WO2025214269A1 - Chimeric antigen receptor targeting gcc and use thereof - Google Patents

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

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

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

Recent data show that colorectal cancer (CRC) has a high incidence and mortality rate in both men and women worldwide, ranking third in both categories. 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].

Depending on the stage of diagnosis, the 5-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 no clinically 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 further decreases as the degree of CRC progression increases, and the proportion of clinical stage IV patients can drop to 4%. The majority of patients are microsatellite stable (MSS) with no mutations in the MMR gene, 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.

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 within the intestinal epithelium has the potential to reduce the toxic side effects of targeted drugs on normal tissues.

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.

At the 2023 ESMO (European Society for Medical Oncology) Annual Meeting, Shanghai Stansay Biotechnology Co., Ltd. reported safety and efficacy data from a GUCY2C-CD19 CAR-T product in 21 patients across two dose-escalation groups at five clinical centers in China for patients with advanced colorectal cancer. Thirteen patients were enrolled in the 1st dose group (1×10 ⁶ cells/kg) and eight patients were enrolled in the 2nd dose group (2×10 ⁶ cells/kg). Clinical results showed an objective response rate (ORR) of 15.4% (2/13) in the 1st dose group and 50% (4/8) in the 2nd dose group. The most common adverse events in the trial patients were cytokine release syndrome (CRS) and diarrhea. Two patients experienced neurotoxicity, which resolved after treatment with corticosteroids. Therefore, the development of CAR-T targeting GUCY2C has the potential to provide long-term relief for patients with advanced colorectal cancer.

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.

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

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.

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.

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.Huebn er,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.Eshle man,B.Vogelstein,L.A.Diaz,Jr.,PD-1Blockade in Tumors with Mismatch-Repair Deficiency,N Engl J Med 372(26)(2015)2509-20.

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-CD3T-Cell Engaging Bispe cific Construct(PF-07062119) for the Treatment of Gastrointestinal Cancers, Clin Cancer Res 26(9)(2020)2188-2202.

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 36Cancers in 185Countries,CA Cancer J Clin 71(3)(2021)209-249.

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

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.1989Dec; 86(24):10024-8.

Document 9: Khalil DN, Smith EL, Brentjens RJ, Wolchok JD. The future of cancer treatment: immunomodulation, CARs and combination immunotherapy. Nat Rev Clin Oncol2016; 13(6):394.

Summary of the Invention

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.

On the one hand, the present invention relates to a chimeric antigen receptor that specifically targets GCC, which includes: 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.

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.

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.

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.

The entire contents of the documents listed in this application are incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

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).

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

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

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

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.

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.

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

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

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.

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

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.

Figure 12 shows the killing curves of CAR-T with different signal domains against HCT116-GCC low-expressing cells (E:T=1:3).

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-expressing cells.

Figure 14 shows the in vitro proliferation folds of resting T cells (Figure 14A) and CAR-T cells (Figure 14B) with different promoters.

FIG15 shows the CAR positive rates of different promoters.

Figure 16 shows the killing curves of CAR-T combined with different promoters against HCT116-GCC low-expressing cells.

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-expressing cells.

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-expressing cells.

Figure 19 shows the cytokine secretion levels of CAR-T with different promoters after stimulation of HCT116-GCC low-expressing cells for 24 hours.

Figure 20 shows the cytokine secretion levels of CAR-T cells with different promoters after culture in IL-2-free medium for 24 hours.

Figure 21 shows the cell proliferation (Figure 21A) and cell viability (Figure 21B) of CAR-T cells with different promoters in IL-2-free culture medium.

Figure 22 shows the tumor inhibition ability of truncated EF1α-CAR-T-1 in NSG tumor-bearing mice (Figure 22A) and the CAR-T expansion ability in peripheral blood (Figure 22B).

FIG23 shows the CT images of patient 01.

FIG24 shows the number of peripheral blood CAR-T cells and plasma cytokine levels of patient 01.

FIG25 shows tumor marker levels of patient 01.

FIG26 shows a CT image of Patient 02.

FIG27 shows the number of peripheral blood CAR-T cells and plasma cytokine levels of patient 02.

FIG28 shows tumor marker levels of patient 02.

FIG29 shows the CAR positivity rate and CAR mean fluorescence intensity of CAR-T-1 and CAR-T-7.

Figure 30 shows the real-time killing effects of CAR-T-1 and CAR-T-7.

Figure 31 shows the repeated stimulation and killing effects of CAR-T-1 and CAR-T-7.

DETAILED DESCRIPTION

1. Design of GCC CAR Molecules

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.

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 further preferably 100% identical to the amino acid sequence shown in SEQ ID NO: 7, and 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.

In a more specific embodiment, the nucleotide sequence encoding the VH is shown in SEQ ID NO: 27, and the nucleotide sequence encoding the VL is shown in SEQ ID NO: 28. 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.

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.

The transmembrane region used in the GCC CAR molecule of the present invention is the CD8 transmembrane region or the 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.

The costimulatory signal domain used in the GCC CAR molecule of the present invention is a CD28 costimulatory signal domain or a 4-1BB costimulatory signal domain, wherein the amino acid sequence of the CD28 costimulatory signal 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 signal 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.

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 further preferably 100% identical to the amino acid sequence shown in SEQ ID NO: 17.

The chimeric antigen receptor of the present invention may or may not contain other genes such as tags, cytokines, or regulatory genes as needed. Tags include, for example, Strep tag II tags.

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

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

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

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

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

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

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

In other embodiments, the six chimeric antigen receptors described above further comprise a protective peptide.

In a more specific embodiment, the protective peptide is the amino acid sequence shown in SEQ ID NO: 24.

In a more specific embodiment, the present invention further provides the following seventh chimeric antigen receptor:

CAR-7 contains the protective peptide shown in SEQ ID NO: 24, GCC scFv, CD8 hinge region, CD8 transmembrane region, CD28 co-stimulatory signal domain, and CD3ζ intracellular signal domain.

In a more specific embodiment, CAR-1 to CAR-7 of the present invention are as follows:

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;

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;

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;

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;

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;

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.

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 shown in SEQ ID NO: 25.

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.

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.

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: 29 to 34, respectively.

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.

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.

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.

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

Table 1 Amino acid sequence

Table 2 Nucleotide sequences

Example

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

(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 the pLenti6.3/V5 plasmid carrying the CAR-1, CAR-2, CAR-3, CAR-4, or CAR-5 gene.

(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.

1.2 CAR-T cell preparation and proliferation detection

(1) Single-sample blood washing: Single-sample blood samples from healthy volunteers were washed twice with physiological saline, then resuspended in X-VIVO 15 (Lonza) medium and counted. Based on the count results, cells were allowed to adhere to the wall at a density of 1 × 10 ⁸ cells/30 mL for 2 hours.

(2) Sorting and activation of T cells: Collect cells that have been attached for 2 hours, centrifuge at 400g for 5 minutes, count, and take 2×10 ⁵ cells to stain 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 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. Then, CD3-positive T cells were 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 culture medium containing 500IU/mL IL-2) and expanded and cultured at a density of 1.5×10 ⁶ cells/mL.

(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.

(4) After 4 days of culture, the cells were collected, Dynabeads were removed using a magnetic rack, centrifuged at 400 g for 5 min, resuspended in complete medium, counted, and cultured at a density of 3 × 10 ⁵ cells/mL.

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

The results of resting T cell proliferation in vitro are shown in Figure 1A. Before Day 10, the T cell proliferation rates of the five groups were similar. By Day 12, the T cell proliferation rate in the CAR-T-5 group was significantly faster, while the proliferation rate in the CAR-T-2 group was slightly lower. CAR positivity was detected using GCC protein conjugated to a PE fluorescent molecule (prepared by Beijing Yimi Shenzhou Pharmaceutical Technology Co., Ltd. after expressing and purifying GCC recombinant protein in CHO cells and then conjugating it to a PE fluorescent molecule). The proliferation fold of CAR-T cells in each group was calculated, as shown in Figure 1B. Before Day 10, the CAR-T cell proliferation rates of the five groups were similar, but by Day 12, the proliferation rate of CAR-T-5 was significantly increased, while there were no significant differences in the other four groups.

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

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 taken from each group. 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, CA 94538, USA).

The results, as shown in Figure 2A, show an increasing trend in the CAR-T cell positivity rate across all groups 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 was highest before Day 12 for CAR-T-1, indicating that CAR-T-1 cells had the highest levels of CAR molecules on their surface.

Example 3: In vitro evaluation of the cytotoxicity of GCC CAR-T cells with different hinge and transmembrane regions

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.

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 minutes, 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. HCT-116 wild-type tumor cells had no GCC-positive signal, "HCT-116-GCC low expression" cells had moderate GCC-positive signals, and "HCT-116-GCC high expression" cells showed the strongest GCC-positive signal.

3.1 Real-time killing experiment

(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 in conjunction with a real-time killing instrument (Agilent xCELLigence RTCA SP), and the plate 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 2×10 ⁵ cells/mL with IMDM complete medium containing 10% FBS. The E-plate 96 was removed and 100 μL (2×10 ⁴ cells/well) of tumor cell suspension was added to each well, and the plate was placed at room temperature for 30 min. The E-plate 96 was placed on the RTCA Station in the incubator to monitor and record the cell proliferation curve in real time.

(2) Addition of CAR-T cells: CAR-T cells on Day 8 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. 1×10 ⁶ T cells were taken and 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. The CAR positive rate was detected using a full-spectrum flow cytometer (Northern Lights, N7-00008-0A, Cytek Biosciences, Fremont, CA 94538, USA); the E-plate 96-well plate was removed and 50 μL of supernatant was aspirated from each well. According to the measured CAR positive rate, the required cell amount of CAR-T cells for each group was calculated according to E:T=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 was added to 100 μL. 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.

(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 were significant differences compared with CAR-T-4 and CAR-T-5.

3.2 Repeated stimulation killing experiment

(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 using IMDM complete medium containing 10% FBS. 500 μL of tumor cell suspension was added to each well of a 48-well plate and cultured overnight.

(2) Addition of CAR-T cells: 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 and incubate 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. Use full spectrum flow cytometer (Northern Lights, N7-00008-0A, Cytek Biosciences, Fremont, CA 94538, USA) to detect CAR positive rate. Take out the 48-well plate, calculate the required amount of CAR-T cells for each group according to the measured CAR positive rate, and calculate the required amount of CAR-T cells for each group according to E:T=1:1. Supplement T cells to each group until the total number of T cells in each group is consistent. Finally, add X-VIVO 15 to 500μL. After preparation, each group of CAR-T cells was added to each well. The untransfected T cell group was added with an equal amount of total T cells as that of each group of CAR-T, and X-VIVO 15 to 500 μL was added.

(3) Every 2 to 3 days, the cells were observed under a microscope. 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 incubated with APC-labeled CD3 antibody (Biolegend) and PE-labeled GCC protein (prepared by Beijing Yimiao Shenzhou Pharmaceutical Technology Co., Ltd.) 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, ACEA Biosciences, San Diego, CA, USA).

The results of repeated killing are shown in Figure 5. After 10 rounds of repeated target cell stimulation, CAR-T-1, CAR-T-2, and CAR-T-3 showed excellent killing and CAR-T expansion capabilities, all significantly superior to CAR-T-4 and CAR-T-5. After 12 rounds of repeated target cell stimulation, as shown in Figure 6, CAR-T-1 maintained the strongest ability to continuously kill tumors, with the largest number of expanded CAR-T cells and the best persistence, significantly superior to CAR-T-2 and CAR-T-3.

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

4.1 Tumor cell plating:

HCT116-GCC low-expressing tumor cells were digested with trypsin (Hyclone) and counted. The cell density was then adjusted to 1×10 ⁵ cells/mL using IMDM complete medium containing 10% FBS. 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.

4.2 CAR-T cell co-incubation:

Cells were harvested, centrifuged at 400 g for 5 min, and resuspended in X-VIVO 15 medium. The cell density was adjusted to 1 × 10 ⁶ cells/mL. CAR positivity was 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 volume of CAR-T cells for each group was calculated based on the measured CAR positivity, using an E:T ratio of 1:1. T cells were supplemented to maintain the same total T cell count for each group, and finally, X-VIVO 15 was added to 500 μL. After preparation, CAR-T cells from each group were added to each well. An equal number of total T cells was added to the untransfected T cell group, and X-VIVO 15 was added to 500 μL. After 24 hours, the cells were gently pipetted in the wells (try not to agitate tumor cells), and the plate was centrifuged at 400 g for 5 min. 50 μL of supernatant was collected and the levels of TNF, IFN-γ, and IL-2 in each group were detected using Cytometric Bead Array (CBA) kits (BD Biosciences).

The results are shown in Figure 7. Compared with CAR-T-4 and CAR-T-5, the 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.

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

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). 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.

The results, as shown in Figure 8, showed high secretion levels of TNF-α, IFN-γ, and IL-2 in the CAR-T-1, CAR-T-2, and CAR-T-3 groups, with CAR-T-1 producing the highest secretion and possessing the highest basal signal intensity. The results of resting cell proliferation, shown in Figure 9, showed significant cell proliferation in all three groups, while CAR-T-4 and CAR-T-5 showed little proliferation and later cell death. This indicates that CAR-T-1, CAR-T-2, and CAR-T-3 exhibit significant basal signals, with CAR-T-1 exhibiting the strongest basal signal, while CAR-T-4 and CAR-T-5 exhibit no basal signal. This suggests that when CAR-T cells are infused back into the body, CAR-T-1 exhibits a more rapid tumor antigen response and enhanced survival, potentially improving therapeutic efficacy.

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

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.

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 days of culture, the cells were collected, centrifuged and counted. 2×10 ⁵ cells were taken and incubated with APC-labeled CD3 antibody (Biolegend) and GCC protein conjugated with PE fluorescent molecule (prepared by Beijing Yimiao Shenzhou Pharmaceutical Technology Co., Ltd.) at room temperature in the dark for 15 minutes. The expression of CAR in each group of cells was detected using a full-spectrum flow cytometer (Northern Lights, N7-00008-0A, Cytek Biosciences, Fremont, CA 94538, USA).

CAR-T cells were expanded and cultured in X-VIVO 15 medium containing 500 IU/mL for 11 days, with passages counted 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.

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.

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

7.1 Real-time Killing

(1) Tumor cell plating: Add 50 μL of IMDM complete medium containing 10% FBS to the wells of a 96-well plate (E-plate 96) used with a real-time killing instrument (Agilent xCELLigence RTCA SP), place it on the RTCA Station, and detect the baseline; digest the HCT116-GCC low-expressing tumor cells with trypsin, count them, and then adjust the cell density to 2×10 ⁵ cells/mL with IMDM complete medium containing 10% FBS; remove the E-plate 96, add 100 μL (2×10 ⁴ cells/well) of tumor cell suspension to the wells, and place them at room temperature for 30 minutes; place the E-plate 96 on the RTCA Station in the incubator, and detect the cell proliferation curve after 24 hours.

(2) Addition of CAR-T cells: CAR-T cells on Day 11 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. 1×10 ⁶ T cells were taken and incubated with APC-labeled CD3 antibody (Biolegend) and GCC protein conjugated with PE fluorescent molecule (prepared by Beijing Yimiao Shenzhou Pharmaceutical Technology Co., Ltd.) at room temperature in the dark for 15 minutes. The CAR positive rate was detected using a full-spectrum flow cytometer (Northern Lights, N7-00008-0A, Cytek Biosciences, Fremont, CA 94538, USA). The E-plate 96-well plate was removed and 50 μL of supernatant was aspirated from each well. According to the measured CAR positive rate, the required volume of CAR-T cells for each group was calculated according to E:T = 1:3 and E:T = 1:6. To maintain a consistent total T cell count in each group, each group needs to be supplemented with T cells, and finally, 15 to 100 μL of X-VIVO is added. After preparation, each group of CAR-T cells is added to each well. The number of cells in the untransfected T cell group is consistent with the total T cell count in each CAR-T group, and 15 to 100 μL of X-VIVO is added.

(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.

The killing results are shown in Figure 12. 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 signal domain is better than that of CAR-T containing the 4-1BB costimulatory signal domain.

7.2 Repeated Stimulation Killing

(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. 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.

(2) CAR-T cell co-incubation: 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 and incubate 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. Then, use a full-spectrum flow cytometer (Northern Lights, N7-00008-0A, Cytek Biosciences, Fremont, CA 94538, USA) to detect the CAR positive rate. Take out the 48-well plate and calculate the required volume of CAR-T cells for each group according to the measured CAR positive rate at E:T=1:3. In order to keep the total number of T cells in each group consistent, it is necessary to supplement T cells to each group and finally add X-VIVO 15 to 500μL. After preparation, each group of CAR-T cells was added to each well. The untransfected T cell group was added with an equal amount of total T cells as the CAR-T group, and X-VIVO 15 to 500 μL was added.

(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 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).

The results of repeated stimulation and killing are shown in Figure 13. 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 signal domain has better long-term killing ability and persistence than CAR-T containing the 4-1BB co-stimulatory signal domain, that is, it has a better ability to resist tumor recurrence.

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

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

8.1 Construction of Lentiviral Transfer Plasmid and Lentivirus Preparation

(1) 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. Lentivirus transfer plasmids were constructed and lentivirus was prepared according to the same method as in Example 1.

8.2 CAR-T Cell Preparation and Proliferation Detection

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. The in vitro proliferation of T cells and CAR-T cells is shown in Figure 14. 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 day 6 to day 12 of culture, the CAR positive rate of each group of CAR-T was detected. The results are shown in Figure 15. The CAR positive rate of truncated EF1α-CAR-T-1 and MND-CAR-T-1 is the highest.

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

9.1 Real-time Killing

(1) Tumor cell plating: Add 50 μL of IMDM complete medium containing 10% FBS to the wells of a 96-well plate (E-plate 96) used with a real-time killing instrument (Agilent xCELLigence RTCA SP), place it on the RTCA Station, and detect the baseline; digest the HCT116-GCC low-expressing tumor cells with trypsin, count them, and then adjust the cell density to 2× ¹⁰⁵ cells/mL with IMDM complete medium containing 10% FBS; remove the E-plate 96, add 100 μL (2× ¹⁰⁴ cells/well) of tumor cell suspension to each well, and place it at room temperature for 30 minutes; place the E-plate 96 on the RTCA Station in the incubator to monitor the cell proliferation curve in real time.

(2) Addition of CAR-T cells: CAR-T cells on Day 8 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. 1×10 ⁶ T cells were taken and 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. The CAR positive rate was detected using a full-spectrum flow cytometer (Northern Lights, N7-00008-0A, Cytek Biosciences, Fremont, CA 94538, USA). The E-plate 96-well plate was removed and 50 μL of supernatant was aspirated from each well. According to the measured CAR positive rate, the required volume of CAR-T cells for each group was calculated according to E:T=1:3. T cells were supplemented to each group until the total number of T cells in each group was consistent, and finally X-VIVO 15 was added to 100 μL. 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 CAR-T group, and X-VIVO 15 to 100 μL was added.

(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.

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.

9.2 Repeated Stimulation Killing

(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.

(2) CAR-T cell co-incubation: 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 and incubate 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. Use full spectrum flow cytometer (Northern Lights, N7-00008-0A, Cytek Biosciences, Fremont, CA 94538, USA) to detect CAR positivity. Take out the 48-well plate and calculate the required volume of CAR-T cells for each group according to the measured CAR positivity rate at E:T=1:1. Supplement T cells to each group until the total number of T cells in each group is consistent, and finally add X-VIVO 15 to 500μL. After preparation, each group of CAR-T cells was added to each well. The untransfected T cell group was added with an equal amount of total T cells as that of each group of CAR-T, and X-VIVO 15 to 500 μL was added.

(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 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 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, ACEA Biosciences, San Diego, CA, USA).

The results after six rounds of repeated target cell stimulation are shown in Figure 17. Truncated EF1α-CAR-T-1, full-length EF1α-CAR-T-1, CMV-CAR-T-1, and MND-CAR-T-1 demonstrated superior cytotoxicity and a high number of expanded CAR-T cells. There were no significant differences between the groups, but all were significantly superior to PGK300-CAR-T-1 and PGK400-CAR-T-1. After a further 10 rounds of repeated target cell stimulation, the results are shown in Figure 18. Truncated EF1α-CAR-T-1, full-length EF1α-CAR-T-1, and CMV-CAR-T-1 demonstrated superior continuous tumor killing abilities, while MND-CAR-T-1 demonstrated reduced tumor cell killing and a low number of expanded CAR-T cells. Overall, the truncated EF1α-CAR-T-1, full-length EF1α-CAR-T-1 and CMV-CAR-T-1 have stronger abilities to continuously kill tumor cells, with larger numbers of CAR-T amplified and better persistence, further proving that the three promoters, namely truncated EF1α, full-length EF1α and CMV, can effectively regulate CAR-T-1 gene expression and anti-tumor function.

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

(1) Tumor cell plating: HCT116-GCC low-expressing tumor cells were digested with trypsin and counted. The cell density was then 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.

(2) CAR-T cell co-incubation: 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. At the same time, use a full-spectrum flow cytometer (Northern Lights, N7-00008-0A, Cytek Biosciences, Fremont, CA 94538, USA) to detect the CAR positive rate; take out the 48-well plate, and calculate the required volume of CAR-T cells for each group according to the measured CAR positive rate at E:T=1:1. In order to make each group able to be compared in parallel, T cells were added until the total T cells in each experimental group were consistent, and finally X-VIVO 15 was added to 500μL. After the preparation was completed, each group of CAR-T cells was added to each well, and the untransfected T cell group was added with an equal amount of total T cells as each group of CAR-T, and X-VIVO 15 was added to 500μL.

(3) After 24 hours, gently pipette the cells in the wells (try not to blow up the tumor cells), and then centrifuge the wells at 400g for 5 minutes. Take 50μL 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.

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.

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

Each group of CAR-T cells was cultured in X-VIVO 15 (without exogenous IL-2 supplementation). Cytokines and proliferation rates were measured after 24 hours. Cell counts and statistical analysis were performed every 2-3 days thereafter to reflect the basal signal strength of the CAR-T cells. The results are shown in Figure 20. The truncated EF1α-CAR-T-1, full-length EF1α-CAR-T-1, and CMV-CAR-T-1 groups secreted higher levels of cytokines, including TNF-α, IFN-γ, and IL-2, with CMV-CAR-T-1 producing the highest secretion level. In vitro cell proliferation results are shown in Figure 21. Significant cell proliferation was observed in the truncated EF1α-CAR-T-1, full-length EF1α-CAR-T-1, and CMV-CAR-T-1 groups, while the MND-CAR-T-1 group had a lower level of cell proliferation. Cells in the PGK300-CAR-T-1 and PGK400-CAR-T-1 groups showed no cell proliferation and subsequently underwent cell death. 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.

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

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.

CAR-T-1 cells were prepared according to the above-mentioned CAR-T cell preparation method. When sufficient cells were cultured, they were resuspended in cryopreservative solution and stored in liquid nitrogen until use. Tumor tissue from colorectal cancer patients with high GCC expression detected by immunohistochemistry was obtained and cut into 2-3 ^mm³ fragments. The cells were inoculated into 6-8 week-old NSG mice. A small incision was made in the lower back of each mouse, and the tumor tissue sample was then subcutaneously transplanted 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 the successful establishment of the mouse xenograft model. When the tumors grew to 100 mm³, each group of mice was injected with CAR-T-1 cells (5× ¹⁰⁶ CAR-T cells/mouse) through the tail vein. The other two groups of mice were injected with the same volume of Dulbecco's phosphate buffered saline (DPBS) and the same number of T cells but without lentivirus transduction of control T cells. The tumor size of mice was measured 1 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 detected on days 2, 6, 14, 21, and 28 after CAR-T cell injection.

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, CAR-T-1 cells slowed tumor growth 3 days after infusion and effectively reduced tumor burden within 26 days of detection, showing significant anti-tumor activity. Figure 22B shows the number of CAR-T-1 cells in the peripheral blood of mice at different times. A high level of CAR-T cells was detected 10 days after infusion, and then decreased and maintained at a certain level.

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

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.

Human peripheral blood mononuclear cells (PBMCs) were obtained from patients. Based on cell count results and the T cell ratio, CD3/CD28 Dynabeads (Thermo) were added at a ratio of 1.5:1 (bead:T cell ratio). The cells were placed on a sample sorter and gently shaken for 30 minutes. The beads were then adsorbed to the beads using a magnetic stand (Invitrogen) to isolate CD3-positive T cells. To fully activate the T cells, they were resuspended in complete culture medium (X-VIVO 15 + 500 IU/mL IL-2) and expanded at a density of 1.5 × ^10⁶ cells/mL. After 24 hours of magnetic bead activation, the cells were harvested and counted. Based on the count results, T cells were infected with lentivirus at an MOI of 2. 24 hours after infection, the cells were centrifuged at 400 g for 5 minutes and replaced with fresh complete culture medium for continued culture. On day 8 of culture, cells were harvested, Dynabeads removed using a magnetic rack, and centrifuged at 400 g for 5 minutes. The CAR-T cells were washed and cryopreserved using transfusion-safe freezing medium. The cell suspension was placed in cryopreservation bags, which were then cooled to -90°C and transferred to a vapor phase liquid nitrogen tank for storage. Before patient transfusion, the frozen CAR-T cells were transported to the hospital and transfused within 30 minutes after resuscitation. The transfusion dose was 1.2 × 10 ⁹ CAR-T cells.

All patients received fludarabine and cyclophosphamide-based conditioning therapy before CAR-T cell infusion. After CAR-T cell infusion, patients were closely observed for at least 2 hours. Peripheral blood was collected regularly to monitor CAR-T cell expansion by flow cytometry, and plasma cytokine levels were monitored using the BD ^™ Cytometric Bead Array (CBA) Human Th1/Th2/Th17 CBA Kit. The protocol was approved by the hospital conducting the trial. All patients provided written informed consent.

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, CT scan images were obtained, as shown in Figure 23 . Lung lesions 1 and 2, as well as peritoneal metastases, were significantly reduced. Changes in tumor diameter are detailed in Table 3 . CAR-T cells expanded significantly in vivo, reaching a maximum of 3.2×10 ⁷ CAR-Ts/L. Among cytokines, IL-6 levels were significantly elevated, reaching a maximum of 2666.7 pg/mL (Figure 24 ). Carcinoembryonic antigen and cancer antigen levels decreased significantly during treatment (Figure 25 ). This patient was assessed as having a partial response (PR) at month 1 and a complete response (CR) at month 7. No severe CRS (e.g., no more than grade 2 CRS) was observed in Patient 01 during treatment.

Table 3 Changes in tumor size of patient 01

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 the infusion of truncated EF1-α-CAR-T-1 cells, the CT scan image is shown in Figure 26. The lung lesions and liver metastases were significantly reduced, and the total tumor diameter was reduced by more than 30%. The changes in tumor diameter are shown in Table 4. CAR-T was significantly amplified in vivo, reaching a maximum of 1.37×10 ⁸ CAR-T/L. The cytokine IL-6 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, 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.

Table 4 Changes in tumor size of patient 02

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

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

14.1 Construction of Lentiviral Transfer Plasmids and Lentivirus Preparation

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.

14.2 CAR-T Cell Preparation and CAR Expression Rate Detection

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.

Example 15: In vitro evaluation of the cytotoxicity of GCC CAR-T cells with protective peptides

15.1 Real-time Kill

A real-time killing experiment was performed according to the same experimental procedures as in Example 9. The real-time killing results are shown in Figure 30. At E:T = 1:3, the killing rate of CAR-T-7 was faster within 72 hours, indicating that CAR-T-7 has a better anti-tumor effect than CAR-T-1 in the short term.

15.2 Repeated Stimulation Killing

Repeated stimulation and killing experiments were conducted using the same experimental procedures as in Example 9. The results, as shown in Figure 31, show that after two rounds of repeated target cell stimulation, CAR-T-7 demonstrated superior killing ability, significantly outperforming CAR-T-1, but showed no significant advantage in the number of CAR-T cell expansion. Overall, CAR-T-7 demonstrated superior persistence and stronger anti-tumor activity.

Claims (12)

A chimeric antigen receptor targeting GCC, comprising: a 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 costimulatory signaling domain or a 4-1BB costimulatory signaling domain, and a 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 HC CDR1 with the amino acid sequence shown in SEQ ID NO: 1, HC CDR2 with the amino acid sequence shown in SEQ ID NO: 2, and HC CDR3 with the amino acid sequence shown in SEQ ID NO: 3, and the VL comprises LC CDR1 with the amino acid sequence shown in SEQ ID NO: 4, LC CDR2 with the amino acid sequence shown in SEQ ID NO: 5, and LC CDR3 with the amino acid sequence shown in SEQ ID NO: 6. 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. A chimeric antigen receptor according to claim 1 or 2, wherein the scFv that specifically recognizes GCC is at least 85% identical to the amino acid sequence of SEQ ID NO: 9. The chimeric antigen receptor according to any one of claims 1 to 3, comprising any one selected from the following (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 shown 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 shown 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 shown in 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 shown in 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. The chimeric antigen receptor according to any one of claims 1-4 further comprises a protective peptide, which preferably has an amino acid sequence shown in SEQ ID NO: 24. The chimeric antigen receptor according to any one of claims 1-5, which is at least 85% identical to the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23 or SEQ ID NO: 25. A nucleic acid molecule comprising a nucleic acid sequence encoding the chimeric antigen receptor according to any one of claims 1 to 6. 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. 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. 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 medicament for treating GCC-positive tumors. The use according to claim 10, wherein the drug is used to treat digestive tract tumors. The use according to claim 11, wherein the drug is used to treat colorectal cancer, esophageal cancer, gastric cancer or pancreatic cancer.