WO2024040681A1

WO2024040681A1 - Car-t cell and use thereof in treatment of non-small cell lung cancer

Info

Publication number: WO2024040681A1
Application number: PCT/CN2022/122202
Authority: WO
Inventors: 钟晓松; 仝帅
Original assignee: 卡瑞济(北京)生命科技有限公司
Priority date: 2022-08-26
Filing date: 2022-09-28
Publication date: 2024-02-29
Also published as: CN115925985B; CN115925985A

Abstract

The present invention relates to an uPAR-targeted third-generation chimeric antigen receptor, immune effector cells (e.g., T cells, NK cells) engineered to express the chimeric antigen receptor of the present invention, and a use of the engineered immune effector cells in the treatment of uPAR-positive non-small cell lung cancer (NSCLC).

Description

CAR-T cells and their application in the treatment of non-small cell lung cancer

Technical field

The present invention generally relates to third-generation chimeric antigen receptors targeting uPAR, immune effector cells (e.g., T cells, NK cells) engineered to express the chimeric antigen receptors of the invention, and said engineered The use of immune effector cells in the treatment of uPAR-positive non-small cell lung cancer (NSCLC).

Background technique

Urokinase-type plasminogen activator receptor (uPAR, also known as urokinase receptor or CD-87), discovered in 1985, is a cysteine-rich glycosylated single-chain protein, relatively The molecular weight is 50kD-60kD [Casey, J.R., et al., The structure of the urokinase-type plasminogen activator receptor gene. Blood, 1994.84(4):p.1151-6]. The gene encoding uPAR, PLAUR, encodes a protein composed of 335 amino acids. Its N-terminus contains a 22-amino acid secretion signal peptide, and the 30 amino acids at the C-terminus bind to the cell membrane through a glycosylphosphatidylinositol (GPI) anchor [Lv, T.,et al.,uPAR:An Essential Factor for Tumor Development.J Cancer,2021.12(23):p.7026-7040; and Blasi,F.and N.Sidenius,The urokinase receptor:focused cell surface proteolysis,cell adhesion and signaling. FEBS Lett, 2010.584(9):p.1923-30]. uPAR consists of three domains with a length of 81 to 87 amino acids, namely D1, D2 and D3 domains, connected by short linkers [De Lorenzi, V., et al., Urokinase links plasminogen activation and cell adhesion by cleavage of the RGD motif in vitronectin.EMBO Rep, 2016.17(7):p.982-98]. The D1 region binds to urokinase plasminogen activator (uPA). The D3 region connected to the D1 and D2 regions anchors uPAR to the cell membrane surface through GPI.

As a multifunctional protein, uPAR is believed to play a key role in regulating a variety of physiological and pathological conditions, such as wound healing, neutrophil recruitment during inflammation, tumor invasion, and tumor metastasis [Ploug, M., Structure- function relationships in the interaction between the urokinase-type plasminogen activator and its receptor. Curr Pharm Des, 2003.9(19):p.1499-528]. Most normal tissues have little or no detectable uPAR expression. However, uPAR has been found to be expressed in a variety of tumor cell lines and tissues (including colon, breast, ovary, etc.), and it has been confirmed in tumor samples obtained from colon and breast cancer patients that uPAR levels are associated with tumor metastasis potential and disease. Late potential relevance. The increased expression level of uPAR in tumor tissues and its relative loss in normal and resting tissues, as well as the role of uPAR in the regulation of angiogenesis and tumor progression, suggest that uPAR is a potential target for cancer therapy [Pillay, V ., C.R.Dass, and P.F.Choong, The urokinase plasminogen activator receptor as a gene therapy target for cancer. Trends Biotechnol, 2007.25(1):p.33-9].

Chimeric antigen receptor (CAR) is a synthetic molecule that specifically recognizes antigens expressed on the surface of tumor cells to guide immune effector cells (e.g., T cells) genetically engineered to express CAR. , NK cells) to clear tumors (Sampson JH, Choi BD, Sanchez-Perez L et al., EGFRvIIImCAR-modified T-cell therapy cures mice with established intracerebralglioma and generates host immunity against tumor-antigen loss. Clinical cancer research:an official journal of the American Association for Cancer Research. 2014; 20(4):972-984).

Recently, Amor et al. [Amor, C., et al., Senolytic CAR T cells reverse senescence-associated pathologies. Nature, 2020.583(7814): p.127-132] proposed targeting uPAR in their study of aging models. The second-generation CAR molecules are used as anti-aging drugs to eliminate senescent cells in the body caused by multiple factors. Studies have shown that after administration of senescence inducers, second-generation uPAR CAR T cells showed a certain elimination effect on lung cancer tumor cells that were induced to appear aging markers. However, the research of Amor et al. also showed that the in vivo application of the combination of this senescence inducer and the second-generation CAR molecule in a tumor-bearing mouse model had a very limited effect on prolonging the overall survival of the animals. Compared with the control mice, they died within 30 days. , mice that received uPAR CAR T cells also died within 40 days. In addition, the application dosage of senescence-inducing agents and their possible interference with the physiological processes of normal human cells have also raised many issues in terms of safety and effectiveness for the application of this combination strategy in clinical cancer treatment.

According to the latest research, lung cancer remains the most common cause of cancer-related death worldwide. More than 2 million people are diagnosed with lung cancer, and 1.76 million people die from this disease every year [Thai, A.A., et al., Lung cancer. Lancet, 2021.398(10299):p.535-554]. The 5-year survival rate for lung cancer varies by stage and region from 4% to 17%, and the disease remains poorly treatable with conventional treatments such as surgery [Hirsch, F.R., et al., Lung cancer: current therapies and new targeted treatments. Lancet, 2017.389(10066):p.299-311]. Therefore, there is an ongoing need in the field to develop new lung cancer treatments to improve patient outcomes and prognosis.

Summary of the invention

During in-depth research, the inventors found that uPAR showed significant positive expression in the cancer tissues of some non-small cell lung cancer (NSCLC) patients, and found that high expression levels of uPAR were correlated with the low survival rate of NSCLC patients. On this basis, the inventors constructed a uPAR-targeted third-generation CAR molecule; by detecting the selective cytotoxicity of T cells transduced with the CAR molecule to uPAR-positive NSCLC cancer cells in vitro alone, as well as in subcutaneous and metastatic , anti-tumor effects in pre-invasive NSCLC lung cancer animal models and PDX models, confirming the therapeutic efficacy of the third-generation CAR molecule of the present invention. Furthermore, the inventors used high-throughput RNA sequencing to reveal the molecular mechanism of uPAR CAR-T cells of the present invention in the treatment of NSCLC lung cancer, and obtained a series of gene expression patterns that can be used to predict the therapeutic efficiency of CAR-T cells; and then A combination therapy of this third-generation CAR-T cell and PD-1 blocker was proposed, and the improved efficacy was confirmed in the PDX model of NSCLC. Based on these studies, the present inventors thus established the present invention.

Therefore, in a first aspect, the invention provides a third-generation chimeric antigen receptor (CAR) polypeptide targeting uPAR, the chimeric antigen receptor polypeptide, from the N-terminus to the C-terminus, comprising:

(i) The extracellular antigen-binding domain that specifically binds uPAR;

(ii) optionally, a hinge/spacer region;

(iii) Transmembrane domain;

(iv) a combination of a CD28 costimulatory domain and a 4-1BB costimulatory domain; and

(v) CD3ζ signaling domain,

Preferably, the uPAR extracellular antigen binding domain consists of the optimized nucleotide sequence of SEQ ID NO: 1 or a nucleoside having at least 95%, 96%, 97%, 98%, 99% or 99.5% identity thereto. Acid sequence encoding.

In some embodiments, the extracellular antigen binding domain that specifically binds uPAR is an antibody or antibody fragment, especially a scFv,

Preferably, the antigen-binding domain includes: LCDR1-3 in the VL amino acid sequence of SEQ ID NO:3 and HCDR1-3 in the VH amino acid sequence of SEQ ID NO:4 (especially the CDR sequence defined by Kabat, or LCDR1-3 and HCDR1-3 sequences shown in SEQ ID NOs: 13-18), further preferably, comprise SEQ ID NOs: 3 or have at least 90%, 92%, 95%, 96%, 97%, A VL having an amino acid sequence of 98%, 99% or more identity and/or comprising SEQ ID NO: 4 or having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more The VH of the amino acid sequence of the same identity, more preferably, the VL of SEQ ID NO:3 and the VH of SEQ ID NO:4, and further preferably, the antigen-binding domain is composed of SEQ ID NO:2 or has the same An anti-uPAR scFv having an amino acid sequence of at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identity.

In some embodiments, the chimeric antigen receptor polypeptide further comprises a hinge or spacer region between the extracellular antigen binding domain and the transmembrane domain. In some embodiments, the hinge/spacer is selected from: a hinge region from an IgG or a spacer from a CD8α or CD28 extracellular region, and is preferably a human CD8α spacer or a CD28 spacer. In other embodiments, the hinge region/spacer region comprises, or is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more different from, the amino acid sequence of SEQ ID NO: 6 The same amino acid sequence, or the amino acid sequence that differs from it by no more than 1-5 amino acid residue modifications (eg, substitutions, insertions and/or deletions).

In some embodiments, the transmembrane domain is selected from the group consisting of: transmembrane domains of CD4, CD8, CD28 and CD3ζ, and is preferably a human CD8 transmembrane domain or a CD28 transmembrane domain. In other embodiments, the transmembrane domain comprises, or is at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, or different from the amino acid sequence of SEQ ID NO: 7 or 22. The above identical amino acid sequence, or the amino acid sequence that differs from it by no more than 1-5 amino acid residue modifications (eg, substitutions, insertions and/or deletions).

In some embodiments, the CD28 costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 11, or is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more different therefrom. The same amino acid sequence, or an amino acid sequence that differs from it by no more than 1-5 amino acid residue modifications (eg, substitutions, insertions and/or deletions).

In some embodiments, the 4-1BB costimulatory domain comprises the amino acid sequence shown in SEQ ID NO: 10, or is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% identical thereto. or an amino acid sequence with the same identity as above, or an amino acid sequence that differs from it by no more than 1-5 amino acid residue modifications (eg, substitutions, insertions and/or deletions).

In some embodiments, the CD3ζ signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 12, or is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more different therefrom. The same amino acid sequence, or an amino acid sequence that differs from it by no more than 1-5 amino acid residue modifications (eg, substitutions, insertions and/or deletions).

In some embodiments, the CAR polypeptide, from N-terminus to C-terminus, includes:

(a) Anti-uPAR scFv shown in SEQ ID NO:2;

(b) The CD28 spacer region shown in SEQ ID NO:6 and the CD28 transmembrane domain of SEQ ID NO:7;

(c) a combination of the CD28 costimulatory domain shown in SEQ ID NO: 11 and the 4-1BB costimulatory domain shown in SEQ ID NO: 10; and

(iv) the CD3ζ signaling domain shown in SEQ ID NO:12,

Preferably, the CAR polypeptide comprises SEQ ID NO: 21 or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

In a second aspect, the invention provides nucleic acid molecules encoding chimeric antigen receptor polypeptides described herein, vectors comprising nucleic acids encoding CAR polypeptides described herein, and cells comprising, or expression of, CAR nucleic acid molecules or vectors described herein The cells of the CAR polypeptides described herein are preferably autologous T cells or allogeneic T cells.

In one embodiment, the present invention uses human PBMC to prepare primary CAR-T cells. CAR-T cells transduced with the CAR molecules of the present invention have effector functions in vitro and have the activity of continuously killing target cells in vitro. In yet another embodiment, the CAR-T cells transduced with the CAR molecules of the present invention also have the function of killing tumor cells in vivo, and in animal individuals with subcutaneous, pre-invasive, and/or metastatic NSCLC lung cancer, they exhibit Significant anti-tumor activity.

In a third aspect, the invention provides a method of producing cells, e.g., immune effector cells, comprising converting a nucleic acid molecule (e.g., an RNA molecule, such as an mRNA molecule) encoding a CAR polypeptide described herein, or comprising a nucleic acid molecule encoding a CAR polypeptide described herein. The vector of the nucleic acid molecule of the CAR polypeptide introduces (eg, transduces) immune effector cells.

In some embodiments, the immune effector cells are T cells, NK cells, for example, the T cells are autologous T cells or allogeneic T cells, for example, the immune effector cells are T cells isolated from human PBMC, Prepared after NK cells.

In some embodiments, a retrovirus is used to introduce the nucleic acid encoding the CAR molecule of the present invention into primary T cells, thereby obtaining the CAR-T cells of the present invention.

In some embodiments, the CAR-T cells of the invention exhibit differentially expressed genes related to anti-tumor activity after contact with target tumor cells. In some embodiments, genes associated with BP, MF and CC are up-regulated upon exposure of the CAR-T cells to target tumors: cellular response to interferon-γ, immune response, inflammatory response, and tumor necrosis factor activation Receptor activity. In other embodiments, genes associated with the following BP, MF and CC are down-regulated upon exposure of the CAR-T cells to target tumors: gene expression regulation, DNA replication, mitotic cell cycle G1/S transition, protein binding and spindle Spindle pole. In some embodiments, the therapeutic response of a patient receiving CAR-T cell therapy can be predicted by monitoring the up-regulation of gene expression and/or the down-regulation of gene expression. In some embodiments, upregulation of expression of genes selected from the group consisting of IL2, IL9, IFN-γ, TNFRSF9 and IL17A genes and chemokine genes such as CXCL1, CXCL5 and CXCL8 is monitored to indicate a patient's treatment response. In other embodiments, upregulation of expression of selected genes: PD-1, PD-L2, and/or Lag-3 is monitored to indicate a patient's likelihood of relapse.

In a fourth aspect, the invention provides a method comprising a pharmaceutically acceptable carrier and a chimeric antigen receptor polypeptide described herein, a CAR-encoding nucleic acid molecule described herein, an immune effector cell described herein, or a chimeric antigen receptor polypeptide described herein. Pharmaceutical compositions of CAR-T cells. In some preferred embodiments, the pharmaceutical composition further includes a PD-1 inhibitor or a PD-L1 inhibitor, preferably an anti-PD-1 antibody. Preferably, the pharmaceutical composition is provided in the form of a pharmaceutical combination, wherein , the CAR-T cells and PD-L1 inhibitors described herein are included in separate formulations in a manner that facilitates separate, sequential and/or simultaneous administration.

In a fifth aspect, the invention provides the use of engineered immune effector cells in the preparation of a medicament for the treatment of uPAR-positive non-small cell lung cancer (NSCLC) in an individual in need thereof and the use of the engineered immune effector cells to treat uPAR A method of expressing positive non-small cell lung cancer (NSCLC), wherein the engineered immune effector cells comprise a uPAR-targeting chimeric antigen receptor polypeptide described herein.

In some embodiments, the immune effector cells are T cells, eg, autologous T cells or allogeneic T cells.

In some embodiments, the NSCLC is large cell lung cancer, adenocarcinoma, or squamous cell carcinoma.

In some embodiments, the subject has preinvasive NSCLC lung cancer or in situ NSCLC lung cancer. In other embodiments, the individual has metastatic NSCLC cancer, such as brain metastases of NSCLC.

In some embodiments, the non-small cell lung cancer is Stage I, Stage II, Stage III, or Stage IV lung cancer.

In some embodiments, the individual is Asian, such as Chinese.

In some embodiments, the individual is an adult individual over 30 years old, or an elderly individual over 60 years old.

In some embodiments, the methods and uses further include determining uPAR positivity in a tumor sample (e.g., tumor biopsy) from the individual prior to administration of the CAR-T cells, such as by immunohistochemical staining. Percentage of expressing cells (ie, uPAR positive rate of tumor). In one embodiment, preferably, approximately 25-80% or more of the cells in the tumor sample exhibit positive expression of uPAR on the cell surface as determined by immunohistochemical staining.

In some embodiments, the methods and uses include administering to the individual one or more doses of a CAR-T cell described herein, e.g., the doses may be administered continuously or at intervals. In some preferred embodiments, the treatment methods and uses described herein further include administering to the individual an immune checkpoint inhibitor, such as a PD-1 or PD-L1 inhibitor or a LAG-3 inhibitor, e.g., Before, during and/or after the administration of the CAR-T cells, one or more doses of the inhibitor, especially a PD-1 inhibitor, such as an anti-PD-1 antibody, are administered.

Brief description of the drawings

The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the following drawings. For the purpose of illustrating the invention, there is shown in the drawing a presently preferred embodiment. It is to be understood, however, that this invention is not limited to the precise arrangements and instrumentalities illustrated in the drawings.

Figure 1 shows that lung cancer patients with relatively high uPAR expression in their tumors have significantly lower survival rates (p<0.001, Log-Rank test).

Figure 2 shows: Immunohistochemical detection of uPAR levels in lung cancer tumors, of which: (a) 4 cases were positive and (b) 8 cases were negative. Shown are 100x magnification of representative immunohistochemically stained sections and 200x magnification of local areas.

Figure 3 shows a schematic diagram of a chimeric antigen receptor (CAR) targeting uPAR, which consists of a uPAR scFv, hinge and transmembrane (TM) regions from CD28, costimulatory domains from CD28 and 4-1BB, and Signaling domain composition of CD3ζ. Among them, SD represents the splice donor site; SA represents the splice acceptor site; LTR represents the long terminal repeat sequence.

Figure 4 shows the quantification of positive CAR T cells by flow cytometry after transduction of T cells with a CAR-encoding retroviral vector. Where, GAM represents goat anti-mouse IgG (Fab specific) F(ab')2 fragment-FITC antibody (GAM, Sigma) staining.

Figure 5 shows the in vitro activity of uPAR CAR-T on uPAR-positive tumor cells. (a) APC-labeled monoclonal antibody was used to stain uPAR expressed on the surface of cancer cells. The human lung cancer cell line H460 has a uPAR positive rate of 95.4%; in the human lung cancer cell line A549 that is genetically modified to overexpress uPAR, the uPAR positive rate is 83.5%. (b) CFSE-labeled CAR-T cells and uPAR-positive tumor cells were co-cultured at an E:T ratio of 2:1 for 12 days. T cells were stimulated with fresh tumor cells every three days, and T cells were counted each time before adding tumor cells to determine the T cell expansion fold. (c) After CAR-T cells were co-cultured with uPAR-positive tumor cells for 6 hours (E:T=10:1), the CD107a level on the CAR-T cells was measured by flow cytometry. (d) After CAR-T cells were co-cultured with uPAR-positive tumor cells for 24 hours (E:T=10:1), the supernatants were collected and IFN-γ levels were assessed by ELISA. The results in Figure 5(b) and (d) were analyzed by student t test, and p<0.05 was considered significant. * represents p<0.05, ** represents p<0.01, and *** represents p<0.001.

Figure 6 shows the in vitro activity of uPAR CAR-T on uPAR-positive tumor cells. (a) In an in vitro killing assay, NGFR CAR-T cells or uPAR CAR-T cells were compared with different target cells expressing luciferase (H460 and uPAR ⁺ A549) at different E:T ratios (1:1 , 2.5:1, 5:1 and 10:1) for 24 hours, and the IVIS imaging system was used to detect the lysis rate of tumor cells. (b) In an impedance-based tumor cell killing assay, tumor cells and T cells were co-cultured, and the xCELLigence impedance system was used to monitor the continuous graphical output of cell index values up to the 12-hour time point from the start time of the co-culture.

Figure 7 shows: In vivo tumor model. (a) 2 x ¹⁰ eGFP-Luc-H460 cells (luciferase- and eGFP-labeled H460 tumor cells) were injected subcutaneously into the left axilla of 6-8 week old female NOD-SCID mice. Three days later, tumor-bearing mice were treated with 2x 10 ⁷ uPAR CAR-T cells injected directly into tumors for three consecutive days, and non-transduced T cells (NT) were used as controls. (b) Fluorescent image of tumor burden in mice. Mice treated with uPAR CAR-T showed significantly prolonged survival compared with control mice, with some mice surviving for more than 84 days. (c) Apply the IVIS imaging system to obtain quantitative bioluminescence imaging data of all mice (i.e., the absolute number of photons emitted from the animal's body surface per unit time, unit area, and unit radian (photons/sec/cm ² /sr)). Higher values indicate greater tumor burden. (d) Overall survival of lung cancer xenograft-bearing mice was measured using the Kaplan-Meier method and compared between groups using Cox proportional hazards regression analysis. A p value less than 0.05 was considered significant.

Figure 8 shows: From a lung xenograft mouse model of tumor recurrence after uPAR CAR T cell therapy, tumor cells were isolated and co-cultured with uPAR CAR-T cells in vitro to confirm the anti-tumor activity of CAR-T cells. "Control mouse cells" means that tumor cells are isolated from mice in the non-CAR-T treatment group; "relapse mouse cells" means that tumor cells are isolated from mice in the CAR-T-treated group. NT represents untransduced T cells. (a) After co-cultivation with E:T=10:1 for 24 hours, the IFN-γ level in the culture supernatant was detected by ELISA. ** indicates p<0.01. (b) After co-culture for 6 hours with E:T=10:1, the expression level of CD107a on uPAR CAR T cells was detected by flow cytometry. (c) In the in vitro killing test, after uPAR CAR-T cells were co-cultured with different target tumor cells for 24 hours (E:T=1:1, 2.5:1, 5:1 and 10:1), the tumor cells were detected lysis rate.

Figure 9 shows: In vivo tumor model. (a) 2× ¹⁰ eGFP-Luc-H460 cells (luciferase- and eGFP-labeled H460 tumor cells) were orthotopically implanted into the lung parenchyma of 6-8 week old female NOD-SCID mice via pleural injection. Three days later, tumor-bearing mice were treated with intraperitoneal (ip) injection of 2x 10 ⁷ uPAR CAR-T cells into tumors for three consecutive days, and non-transduced T cells (NT) were used as controls. (b) Fluorescent image of tumor burden in mice. Mice treated with uPAR CAR-T showed significantly prolonged survival relative to control mice. (c) Apply the IVIS imaging system to obtain quantitative bioluminescence imaging data of all mice (i.e., the absolute number of photons emitted from the animal's body surface per unit time, unit area, and unit arc (p/sec/cm ² /sr)). Higher values indicate greater tumor burden. (d) Overall survival of lung cancer orthotopic xenograft-bearing mice was measured using the Kaplan-Meier method and compared between groups using Cox proportional hazards regression analysis. A p value less than 0.05 was considered significant.

Figure 10 shows: From an orthotopic xenograft mouse model of tumor recurrence after uPAR CAR T cell therapy, tumor cells were isolated and co-cultured with uPAR CAR-T cells in vitro to confirm the anti-tumor activity of CAR-T cells. "Control mouse cells" means that tumor cells are isolated from mice in the non-CAR-T treatment group; "relapse mouse cells" means that tumor cells are isolated from mice in the CAR-T-treated group. NT represents untransduced T cells. (a) After co-cultivation with E:T=10:1 for 24 hours, the IFN-γ level in the culture supernatant was detected by ELISA. *** indicates p<0.001. (b) After co-culture for 6 hours with E:T=10:1, the expression level of CD107a on uPAR CAR T cells was detected by flow cytometry. (c) In the in vitro killing test, after uPAR CAR-T cells were co-cultured with different target tumor cells for 24 hours (E:T=1:1, 2.5:1, 5:1 and 10:1), the tumor cells were detected lysis rate.

Figure 11 shows: In vivo tumor model. (a) 2 x ¹⁰ eGFP-Luc-H460 cells (luciferase- and eGFP-labeled H460 tumor cells) were implanted intracranially into 6-8 week old female NOD-SCID mice. Three days later, 2x 10 ⁷ uPAR CAR-T cells were injected intravenously (iv) into the tumor-bearing mice for three consecutive days, and untransduced T cells (NT) were used as a control. (b) Fluorescent image of tumor burden in mice. Mice treated with uPAR CAR-T showed significantly prolonged survival relative to control mice. (c) Apply the IVIS imaging system to obtain quantitative bioluminescence imaging data of all mice (i.e., the absolute number of photons emitted from the animal's body surface per unit time, unit area, and unit arc (p/sec/cm ² /sr)). Higher values indicate greater tumor burden. (d) Overall survival of lung cancer intracranial xenograft-bearing mice was measured using the Kaplan-Meier method and compared between groups using Cox proportional hazards regression analysis. A p value less than 0.05 was considered significant.

Figure 12 shows: From an intracranial xenograft mouse model of tumor recurrence after uPAR CAR T cell therapy, tumor cells were isolated and co-cultured with uPAR CAR-T cells in vitro to confirm the anti-tumor activity of CAR-T cells. "Control mouse cells" means that tumor cells are isolated from mice in the non-CAR-T treatment group; "relapse mouse cells" means that tumor cells are isolated from mice in the CAR-T-treated group. NT represents untransduced T cells. (a) After co-cultivation with E:T=10:1 for 24 hours, the IFN-γ level in the culture supernatant was detected by ELISA. ** indicates p<0.01. (b) After co-culture for 6 hours with E:T=10:1, the expression level of CD107a on uPAR CAR T cells was detected by flow cytometry. (c) In the in vitro killing test, uPAR CAR-T cells were co-cultured with different target cells for 24 hours (E:T=1:1, 2.5:1, 5:1 and 10:1), and the lysis of tumor cells was detected. Rate.

Figure 13 shows: Gene Ontology Enrichment Analysis of differentially expressed genes after co-culture of uPAR CAR-T cells and tumor cells. Volcano plot of differentially expressed genes (DEGs) (i.e., genes with fold change (FC) >2 in expression levels before and after co-culture and adjusted p-value <0.05). The horizontal axis represents fold change, and the vertical axis represents adjusted p-value.

Figure 14 shows: Using the online bioinformatics tool: DAVID Bioinformatics Resources 6.8, GO analysis was performed on the differentially expressed genes between CAR-T cells before co-culture and CAR-T cells after co-culture. Fisher's exact test was used for this gene enrichment analysis. BP stands for: biological process; CC stands for: cellular component; MF stands for: molecular function.

Figure 15 shows: (a) PPI analysis of differentially expressed genes between CAR-T cells before and after co-culture. Protein-protein interaction plot of genes upregulated in CAR-T cells after co-culture relative to CAR-T cells before co-culture. (b) Volcano plot of differentially expressed genes in CAR-T cells co-cultured for 30 minutes relative to CAR-T cells without co-culture. (c) Comparing CAR-T cells co-cultured for 30 minutes with CAR-T cells co-cultured for 4 hours, 133 genes were up-regulated in both, and 22 genes were down-regulated in both.

Figure 16 shows the upregulated gene expression in CAR-T cells after co-culture with tumor cells. (a) H460 cells and CAR-T cells were co-cultured at an E:T ratio of 2:1 for 30 minutes and 4 hours. RT-qPCR was then performed on CAR-T cells to determine IL2, IL9, IFN-γ, TNFRSF9 and IL17A levels. (b) H460 cells and CAR-T cells were co-cultured at an E:T ratio of 2:1 for 30 minutes and 4 hours. RT-qPCR was then performed on CAR-T cells to determine CXCL1, CXCL5, and CXCL8 levels.

Figure 17 shows the upregulated gene expression in CAR-T cells after co-culture with tumor cells. (a) H460 cells and CAR-T cells were co-cultured at an E:T ratio of 2:1 for 30 minutes and 4 hours. NT represents untransduced T cells. RT-qPCR was then performed on CAR-T cells to determine PD-1 and PDCD1LG2 levels; and on H460 cells, PD-L1 levels were determined. (b) After uPAR CAR-T cells were co-cultured with H460 cells and uPAR+A549 cells at E:T=10:1 for 48 hours, the expression of PD-1, Lag-3 and Tim-3 of CAR-T cells was detected by flow cytometry level.

Figure 18 shows: PD-1/PD-L1 inhibits the anti-tumor activity of uPAR CAR-T cells. (a) and (b) H460 cells were transiently transfected with siRNA to reduce PD-L1 expression. H460-si-PD-L1-NC represents: H460 cells treated with PD-L1 with control siRNA; H460-si-PD-L1-#1 and #2 respectively represent: using two siRNAs with different sequences targeting PD-L1 Treated H460 cells. (a) As detected by PCR, cells have significantly reduced PD-L1 mRNA expression levels after siRNA knockdown treatment. (b) Through flow cytometry, the cell surface PD-L1 expression of tumor cells decreased after siRNA knockdown treatment. (c) Co-culture uPAR CAR-T cells with tumor cells with and without knockdown of PD-L1 at E:T=10:1, and detect the level of secreted IFN-γ. (d) At E:T=2.5:1, co-culture uPAR CAR-T cells with tumor cells with and without knockdown of PD-L1, and conduct an in vitro killing test. The lysis rate of tumor cells was determined by detecting luciferase activity. The results were subjected to one-way analysis of variance, and statistical significance was set at p<0.05. *p<0.05, **P<0.01, ***p<0.001. (e) After co-culturing uPAR CAR-T cells with tumor cells with and without PD-L1 knockdown at E:T=10:1 for 24 hours, the CD107a expression level of CAR-T cells was detected.

Figure 19 shows that PD-1 antibody combined with CAR-T cell treatment significantly inhibited tumor growth in the PDX model of lung cancer. The results showed that the subcutaneous tumor tissue of mice after CAR-T treatment was significantly smaller than that of the untreated group, and the subcutaneous tumor tissue of mice in the CAR-T cell combined with PD-1 antibody treatment group was significantly smaller than that of the CAR-T treatment group alone. significantly reduced. (a) Schematic diagram of in vivo lung cancer PDX model generation. (b) and (c) Measurement of tumor volume: After the mice were suddenly killed by neck rupture, the tumor tissue was taken and measured with a vernier caliper. The volume calculation formula is: length x width x width/2. (d) Measurement of tumor weight: After the mice were suddenly killed by neck fracture, the tumor tissue was taken and measured on a 1/10000 analytical balance to measure the tumor weight. * means: p<0.05; ** means: p<0.01; *** means: p<0.005.

Detailed description of the invention

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Furthermore, the materials, methods, and examples described herein are illustrative only and not intended to be limiting. Other features, objects and advantages of the invention will be apparent from the description and drawings, and from the appended claims.

definition

For the purpose of interpreting this specification, the following definitions will be used and, wherever appropriate, terms used in the singular may also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

The term "about" when used in conjunction with a numerical value is intended to encompass a range of numerical values having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

As used herein, the term "and/or" means any one of the options or two or more of the options.

When the term "comprises" or "includes" is used herein, it also encompasses a combination of the stated elements, integers, or steps unless otherwise specified. For example, when reference is made to an antibody variable region that "comprises" a particular sequence, it is also intended to encompass antibody variable regions that consist of that particular sequence.

The terms "chimeric receptor", "chimeric antigen receptor" or "CAR" are used interchangeably herein and refer to a recombinant protein that contains at least an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. Peptides.

The term "stimulatory molecule" refers to a molecule expressed by a T cell that provides a primary cytoplasmic signaling sequence that modulates the TCR complex in a stimulatory manner in at least some aspect of the T cell signaling pathway. Primary activation. In one embodiment, the primary signal is initiated, for example, by binding of a TCR/CD3 complex to a peptide-loaded MHC molecule and results in the mediation of a T cell response, including but not limited to proliferation, activation, differentiation, and the like. In particular CARs of the invention, the intracellular signaling domain in any one or more CARs of the invention comprises an intracellular signaling sequence, for example, the primary signaling sequence of CD3ζ.

The term "CD3ζ" is defined as the protein provided by GenBan accession number BAG36664.1 or its equivalent, and "CD3ζ signaling sequence" is defined as the amino acid residues from the cytoplasmic domain of the CD3ζ chain that are sufficient to functionally Propagate primary signals necessary for T cell activation. In one embodiment, the cytoplasmic domain of CD3ζ comprises residues 52 to 164 of GenBank accession number BAG36664.1 or as a functional ortholog thereof from a non-human species (e.g., mouse, rodent, equivalent residues of monkeys, apes, etc.). In one embodiment, the "CD3ζ signaling sequence" is the sequence provided in SEQ ID NO: 12 or a variant thereof.

The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand and thereby mediates a costimulatory response of the T cell (eg, but not limited to, T cell proliferation). Costimulatory molecules are cell surface molecules other than the antigen receptor or its ligand required for an effective immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, OX40 , CD40, GITR, 4-1BB (ie CD137), CD27 and CD28. In some embodiments, the "costimulatory molecule" is CD28, 4-1BB (ie, CD137). As used herein, "costimulatory domain" refers to the intracellular portion of the costimulatory molecule.

The term "4-1BB" refers to a TNFR superfamily member having the amino acid sequence provided as GenBank accession number AAA62478.2 or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.) ; and "4-1BB costimulatory signaling domain" is defined as amino acid residues 214-255 of GenBank accession number AAA62478.2 or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.) . In one embodiment, the "4-1BB costimulatory domain" is the sequence provided as SEQ ID NO: 10 or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.).

The term "CD28" refers to the amino acid sequence provided under UniProtKB-P10747 accession number or equivalent residues from a non-human species (eg, mouse, rodent, monkey, ape, etc.). As used herein, the term "CD28 costimulatory domain" is defined as derived from the cytoplasmic region of CD28, e.g., amino acid residues 180-220 of UniProtKB-P10747 or from a non-human species (e.g., mouse, rodent, monkey, ape etc.) equivalent residues. In one embodiment, the "CD28 costimulatory domain" is the sequence provided as SEQ ID NO: 11 or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). As used herein, the term "CD28 transmembrane domain" is defined as the transmembrane region from CD28, e.g., amino acid residues 153-179 of UniProtKB-P10747 or from a non-human species (e.g., mouse, rodent, monkey, ape etc.) equivalent residues. In one embodiment, the "CD28 transmembrane domain" is the sequence provided as SEQ ID NO: 7 or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). In this article, the term "CD28 hinge domain", used interchangeably with the term "CD28 spacer", is defined as a hinge domain derived from the extracellular region of CD28, such as amino acid residues 114-152 of UniProtKB–P10747 or from non- Equivalent residues in human species (e.g., mouse, rodent, monkey, ape, etc.). In one embodiment, the "CD28 spacer" is the sequence provided as SEQ ID NO: 6 or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.).

The terms "amino acid change" and "amino acid modification" are used interchangeably and refer to the addition, deletion, substitution and other modifications of amino acids. Any combination of amino acid additions, deletions, substitutions, and other modifications can be made, provided that the final polypeptide sequence has the desired properties. In some embodiments, the substitution of amino acids is a non-conservative amino acid substitution, i.e., one amino acid is replaced with another amino acid having different structural and/or chemical properties. Amino acid substitutions include substitutions with non-naturally occurring amino acids or naturally occurring amino acid derivatives of the twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxy Lysine) substitution.

The terms "conservative sequence modification" and "conservative sequence change" refer to amino acid modifications or changes that do not significantly affect or change the characteristics of the parent polypeptide containing the amino acid sequence or its constituent elements. Such conservative modifications include amino acid substitutions, additions and deletions. Conservative modifications, especially conservative substitutions, can be introduced into the CAR fusion polypeptide of the invention or its constituent elements (e.g., CAR or Survivin) by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. . A conservative substitution is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include those with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., Glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenyl Alanine, tryptophan, histidine) amino acids.

"Percent identity (%)" of an amino acid sequence/nucleotide sequence means that the candidate sequence is compared to the specific amino acid/nucleotide sequence shown in this specification and, if necessary, to achieve maximum sequence identity. After introducing gaps, and in the case of amino acid sequences, without considering any conservative substitutions as part of the sequence identity, the number of amino acid residues/core in the candidate sequence is identical to the specific amino acid/nucleotide sequence shown in this specification. Percentage of amino acid/nucleotide residues whose nucleotide residues are identical. In some embodiments, the present invention contemplates variants of the fusion polypeptides or nucleic acid molecules of the invention, or constituent elements thereof, that are relative to the fusion polypeptides or nucleic acid molecules, or constituent elements thereof, specifically disclosed herein (e.g., CAR polypeptides/ The sequence of the encoding nucleic acid, or Survivin protein/encoding nucleic acid) has a substantial degree of identity, for example, an identity of at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or higher. The variants may contain conservative modifications. For the purposes of this invention, percent identity is determined using the publicly available BLAST tool at https://blast.ncbi.nlm.nih.gov , using default parameters.

As used herein, the expression "variant" or "functional variant" polypeptide or protein means that the polypeptide or protein has substantially the same sequence or significant sequence identity as compared with the reference polypeptide or protein. and maintain the desired biological activity of the reference polypeptide or protein.

The term "vector" as used herein when referring to nucleic acids refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

The term "lentivirus" refers to a genus of the family Retroviridae. Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells; they can deliver significant amounts of genetic information to host cells, making them one of the most efficient methods of gene delivery vectors. HIV, SIV and FIV are examples of lentiviruses.

The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, including in particular self-inactivating lentiviral vectors as provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that may be used clinically include, but are not limited to, those from Oxford BioMedica

Gene delivery technology, LENTIMAX ^TM vector system from Lentigen, etc. Non-clinical types of lentiviral vectors are also available and known to those skilled in the art.

The term "immune effector cells" refers to cells involved in an immune response, such as in promoting an immune effector response. Examples of immune effector cells include T cells, eg, alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.

Chimeric Antigen Receptor (CAR) of the Invention

Through in-depth research, the inventors found that in the treatment of non-small cell lung cancer, the use of immune cells (for example, CAR-T cells and CAR-NK cells) engineered to express third-generation CAR polypeptides optimized to target uPAR can Effectively achieve anti-tumor immunity in uPAR-positive pre-invasive/in situ NSCLC lung cancer and metastatic NSCLC lung cancer. Based on this, the present invention provides optimized third-generation CAR polypeptides targeting uPAR, immune cells based on the CAR polypeptides, and their effects alone or in combination with other anti-cancer drugs (especially PD-1 inhibitors) in the treatment of NSCLC patients. use.

The CAR and its components of the present invention will be described in detail below. A person skilled in the art can understand that, unless the context clearly indicates otherwise, any technical features mentioned in describing components and any combination thereof are within the scope of consideration of the present invention; and, a person skilled in the art can understand , unless the context clearly indicates otherwise, any CAR-based embodiment of the invention (including, but not limited to, CAR-encoding nucleic acids, CAR-based immune cells and uses thereof) may also include any such combination of features.

In a first aspect, the present invention provides a third-generation chimeric antigen receptor (CAR) polypeptide targeting uPAR, the chimeric antigen receptor polypeptide, from the N-terminus to the C-terminus, comprising:

(i) The extracellular antigen-binding domain that specifically binds uPAR;

(ii) optionally, a hinge/spacer region;

(iii) Transmembrane domain;

(v) CD3ζ signaling domain.

Depending on the uPAR antigen to be targeted, the CAR of the invention can be constructed to include an appropriate antigen-binding domain specific for that antigen target to confer specific recognition and binding to the CAR molecule and the CAR-T cells containing the CAR molecule. ability to target antigens. In one embodiment, the extracellular antigen-binding domain of the CAR molecule according to the invention is a polypeptide molecule with binding affinity for the uPAR target antigen, such as an antibody or antibody fragment that specifically binds uPAR or a ligand from this antigen receptor. body fragments. In one embodiment, a CAR according to the invention comprises an antigen-binding domain derived from an antibody or antibody fragment. In yet another embodiment, the antigen binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL). In a preferred embodiment, the antigen-binding domain comprises a scFv consisting of VL and VH linked via a linker.

scFv can be generated by linking the VH and VL regions together using flexible polypeptide linkers according to methods known in the art. In some embodiments, scFv molecules comprise flexible polypeptide linkers of optimized length and/or amino acid composition. In some embodiments, the scFv comprises a linker between its VL and VH regions, wherein the linker comprises at least 5,6,7,8,9,10,11,12,13,14,15,16,17 ,18,19,20,25,30,35,40,45,50 or more amino acid residues. The linker sequence may contain any naturally occurring amino acid. In one embodiment, the peptide linker of the scFv consists of amino acids such as glycine and/or serine residues used alone or in combination to link the variable heavy chain and variable light chain regions together. In one embodiment, the flexible polypeptide linker is a Gly/Ser linker and, for example, comprises the amino acid sequence (Gly-Gly-Gly-Ser)n, where n is a positive integer equal to or greater than 1. For example, n=1, n=2, n=3. n=4, n=5 and n=6, n=7, n=8, n=9 and n=10. In one embodiment, flexible polypeptide linkers include, but are not limited to, (Gly4Ser)4 or (Gly4Ser)3. In another embodiment, the linker includes multiple repeats of (Gly2Ser), (GlySer) or (Gly3Ser). In yet another embodiment, the linker comprises the GSTGSSGKPGSGEGSTKG amino acid sequence. In one embodiment, the scFv used in the present invention contains from N-terminus to C-terminus: VL-linker-VH; or VH-linker-VL.

The CAR polypeptides of the invention comprise at least one transmembrane domain, which can be derived from natural or synthetic sources. For example, the transmembrane domain may be derived from a membrane-binding or transmembrane protein, such as that from CD3ζ, CD4, CD28, CD8 (eg, CD8α, CD8β). In the chimeric antigen receptor (CAR) polypeptides of the invention, the transmembrane domain confers membrane attachment to the CAR polypeptide of the invention. In some embodiments, the transmembrane domain in the CAR of the invention can be connected to the extracellular region of the CAR via a hinge region/spacer region. Regarding the transmembrane region and hinge region/spacer region that can be used in CAR polypeptides, see, for example, Kento Fujiwara et al., Cells 2020, 9, 1182; doi:10.3390/cells9051182.

The cytoplasmic domain included in the CAR polypeptide of the present invention includes an intracellular signaling domain. The intracellular signaling domain is capable of activating at least one immune effector function of the immune cells into which the CAR of the present invention has been introduced. The immune effector function includes, but is not limited to, for example, enhancing or promoting the function or response of immune attack target cells. The effector function of T cells may be, for example, cytolytic activity or auxiliary activity, including secretion of cytokines.

Examples of cytoplasmic domains useful in CAR polypeptides of the invention include cytoplasmic domains of T cell receptors (TCRs) and/or co-receptors that function to initiate signal transduction upon binding of the extracellular domain to the target antigen. sequences, as well as any derivatives or variants of these sequences and any recombinant sequences having the same functional capabilities. Activation of T cells is mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (i.e., primary intracellular signaling domains) and those that act in an antigen-independent manner to provide co-activation Those sequences that stimulate the signal (i.e., secondary cytoplasmic domains, e.g., costimulatory domains). In one embodiment, a CAR polypeptide of the invention comprises a cytoplasmic domain that provides a primary intracellular signaling domain, e.g., the intracellular signaling domain of CD3ζ, and further comprises a secondary signaling domain, e.g., from 4-1BB (also known as CD137) and the costimulatory domain of CD28. In one embodiment, the cytoplasmic region of the CAR polypeptide of the present invention contains sequentially tandem CD28 and 4-1BB costimulatory domains and a CD3ζ intracellular signaling domain to ensure effective activation of uPAR-positive pre-invasive/in situ NSCLC. Anti-tumor immunity is achieved in lung cancer and metastatic NSCLC lung cancer.

In some embodiments, the CAR polypeptide of the invention may comprise a signal peptide or leader sequence located at the N-terminus of the extracellular antigen-binding domain. Through the signal peptide/leader sequence, the nascent CAR polypeptide can be guided to the endoplasmic reticulum of the cell and then anchored on the cell membrane. A signal peptide/leader sequence of any eukaryotic origin may be used, such as a signal peptide/leader sequence of mammalian or human secretory protein origin.

In some embodiments, chimeric antigen receptor (CAR) polypeptides according to the invention comprise an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic domain.

In one embodiment, the antigen-binding domain of the CAR polypeptide of the invention is an antibody or antigen-binding fragment thereof that specifically binds uPAR. In one embodiment, the antibody or antigen-binding fragment thereof is a murine, human or humanized antibody or antigen-binding fragment thereof. In one embodiment, the antigen-binding domain comprises: the heavy chain complementarity determining region 1 (HC CDR1) of the heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 4, the heavy chain complementarity determining region 2 (HC CDR2) and heavy chain complementarity determining region 3 (HC CDR3), such as the HCDR1-3 amino acid sequence of SEQ ID NO:16-18; and/or the light chain variable region (VL) amino acid sequence of SEQ ID NO:3. Chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2) and light chain complementarity determining region 3 (LC CDR3), such as the LCDR1-3 amino acid sequence of SEQ ID NO: 13-15. In one embodiment, the antigen binding domain comprises a heavy chain variable region and a light chain variable region, wherein,

The heavy chain variable region includes: i) the amino acid sequence of SEQ ID NO:4; ii) having at least one, two or three modifications but no more than 30, 20 or 10 modifications to the amino acid sequence of SEQ ID NO:4 Modified amino acid sequence; or iii) an amino acid sequence having 95-99% identity with the heavy chain variable region amino acid sequence of SEQ ID NO: 4; and/or

The light chain variable region comprises: i) the amino acid sequence of SEQ ID NO:3; ii) having at least one, two or three modifications but no more than 30, 20 or 10 modifications to the amino acid sequence of SEQ ID NO:3 A modified amino acid sequence; or iii) an amino acid sequence having 95-99% identity with the heavy chain variable region amino acid sequence of SEQ ID NO:3.

In one embodiment, the antigen binding domain comprises: i) the amino acid sequence of SEQ ID NO: 2; ii) at least one, two or three modifications to SEQ ID NO: 2 but no more than 30, 20 or 10 modified amino acid sequences; or iii) an amino acid sequence that is 95-99% identical to SEQ ID NO: 2.

In one embodiment, the transmembrane domain comprises a transmembrane domain of a protein selected from: CD4, CD8α, CD28, CD3ζ, TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD9, CD16, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, CD66d, alpha, beta or zeta chain of T cell receptor, MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor , integrins, and activating NK cell receptors. In one embodiment, the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of CD4, CD8α, CD28 and CD3ζ. In one embodiment, the transmembrane domain comprises: i) the amino acid sequence of SEQ ID NO: 7; ii) comprising at least one, two or three modifications but no more than 5 modifications of the amino acid sequence of SEQ ID NO: 7 an amino acid sequence; or iii) an amino acid sequence having 95-99% sequence identity with SEQ ID NO:7. In one embodiment, the transmembrane domain comprises: i) the amino acid sequence of SEQ ID NO: 22; ii) comprising at least one, two or three modifications but no more than 5 modifications of the amino acid sequence of SEQ ID NO: 22 an amino acid sequence; or iii) an amino acid sequence having 95-99% sequence identity with SEQ ID NO: 22.

In one embodiment, the cytoplasmic domain comprises a functional signaling domain of a protein selected from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b or CD66d. In one embodiment, the cytoplasmic domain comprises the functional signaling domain of the CD3ζ protein (also referred to herein as the CD3ζ signaling domain). In one embodiment, the cytoplasmic domain comprises: i) the amino acid sequence of SEQ ID NO: 12; ii) comprising at least one, two or three modifications but no more than 20 of the amino acid sequence of SEQ ID NO: 12, 10 or 5 modified amino acid sequences; or iii) an amino acid sequence having 95-99% sequence identity with SEQ ID NO: 12.

In one embodiment, the cytoplasmic domain further comprises a costimulatory domain of two proteins selected from: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, Signaling lymphocyte activation molecule (SLAM protein), activated NK cell receptor, CD8, ICOS, DAP10, DAP12, OX40, CD40, GITR, 4-1BB (ie CD137), CD27 and CD28. In one embodiment, the cytoplasmic domain comprises the costimulatory domain of two proteins selected from the group consisting of: CD28, CD27, 4-1BB, ICOS and the costimulatory domain of OX40. In one embodiment, the cytoplasmic domain comprises a combination of costimulatory domains of CD28 and 4-1BB. In one embodiment, the cytoplasmic domain comprises a CD28 costimulatory domain and a 4-1BB costimulatory domain, wherein the CD28 costimulatory domain comprises: i) the amino acid sequence of SEQ ID NO: 11; ii) comprising SEQ ID NO : an amino acid sequence with at least one, two or three modifications but not more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO:11; or iii) 95-99% identity with the amino acid sequence of SEQ ID NO:11 The amino acid sequence; and wherein the 4-1BB costimulatory domain includes: i) the amino acid sequence of SEQ ID NO:10; ii) including at least one, two or three modifications of but not more than the amino acid sequence of SEQ ID NO:10 20, 10 or 5 modified amino acid sequences; or iii) an amino acid sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 10.

In one embodiment, the CAR polypeptide further comprises a hinge or spacer region disposed between said transmembrane domain and said extracellular antigen binding domain. In one embodiment, the hinge/spacer region is selected from the group consisting of a GS hinge, a CD8 hinge, an IgG4 hinge, an IgD hinge, a CD16 hinge, and a CD64 hinge. In one embodiment, the CAR polypeptide comprises a hinge region from the extracellular region of CD28. In one embodiment, the hinge region/spacer region comprises: i) the amino acid sequence of SEQ ID NO: 6; ii) comprising at least one, two or three modifications but no more than 5 of the amino acid sequence of SEQ ID NO: 6 A modified amino acid sequence; or iii) an amino acid sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 6. In this document, the expressions "hinge", "hinge region" and "hinge domain" are used interchangeably.

In one embodiment, the CAR polypeptide further comprises a leader or signal peptide, such as the signal peptide from human granulocyte-macrophage colony-stimulating factor receptor alpha chain (GM-CSFRa).

In one embodiment, a CAR polypeptide according to the present invention comprises: i) the amino acid sequence of SEQ ID NO:21; ii) having at least one, two or three modifications but not more than 30 to the amino acid sequence of SEQ ID NO:21 , 20 or 10 modified amino acid sequences; or iii) an amino acid sequence having at least 95-99% identity with the amino acid sequence of SEQ ID NO: 21.

Nucleic acid molecules and vectors encoding the CAR of the present invention and cells expressing the CAR of the present invention

The invention provides nucleic acid molecules encoding the CAR constructs described herein. In one embodiment, the nucleic acid molecules are provided as DNA constructs. Constructs encoding the CAR of the invention can be obtained using recombinant methods well known in the art. Alternatively, the nucleic acid of interest may be produced synthetically rather than by genetic recombination methods.

The invention also provides vectors into which the nucleic acid molecule(s) of the invention or the nucleic acid construct of the invention are inserted. Expression of the nucleic acid encoding the CAR can be achieved by operatively linking the nucleic acid encoding the CAR polypeptide to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration in eukaryotic organisms. Common cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters for regulating expression of the desired nucleic acid sequence. Numerous virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide convenient platforms for gene delivery systems. The nucleic acid constructs of the invention can be inserted into vectors and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells in vivo or ex vivo. Numerous retroviral systems are known in the art. In some embodiments, lentiviral vectors are used.

Vectors derived from retroviruses (e.g., lentiviruses) are suitable tools for long-term gene transfer because they allow long-term, stable integration of the transgene and its propagation in progeny cells. Lentiviral vectors have the additional advantage over vectors derived from onco-retroviruses (such as murine leukemia virus) in that they can transduce non-proliferating cells, such as hepatocytes. They also have the additional advantage of being low immunogenicity. Retroviral vectors may also be, for example, gamma retroviral vectors. A gamma retroviral vector may, for example, comprise a promoter, a packaging signal (ψ), a primer binding site (PBS), one or more (eg, two) long terminal repeats (LTR), and a transgene of interest, e.g., encoding a CAR genes. Gamma retroviral vectors can lack viral structural genes such as gag, pol and env.

An example of a promoter capable of expressing a CAR transgene in mammalian T cells is the EF1a promoter. The native EF1a promoter drives expression of the alpha subunit of the elongation factor-1 complex, which is responsible for enzymatic delivery of aminoacyl tRNA to ribosomes. The EF1a promoter has been used extensively in mammalian expression plasmids and has been shown to efficiently drive CAR expression from transgenes cloned into lentiviral vectors. See, eg, Milone et al., Mol. Ther. 17(8):1453–1464 (2009).

Another example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a constitutively strong promoter sequence capable of driving high-level expression of any polynucleotide sequence to which it is operably linked. However, other constitutive promoter sequences may also be used, including, but not limited to, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) Long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to the actin promoter , myosin promoter, elongation factor-1α promoter, hemoglobin promoter and creatine kinase promoter. Additionally, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention.

In some embodiments, the invention provides methods of expressing the CAR constructs of the invention in mammalian immune effector cells (eg, mammalian T cells or mammalian NK cells) and immune effector cells generated thereby.

A source of cells (eg, immune effector cells, eg, T cells or NK cells) is obtained from the subject. The term "subject" is intended to include living organisms (eg, mammals) that can elicit an immune response. T cells can be obtained from numerous sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors.

T cells can be obtained from blood components collected from a subject using any technique known to those skilled in the art, such as Ficoll ^™ isolation. In a preferred aspect, cells from the individual's circulating blood are obtained by apheresis. Apheresis products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction and to place the cells in a suitable buffer or culture medium for subsequent processing steps. In one aspect of the invention, cells are washed with phosphate buffered saline (PBS).

Specific T cell subsets, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated through positive or negative selection techniques. For example, in one embodiment, by conjugating beads with anti-CD3/anti-CD28 (e.g.

M-450CD3/CD28T) was incubated for a period of time sufficient to positively select the desired T cells, and the T cells were isolated. In some embodiments, the time period is between about 30 minutes and 36 hours or longer. Longer incubation times can be used to isolate T cells in any situation where small numbers of T cells are present, such as for isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals. Additionally, using longer incubation times can increase the efficiency of capturing CD8+ T cells. Thus, by simply shortening or extending this time, allowing T cells to bind to CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells, one can preferentially select at the beginning of the culture or at other time points during the culture process T cell subsets.

Enrichment of T cell populations through a negative selection process can be accomplished using a combination of antibodies directed against surface markers unique to the negatively selected cells. One method is to sort and/or select cells by means of negative magnetic immunoadhesion or flow cytometry, which uses cells present on the negatively selected cells. Mixture of monoclonal antibodies to surface markers.

In some embodiments, the immune effector cells may be allogeneic immune effector cells, such as T cells or NK cells. For example, the cells may be allogeneic T cells, e.g., allogeneic that lack functional T cell receptor (TCR) and/or human leukocyte antigen (HLA) (e.g., HLA class I and/or HLA class II) expression. T cells.

In some embodiments, cells transduced with a nucleic acid encoding a CAR of the invention are propagated, for example, the cells are propagated in culture for 2 hours to about 12 days. The effector function of the CAR-expressing immune effector cells obtained after in vitro proliferation can be tested as described in the Examples.

The expression of the CAR polypeptide of the present invention, which has a combination of CD28 and 4-1BB costimulatory domains for uPAR in immune effector cells, can significantly promote the overall survival of individual tumor-bearing animals. This may be due to the fact that the combined use of two costimulatory domains prolongs the survival time of CAR-T cells while promoting the proliferation and anti-tumor activity of CAR-T cells. By co-culturing the CAR-T cells of the present invention and tumor cells, high-throughput sequencing is performed to detect the differentially expressed genes of the CAR-T cells of the present invention. RNA extraction, cDNA library construction, and sequencing were all performed in strict accordance with transcriptome sequencing standards. Use the online bioinformatics tool DAVID bioinformatics Resources 6.8 to perform gene ontology (GO) analysis on the differentially expressed genes of the CAR-T cells of the present invention. Data visualization and analysis were handled by custom R studio scripts following packages (ggplot2 and Tree map). Gene enrichment analysis was performed using Fisher's exact test. GO analysis results show that most of the differentially expressed genes in uPAR-CD28.4-1BBζCAR-T cells of the present invention are located in the extracellular region and cell membrane, and are related to immune response, inflammatory response and Genes related to the cellular response to interferon γ were significantly up-regulated, and the gene enrichment levels in these three biological processes reached approximately 10 times, approximately 12 times, and approximately 2.5 times, respectively. This suggests that the third generation CAR-T cells of the present invention are effectively activated after contact with target NSCLC cancer cells, supporting the strong anti-tumor function of the CAR-T cells of the present invention in vivo.

Uses of immune effector cells expressing the CAR polypeptide of the present invention and therapeutic methods using immune effector cells expressing the CAR polypeptide of the present invention

In recent decades, CAR-T cell therapy has become a new method of adoptive cellular immunotherapy. However, in solid tumors such as NSCLC, the biological heterogeneity of the tumor itself makes the construction of CAR-T cells more complex, and there is a continuing need for molecules that target different tumor antigens to improve the treatment of individual patients.

The third-generation optimized CAR molecules targeting uPAR constructed by the present inventors have demonstrated significant anti-tumor immune effects in vitro and in various animal tumor-bearing models in vivo. Based on this, in yet another aspect, the present invention provides the use of engineered immune effector cells in the preparation of a medicament for the treatment of uPAR-positive non-small cell lung cancer (NSCLC) in an individual in need thereof and the utilization of the engineered immune effector Methods of cell therapy for uPAR-positive non-small cell lung cancer (NSCLC), wherein the engineered immune effector cells comprise a chimeric antigen receptor polypeptide targeting uPAR as described herein.

As used herein, the terms "individual" or "subject" or "patient" are used interchangeably and include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). mouse). In particular, the individual or subject is a human being.

The terms "tumor" and "cancer" are used interchangeably herein. The term "anti-tumor immunity" refers to biological effects that can be manifested in various ways, including but not limited to, for example, reduction in tumor volume, reduction in the number of cancer cells, reduction in the number of metastases, and improvement in the expected life span of tumor-bearing individuals. Prolongation, reduction of cancer cell proliferation, reduction of cancer cell survival, or improvement of various physiological symptoms associated with cancerous conditions. "Anti-tumor immunity" can also be presented by the ability of peptides, polypeptides, cells and antibodies to prevent cancer from arising in the first place. In some embodiments, the CAR immune effector cells of the invention are administered in the treatment of uPAR-positive NSCLC patients to provide anti-tumor immune effects.

As used herein, "treating" means slowing, interrupting, retarding, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desired therapeutic effects include, but are not limited to, preventing the emergence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the CAR immune effector cells of the invention are used to delay disease development or to slow the progression of disease.

The term "therapeutically effective amount" refers to an amount or dosage of the CAR immune effector cells of the present invention that produces the desired effect in a patient in need of treatment or prevention after administration to the patient in single or multiple doses. The effective amount can be readily determined by the attending physician, who is one of ordinary skill in the art, by considering various factors such as: species of mammal; weight, age, and general health; the specific disease involved; the extent or severity of the disease; the individual The patient's response; the specific CAR immune effector cells administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy. A therapeutically effective amount may also be an amount in which any toxic or detrimental effects of the CAR immune effector cells are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (eg, tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, relative to an untreated subject. 60% or 70% and still more preferably at least about 80% or 90%. The ability of CAR immune effector cells to inhibit a measurable parameter (eg, cancer) can be evaluated in animal model systems that are predictive of efficacy in human tumors.

In one embodiment, the invention provides a method of treating uPAR-positive NSCLC cancer in a patient, wherein the method comprises administering to the patient a therapeutically effective amount of a CAR-T cell described herein (and optionally in combination with other anti-cancer agents). , such as a combination of anti-PD-1 or anti-PD-L1 antibodies).

In some embodiments, the non-small cell lung cancer is early-stage non-small cell lung cancer, non-metastatic non-small cell lung cancer, primary non-small cell lung cancer, resected non-small cell lung cancer, advanced non-small cell lung cancer, locally advanced non-small cell lung cancer, cell lung cancer, metastatic non-small cell lung cancer, unresectable non-small cell lung cancer, non-small cell lung cancer in remission, recurrent non-small cell lung cancer, non-small cell lung cancer in the adjuvant setting, or non-adjuvant Treat any type of non-small cell lung cancer.

In one embodiment, the non-small cell lung cancer is adenocarcinoma. In another embodiment, the non-small cell lung cancer is squamous cell carcinoma. In one embodiment, the non-small cell lung cancer is large cell carcinoma. In one embodiment, the patient has received at least one prior treatment. In one embodiment, the prior therapy is surgical treatment for the treatment of cancer. In one embodiment, the non-small cell lung cancer is invasive stage, or carcinoma in situ. In another embodiment, the non-small cell lung cancer is locally advanced lung cancer. In another embodiment, the non-small cell lung cancer is metastatic, particularly brain metastases of NSCLC. In some embodiments, the non-small cell lung cancer is Stage I, Stage II, Stage III, or Stage IV lung cancer. In some embodiments, the patient is Asian, such as Chinese. In some embodiments, the patient is an adult individual between 30 and 50 years old, or between 30 and 55 years old, or an elderly individual over 60 years old, or over 65 years old.

In some embodiments, the methods of the invention further include, prior to administering the CAR-T therapy described herein, selecting a patient for treatment of the invention. In one embodiment, the selecting includes detecting uPAR expression levels in a sample from the subject/patient.

The term "subject/patient sample" refers to a collection of cells, tissues or body fluids obtained from a patient or subject. The source of the tissue or cell sample may be solid tissue, like from fresh, frozen and/or preserved organ or tissue samples or biopsy or aspiration samples; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid) ), peritoneal fluid (ascites), or interstitial fluid; cells from a subject at any time during pregnancy or development. Tissue samples may contain compounds that are not naturally associated with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. Examples of tumor samples herein include, but are not limited to, tumor biopsies, fine needle aspirates, bronchial lavage fluid, pleural fluid (pleural effusion), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating Plasma proteins in ascites fluid, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples such as formalin-fixed, paraffin-embedded tumor samples or frozen tumors sample.

In some embodiments, uPAR expression levels on the surface of tumor cells can be determined from a tumor sample (eg, a tumor biopsy) from a subject or patient. In some specific embodiments, the term "uPAR high expression" refers to the expression of cells in a suspected cancerous tissue or a cancerous tissue compared with a normal tissue, such as an adjacent normal tissue (i.e., a paired tissue) of a tumor tissue to be tested. Higher (increased) expression levels of the target antigen (ie, uPAR) can be detected on the surface. The assessment of expression levels can be qualitative or quantitative. In other words, an unknown sample can be evaluated as having positive or negative expression compared to a known reference standard. Alternatively, the percentage of positive cells can be expressed quantitatively, where, for example, cells can be counted and scored for uPAR expression levels. In a specific embodiment, the reference tissue or cells used for the comparison is a normal or non-cancerous tissue or cell, which may be obtained or derived from a healthy individual (e.g., lung tissue or cells from the individual), or normal tissue or cells obtained from an individual with cancer or suspected cancer to be diagnosed and/or treated (eg, lung tissue or cells from the individual).

In one embodiment, thus, the treatment methods of the present invention further include identifying a subject or patient with a uPRAR-positive tumor from a tumor sample (eg, a tumor biopsy) from the subject or patient, and optionally The tumor's uPAR positivity (i.e., the percentage of uPAR-positive tumor cells) is determined qualitatively or quantitatively.

In some embodiments, the methods described herein include administering to an NSCLC patient a CAR-T cell described herein (and optionally, other anti-cancer agents, such as in combination with an anti-PD-1 or anti-PD-L1 antibody) , wherein the patient expresses elevated uPAR levels (eg, relative to normal tissue samples) in a tumor tissue sample (eg, a squamous or non-squamous tumor tissue sample). In some embodiments, the methods described herein include administering to an NSCLC patient a CAR-T cell described herein (and optionally, other anti-cancer agents, such as in combination with an anti-PD-1 or anti-PD-L1 antibody) , wherein the patient's tumor tissue sample (e.g., squamous or non-squamous tumor tissue sample) has 1% to 50%, or 20% to 50%, 60%, 70% or more, or 30% to 50% , 60%, 70% or more, or a percentage of uPAR-positive cells from 50% to 60%, 70%, 80% or more. In some embodiments, the patient has a uPAR-positive cell percentage of 50% or greater in a tumor tissue sample (eg, a squamous or non-squamous tumor tissue sample).

In other embodiments, the present invention also provides a method by administering a CAR-T cell as described herein (and optionally, other anti-cancer agents, such as in combination with an anti-PD-1 or anti-PD-L1 antibody) A method of treating uPAR-positive NSCLC cancer in a patient, wherein the method further includes the step of evaluating a biomarker in a biological sample obtained from the patient during treatment. In some embodiments, the biological sample is a blood sample. In some embodiments, the biomarker is selected from one or more of the following: IL2, IL9, IFN-γ, TNFRSF9 and IL17A genes, and optionally chemokine genes such as CXCL1, CXCL5 and CXCL8, wherein said organism An increase in the level of a marker relative to before administration of a treatment of the invention or during treatment can be used, for example, to indicate a patient's responsiveness to treatment. In other embodiments, the biomarker is the expression level of PD-1, PD-L2 and/or Lag-3 on tumor infiltrating lymphocytes isolated from the patient, and/or PD-1 on tumor cells isolated from the patient. Expression levels of L1, where the level of the biomarker increases relative to before administration of a treatment of the invention or during treatment, may be used, for example, to indicate a patient's likelihood of relapse.

In a preferred embodiment, the methods of the invention comprise co-administering a CAR-T cell described herein with another anti-cancer agent (eg, an anti-PD-1 antibody). As used herein, "conjunctive" or "combination" administration means that two (or more) different treatments are delivered to a subject during the course of the subject suffering from a condition, e.g., while the subject is suffering from a condition. Two or more treatments are delivered after a disease has been diagnosed and before the condition is cured or eliminated or treatment is discontinued for other reasons. In some embodiments, delivery of one treatment occurs while delivery of the second treatment is initiated, thus there is an overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "parallel delivery." In other embodiments, delivery of one treatment ends before delivery of another treatment begins. In some embodiments of either case, the treatment is more effective due to combined administration. In some embodiments, coadministration results in a reduction in symptoms, or other parameters associated with the condition, that are better than those observed with one treatment in the absence of the other treatment. The effects of two treatments can be partially additive, fully additive, or more than additive.

In some embodiments, the treatment methods and uses described herein further include administering to the subject an immune checkpoint inhibitor, such as a PD-1 or PD-L1 inhibitor or a LAG-3 inhibitor, e.g., in the One or more doses of the inhibitor, especially a PD-1 inhibitor, such as an anti-PD-1 antibody, are administered before, during and/or after the administration of the CAR-T cells. In some embodiments, anti-PD-1 antibody treatments include, but are not limited to: nivolumab, pembrolizumab, ipilimumab, JS001, TSR-042, pembrolizumab, pidilizumab, BGB-A317, SHR-1210, REGN2810, MDX-1106, PDR001, anti-PD-1 from clone RMP1-14; and anti-PD-1 antibodies disclosed in U.S. Patent No. 8,008,449 , durvalumab, atezolizumab, avelumab and their fragments, derivatives, variants and biosimilars.

In some embodiments, in the treatment methods and uses according to the invention, the individual has received prior cancer treatment prior to administration of the CAR-T cells. In one embodiment, the prior treatment is surgical treatment of lung cancer. In other embodiments, the prior treatment is chemotherapy and/or radiation therapy. In still other embodiments, the individual has been treated with a chemotherapeutic agent or a radiotherapeutic agent but is currently (e.g., one week, two weeks, three weeks, one month or two prior to the administration of the CAR-T cells) months) have not received chemotherapy or radiotherapy. In yet other embodiments, the individual has not received a senescence-inducing treatment within, eg, one week, two weeks, three weeks, one month, or two months prior to administration of the CAR-T cells. In some embodiments, the senescence-inducing treatment is chemotherapy and/or radiotherapy that induces an increase in cell surface uPAR expression upon exposure to cancer cells, for example, doxorubicin, ionizing radiation, MEK inhibitors, and CDK4/6 inhibitors. Combination therapy, combination therapy with CDC7 inhibitors and mTOR inhibitors.

In one aspect, the invention also provides pharmaceutical compositions and pharmaceutical combinations comprising a CAR cell, such as an immune effector cell, as described herein, and optionally, a PD-L1 inhibitor; and provides said pharmaceutical compositions and pharmaceutical combinations The use of the combination in the above-mentioned NSCLC treatment method of the present invention, or the use in the preparation of a medicament for use in the method.

It should be understood that the various embodiments/technical solutions and the features of each embodiment/technical solution described in the present invention can be combined with each other arbitrarily, and the various solutions obtained by these mutual combinations are included in the scope of the present invention, just as in Each of these combinations is specifically and individually listed herein, unless the context clearly indicates otherwise.

The following examples are described to aid understanding of the invention. The examples are not intended and should not be construed in any way as limiting the scope of the invention.

Example

Materials and methods

cell lines

Human cancer cell lines H460, A549, and retrovirus packaging cell line PG13 were purchased from the American Type Culture Collection (ATCC). H460, uPAR ⁺ A549 cells expressing eGFP and firefly luciferase were generated by retroviral infection. All these cells were maintained in Dulbecco's modified Eagle medium (Lonza) containing 10% fetal calf serum (Biosera) and 10,000 IU/mL penicillin/10,000 μg/mL streptomycin (EallBio Life Sciences).

immunochemistry

Formalin-fixed, paraffin-embedded (FFPE) tumor specimen sections were deparaffinized and then incubated with rabbit anti-human uPAR antibody (Cell Signaling Technology) overnight at 4°C. After incubation with HRP-conjugated goat anti-rabbit secondary antibody, the signal was detected using a DAB substrate kit (Abcam) following the manufacturer's instructions. Images were collected using a microscope (Nikon). The relative density of uPAR signals was quantified by Image J v1.49 at 100x magnification. If the relative density is greater than 5%, the lung tumor tissue is confirmed to be uPAR positive.

Generation of retroviral vectors encoding uPAR-specific CAR

The CAR molecule (SEQ ID NO:21) containing the optimized uPAR-scFvs coding sequence (SEQ ID NO:1) was synthesized by GeneArt (Invitrogen) and then subcloned into the SFG retroviral vector (addgene). Verification of CAR clones by sequencing. Forty-eight hours after transient transfection, retroviral packaging cell line PG13 was applied to produce retroviral particles.

Generate CAR T cells

Human peripheral blood mononuclear cells (PBMC) were isolated from healthy donors by Lymphoprep (MP Biomedicals) gradient centrifugation. To generate uPAR-CAR T cells, T cells in PBMCs were stimulated with anti-CD3 and anti-CD28 beads and then infected with retroviruses. After 7 days, T cells were tested for CAR expression by flow cytometry and then cultured in X-VIVO ^TM 15 serum-free medium containing 5% GemCell ^TM human serum AB (Gemini Bio) and IL-2 (SL PHARM). Lonza). This study was approved by the Institutional Review Board of Beijing Millennium Hospital, and informed consent was obtained from all participants.

Flow cytometry assay

Flow cytometry was performed on a FACSCanto Plus instrument (BD Biosciences). FlowJo v.10 (FlowJo, LLC) was used for data analysis. APC-labeled mouse anti-human CD3 antibody (BD Biosciences), PE-labeled mouse anti-human CD8 antibody (BD Biosciences), BV421-labeled mouse anti-human CD4 antibody (BD Biosciences) and goat anti-mouse IgG (Fab Transgenic T cells were detected after staining with specific) F(ab')2 fragment-FITC antibody (GAM, Sigma). H460 cells and uPAR ⁺ A549 cells were stained with APC-labeled monoclonal antibody mouse anti-human uPAR (R&D System), and then flow cytometry was performed to examine cell surface uPAR expression. After staining with APC-labeled mouse anti-human CD3 antibody (BD Biosciences) and APC-labeled mouse anti-human CD107a antibody (BD Biosciences), the activation level of CAR-T cells was detected. APC-labeled mouse anti-human CD3 antibody (BD Biosciences), PE-labeled mouse anti-human CD8 antibody (BD Biosciences), BV421-labeled mouse anti-human CD4 antibody (BD Biosciences), BV480-labeled mouse anti-human PD-1 antibody, BV605-labeled mouse anti-human TIM-3 antibody, BV480-labeled mouse anti-human LAG-3 antibody staining, detect changes in PD-1/TIM-3/LAG-3 levels of CAR-T cells, use PE-labeled mouse anti-human PD-L1 antibody staining was used to detect changes in cell PD-L1 levels.

T cell proliferation assay

1x10^6 H460 cells were seeded in a 6-well plate and cultured overnight to allow the H460 cells to fully adhere. Then, CAR-T cells and H460 cells were co-cultured for 12 days at an E:T ratio of 2:1. Stimulate T cells with fresh H460 cells every 3 days and count T cells before adding H460 cells.

Detection of IFN-γ cytokine secretion levels

CAR-T cells were co-cultured with H460, uPAR ⁺ A549 cells and cells isolated from tumor tissue at an E:T ratio of 10:1 for 24 hours. The supernatant was collected and subjected to IFN-γ detection. IFN-γ levels were measured using the Human IFN-γ DuoSet ELISA kit (Development Systems) according to the manufacturer's instructions.

Tumor cell killing test in vitro

NGFR CAR-T cells or uPAR CAR-T cells, with H460, uPAR ⁺ A549, and cells isolated from tumor tissue blocks at 0:1, 1:1, 2.5:1, 5:1, or 10:1 ratio (E:T), co-cultured in X-VIVO ^TM 15 medium for 24 hours. Using an IVIS imaging system (IVIS, Xenogen, Alameda, CA, USA), luciferase activity was monitored and the percentage of target tumor cell lysis caused by CAR-T cells was determined relative to controls. Calculation method: lysis percentage = 1-(fluorescence intensity after co-culture/fluorescence intensity after co-culture at a ratio of 0:1).

Impedance-based tumor cell killing assay

Assess ongoing tumor cell death over 24 hours using the xCELLigence Impedance System. Tumor cells were seeded at 10,000 cells per well in a 96-well resistor-bottomed plate with integrated resistors on the bottom. Experiments were performed in triplicate. After 24 hours, 100,000 T cells (E:T=10:1) were added. NT cells (untransduced T cells) served as controls. Cell index values related to tumor cell adhesion were normalized relative to NT controls. By measuring the electrical impedance caused by tumor cell adhesion, normalized cell index values based on impedance measurements were collected every 15 minutes.

Xenograft mouse model with subcutaneous injection of lung cancer cells

NOD-SCID mice aged 6 to 8 weeks were purchased from Charles River Laboratories. Female NOD-SCID mice were injected subcutaneously with 2x10 ⁶ H460-eGFP-Luc cells into the left flank to construct a xenograft mouse model. 3 days after tumor cell injection, 2x10 ⁷ CAR T cells were injected directly into the tumor for 3 consecutive days. Tumor development was monitored using an IVIS imaging system (IVIS, Xenogen, Alameda, CA, USA), and mice were sacrificed when the tumor diameter reached 20 mm. All experiments, including mice, were approved by the Institutional Review Board of Beijing Millennium Hospital.

bioluminescence imaging

Mice were anesthetized with 3% isoflurane in 100% oxygen, injected with 4.5 mg/kg D-luciferin in 300 μL saline, and imaged 10 min later using an optical imaging platform (Spectral Instruments Imaging). Images were taken every 5 minutes until the photon count peaked.

RNA high-throughput sequencing

use

^UltraTM RNA Library Preparation Kit (#E7530L, NEB), following the manufacturer's instructions, uses samples containing 2 μg of total RNA each as input material to generate sequencing libraries. Briefly, mRNA was purified from total RNA using magnetic beads with attached poly-T oligonucleotides. Fragmentation was performed using divalent cations in NEB Next First Strand Synthesis Reaction Buffer (5X) at elevated temperatures. First-strand cDNA was synthesized using random hexamer primers and RNase H. The second strand of cDNA is then synthesized using buffer, dNTPs, DNA polymerase I, and RNase H. Library fragments were purified using the QiaQuick PCR kit, eluted with EB buffer, and then subjected to end repair, A-tailing, and adapter addition. To construct libraries, products were recovered and PCR performed. Samples with index codes were classified on the cBot Cluster Generation system using TruSeq SR Cluster Kit v3-cBot-HS (Illumina Inc.) according to the manufacturer's protocol. Subsequently, the library was sequenced on the Illumina NovaSeq 6000 System platform (Illumina Inc.).

real-time reverse transcription polymerase chain reaction

Total RNA was extracted from cells using TRIzol reagent (Invitrogen) following the manufacturer's instructions. RNA quantity and purity were measured using a Nanodrop One spectrophotometer (Thermo Fisher Scientific). Only samples with appropriate absorbance measurements (A260/A280 of approximately 2.0 and A260/A230 of 1.9-2.2) were considered for inclusion in this study. cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), and then amplified using SYBR Green PCR Master Mix (Thermo Fisher Scientific) and gene-specific primers. GAPDH was used as an internal control. Relative gene expression was calculated using the 2-ΔΔCt method.

RNA interference

H460 cells were transfected with small interfering RNA (siRNA) using Lipofectamine 3000 (Thermo Fisher Scientific) for 48 hours. Cells were then collected for RNA and protein extraction. Sequence used for siRNA:

Si-NC, ACGUGACACGUUCGGAGAA (as control); si-PD-L1, UCUCUCUUGGAAUUGGUGG (targeting PD-L1).

PDX modeling and treatment

Tumor tissues from uPAR-positive lung adenocarcinoma patients were inoculated into BALB/C-nu/nu mice to perform PDXs (P0) modeling. Three weeks later, tumor tissues were isolated from PDXs (P0) and re-inoculated into BALB/C-nu/nu mice to establish the PDXs (P1) model. See Figure 19a. Three days after PDXs (P1) modeling, tumor-bearing mice were treated with NT (non-transduced T cells) control, uPAR CAR-T cells alone, or uPAR CAR-T cells in combination with PD-1 antibodies for three consecutive days. mouse. As shown in Figures 19b-19d, PD-1 antibody combination therapy inhibited tumor growth significantly better than CAR-T cells alone.

Orthotopic xenograft model

Transpleural injection for orthotopic xenotransplantation. Mice were anesthetized with 3% isoflurane and kept in right-sided decubitus position. A 5 mm skin incision was made caudal to the left scapula, the fat and muscle tissue were dissected, and the left rib cage was exposed. Observe the movement of the left lung, insert a 31-gauge needle syringe containing 30 μL of H460 cell suspension and 50% Matrigel in PBS solution into the lung parenchyma through the sixth intercostal space to a depth of 3 mm; then inject the cells directly into the left lung. Skin incisions were closed with 3M ^™ Vetbond ^™ tissue adhesive (3M, St. Paul, MN, USA) and mice were kept warm until complete recovery.

Intracranial implantation of lung cancer cells to establish xenograft mouse model

Anesthetize 6- to 8-week-old NOD-SCID mice with a mixed ketamine/xylazine solution. The animal was fixed in a stereotaxic headstand, a 1cm incision was made in the midline of the scalp, a hole was drilled in the skull, and ^2x10 H460-eGFP-luc cells in 5μl PBS were injected into the left striatum using a 10μL BD syringe (coordinates: 2.5 mm lateral to bregma and 0.5 mm posterior to bregma), thereby delivering tumor cells into the brain parenchyma at a depth of 3.5 mm. The drilled hole in the skull is sealed with bone wax, and the incision is closed with medical glue (COMPONT). Three days after tumor cell injection, ^3x10 CAR-T cells were injected through the tail vein, and tumor growth was monitored using the IVIS in vivo imaging system (IVIS, Xenogen, Alameda, CA, USA). All mouse experiments were approved by the Institutional Review Board of Beijing Millennium Hospital.

Statistical Analysis

All experiments were performed at least in triplicate. Statistical analysis was performed using GraphPad Prism version 8.0.2 (GraphPad software). Data are expressed as mean ± standard deviation. Use appropriate statistical tests to test for differences between means. Overall survival of mice with tumor xenografts was measured using the Kaplan-Meier method and compared between groups using Cox proportional hazards regression analysis. Statistical significance was set at p<.05.

Example 1: uPAR is associated with poor survival in lung cancer patients

Through the Human Protein Atlas database ( http://www.proteinatlas.org/ ), we found that the survival rate of lung cancer patients with high uPAR expression was significantly lower than that of patients with low uPAR expression (Figure 1, from a total of 994 lung cancer patient case information ).

In order to detect the specific expression of uPAR in different lung cancer patients and its relationship with patient survival rate, we further detected uPAR levels in tumor samples of 12 Chinese lung cancer patients through immunohistochemistry (Figures 2a and 2b and Table 1 shown). The median age of enrolled patients was 61.5 years (range: 49-73 years). All patients had undergone surgical treatment. The demographic and clinical characteristics of the patients are shown in Table 1. Based on the relative density of uPAR signals quantified on tumor specimen sections, 33.3% of patients' lung tumor tissues were uPAR-positive and the remainder were uPAR-negative.

Table 1. Demographic and clinical characteristics of patients.

患者编号patient number	性别gender	年龄(岁)age)	肿瘤类型Tumor type	肿瘤分期Tumor staging	uPAR uPAR	状态state

#1#1	男male	6565	腺癌Adenocarcinoma	IVIV	阴性 Negative	死亡die

#2#2	女female	7373	鳞状细胞癌squamous cell carcinoma	II	阳性Positive	死亡die
#3#3	男male	6464	鳞状细胞癌squamous cell carcinoma	IIIIII	阴性Negative	存活survive
#4#4	男male	5959	腺癌Adenocarcinoma	IIII	阴性Negative	存活survive
#5#5	男male	4949	鳞状细胞癌squamous cell carcinoma	IVIV	阴性Negative	存活survive
#6#6	男male	6464	鳞状细胞癌squamous cell carcinoma	IIIIII	阴性Negative	存活survive
#7#7	男male	6969	腺癌Adenocarcinoma	IIII	阳性Positive	存活survive
#8#8	男male	5151	小细胞癌small cell carcinoma	IIIIII	阴性Negative	死亡die

#9#9	男male	4141	小细胞癌small cell carcinoma	NANA	阴性 Negative	死亡die

#10#10	女female	5757	NANA	NANA	阳性Positive	死亡die
#11#11	男male	5858	腺癌Adenocarcinoma	II	阴性Negative	存活survive
#12#12	男male	6868	鳞状细胞癌squamous cell carcinoma	IIII	阳性Positive	死亡die

Example 2: uPAR CAR-T cells exhibit significant anti-tumor activity in vitro

In order to generate uPAR-specific CAR-T cells, a third-generation (CD28.4-1BBζ) CAR based on uPAR-specific mAb was developed (Figure 3), and a retroviral vector encoding the third-generation CAR molecule was constructed. Afterwards, T cells isolated from peripheral blood mononuclear cells of healthy donors were stimulated with anti-CD3 and anti-CD28 beads; and infected with the constructed retrovirus. Seven days after transduction, flow cytometry assay was performed to check the transduction efficiency. As shown in Figure 4, approximately 60% of T cells were CAR positive.

The human lung cancer cell line H460, which is known to express high levels of uPAR, and another lung cancer cell line A549 (uPAR ⁺ A549), which artificially overexpresses uPAR, were used as representatives of tumor cell lines with high uPAR expression. Figure 5a shows the results of flow cytometry of cell surface uPAR expression levels for these two cell lines. The constructed uPAR CAR-T cells were co-cultured with these tumor cells (H460 and uPAR ⁺ A549) that highly express uPAR at an effector-target ratio (E:T ratio) of 1:1 to 10:1 to examine the efficacy of CAR T cells. In vitro proliferation and antitumor activity. PBMC cells and NGFR CAR-T cells were used as controls. NGFR CAR-T cells targeting the unrelated antigen NGFR are constructed in the same way as uPAR CAR-T cells. As shown in Figure 5b, co-cultured at a low E:T ratio (2:1), the constructed uPAR CAR-T cells showed good viability and proliferation ability after contact with target tumor cells. As shown in Figure 5c to Figure 5d, after co-culturing uPAR CAR-T cells with target tumor cells for 6 hours at a high E:T ratio (10:1), high levels of auto-cocultured uPAR CAR-T cells were detected CD107a expression (Fig. 5c) and IFN-γ secretion (Fig. 5d). Furthermore, as shown in Figure 6a, compared with control NGFR CAR-T cells, uPAR CAR-T cells caused significant tumor target cell lysis, with a tumor cell lysis rate greater than 60%, and the tumor cell lysis rate is greater than 80% at high E:T ratio (10:1). The results of the real-time cell growth monitoring (RTCA) system also showed that uPAR CAR-T cells inhibited the growth of tumor cells compared with control PBMC cells and NGFR CAR-T cells (Figure 6b).

Example 3: uPAR CAR-T cells display therapeutic efficacy in vivo

To examine the anti-tumor activity of uPAR CAR-T cells in vivo, H460-Luc cells were subcutaneously injected into NOD-SCID mice to generate a lung xenograft mouse model. As shown in Figure 7a. On days 1/2/3, CAR-T cells were directly injected into the tumor, and non-transduced T cells (NT) were used as controls to monitor tumor growth for 84 days. The results showed that the survival period of mice treated with uPAR CAR-T cells was significantly prolonged compared with mice in the NT group (Figures 7b, 7c and 7d).

Tumor recurrence was observed in some mice treated with uPAR CAR-T cells. In order to confirm whether the constructed CAR-T cells still have anti-tumor effects on relapsed tumors, we then isolated tumor cells from mice with relapsed tumors and conducted in vitro cell testing. As shown in Figure 8a-c, uPAR CAR-T still showed excellent anti-tumor ability. This shows that the cause of tumor recurrence may not be the off-target effect of CAR-T cells, but may be due to the survival cycle of CAR-T cells themselves in mice and the tumor immunosuppressive microenvironment that limits the anti-tumor effect of CAR-T cells in vivo. activity, leading to tumor recurrence.

Example 4: uPAR CAR-T cells display therapeutic efficacy in vivo

In order to further simulate the actual clinical situation of lung cancer, we constructed an orthotopic xenograft mouse model and an intracranial metastasis cancer xenograft mouse model to examine the effect of the constructed uPAR CAR-T cells on pre-invasive NSCLC that has not spread. Therapeutic efficacy of lung cancer and metastatic NSCLC lung cancer. Similar good treatment results were obtained in these mouse models. The constructed uPAR CAR-T cells inhibited tumor growth in vivo and showed excellent anti-tumor activity in vitro (Figure 9-12). The statistical results of the Wilcoxon rank sum test are shown in Table 2.

Table 2

Radiation rate P values are based on the Wilcoxon rank sum test. EXCEL Kaplan-Meier and non-parametric Wilcoxon p-values were used for survival curve comparisons. NT = untransduced T cells; CAR-T = uPAR CAR-T cells; subcutaneous = subcutaneous vaccination model; lung = pre-infiltration model; brain = metastasis model.

Example 5: Analysis of differentially expressed genes using high-throughput RNA sequencing

In order to explore why uPAR CAR-T cells have tumor suppressive activity in vivo and in vitro, high-throughput RNA sequencing was used to detect differentially expressed genes between CAR-T cells before and after co-culture with H460 cells. Compared with CAR-T cells without co-culture, 1280 up-regulated genes and 664 down-regulated genes were found in CAR-T cells co-cultured with H460 cells for 4 hours (Figure 13). Gene Ontology analysis was performed on the top 400 differentially expressed genes with the largest expression differences. The results showed that in CAR-T co-cultured with target tumor cells, the up-regulated genes were related to the following biological processes (BP), molecular functions (MF) and cellular components (CC): cell response to interferon-γ, Immune response, inflammatory response, and tumor necrosis factor-activated receptor activity; while the expression of genes related to the following aspects: gene expression regulation, DNA replication, mitotic cell cycle G1/S transition, protein binding, and spindle polar bodies ( spindle pole) (Figure 14). In addition, in CAR-T cells co-cultured with tumor cells, most of the up- and down-regulated genes were enriched in the extracellular region and cell membrane. After protein-protein interaction (PPI) analysis, we found a cluster of 30 genes located in the center of the upregulated PPI network, including CD274 (i.e., PD-L1) and PDCD1, LG2 (i.e., PD-L2), IL2, IL9, IFN-γ, TNFRSF9, and Th17A pathway-related chemokine genes CXCL1, CXCL5, and CXCL8 (Fig. 15a). Furthermore, we also found that in CAR-T cells co-cultured for 30 minutes and CAR-T cells co-cultured for 4 hours, 133 genes were up-regulated in both, and 22 genes were down-regulated in both ( Figures 15b and 15c).

To confirm the RNA-seq results, we collected CAR-T cells after co-cultured with H460 cells for 30 minutes and 4 hours, and then detected some candidate genes by RT-qPCR. The results showed that compared with before co-culture of H460 cells, after co-culture of CAR-T cells and H460 cells, the expression of IL2, IL9, IFN-γ, TNFRSF9 and IL17A genes, as well as Th17A-related chemokine genes such as CXCL1, CXCL5 and CXCL8 Significantly increased (Figure 16a and Figure 16b).

Example 6: The anti-tumor activity of CAR-T cells is limited by the PD-1/PD-L1 axis

It has been reported that T cells are inhibited by multiple mechanisms at tumor sites, among which functional inhibition mediated by the PD-1/PD-L1 axis plays a key role [15-17]. To understand whether the designed third-generation uPAR CAR-T cells would be restricted by the PD-1/PD-L1 axis, we detected PD-1 mRNA in CAR-T cells co-cultured with H460 cells at two different time points. level. As shown in Figure 17a, compared with CAR-T cells without co-culture, CAR-T cells co-cultured with H460 cells for 30 minutes and 4 hours expressed significantly increased PD-1 and PDCD1LG2; moreover, compared with CAR-T cells without co-culture; Compared with co-cultured tumor cells, H460 cells after co-culture also expressed significantly increased PD-L1. In addition, a significant increase in PD-1 and Lag-3 levels was also observed after 48 hours of co-culture of CAR-T cells with H460 cells and uPAR+A549 cells (Figure 17b).

To further confirm the inhibition of the PD-1/PD-L1 axis on the anti-tumor activity of CAR-T cells, we knocked down PD-L1 in tumor cells through siRNA (as shown in Figures 18a and 18b), and then performed in vitro killing test and compared with tumor cells without knockdown of PD-L1. As shown in Figure 18d, after PD-L1 was knocked down in tumor cells, an increase in the tumor lytic activity of co-cultured CAR-T cells (E:T=2.5:1) was observed, and as shown in Figures 18c and 18e showed that the secretion level of IFN-γ and the level of CD107a on the cell membrane surface also increased (E:T=10:1).

Example 7: PD-L1 antibody combined with uPAR CAR T cells has a therapeutic effect on lung cancer patient-derived xenograft (PDX) model

Patient-derived xenograft (PDX) models, constructed by injecting primary tumor biopsies from lung cancer patients rather than human cell lines, are used to evaluate CAR-T cell efficacy in vivo. Tumor tissues from lung adenocarcinoma patients were inoculated into BALB/C-nu/nu mice to perform PDXs (P0) modeling. Three weeks later, tumor tissues were isolated from PDXs (P0) and re-inoculated into BALB/C-nu/nu mice to establish the PDXs (P1) model. See Figure 19a. Three days after PDXs (P1) modeling, tumor-bearing mice were treated with NT (non-transduced T cells) control, uPAR CAR-T cells alone, or uPAR CAR-T cells in combination with PD-1 antibodies for three consecutive days. mouse. As shown in Figures 19b-19d, PD-1 antibody combination therapy inhibited tumor growth significantly better than CAR-T cells alone.

discuss

Lung cancer is a major public health problem worldwide and has been the most commonly diagnosed cancer over the past few decades, and up to 40% of lung cancer patients will develop brain metastases [Schabath, M.B. and M.L. Cote, Cancer Progress and Priorities: Lung Cancer. Cancer Epidemiol Biomarkers Prev, 2019.28(10):p.1563-1579]. Lung cancer can be histologically divided into two main subtypes: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) [Thai, A.A., et al., Lung cancer. Lancet, 2021.398(10299):p.535 -554]. NSCLC accounts for approximately 85% of diagnosed lung cancer cases and can be further divided into adenocarcinoma, squamous cell carcinoma, and large cell carcinoma [Connolly, B.M., et al., Selective abrogation of the uPA-uPAR interaction in vivo reveals a novel role in suppression of fibrin-associated inflammation. Blood, 2010.116(9):p.1593-603; and Amor, C., et al., Senolytic CAR T cells reverse senescence-associated pathologies. Nature, 2020.583(7814): p.127- 132]. Although surgical treatment, chemotherapy, and radiotherapy have made some progress in the treatment of NSCLC in the past 20 years, resulting in increased survival time of NSCLC patients, the prognosis of NSCLC has not been stable due to tumor mutation burden and the heterogeneity of the disease. No significant improvement. Therefore, it is imperative to explore new strategies to prolong patient survival.

CAR-T cell therapy has been clinically successful in the treatment of blood cancers, but its application in solid tumors such as lung cancer is not ideal [Larson, R.C. and M.V.Maus, Recent advances and discoveries in the mechanisms and functions of CAR T cells .Nat Rev Cancer, 2021.21(3):p.145-161; Rosenberg, S.A.and N.P. Restifo, Adoptive cell transfer as personalized immunotherapy for human cancer.Science, 2015.348(6230):p.62-8; and Leko, V .and S.A.Rosenberg,Identifying and Targeting Human Tumor Antigens for T Cell-Based Immunotherapy of Solid Tumors.Cancer Cell, 2020.38(4):p.454-472]. Currently, the design of CAR molecules for solid tumors has been proposed for different specific tumor types and target antigens, ranging from the affinity and specificity of the extracellular antigen domain of CAR-T to the properties of the intracellular signaling domain. , quantity and arrangement, may potentially affect the efficacy of treatment. On the other hand, cell-based in vitro and ex vivo tests used in preclinical studies, although they can reflect the activity of candidate CAR-T cells to a certain extent, are still difficult to reflect the in vivo therapeutic efficacy of CAR-T cells. Therefore, predicting the efficacy of CAR-T cells often needs to be based on appropriate animal models. As a result of these many factors, in the treatment of solid tumors such as lung cancer, the design of CAR-T molecules and their efficacy evaluation are still a challenge that the CAR-T treatment field continues to face.

The biological heterogeneity of solid tumors is an important factor leading to the failure of CAR-T therapy. In this regard, several methods have been proposed to improve clinical efficacy and safety. For example, one way is to treat patients with drugs that increase the expression of target antigens on cancer cells before CAR-T treatment to improve CART efficacy. Another is to engineer CAR molecules to enhance their T cell activity against cancer cells that present a lower density of target antigens. The former is limited by the availability of such drugs and their associated efficacy/toxicity. The latter puts forward higher requirements for the design of CAR molecules.

In a series of studies dedicated to the treatment of lung cancer, we found that there are uPAR-positive and -negative patient subgroups in non-small cell lung cancer patients, and high uPAR expression is associated with the survival rate of human NSCLC lung cancer patients. Based on this, we constructed a third-generation CAR molecule targeting uPAR and evaluated its ability to inhibit NSCLC tumors in vitro and in vivo. T cells transduced with the CARs showed excellent anti-NSCLC tumor activity in vitro and in various xenograft mouse models, thus providing a powerful candidate tool for clinical lung cancer treatment.

The anti-tumor efficiency of CAR-T cells is affected by many factors, such as the affinity of the target antigen, the degree of terminal differentiation of CAR-T cells, as well as off-target toxicity and longevity in vivo [Wang, E., et al., Improving the therapeutic index in adoptive cell therapy:key factors that impact efficacy.J Immunother Cancer, 2020.8(2)]. In our study, after CAR-T cells were stimulated by tumor cells, the expression of IL2, IL9 and IFN-γ was up-regulated. In addition, we also noticed that the expression of IL17A and its related chemokines such as CXCL1, CXCL5 and CXCL8 was also increased. The increased expression of these factors can partially explain the strong anti-tumor function of the CAR-T cells of the present invention.

On the other hand, in xenograft mouse model experiments, we also noticed that despite the strong anti-tumor ability of the designed CAR-T cells, most of the mice tested within a period of time after CAR-T cell treatment The tumors were reduced in size to the point where they were barely detectable, but the tumors still recurred in some mice after about a month. Our in vitro experiments on tumor cells isolated from recurrent tumors showed that the CAR-T cells used still maintained their lytic activity against these isolated tumor cells. This suggests that the efficacy of CAR-T cells in the body is somewhat limited, which may be due to various reasons, such as the tumor immunosuppressive microenvironment and the short survival period of CAR-T cells in the body.

In order to further improve the in vivo therapeutic efficacy of this third-generation CAR-T cell for NSCLC, we studied the expression changes of the PD-1/PD-L1 axis on CAR-T cells and tumor cells after tumor cells stimulated CAR-T cells. The results showed that both PD-1 and PD-L1 were significantly up-regulated, and PDCD1LG2 (programmed cell death protein 1 ligand 2, PD-L2) was also up-regulated. PDCD1LG2 is the second ligand of PD-1 and inhibits T cell activation. It has also been reported that this molecule is one of the important reasons for the inhibition of T cell function [Latchman, Y., et al., PD-L2 is a second ligand for PD- 1and inhibits T cell activation. Nat Immunol, 2001.2(3):p.261-8]. Therefore, the upregulation of the PD-1 axis can explain, to a certain extent, the tumor recurrence in tumor-bearing mice treated with CAR-T cells of the present invention after 1 month. Our PD-L1 knockout studies and PDX model studies further support the use of PD-1 antibodies in combination with CAR-T therapy of the present invention as a strategy to overcome the immunosuppressive effects of PD-1/PD-L1. In our study, we also found that the expression of immunosuppressive LAG-3 also increased during the CAR-T cell treatment of the present invention, which suggests that LAG-3 antibodies can be used in combination with CAR T cells in the treatment of lung cancer to enhance Efficacy.

In summary, we generated a third-generation uPAR CAR molecule with therapeutic effects both in vitro and in vivo. The engineered CAR molecule of the present invention can induce enhanced T cell activity on NSCLC cancer cells presenting uPAR antigen without combining it with senescence-inducing agents, and has achieved significantly good therapy in various NSCLC lung cancer animal models, achieving significant The overall survival of tumor-bearing animals was improved, thus providing a powerful treatment option for patients with uPAR-positive NSCLC lung cancer.

The above-described embodiments only describe the preferred modes of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, those of ordinary skill in the art can make various modifications to the technical solutions of the present invention. All deformations and improvements shall fall within the protection scope determined by the claims of the present invention.

Some embodiments of the invention:

1. A chimeric antigen receptor (CAR) polypeptide targeting uPAR, the chimeric antigen receptor polypeptide, from the N-terminus to the C-terminus, includes:

(i) The extracellular antigen-binding domain that specifically binds uPAR;

(ii) optionally, a hinge/spacer region;

(iii) Transmembrane domain;

(v) CD3ζ signaling domain.

2. The CAR polypeptide of embodiment 1, wherein the extracellular antigen-binding domain that specifically binds uPAR is an antibody or antibody fragment, especially a scFv,

Preferably, the antigen-binding domain includes: LCDR1-3 in the VL amino acid sequence of SEQ ID NO:3 and HCDR1-3 in the VH amino acid sequence of SEQ ID NO:4 (especially the CDR sequence defined by Kabat, or LCDR1-3 and HCDR1-3 sequences shown in SEQ ID NOs: 13-18), further preferably comprising the VL of SEQ ID NO: 3 and the VH of SEQ ID NO: 4,

More preferably, the antigen-binding domain is one comprising SEQ ID NO: 2 or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identity thereto. scFv.

3. The CAR polypeptide of any one of embodiments 1-2, wherein the hinge region/spacer is selected from: a hinge region from IgG or a spacer from CD8α or CD28 extracellular region, and is preferably a human CD8α spacer Or a CD28 spacer, for example, a CD28 spacer comprising the amino acid sequence shown in SEQ ID NO: 6.

4. The CAR polypeptide of any one of embodiments 1-3, wherein the transmembrane domain is selected from the group consisting of: transmembrane domains of CD4, CD8, CD28 and CD3ζ, and is preferably human CD8 transmembrane domain or CD28 transmembrane domain. Membrane domain, alternatively, wherein said transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 7 or 22.

5. The CAR polypeptide of any one of embodiments 1-4, wherein the CD28 costimulatory domain comprises the amino acid sequence shown in SEQ ID NO: 11.

6. The CAR polypeptide of any one of embodiments 1-5, wherein the 4-1BB costimulatory domain comprises the amino acid sequence shown in SEQ ID NO: 10.

7. The CAR polypeptide of any one of embodiments 1-6, wherein the CD3ζ signaling domain comprises the amino acid sequence shown in SEQ ID NO: 12.

8. The CAR polypeptide of any one of embodiments 1-6, wherein the CAR polypeptide, from the N-terminus to the C-terminus, includes:

(a) Anti-uPAR scFv shown in SEQ ID NO:2;

(b) The CD28 spacer region shown in SEQ ID NO:6 and the CD28 transmembrane domain shown in SEQ ID NO:7;

(iv) the CD3ζ signaling domain shown in SEQ ID NO:12,

9. A nucleic acid molecule, characterized in that it encodes the chimeric antigen receptor polypeptide described in any one of embodiments 1-8, preferably, wherein the uPAR extracellular antigen binding structure of the chimeric antigen receptor polypeptide A domain is encoded by the nucleotide sequence of SEQ ID NO: 1, or by a nucleotide sequence that is at least 95%, 96%, 97%, 98%, 99% or 99.5% identical thereto.

10. A recombinant vector, characterized by comprising the nucleic acid molecule described in Embodiment 9, for example, the vector is selected from the group consisting of DNA vectors, RNA vectors, lentiviral vectors, adenoviral vectors or retroviral vectors, preferably, Retroviral vectors.

11. A host cell, characterized by comprising the chimeric antigen receptor polypeptide described in any one of embodiments 1-8, the nucleic acid molecule described in embodiment 9, or the vector described in embodiment 10, wherein The cells are preferably immune effector cells, such as T cells or NK cells, for example, the T cells are autologous or allogeneic T cells.

12. A CAR-T cell, wherein the cell comprises the chimeric antigen receptor polypeptide of any one of embodiments 1-8 or the nucleic acid molecule of embodiment 9.

13. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the chimeric antigen receptor polypeptide described in any one of embodiments 1-8, the nucleic acid molecule described in embodiment 9, or the nucleic acid molecule described in embodiment 11 recombinant cells, or the CAR-T cells described in Embodiment 12.

14. The pharmaceutical composition of embodiment 13, further comprising a PD-1 inhibitor or PD-L1 inhibitor, preferably an anti-PD-1 antibody.

15. Use of an engineered immune effector cell in the preparation of a medicament for the treatment of uPAR-positive non-small cell lung cancer (NSCLC) in an individual in need thereof, wherein the engineered immune effector cell comprises embodiments 1-8 The uPAR-targeting chimeric antigen receptor polypeptide of any one of the above.

16. A method of treating non-small cell lung cancer (NSCLC), comprising administering to an individual in need thereof an engineered immune effector comprising the uPAR-targeting chimeric antigen receptor polypeptide of any one of embodiments 1-8 cell.

17. Use according to embodiment 15 or method according to embodiment 16, wherein the immune effector cells are T cells.

18. The use or method according to embodiment 17, wherein the NSCLC is large cell lung cancer, adenocarcinoma or squamous cell carcinoma.

19. The use or method of embodiment 17, wherein the individual has preinvasive cancer or carcinoma in situ, or metastatic cancer, such as brain metastasis.

20. Use or method according to embodiment 17, wherein said individual is a subrace of human, such as Chinese.

21. The use or method according to embodiment 17, wherein the individual is an adult individual over 30 years old, or an adult individual over 60 years old.

22. The use or method according to embodiments 17-18, further comprising administering to said individual an immune checkpoint inhibitor, such as a PD-1 or PD-L1 inhibitor or a LAG-3 inhibitor, for example, in said One or more doses of a PD-1 inhibitor, especially an anti-PD-1 antibody, are administered before, during and/or after the CAR-T cell administration.

23. The use or method according to embodiments 17-19, wherein the uPAR positive expression rate of the tumor is determined by immunohistochemical staining on a tumor biopsy from the individual prior to administration of the CAR-T cells,

Preferably, the individual has NSCLC tumors with a uPAR positive expression rate of 25% to 80% or more, that is, approximately 25-80% or more of the tumor cells exhibit uPAR positive expression on the cell surface.

sequence list

Claims

A chimeric antigen receptor (CAR) polypeptide targeting uPAR, the chimeric antigen receptor polypeptide, from the N-terminus to the C-terminus, includes:

(i) The extracellular antigen-binding domain that specifically binds uPAR;

(ii) optionally, a hinge/spacer region;

(iii) Transmembrane domain;

(iv) a combination of a CD28 costimulatory domain and a 4-1BB costimulatory domain; and

(v) CD3ζ signaling domain,

Preferably, wherein the extracellular antigen-binding domain that specifically binds uPAR is an antibody or antibody fragment, especially scFv,

Preferably, the antigen-binding domain includes: LCDR1-3 in the VL amino acid sequence of SEQ ID NO:3 and HCDR1-3 in the VH amino acid sequence of SEQ ID NO:4 (especially the CDR sequence defined by Kabat, or the LCDR1-3 and HCDR1-3 sequences shown in SEQ ID NOs: 13-18), further preferably, comprising the VL of SEQ ID NO: 3 and the VH of SEQ ID NO: 4,

More preferably, the antigen-binding domain is one comprising SEQ ID NO: 2 or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identity thereto. scFv.
The CAR polypeptide of claim 1, wherein the hinge region/spacer is selected from: a hinge region from IgG or a spacer from CD8α or CD28 extracellular region, and is preferably a human CD8α spacer or a CD28 spacer, for example, A CD28 spacer region comprising the amino acid sequence shown in SEQ ID NO: 6; and/or

Wherein, the transmembrane domain is selected from: the transmembrane domain of CD4, CD8, CD28 and CD3ζ, and is preferably a human CD8 transmembrane domain or a CD28 transmembrane domain, or wherein the transmembrane domain comprises The amino acid sequence shown in SEQ ID NO:7 or 22.
The CAR polypeptide of any one of claims 1-2, wherein the CD28 costimulatory domain comprises the amino acid sequence shown in SEQ ID NO: 11; and/or wherein the 4-1BB costimulatory domain comprises SEQ ID NO :10 shows the amino acid sequence.
The CAR polypeptide of any one of claims 1-3, wherein the CD3ζ signaling domain comprises the amino acid sequence shown in SEQ ID NO: 12;

Preferably, the CAR polypeptide comprises an amino acid sequence having the amino acid sequence shown in SEQ ID NO: 20 or having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identity therewith. cytoplasmic domain.
The CAR polypeptide of any one of claims 1 to 4, wherein the CAR polypeptide, from the N-terminus to the C-terminus, includes:

(a) Anti-uPAR scFv shown in SEQ ID NO:2;

(b) The CD28 spacer region shown in SEQ ID NO:6 and the CD28 transmembrane domain shown in SEQ ID NO:7;

(c) a combination of the CD28 costimulatory domain shown in SEQ ID NO: 11 and the 4-1BB costimulatory domain shown in SEQ ID NO: 10; and

(iv) the CD3ζ signaling domain shown in SEQ ID NO:12,

Preferably, the CAR polypeptide comprises SEQ ID NO: 21 or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identity thereto.
A nucleic acid molecule, characterized in that it encodes the chimeric antigen receptor polypeptide according to any one of claims 1 to 5, preferably, wherein the uPAR extracellular antigen binding domain of the chimeric antigen receptor polypeptide is composed of The nucleotide sequence of SEQ ID NO: 1 encodes, or is encoded by, a nucleotide sequence that is at least 95%, 96%, 97%, 98%, 99% or 99.5% identical thereto.
A recombinant vector, characterized by comprising the nucleic acid molecule of claim 6, for example, the vector is selected from the group consisting of DNA vectors, RNA vectors, lentiviral vectors, adenovirus vectors or retroviral vectors, preferably, reverse transcription Viral vectors.
A CAR-T cell, wherein the cell contains the chimeric antigen receptor polypeptide of any one of claims 1-5 or the nucleic acid molecule of claim 6.
A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the chimeric antigen receptor polypeptide described in any one of claims 1-5, the nucleic acid molecule described in claim 6, or the nucleic acid molecule described in claim 8 CAR-T cells,

Preferably, the pharmaceutical composition further includes a PD-1 inhibitor or PD-L1 inhibitor, preferably an anti-PD-1 antibody.
Use of an engineered immune effector cell in the preparation of a medicament for the treatment of uPAR-positive non-small cell lung cancer (NSCLC) in an individual in need thereof, wherein the engineered immune effector cell comprises any one of claims 1-5 The uPAR-targeting chimeric antigen receptor polypeptide or the nucleic acid molecule of claim 6,

Preferably, wherein the immune effector cells are T cells,

Preferably, wherein the NSCLC is large cell lung cancer, adenocarcinoma or squamous cell carcinoma;

Preferably, wherein said individual has pre-invasive cancer or carcinoma in situ, or metastatic cancer, such as brain metastasis;

Preferably, further comprising administering an immune checkpoint inhibitor, such as a PD-1 or PD-L1 inhibitor or a LAG-3 inhibitor, to the individual, for example, before, during and/or during the administration of the CAR-T cells. or thereafter, administration of one or more doses of a PD-1 inhibitor, especially an anti-PD-1 antibody;

Preferably, wherein, before administering the CAR-T cells, the uPAR positive expression rate of the tumor is determined by immunohistochemical staining on a tumor biopsy from the individual,

Preferably, the individual has NSCLC tumors with a uPAR positive expression rate of 25% to 80% or more, that is, approximately 25-80% or more of the tumor cells exhibit uPAR positive expression on the cell surface.