CN115925985B

CN115925985B - CAR-T cells and their use in the treatment of non-small cell lung cancer

Info

Publication number: CN115925985B
Application number: CN202211031559.1A
Authority: CN
Inventors: 钟晓松; 仝帅
Original assignee: Carrizi Beijing Life Technology Co ltd
Current assignee: Carrizi Beijing Life Technology Co ltd
Priority date: 2022-08-26
Filing date: 2022-08-26
Publication date: 2023-10-31
Anticipated expiration: 2042-08-26
Also published as: CN115925985A; WO2024040681A1

Abstract

The present invention relates to tertiary 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 the 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 use in the treatment of non-small cell lung cancer

Technical Field

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

Background

Urokinase-type plasminogen activator receptor (uPAR, also known as urokinase receptor or CD-87), a cysteine-rich glycosylated single chain protein, was found in 1985 to have a relative molecular weight of 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 coding gene PLAUR of uPAR codes for a protein consisting of 335 amino acids, whose N-terminus comprises a secretion signal peptide of 22 amino acids, while the 30 amino acids of the C-terminus are bound to the cell membrane via a Glycosyl Phosphatidylinositol (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 is made up of three domains of 81 to 87 amino acids in length, namely D1, D2 and D3 domains, joined 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-type plasminogen activator (uPA). The D3 region, which is linked to the D1 and D2 regions, anchors uPAR to the cell membrane surface via GPI.

As a multifunctional protein uPAR is believed to have 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 [ plurog, m., structure-function relationships in the interaction between the urokinase-type plasminogen activator and its receiver, 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 has been demonstrated in tumor samples obtained from colon and breast cancer patients to be potentially associated with tumor metastasis potential and advanced disease. Increased levels of expression of uPAR in tumor tissue and its relative lack of expression in normal, resting tissue, and the role of uPAR in angiogenesis regulation and tumor progression suggest that uPAR is a potential target for cancer treatment [ pilay, v., c.r.dass, and p.f. choong, the urokinase plasminogen activator receptor as a gene therapy target for cancer trends biotechnology, 2007.25 (1): p.33-9].

Chimeric antigen receptor (Chimeric antigen receptor, CAR) is an artificially synthesized molecule that directs immune effector cells (e.g., T cells, NK cells) genetically engineered to express CAR to clear tumors by specifically recognizing antigens expressed on the tumor cell surface (Sampson JH, choi BD, sanchez-Perez L et al, egfrvllimcar-modified T-cell therapy cures mice with established intracerebralglioma and generates host immunity against tumor-anti 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 methods Nature,2020.583 (7814): p.127-132] in the study of aging models, proposed the use of a second-generation CAR molecule targeting uPAR as an anti-aging drug to eliminate aging cells in vivo due to a variety of factors. Studies have shown that following administration of senescence-inducing agents, the second generation uPAR CAR T cells exhibit some clearance of lung cancer tumor cells induced to develop a marker of aging. However, the study by Amor et al also showed that the combination of this senescence-inducing agent with a second generation CAR molecule was used in vivo in a tumor-bearing mouse model with very limited prolongation of overall survival of the animals, and that mice receiving uPAR CAR T cells died within 40 days relative to 30 days in control mice. In addition, the dose of the senescence inducer and the possible interference of the physiological process of normal human cells also present a number of safety and efficacy issues for the application of the combination strategy in clinical cancer therapy.

According to the current state of the art, lung cancer is still the most common cause of cancer-related death worldwide. Over 200 tens of thousands of people are diagnosed with Lung cancer, with 176 tens of thousands dying each year from the disease [ Thai, a.a., et al, lung cancer, lancet,2021.398 (10299): p.535-554]. The 5-year survival rate of Lung cancer varies from 4% to 17% due to stage and regional differences, and the disease still cannot be well treated by 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]. Accordingly, there is a continuing need in the art to develop new lung cancer therapies to improve patient outcome and prognosis.

Summary of The Invention

In intensive studies, the present inventors found that uPAR appears to be significantly positive expressed in cancer tissues of a part of non-small cell lung cancer (NSCLC) patients, and found that high expression levels of uPAR are associated with low survival of NSCLC patients. Based on the above, the inventor constructs a uPAR targeting third-generation CAR molecule; the therapeutic utility of the third generation CAR molecules of the invention was confirmed by detecting selective cytotoxicity of T cells transduced with the CAR molecules alone on uPAR positive NSCLC cancer cells in vitro, and anti-tumor effects in subcutaneous, metastatic, pre-invasive NSCLC lung cancer animal models and PDX models. Further, the inventors have disclosed the molecular mechanism of the uPAR CAR-T cells of the invention in NSCLC lung cancer treatment using high throughput RNA sequencing, and obtained a series of gene expression patterns useful for predicting CAR-T cell therapeutic efficiency; furthermore, a combination therapy of the third generation CAR-T cells and PD-1 blockers was proposed, and an improvement in therapeutic efficacy was confirmed in the PDX model of NSCLC. Based on these studies, the present inventors have thus established the present invention.

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

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

(ii) Optionally, a hinge/spacer;

(iii) A transmembrane domain;

(iv) A combination of a CD28 co-stimulatory domain and a 4-1BB co-stimulatory domain; and

(v) A CD3 zeta signaling domain,

preferably, the uPAR extracellular antigen binding domain is encoded by the optimized nucleotide sequence of SEQ ID NO. 1 or a nucleotide sequence having at least 95%, 96%, 97%, 98%, 99% or 99.5% identity thereto.

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

preferably, the antigen binding domain comprises: LCDR1-3 of the VL amino acid sequence of SEQ ID NO. 3 and HCDR1-3 of the VH amino acid sequence of SEQ ID NO. 4 (especially the Kabat defined CDR sequences, or the LCDR1-3 and HCDR1-3 sequences shown in SEQ ID NOs: 13-18), more preferably the antigen binding domain is an anti-uPAR comprising SEQ ID NO. 2 or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identity thereto and/or comprising SEQ ID NO. 4 or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identity thereto, more preferably comprising the VL of SEQ ID NO. 3 and the VH of SEQ ID NO. 4, more preferably the antigen binding domain is an anti-uPAR comprising SEQ ID NO. 2 or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

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 the group consisting of: hinge region from IgG or spacer region from CD8 a or CD28 extracellular region, and preferably human CD8 a spacer region or CD28 spacer region. In other embodiments, the hinge/spacer region comprises the amino acid sequence of SEQ ID NO. 6, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identity thereto, or an amino acid sequence differing therefrom by NO more than 1-5 amino acid residue modifications (e.g., substitutions, insertions and/or deletions).

In some embodiments, the transmembrane domain is selected from the group consisting of: the transmembrane domains of CD4, CD8, CD28 and CD3 zeta, and preferably are human CD8 transmembrane domains or CD28 transmembrane domains. In other embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO 7 or 22, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identity thereto, or an amino acid sequence that differs therefrom by NO more than 1-5 amino acid residue modifications (e.g., substitutions, insertions and/or deletions).

In some embodiments, the CD28 co-stimulatory domain comprises the amino acid sequence of SEQ ID NO. 11, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identity thereto, or an amino acid sequence differing therefrom by NO more than 1-5 amino acid residue modifications (e.g., 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 an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identity thereto, or an amino acid sequence differing therefrom by NO more than 1-5 amino acid residue modifications (e.g., substitutions, insertions and/or deletions).

In some embodiments, the CD3 zeta signaling domain comprises the amino acid sequence shown in SEQ ID NO. 12, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identity thereto, or an amino acid sequence differing therefrom by NO more than 1-5 amino acid residue modifications (e.g., substitutions, insertions and/or deletions).

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

(a) An anti-uPAR scFv shown in SEQ ID NO. 2;

(b) A CD28 spacer shown in SEQ ID NO. 6 and a CD28 transmembrane domain of SEQ ID NO. 7;

(c) A combination of the CD28 co-stimulatory domain of SEQ ID NO. 11 and the 4-1BB co-stimulatory domain of SEQ ID NO. 10; and

(iv) A CD3 zeta 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 a nucleic acid molecule encoding a chimeric antigen receptor polypeptide described herein, a vector comprising a nucleic acid encoding a CAR polypeptide described herein, and a cell comprising a CAR nucleic acid molecule or vector described herein, or a cell expressing a CAR polypeptide described herein, preferably the cell is an autologous T cell or an allogeneic T cell.

In one embodiment, the invention prepares primary CAR-T cells using human PBMCs. The CAR-T cells transduced with the CAR molecules of the invention have an in vitro effector function and have the activity of continuously killing target cells in vitro. In yet another embodiment, CAR-T cells transduced with CAR molecules of the invention also have the function of killing tumor cells in vivo, exhibiting significant anti-tumor activity in animal subjects with subcutaneous, pre-invasive, and/or metastatic NSCLC lung cancer.

In a third aspect, the invention provides a method of producing a cell, e.g., an immune effector cell, comprising introducing (e.g., transducing) a nucleic acid molecule (e.g., an RNA molecule, such as an mRNA molecule) encoding a CAR polypeptide described herein, or a vector comprising a nucleic acid molecule encoding a CAR polypeptide described herein, into an immune effector cell.

In some embodiments, the immune effector cells are T cells, NK cells, e.g., the T cells are autologous T cells or allogeneic T cells, e.g., the immune effector cells are prepared after isolation of T cells, NK cells from human PBMCs.

In some embodiments, a nucleic acid encoding a CAR molecule of the invention is introduced into a primary T cell with a retrovirus, resulting in a CAR-T cell of the invention.

In some embodiments, the CAR-T cells of the invention exhibit a differentially expressed gene that is associated with anti-tumor activity upon contact with a target tumor cell. In some embodiments, genes associated with BP, MF and CC are upregulated following exposure of the CAR-T cell to a target tumor: the response of cells to interferon-gamma, immune response, inflammatory response, and tumor necrosis factor-activated receptor activity. In other embodiments, genes associated with BP, MF and CC below are down-regulated in expression following contact of the CAR-T cell with a target tumor: gene expression regulation, DNA replication, mitotic cell cycle G1/S switching, protein binding and spindle pole (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, the up-regulation of expression of a gene selected from the group consisting of: IL2, IL9, IFN-gamma, TNFRSF9 and IL17A genes, as well as chemokine genes such as CXCL1, CXCL5 and CXCL8, to indicate the therapeutic response of the patient. In other embodiments, the up-regulation of expression of a gene selected from the group consisting of: PD-1, PD-L2 and/or Lag-3 to indicate the likelihood of recurrence in the patient.

In a fourth aspect, the invention provides a pharmaceutical composition 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 CAR-T cell described herein. In some preferred embodiments, the pharmaceutical composition further comprises a PD-1 inhibitor or a PD-L1 inhibitor, preferably an anti-PD-1 antibody, preferably the pharmaceutical composition is provided in a pharmaceutical combination, wherein the CAR-T cells and PD-L1 inhibitor described herein are contained in separate formulations in a manner that facilitates separate, sequential and/or simultaneous administration.

In a fifth aspect, the present invention provides the use of an engineered immune effector cell comprising a chimeric antigen receptor polypeptide targeting uPAR as described herein in the manufacture of a medicament for treating uPAR positive non-small cell lung cancer (NSCLC) in an individual in need thereof and a method of treating uPAR positive non-small cell lung cancer (NSCLC) using the engineered immune effector cell.

In some embodiments, the immune effector cell is a T cell, e.g., an autologous T cell or an allogeneic T cell.

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

In some embodiments, the subject has pre-infiltration NSCLC lung cancer or orthotopic NSCLC lung cancer. In other embodiments, the subject has metastatic NSCLC cancer, e.g., brain metastasis of NSCLC.

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

In some embodiments, the individual is an asian, such as a chinese.

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

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

In some embodiments, the methods and uses comprise administering to the subject one or more doses of the CAR-T cells described herein, e.g., the doses can be administered continuously or at intervals. In some preferred embodiments, the methods of treatment and uses described herein further comprise administering to the individual an immunodetection point inhibitor, e.g., a PD-1 or PD-L1 inhibitor or LAG-3 inhibitor, e.g., one or more doses of the inhibitor, particularly a PD-1 inhibitor, e.g., an anti-PD-1 antibody, prior to, during, and/or after administration of the CAR-T cells.

Brief Description of Drawings

The preferred embodiments of the present invention described in detail below will be better understood when read in conjunction with the following drawings. For the purpose of illustrating the invention, there is shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

Fig. 1 shows: lung cancer patients with relatively high uPAR expression in tumors have significantly lower survival (p <0.001, log-Rank test).

Fig. 2 shows: immunohistochemistry detects uPAR levels in lung cancer tumors, wherein: (a) 4 positives and (b) 8 negatives. A 100-fold magnification of a representative immunohistochemical stained section and a 200-fold magnification of its local area are shown.

Fig. 3 shows: a schematic of a Chimeric Antigen Receptor (CAR) targeting uPAR, consisting of uPAR scFv, hinge and Transmembrane (TM) regions from CD28, co-stimulatory domains from CD28 and 4-1BB, and signaling domains from CD3 ζ. Wherein SD represents a splice donor site; SA represents a splice acceptor site; LTR means long terminal repeat.

Fig. 4 shows: positive CAR T cells were quantified by flow cytometry after transduction of T cells with CAR-encoding retroviral vectors. Wherein GAM represents goat anti-mouse IgG (Fab-specific) F (ab') 2 fragment-FITC antibody (GAM, sigma) staining.

Fig. 5 shows: in vitro Activity of uPAR CAR-T on uPAR positive tumor cells. (a) The uPAR expressed on the surface of cancer cells was stained with APC-labeled monoclonal antibodies. Human lung cancer cell line H460 has a uPAR positive rate of 95.4%; in the human lung cancer cell line A549 with transgenic overexpression of uPAR, the uPAR positive rate was 83.5%. (b) CFSE-labeled CAR-T cells were co-cultured with uPAR positive tumor cells at an e:t ratio of 2:1 for 12 days. T cells were stimulated with fresh tumor cells every three days and counted each time before tumor cells were added to determine T cell fold expansion. (c) CD107a levels on CAR-T cells were detected by flow cytometry assays after 6 hours of co-culture of CAR-T cells with uPAR positive tumor cells (E: t=10:1). (d) After 24 hours of co-culture of CAR-T cells with uPAR positive tumor cells (E: t=10:1), supernatants were collected and IFN- γ levels were assessed by ELISA. The results of fig. 5 (b) and (d) were analyzed by student t test, with p <0.05 considered significant. * P <0.05 and p <0.01, p <0.001.

Fig. 6 shows: 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 are combined with luciferase-expressing different target cells (H460 and uPAR ⁺ A549 For 24 hours at different E:T ratios (1:1, 2.5:1, 5:1, and 10:1), tumor cell lysis was examined using an IVIS imaging system. (b) In an impedance-based tumor cell killing assay, tumor cells were co-cultured with T cells, using an xcelligent impedance system, and from the start time of the co-culture, a continuous graphical output of cell index values up to the 12 hour time point was monitored.

Fig. 7 shows: in vivo tumor model. (a) Will be 2x 10 ⁶ Individual eGFP-Luc-H460 cells (luciferase and eGFP-labeled H460 tumor cells) were subcutaneously injected under the left underarm of 6-8 week old female NOD-SCID mice. After three days, 2x 10 will be three days in succession ⁷ uPAR CAR-T cells were injected directly into tumors to treat tumor-bearing mice, and non-transduced T cells (NTs) were used as controls. (b) tumor burden fluorescence profile of mice. Mice treated with uPAR CAR-T showed significant prolongation of survival relative to control mice, with some mice surviving for more than 84 days. (c) Application IVThe IS imaging system acquires quantitative bioluminescence imaging data (i.e., absolute photon count (photons/sec/cm) emitted from the animal body surface per unit time, unit area, unit radian) of all mice ² /sr)). Higher values indicate greater tumor burden. (d) The overall survival of lung cancer xenograft tumor-bearing mice was measured using the Kaplan-Meier method and compared between groups using Cox proportional hazards regression analysis. p values less than 0.05 were considered significant.

Fig. 8 shows: tumor cells were isolated from a lung xenograft mouse model of tumor recurrence following uPAR CAR T cell therapy and co-cultured with uPAR CAR-T cells in vitro to confirm the anti-tumor activity of the CAR-T cells. "control mouse cells" means: tumor cells were isolated from mice in the group untreated with CAR-T; "recurrent mouse cells" means: tumor cells were isolated from CAR-T treated mice. NT represents non-transduced T cells. (a) After 24 hours of co-culture at t=10:1, the IFN- γ levels in the culture supernatants were detected by ELISA. * Represents p <0.01. (b) CD107a expression levels on uPAR CAR T cells were flow tested after 6 hours co-culture at E: t=10:1. (c) In an in vitro killing assay, tumor cell lysis was measured after 24 hours of co-culture of uPAR CAR-T cells with different target tumor cells (E: t=1:1, 2.5:1, 5:1 and 10:1).

Fig. 9 shows: in vivo tumor model. (a) Will be 2x 10 ⁶ Individual eGFP-Luc-H460 cells (luciferase and eGFP-labeled H460 tumor cells) were implanted in situ into lung parenchyma of 6-8 week old female NOD-SCID mice by pleural injection. After three days, 2x 10 will be three days in succession ⁷ uPAR CAR-T cells were injected intraperitoneally (i.p.) into tumors to treat tumor-bearing mice, and non-transduced T cells (NTs) were used as controls. (b) tumor burden fluorescence profile of mice. Mice treated with uPAR CAR-T showed significant prolongation of survival relative to control mice. (c) Obtaining quantitative bioluminescence imaging data (i.e., absolute photon count (p/sec/cm) per unit time, unit area, unit radian from animal body surface using an IVIS imaging system ² /sr)). Higher values indicate greater tumor burden. (d) The overall survival of lung cancer in situ xenograft tumor-bearing mice was measured using the Kaplan-Meier method and usingCox proportional risk regression analysis performed the group comparisons. p values less than 0.05 were considered significant.

Fig. 10 shows: tumor cells were isolated from an in situ xenograft mouse model of tumor recurrence following uPAR CAR T cell therapy and co-cultured with uPAR CAR-T cells in vitro to confirm the anti-tumor activity of the CAR-T cells. "control mouse cells" means: tumor cells were isolated from mice in the group untreated with CAR-T; "recurrent mouse cells" means: tumor cells were isolated from CAR-T treated mice. NT represents non-transduced T cells. (a) After 24 hours of co-culture at t=10:1, the IFN- γ levels in the culture supernatants were detected by ELISA. * Represents p <0.001. (b) CD107a expression levels on uPAR CAR T cells were flow tested after 6 hours co-culture at E: t=10:1. (c) In an in vitro killing assay, tumor cell lysis was measured after 24 hours of co-culture of uPAR CAR-T cells with different target tumor cells (E: t=1:1, 2.5:1, 5:1 and 10:1).

Fig. 11 shows: in vivo tumor model. (a) Will be 2x 10 ⁵ Individual 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, tumor-bearing mice were injected intravenously (i.v.) for 2x 10 three consecutive days ⁷ uPAR CAR-T cells and non-transduced T cells (NTs) were used as controls. (b) tumor burden fluorescence profile of mice. Mice treated with uPAR CAR-T showed significant prolongation of survival relative to control mice. (c) Obtaining quantitative bioluminescence imaging data (i.e., absolute photon count (p/sec/cm) per unit time, unit area, unit radian from animal body surface using an IVIS imaging system ² /sr)). Higher values indicate greater tumor burden. (d) The overall survival of lung cancer intracranial xenograft tumor-bearing mice was measured using the Kaplan-Meier method and compared between groups using Cox proportional hazards regression analysis. p values less than 0.05 were considered significant.

Fig. 12 shows: tumor cells were isolated from an intracranial xenograft mouse model of tumor recurrence following uPAR CAR T cell therapy and co-cultured with uPAR CAR-T cells in vitro to confirm the anti-tumor activity of the CAR-T cells. "control mouse cells" means: tumor cells were isolated from mice in the group untreated with CAR-T; "recurrent mouse cells" means: tumor cells were isolated from CAR-T treated mice. NT represents non-transduced T cells. (a) After 24 hours of co-culture at t=10:1, the IFN- γ levels in the culture supernatants were detected by ELISA. * Represents p <0.01. (b) CD107a expression levels on uPAR CAR T cells were flow tested after 6 hours co-culture at E: t=10:1. (c) In an in vitro killing assay, tumor cell lysis was measured after 24 hours of co-culture of uPAR CAR-T cells with different target cells (E: t=1:1, 2.5:1, 5:1 and 10:1).

Fig. 13 shows: analysis of gene ontology enrichment of differentially expressed genes after co-culture of uPAR CAR-T cells with tumor cells (Gene Ontology Enrichment Analysis). Volcanic plot of Differentially Expressed Gene (DEG) (i.e., gene with fold change in expression level (FC) >2 before and after co-cultivation and adjusted p-value < 0.05). The horizontal axis represents the fold change, and the vertical axis represents the adjusted p-value.

Fig. 14 shows: using an online bioinformatics tool: DAVID Bioinformatics Resources 6.8.8 GO analysis was performed on the differentially expressed genes between pre-co-cultured and post-co-cultured CAR-T cells. Fisher's exact assay was used for this gene enrichment analysis. BP represents: a biological process; CC represents: a cellular component; MF represents: molecular function.

Fig. 15 shows: (a) PPI analysis was performed on differentially expressed genes between CAR-T cells before and after co-culture. Protein-protein interaction profile of genes up-regulated in CAR-T cells after co-culture relative to CAR-T cells before co-culture. (b) Volcanic map of differentially expressed genes of CAR-T cells co-cultured for 30 minutes relative to CAR-T cells not co-cultured. (c) The 30 min co-cultured CAR-T cells had 133 up-regulated genes in both and 22 down-regulated genes in both compared to the 4 hr co-cultured CAR-T cells.

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

Fig. 17 shows: after co-culture with tumor cells, the CAR-T cells up-regulate gene expression. (a) H460 cells were co-cultured with CAR-T cells at a E:T ratio of 2:1 for 30 minutes and 4 hours. NT represents non-transduced T cells. RT-qPCR was then performed on CAR-T cells to determine PD-1 and PDCD1LG2 levels; and the PD-L1 level was measured on H460 cells. (b) After uPAR CAR-T cells were co-cultured with H460 cells and upar+a549 cells for 48 hours at E: t=10:1, PD-1, lag-3, and Tim-3 expression levels of CAR-T cells were flow-tested.

Fig. 18 shows: PD-1/PD-L1 inhibits the anti-tumor activity of uPAR CAR-T cells. (a) And (b) transiently transfecting the H460 cells with the siRNA to reduce expression of PD-L1. H460-si-PD-L1-NC represents: treating the H460 cells of PD-L1 with a control siRNA; h460-si-PD-L1- #1 and #2 represent respectively: h460 cells treated with two siRNAs directed against different sequences of PD-L1. (a) By PCR detection, cells had significantly reduced PD-L1 mRNA expression levels following siRNA knockdown treatment. (b) Through flow-through detection, after siRNA knockdown treatment, cell surface PD-L1 expression of tumor cells decreased. (c) uPAR CAR-T cells were co-cultured with tumor cells knocked down and non-knocked down with PD-L1 at E: t=10:1, and secreted IFN- γ levels were detected. (d) In vitro killing experiments were performed with E:T=2.5:1, co-culturing uPAR CAR-T cells with tumor cells knocked down and non-knocked down PD-L1. The lysis rate of tumor cells was determined by detecting luciferase activity. Results were analyzed by one-way variance analysis, with statistical significance set at p <0.05.* P <0.05, < P <0.01, < P <0.001. (e) CD107a expression levels of CAR-T cells were detected 24 hours after co-culturing uPAR CAR-T cells with tumor cells knocked down and non-knocked down PD-L1 at E: t=10:1.

Fig. 19 shows: PD-1 antibodies in combination with CAR-T cell therapy significantly inhibited tumor growth in lung cancer PDX models. The results show that the mice treated with CAR-T had significantly reduced subcutaneous tumor tissue relative to the untreated group, and that the mice treated with CAR-T cells in combination with PD-1 antibodies had significantly reduced subcutaneous tumor tissue relative to the CAR-T alone treated group. (a) in vivo lung cancer PDX model generation schematic. (b) and (c) measuring tumor volume: tumor tissue is taken from a mouse after sudden death of the neck and is measured by a vernier caliper, and the volume calculation formula is as follows: length x width/2. (d) measuring tumor weight: tumor weight measurements were made on a ten-thousandth analytical balance of tumor tissue taken after sudden cervical death of the mice. * The representation is: p <0.05; * Represents: p <0.01; * Represents: p <0.005.

Detailed Description

Unless defined otherwise, 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. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

Definition of the definition

For purposes of explaining the present specification, the following definitions will be used, and terms used in the singular form may also include the plural, and vice versa, as appropriate. 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 numerical values within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value.

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

In this document, the terms "comprises" or "comprising" when used herein, unless otherwise indicated, also encompass the circumstance of consisting of the recited elements, integers or steps. For example, when referring to an antibody variable region "comprising" a particular sequence, it is also intended to encompass antibody variable regions consisting of that particular sequence.

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

The term "stimulatory molecule" refers to a molecule expressed by a T cell that provides a primary cytoplasmic signaling sequence that modulates primary activation of the TCR complex in a stimulatory manner in at least some aspect of the T cell signaling pathway. In one embodiment, the primary signal initiates and results in mediating T cell responses including, but not limited to, proliferation, activation, differentiation, etc., e.g., through binding of the TCR/CD3 complex to peptide-loaded MHC molecules. In a particular CAR of the invention, the intracellular signaling domain in any one or more CARs of the invention comprises an intracellular signaling sequence, e.g., a primary signaling sequence of cd3ζ.

The term "CD3 zeta" is defined as the protein provided by genbank accession No. BAG36664.1 or an equivalent thereof, and "CD3 zeta signaling sequence" is defined as an amino acid residue from the cytoplasmic domain of the CD3 zeta chain sufficient to functionally propagate the primary signal necessary for T cell activation. In one embodiment, the cytoplasmic domain of cd3ζ comprises residues 52 through 164 of GenBank accession No. BAG36664.1 or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.) as functional orthologs thereof. In one embodiment, the "CD3 zeta signaling sequence" is the sequence provided in SEQ ID NO. 12 or a variant thereof.

The term "costimulatory molecule" refers to an cognate binding partner on a T cell that specifically binds to a costimulatory ligand and thereby mediates the costimulatory response of the T cell (e.g., without limitation, T cell proliferation). Costimulatory molecules are other cell surface molecules in addition to antigen receptors or their ligands that are required for effective immune responses. 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), activation of NK cell receptors, OX40, CD40, GITR, 4-1BB (i.e., CD 137), CD27, and CD28. In some embodiments, the "costimulatory molecule" is CD28, 4-1BB (i.e., CD 137). In this context, a "co-stimulatory domain" refers to the intracellular portion of a co-stimulatory molecule.

The term "4-1BB" refers to a TNFR superfamily member having the amino acid sequence provided as GenBank accession No. AAA62478.2 or an equivalent residue 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 No. AAA62478.2 or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). In one embodiment, a "4-1BB costimulatory domain" is a sequence provided as SEQ ID NO 10 or an equivalent residue 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 (e.g., mouse, rodent, monkey, ape, etc.). The term "CD28 co-stimulatory domain" is defined herein as amino acid residues 180-220 from the cytoplasmic region of CD28, e.g., uniProtKB-P10747, or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). In one embodiment, a "CD28 co-stimulatory domain" is a sequence provided as SEQ ID NO. 11 or an equivalent residue from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). The term "CD28 transmembrane domain" is defined herein as amino acid residues 153-179 from the transmembrane region of CD28, e.g., uniProtKB-P10747, or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). In one embodiment, a "CD28 transmembrane domain" is a sequence provided as SEQ ID NO 7 or an equivalent residue from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). The term "CD28 hinge domain", used interchangeably herein with the term "CD28 spacer", is defined as amino acid residues 114-152 from the extracellular region of CD28, e.g., uniProtKB-P10747, or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). In one embodiment, a "CD28 spacer" is a sequence provided as SEQ ID NO. 6 or an equivalent residue from a non-human species (e.g., mouse, rodent, monkey, ape, etc.).

The terms "amino acid change" and "amino acid modification" are used interchangeably to refer to additions, deletions, substitutions and other modifications of amino acids. Any combination of amino acid additions, deletions, substitutions and other modifications may be made provided that the final polypeptide sequence has the desired properties. In some embodiments, the amino acid substitution is a non-conservative amino acid substitution, i.e., substitution of one amino acid with another amino acid having a different structure and/or chemical property. 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-hydroxylysine).

The terms "conservative sequence modification", "conservative sequence change" refer to an amino acid modification or change that does not significantly affect or alter the characteristics of a parent polypeptide containing the amino acid sequence or its constituent elements. Such conservative modifications include amino acid substitutions, additions and deletions. Conservative modifications, particularly conservative substitutions, may be introduced into the CAR fusion polypeptides of the invention or constituent elements thereof (e.g., CAR or Survivin) by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative substitutions are amino acid substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

"percent (%) identity" of amino acid sequence/nucleotide sequence means that the candidate sequence is identical to that shown in the present specificationAfter alignment and, if necessary, introduction of gaps to achieve the maximum percent sequence identity, and in the case of amino acid sequences, without regard to any conservative substitutions as part of sequence identity, the percentage of amino acid/nucleotide residues in the candidate sequence that are identical to the amino acid residues/nucleotide residues of the particular amino acid/nucleotide sequence shown in the specification. In some embodiments, the invention contemplates variants of the fusion polypeptides or nucleic acid molecules of the invention or constituent elements thereof that have substantial identity, e.g., at least 80%,85%,90%,95%,97%,98% or 99% or more identity, relative to the sequence of the fusion polypeptides or nucleic acid molecules or constituent elements thereof (e.g., CAR polypeptides/encoding nucleic acids, or Survivin proteins/encoding nucleic acids) specifically disclosed herein. The variant may comprise conservative modifications. According to the object of the invention, the percentage identity applieshttps:// blast.ncbi.nlm.nih.govThe above publicly available BLAST tools are determined using default parameters.

As used herein, the expression "variant" or "functional variant" polypeptide or protein refers to a polypeptide or protein that has substantially the same sequence or significant sequence identity as a reference polypeptide or protein and retains the desired biological activity of the reference polypeptide or protein.

The term "vector" as used herein when referring to a nucleic acid 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 and that bind to 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 operatively linked. Such vectors are referred to herein as "expression vectors".

The term "lentivirus" refers to a genus of the retrovirus family (Retroviridae). Lentiviruses are unique among retroviruses in being able to infect non-dividing cells; they can deliver significant amounts of genetic information to host cells, so they are one of the most efficient methods of gene delivery vehicles. HIV, SIV and FIV are all examples of lentiviruses.

The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome and includes, inter alia, as Milone et al, mol. Ther.

17 (8) self-inactivating lentiviral vectors provided in 1453-1464 (2009). Other examples of lentiviral vectors that may be used clinically include, for example, but are not limited to, those from Oxford BioMedicaGene delivery technology, LENTIMAX from Lentigen ^TM Carrier systems, and the like. Non-clinical types of lentiviral vectors are also available and known to those skilled in the art.

The term "immune effector cell" refers to a cell that is involved in an immune response, e.g., involved in promoting an immune effector response. Examples of immune effector cells include T cells, e.g., 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 Receptors (CARs) of the invention

The inventors found in intensive studies that, in the treatment of non-small cell lung cancer, immune cells engineered to express a tertiary CAR polypeptide optimized to target uPAR (e.g., CAR-T cells and CAR-NK cells) can effectively achieve an antitumor immune effect in uPAR positive pre-infiltration/in situ NSCLC lung cancer as well as metastatic NSCLC lung cancer. Based on this, the invention provides a third generation CAR polypeptide optimized for targeting uPAR, immune cells based on said CAR polypeptide, and their use in the treatment of NSCLC patients, alone or in combination with other anti-cancer drugs (in particular PD-1 inhibitors).

The CAR of the present invention and its components are described in detail below. It will be appreciated by those skilled in the art that any feature or combination of features mentioned in the description of the components are contemplated by the present invention unless the context clearly indicates otherwise; also, it will be understood by those skilled in the art that any embodiment of the present invention that is CAR-based (including, but not limited to, CAR-encoding nucleic acids, CAR-based immune cells, and uses thereof) can also comprise any such combination of features, unless the context clearly indicates to the contrary.

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

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

(ii) Optionally, a hinge/spacer;

(iii) A transmembrane domain;

(v) CD3 zeta signaling domain.

Depending on the uPAR antigen to be targeted, the CARs of the invention can be constructed to include an appropriate antigen binding domain specific for the antigen target to confer to the CAR molecule, as well as to CAR-T cells comprising the CAR molecule, the ability to specifically recognize and bind to the target antigen. In one embodiment, the extracellular antigen-binding domain of a CAR molecule according to the invention is a polypeptide molecule having binding affinity for a uPAR target antigen, e.g. an antibody or antibody fragment that specifically binds uPAR or a fragment of a ligand from the antigen receptor. 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 an scFv made from a VL and a VH linked via a linker.

scFv can be produced by joining VH and VL regions together using flexible polypeptide linkers according to methods known in the art. In some embodiments, the scFv molecule comprises a flexible polypeptide linker having an 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 comprise 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 join the variable heavy 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, the flexible polypeptide linker includes, but is not limited to, (Gly 4 Ser) 4 or (Gly 4 Ser) 3. In another embodiment, the linker comprises multiple repeats of (Gly 2 Ser), (GlySer) or (Gly 3 Ser). In yet another embodiment, the linker comprises the GSTSGSGKPGSGEGSTKG amino acid sequence. In one embodiment, the scFv for use in the invention comprises 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 may be derived from natural or synthetic sources. For example, the transmembrane domain may be derived from a membrane-bound protein or a transmembrane protein, such as a transmembrane domain from cd3ζ, CD4, CD28, CD8 (e.g., CD8 a, CD8 β). 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 a CAR of the invention can be linked to the extracellular region of the CAR by a hinge/spacer. For transmembrane and hinge/spacer regions useful in CAR polypeptides, see, e.g., kento Fujiwara et al, cells 2020,9,1182; doi 10.3390/cells9051182.

The cytoplasmic domain comprised in the CAR polypeptides of the invention comprises an intracellular signaling domain. The intracellular signaling domain is capable of activating at least one immune effector function of an immune cell into which the CAR of the invention is introduced. Such immune effector functions include, but are not limited to, for example, enhancing or promoting the function or response of an immune-attacking target cell. The effector function of T cells may be, for example, cytolytic activity or helper activity, including secretion of cytokines.

Examples of cytoplasmic domains for use in the CAR polypeptides of the invention include cytoplasmic sequences of T Cell Receptors (TCRs) and/or co-receptors that can function to initiate signal transduction upon binding of the extracellular domain to a target antigen, 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 different classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation by the TCR (i.e., primary intracellular signaling domains) and those that function in an antigen-independent manner to provide a costimulatory signal (i.e., secondary cytoplasmic domains, e.g., costimulatory domains). In one embodiment, the CAR polypeptide of the invention comprises a cytoplasmic domain that provides a primary intracellular signaling domain, e.g., an intracellular signaling domain of cd3ζ, and further comprises a secondary signaling domain, e.g., a combination of costimulatory domains from 4-1BB (also known as CD 137) and CD 28. In one embodiment, the cytoplasmic region of the CAR polypeptides of the invention comprises CD28 and 4-1BB co-stimulatory domains and a cd3ζ intracellular signaling domain in sequential tandem to ensure effective anti-tumor immunity in uPAR positive pre-invasive/orthotopic NSCLC lung cancer as well as metastatic NSCLC lung cancer.

In some embodiments, a CAR polypeptide of the invention may comprise a signal peptide or leader sequence located N-terminal to an extracellular antigen-binding domain. Through the signal peptide/leader sequence, the nascent CAR polypeptide can be directed to the endoplasmic reticulum of the cell and then anchored to the cell membrane. Any eukaryotic-derived signal peptide/leader sequence may be used, such as a mammalian or human secretory protein-derived signal peptide/leader sequence.

In some embodiments, a Chimeric Antigen Receptor (CAR) polypeptide according to the invention comprises 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 heavy chain complementarity determining region 1 (HC CDR 1), heavy chain complementarity determining region 2 (HC CDR 2) and heavy chain complementarity determining region 3 (HC CDR 3) of the heavy chain variable region (VH) amino acid sequence of SEQ ID NO:4, e.g., the HCDR1-3 amino acid sequences of SEQ ID NO: 16-18; and/or the light chain complementarity determining region 1 (LC CDR 1), light chain complementarity determining region 2 (LC CDR 2) and light chain complementarity determining region 3 (LC CDR 3) of the light chain variable region (VL) amino acid sequence of SEQ ID NO:3, e.g., the LCDR1-3 amino acid sequences 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 comprises: 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; or iii) an amino acid sequence having 95 to 99% identity to 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; or iii) an amino acid sequence having 95-99% identity to 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) having at least one, two or three modifications but not more than 30,20 or 10 modifications of the amino acid sequence of SEQ ID NO. 2; or iii) a sequence which hybridizes with SEQ ID NO:2 having an amino acid sequence of 95-99% identity.

In one embodiment, the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of CD4, CD8 a, CD28, CD3 zeta, TCR zeta, fcRgamma, fcRbeta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD9, CD16, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), fcεRI, CD66d, the alpha, beta or zeta chain of a T cell receptor, an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, and an NK cell receptor activation. In one embodiment, the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of CD4, CD8 a, CD28 and CD3 zeta. In one embodiment, the transmembrane domain comprises i) the amino acid sequence of SEQ ID NO. 7; ii) at least one, two or three modified but not more than 5 modified amino acid sequences comprising the amino acid sequence of SEQ ID NO. 7; or iii) an amino acid sequence having 95-99% sequence identity to SEQ ID NO. 7. In one embodiment, the transmembrane domain comprises i) the amino acid sequence of SEQ ID NO. 22; ii) at least one, two or three modified but not more than 5 modified amino acid sequences comprising the amino acid sequence of SEQ ID NO. 22; or iii) an amino acid sequence having 95-99% sequence identity to SEQ ID NO. 22.

In one embodiment, the cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of TCR zeta, fcRgamma, fcRbeta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In one embodiment, the cytoplasmic domain comprises a functional signaling domain of a CD3 zeta protein (also referred to herein as a CD3 zeta signaling domain). In one embodiment, the cytoplasmic domain comprises i) the amino acid sequence of SEQ ID NO. 12; ii) at least one, two or three modified but not more than 20, 10 or 5 modified amino acid sequences comprising the amino acid sequence of SEQ ID NO. 12; or iii) an amino acid sequence having 95-99% sequence identity to SEQ ID NO. 12.

In one embodiment, the cytoplasmic domain further comprises a costimulatory domain of two proteins selected from the group consisting of: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), activating NK cell receptors, CD8, ICOS, DAP10, DAP12, OX40, CD40, GITR, 4-1BB (i.e., CD 137), CD27, and CD28. In one embodiment, the cytoplasmic domain comprises a co-stimulatory domain of two proteins selected from the group consisting of: the costimulatory domains of CD28, CD27,4-1BB, ICOS and OX 40. In one embodiment, the cytoplasmic domain comprises a combination of co-stimulatory domains of CD28 and 4-1 BB. In one embodiment, the cytoplasmic domain comprises a CD28 co-stimulatory domain and a 4-1BB co-stimulatory domain, wherein the CD28 co-stimulatory domain comprises i) the amino acid sequence of SEQ ID NO. 11; ii) at least one, two or three modified but not more than 20, 10 or 5 modified amino acid sequences comprising the amino acid sequence of SEQ ID NO. 11; or iii) an amino acid sequence having 95-99% identity to the amino acid sequence of SEQ ID NO. 11; and wherein the 4-1BB costimulatory domain comprises i) the amino acid sequence of SEQ ID NO. 10; ii) at least one, two or three modified but not more than 20, 10 or 5 modified amino acid sequences comprising the amino acid sequence of SEQ ID NO. 10; or iii) an amino acid sequence having 95-99% identity to the amino acid sequence of SEQ ID NO. 10.

In one embodiment, the CAR polypeptide further comprises a hinge or spacer region disposed between the transmembrane domain and the extracellular antigen binding domain. In one embodiment, the hinge/spacer 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 CD 28. In one embodiment, the hinge/spacer region comprises i) the amino acid sequence of SEQ ID NO. 6; ii) at least one, two or three modified but not more than 5 modified amino acid sequences comprising the amino acid sequence of SEQ ID NO. 6; or iii) an amino acid sequence having 95-99% identity to the amino acid sequence of SEQ ID NO. 6. The expressions "hinge", "hinge region" and "hinge domain" are used interchangeably herein.

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

In one embodiment, a CAR polypeptide according to the invention comprises i) the amino acid sequence of SEQ ID NO. 21; 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. 21; or iii) an amino acid sequence having at least 95-99% identity to the amino acid sequence of SEQ ID NO. 21.

Nucleic acid molecules encoding the CARs of the invention, vectors, and cells expressing the CARs of the invention

The invention provides nucleic acid molecules encoding the CAR constructs described herein. In one embodiment, the nucleic acid molecule is provided as a DNA construct. Constructs encoding the CARs 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 has been inserted. Expression of the nucleic acid encoding the CAR can be achieved by operably linking the nucleic acid encoding the CAR polypeptide to a promoter, and incorporating the construct into an expression vector. Vectors may be suitable for replication and integration in eukaryotes. Common cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters for regulating the expression of the desired nucleic acid sequence. Numerous virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The nucleic acid construct of the invention may be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus may then be isolated and delivered to cells of the subject 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 achieving long-term gene transfer, as they allow long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have the additional advantage over vectors derived from cancer-retroviruses (e.g., murine leukemia virus) in that they can transduce non-proliferative cells, such as hepatocytes. They also have the additional advantage of low immunogenicity. The retroviral vector may also be, for example, a gamma retroviral vector. The gamma retroviral vector may, for example, comprise a promoter, a packaging signal (ψ), a Primer Binding Site (PBS), one or more (e.g., two) Long Terminal Repeats (LTRs), and a transgene of interest, e.g., a gene encoding a CAR. The gamma retroviral vector may 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 the expression of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNA to the ribosome. The EF1a promoter has been widely used in mammalian expression plasmids and has been shown to be effective in driving the expression of CARs from transgenes cloned into lentiviral vectors. See, e.g., 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 constitutive strong promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. However, other constitutive promoter sequences may also be used, including but not limited to monkey virus 40 (SV 40) 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 actin promoter, myosin promoter, elongation factor-1 alpha promoter, hemoglobin promoter, and creatine kinase promoter. In addition, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention.

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

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

Any technique known to those skilled in the art (e.g., ficoll ^TM Isolation) to obtain T cells from blood components collected from a subject. In a preferred aspect, cells from circulating blood of an individual are obtained by apheresis. Single miningThe products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, cells collected by apheresis may be washed to remove plasma fractions and to place the cells in a suitable buffer or medium for subsequent processing steps. In one aspect of the invention, the 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 by positive or negative selection techniques. For example, in one embodiment, the conjugate is provided by a bead conjugated to an anti-CD 3/anti-CD 28 (e.g. M-450CD3/CD 28T) for a period of time sufficient to positively select the desired T cells, and isolating the T cells. In some embodiments, the period of time is between about 30 minutes and 36 hours or more. Longer incubation times can be used to isolate T cells in any situation where a small number of T cells are present, such as for isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals. In addition, the use of longer incubation times can increase the efficiency of cd8+ T cell capture. Thus, by simply shortening or lengthening this time, allowing T cells to bind to CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells, T cell subsets can be preferentially selected at the beginning of the culture or at other points in time during the culture process.

Enrichment of the T cell population can be accomplished by a negative selection process with 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 a negative magnetic immunoadhesion method or flow cytometry using a monoclonal antibody mixture directed against cell surface markers present on the negatively selected cells.

In some embodiments, the immune effector cell may be an allogeneic immune effector cell, e.g., a T cell or NK cell. For example, the cells may be allogeneic T cells, e.g., allogeneic T cells lacking functional T Cell Receptors (TCRs) and/or expression of Human Leukocyte Antigens (HLA) (e.g., HLA class I and/or HLA class II).

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

Expression in immune effector cells of the CAR polypeptides of the invention having a combination of CD28 and 4-1BB co-stimulatory domains for uPAR can significantly promote overall survival of tumor bearing animal individuals. This may benefit from the combined use of two co-stimulatory domains that extend the survival time of the CAR-T cells, while promoting proliferation and anti-tumor activity of the CAR-T cells. The differential expression genes of the CAR-T cells of the invention are detected by high throughput sequencing after co-culturing the CAR-T cells of the invention with tumor cells. RNA extraction, cDNA library construction and sequencing were all performed strictly according to transcriptome sequencing standards. Gene Ontology (GO) analysis was performed on genes differentially expressed by CAR-T cells of the invention using on-line bioinformatics tools DAVID bioinformatics Resources 6.8.8. Data visualization and analysis were processed by custom R studio scripts in terms of packages (ggplot 2 and Tree map). The gene enrichment analysis uses Fisher's exact test. The GO analysis results show that most of the differentially expressed genes in uPAR-CD28.4-1BB ζcar-T cells of the invention are localized in the extracellular region and cell membrane and that the genes associated with the immune, inflammatory and interferon gamma cellular responses are significantly up-regulated, with the extent of gene enrichment over these three biological processes being about 10-fold, about 12-fold and about 2.5-fold, respectively, compared to prior to contacting the target tumor cells. This suggests that the third generation CAR-T cells of the invention are effectively activated upon contact with target NSCLC cancer cells, supporting the strong anti-tumor function of CAR-T cells of the invention in vivo.

Use of immune effector cells expressing a CAR polypeptide of the invention and methods of treatment using immune effector cells expressing a CAR polypeptide of the invention

In recent decades, CAR-T cell therapy has become a new approach to adoptive cell immunotherapy. However, in solid tumors such as NSCLC, the construction of CAR-T cells is more complicated due to the biological heterogeneity of the tumor itself, and there is a continuing need for molecules targeting different tumor antigens for improving the treatment of individual patients.

The three-generation optimized CAR molecule targeting uPAR constructed by the inventor has obvious anti-tumor immunity effect in-vitro and various in-vivo animal tumor-bearing models. Based on this, in a further aspect, the present invention provides the use of an engineered immune effector cell comprising a chimeric antigen receptor polypeptide targeting uPAR as described herein for the manufacture of a medicament for treating uPAR positive non-small cell lung cancer (NSCLC) in an individual in need thereof and a method of treating uPAR positive non-small cell lung cancer (NSCLC) with said engineered immune effector cell.

The terms "individual" or "subject" or "patient" are used interchangeably herein, 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). In particular, the individual or subject is a human.

The terms "tumor" and "cancer" are used interchangeably herein. The term "anti-tumor immunity" refers to a biological effect that may be exhibited by a variety of means including, but not limited to, for example, a reduction in tumor volume, a reduction in the number of cancer cells, a reduction in the number of metastases, an increase in the life span of an intended tumor-bearing individual, a reduction in cancer cell proliferation, a reduction in cancer cell survival, or an improvement in various physiological symptoms associated with a cancerous condition. "anti-tumor immunity" may also be represented by the ability of peptides, polypeptides, cells and antibodies to prevent the appearance of cancer at a first location. In some embodiments, the CAR immune effector cells of the invention are administered in the treatment of uPAR positive NSCLC patients to provide an anti-tumor immune effect.

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

The term "therapeutically effective amount" refers to an amount or dose of CAR immune effector cells of the invention that, upon administration to a patient in single or multiple doses, produces a desired effect in a patient in need of treatment or prevention. The effective amount can be readily determined by the attending physician as a person skilled in the art by considering a number of factors: species such as mammals; body weight, age, and general health; specific diseases involved; the extent or severity of the disease; response of individual patients; specific CAR immune effector cells administered; mode of administration; the bioavailability characteristics of the administration formulation; a 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 effect of the CAR immune effector cells is less than a therapeutically beneficial effect. The "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 50%, 60% or 70% and still more preferably by at least about 80% or 90% relative to an untreated subject. The ability of a CAR immune effector cell to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system that predicts efficacy in a human tumor.

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, e.g., anti-PD-1 or anti-PD-L1 antibodies).

In some embodiments, the non-small cell lung cancer is any one of 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, metastatic non-small cell lung cancer, unresectable non-small cell lung cancer, advanced non-small cell lung cancer, recurrent non-small cell lung cancer, non-small cell lung cancer in an adjuvant setting, or non-adjuvant setting.

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 a large cell cancer. In one embodiment, the patient has received at least one prior treatment. In one embodiment, the prior therapy is a surgical treatment for treating cancer. In one embodiment, the non-small cell lung cancer is invasive, 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 metastasis of NSCLC. In some embodiments, the non-small cell lung cancer is stage I, II, III, or IV lung cancer. In some embodiments, the patient is an asian, such as a chinese. In some embodiments, the patient is an adult 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 comprise, prior to administering the CAR-T treatment described herein, selecting a patient for treatment of the invention. In one embodiment, the selecting comprises 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 an organ or tissue sample or biopsy or puncture sample from fresh, frozen and/or preserved; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time of gestation or development of a subject. Tissue samples may contain compounds that are not naturally intermixed with the 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, broncholavages, pleural fluid (hydrothorax), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, 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 tumor samples.

In some embodiments, the level of uPAR expression on the surface of a tumor cell can be determined by a tumor sample (e.g., a tumor biopsy) from a subject or patient. In some specific embodiments, the term "uPAR high expression" refers to a higher (increased) level of expression of a target antigen (i.e., uPAR) detectable at the cell surface of a tissue suspected of being cancerous or cancerous tissue as compared to normal tissue, e.g., adjacent normal tissue to the tumor tissue to be tested (i.e., mating tissue). The assessment of the expression level may be qualitative or quantitative. In other words, an unknown sample may be evaluated as having positive or negative expression compared to a known reference standard. Alternatively, the percentage of positive cells can be quantitatively expressed, where, for example, cells can be counted and scored for uPAR expression levels. In a specific embodiment, the reference tissue or cell 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 from normal tissue or cells of an individual for cancer or suspected cancer to be diagnosed and/or treated (e.g., lung tissue or cells from the individual).

In one embodiment, the method of treatment of the invention thus further comprises determining a subject or patient having a uPAR positive tumor from a tumor sample (e.g., a tumor biopsy) from the subject or patient, and optionally qualitatively or quantitatively determining the uPAR positive rate (i.e., the percentage of uPAR positive tumor cells) of the tumor.

In some embodiments, the methods described herein comprise administering the CAR-T cells described herein (and optionally, other anti-cancer agents, e.g., in combination with an anti-PD-1 or anti-PD-L1 antibody) to a NSCLC patient, wherein the patient expresses elevated uPAR levels (e.g., relative to a normal tissue sample) in a tumor tissue sample (e.g., a squamous or non-squamous tumor tissue sample). In some embodiments, the methods described herein comprise administering the CAR-T cells described herein (and optionally, other anti-cancer agents, e.g., in combination with an anti-PD-1 or anti-PD-L1 antibody) to a NSCLC patient, wherein a tumor tissue sample (e.g., a squamous or non-squamous tumor tissue sample) of the patient has a uPAR positive cell percentage of 1% to 50%, or 20% to 50%, 60%, 70% or more, or 30% to 50%, 60%, 70% or more, or 50% to 60%, 70%, 80% or more. In some embodiments, the patient has a percentage of uPAR positive cells in a tumor tissue sample (e.g., a squamous or non-squamous tumor tissue sample) of 50% or greater.

In other embodiments, the invention also provides a method of treating uPAR positive NSCLC cancer in a patient by administering the CAR-T cells described herein (and optionally, other anti-cancer agents, e.g., in combination with an anti-PD-1 or anti-PD-L1 antibody), wherein the method further comprises the step of assessing 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-gamma, TNFRSF9, and IL17A genes, and optionally chemokine genes such as CXCL1, CXCL5, and CXCL8, wherein an increase in the level of the biomarker relative to prior to or during administration of the treatment of the invention can be used, for example, to indicate therapeutic responsiveness of a patient. 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 the expression level of PD-L1 on tumor cells isolated from the patient, wherein an increase in the level of the biomarker relative to prior to or during administration of the treatment of the invention can be used, for example, to indicate the likelihood of recurrence in the patient.

In a preferred embodiment, the methods of the invention comprise administering a CAR-T cell described herein in combination with another anti-cancer agent (e.g., an anti-PD-1 antibody). As used herein, "co-administration" or "combined" administration means that two (or more) different treatments are delivered to a subject during the course of the subject suffering from a disorder, e.g., two or more treatments are delivered after the subject has been diagnosed as suffering from a disorder and before the disorder heals or eliminates or otherwise ceases treatment. In some embodiments, delivery of one treatment is still performed at the beginning of delivery of the second, whereby there is overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "parallel delivery. In other embodiments, the delivery of one treatment ends before the delivery of another treatment begins. In some embodiments of either case, the treatment is more effective due to the combined administration. In some embodiments, the combination administration results in a reduction in symptoms, or other parameter associated with the disorder, that is superior to the parameter observed with one treatment in the absence of another treatment. The effect of both treatments may be partial addition, complete addition or higher than addition.

In some embodiments, the methods of treatment and uses described herein further comprise administering to the individual an immunodetection point inhibitor, e.g., a PD-1 or PD-L1 inhibitor or LAG-3 inhibitor, e.g., one or more doses of the inhibitor, particularly a PD-1 inhibitor, e.g., an anti-PD-1 antibody, prior to, during, and/or after administration of the CAR-T cells. In some embodiments, anti-PD-1 antibody treatment includes, but is not limited to: nivolumab (nivolumab), pembrolizumab (pembrolizumab), ipilimumab (ipilimumab), JS001, TSR-042, pidillizumab (pidilizumab), BGB-a317, SHR-1210, reg 2810, MDX-1106, PDR001, anti-PD-1 from clone RMP 1-14; and anti-PD-1 antibodies, divalizumab (durvalumab), atezolizumab (atezolizumab), avistuzumab (avelumab) and fragments, derivatives, variants, and biological analogs thereof disclosed in U.S. patent No. 8,008,449.

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

In one aspect, the invention also provides pharmaceutical compositions and pharmaceutical combinations comprising a CAR cell described herein, e.g., an immune effector cell, and optionally a PD-L1 inhibitor; and to the use of said pharmaceutical compositions and pharmaceutical combinations in the methods of treatment of NSCLC as described above or in the manufacture of a medicament for use in said methods.

The various embodiments/technical solutions described herein and features in the various embodiments/technical solutions should be understood to be arbitrarily combined with each other, and the various solutions obtained by these combinations are included in the scope of the present invention as if the combinations were specifically and individually listed herein unless the context clearly indicates otherwise.

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

Examples

Materials and methods

Cell lines

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

Immunohistochemistry

Formalin-fixed, paraffin-embedded (FFPE) tumor specimens were sectioned for deparaffinization, and then incubated with rabbit anti-human uPAR antibody (Cell Signaling Technology) overnight at 4 ℃. After incubation with HRP conjugated goat anti-rabbit secondary antibody, the signal was detected using DAB substrate kit (Abcam) according to the manufacturer's instructions. Images were acquired using a microscope (nikon). The relative density of uPAR signal was quantified by Image J v1.49 at 100-fold magnification. The relative density was greater than 5%, confirming that lung tumor tissue was uPAR positive.

Production of retroviral vectors encoding uPAR-specific CARs

The CAR molecule (SEQ ID NO: 21) comprising the optimized uPAR-scFvs coding sequence (SEQ ID NO: 1) was synthesized from GeneArt (Invitrogen) and then subcloned into the SFG retroviral vector (adedge). The cloning of the CAR was verified by sequencing. After 48 hours of transient transfection, retroviral packaging cell line PG13 was used to generate retroviral particles.

Generating CAR T cells

Human Peripheral Blood Mononuclear Cells (PBMCs) were isolated from healthy donors by gradient centrifugation at Lymphoprep (MP Biomedicals). To generate uPAR-CAR T cells, T cells in PBMCs were stimulated with anti-CD 3 and anti-CD 28 beads, then infected with retroviruses. After 7 days, T cells were examined for CAR expression by flow cytometry and then examined for CAR expression in a cell containing 5% gemcell ^TM X-VIVO of human serum AB (Gemini Bio) and IL-2 (SL PHARM) ^TM 15 in serum-free medium (Lonza). The study was approved by the Beijing century jar hospital institutional review board and informed consent was obtained from all participants.

Flow cytometry assays

Flow cytometry was performed on a FACSCanto Plus instrument (BD Biosciences). FlowJo v.10 (FlowJo, LLC) was used for data analysis. Transgenic T cells were detected after staining with APC-labeled mouse anti-human CD3 antibody (BD Biosciences), PE-labeled mouse anti-human CD8 antibody (BD Biosciences), BV 421-labeled mouse anti-human CD4 antibody (BD Biosciences) and goat anti-mouse IgG (Fab-specific) F (ab') 2 fragment-FITC antibody (GAM, sigma). H460 cells and uPAR ⁺ A549 cells were labeled 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 antibodies (BD Biosciences), APC-labeled mouse anti-human CD107a antibodies (BD Biosciences), the CAR-T cell activation level was detected. The change in the level of CAR-T cells PD-1/TIM-3/LAG-3 was detected by staining with an APC-labeled mouse anti-human CD3 antibody (BD Biosciences), a PE-labeled mouse anti-human CD8 antibody (BD Biosciences), a BV 421-labeled mouse anti-human CD4 antibody (BD Biosciences), a BV 480-labeled mouse anti-human PD-1 antibody, a BV 605-labeled mouse anti-human TIM-3 antibody, a BV 480-labeled mouse anti-human LAG-3 antibody, staining with a PE-labeled mouse anti-human PD-L1 antibody, and detecting the change in the level of cell PD-L1.

T cell proliferation assay

After 1x10≡6H 460 cells were seeded in 6-well plates and cultured overnight to allow H460 cells to adhere well, CAR-T cells were co-cultured with H460 cells at an E:T ratio of 2:1 for 12 days. T cells were stimulated with fresh H460 cells every 3 days, and counted prior to addition of H460 cells.

IFN-gamma cytokine secretion level assay

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

In vitro killing test of tumor cells

NGFR CAR-T cells or uPAR CAR-T cells, with H460,uPAR ⁺ A549, and cells isolated from tumor tissue mass at a ratio of 0:1, 1:1, 2.5:1, 5:1, or 10:1 (effective target ratio, E: T), in X-VIVO ^TM Co-culture in 15 medium for 24 hr. Luciferase activity was monitored using an IVIS imaging system (IVIS, xenogen, alameda, CA, USA) and the percent target tumor cell lysis by CAR-T cells was determined relative to the control. The calculation mode is as follows: percent lysis = 1- (fluorescence intensity after co-cultivation/fluorescence intensity after co-cultivation at a ratio of 0:1).

Impedance-based tumor cell killing assay

Sustained tumor cell death was assessed over a 24 hour period using the xcelligent impedance system. Tumor cells were seeded at 10,000 cells per well in a 96-well plate (resistor-bound plate) with resistors integrated at the bottom. Experiments were performed in triplicate. After 24 hours, 100,000T cells (E: t=10:1) were added. NT cells (non-transduced T cells) served as controls. Cell index values associated with tumor cell adhesion were normalized relative to NT controls. By measuring the current impedance caused by tumor cell adhesion, the impedance measurement-based normalized cell index values were collected every 15 minutes.

Xenograft mouse model for subcutaneous lung cancer cells

NOD-SCID mice of 6 to 8 weeks of age were purchased from Charles River laboratories. Left flank of female NOD-SCID mice, subcutaneously injected 2X10 ⁶ H460-eGFP-Luc cells were used to construct a xenograft mouse model. Tumor cells were injected 3 days later, 2X10 ⁷ Individual CAR T cells were injected directly into the tumor for 3 consecutive days. Tumor progression was monitored using an IVIS imaging system (IVIS, xenogen, alameda, CA, USA) and mice were sacrificed when tumor diameters reached 20 mm. All experiments, including mice, were approved by the examination committee of the Beijing century jar hospital institution.

Bioluminescence imaging

Mice were anesthetized with 3% isoflurane in 100% oxygen, injected with 4.5mg/kg D-fluorescein in 300 μl saline, and imaged after 10 minutes using an optical imaging platform (Spectral Instruments Imaging). Images were taken every 5 minutes until photon counts reached a peak.

RNA high throughput sequencing

UsingUltra ^TM RNA library preparation kit (#E7530L, NEB), sequencing library was generated using samples each containing 2. Mu.g of total RNA as input material according to the manufacturer's instructions. Briefly, mRNA was purified from total RNA using magnetic beads attached with poly-T oligonucleotides. Fragmentation was performed using divalent cations at elevated temperature in NEB Next first strand synthesis reaction buffer (5X). First strand cDNA was synthesized using random hexamer primers and RNase H. The cDNA second strand was then synthesized using buffer, dNTPs, DNA polymerase I and RNase H. Library fragments were purified using the qiagquick PCR kit, eluted with EB buffer, followed by end repair, addition of a tail and addition of adaptors. To construct the library, the product was recovered and subjected to PCR. Samples with index codes were classified on the cBot Cluster Generation system using the TruSeq SR Cluster kit v3-cBot-HS (Illumina inc.) according to the manufacturer's protocol. Subsequently, the library was sequenced on an Illumina NovaSeq 6000System platform (Illumina inc.). / >

Real-time reverse transcription polymerase chain reaction

Total RNA was extracted from cells using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The amount and purity of RNA was measured using a Nanodrop One spectrophotometer (Thermo Fisher Scientific). Only samples with appropriate absorbance measurements (A260/A280 of about 2.0 and A260/A230 of 1.9-2.2) were considered for this study. cDNA was synthesized using the 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. The relative gene expression was calculated using the 2- ΔΔct method.

RNA interference

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

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

PDX modeling and treatment

Tumor tissue from uPAR positive lung adenocarcinoma patients was inoculated into BALB/C-nu/nu mice for PDXs (P0) modeling. Three weeks later, tumor tissue was isolated from PDXs (P0) and re-vaccinated into BALB/C-nu/nu mice for modeling PDXs (P1). See fig. 19 a. Tumor-bearing mice were treated three days after modeling of PDXs (P1) with NT (non-transduced T cells) controls, uPAR CAR-T cells alone, or uPAR CAR-T cells in combination with PD-1 antibodies for three consecutive days. As shown in fig. 19b-19d, PD-1 antibody combination treatment inhibited tumor growth significantly better than CAR-T cell alone.

In situ xenograft model

Transpleural injection was used for in situ xenograft. Mice were anesthetized with 3% isoflurane and kept in the right lateral position. A5 mm skin incision was made on the caudal side of the left scapula, dissecting fat and muscle tissue, exposing the left rib cavity. Observing left lung movement, a 31 gauge needle syringe containing 30 μ L H460 cell suspension and 50% matrigel in PBS was inserted into the lung parenchyma through the sixth intercostal space to a depth of 3mm; cells were then injected directly into the left lung. By 3M ^TM Vetbond ^TM Tissue adhesive (3 m, st. Paul, MN, USA) closes the skin incision and keeps the mice warm until complete recovery.

Construction of xenograft mouse model by intracranial implantation of lung cancer cells

NOD-SCID mice of 6 to 8 weeks of age were anesthetized with ketamine/xylazine mixed solution. Animals were fixed in stereotactic head frames, 1cm was cut at the scalp midline, a burr hole was drilled in the skull, and 5 μl of 2x10 in PBS using a 10 μl BD syringe ⁵ H460-eGFP-luc cells were injected into the left striatum (coordinates: 2.5mm outside the bregma and 0.5mm behind the bregma), thereby delivering tumor cells into the brain parenchyma to a depth of 3.5 mm. The burr hole on the skull was sealed with bone wax and the incision was closed with medical glue (component). Three days after tumor cell injection, 3x10 by tail vein injection ⁷ CAR-T cells were isolated and imaged using an IVIS in vivo imaging system (IVIS, xenogen, alameda, CA, USA) to monitor tumor growth. All mice experiments were approved by the examination committee of the hospital agency of the Beijing century jar.

Statistical analysis

All experiments were performed in at least triplicate. Statistical analysis was performed using GraphPad Prism version 8.0.2 (GraphPad software). Data are expressed as mean ± standard deviation. The differences between the averages were examined using a suitable statistical test method. The overall survival of tumor xenograft bearing mice was measured using the Kaplan-Meier method and compared between groups using Cox proportional hazards regression analysis. Statistical significance was set to p <.05.

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

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

To examine the specific expression of uPAR in different lung cancer patients and its relationship to patient survival, we further examined uPAR levels in tumor samples of 12 chinese lung cancer patients by immunohistochemistry (as shown in fig. 2a and 2b and table 1). The median age of the patients entered group was 61.5 years (range: 49-73 years). All patients received surgical treatment. Demographic and clinical characteristics of the patients are shown in table 1. Based on the relative densities of uPAR signals quantified on tumor specimen sections, 33.3% of patients had lung tumor tissues that were uPAR positive, with the remainder being uPAR negative.

Table 1 demographic and clinical characteristics of the patients.

Patient numbering

Sex (sex)

Age (age)

Tumor type

Staging of tumors

uPAR

Status of

#1

Man's body

65

Adenocarcinoma of gland

IV

Negative of

Death of

#2

Female

73

Squamous cell carcinoma

I

Positive and negative

Death of

#3

Man's body

64

Squamous cell carcinoma

III

Negative of

Survival of

#4

Man's body

59

Adenocarcinoma of gland

II

Negative of

Survival of

#5

Man's body

49

Squamous cell carcinoma

IV

Negative of

Survival of

#6

Man's body

64

Squamous cell carcinoma

III

Negative of

Survival of

#7

Man's body

69

Adenocarcinoma of gland

II

Positive and negative

Survival of

#8

Man's body

51

Small cell carcinoma

III

Negative of

Death of

#9

Man's body

41

Small cell carcinoma

NA

Negative of

Death of

#10

Female

57

NA

Positive and negative

Death of

#11

Man's body

58

Adenocarcinoma of gland

I

Negative of

Survival of

#12

Man's body

68

Squamous cell carcinoma

II

Positive and negative

Death of

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

To generate uPAR-specific CAR-T cells, a tertiary (CD 28.4-1BB ζ) CAR based on uPAR-specific mAb was developed (fig. 3) and retroviral vectors encoding the tertiary CAR molecule were constructed. T cells isolated from peripheral blood mononuclear cells of healthy donors were then stimulated with anti-CD 3 and anti-CD 28 beads; and infected with the constructed retrovirus. After 7 days of transduction, flow cytometry assays were performed to check transduction efficiency. As shown in fig. 4, approximately 60% of T cells were CAR positive.

Human lung cancer cell line H460, which is known to express high levels of uPAR, was used, as well as another lung cancer cell line A549 (uPAR ⁺ A549 As representative of tumor cell lines with high uPAR expression. FIG. 5a shows the results of a flow-through examination of the cell surface uPAR expression levels of these two cell lines. The constructed uPAR CAR-T cells were combined with these tumor cells that highly expressed uPAR (H460 and uPAR ⁺ A549 Co-culture at an effective target ratio (E: T ratio) of 1:1 to 10:1 to examine CAR T cells for in vitro proliferation and anti-tumor activity. PBMC cells and NGFR CAR-T cells were used as controls. NGFR CAR-T cells targeting the unrelated antigen NGFR were constructed in the same manner as uPAR CAR-T cells. As shown in fig. 5b, co-cultured at low E: T ratio (2:1), the constructed uPAR CAR-T cells showed good viability and proliferative capacity after contacting target tumor cells. As shown in fig. 5 c-5 d, high levels of CD107a expression (fig. 5 c) and IFN- γ secretion (fig. 5 d) were detected from the co-cultured uPAR CAR-T cells after co-culturing the uPAR CAR-T cells with target tumor cells for 6 hours at a high E: T ratio (10:1). Furthermore, as shown in fig. 6a, significant tumor target cell lysis was induced by uPAR CAR-T cells compared to control NGFR CAR-T cells, with tumor cell lysis greater than 60% at low E: T ratio (1:1) and 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 to control PBMC cells and NGFR CAR-T cells (fig. 6 b).

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

To examine the antitumor 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 fig. 7 a. On day 1/2/3, CAR-T cells were injected directly into the tumor and tumor growth was monitored for 84 days with non-transduced T cells (NTs) as controls. The results showed that mice treated with uPAR CAR-T cells had significantly longer survival compared to the NT group mice (fig. 7b,7c and 7 d).

Tumor recurrence was observed in some mice treated with uPAR CAR-T cells. To confirm whether the constructed CAR-T cells still have anti-tumor effect on recurrent tumors, we then isolated tumor cells from mice with recurrent tumors and performed in vitro cell tests. As shown in fig. 8a-c, uPAR CAR-T still exhibited excellent anti-tumor ability. This suggests that the cause of tumor recurrence may not be CAR-T cell off-target effects, more likely because the survival cycle of CAR-T cells themselves in mice and the tumor immunosuppressive microenvironment limit the in vivo anti-tumor activity of CAR-T cells, resulting in tumor recurrence.

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

To further simulate the actual clinical situation of lung cancer, we constructed an in situ xenograft mouse model and an intracranial metastatic cancer xenograft mouse model to investigate the efficacy of the constructed uPAR CAR-T cells in treating undiffused pre-invasive NSCLC lung cancer and metastatic NSCLC lung cancer, respectively. In these mouse models, similar good therapeutic results were obtained. The constructed uPAR CAR-T cells inhibited tumor growth in vivo while exhibiting excellent anti-tumor activity in vitro (FIGS. 9-12). The statistics of the Wilcoxon rank sum test are shown in table 2.

TABLE 2

The emissivity P value is based on the Wilcoxon rank sum test. EXCEL Kaplan-Meier and nonparametric Wilcoxon p values were used for survival curve comparison. NT = non-transduced T cells; CAR-T = uPAR CAR-T cells; subcutaneous = subcutaneous inoculation model; lung = pre-infiltration model; brain = metastasis model.

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

To explore the reasons why uPAR CAR-T cells have tumor inhibitory 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. 1280 up-regulated genes and 664 down-regulated genes were found in CAR-T cells co-cultured with H460 cells for 4 hours compared to CAR-T cells not co-cultured (fig. 13). Gene Ontolog analysis was performed on the first 400 differentially expressed genes with the greatest differences in expression. As a result, it was found that in CAR-T co-cultured with target tumor cells, genes whose expression was up-regulated were associated with the following Biological Processes (BP), molecular Functions (MF) and Cellular Components (CC): cell response to interferon-gamma, immune response, inflammatory response, and tumor necrosis factor-activated receptor activity; whereas the gene expression associated with the following aspects is relatively low: gene expression regulation, DNA replication, mitotic cell cycle G1/S switching, protein binding and spindle pole (FIG. 14). Furthermore, in CAR-T cells co-cultured with tumor cells, up-and down-regulated genes are mostly enriched in extracellular regions and cell membranes. After protein-protein interaction (PPI) analysis, we found that one cluster comprising 30 genes was located in the center of the upregulated PPI network, including CD274 (i.e. PD-L1) and PDCD1LG2 (i.e. PD-L2), IL2, IL9, IFN- γ, TNFRSF9 and Th17A pathway-associated chemokine genes CXCL1, CXCL5 and CXCL8 (fig. 15 a). Further, 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 (fig. 15b and 15 c).

To confirm the RNA-seq results, we collected CAR-T cells after co-culturing them with H460 cells for 30 min and 4 hours, and then detected some candidate genes by RT-qPCR. As a result, it was found that the expression of IL2, IL9, IFN- γ, TNFRSF9 and IL17A genes and Th 17A-related chemokine genes such as CXCL1, CXCL5 and CXCL8 was significantly increased after co-culture of CAR-T cells with H460 cells compared to before co-culture of H460 cells (fig. 16a and 16 b).

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

T cells have been reported to be inhibited at tumor sites by a variety of mechanisms, with inhibition of PD-1/PD-L1 axis-mediated function playing a key role [15-17]. To understand whether the designed three-generation uPAR CAR-T cells would be limited by the PD-1/PD-L1 axis, we examined the mRNA levels of PD-1 in CAR-T cells co-cultured with H460 cells at two different time points. As shown in fig. 17a, CAR-T cells after 30 minutes and 4 hours of co-culture with H460 cells expressed significantly increased PD-1 and PDCD1LG2 compared to CAR-T cells without co-culture; moreover, H460 cells after co-culture also expressed significantly increased PD-L1 compared to tumor cells without co-culture. In addition, a significant increase in the levels of PD-1 and Lag-3 was also observed after 48 hours of co-culture of CAR-T cells with H460 cells and uPAR+A549 cells (FIG. 17 b).

To further confirm the inhibition of CAR-T cell antitumor activity by the PD-1/PD-L1 axis, we knockdown PD-L1 in tumor cells by siRNA (as shown in fig. 18a and 18 b), and then performed in vitro killing assays and compared to tumor cells that were not knockdown of PD-L1. As shown in fig. 18d, after PD-L1 in tumor cells was knocked down, an increase in tumor lysis activity of co-cultured CAR-T cells was observed (E: t=2.5:1), and as shown in fig. 18c and 18E, IFN- γ secretion levels and CD107a levels on the cell membrane surface were also increased (E: t=10:1).

Example 7: PD-L1 antibodies in combination with uPAR CAR T cells have therapeutic effects on lung cancer patient-derived xenograft (PDX) models

Patient-derived xenograft (PDX) models, constructed by injecting primary tumor biopsies from lung cancer patients instead of human cell lines, were used to assess CAR-T cell in vivo efficacy. Tumor tissue from lung adenocarcinoma patients was inoculated into BALB/C-nu/nu mice for PDXs (P0) modeling. Three weeks later, tumor tissue was isolated from PDXs (P0) and re-vaccinated into BALB/C-nu/nu mice for modeling PDXs (P1). See fig. 19 a. Tumor-bearing mice were treated three days after modeling of PDXs (P1) with NT (non-transduced T cells) controls, uPAR CAR-T cells alone, or uPAR CAR-T cells in combination with PD-1 antibodies for three consecutive days. As shown in fig. 19b-19d, PD-1 antibody combination treatment inhibited tumor growth significantly better than CAR-T cell alone.

Discussion of the invention

Lung cancer is a major public health problem worldwide, the most frequently diagnosed cancer in the last decades, and brain metastases occur in up to 40% of Lung cancer patients [ 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 major 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 about 85% of established 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 scaling.blood, 2010.116 (9): p.1593-603; and Amor, C., et al, senolytic CAR T cells reverse senescence-associated dynamics Nature,2020.583 (7814): p.127-132]. Although surgical, chemotherapy and radiation therapy have advanced in the treatment of NSCLC over the last 20 years, resulting in increased survival in NSCLC patients, the prognosis of NSCLC has not been significantly improved due to the burden of tumor mutations and the heterogeneity of the disease. Therefore, it is imperative to explore new strategies to extend patient survival.

CAR-T cell therapy has been successful in the clinical treatment of hematological cancers, but is not ideal for use in solid tumors such as lung Cancer [ 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]. It has now been proposed for the design of CAR molecules for solid tumors that the nature, number and arrangement of extracellular antigen domains, from CAR-T to intracellular signaling domains, can potentially have an impact on therapeutic efficacy for different specific tumor types and target antigens. On the other hand, cell-based in vitro and ex vivo assays employed in preclinical studies, while may reflect the activity of candidate CAR-T cells to some extent, still have difficulty reflecting the in vivo therapeutic efficacy of CAR-T cells. Thus, predicting CAR-T cell efficacy often needs to be based on a suitable animal model. However, these numerous factors have led to the design of CAR-T molecules and their evaluation of efficacy in the therapeutic application of solid tumors such as lung cancer, and remain a challenge to which the field of CAR-T therapy is continually facing.

Biological heterogeneity of solid tumors is an important factor in the failure of CAR-T therapy. In this regard, several methods for improving clinical effects and safety have been proposed. For example, one approach is to treat the patient with a drug that increases target antigen expression on cancer cells prior to CAR-T treatment to increase CART efficacy. Alternatively, the CAR molecule is engineered to enhance its T cell activity against cancer cells exhibiting lower target antigen density. The former is limited by the availability of such drugs and their associated efficacy/toxicity. The latter places higher demands on the design of CAR molecules.

In a series of studies directed to lung cancer treatment, we found that there is a subset of uPAR positive and negative patient subpopulations in the non-small cell lung cancer patient population and that high expression of uPAR correlates with survival in 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 exhibit excellent anti-NSCLC tumor activity in vitro and in various xenograft mouse models, thereby providing a powerful candidate tool for clinical lung cancer treatment.

The antitumor efficiency of CAR-T cells is affected by a number of factors, such as the affinity of the targeting antigen, the extent of terminal differentiation of CAR-T cells, and the in vivo off-target toxicity and longevity [ 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, IL2, IL9 and IFN-gamma expression was up-regulated after stimulation of CAR-T cells by tumor cells. Furthermore, we also noted that IL17A and chemokines associated therewith, such as CXCL1, CXCL5 and CXCL8, are also expressed. The increased expression of these factors may explain in part the strong anti-tumor function of the CAR-T cells of the invention.

On the other hand, in xenograft mouse model experiments, we also noted that, despite the strong anti-tumor ability of the designed CAR-T cells, the tumor volume of most of the tested mice was reduced to an extent that was almost undetectable for a period of time after CAR-T cell treatment, but that tumors developed recurrence in some mice after about one month. Whereas in vitro experiments we have performed on tumor cells isolated from recurrent tumors, it has been shown that the CAR-T cells used still retain lytic activity on these isolated tumor cells. This suggests that CAR-T cells have some limitation in efficacy in vivo, as may be a variety of, for example, tumor immunosuppressive microenvironments, short survival of CAR-T cells in vivo.

To further improve the in vivo therapeutic efficacy of NSCLC of the third generation CAR-T cells, we studied the change in expression of the PD-1/PD-L1 axis on CAR-T cells and tumor cells after the tumor cells stimulated the CAR-T cells. The results showed that both PD-1and PD-L1 were significantly upregulated, and PDCD1LG2 (apoptosis protein 1 ligand 2, PD-L2) was also upregulated. PDCD1LG2 is the second ligand of PD-1and inhibits T cell activation, and it has also been reported that this molecule is one of the important causes of 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]. Thus, upregulation of the PD-1 axis may explain to some extent tumor recurrence after 1 month in tumor-bearing mice receiving CAR-T cell therapy of the invention. Our PD-L1 knockout study and PDX model study further supported that PD-1 antibodies were combined with the CAR-T therapies of the invention as a strategy to overcome the immunosuppressive effects of PD-1/PD-L1. In our studies, it was also found that the expression of immunosuppressive LAG-3 was also increased during CAR-T cell therapy of the invention, suggesting that LAG-3 antibodies may be used in combination with CAR T cells in lung cancer therapy to enhance efficacy.

In summary, we have generated a third generation uPAR CAR molecule with therapeutic effects both in vitro and in vivo. The engineered CAR molecule can cause enhanced T cell activity to NSCLC cancer cells presenting uPAR antigen without combining aging inducers, and can obtain remarkably good therapy in various NSCLC lung cancer animal models, and realize remarkable overall survival improvement of tumor-bearing animals, thereby providing a powerful treatment option for uPAR positive NSCLC lung cancer patients.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Some embodiments of the invention:

1. a Chimeric Antigen Receptor (CAR) polypeptide that targets uPAR, comprising, from N-terminus to C-terminus:

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

(ii) Optionally, a hinge/spacer;

(iii) A transmembrane domain;

(v) CD3 zeta 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, in particular an scFv,

preferably, the antigen binding domain comprises: 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 Kabat defined CDR sequences, or LCDR1-3 and HCDR1-3 sequences shown in SEQ ID NOs: 13-18), and more preferably, the VL of SEQ ID NO. 3 and the VH of SEQ ID NO. 4,

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

3. The CAR polypeptide of any one of embodiments 1-2, wherein the hinge/spacer region is selected from the group consisting of: the hinge region from IgG or the spacer region from the extracellular region of CD8 alpha or CD28, and preferably is a human CD8 alpha spacer region or CD28 spacer region, e.g., a CD28 spacer region 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: the transmembrane domains of CD4, CD8, CD28 and CD3 ζ, and preferably the human CD8 transmembrane domain or CD28 transmembrane domain, or wherein said transmembrane domain comprises the amino acid sequence shown in SEQ ID No. 7 or 22.

5. The CAR polypeptide of any of embodiments 1-4, wherein the CD28 co-stimulatory domain comprises the amino acid sequence of SEQ ID No. 11.

6. The CAR polypeptide of any one of embodiments 1-5, wherein the 4-1BB co-stimulatory domain comprises the amino acid sequence of SEQ ID No. 10.

7. The CAR polypeptide of any one of embodiments 1-6, wherein said CD3 zeta 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 comprises, from N-terminus to C-terminus:

(a) An anti-uPAR scFv shown in SEQ ID NO. 2;

(b) A CD28 spacer shown in SEQ ID NO. 6 and a CD28 transmembrane domain shown in SEQ ID NO. 7;

(iv) A CD3 zeta signaling domain shown in SEQ ID NO. 12,

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

10. A recombinant vector comprising a nucleic acid molecule according to embodiment 9, e.g. selected from the group consisting of DNA vectors, RNA vectors, lentiviral vectors, adenoviral vectors or retroviral vectors, preferably retroviral vectors.

11. A host cell comprising the chimeric antigen receptor polypeptide of any one of embodiments 1-8, the nucleic acid molecule of embodiment 9, or the vector of embodiment 10, wherein the cell is preferably an immune effector cell, such as a T cell or NK cell, e.g., the T cell is an autologous T cell or an allogeneic T cell.

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 of any one of embodiments 1-8, the nucleic acid molecule of embodiment 9, the recombinant cell of embodiment 11, or the CAR-T cell of embodiment 12.

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

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

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

17. The use according to embodiment 15 or the method according to embodiment 16, wherein the immune effector cell is a T cell.

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 according to embodiment 17, wherein the individual has a pre-invasive or carcinoma in situ, or metastatic cancer, such as brain metastasis.

20. The use or method according to embodiment 17, wherein the individual is a subspecies human, e.g. a chinese.

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

22. The use or method according to embodiments 17-18, further comprising administering to the individual an immunodetection point inhibitor, e.g., a PD-1 or PD-L1 inhibitor or LAG-3 inhibitor, e.g., one or more doses of a PD-1 inhibitor, particularly an anti-PD-1 antibody, before, during and/or after administration of the CAR-T cells.

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

preferably, the individual has a NSCLC tumor with a uPAR positive expression rate of 25% to 80% or more, i.e., about 25-80% or more of the tumor cells exhibit uPAR positive expression on the cell surface.

Sequence listing

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Claims

1. A Chimeric Antigen Receptor (CAR) polypeptide targeting uPAR consisting of the sequence of amino acids 23 to 529 of SEQ ID No. 21.

2. A nucleic acid molecule encoding the chimeric antigen receptor polypeptide of claim 1.

3. A recombinant vector comprising the nucleic acid molecule of claim 2.

4. The recombinant vector according to claim 3, wherein the vector is selected from the group consisting of a DNA vector and an RNA vector.

5. A recombinant vector according to claim 3, wherein the vector is selected from lentiviral, adenoviral or retroviral vectors.

6. The recombinant vector of claim 5, wherein the vector is a retroviral vector.

7. A host cell comprising the chimeric antigen receptor polypeptide of claim 1, the nucleic acid molecule of claim 2, or the recombinant vector of any one of claims 3-6.

8. The host cell of claim 7, wherein the cell is an immune effector cell.

9. The host cell of claim 8, wherein the cell is a T cell or NK cell.

10. The host cell of claim 9, wherein the T cell is an autologous T cell or an allogeneic T cell.

11. A CAR-T cell, wherein the cell comprises the chimeric antigen receptor polypeptide of claim 1 or the nucleic acid molecule of claim 2.

12. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the chimeric antigen receptor polypeptide of claim 1, the nucleic acid molecule of claim 2, the host cell of any one of claims 7-10, or the CAR-T cell of claim 11.

13. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition further comprises a PD-1 inhibitor or a PD-L1 inhibitor.

14. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition further comprises an anti-PD-1 antibody.

15. Use of an engineered immune effector cell in the manufacture of a medicament for treating uPAR positive non-small cell lung cancer (NSCLC) in an individual in need thereof, wherein the engineered immune effector cell comprises a chimeric antigen receptor polypeptide that targets uPAR according to claim 1.

16. The use of claim 15, wherein the immune effector cell is a T cell.

17. The use of claim 16, wherein the NSCLC is large cell lung cancer, adenocarcinoma, or squamous cell carcinoma.

18. The use of claim 16, wherein the individual has a pre-invasive or carcinoma in situ, or metastatic carcinoma.

19. The use of claim 18, wherein the individual has brain metastasis of NSCLC.

20. The use of claim 16, wherein the individual is a stateless person.

21. The use of claim 20, wherein the individual is a chinese.

22. The use of claim 16, wherein the individual is an adult individual over 30 years old.

23. The use of claim 16, wherein the individual is an adult individual over 60 years old.

24. The use of any one of claims 16-23, wherein the medicament further comprises an immunodetection point inhibitor.

25. The use of claim 24, wherein the immunodetection point inhibitor is a PD-1 or PD-L1 inhibitor or LAG-3 inhibitor.

26. The use of claim 25, wherein the engineered immune effector cell is a CAR-T cell, and wherein the medicament is formulated in a form that one or more doses of a PD-1 inhibitor are administered before, during and/or after administration of the CAR-T cell.

27. The use of claim 26, wherein the PD-1 inhibitor is an anti-PD-1 antibody.

28. The use of claim 16, wherein the engineered immune effector cell is a CAR-T cell, and wherein the uPAR positive expression rate of a tumor is determined on a tumor biopsy from an individual by immunohistochemical staining prior to administration of the CAR-T cell.

29. The use of claim 28, wherein the individual has NSCLC tumors with a uPAR positive expression rate of 25% to 80% or more, i.e., 25-80% or more of the tumor cells exhibit uPAR positive expression on the cell surface.