Chimeric antigen receptor targeting GCC and use thereof

Abstract

Provided is a chimeric antigen receptor targeting GCC, comprising: an scFv that specifically recognizes GCC, a CD8 hinge region or a CD28 hinge region, a CD8 transmembrane region or a CD28 transmembrane region, a CD28 co-stimulatory signal domain or a 4-1BB co-stimulatory signal domain, and a CD3ζ signal domain; the scFv that specifically recognizes GCC comprises a heavy chain variable region VH and a light chain variable region VL, the VH comprising an HC CDR1 having the amino acid sequence shown in SEQ ID NO: 1, an HC CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and an HC CDR3 having the amino acid sequence shown in SEQ ID NO: 3, and the VL comprising an LC CDR1 having the amino acid sequence shown in SEQ ID NO: 4, an LC CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and an LC CDR3 having the amino acid sequence shown in SEQ ID NO: 6.

Description

Chimeric antigen receptor targeting GCC and its use

[0001] This application claims priority to a Chinese application filed on April 8, 2024, with application number 2024104170823, entitled “Chimeric Antigen Receptor Targeting GCC and Uses Thereof.” The entire contents of that priority application are incorporated herein by reference. The contents of all references cited in this application are incorporated herein by reference. Technical Field

[0002] The present application relates to a chimeric antigen receptor targeting GCC, a recombinant vector comprising the chimeric antigen receptor, an engineered cell, and uses thereof in drug preparation. Background Art

[0003] Recent data show that colorectal cancer (CRC) has a high incidence and mortality rate in both men and women worldwide, ranking third among all cancers. In the United States, the incidence rate for colorectal cancer is 38.7 per 100,000 people, while the mortality rate is 13.9. In China, there were approximately 310,000 new cases of colorectal cancer in 2018, accounting for 12.2% of all new cancers; the incidence rate was 23.7 per 100,000 people, and the mortality rate was 10.9 per 100,000 people [Reference 1].

[0004] Depending on the stage of diagnosis, the five-year survival rate for colorectal cancer can drop from approximately 90% in stage I to less than 10% in stage IV [Reference 2]. A small number of patients without KRAS mutations can be treated with a combination of chemotherapy and the targeted drugs Avastin and Erbitux, but there are currently no effective treatment options for patients with KRAS or BRAF mutations. The immune checkpoint inhibitor PD-1 antibody has become a first-line treatment for patients with metastatic colorectal cancer, but it is limited to patients with microsatellite instability (MSI), which accounts for approximately 15% of all colorectal cancer patients. This proportion decreases further as CRC progresses, reaching 4% in clinical stage IV. The majority of patients have microsatellite stable (MSS) tumors without MMR gene mutations, accounting for 85% [Reference 3]. PD-1 drugs have a good immune response in MSI patients and significantly improve survival rates, but they are almost ineffective in MSS patients [Reference 4], which greatly limits the scope of application of this drug.

[0005] Guanylyl cyclase 2C (GUCY2C or GCC) is a single-pass transmembrane protein expressed in the apical membrane of enterocytes that converts guanosine triphosphate to cyclic guanosine monophosphate in response to guanylin, uroguanylin or other ligands. GCC is highly specific in its expression profile in normal tissues or organs, mainly concentrated in the intestinal system, and mainly expressed at the top of the cell [5], so the expression characteristics of GCC on the luminal surface of the intestinal epithelium will be expected to reduce the toxic side effects of targeted drugs on normal tissues.

[0006] In the case of disease, GCC is expressed in tubular adenomas, inflammatory bowel disease, precancerous lesions of colorectal cancer, primary and metastatic colorectal cancer, and metastases of these diseases in lymph nodes and liver. In addition, GCC is also expressed in all esophageal and gastrointestinal metaplasia-induced dysplasia and adenocarcinoma [6]. On the contrary, tumors not associated with intestinal metaplasia, including gastric signet ring cell carcinoma, do not express GCC. In one study, researchers evaluated the expression of GCC protein in 627 gastrointestinal tumors, and the results showed that 98% of colorectal cancers expressed GCC, with the highest positive rate [7]. Among other gastrointestinal malignancies, 59% of esophageal cancers, 68% of gastric cancers, and 64% of pancreatic cancers all expressed GCC. Therefore, GCC can be used as a drug target for targeted therapy of the above-mentioned cancers.

[0007] For patients with advanced colorectal cancer, Shanghai Sunesis Biotechnology Co., Ltd. reported at the 2023 ESMO (European Society for Medical Oncology) Annual Meeting the safety and efficacy data of GUCY2C-CD19 CAR-T product from two dose escalation trials of 21 patients from five clinical centers in China. Among them, 13 patients were in the first dose group (1 x 10 6 cells / kg), and 8 patients were in the second dose group (2 x 10 6 cells / kg). The clinical results showed that the objective response rate (ORR) of the first dose group was 15.4% (2 / 13), and the objective response rate of the second dose group was 50% (4 / 8). The most common adverse events in the patients were cytokine release syndrome (CRS) and diarrhea, and two patients had neurotoxicity, which was relieved after treatment with corticosteroids. Therefore, the development of CAR-T targeting GUCY2C is expected to long-term remission of advanced colorectal cancer patients.

[0008] Although GCC19 CAR-T of Sunesis has achieved certain efficacy, this method will kill normal B lymphocytes in the human body, cause B lymphocyte deficiency, and thus cause impaired humoral immunity, leading to the human body being easily infected by pathogens and increasing the cost of care. Therefore, the demand for developing more safe and effective CAR-T cell drugs targeting GCC is still urgent.

[0009] Document 1 : R. L. Siegel, K. D. Miller, A. Jemal, Cancer statistics, 2020, CA Cancer J Clin 70(1) (2020) 7-30.

[0010] Document 2: R. L. Siegel, K. D. Miller, A. Goding Sauer, S. A. Fedewa, L. F. Butterly, J. C. Anderson, A. Cercek, R. A. Smith, A. Jemal, Colorectal cancer statistics, 2020, CA Cancer J Clin 70(3) (2020) 145-164.

[0011] Document 3: R. Gupta, S. Sinha, R. N. Paul, The impact of microsatellite stability status in colorectal cancer, Curr Probl Cancer 42(6) (2018) 548-559.

[0012] Document 4: D. T. Le, J. N. Uram, H. Wang, B. R. Bartlett, H. Kemberling, A. D. Eyring, A. D. Skora, B. S. Luber, N. S. Azad, D. Laheru, B. Biedrzycki, R. C. Donehower, A. Zaheer, G. A. Fisher, T. S. Crocenzi, J. J. Lee, S. M. Duffy, R. M. Goldberg, A. de la Chapelle, M. Koshiji, F. Bhaijee, T. Huebner, R. H. Hruban, L. D. Wood, N. Cuka, D. M. Pardoll, N. Papadopoulos, K. W. Kinzler, S. Zhou, T. C. Cornish, J. M. Taube, R. A. Anders, J. R. Eshleman, B. Vogelstein, L. A. Diaz, Jr., PD-1 Blockade in Tumors with Mismatch-Repair Deficiency, N Engl J Med 372(26) (2015) 2509-20.

[0013] Document 5: D. Mathur, A. R. Root, B. Bugaj-Gaweda, S. Bisulco, X. Tan, W. Fang, J. C. Kearney, J. Lucas, M. Guffroy, J. Golas, C. M. Rohde, C. Stevens, C. Kamperschroer, K. Kelleher, R. F. Lawrence-Henderson, E. Upeslacis, J. Yao, J. Narula, E. R. LaVallie, D. R. Fernandez, B. S. Buetow, E. Rosfjord, L. Bloom, L. E. King, L. Tchistiakova, A. Nguyen, P. Sapra, A Novel GUCY2C-CD3 T-Cell Engaging Bispecific Construct (PF-07062119) for the Treatment of Gastrointestinal Cancers, Clin Cancer Res 26(9) (2020) 2188-2202.

[0014] Document 6: H. Sung, J. Ferlay, R. L. Siegel, M. Laversanne, I. Soerjomataram, A. Jemal, F. Bray, Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries, CA Cancer J Clin 71(3) (2021) 209-249.

[0015] Document 7: H. Danaee, T. Kalebic, T. Wyant, M. Fassan, C. Mescoli, F. Gao, W. L. Trepicchio, M. Rugge, Consistent expression of guanylyl cyclase-C in primary and metastatic gastrointestinal cancers, PLoS One 12(12) (2017) e0189953.

[0016] Document 8: Gross G, Waks T, Eshhar Z. Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity. Proc Natl Acad Sci U S A. 1989 Dec; 86(24): 10024-8.

[0017] Document 9: Khalil DN, Smith EL, Brentjens RJ, Wolchok JD. The future of cancer treatment: immunomodulation, CARs and combination immunotherapy. Nat Rev Clin Oncol 2016; 13(6): 394. SUMMARY

[0018] The present application aims to provide a new chimeric antigen receptor specifically targeting GCC, which has strong and sustained killing ability to tumor cells with low antigen density, can enhance the persistence of CAR-T in vivo, and enhance the anti-tumor effect.

[0019] In one aspect, the present application relates to a chimeric antigen receptor specifically targeting GCC, which comprises: a scFv specifically recognizing GCC, a CD8 hinge region or a CD28 hinge region, a CD8 transmembrane region or a CD28 transmembrane region, a CD28 costimulatory signaling domain or a 4-1BB costimulatory signaling domain, a CD3 zeta signaling domain; wherein the scFv specifically recognizing GCC comprises a heavy chain variable region VH and a light chain variable region VL, the VH comprises an HC CDR1 of an amino acid sequence shown in SEQ ID NO: 1, an HC CDR2 of an amino acid sequence shown in SEQ ID NO: 2, and an HC CDR3 of an amino acid sequence shown in SEQ ID NO: 3, and the VL comprises an LC CDR1 of an amino acid sequence shown in SEQ ID NO: 4, an LC CDR2 of an amino acid sequence shown in SEQ ID NO: 5, and an LC CDR3 of an amino acid sequence shown in SEQ ID NO: 6.

[0020] The present application also relates to a nucleic acid molecule comprising a nucleic acid sequence encoding the chimeric antigen receptor of the present application, a recombinant vector comprising the nucleic acid molecule, and an engineered cell comprising the chimeric antigen receptor, the nucleic acid molecule, or the recombinant vector.

[0021] The present invention also relates to the use of the chimeric antigen receptor, nucleic acid molecule, recombinant vector or engineered cell in the preparation of a drug for treating GCC-positive tumors.

[0022] The present invention further relates to a method for treating GCC-positive tumors, which comprises administering an effective amount of the chimeric antigen receptor, nucleic acid molecule, recombinant vector or engineered cell of the present invention to a patient in need thereof.

[0023] The entire contents of the documents listed in this application are incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 shows the in vitro proliferation fold of resting T cells (Figure 1A) and the in vitro proliferation fold of CAR-T cells (Figure 1B).

[0025] Figure 2 shows the CAR positive rate (Figure 2A) and CAR mean fluorescence intensity (Figure 2B) of cells in each group.

[0026] FIG3 shows the GCC expression levels of HCT116 cell lines at different antigen densities.

[0027] Figure 4 shows the killing curves of CAR-T in each group against HCT116-GCC low-expressing cells.

[0028] Figure 5 shows the residual amount of tumor cells (Figure 5A) and the number of CAR-T cells (Figure 5B) after 10 rounds of stimulation of HCT116-GCC low-expressing cells.

[0029] Figure 6 shows the residual amount of tumor cells (Figure 6A) and the number of CAR-T cells (Figure 6B) after 12 rounds of stimulation of HCT116-GCC low-expressing cells.

[0030] Figure 7 shows the cytokine secretion levels of each group of CAR-T cells under stimulation with HCT116-GCC low-expressing cells.

[0031] Figure 8 shows the cytokine secretion levels of each group of CAR-T cells after culture in IL-2-free medium for 24 hours.

[0032] Figure 9 shows the cell proliferation (Figure 9A) and cell viability (Figure 9B) of each group of CAR-T cells in IL-2-free culture medium.

[0033] Figure 10 shows the in vitro proliferation folds of resting T cells (Figure 10A) and CAR-T cells (Figure 10B) in different signal domains.

[0034] FIG11 shows the CAR positivity rate ( FIG11A ) and the average fluorescence intensity ( FIG11B ) of CAR-T cells with different signal domains detected by flow cytometry.

[0035] Figure 12 shows the killing curve of different signal domain CAR-T against HCT116-GCC low expression cells (E:T = 1:3).

[0036] Figure 13 shows the residual amount of tumor cells (Figure 13A) and the number of CAR-T cells (Figure 13B) after 7 rounds of stimulation of HCT116-GCC low expression cells.

[0037] Figure 14 shows the proliferation fold of T cells in vitro (Figure 14A) and the proliferation fold of CAR-T cells in vitro (Figure 14B) with different promoters.

[0038] Figure 15 shows the CAR positive rate with different promoters.

[0039] Figure 16 shows the killing curve of CAR-T combined with different promoters against HCT116-GCC low expression cells.

[0040] Figure 17 shows the residual amount of tumor cells (Figure 17A) and the number of CAR-T cells (Figure 17B) after 6 rounds of stimulation of HCT116-GCC low expression cells.

[0041] Figure 18 shows the residual amount of tumor cells (Figure 18A) and the number of CAR-T cells (Figure 18B) after 10 rounds of stimulation of HCT116-GCC low expression cells.

[0042] Figure 19 shows the cytokine secretion level of different promoter CAR-T after 24 hours of stimulation of HCT116-GCC low expression cells.

[0043] Figure 20 shows the cytokine secretion level of different promoter CAR-T cultured in IL-2-free medium for 24 hours.

[0044] Figure 21 shows the cell proliferation (Figure 21A) and cell viability (Figure 21B) of different promoter CAR-T in IL-2-free medium.

[0045] Figure 22 shows the tumor inhibition ability (Figure 22A) and CAR-T expansion ability in peripheral blood (Figure 22B) of truncated EF1a-CAR-T-1 in NSG tumor-bearing mice.

[0046] Figure 23 shows the CT image of patient 01.

[0047] Figure 24 shows the number of CAR-T cells in peripheral blood and the level of cytokines in plasma of patient 01.

[0048] Figure 25 shows the level of tumor markers of patient 01.

[0049] Figure 26 shows the CT image of patient 02.

[0050] Figure 27 shows the number of peripheral blood CAR-T cells and cytokine levels in plasma of patient 02.

[0051] Figure 28 shows the tumor marker levels of patient 02.

[0052] Figure 29 shows the CAR positive rate and CAR mean fluorescence intensity of CAR-T-1 and CAR-T-7.

[0053] Figure 30 shows the real-time killing effect of CAR-T-1 and CAR-T-7.

[0054] Figure 31 shows the repeated stimulation killing effect of CAR-T-1 and CAR-T-7. DETAILED DESCRIPTION

[0055] 1. Design of GCC CAR molecule

[0056] The scFv sequence of the GCC CAR molecule of the present application is derived from a monoclonal antibody specifically recognizing GCC target (refer to patents WO2011050242A1 and WO2019178580A1). The CDRs of the antibody and antigen-binding fragment disclosed in the present application are defined or identified by Kabat numbering. The antibody or antigen-binding fragment comprises a heavy chain variable region VH and a light chain variable region VL, the VH comprises HC CDR1 of the amino acid sequence shown in SEQ ID NO: 1, HC CDR2 of the amino acid sequence shown in SEQ ID NO: 2 and HC CDR3 of the amino acid sequence shown in SEQ ID NO: 3, and the VL comprises LC CDR1 of the amino acid sequence shown in SEQ ID NO: 4, LC CDR2 of SEQ ID NO: 5 and LC CDR3 of the amino acid sequence shown in SEQ ID NO: 6.

[0057] In a specific embodiment, in the scFv of the GCC CAR molecule of the present application, the VH is at least 85%, preferably at least 90%, more preferably at least 95%, and further preferably 100% identical to the amino acid sequence shown in SEQ ID NO: 7, and the VL is at least 85%, preferably at least 90%, more preferably at least 95%, and further preferably 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0058] In a more specific embodiment, the nucleotide sequence encoding the above-mentioned VH is shown in SEQ ID NO: 27, and the nucleotide sequence encoding the above-mentioned VL is shown in SEQ ID NO: 28. In a more specific embodiment, the scFv specifically recognizing GCC used in the present application is at least 85% identical, preferably at least 90%, more preferably at least 95%, and further preferably 100% identical to the amino acid sequence of SEQ ID NO: 9.

[0059] The hinge region used in the GCC CAR molecule of the present application is a CD8 hinge region or a CD28 hinge region, wherein the amino acid sequence of the CD8 hinge region is at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably 100% identical to the amino acid sequence set forth in SEQ ID NO: 10, and the amino acid sequence of the CD28 hinge region is at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably 100% identical to the amino acid sequence set forth in SEQ ID NO: 11.

[0060] The transmembrane region used in the GCC CAR molecule of the present application is a CD8 transmembrane region or a CD28 transmembrane region, wherein the amino acid sequence of the CD8 transmembrane region is at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably 100% identical to the amino acid sequence set forth in SEQ ID NO: 13, and the amino acid sequence of the CD28 transmembrane region is at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably 100% identical to the amino acid sequence set forth in SEQ ID NO: 14.

[0061] The co-stimulatory signaling domain used in the GCC CAR molecule of the present application is a CD28 co-stimulatory signaling domain or a 4-1BB co-stimulatory signaling domain, wherein the amino acid sequence of the CD28 co-stimulatory signaling domain is at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably 100% identical to the amino acid sequence set forth in SEQ ID NO: 15, and the amino acid sequence of the 4-1BB co-stimulatory signaling domain is at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably 100% identical to the amino acid sequence set forth in SEQ ID NO: 16.

[0062] The amino acid sequence of the CD3 zeta signaling domain used in the GCC CAR molecule of the present application is at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably 100% identical to the amino acid sequence set forth in SEQ ID NO: 17.

[0063] In the chimeric antigen receptor of the present application, other genes such as a tag, a cytokine, or a regulatory gene can be included or not included as needed. The tag can be exemplified by a Strep tag II tag or the like.

[0064] In a more specific embodiment, the present application provides the following six chimeric antigen receptors:

[0065] CAR-1 comprises a GCC scFv, a CD8 hinge region, a CD8 transmembrane region, a CD28 co-stimulatory signaling domain, and a CD3 zeta intracellular signaling domain.

[0066] CAR-2 comprises a GCC scFv, a CD28 hinge region, a CD28 transmembrane region, a CD28 costimulatory signaling domain, a CD3 zeta intracellular signaling domain;

[0067] CAR-3 comprises a GCC scFv, a CD8 hinge region, a CD28 transmembrane region, a CD28 costimulatory signaling domain, a CD3 zeta intracellular signaling domain;

[0068] CAR-4 comprises a GCC scFv, an IgG4 hinge region, a CD28 transmembrane region, a CD28 costimulatory signaling domain, a CD3 zeta intracellular signaling domain;

[0069] CAR-5 comprises a GCC scFv, an IgG4 hinge region, a CD8 transmembrane region, a CD28 costimulatory signaling domain, a CD3 zeta intracellular signaling domain;

[0070] CAR-6 comprises a GCC scFv, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB costimulatory signaling domain, a CD3 zeta intracellular signaling domain.

[0071] In other embodiments, the six chimeric antigen receptors described above further comprise a shield peptide.

[0072] In more specific embodiments, the shield peptide is the amino acid sequence set forth in SEQ ID NO: 24.

[0073] In more specific embodiments, the present application further provides a seventh chimeric antigen receptor as follows:

[0074] CAR-7 comprises a shield peptide set forth in SEQ ID NO: 24, a GCC scFv, a CD8 hinge region, a CD8 transmembrane region, a CD28 costimulatory signaling domain, a CD3 zeta intracellular signaling domain.

[0075] In more specific embodiments, the CAR-1 to CAR-7 of the present application are as follows, respectively:

[0076] the amino acid sequence of CAR-1 is at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably 100% identical to the amino acid sequence set forth in SEQ ID NO: 18;

[0077] the amino acid sequence of CAR-2 is at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably 100% identical to the amino acid sequence set forth in SEQ ID NO: 19;

[0078] the amino acid sequence of CAR-3 is at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably 100% identical to the amino acid sequence set forth in SEQ ID NO: 20;

[0079] The amino acid sequence of CAR-4 is at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably 100% identical to the amino acid sequence set forth in SEQ ID NO: 21;

[0080] The amino acid sequence of CAR-5 is at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably 100% identical to the amino acid sequence set forth in SEQ ID NO: 22;

[0081] The amino acid sequence of CAR-6 is at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably 100% identical to the amino acid sequence set forth in SEQ ID NO: 23.

[0082] The amino acid sequence of CAR-7 is at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably 100% identical to the amino acid sequence set forth in SEQ ID NO: 25.

[0083] The present application also relates to a nucleic acid molecule comprising a nucleic acid sequence encoding the chimeric antigen receptor as described above.

[0084] The present application also relates to a recombinant vector comprising the nucleic acid molecule as described above. The vector includes, but is not limited to, lentivirus, adenovirus, adeno-associated virus, retrovirus, transposon, etc.

[0085] The recombinant vector of the present application can also optionally further comprise a promoter, such as truncated EF1a promoter, full-length EF1a promoter, CMV promoter, MND promoter, truncated PGK promoter PGK300 promoter, full-length PGK promoter PGK400 promoter, preferably truncated EF1a promoter, full-length EF1a promoter, CMV promoter. The nucleotide sequences of the above promoters correspond to SEQ ID NOs: 29-34, respectively.

[0086] The present application also relates to an engineered cell comprising the chimeric antigen receptor, the nucleic acid molecule, or the recombinant vector as described above. The cell is preferably an immune cell, including, for example, T cell, B cell, gd T cell, NK cell, NKT cell, monocyte, macrophage, etc.

[0087] The present application further relates to the use of the chimeric antigen receptor, the nucleic acid molecule, the recombinant vector, and the engineered cell as described above in the preparation of a medicament for treating a tumor positive for GCC. Specific examples of the tumor include, but are not limited to, a digestive tract tumor, such as colorectal cancer, esophageal cancer, gastric cancer, or pancreatic cancer, etc.

[0088] The present invention further relates to a method for treating GCC-positive tumors, comprising administering an effective amount of a chimeric antigen receptor, nucleic acid molecule, recombinant vector, or engineered cell of the present invention to a patient in need thereof. The term "treat" encompasses both alleviation and cure. Specific examples of GCC-positive tumors include, but are not limited to, digestive tract tumors, such as colorectal cancer, esophageal cancer, gastric cancer, or pancreatic cancer.

[0089] The amino acid sequences and nucleotide sequences described in this application are shown in Tables 1 and 2 below, respectively.

[0090] Table 1 Amino acid sequence

[0091] Table 2 Nucleotide sequences

[0092] Example

[0093] Example 1: Construction of GCC CAR molecules with different hinge and transmembrane regions and differences in in vitro proliferation

[0094] 1.1 Construction of lentiviral transfer plasmids and lentivirus preparation

[0095] (1) The amino acid sequences of CAR-1, CAR-2, CAR-3, CAR-4, and CAR-5 are shown in Table 1. XbaI and SalI restriction sites were added to the 5' and 3' ends of the genes of CAR-1, CAR-2, CAR-3, CAR-4, and CAR-5, respectively, and these genes were synthesized by gene synthesis (Beijing Bomade Gene Technology Co., Ltd.). The above five gene plasmids were double-digested with XbaI and SalI, and the vector pLenti6.3 / V5 (Thermo Fisher, Waltham, MA, USA) containing the truncated EF1α promoter was also double-digested with XbaI and SalI. The gene fragments and plasmid fragments after double digestion were purified using a DNA gel recovery kit and then ligated with T4 DNA ligase to obtain pLenti6.3 / V5 plasmids carrying the CAR-1, CAR-2, CAR-3, CAR-4, or CAR-5 genes.

[0096] (2) The lentiviral packaging plasmids pLP / VSVG, pLP1 / MDK, and pLP2 / RSK (Thermo Fisher, Waltham, MA, USA) and the lentiviral transfer plasmid obtained in step (3) were transfected into HEK293T cells using Lipofectamine 3000 (Thermo Fisher, Waltham, MA, USA). After 48 hours, the culture medium was collected and centrifuged at 300 g to remove cell debris. The cells were then ultracentrifuged at 25,000 rpm for 3 hours. The precipitate was dissolved in 1 mL of saline to obtain the desired lentiviral vector.

[0097] 1.2 CAR-T cell preparation and proliferation detection

[0098] (1) Washing of single blood: Wash the single blood of healthy volunteers twice with physiological saline, then resuspend it in X-VIVO 15 (Lonza) medium and count it. 8 The cells were allowed to adhere to the wall at a density of 10 cells / 30 mL for 2 hours.

[0099] (2) T cell sorting and activation: cells adhered for 2 hours were collected, centrifuged at 400 g for 5 min, counted, and 2 × 10 5 The cells were stained with CD3APC (Biolegend) antibody to detect the proportion of T cells. According to the cell counting results and T cell ratio, CD3 / CD28Dynabeads (Thermo) were added according to the ratio of magnetic beads: cells = 1.5:1 (quantity ratio), and placed on a sample sorter (Thermo, pR5-5 / 12) and gently shaken for 30 minutes. After that, CD3-positive T cells were sorted by the adsorption of magnetic beads using a magnetic stand (Invitrogen). In order to fully activate the T cells, they were resuspended in complete culture medium (X-VIVO 15 culture medium containing 500IU / mL IL-2) and 4% paraformaldehyde was added at 1.5×10 6 The cells were expanded and cultured at a density of 10 cells / mL.

[0100] (3) After 24 hours of magnetic bead activation, cells were collected, centrifuged, and counted. Based on the counting results, the cells were grouped and infected with CAR-1, CAR-2, CAR-3, CAR-4, and CAR-5 lentivirus at an MOI of 2 to obtain CAR-T-1, CAR-T-2, CAR-T-3, CAR-T-4, and CAR-T-5. After 24 hours of viral infection, the cells were centrifuged at 400 g for 5 minutes and replaced with fresh complete medium for continued culture.

[0101] (4) After 4 days of culture, collect the cells, remove the Dynabeads using a magnetic stand, centrifuge at 400g for 5 min, resuspend with complete medium, then count, and continue to culture at a density of 3x10 5 cells / mL.

[0102] (5) Cell expansion: CAR-T cells were expanded in complete medium for 12 days, and passaged every 2 days for counting.

[0103] The results of the in vitro proliferation of resting T cells are shown in Figure 1A. Before Day 10, the proliferation rates of the T cells in the five groups were not significantly different. By Day 12, the proliferation rate of the T cells in the CAR-T-5 group was significantly faster, and the proliferation rate of the CAR-T-2 group was slightly lower. The CAR positive rate was detected using GCC protein conjugated with PE fluorescent molecules (prepared by Beijing Yimiaoshizhao Biotechnology Co., Ltd. after expressing and purifying GCC recombinant protein in CHO cells). The proliferation rate of CAR-T cells in each group was calculated, and the results are shown in Figure IB. Before Day 10, the proliferation rates of CAR-T cells in the five groups were similar, but by Day 12, the proliferation rate of CAR-T-5 was significantly increased, and there was no significant difference in the other four groups.

[0104] Example 2: Detection of CAR expression rate of GCC CAR-T cells with different hinge regions and transmembrane regions

[0105] At different time points (Day 6, Day 8, Day 10, Day 12) in Example 1, 2x10 5 cells were taken from each group, incubated with APC-labeled CD3 antibody (Biolegend) and GCC protein conjugated with PE fluorescent molecules (prepared by Beijing Yimiaoshizhao Biotechnology Co., Ltd.) at room temperature for 15 minutes, and then the CAR expression of each group of cells was detected using a full-spectrum flow cytometer (Northern Lights, N7-00008-0A, Cytek Biosciences, Fremont, CA 94538, USA).

[0106] The results are shown in Figure 2A. The CAR positive rate of each group of CAR-T cells showed an upward trend with time, with CAR-T-3 and CAR-T-4 being slightly lower, but still above 50%. As shown in 2B, the average fluorescence intensity of CAR of each group of CAR-T was the highest in CAR-T-1 before Day 12, indicating that the CAR-T-1 cell surface had the highest CAR molecule level.

[0107] Example 3: Evaluation of in vitro killing function of GCC CAR-T cells with different hinge regions and transmembrane regions

[0108] To screen CAR-T with high sensitivity to tumor GCC antigen, the present experiment used "HCT116-GCC low expression" cells as target cells, which were a cell line with low GCC expression intensity sorted by flow cytometry after HCT-116 wild type tumor cells were infected with a lentivirus containing a full-length human GCC gene. Specifically, HCT-116 wild type tumor cells (not expressing human GCC) were purchased from the Cell Resource Center of Institute of Basic Medicine, Chinese Academy of Medical Sciences. The full-length human GCC sequence (NCBI sequence number: NM_004963.3) was synthesized by gene synthesis method, and the DNA sequence containing the full-length human GCC was cloned into the lentivirus vector pLenti6.3 / V5, and the lentivirus containing the full-length human GCC was prepared according to the method of Example 1, step 1.1(2), and used to infect HCT-116 wild type cells. After 48 hours, the cells were incubated with Alexa 488-labeled GCC antibody (R&D Systems) at room temperature for 20 minutes, and the GCC expression of the cells was detected by flow cytometry (BD Arial II), and "HCT-116-GCC low expression" cells with low GCC expression intensity and "HCT-116-GCC high expression" cells with high GCC expression intensity were sorted; the sorted cells were respectively expanded and cultured to sufficient quantity.

[0109] After HCT-116 wild type tumor cells, HCT116-GCC low expression cells and HCT116-GCC high expression cells were incubated with Alexa 488-labeled GCC antibody (R&D Systems) at room temperature for 20 minutes, the GCC expression was detected by flow cytometry (NoveCyte 2060R, ACEA Biosciences, San Diego, CA, USA) to verify the GCC expression intensity of the two sorted cells, and the results are shown in Figure 3. HCT-116 wild type tumor cells had no GCC positive signal, "HCT-116-GCC low expression" cells had moderate GCC positive signal, and "HCT-116-GCC high expression" cells showed the strongest GCC positive signal.

[0110] 3.1 Real-time killing experiment

[0111] (1) Tumor cell plating: 50 μL of IMDM complete medium containing 10% FBS was added to each well of the 96-well E-plate 96, which was placed on the RTCA Station to detect the baseline. HCT-116-GCC tumor cells were digested with trypsin (Hyclone), counted, and then the cell density was adjusted to 2 x 10 5 4

[0112] (2) CAR-T cell addition: CAR-T cells on Day 8 were collected, centrifuged at 400 g for 5 min, resuspended with X-VIVO 15 medium, and adjusted to a cell density of 1 x 10 6 6

[0113] ​​​​(3) The E-Plate 96-well plate was placed back into the real-time killing instrument to monitor the killing effect of CAR-T on tumor cells in real time. The vertical axis "normalized tumor cell index" represents the killing efficiency. The lower the value, the higher the killing efficiency. The results are shown in Figures 4A and 4B. When E:T = 1:3, CAR-T-4 and CAR-T-5 had no killing effect on target cells, while CAR-T-1, CAR-T-2, and CAR-T-3 were able to kill tumor cells well. As shown in Figure 4C, the experimental data of CAR-T and tumor cells co-incubated for 20 hours were selected for statistical analysis. There was no significant difference between CAR-T-1, CAR-T-2, and CAR-T-3, but there was a significant difference compared with CAR-T-4 and CAR-T-5.

[0114] 3.2 Repeated stimulation killing experiment

[0115] (1) Tumor cell plating: HCT116-GCC low-expressing tumor cells were digested with trypsin (Hyclone), counted, and then the cell density was adjusted to 1×10 5 cells / mL, 500 μL of tumor cell suspension was added to each well of a 48-well plate and cultured overnight.

[0116] (2) CAR-T cell addition: Collect cells, centrifuge at 400g for 5 minutes, resuspend in X-VIVO 15 medium, and adjust the cell density to 1×10 6 cells / mL, take 1×10 6 T cells were incubated with APC-labeled CD3 antibodies (Biolegend) and GCC protein conjugated with PE fluorescent molecules (prepared by Beijing Yimiao Shenzhou Pharmaceutical Technology Co., Ltd.) at room temperature in the dark for 15 minutes. CAR positivity was then determined using a full-spectrum flow cytometer (Northern Lights, N7-00008-0A, Cytek Biosciences, Fremont, CA 94538, USA). The 48-well plate was removed and the required number of CAR-T cells for each group was calculated based on the measured CAR positivity rate, using an E:T ratio of 1:1. T cells were supplemented to each group until the total number of T cells in each group was consistent, and finally, X-VIVO 15 to 500 μL was added. After preparation, each group of CAR-T cells was added to each well. The untransfected T cell group was added with an equal number of total T cells as each CAR-T group, and X-VIVO 15 to 500 μL was added.

[0117] (3) Every 2-3 days, observe the cells under a microscope, and when all the tumor cells in the previous round are killed, collect the CAR-T cells in the well and add them to the tumor cells prepared the day before to repeat the stimulation experiment. When the CAR-T cells cannot completely kill the tumor cells, use trypsin (Hyclone) to digest and collect all the cells, and use APC-labeled CD3 antibody (Biolegend) and PE-labeled GCC protein (prepared by Beijing Yimiaoshizhao Biotechnology Co., Ltd.) to incubate at room temperature for 15 minutes in the dark for staining. Then, use a flow cytometer (NoveCyte 2060R, ACEA Biosciences, San Diego, CA, USA) to count the number of residual tumor cells and the number of CAR-T cell expansion after multiple rounds of target cell stimulation.

[0118] The repeated killing results are shown in FIG. 5. After 10 rounds of target cell stimulation, CAR-T-1, CAR-T-2, and CAR-T-3 had good killing ability and good CAR-T expansion ability, which were significantly better than CAR-T-4 and CAR-T-5. Further, after 12 rounds of target cell stimulation, the results are shown in FIG. 6. CAR-T-1 maintained the strongest continuous tumor killing ability, and the number of CAR-T cell expansion was the largest, and the persistence was the best, which were significantly better than CAR-T-2 and CAR-T-3.

[0119] Example 4: Cytokine secretion levels of GCC CAR-T cells with different hinge regions and transmembrane regions under target cell stimulation

[0120] 4.1 Tumor cell plating:

[0121] HCT116-GCC low expression tumor cells were digested with trypsin (Hyclone), counted, and then the cell density was adjusted to 1 x 10 5 500 μL of tumor cell suspension was added to each well of a 48-well plate, and the tumor cells were cultured overnight to adhere.

[0122] 4.2 CAR-T cell co-incubation:

[0123] The cells were collected, centrifuged at 400g for 5 min, resuspended with X-VIVO 15 medium, and the cell density was adjusted to 1 x 10 6Cells / mL, and the CAR positive rate was detected using a full-spectrum flow cytometer (Northern Lights, N7-00008-0A, Cytek Biosciences, Fremont, CA 94538, USA); the 48-well plate was removed, and the volume of CAR-T cells required for each group was calculated according to the measured CAR positive rate at E:T = 1:1. The total T cells in each group were supplemented to keep consistent, and finally X-VIVO 15 was supplemented to 500 μL. After the preparation was completed, the CAR-T cells in each group were added to each well, and the untransfected T cell group was added with the same amount of total T cells as each CAR-T group, and X-VIVO 15 was supplemented to 500 μL. After 24 hours, the cells in the wells were gently blown (as much as possible without blowing up the tumor cells), and then the plate was centrifuged at 400 g for 5 min. 50 μL of supernatant was taken, and the levels of TNF, IFN-γ, and IL-2 cytokines in each group were detected using a Cytometric Bead Array (CBA) kit (BD Biosciences).

[0124] The results are shown in FIG. 7. Compared with CAR-T-4 and CAR-T-5, CAR-T-1, CAR-T-2, and CAR-T-3 significantly secreted more TNF-a, IFN-γ, and IL-2 cytokines, indicating that the three groups of CAR-T cells can effectively recognize the GCC target antigen and activate CAR-T cells to perform killing functions, while CAR-T-4 and CAR-T-5 cannot effectively recognize the GCC target antigen.

[0125] Example 5: Tonic signaling level of GCC CAR-T cells with different hinge regions and transmembrane regions during in vitro culture

[0126] Tonic signaling is an indicator for measuring the ability of CAR-T cells to initiate downstream signals to promote cell proliferation and differentiation in the absence of exogenous antigen stimulation. Moderate tonic signaling is crucial for maintaining the effector function and survival of CAR-T. The CAR-T of each group on Day 8 was cultured in X-VIVO 15 (without exogenous addition of IL-2), and the levels of cytokines were detected after 24 hours, and cell counting was performed every 2-3 days to comprehensively analyze the strength of the tonic signaling of CAR-T.

[0127] The results are shown in Figure 8. The cytokines TNF-a, IFN-g, and IL-2 secretion levels of the CAR-T-1, CAR-T-2, and CAR-T-3 groups were higher, among which CAR-T-1 was the highest, with the highest basal signal strength. The results of the resting cell proliferation are shown in Figure 9. The CAR-T-1, CAR-T-2, and CAR-T-3 groups all showed obvious cell expansion, while the CAR-T-4 and CAR-T-5 groups had no cell expansion and the cells died at a later stage. This indicates that CAR-T-1, CAR-T-2, and CAR-T-3 have obvious basal signals, among which CAR-T-1 has the strongest basal signal, while CAR-T-4 and CAR-T-5 do not have basal signals. This result indicates that when CAR-T is returned to the body, CAR-T-1 has faster tumor antigen response ability and stronger survival ability, which is beneficial to improve the treatment effect.

[0128] Example 6: CAR-T cell proliferation detection of GCC CAR molecules containing different co-stimulatory signal domains

[0129] Based on the CAR-T-1 with the best in vitro killing function, the functions of CAR-T with two co-stimulatory signal domains (CD28 and 4-1BB) were compared.

[0130] The amino acid sequence of CAR-6 is shown in Table 1. CAR-T-1 and CAR-T-6 containing CAR-6 were prepared according to the method in Example 1. On the 5th and 11th days of culture, the cells were collected, centrifuged and counted. 2x10 5 The cells were incubated with APC-labeled CD3 antibody (Biolegend) and GCC protein coupled with PE fluorescent molecules (prepared by Beijing Yimiaoshizhou Biotechnology Co., Ltd.) at room temperature for 15 minutes, and then the CAR expression of each group of cells was detected using a full-spectrum flow cytometer (Northern Lights, N7-00008-0A, Cytek Biosciences, Fremont, CA 94538, USA).

[0131] The CAR-T cells were cultured and expanded in X-VIVO 15 medium containing 500 IU / mL for 11 days, and passaged and counted every 3 days. The CAR-T cells were collected and centrifuged, resuspended with 1-2 mL of X-VIVO 15 medium, diluted with 10 μL of cell culture solution, stained with trypan blue at a volume ratio of 1:1, and the cell viability and viable cell concentration were recorded by a cell counter. The total cell number of each group was calculated to statistically analyze the proliferation of each group of cells.

[0132] The cell proliferation results are shown in Figure 10. The T cell proliferation rates and CAR-T cell proliferation rates of the CAR-T-1 and CAR-T-6 groups were similar, with no significant difference. As shown in Figure 11, CAR expression in each CAR-T cell group was detected on days 5 and 11. The CAR positivity rate of CAR-T-1 was slightly higher than that of CAR-T-6, but the mean fluorescence intensity of CAR expression was slightly lower than that of CAR-T-6.

[0133] Example 7: Evaluation of the in vitro killing function of GCC CAR-T cells with different costimulatory signaling domains

[0134] 7.1 Real-time Killing

[0135] (1) Tumor cell plating: 50 μL of IMDM complete medium containing 10% FBS was added to the wells of a 96-well plate (E-plate 96) used with a real-time killing instrument (Agilent xCELLigence RTCA SP), placed on the RTCA Station, and baseline was detected; HCT116-GCC low-expressing tumor cells were digested with trypsin, counted, and then the cell density was adjusted to 2×10 with IMDM complete medium containing 10% FBS. 5 cells / mL; take out E-plate 96 and add 100 μL (2×10 4 The tumor cell suspension (cells / well) was placed at room temperature for 30 minutes; the E-plate 96 was placed on the RTCA Station in the incubator, and the cell proliferation curve was detected after 24 hours.

[0136] (2) CAR-T cell addition: CAR-T cells were collected on Day 11, centrifuged at 400 g for 5 min, resuspended in X-VIVO 15 medium, and the cell density was adjusted to 1×10 6 cells / mL, take 1×10 6T cells, and the APC-labeled CD3 antibody (Biolegend) and GCC protein coupled with PE fluorescent molecules (prepared by Beijing Yimiaoshengzhou Medical Technology Co., Ltd.) were incubated at room temperature for 15 minutes in the dark. The CAR positive rate was detected using a full-spectrum flow cytometer (Northern Lights, N7-00008-0A, Cytek Biosciences, Fremont, CA 94538, USA); 50 μL of supernatant was removed from each well of the E-plate 96-well plate. According to the measured CAR positive rate, the required volume of each group of CAR-T cells was calculated according to E:T = 1:3 and E:T = 1:6. To keep the total T cells consistent in each group, T cells need to be supplemented to each group, and finally X-VIVO 15 is added to 100 μL. After the preparation is completed, each group of CAR-T cells is added to each well, and the number of cells in the untransfected T cell group is consistent with the total T cells in each group of CAR-T, and X-VIVO 15 is added to 100 μL.

[0137] (3) Put the E-Plate 96-well plate back into the real-time killing instrument, and monitor the killing effect of CAR-T on tumor cells. The vertical axis "normalized tumor cell index" represents the killing efficiency, and the lower the value, the higher the killing efficiency.

[0138] The killing results are shown in FIG. 12, and the tumor cell killing ability of CAR-T-1 is significantly better than that of CAR-T-6, indicating that the CAR-T containing the CD28 costimulatory signaling domain has a better killing efficiency than the CAR-T containing the 4-1BB costimulatory signaling domain.

[0139] 7.2 Repeated stimulation killing

[0140] (1) Tumor cell plating: HCT116-GCC low expression tumor cells were digested with trypsin (Hyclone), counted, then resuspended with IMDM complete medium containing 10% FBS, and the cell density was adjusted to 1 × 10 5 cells / mL, 500 μL of tumor cell suspension was added to each well of the 48-well plate, and incubated overnight.

[0141] (2) CAR-T cell co-incubation: collect cells, centrifuge at 400g for 5 min, resuspend with X-VIVO 15 medium, and adjust the cell density to 1 × 10 6 cells / mL, take 1 × 10 6T cells, and PE fluorescent molecule-coupled GCC protein (prepared by Beijing Yimiaoshengzhou Medical Technology Co., Ltd.) were incubated at room temperature in the dark for 15 minutes, and then the CAR positive rate was detected using a full-spectrum flow cytometer (Northern Lights, N7-00008-0A, Cytek Biosciences, Fremont, CA 94538, USA). The 48-well plate was removed, and the required volume of CAR-T cells was calculated for each group according to the measured CAR positive rate, with E:T = 1:3. To keep the total T cells consistent in each group, T cells needed to be supplemented to each group, and finally X-VIVO 15 was added to 500 μL. After the preparation was completed, the CAR-T cells in each group were added to each well, and the same amount of total T cells as the CAR-T group was added to the untransfected T cell group, and X-VIVO 15 was added to 500 μL.

[0142] (3) Every 2-3 days, when all the tumor cells in the previous round were killed, the CAR-T cells in the wells were collected and added to the tumor cells prepared the day before for repeated stimulation experiments. When the CAR-T cells could not completely kill the tumor cells, trypsin (Hyclone) was used for digestion, and all the cells were collected and stained using APC-labeled CD3 antibody (Biolegend) and PE-labeled CAR antibody (prepared by Beijing Yimiaoshengzhou Medical Technology Co., Ltd.) incubated at room temperature in the dark for 15 minutes. Then the number of residual tumor cells and the number of CAR-T cells in each well after multiple rounds of target cell stimulation were counted by flow cytometry (NovoCyte 2060R, ACEA Biosciences, San Diego, CA, USA).

[0143] The results of repeated stimulation killing are shown in FIG. 13. After 7 rounds of target cell stimulation, CAR-T-1 had stronger continuous killing ability of tumor cells than CAR-T-6, and the number of CAR-T cell expansion was larger, indicating that the CAR-T containing the CD28 costimulatory signaling domain had better long-term killing ability and persistence than the CAR-T containing the 4-1BB costimulatory signaling domain, i.e., had better ability to resist tumor recurrence.

[0144] Example 8: Construction of GCC CAR molecules with different promoters and preparation of CAR-T cells

[0145] Based on the CAR-T-1 molecule, the design and screening of the promoter of the lentiviral vector were performed.

[0146] 8.1 Construction of lentiviral transfer plasmid and preparation of lentivirus

[0147] (1) The chimeric genes of GCC CAR of full-length EF1a-CAR-T-1, truncated EF1a-CAR-T-1, CMV-CAR-T-1, MND-CAR-T-1, PGK300-CAR-T-1 and PGK400-CAR-T-1 with restriction enzyme sites Xbal and Sail at both ends were synthesized by gene synthesis method (Beijing Bomeide Gene Technology Co., Ltd.), and the specific sequences of the promoters are shown in Table 2. The lentiviral transfer plasmid was constructed and the lentivirus was prepared according to the same method as in Example 1.

[0148] 8.2 Preparation of CAR-T cells and proliferation detection

[0149] The CAR-T cells were prepared according to the same method as in Example 1. The CAR-T cells were expanded in complete culture medium for 12 days, and the cell count was counted every 2 days. The in vitro proliferation of T cells and CAR-T cells is shown in Figure 14. With the extension of culture time, the proliferation rate of full-length EF1a-CAR-T-1 is the fastest, and the proliferation rate of PGK300-CAR-T-1 is the slowest. From the 6th day to the 12th day of culture, the CAR positive rate of each group of CAR-T was detected, and the results are shown in Figure 15. The CAR positive rate of truncated EF1a-CAR-T-1 and MND-CAR-T-1 is the highest.

[0150] Example 9: Evaluation of in vitro killing function of GCC CAR-T cells with different promoters

[0151] 9.1 Real-time killing

[0152] (1) Tumor cell plating: 50 μL of IMDM complete medium containing 10% FBS was added to the E-plate 96 hole, which was placed on the RTCA Station, and the baseline was detected. HCT116-GCC low expression tumor cells were trypsinized, counted, and then the cell density was adjusted to 2 x 10 5 cells / mL with IMDM complete medium containing 10% FBS; take out the E-plate 96, add 100 μL (2 x 10 4 cells / well) tumor cell suspension to each well, and place at room temperature for 30 min; place the E-plate 96 on the RTCA Station in the incubator, and monitor the cell proliferation curve in real time.

[0153] (2) CAR-T cell addition: collect CAR-T cells on Day 8, centrifuge at 400g for 5 min, resuspend with X-VIVO 15 medium, and adjust the cell density to 1 x 10 6cells / mL, take 1×10 6 T cells were incubated with an APC-labeled CD3 antibody (Biolegend) and a GCC protein conjugated to a PE fluorescent molecule (manufactured by Beijing Yimiao Shenzhou Pharmaceutical Technology Co., Ltd.) at room temperature in the dark for 15 minutes. CAR positivity was then determined using a full-spectrum flow cytometer (Northern Lights, N7-00008-0A, Cytek Biosciences, Fremont, CA 94538, USA). The 96-well E-plate was removed and 50 μL of supernatant was aspirated from each well. Based on the measured CAR positivity, the required volume of CAR-T cells for each group was calculated using an E:T ratio of 1:3. T cells were supplemented to maintain the same total T cell count in each group, and finally, 15 to 100 μL of X-VIVO was added. After preparation, CAR-T cells from each group were added to each well. The number of cells in the untransfected T cell group was kept consistent with the total T cell count in each CAR-T group, and 15 to 100 μL of X-VIVO was added.

[0154] (3) Place the E-Plate 96-well plate back into the real-time killing instrument to monitor the killing effect of CAR-T on tumor cells. The vertical axis "normalized tumor cell index" represents the killing efficiency. The lower the value, the higher the killing efficiency.

[0155] The real-time killing results are shown in Figure 16. At E:T = 1:3, the killing rates of truncated EF1α-CAR-T-1, full-length EF1α-CAR-T-1 and CMV-CAR-T-1 were faster than those of other groups of CAR-T, but there was no significant difference among the three groups, indicating that the three promoters, truncated EF1α, full-length EF1α and CMV, can effectively regulate the gene expression and anti-tumor function of CAR-T-1, while the PGK300 and PGK400 promoters cannot enable CAR-T-1 to achieve effective anti-tumor function.

[0156] 9.2 Repeated Stimulation Killing

[0157] (1) Tumor cell plating: HCT116-GCC tumor cells were digested with trypsin (Hyclone), counted, and then the cell density was adjusted to 1×10 5 cells / mL, 500 μL of tumor cell suspension was added to each well of a 48-well plate and cultured overnight.

[0158] (2) CAR-T cell co-incubation: The cells were collected, centrifuged at 400 g for 5 min, resuspended in X-VIVO 15 medium, and the cell density was adjusted to 1×10 6 cells / mL, take 1×10 6T cells, and PE fluorescent molecule-coupled GCC protein (prepared by Beijing Yimiaoshengzhou Biotechnology Co., Ltd.) were incubated at room temperature in the dark for 15 minutes, and then the CAR positive rate was detected using a full-spectrum flow cytometer (Northern Lights, N7-00008-0A, Cytek Biosciences, Fremont, CA 94538, USA). The 48-well plate was removed, and the required volume of CAR-T cells was calculated for each group according to the measured CAR positive rate at an E:T ratio of 1:1. The total number of T cells in each group was kept consistent, and X-VIVO 15 was finally supplemented to 500 μL. After the preparation was completed, each group of CAR-T cells was added to each well, and the untransfected T cell group was added to the same amount of total T cells as each group of CAR-T cells, and X-VIVO 15 was supplemented to 500 μL.

[0159] (3) Every 2-3 days, when all the tumor cells in the previous round were killed, the CAR-T cells in the wells were collected and added to the tumor cells plated the day before for repeated stimulation experiments. When the CAR-T cells could not completely kill the tumor cells, trypsin (Hyclone) was used for digestion, and all the cells were collected and stained using APC-labeled CD3 antibody (Biolegend) and PE-labeled GCC protein (prepared by Beijing Yimiaoshengzhou Biotechnology Co., Ltd.) incubated at room temperature in the dark for 15 minutes. The number of residual tumor cells and the number of CAR-T cell expansion after multiple rounds of target cell stimulation were then counted by flow cytometry (NovoCyte 2060R, ACEA Biosciences, San Diego, CA, USA).

[0160] The results after 6 rounds of repeated stimulation of target cells are shown in Figure 17. The truncated EF1a-CAR-T-1, full-length EF1a-CAR-T-1, CMV-CAR-T-1 and MND-CAR-T-1 have better killing ability, and the number of CAR-T expansion is more, and there is no significant difference between each group, but they are significantly better than PGK300-CAR-T-1 and PGK400-CAR-T-1. After further 10 rounds of repeated stimulation of target cells, the results are shown in Figure 18. The truncated EF1a-CAR-T-1, full-length EF1a-CAR-T-1 and CMV-CAR-T-1 have good continuous killing ability of tumor cells, while the killing of MND-CAR-T-1 to tumor cells is weakened, and the number of CAR-T expansion is less. In summary, the truncated EF1a-CAR-T-1, full-length EF1a-CAR-T-1 and CMV-CAR-T-1 have strong continuous killing ability of tumor cells, and the number of CAR-T expansion is more, and the persistence is better, which further proves that the three promoters of truncated EF1a, full-length EF1a and CMV can effectively regulate the expression of CAR-T-1 gene and the anti-tumor function.

[0161] Example 10: Cytokine secretion levels of GCC CAR-T cells with different promoters

[0162] (1) Tumor cell plating: HCT116-GCC low expression tumor cells were trypsinized, counted, and then the cell density was adjusted to 1 x 10 5 cells / mL, 500 μL of tumor cell suspension was added to each well of a 48-well plate, and the tumor cells were cultured overnight to adhere.

[0163] (2) CAR-T cell co-incubation: collect cells, centrifuge at 400g for 5 min, resuspend with X-VIVO 15 medium, and adjust the cell density to 1 x 10 6 cells / mL, and use a full-spectrum flow cytometer (Northern Lights, N7-00008-0A, Cytek Biosciences, Fremont, CA 94538, USA) to detect the CAR positive rate; remove the 48-well plate, and according to the measured CAR positive rate, calculate the required volume of CAR-T cells added for each group according to E:T = 1:1. To enable parallel comparison of each group, the total T cells in each experimental group were kept consistent, and finally X-VIVO 15 was added to 500 μL. After preparation, add each group of CAR-T cells to each well, and add the same amount of total T cells to each group of CAR-T cells, and add X-VIVO 15 to 500 μL.

[0164] (3) After 24 hours, gently pipette the cells in the wells (try not to blow up the tumor cells), and then centrifuge the plate at 400g for 5 minutes. Take 50 μL of the supernatant and use Cytometric Bead Array (CBA) kits (BD Biosciences) to detect the cytokines TNF, IFN-γ, and IL-2 in each group.

[0165] The test results are shown in Figure 19. The truncated EF1α-CAR-T-1, full-length EF1α-CAR-T-1 and CMV-CAR-T-1 can all secrete more cytokines TNF-α, IFN-γ, and IL-2, indicating that the three groups of CAR-T cells can effectively recognize GCC target antigens and activate CAR-T cells.

[0166] Example 11: Basal signaling levels of GCC CAR-T cells with different promoters during in vitro culture

[0167] Each group of CAR-T was cultured in X-VIVO 15 (without exogenous IL-2 addition), and cytokines and proliferation rates were detected after 24 hours. Cell counting and statistical analysis were performed every 2-3 days thereafter to reflect the basal signal strength of CAR-T. The results are shown in Figure 20. The secretion levels of cytokines TNF-α, IFN-γ, and IL-2 in the three groups of truncated EF1α-CAR-T-1, full-length EF1α-CAR-T-1, and CMV-CAR-T-1 were high, among which CMV-CAR-T-1 had the highest secretion level. The results of in vitro cell proliferation are shown in Figure 21. The truncated EF1α-CAR-T-1, full-length EF1α-CAR-T-1, and CMV-CAR-T-1 groups showed obvious cell proliferation, while the proliferation level of the MND-CAR-T-1 group was low. The cells in the PGK300-CAR-T-1 and PGK400-CAR-T-1 groups did not proliferate, and the cells died in the later stages. This indicates that there are obvious basal signals in truncated EF1α-CAR-T-1, full-length EF1α-CAR-T-1 and CMV-CAR-T-1, among which CMV-CAR-T-1 is the strongest.

[0168] Example 12: Tumor Suppression and In Vivo Expansion Ability of CAR-T-1 Cells in a Subcutaneous Colorectal Cancer PDX Mouse Model

[0169] This example takes the detection of the tumor inhibition ability and in vivo expansion ability of CAR-T (truncated EF1α-CAR-T-1) containing the above-mentioned scFv (promoter is truncated EF1α), CD8 hinge region, CD8 transmembrane region, CD28 co-stimulatory signal domain, and CD3ζ intracellular signal domain in PDX tumor-bearing mice as an example.

[0170] CAR-T-1 cells were prepared according to the above CAR-T cell preparation method, and when the cells were cultured to a sufficient amount, the cells were resuspended in a cryopreservation solution and stored in liquid nitrogen for later use. The tumor tissues of patients with colorectal cancer with high expression of GCC were taken for immunohistochemical detection, and were cut into 2-3 mm 3 fragments. The tumor tissue samples were transplanted subcutaneously into the surgical area of 6-8 week old NSG mice after creating a small incision on the lower back of each mouse. After 7 days, the mice were analyzed by luciferase live imaging (Lumina II small animal live imaging system, PerkinElmer, USA) to verify whether the mouse xenograft model was successfully constructed. When the tumor grew to 100 mm 3 in diameter, CAR-T-1 cells (5 x 10 6 cells / mouse) were injected into the tail vein of each mouse in each group, and another two groups of mice were injected with the same volume of Dulbecco's phosphate buffered saline (DPBS) and the same number of T cells of untransfected control T cells, respectively. The tumor size was measured 1 day before CAR-T cell injection and on days 3, 7, 12, 15, 18, 22, and 26 after CAR-T cell injection, and the number of CAR-T cells in the peripheral blood was detected on days 2, 6, 14, 21, and 28 after CAR-T cell injection.

[0171] The specific results are shown in Figure 22A. Compared with the tumor burden of mice in the DPBS group and the control T cell group, the CAR-T-1 cells slowed down the speed of tumor growth after 3 days of reinfusion, effectively reduced the tumor burden within 26 days of detection, and had significant anti-tumor activity. Figure 22B shows the number of CAR-T-1 cells in the peripheral blood of mice at different times. Higher levels of CAR-T cells were detected after 10 days of reinfusion, and then decreased and maintained at a certain level.

[0172] Example 13: CAR-T cell expansion and anti-tumor activity in patients

[0173] The purpose of the clinical study design is to evaluate the safety and efficacy of infusing autologous truncated EF1-alpha-CAR-T-1 cells into patients, and the tumor target is GCC.

[0174] Human peripheral blood mononuclear cells (PBMCs) were obtained from patients, and according to the cell count results and T cell ratio, CD3 / CD28 Dynabeads (Thermo) were added at a ratio of 1.5:1 (number ratio) and gently shaken on the sample sorter for 30 min. Then, CD3-positive T cells were sorted by using the adsorption of magnetic beads (Invitrogen). To fully activate the T cells, resuspend them with complete medium (X-VIVO 15 + 500 IU / mL IL-2) at a concentration of 1.5 x 10 6The cells were expanded at a density of 1 x 105cells / mL. After 24 hours of magnetic bead activation, the cells were collected and counted. According to the counting results, the T cells were infected with lentivirus at MOI = 2. After 24 hours of viral infection, the cells were centrifuged at 400g for 5 min, and fresh complete medium was replaced for continued culture. On the 8thday of culture, the cells were collected, the Dynabeads were removed using a magnetic stand, the CAR-T cells were centrifuged at 400g for 5 min, and washed, and cryopreservation was performed using a reinfusable cryopreservation solution. The cell suspension was loaded into a cryopreservation bag, which was then gradient-cooled to -90°C and transferred to a gas-phase liquid nitrogen tank for storage. Before patient reinfusion, the frozen CAR-T was transported to the hospital, and patient reinfusion was completed within 30 minutes after resuscitation. The reinfusion dose was 1.2 x 10 9 CAR-T cells.

[0175] All patients received conditioning treatment based on fludarabine and cyclophosphamide before CAR-T reinfusion, and the patients were closely observed for at least 2 hours after CAR-T reinfusion. The peripheral blood of the patients was collected regularly, and the CAR-T expansion was monitored by flow cytometry using a BD TM Cytometric Bead Array (CBA) Human Th1 / Th2 / Th17 CBA Kit was used to monitor the levels of cytokines in the plasma. The protocol was approved by the hospital conducting the test. All patients were provided with written informed consent.

[0176] Patient 01 was diagnosed with advanced metastatic colorectal cancer, with abdominal aortic, right iliac blood vessel para-lymph node metastasis, pelvic, peritoneal, anterior wall of the uterus metastasis, and multiple nodular metastatic lesions in both lungs. After 28 days of infusion of truncated EF1-a-CAR-T-1 cells, CT scan images showed that lung lesions 1 and 2 and peritoneal metastatic lesions were significantly reduced, and the tumor diameter changes are shown in Table 3. CAR-T expanded significantly in vivo, with a maximum of 3.2 x 10 7 CAR-T / L, and the level of IL-6, a cytokine, was significantly elevated, with a maximum of 2666.7 pg / mL, as shown in Figure 24. The levels of carcinoembryonic antigen and cancer antigen decreased significantly during treatment, as shown in Figure 25. This patient was rated as PR (partial remission) in the first month and CR (complete remission) in the 7thmonth. No severe CRS (e.g., no more than grade 2 CRS) was observed in patient 01 during treatment.

[0177] Table 3 Changes in tumor size in patient 01

[0178] Patient 02 was diagnosed with advanced metastatic colorectal cancer with mesenteric lymph node metastasis, retroperitoneal lymph node metastasis, lung metastasis, and liver metastasis. Three months after infusion of truncated EF1-α-CAR-T-1 cells, CT scan images were obtained, as shown in Figure 26. The lung lesions and liver metastases were significantly reduced, with the total tumor diameter reduced by more than 30%. The changes in tumor diameter are shown in Table 4. CAR-T cells significantly expanded in vivo, reaching a maximum of 1.37×10 8 CAR-T / L, IL-6 in cytokines was significantly improved, reaching a maximum of 3500pg / mL, and the results are shown in Figure 27. Carcinoembryonic antigen and cancer antigen decreased significantly during treatment, and the results are shown in Figure 28. The patient was rated as PR (partial remission) at month 3 and maintained PR (partial remission) for more than 8 months. During treatment, no severe CRS (e.g., no more than grade 2 CRS) was observed in patient 02.

[0179] Table 4 Changes in tumor size of patient 02

[0180] Example 14: Construction of GCC CAR molecules with protective peptides and preparation of CAR-T cells

[0181] Based on the above-mentioned CAR-T-1 molecule, a protective peptide was designed for the lentiviral vector.

[0182] 14.1 Construction of Lentiviral Transfer Plasmids and Lentivirus Preparation

[0183] A chimeric gene encoding CAR-T-7 with a Strep tag II protective peptide at the N-terminus and restriction enzyme sites XbaI and SalI at both ends was synthesized by gene synthesis (Beijing Bomade Gene Technology Co., Ltd.). The specific amino acid sequence of the protective peptide is shown in Table 1. The lentiviral transfer plasmid was constructed and the lentivirus was prepared according to the same method as in Example 1.

[0184] 14.2 CAR-T Cell Preparation and CAR Expression Rate Detection

[0185] CAR-T cells were prepared using the same method as in Example 1. CAR-T cells were expanded and cultured in complete medium for 8 days, with passages and counts every 2 days. On day 8 of culture, the CAR positivity and mean fluorescence intensity of each CAR-T group were measured. The results are shown in Figure 29. The CAR positivity rate of CAR-T-7 was lower than that of CAR-T-1, but the CAR mean fluorescence intensity was slightly higher.

[0186] Example 15: Evaluation of the in vitro killing function of GCC CAR-T cells with protective peptides

[0187] 15.1 Real-time Kill

[0188] Real-time killing experiments were performed according to the same experimental procedures as in Example 9. The results of real-time killing are shown in Figure 30. At E:T = 1:3, the killing rate of CAR-T-7 is faster within 72 hours, indicating that the anti-tumor effect of CAR-T-7 is better than that of CAR-T-1 in the short term.

[0189] 15.2 Repeat stimulation killing

[0190] Repeat stimulation killing experiments were performed according to the same experimental procedures as in Example 9. The results are shown in Figure 31. After 2 rounds of target cell repeat stimulation, the killing ability of CAR-T-7 is better, significantly better than CAR-T-1, but there is no obvious advantage in the number of CAR-T expansion. In summary, CAR-T-7 has better persistence and stronger anti-tumor function.

Claims

1. A chimeric antigen receptor targeting GCC, comprising: scFv that specifically recognizes GCC, CD8 hinge region or CD28 hinge region, CD8 transmembrane region or CD28 transmembrane region, CD28 costimulatory signaling domain or 4-1BB costimulatory signaling domain, CD3ζ signaling domain; The scFv that specifically recognizes GCC comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises an HC CDR1 with the amino acid sequence shown in SEQ ID NO: 1, an HC CDR2 with the amino acid sequence shown in SEQ ID NO: 2, and an HC CDR3 with the amino acid sequence shown in SEQ ID NO: 3, and the VL comprises an LC CDR1 with the amino acid sequence shown in SEQ ID NO: 4, an LC CDR2 with the amino acid sequence shown in SEQ ID NO: 5, and an LC CDR3 with the amino acid sequence shown in SEQ ID NO:

6.

2. The chimeric antigen receptor of claim 1, wherein the VH is at least 85% identical to the amino acid sequence of SEQ ID NO: 7, and the VL is at least 85% identical to the amino acid sequence of SEQ ID NO: 8; Preferably, the VH comprises the amino acid sequence of SEQ ID NO: 7, and the VL comprises the amino acid sequence of SEQ ID NO:

8. 3 . The chimeric antigen receptor according to claim 1 , wherein the scFv that specifically recognizes GCC is at least 85% identical to the amino acid sequence of SEQ ID NO:

9.

4. The chimeric antigen receptor according to any one of claims 1 to 3, comprising any one selected from the group consisting of (1) to (4): (1) scFv that specifically recognizes GCC, CD8 hinge region, CD8 transmembrane region, CD28 costimulatory signaling domain, and CD3ζ intracellular signaling domain. (2) scFv that specifically recognizes GCC, CD28 hinge region, CD28 transmembrane region, CD28 costimulatory signaling domain, and CD3ζ intracellular signaling domain, (3) scFv that specifically recognizes GCC, CD8 hinge region, CD28 transmembrane region, CD28 costimulatory signaling domain, and CD3ζ intracellular signaling domain, (4) scFv that specifically recognizes GCC, CD8 hinge region, CD8 transmembrane region, 4-1BB costimulatory signaling domain, and CD3ζ intracellular signaling domain; in, The amino acid sequence of the CD8 hinge region is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 10, and the amino acid sequence of the CD28 hinge region is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 11, The amino acid sequence of the CD8 transmembrane region is at least 85% identical to the amino acid sequence shown in SEQ ID NO: 13, and the amino acid sequence of the CD28 transmembrane region is at least 85% identical to the amino acid sequence shown in SEQ ID NO:

14. The amino acid sequence of the CD28 costimulatory signaling domain is at least 85% identical to the amino acid sequence of SEQ ID NO: 15, and the amino acid sequence of the 4-1BB costimulatory signaling domain is at least 85% identical to the amino acid sequence of SEQ ID NO:

16. The amino acid sequence of the CD3ζ intracellular signaling domain is at least 85% identical to the amino acid sequence shown in SEQ ID NO:

17.

5. The chimeric antigen receptor according to any one of claims 1 to 4, further comprising a protective peptide, wherein the protective peptide preferably has the amino acid sequence shown in SEQ ID NO:

24.

6. The chimeric antigen receptor according to any one of claims 1 to 5, which is at least 85% identical to the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23 or SEQ ID NO:

25.

7. A nucleic acid molecule comprising a nucleic acid sequence encoding the chimeric antigen receptor according to any one of claims 1 to 6.

8. A recombinant vector comprising the nucleic acid molecule according to claim 7, optionally further comprising a truncated EF1α promoter, a full-length EF1α promoter, and a CMV promoter; preferably, The nucleotide sequence of the truncated EF1α promoter is shown in SEQ ID NO:

29. The nucleotide sequence of the full-length EF1α promoter is shown in SEQ ID NO:

30. The nucleotide sequence of the CMV promoter is shown in SEQ ID NO: 31; More preferably, the recombinant vector is a recombinant lentiviral vector.

9. An engineered cell comprising the chimeric antigen receptor according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, or the recombinant vector according to claim 8.

10. Use of the chimeric antigen receptor according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the recombinant vector according to claim 8, or the engineered cell according to claim 9 in the preparation of a drug for treating GCC-positive tumors. The use according to claim 10 , wherein the drug is used to treat digestive tract tumors.

12. The use according to claim 11, wherein the medicament is used to treat colorectal cancer, esophageal cancer, gastric cancer or pancreatic cancer.

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