CN116284435A

CN116284435A - EGFRvIII chimeric antigen receptor and uses thereof

Info

Publication number: CN116284435A
Application number: CN202211140506.3A
Authority: CN
Inventors: 钟晓松; 白玥
Original assignee: Carrizi Beijing Life Technology Co ltd
Current assignee: Carrizi Beijing Life Technology Co ltd
Priority date: 2022-09-19
Filing date: 2022-09-19
Publication date: 2023-06-23
Also published as: WO2024060140A1

Abstract

The present invention relates to novel chimeric antigen receptors that specifically bind egfrvlll and uses thereof.

Description

EGFRvIII chimeric antigen receptor and uses thereof

The present invention relates generally to the use of T cells engineered to express an egfrvlll Chimeric Antigen Receptor (CAR) for the treatment of diseases associated with the expression of egfrvlll.

Background

Glioblastoma (GBM) is the most common and invasive primary malignant brain tumor in humans, with a high degree of malignancy in most cases. GBM creates a huge social and medical burden worldwide due to high morbidity, high mortality, and low cure rate, and is almost incurable with traditional therapies, so the overall survival of the patient is far from ideal. In particular, the current standardized therapeutic strategy is the standard treatment regimen for maximum safety margin surgical tumor resection, simultaneous post-operative radiation and chemotherapy, and then six months of temozolomide-assisted chemotherapy. However, the average survival of the patient is only 14.6 months.

Chimeric Antigen Receptor (CAR) T cell therapy is an emerging immunotherapy that can treat a variety of malignancies, including glioblastomas. Immunotherapy of T cells expressing GBM-specific Chimeric Antigen Receptors (CARs) provides a promising therapeutic platform, as it is possible to target tumor tissue exclusively, while preserving normal brain. Challenges facing solid tumor immunotherapy include that normal cell tissue expresses tumor target antigens, that blood brain barrier factors limit the transport of CAR-T cells to the tumor site, and that CAR-T cells sometimes lack long-term persistence at the tumor site.

Some transgenic T cells expressing Chimeric Antigen Receptor (CAR) have shown some effect in preclinical models for glioma treatment, but early clinical tests showed limited anti-glioma activity, and these CAR-T cells target IL13 receptor subunit α2 (IL 13 ra 2), HER2 and EGFR variant III (EGFRvIII) or EPH receptor A2 (EphA 2). While we attempted to solve these problems in an immunodeficient glioblastoma mice model using chimeric antigen receptor constructs based on egfrvlll.

Type III EGF deletion mutant receptor (egfrvlll) is expressed in a variety of tumor types, including glioblastoma multiforme (GBM), but is rarely observed in normal tissues. EGFRvIII is an independent poor prognostic indicator after expression of ≡1 year in 24% to 67% of GBM cases and surviving patients.

Although the Central Nervous System (CNS) is generally considered to have an immune barrier, recent positive results have been achieved for vaccine studies in glioma patients. However, depending on the vaccine efficacy of the complete host immune activity, systemic immunosuppression may be experienced due to tumor expression of immunosuppressive cytokines as well as chemotherapy and radiotherapy. On the other hand, adoptive Cell Transfer (ACT) therapy with autologous T cells, particularly T cells transduced with Chimeric Antigen Receptors (CARs), has shown good promise in hematologic cancer assays. Since 2017, CD 19-targeted CAR-T therapies have been approved by the FDA for clinical use, which is a further major breakthrough in the field of cancer gene therapy (https:// www.fda.gov /) recently reported by intravenous infusion studies in humans where single dose autologous T cells were redirected to egfrvlll by Chimeric Antigen Receptors (CARs) for the first time, it was feasible and safe to make and infuse CAR-modified T Cells (CART) -egfrvlll cells without evidence of extra-tumor toxicity or cytokine release syndrome. One patient in the clinical trial had residual stable disease at 18 months of follow-up.

We constructed third generation Chimeric Antigen Receptor (CAR) T cells, expressed tumor-specific mutations and co-stimulatory CD28BB ζ endodomains of the epidermal growth factor receptor egfrvlll, and evaluated effector function in vitro and in vivo. In cytotoxicity assays, CAR T cells kill egfrvlll positive cells, which promote CAR T cell activation compared to untransformed T cells in vitro and in vivo.

Summary of The Invention

The present invention constructs a third generation CAR against egfrvlll.

Thus, in some aspects, the invention relates to chimeric antigen receptors that bind egfrvlll, and T cells comprising the same.

In some aspects the invention also relates to nucleic acids encoding said chimeric antigen receptor, expression vectors, viruses and T cells comprising the same.

The invention also relates to the use of T cells comprising a chimeric antigen receptor for the treatment of a tumor, preferably said tumor highly expresses egfrvlll, more preferably said tumor is a glioma, most preferably said tumor is a glioblastoma.

In some embodiments, an isolated chimeric antigen receptor is provided comprising, from N-terminus to C-terminus, a sequence of linked: an extracellular binding region, a hinge region/spacer region, a transmembrane region, and an intracellular signaling region comprising a costimulatory domain and a stimulation signaling domain, wherein the extracellular binding region binds egfrvlll.

In some embodiments, the extracellular binding region comprises a heavy chain variable region VH comprising complementarity determining regions HCDR1, HCDR2, and HCDR3 and/or a light chain variable region VL comprising complementarity determining regions LCDR1, LCDR2, and LCDR3. In some specific embodiments, the HCDR1, HCDR2 and HCDR3 are 3 complementarity determining regions of a heavy chain variable region having an amino acid sequence as shown in SEQ ID No. 7 or a sequence having 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID No. 7 and/or the LCDR1, LCDR2 and LCDR3 are 3 complementarity determining regions of a light chain variable region having an amino acid sequence as shown in SEQ ID No. 8 or a sequence having 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID No. 8. In some specific embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise sequences having 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% identity to

SEQ ID NOs

1, 2, 3, 4, 5, and 6, respectively. In some specific embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise sequences having NO more than 3 amino acid changes, NO more than 2 amino acid changes, or NO more than 1 amino acid change as compared to

SEQ ID NOs

1, 2, 3, 4, 5, and 6, respectively.

In some embodiments, the extracellular binding region comprises a heavy chain variable region VH comprising or consisting of the amino acid sequences: the sequence of SEQ ID NO. 7 or a sequence having 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 7.

In some embodiments, the extracellular binding region comprises a light chain variable region VL comprising or consisting of the amino acid sequences: the sequence of SEQ ID NO. 8 or a sequence having 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 8.

In some embodiments, the extracellular binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises or consists of the amino acid sequences of seq id no: the sequence of SEQ ID No. 7 or a sequence having 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 7 and the VL comprises or consists of the amino acid sequences: the sequence of SEQ ID NO. 8 or a sequence having 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 8.

In some embodiments, the extracellular binding region that binds egfrvlll is an antibody or antigen binding fragment thereof, e.g. scFv.

In some embodiments, the scFv comprises or consists of a VH and a VL, preferably wherein the VH comprises or consists of the amino acid sequences of seq id no: the sequence of SEQ ID No. 7 or a sequence having 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 7 and the VL comprises or consists of the amino acid sequences: the sequence of SEQ ID NO. 8 or a sequence having 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 8. In a preferred embodiment, the VH and VL are connected by a linker. In a more preferred embodiment, the sequence of the linker is the sequence shown as SEQ ID NO. 11 or 12 or a sequence having at least 80% identity thereto.

In some embodiments, the scFv comprises or consists of an amino acid sequence selected from the group consisting of the amino acid sequences set forth in any of SEQ ID NO 17-20 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to said sequence.

In some embodiments, the hinge region comprises an IgG4 hinge region and/or a CD8 hinge region. In some embodiments, the hinge region comprises or has at least 80% identity to a sequence as set forth in SEQ ID NO. 13 or 14.

In some embodiments, the transmembrane region comprises a CD28 transmembrane domain, a CD4 transmembrane domain and/or a CD8 transmembrane domain. In some embodiments, the transmembrane region comprises a sequence as set forth in any one of SEQ ID NOS.15-17 or a sequence having at least 80% identity thereto.

In some embodiments, optionally, the transmembrane region is linked to an extracellular binding region via a hinge region.

In some embodiments, the intracellular signaling region comprises a signaling domain (stimulation signaling domain) of cd3ζ and a costimulatory domain. In some embodiments, the CD3 zeta signaling domain comprises or consists of the sequence shown in SEQ ID NO. 20 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the co-stimulatory domain is a functional signaling domain derived from the 4-1BB (CD 137) protein, or a functional signaling domain derived from the CD28 protein, or preferably, the co-stimulatory domain comprises both CD28 and 4-1BB derived. In some embodiments, the 4-1BB (CD 137) signaling domain comprises or consists of the sequence shown in SEQ ID NO. 19 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the CD28 signaling domain comprises or consists of the sequence shown in SEQ ID NO. 18 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

In some embodiments, the costimulatory domain precedes the signaling domain of cd3ζ (i.e., closer to the N-terminus).

In some embodiments, there is provided a humanized Chimeric Antigen Receptor (CAR) polypeptide targeting egfrvlll comprising from N-terminus to C-terminus:

(1) An extracellular binding region that is a humanized anti-egfrvlll scFv sequence, wherein the scFv sequence specifically binds egfrvlll and comprises:

(i) A heavy chain variable region comprising a sequence numbered according to Kabat

(a) A heavy chain complementarity determining region HCDR1 represented by amino acid sequence SEQ ID No. 1, or a variant of said HCDR1 having NO more than 2 amino acid changes or NO more than 1 amino acid change;

(b) An HCDR2 as shown in amino acid sequence SEQ ID No. 2, or a variant of said HCDR2 having NO more than 2 amino acid changes or NO more than 1 amino acid change; and

(c) An HCDR3 as set forth in amino acid sequence SEQ ID No. 3, or a variant of said HCDR3 having NO more than 2 amino acid changes or NO more than 1 amino acid change; and

(ii) A light chain variable region comprising a sequence numbered according to Kabat

(d) A light chain complementarity determining region LCDR1 represented by amino acid sequence SEQ ID NO. 4, or a variant of said LCDR1 having NO more than 2 amino acid changes or NO more than 1 amino acid change;

(e) LCDR2, as shown in amino acid sequence SEQ ID NO. 5, or a variant of said LCDR2 with NO more than 2 amino acid changes or NO more than 1 amino acid change; and

(f) LCDR3, as shown in amino acid sequence SEQ ID NO. 6, or a variant of said LCDR3 with NO more than 2 amino acid changes or NO more than 1 amino acid change;

wherein the amino acid change is an addition, deletion or substitution of an amino acid;

(2) A hinge/spacer region selected from

(i) An IgG4 hinge region (SEQ ID NO: 13), or an IgG4 hinge region having at least 80% sequence identity;

(ii) A CD8 hinge region (SEQ ID NO: 14), or a CD8 hinge region thereof having at least 80% sequence identity.

(3) A transmembrane region (TM) selected from

(i) A CD28 transmembrane domain or variant thereof having 1-5 amino acid modifications, for example, the sequence shown in SEQ ID NO. 15 or a variant thereof having 1-2 amino acid modifications;

(ii) A CD4 transmembrane domain or variant thereof having 1-5 amino acid modifications, e.g., the sequence shown in SEQ ID NO. 16 or a variant thereof having 1-2 amino acid modifications;

(iii) A CD8 transmembrane domain or variant thereof having 1-5 amino acid modifications, e.g., a sequence as set forth in SEQ ID NO. 17 or a variant thereof having 1-2 amino acid modifications;

(4) A Costimulatory Signaling Domain (CSD), which is:

(i) A CD28 costimulatory domain or variant thereof having 1-5 amino acid modifications, e.g.the sequence shown in SEQ ID NO. 18 or a variant thereof having 1-2 amino acid modifications; and

(ii) 4-1BB costimulatory domain or variant thereof with 1-5 amino acid modifications, e.g.the sequence shown in SEQ ID NO. 19 or variant thereof with 1-2 amino acid modifications;

(5) A Stimulatory Signaling Domain (SSD) is a CD3 zeta signaling domain or a variant thereof having 1-10 amino acid modifications, e.g., the sequence shown in SEQ ID NO. 20 or a variant thereof having 1-10, 1-5 amino acid modifications.

In some embodiments, the egfrvlll-targeting humanized Chimeric Antigen Receptor (CAR) polypeptide of the invention comprises from N-terminus to C-terminus:

(i) A heavy chain variable region comprising the sequence of SEQ ID NO. 7 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and

(ii) A light chain variable region comprising the sequence of SEQ ID No. 8 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;

For example, (i) a heavy chain variable region comprising the sequence of SEQ ID NO:7, and

(ii) A light chain variable region comprising the sequence of SEQ ID NO. 8;

(2) A hinge/spacer region selected from

(i) An IgG4 hinge region (SEQ ID NO 13), or an IgG4 hinge region thereof having at least 90%, at least 95% sequence identity;

(ii) A CD8 hinge region (SEQ ID NO 14), or a CD8 hinge region thereof having at least 90%, at least 95% sequence identity.

(3) A transmembrane region (TM) selected from

(i) A CD28 transmembrane domain shown in SEQ ID No. 15 or a variant thereof having 1-3 amino acid modifications;

(ii) A CD4 transmembrane domain shown in SEQ ID No. 16 or a variant thereof having 1-3 amino acid modifications;

(iii) A CD8 transmembrane domain shown in SEQ ID No. 17 or a variant thereof having 1-3 amino acid modifications;

(4) A Costimulatory Signaling Domain (CSD), which is:

(i) A CD28 co-stimulatory domain of SEQ ID No. 18 or a variant thereof having 1-3 amino acid modifications; and

(ii) A 4-1BB costimulatory domain shown in SEQ ID NO. 19 or a variant thereof with 1-3 amino acid modifications;

(5) A Stimulatory Signaling Domain (SSD) that is the CD3 zeta signaling domain shown in SEQ ID NO. 20 or a variant thereof having 1-3 amino acid modifications;

Wherein the amino acid modification is an addition, deletion or substitution of an amino acid.

In some embodiments, the egfrvlll-targeting humanized Chimeric Antigen Receptor (CAR) polypeptide of the invention comprises from N-terminus to C-terminus: :

(i) A heavy chain variable region shown in SEQ ID NO. 7, and

(ii) A light chain variable region shown in SEQ ID NO. 8;

(2) A hinge region shown in SEQ ID NO. 11 or 12;

(3) A transmembrane region (TM) which is the CD28 transmembrane domain shown in SEQ ID NO. 15;

(4) A Costimulatory Signaling Domain (CSD), which is:

(i) A CD28 co-stimulatory domain of SEQ ID NO. 18; and

(ii) A 4-1BB costimulatory domain shown in SEQ ID NO. 19;

(5) CD3 zeta signaling domain shown in SEQ ID NO. 20.

In some embodiments, the humanized Chimeric Antigen Receptor (CAR) polypeptide of the invention that targets egfrvlll further comprises a signal peptide at the N-terminus, such as, but not limited to, a signal peptide having a sequence as shown in SEQ ID No. 21 or having at least 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98% or 99% identity thereto.

In some embodiments, the humanized Chimeric Antigen Receptor (CAR) polypeptide targeting EGFRvIII of the present invention has the amino acid sequence shown in SEQ ID NO. 22 or an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

In some embodiments, the invention provides a nucleic acid encoding any of the foregoing chimeric antigen receptor building elements/full length; in some more specific embodiments, the coding nucleic acid comprises the sequence of SEQ ID NO. 30 and/or 31.

In some embodiments, the invention provides an expression vector comprising the foregoing encoding nucleic acid of the invention.

In some embodiments, the vector is selected from a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector. In some embodiments, the expression vector is derived from a retroviral plasmid; preferably, the retroviral plasmid is SFG.

In some embodiments, the invention provides a virus comprising any of the foregoing vectors.

In some embodiments, the invention provides an immune effector cell transduced with any of the foregoing encoding nucleic acids, or any of the foregoing expression vectors or any of the foregoing viruses, preferably the immune effector cell is a T cell, more preferably a T lymphocyte.

In some embodiments, the invention provides an immune effector cell, the surface of which expresses any of the foregoing chimeric antigen receptors, preferably the immune effector cell is a T cell, more preferably a T lymphocyte.

In some embodiments, the immune effector cells of the invention exhibit enhanced tumor cell killing due to transduction/expression of the chimeric antigen receptor of the invention. In some preferred embodiments, enhanced killing of target cells is manifested by higher anti-tumor cytokine secretion and/or greater direct oncolytic capacity. In some preferred embodiments, higher amounts of anti-tumor cytokine secretion refer to secretion of cytokines (e.g., without limitation, IFN-gamma, IL-2, IL-6, IL-10, IL-17, and/or TNF-alpha, etc.) that are increased 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 0.6 fold, 0.7 fold, 0.8 fold, 0.9 fold, 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold or more relative to non-specific control effector cells. By stronger direct oncolytic capacity is meant that the amount of direct killing of a target tumor cell by an immune effector cell of the invention is increased by 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 0.6 fold, 0.7 fold, 0.8 fold, 0.9 fold, 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold or more relative to an effector cell of a non-specific control.

In some embodiments, the invention provides methods of making an immune effector cell of the invention, comprising introducing the cell with a nucleic acid, expression vector, and/or virus of the invention, and/or allowing the cell to express a chimeric antigen receptor of the invention, e.g., isolating T cells from human PBMCs, and transducing the isolated T cells with the nucleic acid, expression vector, and/or virus.

In some embodiments, the invention provides a method of preventing or treating a tumor (e.g., cancer) or providing anti-tumor immunity in a subject comprising administering to the subject an effective amount of a cell expressing any of the foregoing chimeric antigen receptors, or any of the foregoing T cells.

In other embodiments, the tumor (e.g., cancer) has egfrvlll (e.g., at an elevated level, e.g., at a nucleic acid or protein level) in the patient, preferably the tumor is a glioma, more preferably the tumor is a glioblastoma.

In other embodiments, the cells are administered in combination with one or more other therapies, such as therapeutic modalities and/or other therapeutic agents. In a specific embodiment, the additional therapeutic agent is a chemotherapeutic agent, a cytokine, a cytotoxic agent, a therapeutic monoclonal antibody, a small molecule drug, an immunomodulatory agent (e.g., an immunosuppressant), or any combination thereof. In a preferred embodiment, the other therapeutic agent is a therapeutic monoclonal antibody, more preferably a PD-1 antibody.

In other embodiments, the invention provides the use of a chimeric antigen receptor, nucleic acid, vector, virus or cell of any of the preceding embodiments for the preparation of a medicament. The medicament is used for preventing or treating tumors. Preferably, the tumor has (e.g. elevated levels of e.g. nucleic acid or protein levels) egfrvlll, preferably the tumor is a glioma, more preferably the tumor is a glioblastoma. In other embodiments, the invention provides the medicament, i.e., the invention provides a pharmaceutical composition comprising the chimeric antigen receptor, nucleic acid, vector, virus or cell of any of the preceding embodiments.

In some further embodiments, the medicament is administered alone or in combination with one or more other therapies, such as therapeutic modalities and/or other therapeutic agents. In a specific embodiment, the additional therapeutic agent is a chemotherapeutic agent, a cytokine, a cytotoxic agent, a therapeutic monoclonal antibody, a small molecule drug, an immunomodulatory agent (e.g., an immunosuppressant), or any combination thereof. In a preferred embodiment, the other therapeutic agent is a therapeutic monoclonal antibody, more preferably a PD-1 antibody.

Description of the drawings:

figure 1 egfrvlll CAR-T cells mediate excellent anti-tumor efficacy in vitro. A. After 6 hours of co-culture with U373-EGFRvIII (right panel) or U87-EGFRvIII (left panel), the up-regulation of CD107a by CAR-T cells (nonspecific CARs (top); EGFRvIII CARs (bottom)) was analyzed by flow cytometry. Egfrvlll CARs show degranulation when co-cultured with target cells in both co-culture systems. T cell populations were determined by analysis of CD3 expression. B. Statistics of in vitro experimental data. To evaluate EGFRvIII ⁺ Target cell recognition, non-specific T cells (non-transformed T cell non-specific CAR-T cells) or EGFRvIII ⁺ CAR T cells and EGFRvIII ⁺ Co-culturing target cells. Quantification of CD107a+ cells positive for intracellular staining (ICS) showedEgfrvlll CAR induced T cell activation resulted in significant CD107a expression. C. Quantification of IFN-. Gamma. + intracellular staining (ICS) -positive cells is shown. The effect of T cell transduction on IFN- γ+ cell proportion varies significantly between mock CAR cells and egfrvlll+ CAR T cells. D. Cytokine production triggered by egfrvlll specific CARs. EGFRvIII specific and non-specific T cells (including non-transduced and non-specific CARs) were co-cultured with target cells at a 10:1 E:T ratio, and after 24 hours, IFN-. Gamma.produced by the T cells was determined by ELISA. E. Cytotoxicity of EGFRvIII CARs against target cells was confirmed by RT-PCR. F. Residual status of egfrvlll positive target cells after 24 hours of co-culture (middle), egfrvlll CAR was strongly toxic to target cells (a represents U87-egfrvlll cells, B represents U373-egfrvlll cells). All results are expressed as mean ± SD of at least duplicate. (. P) <0.05,**p<0.005,***p<0.005)。

Figure 2. Egfrvlll CAR-T cells show egfrvlll specific tumor cell killing activity in vitro. A. Impedance-based cytotoxicity assays were measured on effector cells E: target cell t=10: the growth curve of the target cells was measured at 1 and the cell index was used as an inverse measure of the viability of the target cells. Using the xcelligent system (Agilent), the cell index values of tumor cells were continuously graphically output during the co-incubation of tumor cells with CAR T cells and incubation of tumor cells alone, up to a time point of 50 hours. Real-time impedance analysis was performed on mock-and EGFRvIII-CAR-T cells according to the co-culture protocol. Cell index values of U87-EGFRvIII (left) and U373-EGFRvIII (right) cells, depending on the co-culture protocol used. Experiments were performed in duplicate. The red line indicates the growth curve for only target cells and the blue line indicates the growth curve for cytotoxicity experiments. B. Luciferase reporter assay. In a standard 24-hour cytotoxicity assay, EGFRvIII CAR T cells kill U87-EGFRvIII and U373-EGFRvIII cells, in contrast to EGFRvIII negative cells.

Fig. 3 intracranial delivery of egfrvlll CAR T cells significantly prolonged survival of mice. A. U87-EGFRvIII cells (2X 10) ⁵ Left panel) and U373-EGFRvIII cells (2X 10) ⁵ Right panel) in situ implantation into NOD-SCID mice with intracranial effects after implantationCells (1X 10) ⁷ ) And (5) processing. The survival curves for each group were estimated using the Kaplan-Meier product limit estimation method. The main comparative analysis of the curves of each group was performed using the log-rank test. Bioluminescence imaging of u 87-egfrvlll tumors over time, n=4 mice. Bioluminescence imaging of the u 373-egfrvlll tumor over time, n=3 mice.

Figure 4. Homing of egfrvlll CAR-T cells to tumor sites. Immunohistochemical analysis was performed to detect a. Histomorphology, b. Egfrvlll protein, and C.T cells.

Fig. 5. Schematic representation of egfrvlll CAR molecules. B. Flow cytometry detected egfrvlll CAR cell surface expression of PBMC transfectants 9 days after nuclear transfection. GAM expression is typically detected as a low-magnitude population transition of 0.5 logarithmic units of average fluorescence. Egfrvlll car+ T cells were gated on living cells (left), cd4+ (middle), cd8+ (right). Negative staining controls were performed by staining untransfected T cells. C. Cell surface egfrvlll CAR of PBMC transfectants was detected with Western blot 9 days after nuclear transfection. Average expression rate of egfrvlll CAR on D.T cell membrane.

FIG. 6.A. Cell lines stably transfected with green fluorescent protein and luciferase. The green fluorescent protein was performed using a flow cytometer. B. EGFR and EGFRvIII expression was detected by RT-PCR.

FIG. 7.T upregulation of cell activation markers. Mock T cells or B.EGFRvIII-CAR T cells were co-cultured with U87-EGFRvIII or U373-EGFRvIII. Analysis of CD25 by flow cytometry by specific antibody-coupled APCs showed significantly higher expression ratios of CAR-T cells compared to control (mock) cells.

FIG. 8 shows quantification of ICS-positive cells for IFN-. Gamma.+. The effect of A.T cell transduction on IFN- γ+ cell proportion was significantly different between mock CAR cells and egfrvlll+ CAR T cells. Egfrvlll+car T cells cultured alone (i.e. without target cells) or in co-culture with egfrvlll negative glioma cells; B. quantification of IFN-. Gamma. + ICS positive cells was negligible.

Fig. 9 egfrvlll+ cells induced significantly higher levels of various cytokines, especially ifnγ and TNF, than egfrvlll-cells. IL17A did not reach the lowest detection sensitivity and therefore did not account for its changes (not shown). The U373 (tumor cell only) group served as a statistical negative control.

FIG. 10 luciferase reporter assay. In a standard 24-hour cytotoxicity assay, EGFRvIII CAR T cells killed U87-EGFRvIII and U373-EGFRvIII cells, in contrast to EGFRvIII negative cells. A. Luciferase fluorescence intensity detected in co-culture system. B. The control group (egfrvlll negative cells) showed lower cytolytic activity. Relative lysis (%) refers to the ratio of the higher mortality of egfrvlll CAR to its basal mortality (Mock-CAR T cells).

Fig. 11T cells from the same donor transduced egfrvlll CAR or Mock CAR and were cultured overnight with egfrvlll positive or negative tumor cells. A. Proliferation of T cells transduced with CAR. CAR-T cells were grouped by flow cytometry according to B.T cell phenotype or c.cd4 and CD8 gated group. D. Effector cells were co-cultured with target cells at 10:1 E:T. T cell phenotypes were assessed by flow cytometry on day 1 (after stimulation, up) and day 7 (down). We counted the CD3 (left), CD4 (middle), CD8 (right) positive T cell subsets, respectively. After 24 hours antigen stimulation, the exhaustion markers were measured with a flow cytometer.

FIG. 12 anti-tumor response of EGFRvIII CAR-T in vivo. Bioluminescence imaging of tumor growth over time, treatment with intracranial effector cells after implantation. The u 373-egfrvlll cells were implanted subcutaneously with n=5 mice. U251 cells were implanted in situ, n=5 mice. C. Immunohistochemistry of CD3 in the subcutaneous model showed that CAR-T cells were not present in the tumor at the end of the experiment (10X). D. Survival curves for each group were evaluated using the Kaplan-Meier product limit estimation method. The main comparative analysis was performed on the curves of each group using the log-rank test (subcutaneous model, upper; in situ model, lower).

Detailed Description

I. Definition of the definition

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. 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.

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.

As used herein, the terms "comprises" or "comprising" are intended to include the stated elements, integers or steps but do not exclude any other elements, integers or steps. In this document, the terms "comprises" or "comprising" when used herein, unless otherwise indicated, also encompass the instances of the recited elements, integers, or steps in combination. For example, when referring to an element/polypeptide "comprising" a particular sequence, it is also intended to encompass elements/polypeptides that consist of that particular sequence.

The term "EGFRvIII" as used herein refers to the EGFRvIII (EGFR variant type-III) gene, or a protein expressed by the gene. Increased expression of EGFR is often detected in a variety of cancers, including breast cancer, lung cancer, head and neck cancer, and glioblastoma. Spontaneous rearrangement of the EGFR gene was first found in primary human glioblastomas, and in almost all cases, this change was reported in EGFR-amplified tumors. These rearrangements result in three different types of mutants, of which type III is the most common, all known as type III EGF deletion mutant receptor, with exons 2-7 in the mRNA deleted compared to wild type EGFR (epidermal growth factor receptor). These deletions correspond to cDNA nucleotides 275-1075, which encode amino acids 6-276, possibly by alternative splicing or rearrangement. Deletion of 801bp in the extracellular region of the EGFR gene results in-frame truncation of the normal EGFR protein, producing a 145kDa receptor, thereby producing tumor-specific and immunogenic epitopes. Egfrvlll is expressed in a variety of tumor types, including glioblastoma multiforme (GBM), but is rarely observed in normal tissues. EGFRvIII is expressed for > 1 year in 24% to 67% of GBM cases and surviving patients, and EGFRvIII expression is an independent negative prognostic indicator.

The term "anti-egfrvlll antibody", "anti-egfrvlll", "egfrvlll antibody" or "antibody binding to egfrvlll" as used herein refers to an antibody capable of binding (human) egfrvlll with sufficient affinity such that the antibody can be used as a therapeutic agent for targeting (human) egfrvlll. In some embodiments, the (human) egfrvlll antibody binds to (human) egfrvlll with high affinity in vitro or in vivo.

The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen binding fragment.

An "antigen binding fragment" refers to a molecule that is different from an intact antibody, which comprises a portion of the intact antibody and binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to Fv, fab, fab ', fab ' -SH, F (ab ') ₂ The method comprises the steps of carrying out a first treatment on the surface of the dAb (domain antibody); a linear antibody; single chain antibodies (e.g., scFv); single domain antibodies such as VHH; a diabody or fragment thereof; or camelidae antibodies. The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light chain and heavy chain variable regions are contiguous (e.g., via a synthetic linker such as a short flexible polypeptide linker) and are capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein, an scFv may have the VL and VH variable regions, s, in any order (e.g., relative to the N-and C-termini of the polypeptide) The cFv may comprise a VL-linker-VH or may comprise a VH-linker-VL.

When referring to "anti-egfrvlll antibody", "anti-egfrvlll", "egfrvlll antibody" or "antibody binding to egfrvlll" herein, also an antibody fragment, e.g. scFv, that specifically binds EGFR may be referred to, unless otherwise indicated.

"complementarity determining regions" or "CDR regions" or "CDRs" are regions of an antibody variable domain that are hypervariable in sequence and form structurally defined loops ("hypervariable loops") and/or contain antigen-contacting residues ("antigen-contacting points"). CDRs are mainly responsible for binding to the epitope. CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2 and HCDR3, while CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2 and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the exact amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example: chothia (Chothia et al, (1989) Nature 342:877-883, al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", journal of Molecular Biology,273,927-948 (1997)) based on the three-dimensional structure of antibodies and topology of CDR loops, kabat (Kabat et al, sequences of Proteins of Immunological Interest, 4 th edition, U.S. Pat. No. of Health and Human Services, national Institutes of Health (1987)), abM (University of Bath), contact (University College London), international ImMunoGeneTics database (IMGT) (on the world Wide Web) based on neighbor-transmitted clusters (CDR affinity propagation clustering) using a large number of crystal structures.

For example, the residues of each CDR are as follows, according to different CDR determination schemes.

CDRs may also be determined based on having the same Kabat numbering positions as the reference CDR sequences (e.g., any of the exemplary CDRs of the invention).

In the present invention, unless otherwise indicated, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-described ways.

In the present invention, unless otherwise indicated, when referring to residue positions in the antibody variable region, including heavy chain variable region residues and light chain variable region residues, reference is made to numbering positions according to the Kabat numbering system (Kabat et al Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md. (1991)).

In some embodiments, the heavy chain variable region CDRs of an antibody of the invention are determined according to Kabat rules

In some embodiments, the CDRs of the light chain variable region of an antibody of the invention are determined according to Kabat rules.

In some embodiments, the heavy chain variable region CDRs of an antibody of the invention are determined according to Kabat rules; and the light chain variable region CDRs are determined according to Kabat rules.

In another alternative embodiment, the heavy chain variable region CDRs and/or the light chain variable region CDRs of an antibody of the invention are determined according to non-Kabat rules, e.g., according to rules well known in the art as Chothia, abM, contact, IMGT and North, et al.

It should be noted that the boundaries of CDRs of variable regions of the same antibody obtained based on different assignment systems may differ. I.e. the CDR sequences of the same antibody variable region defined under different assignment systems are different. Thus, when referring to defining antibodies with a particular CDR sequence as defined herein, the scope of the antibodies also encompasses antibodies whose variable region sequences comprise the particular CDR sequence, but whose purported CDR boundaries differ from the particular CDR boundaries defined herein by the application of different protocols (e.g., different assignment system rules or combinations).

Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs (under the same assignment system). However, although CDRs vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. The minimum overlap region can be determined using at least two of the Kabat, chothia, abM, contact and North methods, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit may be a sub-portion of the CDR. As will be apparent to those skilled in the art, the residues in the remainder of the CDR sequences can be determined by the structure of the antibody and the protein folding. Thus, the present invention also contemplates variants of any of the CDRs presented herein. For example, in a variant of one CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues according to the Kabat or Chothia definition may be replaced by conserved amino acid residues.

As used herein, a "human-derived" or "mouse-derived" region refers to or consists of an amino acid sequence or coding nucleic acid sequence of the region of the native human or native mouse. A region "derived from" a human or mouse also encompasses a region that is substantially identical to the amino acid sequence or coding nucleic acid sequence of the region of the native human or native mouse, or that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence, and still has the same or similar activity and/or function as the region. For example, when the definition "the cd3ζ chain is derived from human" is used, it is meant that the cd3ζ chain has or consists of the amino acid sequence of a native human cd3ζ chain; or means that the amino acid sequence of the CD3 zeta chain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of the native human CD3 zeta chain and still has the activity and/or function of the native human CD3 zeta chain.

"humanized" antibody refers to an antibody that comprises amino acid residues from a non-human CDR and amino acid residues from a human FR. In some embodiments, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.

"human antibody" or "fully human antibody" may be used interchangeably to refer to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source that utilizes a human antibody repertoire or other human antibody coding sequence. This definition of human antibodies specifically excludes humanized antibodies that comprise non-human antigen binding residues.

As used herein, the term "bind" or "specifically bind" means that the binding is selective for an antigen and distinguishable from unwanted or non-specific interactions. The ability of an antigen binding site to bind to a particular antigen may be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as by Radioimmunoassay (RIA) or biofilm thin layer interferometry or MSD assay or Surface Plasmon Resonance (SPR) or cell binding experiments.

The term "chimeric antigen receptor" or alternatively "CAR" refers to a group of polypeptides that, when in an immune effector cell, provide the cell with specificity for a target cell (typically a cancer cell) and have intracellular signaling. In certain embodiments, the CAR comprises at least one extracellular binding region, a transmembrane region, and an intracellular signaling region. In certain aspects, the sets of polypeptides are contiguous with each other.

The term "intracellular signaling region" refers to a functional portion of a protein that functions by transmitting information within a cell to regulate the activity of the cell via a defined signaling pathway by either producing a second messenger or by acting as an effector in response to such a messenger.

The term "extracellular binding region" refers to the portion of the chimeric antigen receptor that recognizes an antigen, and in some embodiments, the extracellular binding region is an scFv from a monoclonal antibody, or dAb (domain antibody) or VHH.

The term "transmembrane region" refers to the portion of a chimeric antigen receptor that spans the membrane. The transmembrane region may be any protein structure that is thermodynamically stable in the membrane. This is typically an alpha helix comprising several hydrophobic residues. The transmembrane region of any transmembrane protein may be used to provide the transmembrane portion of a chimeric receptor, for example the transmembrane region may be a transmembrane region from CD8 a or CD28, preferably a sequence comprising or having at least 90%, 95%, 96%, 97%, 98% or 99% or more identity to the amino acid sequence of any one of SEQ ID NOs 15 to 17.

The term "CD3- ζ", also known as CD247, is defined as a protein as provided by genbank acc.no. bag36664.1 or an equivalent residue from a non-human species (e.g., mouse, rodent, monkey, ape, etc.); "CD 3-zeta signaling domain" is defined as the amino acid residue from the cytoplasmic domain of CD 3-zeta or a functional derivative thereof sufficient to functionally transmit the initiation signal required for T cell activation. In one embodiment, a "CD 3-zeta signaling domain" comprises a sequence as provided in SEQ ID NO. 20 or a sequence having at least 90%, 95%, 96%, 97%, 98% or 99% or more identity thereto.

The term "4-1BB" refers to a member of the TNFR superfamily having an amino acid sequence as provided by GenBank Acc.No. AAA62478.2, or an equivalent residue from a non-human species such as mouse, rodent, monkey, ape, or the like. In one aspect, the "4-1BB co-stimulatory domain" is defined as amino acid residues 214-255 of GenBank Acc.No. AAA62478.2, or equivalent residues from a non-human species such as mouse, rodent, monkey, ape, etc. In one aspect, a "4-1BB costimulatory domain" is a sequence as provided in SEQ ID NO 19 or a sequence that is identical or has at least 90%, 95%, 96%, 97%, 98% or 99% or more identity to an equivalent residue from a non-human species such as mouse, rodent, monkey, ape, or the like.

The term "CD28" refers to human leukocyte differentiation antigen 28, which is known under the official name CD28, ID number 940, there are 3 isoforms (cDNA sequence/protein sequence) NM_006139.3/NP_006130.1, NM_001243077.1/NP_001230006.1, NM_001243078.1/NP_001230007.1, respectively. As used herein, the term "CD28 co-stimulatory domain" comprises a sequence as provided in SEQ ID No. 18 or a sequence having at least 90%, 95%, 96%, 97%, 98% or 99% or more identity thereto.

The term "retrovirus" as used herein refers to those viruses used as commonly used genetic engineering/gene therapy vectors among retroviruses, i.e., RNA viruses. In a specific embodiment, the retroviral vector used in the present invention is based on the Moloney mouse leukemia virus (SFG) plasmid, also known as "vector pMSGV1". Commonly used retroviral vectors are available and known to those skilled in the art, for example, from Addgene Inc.

The term "therapeutic agent" as described herein encompasses any substance that is effective in preventing or treating a tumor, such as a cancer, including a chemotherapeutic agent, cytokine, cytotoxic agent, therapeutic monoclonal antibody, small molecule drug, or immunomodulatory agent (e.g., immunosuppressant).

The term "cytotoxic agent" is used herein to refer to a substance that inhibits or prevents cellular function and/or causes cell death or destruction.

"chemotherapeutic agents" include chemical compounds useful in the treatment of cancer.

The term "small molecule drug" refers to a low molecular weight organic compound capable of modulating biological processes. A "small molecule" is defined as a molecule having a molecular weight of less than 10kD, typically less than 2kD and preferably less than 1 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptidomimetics, and antibody mimetics. As a therapeutic agent, small molecules may be more cell permeable, less susceptible to degradation, and less prone to eliciting an immune response than large molecules.

The term "immunomodulator" as used herein refers to a natural or synthetic active agent or drug that inhibits or modulates an immune response. The immune response may be a humoral response or a cellular response. Immunomodulators comprise immunosuppressants.

As used herein, an "immunosuppressant," "immunosuppressant drug," or "immunosuppressant" is a therapeutic agent used in immunosuppressive therapy to inhibit or prevent the activity of the immune system.

The term "effective amount" refers to an amount or dose of an antibody or fragment or conjugate or composition or combination 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.

"therapeutically effective amount" means an amount effective to achieve the desired therapeutic result at the desired dosage and for the desired period of time. A therapeutically effective amount is also an amount in which any toxic or detrimental effect of the antibody or antibody fragment or conjugate or composition or combination thereof is less than a therapeutically beneficial effect. The "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70% relative to an untreated subject.

"prophylactically effective amount" means an amount effective to achieve the desired prophylactic result at the desired dosage and for the desired period of time. Typically, since the prophylactic dose is administered in the subject prior to or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

"individual" or "subject" includes 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 some embodiments, the individual or subject is a human.

Calculation of sequence identity between sequences was performed as follows.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, the length of the reference sequences aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequences. Amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

Sequence comparison and calculation of percent identity between two sequences can be accomplished using mathematical algorithms. In a preferred embodiment, the percentage identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) j.mol.biol.48:444-453) algorithm (available at http:// www.gcg.com) which has been integrated into the GAP program of the GCG software package, using the Blossum 62 matrix or PAM250 matrix and the

GAP weights

16, 14, 12, 10, 8, 6 or 4 and the

length weights

1, 2, 3, 4, 5 or 6. In yet another preferred embodiment, the percentage of identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http:// www.gcg.com) using the NWS gapdna.CMP matrix and the

GAP weights

40, 50, 60, 70 or 80 and the

length weights

1, 2, 3, 4, 5 or 6. A particularly preferred set of parameters (and one that should be used unless otherwise indicated) is the Blossum 62 scoring matrix employing gap penalty 12, gap extension penalty 4, and frameshift gap penalty 5. The percent identity between two amino acid sequences or nucleotide sequences can also be determined using PAM120 weighted remainder table, gap length penalty 12, gap penalty 4) using the e.meyers and w.miller algorithm that has been incorporated into the ALIGN program (version 2.0) ((1989) CABIOS, 4:11-17). Additionally or alternatively, the nucleic acid sequences and protein sequences described herein may be further used as "query sequences" to perform searches against public databases, for example, to identify other family member sequences or related sequences.

The term "anti-tumor effect" refers to a biological effect that can be demonstrated by a variety of means including, but not limited to, for example, a decrease in tumor volume, a decrease in tumor cell number, a decrease in tumor cell proliferation, or a decrease in tumor cell survival.

The terms "tumor" and "cancer" are used interchangeably herein to encompass solid tumors and liquid tumors.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. In certain embodiments, cancers suitable for treatment by the antibodies of the invention include gastric or pancreatic cancers, including metastatic forms of those cancers.

The term "tumor" refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancerous" and "tumor" are not mutually exclusive when referred to herein.

The term "glioma" is an abbreviation for glioma, also known as glioma. Glioma is a neoplastic disease of the central nervous system, which refers to a tumor that occurs in the neuroectodermal layer, which results from the cancerous changes of neuronal or mesenchymal cells. Gliomas are the most common primary central nervous system neoplasms in the cranium, accounting for about half of all primary intracranial neoplasms. Glioblastoma is a common type of glioma, also called glioblastoma multiforme (GBM), and is a name of grade IV in the classification of gliomas by the world health organization, and grade IV glioma is the glioma with the highest malignancy. Glioblastoma generally occurs at peak ages of 50-60 years.

The term "combination therapy" refers to the administration of two or more therapeutic agents or modes of treatment (e.g., radiation therapy or surgery) to treat the diseases described herein. Such administration includes co-administration of the therapeutic agents in a substantially simultaneous manner, e.g., in a single capsule with a fixed ratio of active ingredients. Alternatively, such administration includes co-administration of the individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). The powder and/or liquid may be reconstituted or diluted to the desired dosage prior to administration. In addition, such administration also includes the use of each type of therapeutic agent in a sequential manner at about the same time or at different times. In either case, the treatment regimen will provide a beneficial effect of the pharmaceutical combination in treating the disorders or conditions described herein.

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.

As used herein, "preventing" includes inhibition of the occurrence or progression of a disease or disorder or a symptom of a particular disease or disorder. In some embodiments, subjects with a family history of cancer are candidates for prophylactic regimens. Generally, in the context of cancer, the term "prevention" refers to administration of a drug prior to the occurrence of a sign or symptom of cancer, particularly in a subject at risk of cancer.

The term "vector" as used herein 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 a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".

The term "marker" as used herein refers to a substance that is derived from an in vivo or ex vivo sample and whose level of presence in the sample is relatively easy to detect using established or routine experimentation and tools in the art, and which level of presence (e.g., level or absence) is associated with a particular physiological or pathological condition, so that the particular physiological or pathological condition can be inferred or assisted by obtaining the level of presence of the marker. The marker may be any substance present in the body, such as, but not limited to, a nucleic acid, a polysaccharide, a protein, an inorganic or organic small molecule, or a polymer or hybrid thereof (e.g., glycoprotein, phosphorylated protein, methylated nucleic acid sequence, etc.), or a gene encoding the same (e.g., protein). In some embodiments of the invention, the marker is IFN-gamma. In some embodiments of the invention, the particular physiological or pathological state that needs to be inferred refers to the ability of a CAR-T cell of the invention to kill or inhibit a target tumor that is or will be exhibited upon entry into a subject. In some further embodiments, the sample is an in vitro culture supernatant of T cells after transfection of the CAR molecule, or serum of a subject after treatment with CAR-T cells.

The term "IFN-gamma" as used herein refers to a subtype of cytokine interferon (interferon) which is secreted by activated T cells and natural killer cells, particularly type I helper T cells (Th 1 cells), as water soluble dimers. The receptor for IFN-gamma consists of two subunits, which are activated after binding to IFN-gamma, modulating the JAK-STAT pathway. Several studies have shown that IFN-gamma has antiviral, immunomodulatory and antitumor activities.

CD107a is a surface marker for T cells. Cytotoxic particles in CTL and NK cells are mainly present in secretory lysosomes, and CD107a is also known as lysosome-associated membrane protein-1, a protein present on lysosome membranes, and when secretory lysosomes are secreted, lysosomes are bound to cell membranes, CD107a is expressed on cell membranes as lysosome membranes are bound to cell membranes, so that CD107a molecule is a sensitive marker of CTL cell degranulation, is directly related to cytotoxic activity, and can reflect the level of cytotoxic cell killing activity

Chimeric antigen receptor and nucleic acid encoding same

In one aspect of the invention, the invention relates to an isolated Chimeric Antigen Receptor (CAR) molecule comprising (e.g., sequentially linked to) an extracellular binding region that binds egfrvlll, a transmembrane region, and an intracellular signaling region (e.g., comprising a costimulatory domain).

II-1 extracellular binding region

In some embodiments, an extracellular binding region described herein that binds egfrvlll binds to wild type egfrvlll, e.g., human wild type egfrvlll.

The extracellular binding region that binds egfrvlll can be any antigen binding domain that binds egfrvlll: including but not limited to monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and functional fragments thereof, including but not limited to scFv, single domain antibodies, such as heavy chain variable domains (VH), light chain variable domains (VL), and variable domains (VHH) of camelid-derived nanobodies, and function as antigen binding domains in combination with alternative scaffolds known in the art. In some cases it is advantageous that the antigen binding domain is derived from the same species as the end use of the CAR. For example, for use in humans, it may be advantageous for the antigen binding domain of the CAR to comprise residues of the antigen binding domain of a human or humanized antibody or antibody fragment. Thus, in one aspect, the antigen binding domain comprises a human antibody or antibody fragment. In one aspect, the antigen binding domain is an scFv.

In one embodiment, the extracellular binding region that binds egfrvlll comprises one or more (e.g., all 3) light chain complementarity determining region 1 (LCDR 1), light chain complementarity determining region 2 (LCDR 2) and light chain complementarity determining region 3 (LCDR 3) described herein and one or more (e.g., all 3) heavy chain complementarity determining region 1 (HCDR 1), heavy chain complementarity determining region 2 (HCDR 2) and heavy chain complementarity determining region 3 (HC CDR 3) described herein.

In some embodiments, the extracellular binding region that binds egfrvlll of the present invention comprises 3 complementarity determining regions (HCDR) from the heavy chain variable region, HCDR1, HCDR2 and HCDR3.

In some embodiments, the extracellular binding region that binds egfrvlll of the present invention comprises 3 complementarity determining regions (LCDR) from the light chain variable region, LCDR1, LCDR2 and LCDR3.

In some embodiments, the extracellular binding region that binds egfrvlll of the invention comprises 3 complementarity determining regions (HCDR) from a heavy chain variable region and 3 complementarity determining regions (LCDR) from a light chain variable region.

In some aspects, the extracellular binding region of the invention that binds egfrvlll comprises a heavy chain variable region (VH). In some aspects, the extracellular binding region of the invention that binds egfrvlll comprises a light chain variable region (VH). In some aspects, the extracellular binding region of the invention that binds egfrvlll comprises a heavy chain variable region and a light chain variable region (VH). In some embodiments, the heavy chain variable region comprises 3 Complementarity Determining Regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2 and HCDR3. In some embodiments, the light chain variable region comprises 3 Complementarity Determining Regions (CDRs) from the light chain variable region, LCDR1, LCDR2 and LCDR3.

In some embodiments, the heavy chain variable region of the extracellular binding region of EGFRvIII of the present invention

(i) Comprising or consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID No. 7; or alternatively

(ii) Comprising or consisting of an amino acid sequence selected from SEQ ID NO. 7; or alternatively

(iii) An amino acid sequence comprising (preferably amino acid substitutions, more preferably amino acid conservative substitutions) 1 or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence selected from SEQ ID NO. 7 consists of said amino acid sequence, preferably said amino acid changes do not occur in the CDR regions.

In some embodiments, the light chain variable region of the extracellular binding region of EGFRvIII of the present invention

(i) Comprising or consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID No. 8; or alternatively

(ii) Comprising or consisting of an amino acid sequence selected from SEQ ID NO. 8; or alternatively

(iii) An amino acid sequence comprising (preferably amino acid substitutions, more preferably amino acid conservative substitutions) 1 or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence selected from SEQ ID NO. 8 consists of said amino acid sequence, preferably said amino acid changes do not occur in the CDR regions.

In some embodiments, the 3 complementarity determining regions (HCDRs) from the heavy chain variable region of the invention, HCDR1, HCDR2 and HCDR3 are selected from

(i) Three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO. 7, or

(ii) A sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in total on the three HCDR regions relative to any of (i).

In some embodiments, the 3 complementarity determining regions (LCDRs) from the light chain variable region of the invention, LCDR1, LCDR2 and LCDR3 are selected from

(i) Three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO. 8, or

(ii) A sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in total on the three LCDR regions relative to any of (i).

In some embodiments, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO. 1, or HCDR1 comprises an amino acid sequence having one, two, or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO. 1.

In some embodiments, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO. 2, or HCDR2 comprises an amino acid sequence having one, two, or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO. 2.

In some embodiments, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO. 3, or HCDR3 comprises an amino acid sequence having one, two, or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO. 3.

In some embodiments, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO. 4, or LCDR1 comprises an amino acid sequence having one, two, or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO. 4.

In some embodiments, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO. 5, or LCDR2 comprises an amino acid sequence having one, two, or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO. 5.

In some embodiments, LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO. 6, or LCDR3 comprises an amino acid sequence having one, two, or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO. 6.

In some embodiments of the invention, the extracellular binding region of the invention that binds egfrvlll comprises a VH and a VL, wherein said VH and VL comprise or consist of the amino acid sequences shown below, respectively: SEQ ID NOS.7 and 8.

In some embodiments of the invention, the amino acid changes described herein include substitutions, insertions, or deletions of amino acids. Preferably, the amino acids described herein are changed to amino acid substitutions, preferably conservative substitutions.

In a preferred embodiment, the amino acid changes described in the present invention occur in regions outside the CDRs (e.g., in the FR). More preferably, the amino acid changes described herein occur in regions outside the heavy chain variable region and/or outside the light chain variable region.

In some embodiments, the substitutions are conservative substitutions. Conservative substitutions refer to the substitution of one amino acid with another within the same class, e.g., the substitution of one acidic amino acid with another acidic amino acid, the substitution of one basic amino acid with another basic amino acid, or the substitution of one neutral amino acid with another neutral amino acid. Exemplary permutations are shown in the following table:

in certain embodiments, the substitution occurs in the CDR regions of the extracellular binding region of egfrvlll. Typically, the resulting variants will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity) relative to the parent antibody and/or will have certain biological properties of the parent that are substantially preserved. Exemplary substitution variants are affinity matured antibodies.

In some embodiments, the extracellular binding region of egfrvlll of the invention is an scFv fragment of an anti-egfrvlll antibody.

In some embodiments, the scFv fragment comprises a linker. In some embodiments, the linker is a peptide linker. The peptide linker may or may not include predominantly the following amino acid residues: gly, ser, ala or Thr. Useful linkers include glycine-serine polymers including, for example, (GS) n, (GSGGS) n, (GGGGS) n, (GGGS) n and (GGGGS) nG, where n is an integer of at least 1 (and preferably 2, 3, 4, 5, 6, 7, 8, 9, 10). In some embodiments, the linker comprises the amino acid sequence of (GSGGS) 3 (SEQ ID NO: 11). In some embodiments, the linker comprises the amino acid sequence of (GSGGS) 4 (SEQ ID NO: 12).

In some embodiments, the scFv fragment comprises or consists of an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs 9 or 10 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to said sequence.

II-2 transmembrane region

In various embodiments, with respect to the transmembrane region, the CAR can be designed to include a transmembrane region linked to the extracellular domain of the CAR. The transmembrane region may include one or more additional amino acids adjacent to the transmembrane region, such as one or more amino acids associated with the extracellular region of the protein from which the transmembrane is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region) and/or one or more additional amino acids associated with the extracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the intracellular region). In one aspect, the transmembrane region is a region associated with one of the other regions of the CAR used, e.g., in one embodiment, the transmembrane region may be from the same protein from which the signaling domain, co-stimulatory domain, or hinge domain is derived. In another aspect, the transmembrane region is not the same protein from any other region of the CAR. In some cases, the transmembrane region may be selected or modified by amino acid substitution to avoid binding of such region to the transmembrane region of the same or a different surface membrane protein, e.g., to minimize interaction with other members of the receptor complex. In one aspect, the transmembrane region is capable of homodimerizing another CAR on the cell surface of the CAR-expressing cell. In a different aspect, the amino acid sequence of the transmembrane region can be modified or substituted so as to minimize interaction with the binding domain of a natural binding partner present in a cell expressing the same CAR.

The transmembrane region may be derived from natural or recombinant sources. When the source is natural, the domain may be derived from any membrane-bound protein or transmembrane protein. In one aspect, the transmembrane region is capable of signaling to the intracellular region whenever the CAR binds to the target. The transmembrane region particularly used in the present invention may include at least the following transmembrane regions: such as CD28, CD8 (e.g., CD8a, CD8 β), CD4. In one embodiment, the transmembrane domain comprises a transmembrane domain described herein, e.g., having the sequence of any one of SEQ ID NOS: 15-17, an amino acid sequence having at least 1, 2 or 3 modifications (e.g., substitutions) but NO more than 20, 10 or 5 modifications (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequence of any one of SEQ ID NOS: 15-17, or a sequence having 90-99% identity to the amino acid sequence of any one of SEQ ID NOS: 15-17.

In some cases, the transmembrane region can be linked to an extracellular region of the CAR, e.g., an antigen binding domain of the CAR, via a hinge (e.g., a hinge from a human protein). For example, in one embodiment, the hinge can be a human Ig (immunoglobulin) hinge (e.g., an IgG4 hinge, an IgD hinge), a GS linker (e.g., a GS linker as described herein), a KIR2DS2 hinge, or a CD8a hinge. In one embodiment, the hinge region is a human IgG4 or CD8 hinge; in some embodiments, the hinge region sequence has at least 95% sequence identity to a human IgG4 or CD8 hinge; in one embodiment, the hinge region comprises (e.g., consists of) the amino acid sequence of SEQ ID NO. 13 or 14.

II-3 intracellular Signal region

In some embodiments, the CAR molecule comprises an intracellular signaling region. In some embodiments, the intracellular signaling region comprises a costimulatory domain. In one embodiment, the co-stimulatory domain is a functional signaling domain derived from a protein selected from the group consisting of CD28 and/or 4-1BB (CD 137) or a functional variant thereof. In one embodiment, the costimulatory domain comprises or consists of the sequence of SEQ ID NO:18 and/or the sequence of SEQ ID NO: 19. In one embodiment, the costimulatory domain comprises or consists of an amino acid sequence having at least 1, 2 or 3 modifications (e.g., substitutions) but NO more than 20, 10 or 5 modifications (e.g., substitutions, such as conservative substitutions) of the amino acid sequence of SEQ ID NO:18 and/or SEQ ID NO:19, or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO:18 and/or SEQ ID NO: 19.

The intracellular signaling region is generally responsible for activation of at least one of the normal effector functions of the immune cells into which the CAR has been introduced. The term "effector function" refers to a specialized function of a cell. Effector functions of T cells may be, for example, cytolytic activity or helper activity, including secretion of cytokines. Thus, the term "intracellular signaling region" refers to the portion of a protein that transduces an effector function signal and directs a cell to perform a particular function. Although it is generally possible to apply the entire intracellular signal region, it is not necessary in many cases to use the entire chain. In the case of using a truncated portion of the intracellular signaling region, such a truncated portion may be used instead of the complete strand, as long as it transduces the effector function signal. Thus, the term intracellular signal region is meant to include a truncated portion of the intracellular signal region sufficient to transduce an effector function signal.

The intracellular signaling region for the CARs of the invention also includes T Cell Receptor (TCR) and co-receptor cytoplasmic sequences that co-act to elicit signal transduction upon engagement of antigen receptors, as well as any derivatives or variants of these sequences and any recombinant sequences having the same functional capabilities. It is well known that the signal generated by TCR alone is not sufficient to fully activate T cells, and secondary and/or co-stimulatory signals are also required. Thus, T cell activation can be said to be mediated by two different classes of cytoplasmic signaling sequences: those that elicit antigen-dependent primary activation by TCRs (primary intracellular signaling regions) and those that function in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic domains, e.g., costimulatory domains). The primary cytoplasmic signaling domain modulates primary activation of the TCR complex either in a stimulatory manner or in an inhibitory manner. The primary intracellular signaling region acting in a stimulatory manner may contain a signaling motif, referred to as an immune receptor tyrosine-based activation motif or ITAM. Examples of first-order intracellular signal regions containing ITAMs particularly useful in the present invention include those as follows: CD3 ζ. In one embodiment, a CAR of the invention comprises an intracellular signaling region, such as the primary signaling domain of CD3- ζ.

The intracellular signaling region of the CAR may comprise the CD 3-zeta signaling domain itself, or it may be combined with other intracellular signaling regions of the CAR for use in the invention, such as a costimulatory signaling domain. For example, the intracellular signaling region of the CAR may comprise a cd3ζ chain portion and a costimulatory signaling domain, preferably prior to the signaling domain of cd3ζ.

Intracellular signaling regions within the cytoplasmic portion of the CARs of the invention can be linked to each other in random or specified order. Optionally, a short oligopeptide or polypeptide linker, for example, between 2 and 10 amino acids in length (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) may form a link between intracellular signal regions. In one embodiment, glycine-serine diads can be used as suitable linkers. In one embodiment, a single amino acid, such as alanine, glycine, may be used as a suitable linker.

In one aspect, the intracellular signaling region is designed to comprise two or more, e.g., 2, 3, 4, 5 or more co-stimulatory signaling domains. In one embodiment, two or more, e.g., 2, 3, 4, 5 or more co-stimulatory signaling domains are separated by a linker (e.g., as described herein) molecule. In one embodiment, the intracellular signaling region comprises two costimulatory signaling domains. In certain embodiments, the linker molecule is a glycine residue. In certain embodiments, the linker is an alanine residue.

In one aspect, the intracellular signaling region is designed to comprise a signaling domain of CD3- ζ and a signaling domain of CD 28. In one aspect, the intracellular signaling region is designed to comprise a signaling domain of CD 3-zeta and a signaling domain of 4-1 BB. In one aspect, the signaling domain of CD3- ζ comprises or consists of the sequence shown in SEQ ID NO. 20 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

The CAR molecule can also comprise a signal peptide, such as, but not limited to, a signal peptide having a sequence as shown in or having at least 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 21.

Thus, a CAR molecule of the invention may comprise or consist of an extracellular binding region that binds egfrvlll, e.g. an scFv that binds egfrvlll, e.g. an amino acid sequence selected from the group consisting of or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID No. 9 or 10; a hinge region, for example an IgG4 hinge region, for example comprising or consisting of the amino acid sequence of SEQ ID NO. 13, or a CD8 hinge region, for example comprising or consisting of the amino acid sequence of SEQ ID NO. 14; a transmembrane region, for example a CD28 transmembrane region, for example comprising or consisting of the amino acid sequence of SEQ ID NO. 18, or for example a CD8 transmembrane region, for example comprising or consisting of the amino acid sequence of SEQ ID NO. 17, for example a CD4 transmembrane region, for example comprising or consisting of the amino acid sequence of SEQ ID NO. 16; an intracellular signaling region comprising or consisting of a CD28 signaling domain, e.g., comprising or consisting of the amino acid sequence of SEQ ID No. 18, and/or a 4-1BB signaling domain, e.g., comprising or consisting of the amino acid sequence of SEQ ID No. 19, and CD3 ζ, e.g., comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence shown in SEQ ID No. 20; and reporter genes, such as GFP, e.g., T2A-added GFP.

III coding nucleic acids and vectors

The invention provides nucleic acids encoding a CAR comprising a nucleic acid sequence encoding one or more CARs of the invention. In one aspect, the CAR-encoding nucleic acid is provided in the form of a DNA construct.

The CAR encoding nucleic acid may comprise a Kozak sequence at the 5' end to promote stability and translational efficiency. In some embodiments, the Kozak sequence comprises or consists of a nucleic acid sequence of GCCACCATGG (SEQ ID NO: 29).

The invention further provides vectors comprising the CAR transgene. In one aspect, the CAR vector can be directly transduced into a cell, such as a T cell, e.g., a T lymphocyte. In one aspect, the vector is a cloning vector or an expression vector, including, for example, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, microcarriers, double minichromosomes), retroviruses, and lentiviral vector constructs. In one aspect, the vector is capable of expressing the CAR molecule in mammalian T cells. In one aspect, the mammalian T cells are human T cells, e.g., T lymphocytes.

The invention also provides a vector into which is inserted a nucleic acid encoding a CAR of the invention. In some embodiments, the vector is a DNA, RNA, plasmid, adenovirus vector, lentiviral vector, or retroviral vector. In some embodiments, the vector is a lentiviral vector. In other embodiments, the vector is a retrovirus. In a preferred embodiment, the retrovirus is SFG.

In some embodiments, the vector further comprises a promoter.

Briefly, expression of a natural or synthetic nucleic acid encoding a CAR is typically achieved by operably linking a nucleic acid encoding a CAR polypeptide or portion thereof to a promoter, and incorporating the construct into an expression vector. Vectors may be suitable for replication and integration into eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters for regulating expression of the desired nucleic acid sequences.

The expression constructs of the invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. Nucleic acids can be cloned into a wide variety of vectors. For example, the nucleic acid may be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe-producing vectors and sequencing vectors.

Further, the expression vector may be provided to the cell in the form of a viral vector. Viral vector techniques are well known in the art and are described, for example, in Sambrook et al, 2012,MOLECULAR CLONING:A LABORATORYMANUAL,volumes 1-4,Cold Spring Harbor Press,NY) and other virology and molecular biology manuals. Viruses used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In general, suitable vectors contain an origin of replication that is functional in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers.

In one embodiment, a retroviral vector is used.

To assess expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cell may also contain a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expression cells from a population of cells sought to be transfected or infected by the viral vector. In some embodiments, the reporter gene is GFP. In some embodiments, the reporter gene is a luciferase, such as a firefly luciferase.

Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, the vectors may be readily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method known in the art. For example, the expression vector may be transferred into the host cell by physical, chemical or biological means.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art, see, e.g., sambrook et al, 2012,MOLECULAR CLONING:A LABORATORY MANUAL,volumes 1-4,Cold Spring Harbor Press,NY). A preferred method for introducing polynucleotides into host cells is lipofection.

IV. cells

In another aspect, the invention relates to a cell comprising said vector or nucleic acid. In one embodiment, the cell is a human T cell, e.g., a T cell described herein. In one embodiment, the T cell is a T lymphocyte. In one embodiment, the cell is an autologous T cell. In one embodiment, the cells are allogeneic T cells.

Prior to expansion and genetic modification, a cell source (e.g., T cells or NK cells) is obtained from the subject. The term "subject" is meant to include a living organism (e.g., a mammal) in which an immune response may be elicited. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a number of sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. In certain aspects of the invention, a number of T cell lines available in the art may be used. In certain aspects of the invention, T cells may use any of a variety of techniques known to those skilled in the art (e.g., ficoll ^TM Isolation) is obtained from blood units collected from the subject. In a preferred aspect, the cells are obtained from circulating blood of the individual by apheresis.

In one aspect, T cells are isolated from peripheral blood lymphocytes by lysing the erythrocytes and elutriating the depleted monocytes, e.g., by centrifugation through a percoltm gradient or counter-current centrifuge. Specific sub-populations of T cells, such as cd3+, cd28+, cd4+, cd8+, cd45ra+ and cd45ro+ T cells, can be further isolated by positive selection or negative selection techniques. For example, in one aspect, T cells are isolated by culturing with anti-CD3/anti-CD 28 (e.g., 3 x 28) -conjugated beads (such as M-450 CD3/CD 28T) for a period sufficient to be able to select for desired T cells.

In another aspect, the invention relates to a method of producing a cell, comprising transducing a cell (e.g., a T cell) described herein with a vector comprising a nucleic acid encoding a CAR molecule (e.g., a CAR molecule described herein). In one embodiment, the vector is a lentiviral vector as described herein.

V, uses and methods

In one aspect, the invention provides a method of preventing or treating a tumor (e.g., cancer) or providing anti-tumor immunity in a subject, comprising administering to the subject an effective amount of a cell containing a CAR molecule, e.g., a cell expressing a CAR molecule described herein. In one embodiment, the cell is an autologous T cell. In one embodiment, the cells are allogeneic T cells or NK cells. In one embodiment, the subject is a human. In one embodiment, the T cell is a T lymphocyte.

In some embodiments, the tumor (e.g., cancer) patient has an egfrvlll (e.g., at an elevated level, e.g., at a nucleic acid or protein level).

In some embodiments, the tumor, e.g., cancer, includes solid tumors and hematological tumors, and metastatic lesions. In some embodiments, examples of solid tumors include malignant tumors. The cancer may be in early, intermediate or late stages or metastatic cancer.

In some embodiments, the tumor treatment will benefit from inhibiting egfrvlll at the nucleic acid or protein level. In some embodiments, the tumor treatment will benefit from direct killing of tumor cells by the CAR-T cells of the invention. In some embodiments, the tumor treatment will benefit from growth inhibition or killing of tumor cells by cytokines secreted by the CAR-T cells of the invention. In other embodiments, the tumor treatment will benefit from the overall or local modulation of the immune system in vivo by cytokines secreted by the CAR-T cells of the invention, and the growth inhibition or killing of tumor cells by the latter.

In a specific embodiment, the anti-egfrvlll antibodies of the invention are capable of inhibiting tumor cell proliferation, e.g., egfrvlll expressing tumor cells, e.g., glioma cells, e.g., GBM cells.

In some embodiments, the tumor is tumor immune escape.

In some embodiments, the tumor is a cancer, such as a glioma, such as GBM.

The subject may be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., an individual having or at risk of having a disease described herein). In some embodiments, the subject has or is at risk of having a disease described herein (e.g., cancer). In certain embodiments, the subject receives or has received other treatments, such as chemotherapy and/or radiation therapy. In some embodiments, the subject has previously received or is receiving immunotherapy.

In other aspects, the invention provides the use of a CAR molecule or a nucleic acid encoding the same or a vector comprising the same or a cell comprising the same in the manufacture or preparation of a medicament for use as described herein, e.g. for the prevention or treatment of a related disease or disorder as referred to herein.

In some embodiments, the CAR molecule of the invention, or a nucleic acid encoding the same, or a vector comprising the same, or a cell comprising the same, delays the onset of the disorder and/or symptoms associated with the disorder.

In some embodiments, the CAR molecules of the invention, or encoding nucleic acids thereof, or vectors comprising the same, or cells comprising the same, can also be administered in combination with one or more other therapies, e.g., therapeutic modalities and/or other therapeutic agents, for the uses described herein, e.g., for the prevention and/or treatment of the related diseases or disorders mentioned herein.

In some embodiments, the therapeutic means or agent enhances the activity or adaptability of a cell expressing a CAR molecule, or reduces side effects associated with administration of a cell expressing a CAR molecule, or treats a disease associated with egfrvlll.

In some embodiments, the treatment regimen comprises surgery; radiation therapy, localized irradiation, or focused irradiation, among others. In some embodiments, the therapeutic agent is selected from a chemotherapeutic agent, a cytokine, a cytotoxic agent, a vaccine, a therapeutic monoclonal antibody, a small molecule drug, or an immunomodulatory agent. Exemplary immunomodulators include immunosuppressants or anti-inflammatory agents.

Detailed Description

All embodiments of the invention are intended to be illustrative and not limiting. All embodiments of the present invention underwent approval by the Beijing century jar hospital institutional review board, if necessary, and informed consent was obtained for all participants.

Immune effector cells stably expressing the EGFRvIII CAR molecules of the invention have killing effect on human glioma cell lines U87-EGFRvIII and U373-EGFRvIII stably expressing EGFRvIII. In vitro experiments, we found that effector cells were significantly activated and secreted various cytokines associated with immune function, particularly INF-gamma, in the co-culture system. By detecting the presence of target cells, tumor lysis can be assessed more intuitively. We used a variety of methods to assess oncolytic effects including RT-PCR, RTCA and luciferase reporter experiments. These methods all demonstrate that egfrvlll CAR has a stable, powerful and specific oncolytic effect. Most importantly, egfrvlll CAR shows similar cytolytic potential compared to other published reports in an ectopic graft orthotopic model. Although there were differences in the evaluation system, we constructed third generation CAR-T cells that exhibited stable cytolysis.

The sequence SEQ ID NO numbers (all CDRs are defined using the Kabat rules) of exemplary scFv's of the invention

	ScFv
		HCDR1
	1
		HCDR2	2
HCDR3	3
		VH	7
LCDR1	4
		LCDR2	5
LCDR3	6
		VL	8
scFv	9 or 10

Example 1 materials and methods

Cell lines and antibodies

Expression of EGFRvIII or wtEGFThe glioma cell line of R, generous donation by the coumarone cancer center Webster K Cavenee, was maintained in DMEM supplemented with 1 μg/ml puromycin. Other glioma cell lines, U87, U251, U373, were obtained from ATCC. All cell lines were stably transfected with green fluorescent protein and luciferase was cultured in DMEM medium supplemented with 10% fetal bovine serum and 1%10000IU/ml penicillin/10000. Mu.g/ml streptomycin (FIG. 6). PG-13 and Phoenix ECO retrovirus producer cell lines were cultured in DMEM medium supplemented with 10% FBS, 2mM glutamine and 1mM sodium pyruvate (Lonza). T cells were cultured in T cell medium consisting of X-VIVO-15 medium plus 5% antibody Serum (SIGMA), 100U/ml IL-2, 100U/ml penicillin, 100. Mu.g/ml streptomycin. All cells were routinely tested for mycoplasma and the results were negative. The following antibodies were used: AF647 binds goat anti-mouse antibody (Jackson ImmunoResearsh, 115-606-072); FITC binds goat anti-mouse IgG (H+L) (GAM) (F2653, sigma-Aldrich); APC binding CD107a (BD Pharmingen) ^TM 560664); APC-R700 binds CD3 (BD-Pharmingen) ^TM 565119); BV421 binds CD4 (BD Pharmingen) ^TM 562424); PE-Cy7 binds CD8 (BD-Pharmingen) ^TM 557746); PE binds CD3 (555340,BD Biosciences); FITC binds to CD3 (Invitrogen, 11-0036-42); PE-Cy7 binds IFN-gamma (Biolegend, 502528); v500 binds CD45 (BD Pharmingen ^TM 560777); FITC binds CD8 (BD Pharmingen) ^TM 555634); PE-CY 5-conjugated CD95 (BD-Pharmingen) ^TM 559773); PE-CY 7-conjugated CCR7 (BD-Pharmingen) ^TM 557648); AF700 binds CD27 (BD Pharmingen) ^TM 560611), BV605 binds CD45RO (bioleged, 304238); APC binding CD25 (BD Pharmingen) ^TM 662525); BV 421-conjugate CD223 (BD-Pharmingen) ^TM 656720); AF647 binds CD366 (BD Pharmingen) ^TM 565558); percp-cy 5.5-conjugated CD279 (BD-Pharmingen) ^TM ,561273)。

Flow cytometer analysis

Cells were washed once with PBS containing 1% fetal bovine serum (FACS buffer) prior to antibody addition. After staining at 4℃for 30 min in the absence of light, the cells were washed once and fixed in 0.5% paraformaldehyde/FACS buffer. For the followingCD107a assay using APC binding CD107a and protein transport inhibitor (BD Pharmingen ^TM 554724) the T cell activation rate was determined. IFN-gamma expression in T cells was determined using IFN-gamma antibodies according to the procedure of the intracellular fixation and permeation buffer set kit (Invitrogen, 88-8824). Flow cytometry was performed using BD-FacsCanto II Plus (BD Biosciences) and analyzed using FlowJo software (Tree star, inc. Ashland, OR).

Co-culture analysis and cytokine production

Freshly harvested PBMCs transduce egfrvlll-28 BB ζcar and co-cultured with egfrvlll expressing cell lines or other cell lines for restimulation. Cells were added in a 10:1 ratio to (1X 10) in U-shaped bottom 6-well microplates ⁶ CAR-T：1×10 ⁵ Target cells), after 24 hours, the supernatant was collected and flash frozen in liquid nitrogen and then stored at-80 ℃ until the experiment was used. Through enzyme-linked immunosorbent assay

ELISA kit, development catalog number: DY 285) or flow cytometry analysis (human INF- γflex kit, BD,558269; human Th1/Th2/Th17 cytokine kit, 560484) measures IFN-gamma production and other cytokine production. All samples were analyzed in triplicate and compared to multiple internal standards using standard curves.

To determine intracellular expression of IFN-gamma, effector T cells (1X 10) of ICCS (intracellular cytokine staining assay) assays ⁶ Cells) and target cells were isolated in 24-well microwell plates at 10:1e:t (effector cells: target cells). Each well contained 1ml of T cell medium and 2. Mu.l of protein transport inhibitor (Invitrogen, 00-4980-93). After 24 hours incubation of the dishes at 37℃cells were collected and intracellular staining was performed to assess IFN-gamma expression.

Luciferase reporter assay

Cells were co-cultured with target cells in 96-well microwell plates at a ratio of E: t=10:1, respectively. Culturing the well plate at 37deg.C for 24 hr, adding appropriate amount of matrix to the culture medium, and providing high sensitivityCooled CCD camera

Xenon) was imaged every three minutes until peak emission values were obtained.

Cell lines artificially expressing EGFRvIII

EGFR and EGFRvIII fragments were amplified by PCR and assembled between constructs by restriction cleavage using appropriately designed primers. The final 196 base pair (bp) DNA fragment of EGFR-encoding product was amplified with a 10. Mu.M concentration of specific primer (forward, 5'-ATGCGACCCCGGGACGCGC-3', SEQ ID NO:23; reverse, 5'-GCCCTTCGCACTTACTACTTGCGG-3', SEQ ID NO: 24), EGFRvIII fragment (150 bp) was amplified with primer (forward, 5'-ATGCGCCTCCGGGACGGCC-3' SEQ ID NO:25; reverse, 5'-CTCATAGTCGGCCCCCAGG-3', SEQ ID NO: 26) and actin was amplified with primer (forward, 5'-ACCTGCCCATACAGG-3', SEQ ID NO:27; reverse, 5'-AGGGCCGGACTCGTCATACT-3', SEQ ID NO: 28).

Glioma cell lines stably expressing egfrvlll (U251, U87 and U373) were constructed and their expression levels were verified (see fig. 6B).

Assessing maintenance of anti-tumor activity of CAR T cells

Real-time cellular analysis (RTCA) is a system based on evaluation of changes in cellular impedance. When cells adhere, the Cell Index (CI) value is zero. First, target cells were seeded onto the E-plate at a density of 10000 cells/well. After 30 minutes incubation at room temperature, the E plate was placed on an RTCA SP station in an incubator (5% CO2;37 ℃) and continuous impedance recordings were performed. On day 1 post-inoculation, CI values were measured on the other day of co-culture with 10-fold effector cells as an indicator of tumor cell density.

Treatment and imaging studies of NOD/SCID mouse glioblastoma

Female NOD/SCID mice 6 to 8 weeks of age were used for the experiment. All animals were housed in a specific pathogen-free environment and were housed strictly in accordance with protocols and standard animal care requirements approved by the Beijing century jar Hospital, university of capital medical science. The pain of the experimental animal is reduced as much as possible.

Scheme 1: after 1 week of adaptive feeding, mice received intracranial (i.c.) implantation of 2×10≡5 egfrvlll target cells or other glioma cell lines using stereotactic coordinates (2.5 mm posterior to bregma, 2.5mm lateral, 3.5mm deep into the parenchyma). The target cells were resuspended slightly in separate microcentrifuge tubes prior to each implantation. On day 6 after intracranial tumor implantation, 2X 10≡7 effector cells (EGFRvIII CAR or non-specific T cells) were injected intravenously.

Scheme 2: for 1 week of adaptive feeding, mice received intracranial (i.c.) implantation of 2×10≡5 egfrvlll target cells or other glioma cell lines using stereotactic coordinates (2.5 mm posterior to bregma, 2.5mm lateral, 3.5mm deep into the parenchyma). Mice recovered 3 days after the first surgery, and were injected intracerebrally with 1×10≡7 effector cells (EGFRvIII CAR or nonspecific T cells) (0.3 mm before bregma point, 2mm left, 3.5mm deep into the parenchyma).

Scheme 3: the subcutaneous model uses 2X 10-6U 373-EGFRvIII cells with a final volume of 100. Mu.L. The right side of each NOD/SCID mouse was injected with a tumor cell suspension. The progress of subcutaneous xenografts was measured with vernier calipers and calculated according to the formula v=length×width×depth/2 to ensure that the size of each group of subcutaneous models was approximately the same, 2×10≡7 effector cells (egfrvlll CAR or non-specific T cells) were injected intravenously on day 8.

Tumor progression is followed by bioluminescence imaging (BLI) and imaging datasets are acquired using the Xenogen IVIS imaging system (Xenogen) and in vivo imaging software (Xenogen). The disease-modifying mice were then euthanized for further histological analysis. Briefly, during intraperitoneal injection of D-fluorescein (150 mg/kg; 200. Mu.l PBS) 10 minutes prior to imaging, mice were anesthetized with 1-2% isoflurane. In the CCD camera with high sensitivity and cooling

Xenogen) was imaged every five minutes until peak emission values were obtained and a decrease in fluorescence signal was observed for all mice.

Immunohistological staining

To assess tumor structure and growth pattern, NOD/SCID mice received an intra-brain or subcutaneous tumor implant and were euthanized prior to death. The brains were harvested and fixed in 4% frozen PFA at 4 ℃ for 12 hours prior to paraffin embedding. 5 μm coronal sections were sectioned and immunostained with rabbit monoclonal anti-human CD3 antibody and rabbit monoclonal anti-human EGFRvIII antibody at room temperature. For anatomical assessment, brains of every 10 serial sections were stained with hematoxylin and eosin (H & E). The image is magnified 10 times, 40 times under an optical microscope.

Example 2 production of EGFRvIII specific CAR T cells

Construction and expression of third generation anti-EGFRvIII specific CAR-T cells

The third generation of anti-EGFRvIII CAR (FIG. 5A) was generated by the addition of the CD28-4-1BB intracellular domain between the transmembrane region and the signaling domain molecule of CD3 zeta. The CD28 transmembrane domain fragment was amplified by PCR and assembled between the constructs by restriction enzyme cleavage using appropriately designed primers with the amino acid sequence SEQ ID NO. 15. The EGFRvIII binding moiety is derived from scFv and has the amino acid sequence shown in SEQ ID NO. 9. Co-stimulatory domain and CD3 zeta signaling domain sequences from CD28.4-1BB are shown in SEQ ID NOs 18, 19 and 20, respectively. ScFv is linked to the transmembrane domain by a hinge region (SEQ ID NO: 13).

The retroviral vector retro-SFG-IgG4-CD28-4-1BB-CD3 zeta of the third generation CAR-T was obtained from the retroviral vector SFG (Addgene Corp., also referred to as "vector pMSGV 1"), double digested with SmaI/MluI, then the nucleotide sequence of the aforementioned anti-EGFRvIII scFv sequence was ligated to the vector by cloning technique, after ligation for 1h at 37℃the transformation was applied to an ampicillin-resistant LB plate, and the recombinant vector was identified by subcloning, plasmid extraction and sequencing after cleavage verification, thus constructing the retroviral vector retro-SFG-EGFRvIII scFv-IgG4-CD28-4-1BB-CD3 zeta of the third generation CAR-T (as shown in FIG. 5A).

Viral packaging was performed using the constructed egfrvlll specific CAR retroviral vector. Specifically, 10. Mu.g of the retroviral vector retro-SFG-EGFRvIII scFv-IgG4-CD28-4-1BB-CD3 zeta was transfected into Phoenix ECO cells by the calcium phosphate reagent. After 48h, the supernatant was collected and used to transduce retroviral PG13 packaging cell clones. Retroviral supernatant was generated from the highest titer PG13 packaging cell clone, passing 0.45 μm

-HP needle filter to obtain retrovirus stock.

The virus stock was ultracentrifuged at 4℃and 32000r/min for 2 hours, and the retrovirus pellet was dissolved in the X-VIVO culture broth. Obtaining retrovirus concentrated solution, sub-packaging and freezing at-80 ℃.

The retroviral concentrates were subjected to titer detection. 0.5X10 of 96-well tip bottom plate is paved in each well ⁶ mu.L/well of Jurkat cells, retrovirus concentrate was diluted 100-fold and 400. Mu.L, 40. Mu.L and 10. Mu.L of each well were added in the proportions 1:50, 1:500 and 1:2000. Centrifuge at 32℃and 1200 Xg for 90min. After 4h cells were washed once with DPBS and plated on 12-well plates and transduced for 48h for detection by flow cytometry. The results demonstrate that the retroviral concentrate prepared in this example, after 100-fold dilution, is capable of infecting Jurkat cells and expressing a CAR, and therefore, CAR-T cell production was performed using the retroviral concentrate.

The virus was verified by sequencing.

Retroviral transduction

Peripheral Blood Mononuclear Cells (PBMCs) of healthy donors were isolated by gradient centrifugation using Ficoll solution (GE healthcare). Immediately after purification, activation was performed with anti-CD 3/CD 28T cell activator Dynabeads (Invitrogen) at a bead ratio of 1:1. After 48 hours of activation, T cells were transduced with retrovirus supernatant by centrifugation on a reverse transcription connexin (Takara) coated plate to obtain egfrvlll-28 BB ζcar T cells. Transduction efficiency was verified by flow cytometry or western blot after 9 days (fig. 5B). As a control, control T cells were transduced with a vector encoding a CD19 specific CAR gene.

Expression of CAR molecules on T cells

T cell subsets were analyzed with monoclonal antibodies CD3, CD4, CD8 (BD-Pharmingen) at room temperature and CAR expression was detected with FITC-labeled goat anti-mouse IgG (H+L) antibody (Sigma). We also used Western blot to assess expression of CARs, washed cells with PBS, and lysed using RIPA buffer containing protease inhibitors, and the resulting proteins isolated on 15% sds-PAGE gel (Bio-Rad). The isolated protein was then transferred to PVDF membrane and detected using anti-human CD3- ζ monoclonal antibody (BD Pharmingen, clone 8D 3). The bands were visualized using an adelstein imaging system (LI-COR) with untransduced cells as controls.

Example 3T cells expressing EGFRvIII CARs recognize EGFRvIII expressing cells

Peripheral Blood Mononuclear Cells (PBMCs) were obtained from healthy volunteers for transduction of retroviruses encoding third generation egfrvlll CARs. After transduction, surface expression was determined by flow cytometry and western blot, and T cells were found to efficiently express egfrvlll CAR structure on their cell surface (fig. 5).

Next, we transfected and cultured target cells (egfrvlll+) and control tumor cells (egfrvlll-), so that they stably expressed GFP/Luc (fig. 6A). And screening by adopting a flow cytometry to obtain the target cells with GFP/Luc completely expressed. We found that the extent of activation of CARs cells under glioma cell stimulation was relatively weak compared to hematological tumors. The relative efficacy of egfrvlll CAR and non-specific CAR was first compared by measuring the expression of T cell degranulation functional marker CD107 a. As shown in fig. 1A, egfrvlll CAR induced T cell activation was observed to result in significant CD107a expression. To further confirm that egfrvlll CARs can be activated by the corresponding target cells, CD25 expression was assessed by flow cytometry as a T cell activation marker after co-culture with U87-egfrvlll or U373-egfrvlll. The frequency of CAR-T cell expression markers was significantly higher compared to Mock transfected cells, 63.3% and 67.6%, respectively (fig. 7). These data indicate that egfrvlll CAR is indeed activated in the presence of the corresponding antigen.

Then, to assess whether egfrvlll CAR has the potential to kill glioma cells, we examined the level of cytokines, especially the production and release of INF- γ, including secretion and intracellular INF- γ after 24 hours of co-culture. As shown (fig. 1c 1d, fig. 8, fig. 9), we observed that egfrvlll CAR induced T cell activation resulted in a 20-fold increase in IFN- γ production compared to Mock CAR-T cells. CAR T cells also secreted Th1/Th2/Th17 cytokines when co-cultured with egfrvlll+ cell lines (fig. 9).

Finally, antigen-specific reactivity against egfrvlll was determined in a co-culture assay. In contrast, egfrvlll+car T cells did strongly induce target cell death as determined by PCR (fig. 1e 1 f) and luciferase reporter assay (fig. 10), although the percentage of CD107a positive T cells was not high. Importantly, egfrvlll+car T cells observed anti-tumor activity, whereas non-specific T cells did not.

Example 4T cells expressing EGFRvIII+CAR show similar proliferation levels despite high levels of activation

In view of the high level of cytokine production, we tested whether the egfrvlll CAR structure would induce any potential depletion effect that could affect the long-term viability of these cells in vivo.

We assessed the initial T cells defined by CD45RO, CCR7, CD27 and CD95 following in vitro egfrvlll stimulation after initiation of transduction and following 7 days

T cell, tn), stem-like memory T cells (Tscm), central memory T cells (Tcm), effector memory T cells (Tem), or effector T cells (Tte) phenotype. Memory phenotype T cells were higher in egfrvlll CARs than non-specific CARs (fig. 11B, 11D). First, we found that the number of CD8 positive T cells gradually increased, and Tscm and Tcm were gradually replaced by Tem and Tte as the cells were expanded in vitro and the incubation time was prolonged (fig. 11A, 11B, 11C).

In general, antigen-independent CAR-T signaling is characterized by the presence of multiple co-inhibitory receptors expressed on the cell surface (PD-1, lag-3, and Tim-3). We found that antigen activated CAR-T cells expressed at the same level of depletion marker as T cells without egfrvlll, i.e. did not show depletion. Egfrvlll CAR T cells expressed the same level of depletion marker after egfrvlll stimulation compared to T cells expressing non-specific CARs (fig. 11E).

Example 5 monitoring of the cytotoxic Effect of CAR-induced EGFRvIII+ cells Using Real Time Cell Analysis (RTCA)

To evaluate RTCA-based assay methods as a method of measuring the cytotoxic effect of CAR-T cells, the cellular impedance of target cells was measured in real time using the xcelligent system, wherein target cells were divided into co-cultured and non-co-cultured groups with egfrvlll CARs. Tumor cells were seeded in triplicate in 16-well E plates (E plates). After 24 hours of tumor cell attachment, T cells were added at a rate of 10T cells per 1 tumor cell. The electrical impedance was continuously recorded as an indicator of tumor cell density.

Figure 2A shows tumor cell impedance measured according to the co-culture protocol used. When cells were cultured in the medium for 24 hours, the CI values of U87-EGFRvIII and U373-EGFRvIII increased significantly from 0 (CI before co-culture) to 1.76.+ -. 0.14 and 0.97.+ -. 0.03 (CImin), respectively. Then, in the tumor cell group alone, CI was kept steadily increasing by adding fresh medium, eventually reaching 2.42.+ -. 0.03 and 1.52.+ -. 0.02 (CI at the end of the experiment). However, the addition of the same volume of medium (co-culture group) but containing 10 fold-amounts of effector cells resulted in a significant decrease in CI for both application protocols to zero over time. While egfrvlll negative cells under effector cell treatment showed a deficient immune response, the assay value remained stable for longer co-culture time during the experiment, although the CI value was slightly decreased.

Overall, these results indicate that the kinetic profile of cell impedance is affected by specific immune responses, whereas there is no difference in the culture of egfrvlll negative tumor cells.

Example 6 inhibition of tumor growth and prolongation of survival of tumor-bearing mice by EGFRvIII CAR cells in situ xenograft and subcutaneous models

To further investigate the potential therapeutic applications of egfrvlll CAR T cells, we examined their anti-tumor activity in vivo.

First, we performed a subcutaneous model, with no detectable toxicity in 5 mice receiving egfrvlll CAR, all mice maintaining overall body weight (fig. 12A). The therapeutic effect of CAR-T cells was limited, and even unable to prolong survival (fig. 12D). Subsequently, the immunohistochemical results showed that no cd3+ T cells were found in the mouse subcutaneous tumor (fig. 12C), indicating that CAR-T cells injected intravenously from the mouse tail hardly penetrated the subcutaneous tumor.

To obtain results closer to the human glioma treatment process, we established in situ gliomas by gene transfection of egfrvlll expressing U87 and U373 cells expressing firefly luciferase and implantation into NOD-SCID mouse brain. The expression of firefly luciferase enabled us to monitor tumor growth by in vivo bioluminescence imaging. To minimize potential systemic toxicity, we injected CAR-T within the tumor 3 days after tumor cell implantation. As shown in fig. 3, mice bearing U87-egfrvlll or U373-egfrvlll cells showed significantly reduced tumor growth (p < 0.01) after treatment of egfrvlll CAR-T cells with the method of bioluminescence imaging assay compared to Mock CAR-T cell injected mice. Importantly, the reduction in tumor growth did not appear to be much different in mice with grade III and grade iv glioma cell lines or glioblastoma cell lines.

To further elucidate the therapeutic effect of EGFRvIII CAR T cells on GB tumors expressing wtEGFR, we established an in situ model by intracranial implantation of U251 cells into NOD-SCID mice. 6 days after tumor cell implantation, we injected egfrvlll CAR T cells or PBS as vehicle controls within the tumor. As shown in fig. 12B, mice injected with CAR-T cells in a single tumor did not significantly reduce tumor burden compared to the control group. In addition, mice receiving CAR-T cell therapy did not benefit from survival.

Taken together, egfrvlll CAR T cells can effectively target and kill egfrvlll expressing tumor cells in vivo.

Example 7 evaluation of EGFRvIII CAR cell migration and elongation following intracranial injection

We demonstrate the feasibility and safety of intracranial administration in xenograft models. On this basis, we analyzed the distribution of cells within the tumor after injection. Tumor-bearing mouse brain sections injected with mock or egfrvlll-CAR-T cells within the tumors were stained with hematoxylin and eosin (H & E), and the results showed that the tumors did not completely regress (fig. 4A). In the U87-EGFRvIII group, we found that tumor tissue still strongly expressed the target protein molecule, but we surprisingly found very few CD3+ T cells, demonstrating that under antigen stimulation, CAR-T cells can persist in the brain for a long period of time. In contrast, the target protein molecule egfrvlll was also detected in mice carrying U373-egfrvlll cells, but T cells were not found to survive for a long period of time (fig. 4b 4 c).

Patent applications, patents, documents, books, etc. cited, referred to, and/or referenced herein are hereby incorporated by reference in their entirety.

The list of sequences involved in the present invention is as follows:

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Claims

1. An isolated chimeric antigen receptor comprising, from N-terminus to C-terminus, a sequence of linked: an extracellular binding region, a hinge region/spacer region, a transmembrane region and an intracellular signal region comprising a costimulatory domain and a stimulatory signal domain, wherein the extracellular binding region binds to egfrvlll, preferably the extracellular binding region comprises a heavy chain variable region VH and/or a light chain variable region VL, wherein the heavy chain variable region comprises complementarity determining regions HCDR1, HCDR2 and HCDR3, more preferably the HCDR1, HCDR2 and HCDR3 are amino acid sequences as shown in SEQ ID NO:7 or 3 complementarity determining regions as shown in sequences having 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:7, and/or the LCDR1, LCDR2 and LCDR3 are amino acid sequences as shown in SEQ ID NO:8 or have complementarity determining regions as shown in SEQ ID NO:8, 80%, 90%, 98%, 99%, 95% or 3% complementarity determining regions as shown in SEQ ID NO:8 or having 80%, 85%, 90%, 96%, 98%, 99%, 3% or 3% identity to SEQ ID NO: 3, and preferably the most preferably the sequences as shown in SEQ ID NO: 6%, 95%, 3% or 3% and/or 3% complementarity determining regions as shown in SEQ ID NO: 7.

2. The chimeric antigen receptor of claim 1, wherein the extracellular binding region comprises a heavy chain variable region VH and/or a light chain variable region VL, the VH comprising or consisting of the amino acid sequences: the sequence of SEQ ID NO. 7 or a sequence having 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 7; the VL comprises or consists of the amino acid sequence: the sequence of SEQ ID NO. 8 or a sequence having 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 8.

3. The chimeric antigen receptor of claim 1 or 2, wherein the extracellular binding region that binds egfrvlll is an antibody or antigen binding fragment thereof, e.g. an scFv, preferably the scFv comprises a VH and a VL, preferably wherein the VH comprises or consists of the amino acid sequences: the sequence of SEQ ID No. 7 or a sequence having 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 7 and the VL comprises or consists of the amino acid sequences: the sequence of SEQ ID NO. 8 or a sequence having 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 8, optionally, the VH and VL are linked by a linker, preferably the sequence of the linker is as shown in SEQ ID NO. 11 or 12 or a sequence having at least 80% identity thereto.

4. A chimeric antigen receptor according to any one of claims 1 to 3, wherein the transmembrane region comprises a CD28 transmembrane domain, a CD4 transmembrane domain and/or a CD8 transmembrane domain, preferably the transmembrane region comprises a sequence as set forth in any one of SEQ ID NOs 15 to 17 or a sequence having at least 80% identity thereto, optionally the transmembrane region is linked to an extracellular binding region via a hinge region.

5. The chimeric antigen receptor of any one of claim 1-4, wherein the intracellular signaling region comprises a signaling domain of CD3ζ and a costimulatory domain,

preferably, the CD3 zeta signaling domain comprises or consists of the sequence shown in SEQ ID NO. 20 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto,

preferably, the co-stimulatory domain is a functional signaling domain derived from a 4-1BB (CD 137) protein, or a functional signaling domain derived from a CD28 protein, or preferably, the co-stimulatory domain comprises or consists of an amino acid sequence derived from both CD28 and 4-1BB, preferably, the 4-1BB (CD 137) signaling domain comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence shown as SEQ ID NO. 19, preferably, the CD28 signaling domain comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence shown as SEQ ID NO. 18.

6. A humanized Chimeric Antigen Receptor (CAR) polypeptide targeting egfrvlll comprising from N-terminus to C-terminus:

(2) A hinge/spacer region selected from

(3) A transmembrane region (TM) selected from

(4) A Costimulatory Signaling Domain (CSD), which is:

7. A cell expressing the chimeric antigen receptor of any one of claims 1-6 on its surface, preferably the cell is an immune effector cell, more preferably the cell is a T cell, most preferably the cell is a T lymphocyte.

8. Use of a cell according to claim 7 for the preparation of a pharmaceutical composition for preventing or treating a tumor (e.g. cancer) or providing anti-tumor immunity in a subject, preferably the tumor is a glioma, more preferably the tumor is a glioblastoma.

9. A method of preventing or treating a tumor (e.g., cancer) or providing anti-tumor immunity in a subject, comprising administering to the subject an effective amount of a cell expressing the chimeric antigen receptor of any one of claims 1-6, or a cell of claim 7, preferably wherein the tumor (e.g., cancer) has an egfrvlll (e.g., at an elevated level, e.g., at a nucleic acid or protein level) in a patient, preferably the tumor is a glioma, more preferably the tumor is a glioblastoma,

Optionally, wherein the cells are administered in combination with one or more other therapies, such as therapeutic modalities and/or other therapeutic agents.

10. A pharmaceutical composition comprising the chimeric antigen receptor of any one of claims 1-6, and/or the cell of claim 7.