Chimeric antigen receptor targeting GCC and application thereof

Abstract

The invention relates to a chimeric antigen receptor targeting GCC. The chimeric antigen receptor comprises scFv capable of specifically recognizing GCC, a CD8 hinge region or a CD28 hinge region, a CD8 transmembrane region or a CD28 transmembrane region, a CD28 costimulatory signal domain or a 4-1BB costimulatory signal domain and a CD3 zeta signal domain, the scFv for specifically recognizing the 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 as shown in SEQ ID NO: 1, an HC CDR2 of an amino acid sequence as shown in SEQ ID NO: 2 and an HC CDR3 of an amino acid sequence as shown in SEQ ID NO: 3, and the VL comprises an LC CDR1 of an amino acid sequence as shown in SEQ ID NO: 4, an LC CDR2 of SEQ ID NO: 5 and an LC CDR3 of an amino acid sequence as shown in SEQ ID NO: 6.

Description

Technical Field

[0001] 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

[0002] 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].

[0003] 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.

[0004] Guanylyl cyclase 2C (GUCY2C or GCC) is a single-pass transmembrane protein expressed in intestinal epithelial cells. It converts GTP to cyclic GMP in response to guanylin, uroguanylin, or other ligands. GCC expression in normal tissues and organs is highly specific, primarily concentrated in the intestinal tract and expressed primarily at the apical level [Reference 5]. Therefore, GCC's expression on the surface of the intestinal epithelium has the potential to reduce the toxic side effects of targeted drugs on normal tissues.

[0005] In the context of disease, GCC is expressed in tubular adenomas, inflammatory bowel disease, colorectal precancerous lesions, primary and metastatic colorectal cancers, and metastatic lesions of these diseases in the lymph nodes and liver. Furthermore, GCC is expressed in all dysplasias and adenocarcinomas arising from esophageal and gastrointestinal metaplasia [Reference 6]. In contrast, tumors unrelated to intestinal metaplasia, including gastric signet ring carcinoma, do not express GCC. In one study, researchers evaluated GCC protein expression in 627 gastrointestinal tumors and found that 98% of colorectal cancers expressed GCC, the highest positive rate [Reference 7]. Among other gastrointestinal malignancies, 59% of esophageal cancers, 68% of gastric cancers, and 64% of pancreatic cancers expressed GCC. Therefore, GCC could be a potential drug target for the targeted treatment of these cancers.

[0006] For patients with advanced colorectal cancer, Shanghai Stansay Biotechnology Co., Ltd. reported safety and efficacy data of a GUCY2C-CD19 CAR-T product from 21 patients in two dose-escalation trials at five clinical centers in China at the 2023 ESMO (European Society for Medical Oncology) Annual Meeting. 6 There were 13 patients in the 2nd dose group (2×10 6 cells / kg) in 8 patients. Clinical results showed that the objective response rate (ORR) in the level 1 dose group was 15.4% (2 / 13), and the objective response rate in the level 2 dose group was 50% (4 / 8). The most common adverse events in the patients were cytokine release syndrome (CRS) and diarrhea. Two patients developed neurotoxicity, which was relieved after treatment with corticosteroids. Therefore, the development of CAR-T targeting GUCY2C is expected to provide long-term relief for patients with advanced colorectal cancer.

[0007] While Stansay's GCC19 CAR-T has achieved some efficacy, this approach kills the body's normal B lymphocytes, causing B lymphocyte depletion and thus impairing humoral immunity, making the body susceptible to infection by pathogens and increasing the cost of care. Therefore, the need to develop safer and more effective CAR-T cell therapies targeting GCC remains urgent.

[0008] Document 1: RLSiegel, KDMiller, A.Jemal, Cancer statistics, 2020, CA CancerJ Clin 70(1)(2020)7-30.

[0009] Reference 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.

[0010] Reference 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.

[0011] Reference 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.

[0012] Reference 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.

[0013] Reference 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.

[0014] Reference 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.

[0015] Document 8: Gross G, Waks T, Eshhar Z. Expression of immunoglobulin-T-cellreceptor chimeric molecules as functional receptors with antibody-type specificity. Proc Natl Acad Sci US A. 1989Dec; 86(24):10024-8.

[0016] Document 9: Khalil DN, Smith EL, Brentjens RJ, Wolchok JD. The future of cancer treatment: immunomodulation, CARs and combination immunotherapy. Nat Rev ClinOncol2016; 13(6):394. Summary of the Invention

[0017] The present invention aims to provide a new chimeric antigen receptor that specifically targets GCC, which has a strong and sustained killing ability against tumor cells with low antigen density, can enhance the persistence of CAR-T in the body, and enhance the anti-tumor effect.

[0018] On one hand, the present invention relates to a chimeric antigen receptor that specifically targets 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; wherein the scFv that specifically recognizes GCC comprises a heavy chain variable region VH and a light chain variable region VL, 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.

[0019] The present invention also relates to a nucleic acid molecule comprising a nucleic acid sequence encoding the chimeric antigen receptor of the present invention, a recombinant vector comprising the nucleic acid molecule, and an engineered cell comprising the chimeric antigen receptor, nucleic acid molecule or recombinant vector.

[0020] 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.

[0021] 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.

[0022] The entire contents of the documents listed in this application are incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Shows the proliferation fold of resting T cells in vitro ( Figure 1 A) CAR-T cell proliferation times in vitro ( Figure 1 B).

[0024] Figure 2 The CAR positive rate of cells in each group is shown ( Figure 2 A), mean fluorescence intensity of CAR ( Figure 2 B).

[0025] Figure 3 GCC expression levels of HCT116 cell lines with different antigen densities are shown.

[0026] Figure 4 Shows the killing curves of CAR-T in each group against HCT116-GCC low-expressing cells.

[0027] Figure 5 The residual amount of tumor cells after 10 rounds of stimulation of HCT116-GCC low-expressing cells ( Figure 5 A) Number of CAR-T cells ( Figure 5 B).

[0028] Figure 6 The residual amount of tumor cells after 12 rounds of stimulation of HCT116-GCC low-expressing cells ( Figure 6 A) Number of CAR-T cells ( Figure 6 B).

[0029] Figure 7 The cytokine secretion levels of each group of CAR-T cells under stimulation with HCT116-GCC low-expressing cells are shown.

[0030] Figure 8 The cytokine secretion levels of each group of CAR-T cells after culture in IL-2-free medium for 24 hours are shown.

[0031] Figure 9 Shows the cell proliferation of each group of CAR-T in the culture medium without IL-2 ( Figure 9A), cell viability ( Figure 9 B).

[0032] Figure 10 Showing the proliferation fold of resting T cells in vitro with different signaling domains ( Figure 10 A) CAR-T cell proliferation times in vitro ( Figure 10 B).

[0033] Figure 11 The CAR positive rate of CAR-T with different signal domains was shown in flow cytometry ( Figure 11 A), mean fluorescence intensity of CAR ( Figure 11 B).

[0034] Figure 12 Shows the killing curve of CAR-T with different signal domains against HCT116-GCC low-expressing cells (E:T=1:3).

[0035] Figure 13 The residual amount of tumor cells after 7 rounds of stimulation of HCT116-GCC low-expressing cells ( Figure 13 A) Number of CAR-T cells ( Figure 13 B).

[0036] Figure 14 Shows the proliferation fold of resting T cells in vitro with different promoters ( Figure 14 A) CAR-T cell proliferation times in vitro ( Figure 14 B).

[0037] Figure 15 The CAR positive rates of different promoters are shown.

[0038] Figure 16 Shown are the killing curves of CAR-T cells combined with different promoters against HCT116-GCC low-expressing cells.

[0039] Figure 17 The residual amount of tumor cells after 6 rounds of stimulation of HCT116-GCC low-expressing cells ( Figure 17 A) Number of CAR-T cells ( Figure 17 B).

[0040] Figure 18 The residual amount of tumor cells after 10 rounds of stimulation of HCT116-GCC low-expressing cells ( Figure 18 A) Number of CAR-T cells ( Figure 18 B).

[0041] Figure 19 Shows the cytokine secretion levels of CAR-T with different promoters after 24 hours of stimulation of HCT116-GCC low-expressing cells.

[0042] Figure 20 The cytokine secretion levels of CAR-T cells expressing different promoters after culturing in IL-2-free medium for 24 hours are shown.

[0043] Figure 21 Shows the cell proliferation of CAR-T cells with different promoters in IL-2-free culture medium ( Figure 21 A), cell viability ( Figure 21 B).

[0044] Figure 22 The tumor-suppressing ability of truncated EF1α-CAR-T-1 in NSG tumor-bearing mice was shown ( Figure 22 A) CAR-T expansion capacity in peripheral blood ( Figure 22 B).

[0045] Figure 23 Displays the CT image of patient 01.

[0046] Figure 24 Showing the number of peripheral blood CAR-T cells and plasma cytokine levels of patient 01.

[0047] Figure 25 Tumor marker levels of patient 01 are shown.

[0048] Figure 26 Displays the CT image of patient 02.

[0049] Figure 27 Shown are the number of peripheral blood CAR-T cells and plasma cytokine levels of patient 02.

[0050] Figure 28 Tumor marker levels of patient 02 are shown. DETAILED DESCRIPTION

[0051] 1. Design of GCC CAR Molecules

[0052] The scFv sequence of the GCC CAR molecule of the present invention is derived from a monoclonal antibody that specifically recognizes the GCC target (reference patents WO2011050242A1 and WO2019178580A1). The CDRs of the antibodies and antigen-binding fragments disclosed in the present invention 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, wherein the VH comprises an HC CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, an HC CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, and an HC CDR3 having an amino acid sequence as shown in SEQ ID NO: 3, and the VL comprises an LC CDR1 having an amino acid sequence as shown in SEQ ID NO: 4, an LC CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, and an LC CDR3 having an amino acid sequence as shown in SEQ ID NO: 6.

[0053] In a specific embodiment, in the scFv of the GCC CAR molecule of the present invention, VH is at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably 100% identical to the amino acid sequence of SEQ ID NO: 7, and VL is at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0054] In a more specific embodiment, the nucleotide sequence encoding the VH is shown in SEQ ID NO: 25, and the nucleotide sequence encoding the VL is shown in SEQ ID NO: 26. In a more specific embodiment, the scFv specifically recognizing GCC used in the present invention is at least 85% identical to the amino acid sequence of SEQ ID NO: 9, preferably at least 90%, more preferably at least 95%, and even more preferably 100% identical.

[0055] The hinge region used in the GCC CAR molecule of the present invention 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 further preferably 100% identical to the amino acid sequence shown 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 further preferably 100% identical to the amino acid sequence shown in SEQ ID NO: 11.

[0056] The transmembrane region used in the GCC CAR molecule of the present invention 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 further preferably 100% 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%, preferably at least 90%, more preferably at least 95%, and further preferably 100% identical to the amino acid sequence shown in SEQ ID NO: 14.

[0057] The costimulatory signaling domain used in the GCC CAR molecule of the present invention is a CD28 costimulatory signaling domain or a 4-1BB costimulatory signaling domain, wherein the amino acid sequence of the CD28 costimulatory signaling domain 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: 15, and the amino acid sequence of the 4-1BB costimulatory signaling domain 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: 16.

[0058] The amino acid sequence of the CD3ζ signaling domain used in the GCC CAR molecule of the present invention is at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably 100% identical to the amino acid sequence shown in SEQ ID NO: 17.

[0059] The chimeric antigen receptor of the present invention may or may not contain tags, cytokines, regulatory genes, or other genes as needed. Examples of tags include Strep tag II tags.

[0060] In more specific embodiments, the present invention provides the following six chimeric antigen receptors:

[0061] CAR-1 contains GCC scFv, CD8 hinge region, CD8 transmembrane region, CD28 costimulatory signaling domain, and CD3ζ intracellular signaling domain;

[0062] CAR-2 contains GCC scFv, CD28 hinge region, CD28 transmembrane region, CD28 costimulatory signaling domain, and CD3ζ intracellular signaling domain;

[0063] CAR-3 contains GCC scFv, CD8 hinge region, CD28 transmembrane region, CD28 costimulatory signaling domain, and CD3ζ intracellular signaling domain;

[0064] CAR-4 contains GCC scFv, IgG4 hinge region, CD28 transmembrane region, CD28 costimulatory signaling domain, and CD3ζ intracellular signaling domain;

[0065] CAR-5 contains GCC scFv, IgG4 hinge region, CD8 transmembrane region, CD28 costimulatory signaling domain, and CD3ζ intracellular signaling domain;

[0066] CAR-6 contains GCC scFv, CD8 hinge region, CD8 transmembrane region, 4-1BB co-stimulatory signaling domain, and CD3ζ intracellular signaling domain.

[0067] In a more specific embodiment, CAR-1 to CAR-6 of the present invention are as follows:

[0068] 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 of SEQ ID NO: 18;

[0069] 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 of SEQ ID NO: 19;

[0070] 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 of SEQ ID NO: 20;

[0071] 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 of SEQ ID NO: 21;

[0072] 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 of SEQ ID NO: 22;

[0073] 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 shown in SEQ ID NO: 23.

[0074] The present invention also relates to a nucleic acid molecule comprising a nucleic acid sequence encoding the chimeric antigen receptor of the present invention as described above.

[0075] The present invention also relates to a recombinant vector comprising the nucleic acid molecule described above, including but not limited to lentivirus, adenovirus, adeno-associated virus, retrovirus, transposon, and the like.

[0076] The recombinant vector of the present invention may optionally further comprise a promoter, such as a truncated EF1α promoter, a full-length EF1α promoter, a CMV promoter, an MND promoter, a truncated PGK promoter (PGK300 promoter), or a full-length PGK promoter (PGK400 promoter), preferably a truncated EF1α promoter, a full-length EF1α promoter, or a CMV promoter. The nucleotide sequences of the above promoters correspond to SEQ ID NOs: 27-32, respectively.

[0077] The present invention also relates to engineered cells comprising the chimeric antigen receptor, nucleic acid molecule, or recombinant vector of the present invention as described above. The cells are preferably immune cells, including, for example, T cells, B cells, γδT cells, NK cells, NKT cells, monocytes, macrophages, and the like.

[0078] The present invention further relates to the use of the chimeric antigen receptor, nucleic acid molecule, recombinant vector, and engineered cell described above in the preparation of a medicament for treating GCC-positive tumors. Specific examples of such tumors include, but are not limited to, digestive tract tumors, such as colorectal cancer, esophageal cancer, gastric cancer, or pancreatic cancer.

[0079] 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.

[0080] The amino acid sequences and nucleotide sequences described in this application are shown in Tables 1 and 2 below, respectively.

[0081] Table 1 Amino acid sequence

[0082]

[0083]

[0084]

[0085] Table 2 Nucleotide sequences

[0086]

[0087]

[0088]

[0089] Example

[0090] Example 1: Construction of GCC CAR molecules with different hinge and transmembrane regions and differences in in vitro proliferation 1.1 Construction of lentiviral transfer plasmids and lentivirus preparation

[0091] (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 enzyme 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.

[0092] (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.

[0093] 1.2 CAR-T cell preparation and proliferation detection

[0094] (1) Washing of single blood: Wash the single blood of healthy volunteers twice with physiological saline, then resuspend it in X-VIVO15 (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.

[0095] (2) T cell sorting and activation: cells adhered for 2 hours were collected, centrifuged at 400 g for 5 min, counted, and 2 × 10 5The cells were stained with CD3 APC (Biolegend) antibody to detect the proportion of T cells. According to the cell count results and T cell ratio, CD3 / CD28 Dynabeads (Thermo) were added at a 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. CD3-positive T cells were then sorted by 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 medium containing 500 IU / mL IL-2) and cultured at 1.5×10 6 The cells were expanded and cultured at a density of 10 cells / mL.

[0096] (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.

[0097] (4) After 4 days of culture, cells were collected, Dynabeads were removed using a magnetic stand, centrifuged at 400 g for 5 min, resuspended in complete medium, and counted. 5 The cells were cultured at a density of 10 cells / mL.

[0098] (5) Cell expansion: CAR-T cells were expanded and cultured in complete culture medium for 12 days, and passaged and counted every 2 days.

[0099] The results of resting T cell proliferation in vitro were as follows Figure 1 As shown in A, before Day 10, the T cell proliferation rates of the five groups were similar; by Day 12, the T cell proliferation rate of 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 coupled with PE fluorescent molecules (Beijing Yimi Shenzhou Pharmaceutical Technology Co., Ltd. expressed and purified GCC recombinant protein in CHO cells and then coupled with PE fluorescent molecules). The proliferation times of CAR-T cells in each group were calculated. The results are shown in Figure 1. Figure 1 As shown in Figure B, 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 increased significantly, while there were no significant differences in the other four groups.

[0100] Example 2: Detection of CAR expression rates in GCC CAR-T cells with different hinge and transmembrane regions

[0101] Different groups of CAR-T cells at different culture time points (Day 6, Day 8, Day 10, Day 12) in Example 1 were taken, and 2×10 5 T cells were incubated with APC-labeled CD3 antibody (Biolegend) and GCC protein coupled with PE fluorescent molecule (prepared by Beijing Yimiao Shenzhou Pharmaceutical Technology Co., Ltd.) at room temperature in the dark 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).

[0102] The results are as follows Figure 2 As shown in Figure A, the CAR positivity rate of CAR-T cells in each group increased over time, with rates slightly lower for CAR-T-3 and CAR-T-4, but still above 50%. As shown in Figure 2B, the mean fluorescence intensity of CAR in each group of CAR-T cells was highest before Day 12 for CAR-T-1, indicating that CAR molecules were present at the highest levels on the CAR-T-1 cell surface.

[0103] Example 3: Evaluation of the in vitro killing function of GCC CAR-T cells with different hinge and transmembrane regions

[0104] In order to screen out CAR-T with high sensitivity to tumor GCC antigen, this experiment used "HCT116-GCC low expression" cells as target cells. The cells were made by infecting HCT-116 wild-type tumor cells with lentivirus containing the human full-length GCC gene, and then detecting and sorting the cell line with low GCC expression intensity by flow cytometry. Specifically, HCT-116 wild-type tumor cells (not expressing human GCC) were purchased from the Cell Resource Center of the 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, and the DNA sequence containing the full-length human GCC was cloned into the lentiviral vector pLenti6.3 / V5. The lentivirus containing the full-length human GCC was prepared according to the method of step 1.1 (2) of Example 1. The lentivirus was used to infect HCT-116 wild-type cells. After 48 hours, the cells were inoculated with Alexa Fluor. The cells were incubated with 488-labeled GCC antibody (R&D Systems) at room temperature for 20 minutes. The GCC expression of the cells was detected by flow cytometry (BD Arial II), and the "HCT-116-GCC low expression" cells with low GCC expression intensity and the "HCT-116-GCC high expression" cells with high GCC expression intensity were sorted. The sorted cells were expanded and cultured to a sufficient number.

[0105] HCT-116 wild-type tumor cells, HCT116-GCC low-expressing cells and HCT116-GCC high-expressing cells were respectively After incubation with 488-labeled GCC antibody (R&D Systems) at room temperature for 20 min, GCC expression was detected by flow cytometry (NovoCyte 2060R, ACEA Biosciences, San Diego, CA, USA) to verify the GCC expression intensity of the two sorted cells. The results are shown in Figure 3 As shown, 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.

[0106] 3.1 Real-time killing experiment

[0107] (1) Tumor cell plating: 50 μL of IMDM complete medium containing 10% FBS was added to each well of a 96-well plate (E-plate 96) used with a real-time killing instrument (Agilent xCELLigence RTCA SP), placed on the RTCAStation, and baseline detection was performed; HCT-116-GCC tumor cells were digested with trypsin (Hyclone), 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 RTCAStation in the incubator to monitor and record the cell proliferation curve in real time.

[0108] (2) CAR-T cell addition: CAR-T cells were collected on Day 8, 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 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. The CAR positivity rate was 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 rate, the required number of CAR-T cells for each group was calculated using an E:T ratio of 1:3. T cells were supplemented to each group until the total number of T cells in each group was the same, and finally, X-VIVO 15 to 100 μL 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 consistent with the total number of T cells in each CAR-T group, and X-VIVO 15 to 100 μL was added.

[0109] (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 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 as follows Figure 4 As shown in Figures A and 4B, when E:T = 1:3, CAR-T-4 and CAR-T-5 have no killing effect on target cells, while CAR-T-1, CAR-T-2, and CAR-T-3 can all kill tumor cells well. Figure 4 As shown in Figure C, 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 were significant differences compared with CAR-T-4 and CAR-T-5.

[0110] 3.2 Repeated stimulation killing experiment

[0111] (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.

[0112] (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 6T 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, CA94538, 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.

[0113] (3) Every 2 to 3 days, observe the cells under a microscope. When all the tumor cells in the previous round are killed, collect the CAR-T cells in the wells and add them to the tumor cells that were plated one day in advance to repeat the stimulation experiment. When the CAR-T cells cannot completely kill the tumor cells, use trypsin (Hyclone) to digest, collect all the cells, and use APC-labeled CD3 antibody (Biolegend) and PE-labeled GCC protein (prepared by Beijing Yimiao Shenzhou Pharmaceutical Technology Co., Ltd.) to incubate at room temperature in the dark for 15 minutes for staining. Then, the number of residual tumor cells and the number of expanded CAR-T cells after multiple rounds of target cell stimulation were counted by flow cytometry (NovoCyte 2060R, ACEA Biosciences, San Diego, CA, USA).

[0114] Repeated killing results such as Figure 5 As shown in Figure 2, after 10 rounds of repeated stimulation of target cells, CAR-T-1, CAR-T-2, and CAR-T-3 had better killing ability and CAR-T expansion ability, which were significantly better than CAR-T-4 and CAR-T-5. Figure 6 As shown, CAR-T-1 maintains the strongest ability to continuously kill tumors, and the number of CAR-T cells expanded is the largest and the persistence is the best, which is significantly better than CAR-T-2 and CAR-T-3.

[0115] Example 4: Cytokine secretion levels of GCC CAR-T cells with different hinge and transmembrane regions upon target cell stimulation

[0116] 4.1 Tumor cell plating:

[0117] 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 to allow the tumor cells to adhere to the wall.

[0118] 4.2 CAR-T cell co-incubation:

[0119] 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 The CAR-T cell positivity rate was determined using a full-spectrum flow cytometer (Northern Lights, N7-00008-0A, Cytek Biosciences, Fremont, CA94538, USA). The 48-well plate was removed and the required volume of CAR-T cells for each group was calculated based on the measured CAR-T positivity rate, using an E:T ratio of 1:1. T cells were supplemented to each group until the total T cell volume in each group was consistent, and finally, 15 to 500 μL of X-VIVO was added. After preparation, each CAR-T cell group was added to each well. The untransfected T cell group was supplemented with an equal amount of total T cells as the CAR-T cells in each group, and 15 to 500 μL of X-VIVO was added. After 24 hours, the cells in the wells were gently pipetted (try not to agitate tumor cells), and the plate was centrifuged at 400 g for 5 minutes. 50 μL of supernatant was collected and the levels of TNF, IFN-γ, and IL-2 in each group were detected using Cytometric BeadArray (CBA) kits (BD Biosciences).

[0120] The results are as follows Figure 7 As shown in the figure, compared with CAR-T-4 and CAR-T-5, CAR-T-1, CAR-T-2, and CAR-T-3 groups secreted significantly more TNF-α, IFN-γ, and IL-2 cytokines, indicating that these three groups of CAR-T cells can effectively recognize GCC target antigens and activate CAR-T cells to perform killing functions, while CAR-T-4 and CAR-T-5 cannot effectively recognize GCC target antigens.

[0121] Example 5: Basal signaling levels of GCC CAR-T cells with different hinge and transmembrane regions during in vitro culture

[0122] Basal signaling is a measure of the ability of CAR-T cells to initiate downstream signaling, promoting cell proliferation and differentiation, in the absence of exogenous antigen stimulation, relying on their own CAR molecules. Moderate basal signaling is crucial for maintaining CAR-T effector function and survival. Day 8 CAR-T cells in each group were cultured in X-VIVO 15 (without exogenous IL-2 supplementation). Cytokine levels were measured 24 hours later, and cell counts were performed every 2-3 days to comprehensively analyze the basal signal strength of CAR-T cells.

[0123] The results are as follows Figure 8 As shown in the figure, the secretion levels of cytokines TNF-α, IFN-γ, and IL-2 in the CAR-T-1, CAR-T-2, and CAR-T-3 groups were high, among which CAR-T-1 had the highest secretion level and the highest basal signal intensity. Figure 9 As shown, all three groups, CAR-T-1, CAR-T-2, and CAR-T-3, showed significant cell proliferation, while CAR-T-4 and CAR-T-5 showed little cell proliferation and later cell death. This indicates that CAR-T-1, CAR-T-2, and CAR-T-3 have significant basal signals, with CAR-T-1 having the strongest basal signal, while CAR-T-4 and CAR-T-5 have no basal signal. This result suggests that when CAR-T is reinfused into the body, CAR-T-1 has a faster tumor antigen response and stronger survival ability, which is conducive to improving the treatment effect.

[0124] Example 6: CAR-T cell proliferation assay containing GCC CAR molecules with different co-stimulatory signaling domains

[0125] Based on the CAR-T-1 with the best killing function in vitro, the functions of CAR-T with two co-stimulatory signaling domains (CD28 and 4-1BB) were compared.

[0126] The amino acid sequence of CAR-6 is shown in Table 1. CAR-T-1 and CAR-T-6 cells containing CAR-6 were prepared according to the method in Example 1. On the 5th and 11th day of culture, the cells were collected, centrifuged and counted. 2×10 5 After incubation with APC-labeled CD3 antibody (Biolegend) and GCC protein coupled with PE fluorescent molecule (prepared by Beijing Yimiao Shenzhou Pharmaceutical Technology Co., Ltd.) at room temperature in the dark for 15 minutes, the CAR expression of each group of cells was detected using a full-spectrum flow cytometer (Northern Lights, N7-00008-0A, Cytek Biosciences, Fremont, CA94538, USA).

[0127] CAR-T cells were expanded and cultured in X-VIVO 15 medium containing 500 IU / mL for 11 days, with passages and counting every three days. CAR-T cells were harvested and centrifuged, then resuspended in 1-2 ml of X-VIVO 15 medium. Ten microliters of cell culture medium were diluted to a certain multiple and stained with trypan blue at a 1:1 volume ratio. Cell viability and viable cell concentration were recorded using a cell counter, and the total cell count in each group was calculated for statistical analysis of cell proliferation.

[0128] Cell proliferation results Figure 10 As shown in the figure, the T cell proliferation rate and CAR-T cell proliferation rate of the CAR-T-1 and CAR-T-6 groups were relatively close, with no significant difference. Figure 11 As shown in the figure, the CAR expression of CAR-T in each group was detected on the 5th and 11th days. The CAR positive rate of CAR-T-1 was slightly higher than that of CAR-T-6, but the average fluorescence intensity of CAR expression was slightly lower than that of CAR-T-6.

[0129] Example 7: In vitro evaluation of the killing function of GCC CAR-T cells with different costimulatory signaling domains 7.1 Real-time killing

[0130] (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 RTCASP), 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.

[0131] (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 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 for 15 minutes in the dark. CAR positivity was 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 or 1:T ratio of 1:6. To maintain consistent total T cell counts within each group, T cells were supplemented for each group, and finally, 15 to 100 μL of X-VIVO was added. After preparation, each group of CAR-T cells was added to each well. The number of cells in the untransfected T cell group was kept consistent with the total T cells in each group of CAR-T, and 15 to 100 μL of X-VIVO was added.

[0132] (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.

[0133] The killing results are as follows Figure 12 As shown in the figure, the tumor cell killing ability of CAR-T-1 is significantly better than that of CAR-T-6, indicating that the killing efficiency of CAR-T containing the CD28 costimulatory signaling domain is better than that of CAR-T containing the 4-1BB costimulatory signaling domain.

[0134] 7.2 Repeated Stimulation Killing

[0135] (1) Tumor cell plating: HCT116-GCC low-expressing tumor cells were digested with trypsin (Hyclone), counted, and then resuspended in 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 a 48-well plate and cultured overnight.

[0136] (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 were incubated with APC-labeled CD3 antibodies (Biolegend) and GCC protein conjugated with PE fluorescent molecules (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, CA94538, USA). The 48-well plate was removed and the required volume of CAR-T cells for each group was calculated based on the measured CAR positivity, using an E:T ratio of 1:3. To maintain consistent total T cell counts in each group, each group was supplemented with T cells, and finally, X-VIVO 15 was added to 500 μL. After preparation, each group of CAR-T cells was added to each well. The untransfected T cell group was supplemented with an equal number of total T cells as the CAR-T group, and X-VIVO 15 was added to 500 μL.

[0137] (3) Every 2 to 3 days, when all tumor cells in the previous round were killed, the CAR-T cells in the wells were collected and added to the tumor cells that were plated one day in advance for repeated stimulation experiments. When the CAR-T cells could not completely kill the tumor cells, they were digested with trypsin (Hyclone), all cells were collected, and APC-labeled CD3 antibody (Biolegend) and PE-labeled CAR antibody (prepared by Beijing Yimiao Shenzhou Pharmaceutical Technology Co., Ltd.) were incubated at room temperature in the dark for 15 minutes for staining. Afterwards, 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).

[0138] The results of repeated stimulation killing are as follows Figure 13 As shown in the figure, after 7 rounds of target cell stimulation, CAR-T-1 has a stronger ability to continuously kill tumor cells than CAR-T-6, and the number of CAR-T cell proliferation is greater, indicating that CAR-T containing the CD28 co-stimulatory signaling domain has better long-term killing ability and persistence than CAR-T containing the 4-1BB co-stimulatory signaling domain, that is, it has a better ability to resist tumor recurrence.

[0139] Example 8: Construction of GCC CAR molecules with different promoters and preparation of CAR-T cells

[0140] Based on the CAR-T-1 molecule, the promoter of the lentiviral vector is designed and screened.

[0141] 8.1 Construction of Lentiviral Transfer Plasmid and Lentivirus Preparation

[0142] (1) The chimeric genes of GCC CAR of full-length EF1α-CAR-T-1, truncated EF1α-CAR-T-1, CMV-CAR-T-1, MND-CAR-T-1, PGK300-CAR-T-1 and PGK400-CAR-T-1 with restriction enzyme sites XbaI and SalI at both ends were synthesized by gene synthesis (Beijing Bomade Gene Technology Co., Ltd.). The specific sequence of the promoter is shown in Table 2. The lentiviral transfer plasmid was constructed and the lentivirus was prepared according to the same method as in Example 1.

[0143] 8.2 CAR-T Cell Preparation and Proliferation Assay

[0144] CAR-T cells were prepared in the same manner as in Example 1. CAR-T cells were expanded and cultured in complete medium for 12 days, and passaged and counted every 2 days. Figure 14 As shown in Figure 2, as the culture time increases, the proliferation rate of full-length EF1α-CAR-T-1 is the fastest, and that of PGK300-CAR-T-1 is the slowest. From the 6th to the 12th day of culture, the positive rate of CAR in each group of CAR-T was detected, and the results are shown in Figure 2. Figure 15 As shown, truncated EF1α-CAR-T-1 and MND-CAR-T-1 had the highest CAR positive rates.

[0145] Example 9: In vitro evaluation of the killing function of GCC CAR-T cells with different promoters 9.1 Real-time killing

[0146] (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 to monitor the cell proliferation curve in real time.

[0147] (2) CAR-T cell addition: CAR-T cells were collected on Day 8, 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 1×106 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.

[0148] (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.

[0149] Real-time killing results such as Figure 16 As shown in the figure, 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.

[0150] 9.2 Repeated Stimulation Killing

[0151] (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.

[0152] (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 were incubated with an APC-labeled CD3 antibody (Biolegend) and GCC protein conjugated with a PE fluorescent molecule (manufactured by Beijing Yimiao Shenzhou Pharmaceutical Technology Co., Ltd.) at room temperature in the dark for 15 minutes. The CAR positivity rate was then determined using a full-spectrum flow cytometer (Northern Lights, N7-00008-0A, Cytek Biosciences, Fremont, CA94538, USA). The 48-well plate was removed and the required volume 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 T cell count remained the same, and finally, 15 to 500 μL of X-VIVO was added. After preparation, each CAR-T cell group was added to each well. The untransfected T cell group was supplemented with an equal number of total T cells as the CAR-T cells in each group, and 15 to 500 μL of X-VIVO was added.

[0153] (3) Every 2 to 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 that were plated one day in advance for repeated stimulation experiments. When the CAR-T cells could not completely kill the tumor cells, they were digested with trypsin (Hyclone), all the cells were collected, and APC-labeled CD3 antibody (Biolegend) and PE-labeled GCC protein (prepared by Beijing Yimiao Shenzhou Pharmaceutical Technology Co., Ltd.) were incubated at room temperature in the dark for 15 minutes for staining. Afterwards, the number of residual tumor cells and the number of expanded CAR-T cells after multiple rounds of target cell stimulation were counted by flow cytometry (NovoCyte 2060R, ACEABiosciences, San Diego, CA, USA).

[0154] The results after 6 rounds of repeated stimulation of target cells are as follows Figure 17 As shown in the figure, truncated EF1α-CAR-T-1, full-length EF1α-CAR-T-1, CMV-CAR-T-1 and MND-CAR-T-1 had better killing ability and a larger number of CAR-T amplification. There was no significant difference between the groups, but they were all significantly better than PGK300-CAR-T-1 and PGK400-CAR-T-1. After 10 rounds of repeated stimulation of target cells, the results were as follows Figure 18As shown, truncated EF1α-CAR-T-1, full-length EF1α-CAR-T-1, and CMV-CAR-T-1 have strong continuous tumor killing capabilities, while MND-CAR-T-1 has weakened tumor cell killing and a small number of CAR-T cells expanded. Overall, truncated EF1α-CAR-T-1, full-length EF1α-CAR-T-1, and CMV-CAR-T-1 have strong continuous tumor cell killing capabilities, a large number of CAR-T cells expanded, and good persistence, further demonstrating that the truncated EF1α, full-length EF1α, and CMV promoters can all effectively regulate CAR-T-1 gene expression and anti-tumor function.

[0155] Example 10: Cytokine secretion levels of GCC CAR-T cells with different promoters

[0156] (1) Tumor cell plating: HCT116-GCC low-expressing tumor cells were digested with trypsin, 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 to allow the tumor cells to adhere to the wall.

[0157] (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 The CAR-T cell count was measured at 48-well plates (N7-00008-0A, Cytek Biosciences, Fremont, CA94538, USA), and the CAR-T cell positivity rate was determined using a full-spectrum flow cytometer (Northern Lights, N7-00008-0A, Cytek Biosciences, Fremont, CA94538, USA). The 48-well plates were removed and the required volume of CAR-T cells for each group was calculated based on the measured CAR-T positivity rate, using an E:T ratio of 1:1. To enable parallel comparison of each group, T cells were added until the total T cell count in each experimental group remained consistent, and finally, X-VIVO 15 was added to 500 μL. After preparation, each CAR-T cell group was added to each well. The untransfected T cell group was supplemented with an equal number of total T cells as the CAR-T cells in each group, and X-VIVO 15 was added to 500 μL.

[0158] (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.

[0159] Test results such as Figure 19As shown in the figure, 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.

[0160] Example 11: Basal signaling levels of GCC CAR-T cells with different promoters during in vitro culture

[0161] Each group of CAR-T cells was cultured in X-VIVO 15 (without exogenous IL-2), and cytokines and proliferation rates were detected after 24 hours. Cell counts and statistical analysis were performed every 2-3 days to reflect the basal signal strength of CAR-T cells. Figure 20 As shown in Figure 3, the secretion levels of cytokines TNF-α, IFN-γ, and IL-2 in the truncated EF1α-CAR-T-1, full-length EF1α-CAR-T-1, and CMV-CAR-T-1 groups were higher, with CMV-CAR-T-1 having the highest secretion level. Figure 21 As shown, the truncated EF1α-CAR-T-1, full-length EF1α-CAR-T-1, and CMV-CAR-T-1 groups showed significant cell proliferation, while the MND-CAR-T-1 group had a lower level of proliferation. The PGK300-CAR-T-1 and PGK400-CAR-T-1 groups showed no cell proliferation and later cell death. This indicates that there is a clear basal signal in the truncated EF1α-CAR-T-1, full-length EF1α-CAR-T-1, and CMV-CAR-T-1 groups, among which CMV-CAR-T-1 has the strongest signal.

[0162] Example 12: Tumor Suppression and In Vivo Expansion Ability of CAR-T-1 Cells in a Subcutaneous Colorectal Cancer PDX Mouse Model

[0163] 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.

[0164] CAR-T-1 cells were prepared according to the above CAR-T cell preparation method. When the cells reached a sufficient number, they were resuspended in freezing solution and stored in liquid nitrogen for future use. Tumor tissues from patients with colorectal cancer who showed high expression of GCC by immunohistochemistry were obtained and cut into 2-3 mm 3Fragments were inoculated into 6-8 week old NSG mice. A small incision was made in the lower back of each mouse, and then the tumor tissue samples were transplanted subcutaneously into the surgical area. 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 established. When the tumor grew to 100 mm 3 Each group of mice was injected with CAR-T-1 cells (5×10 6 Two groups of mice were injected with the same volume of Dulbecco's phosphate buffered saline (DPBS) and the same number of T cells as control T cells not transfected with the lentivirus. Tumor size was measured one day before CAR-T cell injection and on days 3, 7, 12, 15, 18, 22, and 26 after injection. The number of CAR-T cells in peripheral blood was measured on days 2, 6, 14, 21, and 28 after CAR-T cell injection.

[0165] Specific results such as Figure 22 As shown in A, compared with the tumor burden of mice in the DPBS group and the control T cell group, CAR-T-1 cells slowed down the tumor growth rate 3 days after reinfusion and effectively reduced the tumor burden within 26 days of detection, showing significant anti-tumor activity. Figure 22 B shows the number of CAR-T-1 cells in the peripheral blood of mice at different times. A higher level of CAR-T cells can be detected 10 days after transfusion, and then decreases and maintains at a certain level.

[0166] Example 13: CAR-T cell expansion and anti-tumor activity in patients

[0167] The clinical study was designed to evaluate the safety and efficacy of infusing autologous truncated EF1-α-CAR-T-1 cells into patients with GCC tumors.

[0168] Human peripheral blood mononuclear cells (PBMCs) were obtained from patients. Based on the cell count results and T cell ratio, CD3 / CD28 Dynabeads (Thermo) were added at a ratio of magnetic beads: T cells = 1.5:1 (quantity ratio), and the cells were placed on a sample sorter and gently shaken for 30 minutes. CD3-positive T cells were then sorted using a magnetic stand (Invitrogen) to adsorb the magnetic beads. To fully activate the T cells, they were resuspended in complete culture medium (X-VIVO 15 + 500 IU / mL IL-2) and incubated at 1.5 × 10 6The cells were expanded and cultured at a density of cells / mL. After 24 hours of magnetic bead activation, the cells were collected and counted. According to the counting results, the lentivirus was used to infect T cells at an MOI of 2. 24 hours after the virus infected the cells, they were centrifuged at 400g for 5 minutes, and fresh complete culture medium was replaced and cultured. On the 8th day of culture, the cells were collected, the Dynabeads were removed using a magnetic stand, centrifuged at 400g for 5 minutes, the CAR-T cells were washed, and cryopreserved using a reusable freezing solution. The cell suspension was placed in a freezing bag, and the bag was then gradient-cooled to -90°C and transferred to a gas phase liquid nitrogen tank for storage. Before the patient's reinfusion, the frozen CAR-T was transported to the hospital, and the patient's reinfusion was completed within 30 minutes after recovery. The reinfusion dose is 1.2×10 9 CAR-T cells.

[0169] Patients received fludarabine and cyclophosphamide-based conditioning therapy before CAR-T infusion. After CAR-T infusion, patients were closely observed for at least 2 hours. Peripheral blood was collected regularly and CAR-T amplification was monitored by flow cytometry using BD Biosciences. TM Plasma cytokine levels were monitored using the Human Th1 / Th2 / Th17 Cytometric Bead Array (CBA) Kit. The protocol was approved by the hospital where the trial was conducted. All patients provided written informed consent.

[0170] Patient 01 was diagnosed with advanced metastatic colorectal cancer with metastases to the abdominal aorta, right iliac lymph nodes, pelvic cavity, peritoneum, anterior uterine wall, and multiple nodular metastases in both lungs. 28 days after infusion of truncated EF1-α-CAR-T-1 cells, the CT scan images were as follows: Figure 23 As shown in Table 3, lung lesions 1 and 2 and peritoneal metastases were significantly reduced. The changes in tumor diameter are shown in Table 3. CAR-T cells were significantly expanded in vivo, reaching a maximum of 3.2×10 7 CAR-T / L, IL-6 in cytokines increased significantly, reaching a maximum of 2666.7pg / mL. The results are shown in Figure 24 Carcinoembryonic antigen and cancer antigen decreased significantly during treatment, as shown in Figure 25 This patient was rated as PR (partial response) at month 1 and CR (complete response) at month 7. During treatment, no severe CRS (e.g., no more than grade 2 CRS) was observed in patient 01.

[0171] Table 3 Changes in tumor size of patient 01

[0172]

[0173] Patient 02 was diagnosed with advanced metastatic colorectal cancer with mesenteric lymph node metastasis, retroperitoneal lymph node metastasis, lung metastasis, and liver metastasis. CT scan images were obtained 3 months after infusion of truncated EF1-α-CAR-T-1 cells. Figure 26 As shown in Table 4, lung lesions and liver metastases were significantly reduced, and the total tumor diameter decreased 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 increased, reaching a maximum of 3500pg / mL. The results are shown in Figure 27 Carcinoembryonic antigen and cancer antigen decreased significantly during treatment, as 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.

[0174] Table 4 Changes in tumor size of patient 02

[0175]

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 of the following: (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, which is at least 85% identical to the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO:

23.

6. A nucleic acid molecule comprising a nucleic acid sequence encoding the chimeric antigen receptor according to any one of claims 1 to 5.

7. A recombinant vector comprising the nucleic acid molecule according to claim 6, 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:

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

28. The nucleotide sequence of the CMV promoter is shown in SEQ ID NO: 29; More preferably, the recombinant vector is a recombinant lentiviral vector.

8. An engineered cell comprising the chimeric antigen receptor according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, or the recombinant vector according to claim 7.

9. Use of the chimeric antigen receptor according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the recombinant vector according to claim 7, or the engineered cell according to claim 8 in the preparation of a medicament for treating GCC-positive tumors.

10. The use according to claim 9, wherein the drug is used to treat digestive tract tumors, such as colorectal cancer, esophageal cancer, gastric cancer or pancreatic cancer.

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