architecture and viable tumor cells similar to those in the native tissue. The results showed that anti-GPC1 CAR-T cells were more effective at inducing apoptosis and killing targeted cancer cells in HGSOC patient tissue than UT CD3 T cells.

[0279]

[0349] In summary, these results demonstrate that: i) GPC1 CAR-T cells have antitumor activity against ovarian cancer cell lines in monolayer and 3D spheroid assays; ii) GPC1 CAR-T cells have antitumor activity against primary ovarian cancer cells isolated from HGSOC patients after disease recurrence in monolayer and 3D spheroid assays; and iii) GPC1 CAR-T cells are effective in inducing apoptosis in a PDE assay. Collectively, these findings suggest that GPC1 CAR-T cells may offer a novel immunotherapy for ovarian cancer, particularly HGSOC.

[0280]

[0350] [Table 9] TIFF2025540193000013.tif181149

Claims

1. A chimeric antigen receptor (CAR) comprising an antigen recognition domain, a transmembrane domain, and a signal transduction domain, wherein the antigen recognition domain recognizes glypican-1.

2. The CAR according to claim 1, wherein the antigen recognition domain comprises a binding portion of an antibody that recognizes glypican-1.

3. The CAR according to claim 1 or 2, wherein the portion of the antibody that recognizes glypican-1 is selected from the group consisting of an antigen-binding fragment (Fab), an antibody variable heavy chain, or an antibody variable light chain.

4. The CAR according to any one of claims 1 to 3, wherein the antigen recognition domain is a single-chain variable fragment (scFv) having sequence identity to an antibody that binds to glypican-1.

5. The CAR according to any one of claims 1 to 4, further comprising a linker between the antigen recognition domain and the transmembrane domain.

6. A cell comprising the CAR according to any one of claims 1 to 5.

7. The cell of claim 6 , wherein the cell is an immune cell.

8. The cell according to claim 6 or 7, wherein the cell is a lymphocyte.

9. The cell according to any one of claims 6 to 8, wherein the cell is a CD3+ cell.

10. The cell according to any one of claims 6 to 9, wherein the cell is a CD8+ cell or a CD4+ cell.

11. The cell according to any one of claims 6 to 8, wherein the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

12. 1. A method of diagnosing a subject with ovarian cancer or assessing the prognosis of said subject, comprising determining the amount of glypican-1 in ovarian cells from said subject, wherein an elevated amount of glypican-1 indicates the presence of ovarian cancer and / or indicates a poor prognosis for said subject.

13. 13. The method of claim 12, wherein the ovarian cancer is recurrent ovarian cancer.

14. 14. The method of claim 12 or 13, wherein the ovarian cancer is high-grade serous ovarian cancer.

15. 15. The method of any one of claims 12 to 14, wherein the subject has already been treated for ovarian cancer with one or more of chemotherapy, surgical resection or debulking, or radiation therapy.

16. The method according to any one of claims 12 to 15, wherein the ovarian cancer is recurrent ovarian cancer, and the increase in the amount of glypican-1 is compared with that in cancer tissue before recurrence.

17. The method of any one of claims 12 to 16, wherein the elevated amount of glypican-1 is compared to non-cancerous ovarian tissue.

18. The method of any one of claims 12 to 17, wherein determining the amount of glypican-1 comprises quantifying glypican-1 protein expression and / or mRNA expression.

19. 19. The method of claim 18, wherein quantifying glypican-1 protein expression comprises quantifying surface expression of said glypican-1 protein.

20. 19. The method of claim 18, wherein determining the amount of glypican-1 protein expression comprises quantifying intracellular expression of the glypican-1 protein.

21. The method of any one of claims 18 to 20, wherein the protein expression is determined by an agent that preferentially or selectively binds to glypican 1.

22. The method of claim 21, wherein the agent that binds to glypican-1 is an antibody or a binding fragment thereof.

23. The method of claim 21 , wherein the agent that binds to glypican 1 is a single-chain variable fragment comprising the sequences of the variable light and variable heavy chains of an antibody.

24. The method of claim 22 or 23, wherein the antibody is MIL-38.

25. A method of treating a subject with ovarian cancer, the method comprising killing cells that express glypican-1.

26. 26. The method of claim 25, wherein the glypican-1 expressing cells are determined to express elevated amounts of glypican-1 protein.

27. 27. The method of claim 26, wherein the increased amount of glypican-1 protein expression comprises increased surface expression of the glypican-1 protein.

28. 27. The method of claim 26, wherein the increased amount of glypican-1 protein expression comprises increased intracellular expression of glypican-1 protein.

29. The method of any one of claims 25 to 28, wherein the ovarian cancer is recurrent ovarian cancer.

30. 30. The method of any one of claims 25 to 29, wherein the ovarian cancer is high-grade serous ovarian cancer (HSOC).

31. 31. The method of any one of claims 25 to 30, wherein the subject has already been treated for ovarian cancer with one or more of chemotherapy, surgical resection or debulking, or radiation therapy.

32. The method according to any one of claims 26 to 31, wherein the ovarian cancer is recurrent ovarian cancer, and the increase in the amount of glypican-1 is compared with that in cancer tissue before recurrence.

33. The method of any one of claims 26 to 31, wherein the elevated amount of glypican-1 is compared to non-cancerous ovarian tissue.

34. The method of any one of claims 25 to 33, wherein the cells are killed by administering to the subject an agent that preferentially or selectively binds to glypican 1.

35. The method of claim 34, wherein the agent that binds to glypican-1 is an antibody or a binding fragment thereof.

36. 35. The method of claim 34, wherein the agent that binds to glypican 1 is a single-chain variable fragment comprising the sequences of the variable light and variable heavy chains of an antibody.

37. The method of claim 35 or 36, wherein the antibody is MIL-38.

38. The method of claim 34, wherein the agent that binds to glypican-1 is a cell that expresses a chimeric antigen receptor (CAR).

39. The method according to claim 38, wherein the cell expressing the CAR is a cell according to any one of claims 6 to 11.

40. The method according to any one of claims 25 to 39, comprising carrying out the method according to any one of claims 12 to 24 before killing the cells expressing glypican-1.

Images