Methods for diagnosing and treating ovarian cancer

Abstract

The present application provides methods for the prognosis and / or diagnosis of cancer, in particular ovarian cancer. Also provided are methods of treating ovarian cancer and methods of treating cancer, in particular ovarian cancer, using the chimeric antigen receptor (CAR) and the CAR-expressing cells.

Description

[0001] This application claims priority to Australian provisional patent application 2022903762 filed on December 9, 2022, the entire disclosure of which is incorporated herein by reference. Technical Field

[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 treating glypican-1 positive cancers. Background Art

[0003] The following discussion of the background to the invention is intended to facilitate an understanding of the invention. However, it should be appreciated that the discussion is not an acknowledgement or admission that any of the material referred to was published, known or part of the common general knowledge as of the priority date of any claim in this specification.

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

[0005] Currently, there is a lack of treatment options for ovarian cancer, particularly recurrent ovarian cancer. In addition, there is a lack of biomarkers for diagnosing or indicating the likely prognosis of ovarian cancer. Therefore, there is a need to provide alternatives to current treatments (such as surgery and chemotherapy) for the treatment of ovarian cancer. There is also a need to improve the diagnosis or prognosis of ovarian cancer. Summary of the Invention

[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.

[0007] Therefore, in one aspect, a chimeric antigen receptor (CAR) is provided, which includes an antigen recognition domain, a transmembrane domain, and a signal transduction domain, wherein the antigen recognition domain recognizes glypican -1 (GPC1). When expressed in appropriate cells, CAR can be used for targeting cancer cells expressing glypican -1, particularly ovarian cancer cells.

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

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

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

[0011] The disclosure also provides cells comprising the CAR of the present invention. Chimeric antigen receptor constructs can be transduced into various cell types, and particularly envisioned embodiments are immune cells, such as lymphocytes, CD3+ lymphocytes, CD8+ lymphocytes (such as CD8+ T cells), CD4+ lymphocytes (such as CD4+ T cells), natural killer (NK) cells or NKT cells.

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

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

[0014] In some embodiments, increased expression of the Glypican-1 gene is associated with shorter overall survival. In some embodiments, increased expression of the Glypican-1 gene and / or increased expression of the Glypican-1 protein is associated with shorter progression-free survival.

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

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

[0017] In some embodiments, the method of diagnosing or assessing prognosis is performed on a subject who has previously been treated for ovarian cancer with one or more of the following: chemotherapy, surgical resection or cytoreductive surgery, or radiation therapy.

[0018] In some embodiments, the ovarian cancer is recurrent ovarian cancer and the level of Glypican-1 is elevated compared to cancer tissue prior to recurrence.

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

[0020] In some embodiments, determining the level of Glypican-1 comprises quantifying Glypican-1 protein expression and / or mRNA expression. Glypican-1 protein expression can be surface expression of Glypican-1 protein and / or intracellular expression of Glypican-1 protein, and / or secretory levels of Glypican-1.

[0021] In some embodiments, protein expression is determined by an agent that preferentially or selectively binds to Glypican- 1. Such agents include antibodies or binding fragments of antibodies.

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

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

[0024] The present invention also provides a method of treating a subject having ovarian cancer or preventing ovarian cancer in a subject, the method comprising killing cells expressing Glypican- 1. In some embodiments of the method, the cells expressing Glypican- 1 are determined to express elevated levels of Glypican- 1 protein.

[0025] In some embodiments, the increased expression level of Glypican-1 protein comprises increased surface expression of Glypican-1.

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

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

[0028] In some embodiments of the methods of treatment, the subject has previously been treated for ovarian cancer with one or more of the following: chemotherapy, surgical resection or cytoreductive surgery, or radiation therapy.

[0029] In some embodiments of the method of treating, the ovarian cancer is recurrent ovarian cancer and the level of Glypican-1 is elevated compared to cancerous tissue prior to recurrence and / or compared to non-cancerous ovarian tissue.

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

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

[0032] In some embodiments of treatment, prior to killing the cells expressing Glypican-1, a method of diagnosis or assessing prognosis (as disclosed herein) is performed.

[0033] Also provided is the use of a CAR or agent for Glypican-1 in the preparation of a medicament for preventing or treating ovarian cancer in a subject, preferably wherein the ovarian cancer has increased expression of Glypican-1. Also provided is a method for preventing or treating ovarian cancer in a subject, preferably wherein the ovarian cancer has increased expression of Glypican-1. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] To further understand the aspects and advantages of the present invention, reference should be made to the following detailed description taken in conjunction with the accompanying drawings.

[0035] Figures 1A to 1F : GPC1 expression is elevated in HGSOC tissues compared with non-cancerous tissues.

[0036] (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 were assessed in OSE, benign serous cystadenoma, and (HGSOC) tissues by GPC1 immunohistochemistry (IHC) staining and measured by 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 obtained 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.

[0037] Figures 2A to 2F : High expression of GPC1 is associated with poor prognosis.

[0038] 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. Representative images of (C) HGSOC with low GPC1 protein expression and (D) HGSOC with high GPC1 expression obtained by IHC (scale bar = 50 μm), all images are at the same magnification. Kaplan Meier survival analysis shows the relationship between GPC1 protein expression and HGSOC PFS and OS. The maximum H-index score was used as the cutoff point to stratify samples into high (H-index>70) or low (H-index≤70) GPC1 protein expression groups. (E) PFS, log-rank test, P=0.031, (n=96) (F) OS, log-rank test, p=0.536, (n=100).

[0039] Figures 3A to 3E : GPC1 is highly expressed in relapse tissues compared with tissues at diagnosis of matched HGSOC patients.

[0040] Representative images of GPC1 protein expression in HGSOC tissues obtained by IHC at (AB) diagnosis and (CD) relapse (scale bar = 50 μm). All images are at the same magnification. (E) GPC1 staining in HGSOC tissues at diagnosis (n = 4) and relapse (n = 4) measured using QuPath (*P < 0.05, paired T test).

[0041] Figures 4A to 4F : Expression of GPC1 in ovarian cancer cells.

[0042] Quantification of GPC1 mRNA expression by qRT-PCR in triplicate in (A) ovarian cancer cell lines and (B) primary HGSOC cells from independent experiments (n=4). -Δct Data were analyzed using the PCR method and normalized to the housekeeping gene β-actin. GPC1 protein levels were assessed by western blotting for GPC1 (65 kDa) and β-actin (48 kDa). 20 μg of protein from each ovarian cancer cell line (C) and primary ovarian cancer cells (D) were 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).

[0043] Figures 5A to 5F : Effect of GPC1 CART cell therapy on ovarian cancer survival in a 2D monolayer assay.

[0044] Ovarian cancer cells were seeded at 10,000 cells / well in 96-well plates and treated for 48 hours with medium alone, untransduced (UT) CD3 T cells (blue), or GPC1 CAR-T cells (pink) at ratios of 2:1, 5:1, and 10:1, E:T ratio. Cell survival 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). Compared to UT T-CD3 cells, GPC1 CAR-T cells demonstrated potent cytotoxicity at all concentrations in SKOV3 and COV362 cell lines. Treatment of OV90 and OVCAR3 cells with GPC1 CAR T cells at 5:1 and 2:1 ratios resulted in a significant decrease in cell survival compared to UT CD3 T cells. Cells from both Patient 1 and Patient 3 showed a significant decrease in cell survival at 10:1, but did not show any significant decrease in cell survival at 5:1, and only Patient 3 showed a decrease in cell survival at 2:1.

[0045] Figures 6A to 6C : Effects of GPC1 CAR-T cells on ovarian cancer 3D spheroid cultures.

[0046] Representative images of (A) COV362, (B) SKOV3, and (C) OVCAR-3 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 from three independent experiments (repeated twice) and measured from five randomly selected areas within each well using ImageJ software. Data are expressed as the percentage of spheroid area relative to the control. For all cell lines, a significant decrease in spheroid size was observed between the UT CD3 T cell group and the GPC1 CAR-T cell group. Statistical significance was assessed by one-way ANOVA combined with Tukey's multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, scale bar represents 1000 μm. All images are at the same magnification.

[0047] Figure 7A and Figure 7B : Effects of GPC1 CAR-T cells on primary ovarian cancer cell 3D spheroid cultures.

[0048] Representative images of spheroid cultures 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 from three independent experiments (repeated twice) and measured from five randomly selected areas within each well using ImageJ software. Data are expressed as the percentage of spheroid area relative to the control. For both primary cells, a significant decrease in spheroid size was observed between the control and GPC1 CAR-T cell-treated groups. Statistical significance was assessed by one-way ANOVA combined 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.

[0049] Figures 8A to 8G : Impact of GPC1 CAR-T cells in a patient-derived explant assay.

[0050] GPC1 CART cell therapy induces apoptosis in explants derived from patients with high expression of GPC1. AF cleaved caspase 3 was quantified, and the data were expressed as % of positively stained cells / mm2. The bar graph shows the mean + / - SD. Compared with the non-transduced group, the expression of cleaved caspase 3 in the CAR-T treatment group was significantly increased. Unpaired t-tests (AD) were performed on patients 1-4, but unpaired t-tests (EF) were not performed on patients 5 and 6. G. Representative images of GPC1 expression obtained by immunohistochemistry in non-responsive explant tissues and in explant tissues that responded to GPC1 CAR-T cell therapy. Scale bar = 50 μm, same magnification for all images. GPC1 expression measured by using QuPath's H score was significantly increased in responders compared to non-responders.

[0051] Figure 9 :Development of GPC1 CD3 CAR-T cells

[0052] Flowchart of the established protocol for generating CAR-T cells in Professor Simon Barry's laboratory.

[0053] Figure 10 :Flow cytometric analysis of 98 batches of cells (FACs)

[0054] EGFR expression in CD3 untransduced (UT) T cells and GPC1 CD3 CAR T cells.

[0055] Figure 11 :Flow cytometry analysis of 98 batches of cells (FACs) - cell maturation detection panel

[0056] CD45RA and CD62L markers in CD4 T cell populations and CD8 T cell populations. EMRA (re-expressing CD45RA, CD45RA+CD62L- effector memory cells), Q10: naive phenotype (CD45RA+CD62L+), Q11: central memory (CD45RA-CD62L+), Q12: effector memory (CD45RA-CD62L-)

[0057] Figure 12A and Figure 12B : Flow cytometric analysis (FACs) of two batches of cells. Depletion detection panel.

[0058] (A) Expression of programmed cell death-1 (PD1), LAG3, and TIM in CD4 T cell populations. (B) Expression of programmed cell death-1 (PD1), LAG3, and TIM in CD8 T cell populations.

[0059] Figure 13A and Figure 13B : GPC1 expression in the control

[0060] Representative images of GPC1 protein expression in mouse kidneys used for IHC analysis. (A) Positive control, and (B) negative control.

[0061] Figure 14A and Figure 14B : The relationship between GPC1 and progression-free survival and overall survival was measured using H-index quartiles.

[0062] 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).

[0063] Figures 15A to 15F : Construction of CNA500200, CNA510200, CNA 500300, CNA510300, CNA500400 and CNA510400.

[0064] Six different CAR constructs were generated. These consisted of two different scFv fusion proteins that provided binding domains and three different linker domains. The two scFvs contained: (i) a MIL-38 leader sequence (1) connected to the variable light chain (VL) (2) of the MIL-38 antibody, which was fused to the variable heavy chain (VH) (4) of the MIL-38 antibody via a Whitlow linker (3); and (ii) a MIL-38 leader sequence (1) connected to the variable heavy chain (VH) (4) of the MIL-38 antibody, which was fused to the variable light chain (VL) (2) of the MIL-38 antibody via a Whitlow linker (3). The three linker domains contained: (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 includes a CD28 transmembrane domain (6), a costimulatory domain with a 4-1BB portion (7), an activation domain with a CD3ζ portion (8), a T2A self-1 cleavage site (9) and a truncated EGFR (10). DETAILED DESCRIPTION

[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 an individual. Furthermore, the inventors have demonstrated that GPC1 can serve as a prognostic marker for patients with ovarian cancer, with elevated expression indicating a poor prognosis for the patient.

[0066] Furthermore, the present inventors have demonstrated that CPG1 can target and kill cancer cells, such as ovarian cancer cells, and thus can provide a target for cancer treatment.

[0067] Glypican-1

[0068] Glypicans belong to the heparan sulfate proteoglycan (HSPC) family and are numbered 1 (GPC-1) to 6 (GPC-6).

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

[0070] During embryonic development, GPC1 is primarily expressed in the nervous and skeletal systems, with lower expression levels in adult tissues such as the heart and testis. It participates in organ development by regulating extracellular growth signals and morphogenetic gradient formation.

[0071] Cancer treatment

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

[0073] In some aspects, the invention provides a method of treating a subject having cancer comprising killing cells expressing Glypican-1.

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

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

[0076] Most epithelial ovarian / fallopian tube cancers are of the serous type, which are classified as either low-grade serous carcinoma (LGSC or LSOC) or high-grade serous carcinoma (HGSC or HSOC). These tumors have different genetic alterations and biological properties.

[0077] The present inventors have discovered that Glypican-1 is particularly associated with high-grade serous ovarian cancer. Thus, in some embodiments of the methods of treatment or prevention, the ovarian cancer is epithelial cell ovarian cancer, particularly serous ovarian cancer, 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.

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

[0079] Agents suitable 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, cells expressing chimeric antigen receptors (CARs), bispecific binding molecules comprising bispecific T cell binders and bispecific antibodies, and vaccines designed to elicit an immune response against GPC1.

[0080] Thus, in some embodiments of the methods of treatment or prevention, a subject is administered, or a cell is exposed, to an agent that preferentially or selectively binds to Glypican 1. In some embodiments, such an agent can be an antibody or a binding fragment of an antibody.

[0081] Antibody binding fragments can be derived from antibodies or can be recombinantly produced using the same sequence as the CDR of an antibody or antibody fragment. In fact, these CDRs can be derived from affinity-matured antibodies and therefore may be different from antibodies derived in vivo.

[0082] Antibodies are composed of four chains (two heavy chains and two light chains) and can be divided into Fc (crystallizable portion) and Fab (antigen binding portion) domains. 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 antibodies. However, the Fab portion contains the binding region of the antibody, which is crucial for the specificity of the desired epitope of the antibody.

[0083] Thus, in some embodiments, the antibody fragment is a Fab fragment of an antibody. The Fab fragment can be a single Fab fragment (i.e., the antibody fragment is produced without a disulfide bridge) or a F(ab')2 fragment, which comprises two Fab fragments of an antibody connected by a disulfide bridge. These fragments are typically produced by cleaving the antibody using a digestive enzyme such as pepsin. Methods for preparing such Fabs are well known in the art (see, for example, J. et al., Methods Mol Biol. 2017; 1535: 319-329).

[0084] Antibodies are composed of a total of six CDRs, with the VH and VL chains each comprising three CDRs (within a framework consisting of four framework regions). Single VH and VL chains (each comprising only three CDRs) have been shown to bind specifically with high affinity. Typically, a single binding region is referred to as a single antibody domain (sdAb). Alternatively, the 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 Fab, scFv is not fragmented from an antibody, but is typically recombinantly formed based on the CDRs and framework regions of an antibody. In addition, sdAbs and scFvs can also be recombinantly produced to form the binding portion of a larger fusion protein, which can also contain other parts. Therefore, in some embodiments, the reagent is or includes an scFv or sdAb comprising CDRs from an antibody that binds to GPC1. An scFv can include multiple VH and VL chains linked together to form a multivalent scFv (such as a bivalent scFv or a trivalent scFv).

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

[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 sequences of the variable light chain and the variable heavy chain 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 (such as MIL-38) that binds to Glypican-1.

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

[0088] In some embodiments, the variable heavy chain region has the amino acid sequence of SEQ ID NO: 2, or a variant thereof having sequence identity thereto. In some embodiments, the variable light chain region has the amino acid sequence of SEQ ID NO: 1, or a variant thereof having sequence identity thereto. In some embodiments, the variant of the variable heavy chain or variable light chain 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 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% identity to the variable heavy chain and / or variable light chain of SEQ ID NO:2 and / or SEQ ID NO:1.

[0089] In some embodiments, the heavy chain variable region comprises a heavy chain CDR1 having the amino acid sequence of DYSMN or the amino acid sequence of DYSMN with up to 1, 2, or 3 amino acid modifications, a heavy chain CDR2 having the amino acid sequence as shown in SEQ ID NO:4 or the amino acid sequence as shown in SEQ ID NO:4 with up to 1, 2, or 3 amino acid modifications, and a heavy chain CDR3 having the amino acid sequence of HYDYGGFPY or the amino acid sequence of HYDYGGFPY with up to 1, 2, or 3 amino acid modifications.

[0090] In some embodiments, the variable light chain comprises a light chain CDR1 having the amino acid sequence as shown in SEQ ID NO:3, or the amino acid sequence as shown in SEQ ID NO:3 with up to 1, 2, or 3 amino acid modifications, a light chain CDR2 having the amino acid sequence of TAKTLAD, or the amino acid sequence of TAKTLAD with up to 1, 2, or 3 amino acid modifications, and a light chain CDR3 having the amino acid sequence of QHFWSNPWT, or the amino acid sequence of QHFWSNPWT with up to 1, 2, or 3 amino acid modifications.

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

[0092] In some embodiments, the antibody or antigen-binding fragment comprises a light chain CDR1, CDR2, and CDR3 having the amino acid sequence of SEQ ID NO: 3, TAKTLAD, and QHFWSNPWT, with up to 1, 2, or 3 amino acid modifications.

[0093] Antibodies produced in vivo in a subject can be modified for specificity, avidity, and affinity 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):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 adjacent to hot spot positions defined by A / G-G-C / T-A / T (RGYW) and AG-C / T (AGY) sequences (referring to the DNA encoding the immunoglobulin) can alter the affinity of the produced antibody. Alternatively, methods 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 modification in the CDR regions with every possible mutation. Each variant is then assessed for antigen affinity and specificity. Thus, further modifications can be made to antibodies or binding fragments thereof derived in vivo to produce different but closely related antibodies. Thus, the term "antibody" (and fragments thereof) encompasses antibodies derived in vivo and in vitro molecules that have undergone a mutagenic process to modify the CDR binding site, thus having a unique sequence compared to the antibody produced in vivo. Furthermore, the binding portions of antibodies, in particular the CDRs, can be affinity matured and mutated using techniques well known in the art.

[0094] The term "antibody" also encompasses unconventional antibodies produced by species such as camelids, sharks and agnathans. Thus, the term antibody includes heavy chain antibodies, which include camelid antibodies, IgNAR and variable lymphocyte receptors (VLR). Furthermore, these can be fragmented into their binding portions (such as single binding portions of VNAR - IgNAR) or recombinantly integrated into fusion proteins. Methods for producing and manipulating such unconventional antibodies are well known in the art, see, e.g., Nuttall, S., Methods Mol. Biol, 2012; 911:27-36 and Vincke C. et al., Methods Mol. Biol. 2012; 907:145-76.

[0095] Antibodies that bind to GPC1 can be generated. Furthermore, these antibodies can be affinity matured to optimize long-lasting affinity and avidity. Thus, in some embodiments, the binding domain comprises a sequence identical to the binding region of an antibody that binds to GPC1, or comprises a sequence corresponding to an affinity-matured form of the binding region that binds to GPC1. Although the affinity-matured binding region differs significantly 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.

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

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

[0098] Other well-known conjugates for ADCs include: Monomethyl auristatin E (MMAE)-in brentuximab vedotin (Adcetris TM ) and Enfortumab Vedotin (Padcev TM ) used; monomethyl auristatin F (MMAF) - in Belantamab mafodotin (Blenrep TM ) used in; Calichexamycin - in Gemtuzumab Ozogamicin (Mylotarg TM ) used; Maytansine DM1 - in Trastuzumab Emtansine (Kadcyla TM ) used in; Maytansine DM4 - used in Mirvetuximab Soravtansine; Pyrrolbenzodiazepine (PBD) dimer - used in Rova-T; Camptothecin analogs - used in Sacituzumab Govitecan (Trodelvy TM ) used in; Duocarmycin analogs - used in multi-trastuzumab (Trastuzumab duocarmazine); Camptothecin derivative SN-38 - used in gosatuzumab (Sacituzumab Govitecan (TrodelvyTM ) used; Irinotecan metabolite SN-38- in Trastuzumab Deruxtecan (Enhertu TM ) used in; and topoisomerase I inhibitors - in Trastuzumab Deruxtecan (Enhertu TM ) are used. Any of the listed conjugates can be used in the present invention. Further conjugates and information for preparing such ADCs are provided 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 by reference.

[0099] Antibody drug conjugates of glypican-1 are known in the art. These include those disclosed in 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. 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 references are included herein).

[0100] The treatment or prevention methods provided herein can be used for patients diagnosed with cancer, particularly ovarian cancer. In some embodiments, in a subject, or especially on a subject's cancer cell, the treatment or prevention methods are performed after analyzing the expression of Glypican-1. In some embodiments of the prevention or treatment methods, it is determined that cells expressing Glypican-1 express elevated levels of Glypican-1, particularly Glypican-1 protein or Glypican-1 mRNA. The formation of elevated levels of Glypican-1 is well known in the art and is defined herein. Methods for assessing protein and mRNA levels are well known in the art and these methods are provided herein.

[0101] In some embodiments, the increased expression level of Glypican-1 protein includes increased surface expression of Glypican-1 protein and / or increased intracellular expression of Glypican-1 protein.

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

[0103] In some embodiments of the prophylactic or therapeutic methods, the diagnostic or prognostic methods as described herein are performed before or after treatment.

[0104] The treatment and prevention methods of the present invention can be performed alone or in combination with other cancer treatments, including, but not limited to, chemotherapy, surgical resection or tumor reduction surgery, or radiotherapy.

[0105] In some embodiments, the treatment or prevention methods of the present invention are performed in combination with another treatment (such as immunotherapy) as an adjuvant therapy. In some embodiments, the reagent that binds to Glypican 1 is administered together with an immune checkpoint inhibitor, such as a PD-1 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.

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

[0107] Chimeric antigen receptor

[0108] Chimeric antigen receptor (CAR) is an artificially constructed protein that can induce antigen-specific cellular responses after being expressed on the cell surface. In its most basic form, CAR includes at least three domains. The first domain is an extracellular antigen recognition domain that specifically recognizes an antigen, or more specifically recognizes one or more epitope portions of an antigen. The second domain is an intracellular signal transduction domain that can induce or participate in inducing an intracellular signal transduction pathway. And the third domain is a transmembrane domain that connects the extracellular antigen recognition domain and the intracellular signal transduction domain through the plasma membrane.

[0109] The combination of the first two domains determines the antigen specificity of the CAR and the ability of the CAR to induce the desired cellular response, the latter of which also depends on the host cell of the CAR. For example, after activation of a CAR expressed in a helper T cell, its signal transduction domain contains a CD3 activation domain, which, once activated by its cognate antigen, may induce CD4+ helper T cells to secrete a series of cytokines. In a further example, when expressed in a CD8+ cytotoxic T cell, the same CAR is activated by cells expressing the cognate antigen, which may induce the release of cytotoxins, ultimately leading to the induction of apoptosis of the cells expressing the antigen.

[0110] The third domain (membrane spanning domain) can include a part of the signal transduction domain of CAR, or can be combined with the signal transduction domain of CAR. The membrane spanning domain is typically one or more hydrophobic helices that span the lipid bilayer of the cell and embed CAR into the cell membrane. When combined with the cell, the membrane spanning domain of CAR may be a decisive factor in the expression pattern of CAR. For example, among other things, the membrane spanning domain combined with the CD3 co-receptor can allow CAR to be expressed in naive T cells, while the membrane spanning domain using the CD4 co-receptor can guide CAR to be expressed in helper T cells. The use of the CD8 co-receptor membrane spanning domain can guide expression in cytotoxic T lymphocytes (CTL), while the CD28 membrane spanning domain can allow expression in CTL and helper T cells, and helps to stabilize CAR.

[0111] A further component or part of a chimeric antigen receptor can 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 to the transmembrane domain. Generally, in the art, the linker domain is considered to be an optional domain because some CARs work without a linker domain.

[0112] Therefore, in one aspect, the present invention provides 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 (GPC1).

[0113] As used throughout, the term "recognition" (related to Glypican-1) refers to the ability of a binding domain to bind to a desired epitope of GPC1 or any part of the GPC1 molecule. Preferably, this recognition is selective in that the binding domain binds only or primarily to GPC1. In some embodiments, the binding domain can bind directly to GPC1 or an epitope thereof. In some embodiments, the binding domain can bind indirectly to GPC1 or an epitope thereof, for example, by way of an intermediate or a bispecific molecule (e.g., a fifth-generation CAR). In some embodiments, the antigen recognition domain can bind to a processed form of GPC1. As used in this context, the term "processed form" relates to forms of GPC1 that are typically truncated or digested due to intracellular processing, including forms and epitopes of GPC1 presented on the major histocompatibility complex (e.g., human leukocyte antigen).

[0114] The CAR binding domain can be any suitable domain capable of recognizing GPC1 or its antigens. As used throughout, the term "binding domain" refers to the portion of the CAR that provides specificity for GPC1. In the context of the present invention, the binding domain comprises only a portion of the extracellular region (or extracellular region) of the CAR.

[0115] The binding domain of CAR can include a series of binding molecules. These include antibodies (including unconventional antibodies, such as heavy chain antibodies), antibody binding fragments (as described herein, including scFv, Fab, sdAb) and protein binding scaffolds. In some embodiments, the binding domain includes the variable heavy chain of the antibody bound to GPC1 and / or the binding domain includes the variable light chain of the antibody bound to GPC1 - including antibodies and binding fragments disclosed herein. In some embodiments, the binding domain includes Fab. The antigen recognition domain can also be a fusion protein, such as a single-chain variable fragment (scFv), which has the same sequence as the antibody bound to Glypican-1. In some embodiments, the antigen binding domain includes SEQ ID NO: 28 or 29 (with or without SEQ ID NO: 27 MIL-28 leader), or a functional variant thereof having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 99.5% sequence identity.

[0116] For the avoidance of doubt, the binding domain of the CAR may comprise any antibody or antibody fragment sequence (including CDRs) disclosed herein in relation to an anti-GPC1 antibody (such as those described as possible "agents"), including any possible modifications disclosed herein.

[0117] Antibodies capable of binding to GPC1 are discussed herein, and include MIL-38.

[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 developed that do not include the linker domain, so in this context, the linker domain is generally not considered essential for the function of all CARs.

[0120] It is not desirable to be bound by theory, the joint domain can provide an appropriate molecular length for the extracellular domain (extracellular domain) of CAR to allow the antigen recognition domain to recognize an epitope while forming a correct immune synapse distance between the effector cell expressing CAR and the target cell. In addition, the joint domain can provide appropriate flexibility for the antigen recognition domain so that it is oriented in the correct manner to recognize its epitope.

[0121] Therefore, in some embodiments, the extracellular domain includes a linker domain connecting the binding domain to the transmembrane domain. In some embodiments, the length of the linker domain is at least 12 amino acids. In some embodiments, the length of the linker domain is at least about 12 amino acids. In some embodiments, the length of the linker domain is greater than 12 amino acids. In some embodiments, the length of the linker domain is at least 119 amino acids. In some embodiments, the length of the linker domain is at least about 119 amino acids. In some embodiments, the length of the linker domain is greater than 119 amino acids. In some embodiments, the length of the linker domain is at least 229 amino acids. In some embodiments, the length of the linker domain is at least about 229 amino acids. In some embodiments, the length of the linker domain is greater than 229 amino acids.

[0122] In some embodiments, the length of the connecting domain can be up to 119 amino acids. In some embodiments, the length of the connecting domain can be up to about 119 amino acids. In some embodiments, the length of the connecting domain can be up to 229 amino acids. In some embodiments, the length of the connecting domain can be up to about 229 amino acids.

[0123] Selection of an appropriate linker domain can optimize the potency of the CAR construct based on (i) reducing binding affinity to Fc receptors (such as Fcγ and FcRn receptors), which minimizes "off-target" activation of CAR-expressing cells and (ii) reducing spatial constraints on immune synapse formation (e.g., reducing steric hindrance and optimizing synaptic distance) by enhancing the flexibility of the antigen-binding region.

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

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

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

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

[0128] In some embodiments, the linker domain comprises all or a portion of an immunoglobulin hinge region. As will be 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.

[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 comprise mutations L235D, N297Q).

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

[0131] Table 1 - Possible linker domain components

[0132]

[0133] The hinge region, CH2 and CH3 regions of immunoglobulins (particularly IgG isotype antibodies) can bind to Fc receptors, such as Fcγ receptors and Fc neonatal receptors. The binding of the joint domain of the chimeric antigen receptor can reduce receptor efficacy and may cause off-target killing. Therefore, in some embodiments, the joint domain is designed to reduce or lose its binding ability to the Fc receptor. In some embodiments, the joint domain is the same as an immunoglobulin with reduced Fc receptor binding ability compared to other immunoglobulin isotypes. In some embodiments, the joint domain of the chimeric antigen receptor does not include an amino acid sequence that substantially binds to the Fc receptor.

[0134] The ability of Fc receptors to bind to different IgG isotypes is shown in Table 2 below.

[0135] Table 2 - Fc receptors binding to IgG subtypes

[0136]

[0137]

[0138] In some embodiments, where the linker domain comprises a portion identical to the Fc region of an immunoglobulin, the portion can be modified to reduce binding to Fc receptors. Methods for modifying proteins to reduce binding to Fc receptors are well known in the art. Fcγ receptors primarily bind to the lower hinge region of the immunoglobulin region and the n-terminus of the CH2 region, while neonatal Fc receptors primarily bind to amino acids at the C-terminus of the CH2 region and the n-terminus of the CH3 region. A guide to Fc receptor binding to IgG antibodies can be found in "Antibody Fc: Linking Adaptive and Innate Immunity" Ackerman and Nimmerjahn, Elsevier Science & Technology 2014”, Chapter 7. Therefore, modifications in these regions may alter Fc receptor binding to the linker domain homologous to the Fc portion of an immunoglobulin. A non-exhaustive exemplary list of human IgG1 mutations that have been shown to reduce Fcγ 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, V188A, T190A, L192A, Q194A, D195A, N198A, K200A, K205A , K209A, A210Q, A210S, A210G, P212A, P214A, E216A, K217A, S220A, K221A, A222 T, K243A, Q245A, H251A, D259A, A261Q, E263A, E265A, V286A, S288A, K297A, S307A, E313A, H316A, N317A, H318A, Y319A (numbering corresponds to the sequence shown in Uniprot reference number P01857-1).

[0139] In some embodiments, the linker domain has a sequence selected from 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.

[0140] Transmembrane and intracellular domains

[0141] The transmembrane domain of CAR binds the extracellular part (ectodomain) to the intracellular part (intracellular domain), and its role is mainly structural. Therefore, the transmembrane domain can be composed of any sequence that can anchor and cross the cell lipid bilayer. However, the nature of the transmembrane domain can affect its positioning and expression.

[0142] In a preferred embodiment, the transmembrane domain has sequence identity with the sequence of a molecule involved in T cell synapse formation or T cell signal induction. In some embodiments, the chimeric antigen receptor of the present 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 identical to all or a portion of the transmembrane domain of CD8 or CD28. In some embodiments, the transmembrane domain has sequence identity with all or a portion of the transmembrane domain of CD28. In some embodiments, the transmembrane domain has an amino acid sequence identical to SEQ ID NO: 18 or is a functional variant thereof having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 99.5% sequence identity.

[0143] In addition to the antigen recognition domain, linker domain and transmembrane domain, the chimeric antigen receptor of the present invention comprises an intracellular (intracellular) domain, which comprises a signal transduction portion (signaling domain).

[0144] In addition to the antigen recognition domain, linker domain and transmembrane domain, the chimeric antigen receptor of the present invention comprises an intracellular (intracellular) domain, which comprises a signal transduction portion (signaling domain).

[0145] The intracellular signal transduction domain of the chimeric antigen receptor can be any suitable domain, which can induce or participate in inducing intracellular signal cascades when CAR is activated, which is the result of the antigen recognition domain recognizing the antigen. The signal transduction domain of CAR will be specifically selected according to the expected cell outcome after activating CAR. Although there are many possible signal transduction domains, when used for immunotherapy and cancer therapy, the signal transduction domain can be divided into two major categories according to the receptor from which it is derived, i.e., activation receptors and costimulatory receptors (see further details below). Therefore, in some embodiments, the signal transduction domain includes a portion having the same amino acid sequence as the signal portion of the activation receptor or a functional variant thereof. In some embodiments, the signal transduction domain includes a portion having the same amino acid sequence as the signal portion of the costimulatory receptor or a functional variant thereof.

[0146] As used throughout, when the term "portion" is used for an activating receptor or a co-stimulatory receptor, it refers to any segment of the receptor, including sequences that are responsible for or participate in initiating / inducing an intracellular signal transduction cascade following interaction of the receptor with its cognate antigen or ligand. An example of an intracellular signal transduction cascade initiated / induced by CD3 is outlined below.

[0147] Although not wishing to be bound by theory, the extracellular portion of the TCR is primarily composed of heterodimers of clonal TCRα and TCRβ chains (TCRα / β receptors) or TCRγ and TCRδ chains (TCRγδ receptors). These TCR heterodimers typically lack intrinsic signal transduction capabilities and therefore non-covalently bind to multiple signal transduction subunits of CD3 (primarily CD3ζ, γ, δ, and ε). Each of the γ, δ, and ε chains of CD3 has an intracellular (cytoplasmic) portion that includes a tyrosine-based immunoreceptor activation motif (ITAM), while the CD3ζ chain contains three ITAMs in series. In the presence of MHC, the TCR engages with its cognate antigen and binds to the necessary co-receptors (such as CD4 or CD8), initiating signal transduction, leading to tyrosine kinase (i.e., Lck) phosphorylating two tyrosine residues within the intracellular ITAM of the CD3 chain. Subsequently, a second tyrosine kinase (ZAP-70—itself activated by Lac phosphorylation) is recruited to dually phosphorylate the ITAM. Consequently, several downstream target proteins are activated, ultimately leading to intracellular conformational changes, calcium mobilization, and actin cytoskeleton rearrangements, which, when combined, ultimately lead to the activation of transcription factors and the induction of T cell immune responses.

[0148] As used throughout, the term "activating receptor" relates to a receptor or co-receptor that forms a component of or participates in the formation of the T cell receptor (TCR) complex, or a receptor that is involved in the specific activation of an immune cell as a result of recognition of an antigen or other immunogenic stimulus.

[0149] Non-limiting examples of such activating receptors include T cell receptor-CD3 complexes (CD3ζ, γ, δ and ε), CD4 co-receptors, CD8 co-receptors, Fc receptors or natural killer (NK) cell-associated activating receptors such as LY-49 (KLRA1), natural cytotoxicity receptors (NCR, preferably NKp46, NKp44, NKp30 or NKG2 or CD94 / NKG2 heterodimers). Thus, in some embodiments of the CAR of the present invention, the signal transduction domain includes a signal transduction portion derived from a CD3 co-receptor complex member (preferably at least the signal transduction portion of the CD3ζ chain), a CD4 co-receptor, a CD8 co-receptor, an Fc receptor (FcR) signal transduction portion (preferably a signal transduction portion of FcεRI or FcγRI) or an NK-associated receptor such as LY-49.

[0150] The specific intracellular signal transduction portion of each CD3 chain is well known in the art. See, for example, 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 CAR.

[0151] In some embodiments of the present invention, the signal transduction domain comprises a portion derived from CD3 or having sequence homology with CD3 (preferably the CD3-zeta chain or a portion thereof). In some embodiments, the signal transduction domain comprises a sequence identical to all or part of the intracellular domain of 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 thereof having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 99.5% sequence identity.

[0152] Alternative signaling domains include the intracellular portion of an Fc receptor, which is well known in the art, for example, the intracellular portion of the FcεR1 or FcγRI receptors (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 the CAR, such as CD3ζTM and CD3ζIC (Landmeier S et al., (2007). Gene-Engineered Varicella-Zoster Virus–Reactive CD4+ Cytotoxic TCells 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, JImmunol, 180(10), 7028-38; Patel SD et al., (1999). Impact of chimeric immunereceptor 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-ζvs 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-SignalingγSubunit, J Immunol, 161, 6604-6613). .

[0153] As discussed above, in some embodiments of the chimeric antigen receptors of the invention, the signaling domain includes a portion having an amino acid sequence identical to the signaling portion of a costimulatory receptor.

[0154] As used throughout, the term "co-stimulatory receptor" refers to a receptor or co-receptor that assists in activating immune cells when antigen-specific induction activates the receptor. It will be understood that co-stimulatory receptors do not require the presence of antigens, nor are they antigen-specific, but are typically one of two signals, the other being the activation signal required for inducing immune cell responses. In the context of immune response, co-stimulatory receptors are typically activated by the presence of their expressed ligands on the surface of antigen-presenting cells (APCs) (such as dendritic cells or macrophages). With respect to T cells, co-stimulation is necessary for cell activation, proliferation, differentiation, and survival (all of which are typically within the scope of T cell activation), and presenting antigens to T cells in the absence of co-stimulation may lead to the development of anergy, clonal deletion, and / or antigen-specific tolerance. Importantly, co-stimulatory molecules can notify T cells of the response to antigens encountered at the same time. Typically, the antigens encountered in the context of "positive" co-stimulatory molecules lead to T cell activation and a cellular immune response intended to eliminate cells expressing the antigen. And in the context of "negative" co-receptors, the antigens encountered lead to induction of a state of tolerance to the antigens encountered together.

[0155] Non-limiting examples of T cell costimulatory receptors include CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137), ICOS. In particular, CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS all represent “positive” costimulatory molecules that enhance activation of T cell responses. Thus, in some embodiments of the first aspect of the application, the signal transduction domain comprises a portion derived from any one or more of CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS.

[0156] In some embodiments of the application, the signal transduction domain comprises a portion derived from a CD28, OX40, or 4-1BB costimulatory receptor. In some embodiments, the signal transduction domain comprises a portion of 4-1BB as set forth in SEQ ID NO: 20, or a functional variant thereof having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.5% sequence identity thereto.

[0157] Different portions of costimulatory receptors can be used individually or in combination to form the transmembrane (TM) and intracellular (IC) portions of the CAR. Examples of 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).

[0158] Sequence information for the above-mentioned activating and costimulatory receptors can be readily accessed in various databases. For example, embodiments of the human amino acid, gene, and mRNA sequences for these receptors are provided in Table 3.

[0159] Table 3 - Summary of activating and costimulatory receptor sequence information

[0160]

[0161]

[0162] Although Table 3 is provided with reference to human activating and co-stimulatory receptors, it will be understood by those skilled in the art that homologous and orthologous forms of each receptor exist in most mammals and vertebrates. Therefore, the sequences cited above are provided only as non-limiting examples of receptor sequences included in the CAR of the first aspect of the invention, and homologous and orthologous sequences from any desired species can be used to generate CARs suitable for a given species.

[0163] In some embodiments of the present invention, the transmembrane domain and a portion of the signaling domain have homology to the same molecule. For example, a portion of CD3 comprising a transmembrane domain and a signaling domain can be used. 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.

[0164] In some embodiments of the present invention, the signal transduction domain includes a portion derived from an activating receptor and a portion derived from a costimulatory receptor. Although it is not desired to be bound by theory, in this case, the recognition of the antigen by the antigen recognition domain of CAR will simultaneously induce intracellular activation signal and intracellular costimulatory signal. Therefore, this will simulate the presentation of antigens by APC expressing costimulatory ligands. Alternatively, CAR can have a signal transduction domain comprising a portion from an activating receptor or a costimulatory receptor. In this alternative form, CAR will only induce activation of intracellular signal transduction cascades or costimulatory intracellular signal transduction cascades.

[0165] In some embodiments of the invention, 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.

[0166] In some embodiments, CAR will have a signal transduction domain comprising a portion of a single activating receptor and a portion of multiple costimulatory receptors. In some embodiments, CAR will have a signal transduction domain comprising the same sequence as a portion of multiple activating receptors and a single portion from a single costimulatory receptor. In some embodiments, CAR will have a signal transduction domain comprising the same sequence as a portion of multiple activating receptors and a portion of multiple costimulatory receptors. In some embodiments, CAR will have a signal transduction domain comprising the same sequence as a portion of a single activating receptor and a portion of two costimulatory receptors. In some embodiments, CAR will have a signal transduction domain comprising the same sequence as a portion of a single activating receptor and a portion from three costimulatory receptors. In some embodiments, CAR will have a signal transduction domain comprising the same sequence as a portion of two activating receptors and a portion of a costimulatory receptor. In some embodiments, CAR will have a signal transduction domain comprising the same sequence as a portion of two activating receptors and a portion of a costimulatory receptor. In some embodiments, CAR will have a signal transduction domain comprising the same sequence as a portion of two activating receptors and a portion of two costimulatory receptors. It will be understood that the number of activating receptors and costimulatory receptors has further variations, and the above examples are not considered to be limitations on the possible combinations contained herein.

[0167] In some embodiments of the present invention, the sequence of at least a portion of the transmembrane domain and the signal transduction domain has sequence similarity to portions of different molecules. In some embodiments, the transmembrane domain comprises or consists of the same sequence as all or a portion of the transmembrane domain of CD28, and the signal transduction domain comprises or consists of the same sequence as all or a portion of the intracellular domain of 4-1BB and CD3-ζ chains.

[0168] CAR is currently referred to as generation 1 to generation 5 (see Labanieh L and Mackall CL. (2023), CARimmune 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 disclosure of which is incorporated herein). In some embodiments, the CAR of the present invention is a 3rd generation CAR or higher (i.e., containing an activation domain and two or more costimulatory domains). In some embodiments, CAR is a 4th generation CAR or higher (i.e., TRUCK-T cells are redirected for universal cytokine-mediated killing). In some embodiments, CAR T cells include a terminator receptor to allow removal of CAR T cells after administration.

[0169] Chimeric antigen receptor

[0170] An exemplary chimeric antigen receptor (CAR) of the present invention is prepared using two scFv fusion proteins with two orientations of the variable light chain domain and variable heavy chain domain of the MIL-38 antibody (WO2016 / 168885A1; 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).

[0171] As in Figures 15A to 15F As shown in , two scFv domains were prepared with the following orientations:

[0172] 1. Light chain variable region (2)-Whitlow linker (3) (PMID: 8309948)-heavy chain variable region (4) (represented by number CNA500xxx); and

[0173] 2. Heavy chain variable region (4) - Whitlow linker (3) - light chain variable region (2) (indicated by number CNA510xxx).

[0174] In addition, three different linker domains were used, namely:

[0175] Linker 1 - IgG4 hinge (5) (SEQ ID NO: 15) - (represented by numbering CNA5x02xx);

[0176] Linker 2—IgG4 hinge+IgG4 CH3 (11) (SEQ ID NO: 16)—(represented by numbering CNA5x03xx); and

[0177] Linker 3—IgG hinge+IgG4 CH2 L235D and N297Q mutations+IgG4 CH3 (12) (SEQ ID NO: 17)—(represented by numbering CNA5x04xx).

[0178] The sequences (SEQ ID NOs) and components of the chimeric antigen receptors are provided in Table 4.

[0179] Table 4: Summary of sequence identifiers

[0180]

[0181]

[0182] Further references Figures 15A to 15F , the CAR as a specific embodiment example in the present invention includes the following components: a MIL-38 leader sequence (1), a transmembrane region (6) having a sequence identical to a portion of CD28, a costimulatory domain (7) having a sequence identical to a portion of 4-1BB, and an activation domain (8) having a sequence identical to a portion of CD3ζ. The exemplary CAR also includes a truncated ECF receptor (EGFRt) (10), which allows transduction and expression in analysis cells. EGFRt is connected by a self-cleavage site T2A (9), allowing EGFRt to be separated from CAR. The sequences of EGFRt and T2a are well known in the art and are disclosed in WO / 2022 / 104424.

[0183] In some embodiments, the CAR will comprise an antigen recognition domain specific for GPC1, a linker domain having sequence identity to the hinge region of IgG4, a transmembrane region having sequence identity to the CD28 transmembrane sequence, an intracellular portion having sequence identity to the signaling region of 4-1BB and / or an intracellular portion having sequence identity to the signaling region of CD3ζ, or functional variants of such portions, domains, or regions.

[0184] In some embodiments, the CAR will comprise an antigen recognition domain specific for GPC1, a linker domain having sequence identity to the hinge region of IgG4 combined with the CH3 region of IgG4, a transmembrane region having sequence identity to the CD28 transmembrane sequence, an intracellular portion having sequence identity to the signaling region of 4-1BB and / or an intracellular portion having sequence identity to the signaling region of CD3ζ, or functional variants of such portions, domains, or regions.

[0185] In some embodiments, the CAR will comprise an antigen recognition domain specific for GPC1, a linker domain having sequence identity to the IgG4 hinge region combined with the IgG4 CH2 region (which may comprise L235D and N297Q mutations) and the IgG4 CH3 region, a transmembrane region having sequence identity to the CD28 transmembrane sequence, an intracellular portion having sequence identity to the signaling region of 4-1BB and / or an intracellular portion having sequence identity to the signaling region of CD3ζ, or functional variants of such portions, domains, or regions.

[0186] 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 functional variants thereof.

[0187] It will be understood by those skilled in the art that the CAR receptors described herein may be modified without departing from the scope of the present invention. For example, for SEQ ID NOs: 21, 22, 23, 24, 25, and 26, the preferred function of CAR is to recognize GPC1 and induce intracellular signals to activate T cells expressing CAR. Therefore, portions of the amino acid sequence of the chimeric antigen receptor may be changed without significantly changing the specificity of the CAR and / or the activation of cells (such as T cells) expressing CAR. Such changes may include, but are not limited to, changes in the hinge region of the chimeric antigen receptor, changes in the transmembrane domain, and changes in portions of the activation receptor and / or co-stimulatory receptor comprising the intracellular domain of the chimeric antigen receptor. When making such changes, those skilled in the art will utilize this knowledge and skills to produce viable CARs. Therefore, the scope of these changes does not include those changes that those skilled in the art can immediately identify as causing the abolition of CAR function.

[0188] In some embodiments of the present invention, the chimeric antigen receptor comprises or consists of a variant of SEQ ID NO: 21, 22, 23, 24, 25 or 26, wherein the amino acid sequence of the variant is selected from the group consisting of SEQ ID NO: The amino acid sequences of the group consisting of NO: 21, 22, 23, 24, 25 or 26 have 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% or at least 99.9% sequence identity.

[0189] Genetic modification of nucleic acid constructs and cells

[0190] CAR as described herein can be produced by any method known in the art, but is preferably produced using recombinant DNA technology. Nucleic acids encoding several regions of chimeric antigen receptors can be easily prepared and assembled into complete coding sequences by standard molecular cloning techniques (genomic library screening, PCR, primer-assisted connection, site-directed mutagenesis, etc.) known in the art. Preferably, the resulting coding region is inserted into an expression vector and used to transform a suitable expression host cell line, preferably a T lymphocyte line, and most preferably an autologous T lymphocyte line.

[0191] Therefore, the present invention also provides a nucleic acid molecule or a nucleic acid construct comprising the nucleic acid molecule, which has a nucleic acid sequence encoding the chimeric antigen receptor described above.

[0192] Furthermore, the nucleic acid construct may be an expression vector comprising the nucleic acid sequence encoding the chimeric antigen receptor described above.

[0193] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 23, 24, 25, and 26, or variants of these sequences as previously defined.

[0194] Nucleic acid molecules can comprise any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified or modified RNA or DNA. For example, nucleic acid molecules can include single-stranded and / or double-stranded DNA, DNA as a mixture of single-stranded and / or double-stranded regions, single-stranded and double-stranded RNA and RNA as a mixture of single-stranded and double-stranded regions, hybrid molecules comprising DNA and RNA, which can be single-stranded or more typically double-stranded or a mixture of single-stranded and double-stranded regions. In addition, nucleic acid molecules can comprise triple-stranded regions comprising RNA or DNA or both RNA and DNA. Nucleic acid molecules can also comprise one or more modified bases or a DNA or RNA backbone modified for stability or other reasons. DNA and RNA can be subjected to a variety of modifications; therefore, the term "nucleic acid molecule" encompasses chemical, enzymatic or metabolically modified forms.

[0195] In some embodiments of the invention, the nucleic acid molecule comprises a portion of the nucleotide sequence shown in SEQ ID NO: 7, 8, 9, 10, 11 or 12, which encodes the amino acids shown in SEQ ID NO: 21, 22, 23, 24, 25 and 26, or a functional variant thereof.

[0196] For the avoidance of doubt, it should be understood that functional variants of the relevant portions of SEQ ID NO: 7, 8, 9, 10, 11 or 12 include sequence variants having one or more different nucleic acids but still encoding 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 typically the only codon for methionine and TGG, which is typically the only codon for tryptophan) can be modified to produce a functionally identical molecule. Thus, each silent mutation of a nucleotide sequence encoding a polypeptide is implicit in each described sequence.

[0197] It should be understood that, according to the present invention, the nucleic acid construct may further comprise one or more of the following: an origin of replication for one or more hosts; a selectable marker gene active in one or more hosts; and / or one or more transcription control sequences under the control of which the nucleic acid molecule is expressed.

[0198] As used herein, the term "selectable marker gene" includes any gene that confers a phenotype on a cell expressing the gene, thereby facilitating the identification and / or selection of cells transfected or transduced with a construct.

[0199] "Selectable marker gene" includes any nucleotide sequence that, when expressed in cells transduced with a construct, confers a phenotype on the cell that facilitates identification and / or selection of the transduced cells. A list of nucleotide sequences encoding suitable selectable markers is well known in the art (e.g., Mortesen, RM. and Kingston RE. Curr Protoc Mol Biol, 2009; Unit 9.5). Exemplary nucleotide sequences encoding selectable markers include: adenosine deaminase (ADA) gene; cytosine deaminase (CDA) gene; dihydrofolate reductase (DHFR) gene; histidinol dehydrogenase (hisD) gene; puromycin-N-acetyltransferase (PAC) gene; thymidine kinase (TK) gene; xanthine-guanine phosphoribosyltransferase (XGPRT) gene or antibiotic resistance genes, such as ampicillin resistance gene, puromycin resistance gene, bleomycin resistance gene, hygromycin resistance gene, kanamycin resistance gene and ampicillin resistance gene; fluorescent reporter genes, such as green, red, yellow or blue fluorescent protein encoding genes; and luminescence-based reporter genes, such as luciferase gene, etc., which allow cells to be optically selected using techniques such as fluorescence-activated cell sorting (FACS). Further, in Barese, CN and Dunubar CE, Hum.Gene Ther., 2011; 22 (6): 659-68 pages, cell selection markers for T cells are specifically discussed. These markers include neomycin (NEO) resistance gene, ΔNGFR (non-signaling NGFR), truncated CD34 and truncated non-signaling CD19 (ΔCD19). Embodiments of the present invention (as further described herein) utilize a truncated form of epithelial growth factor receptor (EGFRt). Further techniques have been developed to track CAR T cells comprising modified eDHFD in vivo (see Sellmyer, MA et al. Mol. Ther., 2020; 28 (1): 42-51 pages).

[0200] Furthermore, it should be noted that the selectable marker gene can be a different open reading frame in the construct or can be expressed as a fusion protein with another polypeptide (e.g., CAR).

[0201] As mentioned above, nucleic acid construct can also comprise one or more transcription control sequences.Term " transcription control sequence " should be construed as comprising any nucleic acid sequence that influences the transcription of operably connected nucleic acid.Transcription control sequence can comprise, for example, leader sequence, polyadenylation sequence, promoter, enhancer or upstream activation sequence and transcription terminator.Usually, transcription control sequence comprises at least promoter.As used in this article, term " promoter " has described any nucleic acid that gives, activates or enhances nucleic acid expression in cell.

[0202] In some embodiments, at least one transcription control sequence is operably linked to the nucleic acid molecule of the second aspect of the invention. For the purposes of this specification, a transcription control sequence is considered to be "operably linked" to a given nucleic acid molecule if the transcription control sequence is capable of promoting, inhibiting, or otherwise regulating the transcription of the nucleic acid molecule. Thus, in some embodiments, the nucleic acid molecule is under the control of a transcription control sequence (e.g., a constitutive promoter or an inducible promoter).

[0203] In the cells, tissues or organs where expression occurs, promoters can constitutively or differentially regulate the expression of an operably linked nucleic acid molecule. Thus, promoters can include, for example, constitutive promoters or inducible promoters. A "constitutive promoter" is a promoter that is active under most environmental and physiological conditions. An "inducible" promoter is a promoter that is activated under specific environmental or physiological conditions. The present invention contemplates the use of any promoter that is active in the cell of interest. Therefore, one of ordinary skill in the art will readily determine a variety of promoters.

[0204] Mammalian constitutive promoters can include, but are not limited to, simian virus 40 (SV40), cytomegalovirus (CMV), P-actin, ubiquitin C (UBC), elongation factor-1α (E3A), phosphoglycerate kinase (PGK), and CMV early enhancer / chicken beta-actin (CAGG).

[0205] Inducible promoters can include, but are not limited to chemical induction promoters and physical induction promoters. Chemical induction promoters include promoters with activity that are regulated by chemical compounds such as alcohols, antibiotics, steroids, metal ions or other compounds. Examples of chemical induction promoters include: promoters regulated by tetracycline (e.g., see U.S. Patent No. 5,851,796 and U.S. Patent No. 5,464,758); steroid responsive promoters such as glucocorticoid receptor promoters (e.g., see U.S. Patent No. 5,512,483), ecdysone receptor promoters (e.g., see U.S. Patent No. 6,379,945); and metal responsive promoters such as metallothionein promoters (e.g., see U.S. Patent No. 4,940,661, U.S. Patent No. 4,579,821 and U.S. Patent No. 4,601,978) etc.

[0206] As mentioned above, control sequences can also include terminators. The term "terminator" refers to a DNA sequence at the end of a transcription unit that signals the termination of transcription. A terminator is a 3'-untranslated DNA sequence that typically contains a polyadenylation signal, which helps add the polyadenylation sequence to the 3' end of the primary transcript. Like the promoter sequence, the terminator can be any terminator sequence that is operable in the cell, tissue, or organ in which it is intended to be used. Suitable terminators will be well known to those skilled in the art.

[0207] According to understanding, the nucleic acid construct according to the present invention may further include additional sequences, such as sequences allowing enhanced expression, cytoplasm or membrane transport and localization signals. Specific non-limiting examples include internal ribosome entry site (IRES), N-terminal interleukin-2 signal peptide (Moot R. et al., Mol Ther Oncolytics, 2016; 3: 16026), CSF2RA, IgE leader sequence (WO2017147458), influenza hemagglutinin signal sequence (Quitterer, U. et al., Biochem. Biophys. Res., 2011: 409 (3): .544-579 pages) etc. A review of signal peptides is provided in Owki, H. et al., Eur. J. Cell Biol., 2018; 97 (6): .422-441 pages, which are incorporated herein by reference.

[0208] The present invention extends to all genetic constructs described herein in principle.These constructs may also comprise nucleotide sequences intended for maintaining and / or replicating the genetic construct in eukaryotic organisms and / or integrating the genetic construct or part thereof into the genome of a eukaryotic cell.

[0209] The nucleic acid construct may be in any suitable form, such as a plasmid, phage, transposon, cosmid, chromosome, vector, etc., which, when combined with appropriate control elements, is capable of replicating and transferring the gene sequence contained in the construct between cells.

[0210] Therefore, the term vector includes cloning and expression vectors and viral vectors. In some embodiments, the nucleic acid construct is a vector. In some embodiments, the vector is a viral vector, and therefore the present invention provides a viral vector comprising a nucleic acid molecule or nucleic acid construct encoding the above-mentioned CAR. In some embodiments, the vector is a DNA vector or an mRNA vector.

[0211] In at least some embodiments, the present invention provides nucleic acid molecules or nucleic acid constructs encoding the above-mentioned CAR for use in preparing genetically modified cells. In addition, in at least some embodiments, the present invention provides the use of nucleic acid molecules in preparing vectors for cell transformation, transfection or transduction as described herein. Cells suitable for genetic modification can be allogeneic or autologous.

[0212] In some embodiments, the cells are used in methods for preventing or treating cancer or in the preparation of a medicament. Therefore, in some embodiments, the present invention provides the use of a vector in the preparation of a medicament for preventing or treating cancer, particularly ovarian cancer expressing Glypican-1.

[0213] Methods for intentionally introducing exogenous genetic material (such as nucleic acid constructs) into eukaryotic cells (transfection / transduction) are well known in the art. It is understood that the method most suitable for introducing a nucleic acid construct into a desired host cell depends on many factors, such as the size of the nucleic acid construct, the type of host cell, the expected efficiency of transfection / transduction, and the ultimate expected or desired survival 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 phototransfection; particle-based methods such as "gene gun" delivery, magnetofection or puncture transfection, lipid nanoparticles, or viral transduction.

[0214] A variety of viral transduction techniques for mammalian cells are well known in the art. Common viral vectors include lentiviruses and retroviruses. An exemplary protocol is provided 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).

[0215] In some embodiments, the present invention provides a lentivirus comprising a nucleic acid encoding a chimeric antigen receptor as described herein. In addition, the present invention provides a use of a viral vector, preferably a retrovirus, such as a lentivirus or a gammaretrovirus, in the preparation of a genetically modified cell or medicament for preventing or treating cancer or for killing cells expressing Glypican-1 or aberrantly expressing Glypican-1.

[0216] Cell transduction can result in genomic integration of the DNA encoding the above-mentioned CAR. Alternatively, the DNA can be transiently expressed in the transduced cells. Each of these has advantages and disadvantages. The genomic-integrated DNA is stably expressed and replicated to daughter cells during cell replication. This ensures a significant increase in the body's powerful immune response and CAR-expressing T cells.

[0217] Alternatively, transient transduction (usually achieved by transduction with mRNA) results in transient CAR expression in the cells. This generally results in a lower response but provides the practitioner with more control, allowing them to increase or decrease the "dose" as needed.

[0218] As described above, in some embodiments, the present invention provides the use of a DNA vector or recombinant DNA in preparing a viral vector for gene transduction in a cell. The cell can be any cell, but suitable examples are provided.

[0219] Nucleic acid construct will be selected according to the method required for transfection / transduction.In some embodiments, nucleic acid construct is a viral vector, and the method for introducing nucleic acid construct into host cell is viral transduction. It is well known in the art to use viral transduction to induce CAR expression in PBMC such as T cells (Parker, LL. et al., Hum Gene Ther.2000; 11: 2377-87), and more generally to utilize retroviral system to transduce mammalian cells (Cepko, C. and Pear, W.CurrProtoc Mol Biol.2001, unit 9.9). In some embodiments, nucleic acid construct is a plasmid, cosmid, artificial chromosome et al., and can be transfected into cells by any suitable method known in the art.

[0220] Techniques for selecting / isolating cell subsets are well known in the art. These include fluorescence activated cell sorting (Basu S. et al., J. Vis. Exp. 2010; 41: 1546), techniques using antibodies immobilized on a substrate, such as magnetic cell separation ( ) device to immunomagnetically select cells expressing the desired marker (Zola H. et al., Blood, 2005; 106(9): 3123-6) or use a microfluidic chip. A series of cell markers can 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, CD48, CD49d, CD49f, CD51, CD56, CD57, CD62, CD62L, CD68, CD69, CD62, C D62L, CD66b, CD68, CD69, CD73, CD78, CD79a, CD79b, CD80, CD81, CD83, CD84, CD85g, CD86, CD94, CD103 CD106, CD115, CD117, CD122, CD123, CD126, CD127, CD130, CD138, CD140a, CD140b, CD141, CD152, CD159a, CD160, CD161, CD16 3. CD165, CD169, CD177, CD178, CD183, CD185, CD192, CD193, CD194, CD195, CD196, CD198, CD200, CD200R, CD203c, CD205, CD2 06, CD207, CD209, CD212, CD217, CD218α, CD229, CD244, CD268, CD278, CD279, CD282, CD284, CD289, CD294, CD303, CD304, CD314, CD319, CD324, CD335, CD336, CXCR3, Dectin-1, TcεR1α, Flt3, granzyme A, granzyme B, IL-9, IL-13α1, IL-21R, iNOS, KLRG1, MARCO, MHC class II, RAG, RORγT, Singlec-8, ST2, TCRα / β, TCRγ / δ, TLR4, TLR7, VEGF, ZAP70.

[0221] Of particular note are T cell markers such as 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, CD140b, CD152, CD159a, CD160, CD161, CD165, CD178, CD183, CD185, CD192, CD193, CD194, CD195, CD196, CD198, CD200, CD200R, CD212, CD217, CD218α, CD229, CD244, CD278, CD279, CD294, CD304, CD314, CXCR3, Flt3, granzyme A, granzyme B, IL-9, IL-13α1, IL-21R, KLRG1, MHC class II, RAG, RORγT, ST2, TCRα / β, TCRγ / δ, ZAP70. Particularly preferred cell markers for T cell selection include TCRγ, TCRδ, CD3, CD4 and CD8.

[0222] The isolated cells can then be cultured to alter cell activity, amplify, or activate. Techniques for amplifying and activating cells are well known in the art (Wang X. and Rivière I. Mol. Thera. Oncolytics. 2016; 3: 16015). These include the use of anti-CD3 / CD28 microbeads (Miltenyi Biotec or Thermofisher Scientific-according to the manufacturer's instructions), or other forms of solidified CD3 / CD28 activating antibodies. The activated / genetically modified cells can then be amplified in vitro in the presence of cytokines (such as IL-2, IL-12, IL-15, or IL-17) and then cryopreserved. A review of methods for amplifying CAR T cells is provided in Wang and Rivièra (supra).

[0223] The present invention further provides a genetically modified cell comprising a chimeric antigen receptor, a nucleic acid molecule or a nucleic acid construct as described above. In some embodiments, the genetically modified cell includes a genomic integration form of a nucleic acid molecule or a 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 myeloid 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 αβT cell. In some embodiments, the genetically modified cell is a γδT cell. In some embodiments, the genetically modified cell is a CD3+T cell (such as an initial CD3+T cell or a memory CD3+T cell). In some embodiments, the T cell is a CD4+T cell (such as an initial CD4+T cell or a memory CD4+T cell). In some embodiments, the T cells are CD8+ T cells (such as initial 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.

[0224] Use of CARs in treating or preventing cancer

[0225] In addition to the high expression of GPC-1 identified by the present inventors as ovarian cancer, high expression of GPC-1 has been reported in pancreatic ductal adenocarcinoma, breast cancer, cervical cancer, lung cancer, malignant pleural mesothelioma and glioblastoma (see 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-1antibody–drug conjugate exhibits potent preclinical antitumoractivity against glypican-1–positive uterine cervical cancer. Int J Cancer, 142, 1056–66; Chiu K et al., (2018). mesothelioma from pulmonary adenocarcinoma. Mod Pathol, 31, 1400–3; and Saito T et al., (2017). High expression of glypican-1predicts dissemination and poor prognosis in glioblastomas. World Neurosurg, 105, 282–8).

[0226] Therefore, the CAR of the present invention can be used to treat or prevent any cancer associated with Glypican-1 expression, including 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 the method for treating or preventing cancer is a method for treating or preventing ovarian cancer in a subject, the method comprising administering an anti-GPC1 CAR cell to the subject.

[0227] The present invention also provides a pharmaceutical composition comprising a genetically modified cell containing a chimeric antigen receptor, nucleic acid molecule, or nucleic acid construct as described above, and one or more pharmaceutically acceptable carriers, excipients, or diluents, wherein the pharmaceutical composition is used to prevent or treat cancer, including (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.

[0228] Methods of diagnosis and prognosis

[0229] Also provided is a method for diagnosing or assessing the prognosis of a subject with ovarian cancer, the method comprising determining the level of glypican-1 in ovarian cancer cells or suspected ovarian cancer cells from the subject, wherein an increased level of glypican-1 indicates the presence of ovarian cancer and / or indicates a poor prognosis.

[0230] 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).

[0231] As used herein, "elevated expression of Glypican-1" refers to an increase in Glypican-1 mRNA or protein compared to a control value. In some embodiments, the control value is normal expression. In some embodiments, the control value is a threshold value. In some embodiments, the control value is a precancerous value. In some embodiments where the ovarian cancer is recurrent ovarian cancer, the control value is a value prior to recurrence.

[0232] In some embodiments, normal expression is determined by non-cancerous ovarian cells or, in the case of some ovarian cancers, fallopian tube cells. In some embodiments, these cells are the same type of cell as the cancer cell, such as an epithelial cell.

[0233] In some embodiments, the threshold is a predetermined threshold value. Such a predetermined threshold value can be based on a previous analysis of the subject, or can be based on a population value, such as a median or mean value determined from multiple samples of non-cancerous cells or healthy cells from a population that can be compared to other population members.

[0234] In some embodiments, expression of Glypican-1 is increased by at least or about by at least 5%, or 10%, or 15%, or 20%, or 25%, or 30%, or 35%, or 40%, or 45%, or 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 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.

[0235] In some embodiments, a poor prognosis refers to a lower overall survival rate or a lower progression-free survival rate in a subject. In some embodiments, increased gene expression indicates a shorter overall survival. In some embodiments, increased protein expression indicates a shorter overall survival. In some embodiments, increased gene expression and increased protein expression indicate a shorter overall survival. In some embodiments, increased gene expression indicates a shorter progression-free survival. In some embodiments, increased protein expression indicates a shorter progression-free survival. In some embodiments, increased gene expression and increased protein expression indicate a shorter progression-free survival.

[0236] In some embodiments, a poor prognosis refers to a faster progression of the cancer or a more rapid tumor growth. In some embodiments, a poor prognosis refers to a higher likelihood of progression to a higher stage of the cancer. In some embodiments, a poor prognosis refers to a higher likelihood of the primary cancer metastasizing.

[0237] The stages of ovarian cancer (including fallopian tube and peritoneal cancer) are provided in Table 5.

[0238] Table 5: FIGO staging of ovarian, fallopian tube, and peritoneal cancer

[0239]

[0240]

[0241] In some embodiments, the ovarian cancer is recurrent ovarian cancer.

[0242] In some embodiments, the ovarian cancer is high-grade serous ovarian cancer.

[0243] In some embodiments, the method of diagnosis or prognosis is performed on a subject who has been previously treated for ovarian cancer, including one or more of chemotherapy, surgical resection or cytoreductive surgery, radiation therapy, hormone therapy, or immunotherapy.

[0244] In some embodiments of the methods of diagnosis or prognostic assessment, the ovarian cancer is recurrent ovarian cancer, and the level of Glypican-1 is elevated compared to cancer tissue prior to recurrence.

[0245] In some embodiments of the methods of diagnosis or prognosis, the level of Glypican-1 is elevated compared to non-cancerous ovarian tissue or a comparison tissue not suspected of cancer. Such non-cancerous tissue can be collected from the same individual or another individual. In some embodiments, the comparison tissue is from the same sample and the suspected or confirmed cancer cells. In some embodiments, it is from a different ovary from the same individual.

[0246] In some embodiments of the methods of diagnosis or prognosis, 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, as described herein, which can be used as binding agents for treating cancer. Also included are fusion proteins, such as single-chain variable fragments, which comprise sequences of the variable light chain and variable heavy chain of an antibody.

[0247] Methods for analyzing RNA

[0248] RNA isolation

[0249] Various methods for RNA isolation are well known in the art, and those skilled in the art will select an appropriate method based on their specific requirements and limitations.

[0250] In the field of RNA extraction, at least three main techniques are widely used: organic extraction, such as phenol-guanidine isothiocyanate (GITC)-based solutions, silica membrane-based spin column technology, and paramagnetic particle technology.

[0251] There are many commercially available kits for RNA isolation, such as AxyPrep Multisource Total RNA Miniprep (Axygen), Mini (Qiagen), EasySpin (Citomed), Ilustra RNAspin MiniRNA Isolation Kit (GE), and TRIzol plus RNA purification system (Invitrogen) and EZNA TMTotal RNA Kit II (Omega Bio-Tek). A comparison of the advantages, disadvantages, and performance of each 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, a protocol for RNA isolation is provided in Liu and Harada (2013), RNA Isolation from Mammalian Samples, Current Protocols in Molecular Biology; 103: 4.16.1–4.16.16.

[0252] Reverse transcription polymerase chain reaction (RT-PCR)

[0253] RT-PCR is one of the most sensitive techniques for quantifying specific nucleic acid samples.

[0254] In order to carry out RT-PCR, RNA is extracted from tissue samples and purified. Then, this RNA is reverse transcribed by the reverse transcriptase of retrovirus and converted into complementary DNA (cDNA). Then, cDNA is combined with thermostable DNA polymerase, deoxynucleotides and forward and reverse primers in a buffer solution, and then thermal cycling is performed to denature the double-stranded DNA (separation), anneal the primers to the separated DNA chains, and extend the new DNA copy by DNA polymerase. Repeat this process to increase the sequence chain between the forward and reverse primers, providing a short DNA sequence called amplicon. Amplicon can then be visualized and / or quantified. An exemplary scheme for carrying out RT-PCR is provided in Mitchel, J. (2002) RT-PCR Protocols.Methods in Molecular Biology, Vol.193.

[0255] In situ hybridization

[0256] In situ hybridization allows for the identification and localization of nucleic acids (e.g., RNA) within a biological sample. Thus, unlike some other techniques, in situ hybridization can indicate the tissue distribution of nucleic acids within a sample, rather than simply identifying the presence of a nucleic acid or quantifying its expression.

[0257] In situ hybridization utilizes the hybridization between a target nucleic acid (such as mRNA) and an oligonucleotide (such as cDNA) or RNA probe (riboprobe) between. Each probe is coupled to a detection moiety, such as a radiolabel, enzyme or fluorophore. The hybridization between the complementary probe nucleic acid sequence and the target sequence can then be detected or visualized to determine the location and quantity of the target nucleotide.

[0258] Techniques for performing in situ hybridization are well known in the art. For example: Henley S.R. 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, pages 68-75.

[0259] Nuclease protection assay

[0260] Techniques for performing nuclease protection assays are well known in the art, including: Henttu P. (2001), Quantification of mRNA levels using ribonuclease protection assay. Methods in Molecular Biology; 169, pages 65-79.

[0261] Northern analysis

[0262] RNA samples are purified from tissue or cell samples and then separated by size by gel (e.g. agarose gel) electrophoresis under denaturing conditions (such as in the presence of formaldehyde or glyoxal / DMSO). The size separated RNA is then transferred to a membrane (such as nitrocellulose or nylon membrane). This transfer can be accomplished by techniques such as capillary transfer, vacuum transfer, salt gradient or electrophoretic transfer. The RNA is then cross-linked or immobilized on the membrane, after which it is hybridized to specific labeled probes.

[0263] Northern blotting allows analysis and quantification based on transcript size. This allows analysis of different expression variants of a gene.

[0264] Examples of northern blotting techniques are provided 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.

[0265] RNA microarray

[0266] Microarrays utilize a series of specific oligonucleotide probes immobilized on a solid support. The probe at each specific position has a known sequence that will specifically hybridize to a complementary nucleic acid.

[0267] Nucleic acid samples for microarray analysis are typically prepared by reverse transcribing mRNA isolated from the sample to produce cDNA. Fluorescent labels can be added to the cDNA produced during or after the reverse transcription process.

[0268] Then, under high stringency conditions, the labeled cDNA from the sample to be analyzed is incubated with the immobilized probes on the microarray, and unhybridized cDNA is removed. The fluorescence at each position is then quantified, indicating the amount of hybridized sample nucleic acid complementary to each immobilized probe.

[0269] A series of commercially available microarray chips are well known in the art, including chips manufactured by Affymetrix, Illumina, Agilent, Applied Microarrays, Eppendorf and Arrayit. In addition, microarray protocols are well 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 MicroarrayGene Expression Data.Current Protocols in Molecular Biology, 77:19.6.1-19.6.30.https: / / doi.org / 10.1002 / 0471142727.mb1906s77.

[0270] RNA sequencing (RNA-Seq)

[0271] RNA-Seq uses a new generation sequencing platform to analyze the RNA sequence and expression in cells at any given time. RNA-Seq can be used to analyze total RNA, microRNA, transfer RNA, and mRNA. Messenger RNA is reverse transcribed into cDNA before adapters are connected to the ends of each cDNA. Sequencing can be unidirectional (single-end sequencing) or bidirectional (paired-end sequencing), with the sequences being compared by computer to a reference genome database, or assembled to obtain de novo transcripts. RNA quantification is performed by calculating the number of reads mapped to each locus of the reference genome. A range of tools can be used to quantify counts, including HTSeq, FeatureCounts, Rcount, Maxcounts, FIXSEQ, Cuffquant, Sailfist, and Kallisto.

[0272] Differential expression between two tissues (eg, cancer and non-cancerous) can be calculated by well-known tools including DESeq, edgeR, and Voom+limma.

[0273] Protocols for performing RNA-Seq and analyzing data are well known in the art, including: Kukurba KR and 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.

[0274] Protein analysis methods

[0275] Immunohistochemistry / immunostaining

[0276] One of the most common techniques for protein quantification and localization is immunohistochemistry. This technique includes solidifying and encapsulating the tissue, which is then sliced ​​before incubating with a primary antibody against the protein of interest. These primary antibodies are directly labeled, or can be detected by labeled secondary antibodies. Common markers include enzymes (such as horseradish peroxidase), fluorescent labels, radioactive labels, or conjugates, such as biotin. The markers can then be detected and used to identify the position and / or quantity of the protein of interest.

[0277] Methods for performing IHC are well 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.

[0278] 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 blotting. Protocols for performing these techniques are well known in the art and include: G.和Mechtler K(2006),HPLCtechniques for proteomics analysis—a short overview of latest developments,Briefings in Functional Genomics,5,4,249–260页;Gao Z等人,(2009)Identificationand Verification of the Main Differentially Expressed Proteins in GastricCancer via iTRAQ Combined with Liquid Chromatography-MassSpectrometry.Analalytical Cellular Pathology(Amsterdam),2019:5310684;Lorne F等人(2001),Whole cell ELISA for detection of tumor antigen expression intumor samples,Journal of Immunological Methods,258,1–2,47-53页;Kim,S.M.等人,(2017).Two different protein expression profiles of oral squamous cellcarcinoma analyzed by immunoprecipitation high-performance liquidchromatography.World journal of surgical oncology,15(1),151;Osborne C,BrooksSA.(2006)SDS-PAGE and Western blotting to detect proteins and glycoproteinsof interest in breast cancer research.Methods in Molecular Medicine,120:217-29页;Ni,D.,Xu,P.和Gallagher,S.2016.Immunoblotting and immunodetection.Curr.Protoc.Mol.Biol.114:10.8.1-10.8.37; and Adams, LD and Gallagher, SR (2004), Two-Dimensional Gel Electrophoresis. Current Protocols in Molecular Biology, 67:10.4.1-10.4.23.doi:10.1002 / 0471142727.mb1004s67. .

[0279] Another method for quantitatively expressing protein on and in cells is flow cytometry. In short, a tissue sample is collected and chopped into the tissue of interest, and the tissue is then dissociated, digested, and filtered into a single cell suspension. The cell suspension is then stained with an antibody for the protein of interest (such as Glypican-1) (i.e., using a fluorophore-labeled primary antibody, or a two-step labeling of a primary antibody and a fluorophore-labeled secondary antibody for the primary antibody). For analysis of intracellular staining, cells can be permeabilized (usually after fixation) before staining. Cells are then processed in a flow cytometer to identify cells expressing the protein of interest (such as Glypican-1), and to quantify the protein expression on each cell.

[0280] Methods for performing flow cytometry are well known in the art, including: 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.

[0281] Techniques for simultaneous quantification of mRNA and protein expression are also well known, including REAP-seq and CITE-seq. Techniques well 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) Simultaneousepitope and transcriptome measurement in single cells. Nature Methods, 14(9): 865-868. doi: 10.1038 / nmeth.4380. Epub 2017 Jul 31. PMID: 28759029; PMCID: PMC5669064.

[0282] Analysis System

[0283] The present invention also provides a method for diagnosing or assessing the prognosis of a subject suffering from ovarian cancer, the method comprising:

[0284] obtaining a sample of suspected or confirmed ovarian cancer cells from the subject;

[0285] quantifying the expression of Glypican-1 in the samples and, alternatively, in control samples;

[0286] comparing the quantitative expression of the subject sample to a control sample or control value (as defined herein); and

[0287] An analysis is performed comparing the comparative expression of Glypican-1 in the sample to a control sample or control value, wherein elevated expression in the sample from the subject indicates that the subject has ovarian cancer or a poor prognosis.

[0288] In some embodiments of the above methods, the control value is stored in a computer database or on a computer system.

[0289] The method of the present invention can be carried out with any suitable method well known in the art. However, in some embodiments, the expression of Glypican-1 in the subject's sample is compared with a control value (as defined herein) by a computer system. Preferably, the level of the predetermined control value is stored in a database. This allows the database to be used as a reference for comparison with multiple samples of cancer or suspected cancer.

[0290] In such embodiments, after the expression level of Glypican-1 is quantified, the data is input (e.g., uploaded or entered) into a computer system where the expression level in the sample is compared to a control value stored in a database. The computer system and associated computer-readable media can then perform any desired statistical analysis that can provide a diagnosis of the likelihood that the sample is cancerous and / or provide an indication of the subject's prognosis.

[0291] Therefore, in some embodiments of the present invention, a computer system is provided, comprising a computer processor and a computer readable medium encoded with program instructions executed by the computer processor, to compare the quantitative expression of one or more markers and to a reference standard. Preferably, the reference standard is stored in a database.

[0292] 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 sample suspected of ovarian cancer, and a detection portion comprising one or more nucleic acids configured to hybridize to a Glypican-1 nucleic acid.

[0293] In some embodiments, the detection system can also include a computer system as described herein. In such embodiments, a computer-readable medium or a computer-readable medium encoded with program instructions is executed by a computer processor to process the data relevant to the detection portion, and determine the expression of Glypican-1 in the received sample. In addition, the program instructions can include a control value for Glypican-1 or can process the data relevant to the control sample to determine the control value. The program instructions for processing data are to compare the expression of Glypican-1 in the received sample from the experimenter with the control value, thereby allowing the sample to be evaluated to determine or predict the possibility that the sample is ovarian cancer or to assess the experimenter's prognosis, wherein the level of Glypican-1 increases, prompting the presence of ovarian cancer and / or prompting the experimenter's poor prognosis.

[0294] treat

[0295] If it is determined that the patient has cancer (such as ovarian cancer) or is likely to have cancer by any of the methods for diagnosing or determining prognosis as described herein, appropriate treatment may be suitable. What constitutes appropriate treatment will be determined by those skilled in the art and the treatment that can be used and approved. Currently available treatments include, but are not limited to surgical resection or tumor reduction surgery for cancer, systemic or local chemotherapy, systemic or local immunotherapy (as described herein), radiotherapy or CAR T cell therapy (including CAR as described herein). Therefore, any diagnostic or prognostic method may include a method of treating or a part of a method of treating. For the avoidance of doubt, there is provided herein a method for treating a subject diagnosed with or suspected of having cancer by performing a diagnostic or prognostic method as described herein.

[0296] Example

[0297] The present invention is further described and illustrated in the following examples. These examples are only for the purpose of describing specific embodiments of the present invention and are not intended to limit the scope of the above description and this application or the scope of the invention required in future applications claiming priority to this application.

[0298] Example 1 - Analysis of Glypican-1 (GPC1) Expression in Ovarian Cancer

[0299] Analysis of microarray expression data and immunohistochemistry (IHC) showed that GPC1 was increased in ovarian cancer cells and correlated with negative patient prognosis—including decreased overall survival and progression-free survival.

[0300] Materials and methods

[0301] Microarray analysis

[0302] 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) was used to assess GPC1 mRNA levels in normal tissues (ovarian surface epithelium (n=66), fallopian tube (n=40) based on annotated Gene Expression Omnibus (U133Plus2) data) and HGSOC tissues (n=807). The Kaplan-Meier plotting tool was used to evaluate the relationship between GPC1 mRNA expression (GPC1:202755_s_at&202756_s_at) and progression-free survival (PFS) and overall survival (OS) in HGSOC patients (Fekete JT et al. (2020). Predictive biomarkers of platinum and taxane resistance using the transcriptomic data of 1816ovarian cancer patients. Gynecol Oncol 156, 654-661).

[0303] Immunohistochemistry (IHC)

[0304] IHC was performed on tissue sections of high-grade serous ovarian cancer (HGSOC) (n=37), benign (n=7), normal ovary (n=14), fallopian tube (FT, n=10), and matched HGSOC tissue (n=4) at diagnosis and recurrence. Tissue microarray (TMA) cohort HGSOC patients (n=101) (Ricciardelli C et al., (2017). Keratin5 overexpression is associated with serous ovarian cancer recurrence and chemotherapy resistance. Oncotarget 8, 17819-17832) were also evaluated. The clinical and pathological parameters of these tissues are listed in Tables 6 and 7, respectively.

[0305] Table 6: Clinical and pathological characteristics of the ovarian tissue cohort

[0306]

[0307] Table 7: Clinicopathological characterization of the high-grade serous ovarian cancer TMA cohort

[0308]

[0309] The method was modified 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). After citric acid antigen retrieval, tissue sections were incubated at 4°C with the primary antibody: Rb polyclonal GPC1 (1 / 75, 16700-1-AP, Proteintech TM ) were incubated overnight. Then, the sections were incubated with secondary antibody (biotinylated goat anti-rabbit, 1 / 400, Dako TM , Australia) and then incubated with streptavidin HRP (1 / 500, Dako TM , Australia) and incubated at room temperature for 1 h. TM ) to detect peroxidase activity. Kidney and liver tissues were selected as positive and negative controls, respectively, using the Human Protein Atlas online database. High and low GPC1 immunostaining was observed in mouse kidney and mouse liver, respectively ( Figure 13A and Figure 13B ).

[0310] Immunohistochemical evaluation

[0311] Using Nanozoomer TM Digital Pathology System (Hamamatsu Photonics TM , Japan) to scan IHC slides. Using Qupath TM Software was used to assess the intensity of GPC1 staining in tumor cells (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, with a score range of (0-300) and three thresholds for scoring: weak (1+), moderate (2+), and strong (3+).

[0312] Cell culture

[0313] 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 Type Cell Cultures (ECACC). OVCAR-5 cells were obtained from Dr. Thomas Hamilton (Fox Chase Cancer Center, PA, USA). Cell lines were cultured in a 1% 4% 4% 4% 5% 2% 1% 2% 3% 5% 2% 3% 4% 5% 2% 3% 5% 4% 2% 5% 3% 5% 2% 3% 5% 2% 3% 5% 2% 3% 5% 2% 3% 4% 2% 3% 5 ... TM )) DMEM (Thermo FisherScientific TM ) or RPMI (Thermo Fisher Scientific TM All cells were maintained at 37°C in a 5% CO2 environment.

[0314] Primary HGSOC cells (n=7) were derived from ascites collected from patients with advanced HGSOC at the Royal Adelaide Hospital and cultured as previously described (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 consists of the clinical and pathological parameters of these patients. Primary HGSOC cells were maintained in advanced RPMI (Life Technologies), 10% FBS, 2 mM Glutamax TM (Life Technologies) and antibiotics.

[0315] Table 8: Summary of clinical and pathological characterization of primary cell cohorts and tissue explants

[0316]

[0317] Quantitative real-time reverse transcription-PCR

[0318] Ovarian cancer cells (COV362, COV318, OAW28, OV90, OVCAR3, A2780, OVCAR5) were plated at 30,000 cells per well for 24 hours. TaqMan® gene expression assay was used to detect ovarian cancer cells. TM RNA was isolated from cells (Life Technologies) and reverse transcribed 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 quantified using the Quantstudio 12K Flex Real-Time PCR System. TM (Applied Biosystems) for subsequent PCR analysis. TM Gene Expression Master Mix (2X), GPC1 primer (Hs00892476_m1), nuclease-free water, and cDNA sample were used to prepare 10 μL of solution for PCR. 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 TM ,Lifetechnologies) and use 2 -ΔCT Method to correct it.

[0319] Western blotting

[0320] 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). Then, 20 μg of each sample was 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 at 4°C. TM ) overnight. Subsequently, the membrane was incubated with Rb polyclonal GPC1 (1 / 500, 16700-1-AP, Proteintech TM ) were incubated for 2 h and stained with peroxide-conjugated anti-rabbit IgG (1 / 4000, Millipore TM ) and incubated for 1 hour. TM , GE Healthcare) and visualized protein expression using Chemidoc TM MP Imaging System (Bio-Rad Laboratories TM , Inc) scanned and used Imagelab TM β-actin anti-rabbit antibody (1 / 5000, ab8226, Abcam TM ) as a loading control.

[0321] Statistical analysis

[0322] For the GENT2 database, the Kruskal Wallis test was used in conjunction with the Dunn multiple comparison test. One-way ANOVA was combined with the Tukey multiple comparison test to evaluate the GPC1 H index score measured by Qupath. The KaplanMeier plot database was used to generate survival curves and determine the relationship between GPC1 mRNA and patient outcomes. Kaplan-Meier analysis was performed in conjunction with the log-rank test 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 the H-index score of matching HGSOC patient tissues at diagnosis and recurrence.

[0323] result

[0324] GPC1 is increased in high-grade serous ovarian cancer

[0325] Analysis of the GENT2 database revealed that GPC1 mRNA levels were significantly increased 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) assessment of GPC1 protein levels, as measured by the H-index, was significantly increased in HGSOC compared with benign serous cystadenomas ( Figure 1B However, no differences in the H-index of HGSOC GPC1 were observed when OSE or FT were compared. Representative images show the H-index of HGSOC GPC1 in OSE ( Figure 1C )、FT( Figure 1D ) and in benign serous cystadenoma tissue ( Figure 1E ). Figure 1F ) with high GPC1 cytoplasmic and membrane staining.

[0326] High GPC1 expression is associated with poor patient outcomes

[0327] Survival curves were generated using the Kaplan Meier online plotting tool to investigate the relationship between GPC1 mRNA levels and patient outcomes ( Figures 2A to 2F ). High expression of GPC1 mRNA is associated with PFS ( Figure 2A , hazard ratio (HR) = 1.3, p = 0.0015) and OS ( Figure 2B , HR=1.35, p=0.00026) was significantly associated with a decrease.

[0328] GPC1 protein levels were assessed in a TMA cohort of HGSOC. The median H-index value observed for this cohort was 73.7. Examples of HGSOC tissues with low and high expression of GPC1 protein are shown in Figure 2, respectively. Figure 2C and Figure 2D For the initial Kaplan-Meier survival analysis, the GPC1 H-index was divided into quartiles (Figure 14). For the PFS analysis, there was a separation between the higher quartiles (Q3 and Q4) and the lower quartiles (Q1 & Q2), but no separation was observed in the OS analysis. Using an H-index score with a cutoff of 70 (close to the median), GPC1 levels >70 were associated with decreased PFS ( Figure 2E , P = 0.031), but not associated with OS ( Figure 2F , p = 0.536).

[0329] GPC1 expression increases after relapse

[0330] GPC1 protein levels were assessed in matched HGSOC tissues at diagnosis and at relapse. Examples of GPC1 immunostaining in HGSOC tissues at diagnosis are shown. Figure 3A and Figure 3B In the , and at relapse, matched tissue is shown in Figure 3C and Figure 3D Quantification of IHC staining using QuPath demonstrated elevated GPC1 levels in matched tumor tissue at relapse compared to tissue at diagnosis ( Figure 3E , P = 0.0014, paired T test).

[0331] GPC1 expression in ovarian cancer cells

[0332] Quantitative PCR (qRT-PCR) showed that in all ovarian cancer cell lines ( Figure 4A ) and primary HGSOC cells ( Figure 4B ). The highest GPC1 expression was observed in primary cells from OVCAR5 and patient 4. Western blotting detected a band with a predicted molecular weight of 65 kDa, confirming that GPC1 mRNA was expressed in ovarian cancer cell lines ( Figure 4C ) and primary HGSOC cells ( Figure 4D ) expressed GPC1. Western blot quantification showed that GPC1 protein levels were highest in OAW28 and OV90 ( Figure 4E Primary cells isolated from patients 1 and 3 of HGSOC with recurrent disease expressed the highest levels of GPC1 protein ( Figure 4F Ovarian cancer cell lines (OVCAR3, OV90, COV362, and SKOV3) and primary cells (patients 1 and 3) with a range of GPC1 levels were selected for further in vitro studies.

[0333] Discussion of the results

[0334] These results indicate that: i) GPC1 mRNA and protein expression is elevated in HGSOC compared with noncancerous tissues, but these levels do not necessarily correlate within patients or within cell lines; ii) increased GPC1 mRNA levels are associated with PFS and OS; and iii) high GPC1 protein expression levels in tumor cells are associated with decreased PFS.

[0335] Studies examining GPC1 expression have shown that GPC1 mRNA expression is significantly increased in HGSOC compared to FT, the site of origin of HGSOC. Furthermore, GPC1 protein expression is significantly increased in HGSOC compared to benign tissue.

[0336] Kaplan-Meier plot analysis and the results of the TMA cohort showed that high GPC1 expression was significantly associated with poor outcome in patients with HGSOC. We observed only faint GPC1 staining in the stroma; GPC1 was mainly located in the cell membrane and cytoplasm.

[0337] The results also showed that GPC1 was expressed to varying degrees in ovarian cancer cell lines and primary cells.

[0338] Example 2 - Anti-Glypican-1 Chimeric Antigen Receptor T Cells Effectively Kill Ovarian Cancer Cells

[0339] After demonstrating that GPC1 is expressed in ovarian cancer cells (particularly HGSOC), it was determined whether agents that can be used to induce killing of GPC1-expressing cells could be targeted to ovarian cancer cells. To target GPC1-expressing cells, anti-GPC1 CAR-T cells were developed and used in cancer cell lysis assays. These experiments demonstrated that ovarian cancer cells can be killed by anti-GPC1 agents, such as CAR-T cells.

[0340] Production and characterization of CAR-T cells

[0341] CNA500200CAR-T cells were generated in Professor Simon Barry'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 WO2022 / 104424A). The GPC1 binding domain has been cloned into a second-generation CAR backbone encoding a linker and intracellular domains CD3, CD28, and epidermal growth factor (EGFR) reporter (Jensen MC & Riddle SR (2015), supra, and Wang X et al. (2012), supra).

[0342] Lentivirus was produced by transfecting 293T cells with a third generation self-inactivating lentiviral plasmid 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 11, 323-332). A brief overview of the transduction protocol is shown below. Figure 9 As shown in .

[0343] Batjargal Gundsambuu (Molecular Immunology, University of Adelaide) performed a comprehensive characterization of the CAR-T cells used in the assay using fluorescence-activated cell sorting (FACS). Transduction efficiency was measured by EGFR expression. Within the CD4 T cell population, 80.4% of cells were EGFR-positive, within the CD8 T cell population, 66.3% of cells were positive, and within the total lymphocyte population, 78.3% were positive ( Figure 10 ).

[0344] Cell maturation markers CD45RA and CD62L were also assessed. In the CD4 population, 51.7% of cells showed effector memory T cells (T EM ) phenotype, 32.7% showed central memory T cells (T CM ) phenotype, 8.29% showed effector memory cells re-expressing CD45RA (T EMRA ) phenotype and 7.3% of cells expressed the naive T cell phenotype ( Figure 11 Although the CD8 cell population had a slightly more naive phenotype (19.3%), 20.7% expressed a central memory T cell phenotype, 29.8% expressed an effector memory T cell phenotype, and 20.2% expressed a T cell phenotype. EMRA Phenotype ( Figure 11 ).

[0345] A combination of exhaustion staining assays was also performed using PD1, LAG3, and TIM3 antibodies. FACS analysis showed that in the CD4 population, 1.38% of these cells expressed very low levels of PD1, and only 2.31% expressed LAG3 and TIM3 (Figure 12). In the CD8 population, 3.92% of these cells expressed PD1, and only 1.56% expressed LAG3 and TIM3. These results indicate a low level of T cell exhaustion.

[0346] Materials and methods

[0347] MTT cell viability assay

[0348] SKOV3, COV362, OVCAR3, OV90, and primary HGSOC cells (n=2) were plated at 10,000 cells / well in 96-well plates in their respective growth media. After 24 hours, cells were treated with (i) control medium, (ii) untransduced (UT) CD3 T cells, or (iii) anti-GPC1 CAR T cells (at 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 medium 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).

[0349] Spheroid assay

[0350] SKOV3, COV362, OVCAR3 and primary ovarian cancer cells (n=2) were cultured at 20,000 cells / well in their respective growth media on plates coated with poly-HEMA (30 mg / mL in 95% ethanol, Sigma Aldrich). TM ) were plated on 24-well plates. After 24 h, the cells were cultured with X-VIVO 15 medium (Lonza TM ,5% human serum-Sigma-Aldrich TM , 2mM L-glutamine – Sigma TM , 20 mM HEPES) or UT CD3 T cells or anti-GPC1 CAR-T cells (E:T ratio of 5:1). Spheroid formation was observed within 6 days and used Optical microscope FL imaging system (Life Technologies TM ) Bright field images were taken. As previously described, the spherical area (μm) of spheroids with a diameter greater than 50 μm in each treatment group was measured using Image J 32 software (Image J I.50i, National Institute Health, Bethesda, MD, USA). 2) (n=5 images / well in replicate wells of three independent experiments) (Lokman NA et al. (2019). 4-Methylumbelliferone Inhibits Cancer Stem Cell Activation and Overcomes Chemoresistance in Ovarian Cancer. Cancers (Basel) 11, 1187).

[0351] Patient-derived explant (PDE) assay

[0352] Tissues were collected at the time of surgery and frozen in liquid nitrogen containing 15% DMSO and 25% FBS. Figure 11 ) cut into 1mm 3 The cells were transplanted onto gelatin dental sponges (Spongostan) in CD3 T cell X-VIVO medium (containing cytokines IL-2 (50 U / mL), IL-7 (5 ng / mL) and IL-15 (0.5 ng / mL)). TM ,Johnson&Johnson TM ) and cultured in a humidified atmosphere at 37°C with 5% CO2 using (i) control culture medium, (ii) anti-GPC1 CAR-T cells (2×10 6 / mL) or UT CD3 T cells (2x10 6 / ml) treatment. After 72 h, tissues were collected and fixed with formalin, and then processed for histology. As previously described, apoptosis was measured using cleaved caspase 3 (Ricciardelli C et al., (2018). Novel ex vivo ovarian cancer tissue explant assay for prediction of chemosensitivity and response to novel therapeutics. Cancer Lett 421, 51-58).

[0353] result

[0354] Effects of GPC1 CAR-T cells on ovarian cancer in vitro monolayers

[0355] All ovarian cancer cells and primary cells responded to GPC1 CAR-T cell treatment. Figure 5A )、COV362( Figure 5B ), OV90( Figure 5C ) and SKOV3( Figure 5D ) cell survival was observed to be significantly reduced at E:T ratios of 5:1 and 2:1 compared to UT CD3 T cells. Anti-GPC1 CAR-T cells also reduced COV362( Figure 5B ) and SKOV3 cell( Figure 5D ) survival compared to UT CD3 T cells at 10:1. Patient 1 primary cells showed statistically significant reduced cell survival when incubated with anti-GPC1 CAR-T cells at an E:T ratio of 10:1( Figure 5E ) but not 5:1 and 2:1. Anti-GPC1 CAR-T cells also showed a statistically significant effect on patient 3 primary cell survival at E:T ratios of 10:1 and 2:1 but not 5:1. Figure 5F

[0356] Effect of GPC1 CAR-T cells on ovarian cancer in 3D spheroid format

[0357] Spheroids comprising cell lines COV362( Figure 6A ), SKOV3( Figure 6B ) and OVCAR3( Figure 6C ) responded to anti-GPC1 CAR-T cell treatment at an E:T ratio of 5:1 with a statistically significant reduction in spheroid area. Significant differences between control and anti-GPC1 CAR-T cells were also observed for spheroids comprising COV362( Figure 6A ) and SKOV3( Figure 6B ) cells but not OVCAR3 cells( Figure 6C ). For spheroids composed of cells from patient 3, a statistically significant reduction in spheroid size was observed between UT CD3 T cells and anti-GPC1 CAR-T cells( Figure 7B ) but not for patient 1 spheroids( Figure 7A ) although a reduction in spheroid size was observed for this patient. Treatment with anti-GPC1 CAR-T cells resulted in a statistically significant reduction in spheroid size from patient 1( Figure 7A ) and patient 3( Figure 7B ) compared to control medium. No cells showed a statistically significant difference between UT CD3 T cell treatment and control treatment.

[0358] Effect of GPC1 CAR-T cells on patient-derived explants

[0359] Ovarian cancer tissue from 6 patients was selected for PDE assay( Figures 8A to 8F ​PDE assays were performed by treating explanted patient tissues with UT CD3 T cells or anti-GPC1 CAR-T cells, and then assessing apoptosis in the explant tissues by immunostaining for cleaved caspase-3.

[0360] Patients 1 to 4 had statistically significant increases in cleaved caspase-3 staining ( Figures 8A to 8D ), indicating that cell death was increased in explant tumor tissues after treatment with anti-GPC1 CAR-T cells compared with UTCD3 T cells. Comparison of GPC1 expression in tissues that responded to CAR T cell treatment with tissues that did not respond (e.g., Patients 5 and 6) showed that GPC1 expression levels were lower in explants that did not respond to CAR T cell treatment ( Figure 8G ), indicating that GPC1 expression is associated with treatment outcomes.

[0361] Discussion of the results

[0362] The effects of anti-GPC1 CAR-T cells were evaluated in ovarian cancer cell lines with varying GPC1 expression as well as in two primary cell lines from patients with recurrent ovarian cancer.

[0363] Results from 2D monolayer assays demonstrated that anti-GPC1 CAR-T cells exhibited killing effects in a dose-dependent manner.

[0364] Cancer cell survival was further significantly reduced at the lowest concentration, 2:1, for all ovarian cancer cells except primary cells from Patient 1. No statistically significant effect was observed for OVCAR3 and OV90 at a 10:1 ratio, likely due to increased cytotoxicity resulting from higher numbers of UT CD3 T cells, but the data suggest that increased killing still occurred at these ratios.

[0365] Assessment of CAR-T cell efficacy in 3D spheroid assays allows for precise physiological characterization of the tumor microenvironment, particularly when such structures are formed in malignant ascites from ovarian cancer patients.

[0366] This study demonstrated that anti-GPC1 CAR-T cells exhibited significant antitumor activity against primary ovarian cancer 3D spheroids in vitro.

[0367] The effectiveness of anti-GPC1 CAR-T cells was also investigated in an ex vivo model, using a PDE assay. Importantly, the tissue architecture and viable tumor cells were preserved, as in native tissue. The results showed that, compared to UT CD3 T cells, anti-GPC1 CAR-T cells effectively induced apoptosis in HGSOC patient tissues, indicating that target cancer cells were killed.

[0368] 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 relapse in monolayer and 3D spheroid assays; and iii) GPC1 CAR-T cells can effectively induce apoptosis in the PDE assay. Taken together, these findings suggest that GPC1 CAR-T cells may provide a new immunotherapy against ovarian cancer, particularly HGSOC.

[0369] Table 9 - List of abbreviations

[0370] CAR Chimeric antigen receptor cDNA complementary DNA CT Cycle threshold DMSO dimethyl sulfoxide DNA DNA E:T Effector cell to target cell ratio FACS Fluorescence-activated cell sorting FCS-A Forward scattering area FBS Fetal bovine serum FT oviduct GPC1 Glypican-1 HGSOC High-grade serous ovarian cancer HR Hazard ratio IHC Immunohistochemistry IL interleukins LAG3 lymphocyte activation gene 3 mRNA messenger RNA OS Overall survival OSE Ovarian surface epithelium PCR polymerase chain reaction PD1 Programmed cell death protein 1 PDE determination Patient-derived explant assays PFS Progression-free survival RNA RNA <![CDATA[T CM ]]> Central memory T cells <![CDATA[T EM ]]> Effector memory T cells [CAT EMRA ]]> Effector memory cells re-express CD45RA TIGIT T cell immunoreceptors with Ig and ITIM domains TIM3 T cell immunoglobulin and mucin domain-containing protein 3 TMA Tissue microarray UT Not transduced

[0371] Definition and Qualifications

[0372] The discussion of documents, acts, materials, devices, articles and the like in this specification is included for the purpose of providing a background to the present invention only. It is not intended or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

[0373] In this specification, unless the context requires otherwise, the word "comprise" or "comprises" or "comprising" and other variations will be understood to imply the inclusion of a stated 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.

[0374] It should be further understood that terms such as "comprise," or variations such as "comprises" or "comprising" inherently include within their scope (without limitation) forms of the invention that exclude other elements directly relevant to the invention. Thus, terms such as "consisting of" or "consisting essentially of" may be substituted for terms such as "comprise," "comprises," or "comprising," which have the effect of limiting the scope of the invention to the specifically recited elements. It is worth noting that where the invention is expressly intended to be considered exhaustive, such limitations should be considered solely with respect to the inventive concepts disclosed herein, and other features that are not within the scope of the inventive concepts may be added. Such features or elements may include, but are not limited to, excipients, formulations, additives, diluents, packaging, adjuvants, and co-located features, which should not be excluded by terms such as "consisting of" or "consisting essentially of."

[0375] The cited documents, publications and patents are hereby incorporated by reference in their entirety. The teachings and disclosures of such documents, publications and patents are therefore considered to be part of the disclosure of this specification.

[0376] Unless otherwise indicated herein or clearly contradicted by the context, all methods described herein can be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "such as," "i.e.") provided herein is intended only to better illustrate the example embodiments and does not limit the scope of the claimed invention unless otherwise required. No language in the specification should be construed as indicating that any unclaimed element is essential.

[0377] The description provided herein is relevant to several embodiments that may share common features and characteristics. It should be understood that one or more features of an embodiment can be combined with one or more features of other embodiments. In addition, the combination of a single feature of an embodiment or features can constitute additional embodiments.

[0378] The subject headings used herein are for the convenience of the reader and should not be used to limit the subject matter of the entire disclosure or claims. The subject headings should not be used to interpret the scope of the claims or the limitations of the claims.

[0379] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all steps, features, compositions and compounds referred to or indicated in this specification, whether singly or collectively, and any and all combinations of any two or more steps or features.

[0380] Furthermore, it should be noted that as used herein, the singular forms "a," "an," and "the" include plural referents unless the context dictates otherwise.

[0381] Future patent applications may be filed based on, or as continuations of, or divisions of, the present application. It should be understood that the following claims are not intended to limit the scope of what may be claimed in any such future application. Features may be added or omitted later in the claims to further define or redefine the claimed invention.

[0382] Those skilled in the art will appreciate that although the invention has been described in some detail for purposes of clarity and understanding, various modifications and alterations may be made to the embodiments and methods described herein without departing from the scope of the inventive concepts disclosed in this specification.

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 of 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), a variable heavy chain of an antibody, or a variable light chain of an antibody.

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 with 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 according to 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. A method for diagnosing or assessing the prognosis of a subject with ovarian cancer, wherein the method comprises determining the level of Glypican-1 in ovarian cells from the subject, wherein an elevated level of Glypican-1 indicates the presence of ovarian cancer and / or indicates a poor prognosis for the subject.

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

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

15. The method of any one of claims 12 to 14, wherein the subject has previously been treated for ovarian cancer with one or more of chemotherapy, surgical resection or cytoreductive surgery, 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 level of Glypican-1 is elevated compared to cancer tissue before recurrence.

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

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

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

20. The method of claim 18, wherein determining the level of Glypican-1 protein expression comprises quantifying intracellular expression of 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 chain and the variable heavy chain of an antibody.

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

25. A method of treating a subject having ovarian cancer, wherein the method comprises killing cells that express Glypican-1.

26. The method of claim 25, wherein cells expressing Glypican-1 are determined to express elevated levels of Glypican-1 protein.

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

28. The method of claim 26, wherein the increased expression level of Glypican-1 protein 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. The method of any one of claims 25 to 29, wherein the ovarian cancer is high-grade serous ovarian cancer (HSOC).

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

32. The method of any one of claims 26 to 31, wherein the ovarian cancer is recurrent ovarian cancer and the level of Glypican-1 is elevated compared to cancer tissue before recurrence.

33. The method of any one of claims 26 to 31, wherein the level of Glypican-1 is elevated 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. 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 chain and the variable heavy chain 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 expressing a chimeric antigen receptor (CAR).

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

40. The method of any one of claims 25 to 39, comprising performing the method of any one of claims 12 to 24 prior to killing the cells expressing Glypican-1.

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