WO2024060140A1 - Récepteur antigénique chimérique anti-egfrviii et son utilisation - Google Patents

Récepteur antigénique chimérique anti-egfrviii et son utilisation Download PDF

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WO2024060140A1
WO2024060140A1 PCT/CN2022/120569 CN2022120569W WO2024060140A1 WO 2024060140 A1 WO2024060140 A1 WO 2024060140A1 CN 2022120569 W CN2022120569 W CN 2022120569W WO 2024060140 A1 WO2024060140 A1 WO 2024060140A1
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sequence
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钟晓松
白玥
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卡瑞济(北京)生命科技有限公司
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    • C07K2317/622—Single chain antibody (scFv)
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    • C07K2319/00—Fusion polypeptide
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention generally relates to the use of T cells engineered to express an EGFRvIII chimeric antigen receptor (CAR) for treating diseases associated with the expression of EGFRvIII.
  • CAR chimeric antigen receptor
  • GBM Glioblastoma
  • GBM is the most common and most aggressive primary malignant brain tumor in humans, and most cases are highly malignant. Due to high morbidity, high mortality, and low cure rate, GBM has caused a huge social and medical burden worldwide, and is almost impossible to cure with traditional treatment methods, so the overall survival of patients is very unsatisfactory.
  • the current standard treatment strategy is to maximize safety by surgical resection of the tumor, postoperative concurrent radiotherapy and chemotherapy, and subsequent six months of adjuvant chemotherapy with temozolomide.
  • the average survival time of patients is only 14.6 months.
  • Chimeric antigen receptor (CAR) T cell therapy is an emerging immunotherapy that can treat a variety of malignant tumors, including glioblastoma.
  • Immunotherapy with T cells expressing GBM-specific chimeric antigen receptors (CARs) offers a promising therapeutic platform as it has the potential to specifically target tumor tissue while sparing the normal brain.
  • Challenges faced by solid tumor immunotherapy include the expression of tumor target antigens by normal cell tissues, factors such as the blood-brain barrier that limit the transport of CAR-T cells to tumor sites, and the sometimes lack of long-term persistence of CAR-T cells at tumor sites.
  • CAR chimeric antigen receptors
  • EGF deletion mutant receptor type III (EGFRvIII) is expressed in multiple tumor types, including glioblastoma multiforme (GBM), but is rarely observed in normal tissues. EGFRvIII is expressed in 24% to 67% of GBM cases and in surviving patients ⁇ 1 year later, and expression of EGFRvIII is an independent adverse prognostic indicator.
  • CNS central nervous system
  • ACT adoptive cell transfer
  • CAR-T therapy targeting CD19 has been approved by the FDA for clinical application, which is another major breakthrough in the field of cancer gene therapy (https://www.fda.gov/) Recently, for the first time In vivo intravenous infusion study of single-dose autologous T cells redirected to EGFRvIII via chimeric antigen receptor (CAR) reports that manufacturing and infusing CAR-modified T cells (CART)-EGFRvIII cells is feasible and safe, no Evidence of extratumoral toxicity or cytokine release syndrome. One patient in the clinical trial had residual stable disease at 18 months of follow-up.
  • CAR chimeric antigen receptor
  • CAR T cells expressing tumor-specific mutations of the epidermal growth factor receptor EGFRvIII and the costimulatory CD28BB ⁇ endodomain and evaluated effector function in vitro and in vivo.
  • CAR T cells killed EGFRvIII-positive cells, and EGFRvIII-positive cells promoted CAR T cell activation compared with untransformed T cells in vitro and in vivo.
  • the present invention constructs a third-generation CAR targeting EGFRvIII.
  • the present invention relates to chimeric antigen receptors that bind EGFRvIII, and T cells comprising the same.
  • the invention also relates to nucleic acids encoding said chimeric antigen receptors, expression vectors comprising the same, viruses and T cells.
  • the invention also relates to the use of T cells containing chimeric antigen receptors for the treatment of tumors, preferably the tumor highly expresses EGFRvIII, more preferably the tumor is a glioma, most preferably the tumor is a glioblastoma blastoma.
  • an isolated chimeric antigen receptor comprising sequentially connected from its N-terminus to its C-terminus: an extracellular binding region, a hinge region/spacer region, a transmembrane region and an intracellular signaling region including a costimulatory domain and a stimulatory signal domain, wherein the extracellular binding region binds EGFRvIII.
  • 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, and the light chain variable region
  • the chain variable region contains the complementarity determining regions LCDR1, LCDR2 and LCDR3.
  • the HCDR1, HCDR2 and HCDR3 are amino acid sequences as shown in SEQ ID NO:7 or as having 80%, 85%, 90%, 93%, 95%
  • the LCDR1, LCDR2 and LCDR3 are amino acid sequences such as SEQ ID NO :8 or as shown by a sequence having 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:8
  • Three complementarity determining regions of the chain variable region are amino acid sequences as shown in SEQ ID NO:7 or as having 80%, 85%, 90%, 93%, 95%
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise 80%, 85%, 90%, 93% of the same as SEQ ID NOs: 1, 2, 3, 4, 5 and 6, respectively. Sequences that are %, 95%, 96%, 97%, 98%, 99% or 100% identical. In some specific embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 respectively comprise no more than 3 amino acid changes, no more than 3 amino acid changes compared to SEQ ID NOs: 1, 2, 3, 4, 5 and 6. Sequences with more than 2 amino acid changes or no more than 1 amino acid change.
  • the extracellular binding region comprises a heavy chain variable region VH, the VH comprising or consisting of the following amino acid sequence: the sequence of SEQ ID NO:7 or 80% identical to SEQ ID NO:7 Sequences that are %, 85%, 90%, 93%, 95%, 96%, 97%, 98% or 99% identical.
  • the extracellular binding region comprises a light chain variable region VL, which comprises or consists of the following amino acid sequence: a sequence of SEQ ID NO:8 or a sequence that is 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:8.
  • 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 following amino acid sequence: the sequence of SEQ ID NO: 7 or A sequence that is 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:7, and the VL comprises the following amino acid sequence or consists of the following amino acid sequence Sequence composition: The sequence of SEQ ID NO:8 or a sequence that is 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:8.
  • the extracellular binding region that binds EGFRvIII is an antibody or antigen-binding fragment thereof, such as a scFv.
  • the scFv comprises VH and VL, preferably, wherein the VH comprises or consists of the following amino acid sequence: a sequence of SEQ ID NO:7 or a sequence having 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:7, and the VL comprises or consists of the following amino acid sequence: a sequence of SEQ ID NO:8 or a sequence having 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:8.
  • the VH and VL are connected by a linker.
  • the sequence of the linker is a sequence as shown in SEQ ID NO:11 or 12 or a sequence having at least 80% identity thereto.
  • the scFv comprises an amino acid sequence selected from any one of SEQ ID NO: 17-20 or is at least 90%, 91%, 92%, 93%, 94%, 95% identical to said sequence. %, 96%, 97%, 98% or 99% identity to or consisting of an amino acid sequence.
  • the hinge region comprises an IgG4 hinge region and/or a CD8 hinge region. In some embodiments, the hinge region comprises the sequence set forth in SEQ ID NO: 13 or 14 or a sequence that is at least 80% identical thereto.
  • 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 set forth in any one of SEQ ID NOs: 15-17 or a sequence that is at least 80% identical thereto.
  • the transmembrane region is connected to the extracellular binding region via a hinge region.
  • the intracellular signaling region includes the signaling domain (stimulatory signal domain) and the costimulatory domain of CD3 ⁇ .
  • the CD3 ⁇ signaling domain comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% identical to the sequence set forth in SEQ ID NO: 20 %, 98% or 99% identity to or consisting of an amino acid sequence.
  • the co-stimulatory domain is a functional signaling domain derived from the 4-1BB (CD137) protein, or a functional signaling domain derived from the CD28 protein, or preferably, the co-stimulatory domain The stimulatory domain contains both those derived from CD28 and 4-1BB.
  • the 4-1BB (CD137) signaling domain comprises or is at least 90%, 91%, 92%, 93%, 94%, 95% identical to the sequence set forth in SEQ ID NO: 19 , or consisting of an amino acid sequence that is 96%, 97%, 98% or 99% identical.
  • the CD28 signaling domain comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the sequence set forth in SEQ ID NO: 18 %, 98% or 99% identity to or consisting of an amino acid sequence.
  • the costimulatory domain precedes (i.e., is closer to the N-terminus) the signaling domain of CD3 ⁇ .
  • a humanized chimeric antigen receptor (CAR) polypeptide targeting EGFRvIII comprising from the N-terminus to the C-terminus:
  • Extracellular binding region which is a humanized anti-EGFRvIII scFv sequence, wherein the scFv sequence specifically binds to EGFRvIII and includes:
  • LCDR3 having the amino acid sequence of SEQ ID NO:6, or a variant of said LCDR3 having no more than 2 amino acid changes or no more than 1 amino acid change;
  • amino acid change is the addition, deletion or substitution of amino acids
  • IgG4 hinge region (SEQ ID NO: 13), or an IgG4 hinge region thereof having at least 80% sequence identity;
  • CD8 hinge region (SEQ ID NO:14), or a CD8 hinge region having at least 80% sequence identity thereto.
  • CD28 transmembrane domain or a variant thereof with 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO: 15 or a variant thereof with 1-2 amino acid modifications;
  • CD4 transmembrane domain or a variant thereof with 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO: 16 or a variant thereof with 1-2 amino acid modifications;
  • a CD8 transmembrane domain or a variant thereof having 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO: 17 or a variant thereof having 1-2 amino acid modifications;
  • Costimulatory signaling domain which is:
  • CD28 costimulatory domain or a variant thereof with 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO: 18 or a variant thereof with 1-2 amino acid modifications;
  • Stimulating signal domain which is a CD3 ⁇ signaling domain or a variant thereof with 1-10 amino acid modifications, for example, the sequence shown in SEQ ID NO: 20 or a variant thereof with 1-10, 1 -5 amino acid modified variants.
  • the humanized chimeric antigen receptor (CAR) polypeptide targeting EGFRvIII of the invention includes from N-terminus to C-terminus:
  • Extracellular binding region which is a humanized anti-EGFRvIII scFv sequence, wherein the scFv sequence specifically binds to EGFRvIII and includes:
  • a heavy chain variable region comprising or sharing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with the sequence of SEQ ID NO:7 % identity of the sequence, and
  • a light chain variable region comprising or sharing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with the sequence of SEQ ID NO:8 % identity sequence;
  • IgG4 hinge region (SEQ ID NO 13), or an IgG4 hinge region having at least 90%, at least 95% sequence identity;
  • CD8 hinge region (SEQ ID NO 14), or a CD8 hinge region having at least 90% or at least 95% sequence identity thereto.
  • Costimulatory signaling domain which is:
  • a stimulatory signaling domain which is the CD3 ⁇ signaling domain shown in SEQ ID NO: 20 or a variant thereof having 1-3 amino acid modifications;
  • the amino acid modification is the addition, deletion or substitution of amino acids.
  • the humanized chimeric antigen receptor (CAR) polypeptide targeting EGFRvIII of the present invention includes from N-terminus to C-terminus::
  • Extracellular binding region which is a humanized anti-EGFRvIII scFv sequence, wherein the scFv sequence specifically binds to EGFRvIII and includes:
  • TM Transmembrane region
  • Costimulatory signaling domain which is:
  • the humanized chimeric antigen receptor (CAR) polypeptide targeting EGFRvIII of the present invention also includes a signal peptide located at the N-terminus, such as, but not limited to, having a signal peptide as shown in SEQ ID NO: 21 or therewith.
  • 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 is at least 90%, 93%, 95%, 96% identical thereto. Amino acid sequences with %, 97%, 98%, 99% or greater identity.
  • the invention provides any of the aforementioned chimeric antigen receptor component/full-length coding nucleic acids; in some more specific embodiments, the coding nucleic acids comprise SEQ ID NO: 30 and/or 31 the sequence of.
  • the invention provides an expression vector comprising the aforementioned encoding nucleic acid of the invention.
  • the vector is selected from a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector.
  • the expression vector is derived from a retroviral plasmid; preferably, the retroviral plasmid is SFG.
  • the invention provides a virus comprising any of the aforementioned vectors.
  • the invention provides an immune effector cell transduced with any of the aforementioned coding nucleic acids, or any of the aforementioned expression vectors or any of the aforementioned viruses.
  • the immune effector cell is a T cell, More preferably, they are T lymphocytes.
  • the invention provides an immune effector cell whose surface expresses any of the aforementioned chimeric antigen receptors.
  • the immune effector cell is a T cell, more preferably, a T lymphocyte.
  • the immune effector cells of the invention exhibit enhanced tumor cell killing due to transduction/expression of the chimeric antigen receptor of the invention.
  • enhanced target cell killing is manifested by higher secretion of anti-tumor cytokines and/or stronger direct oncolytic ability.
  • higher anti-tumor cytokine secretion refers to cytokines (such as, but not limited to, IFN- ⁇ , IL-2, IL-6, IL-10, IL-17, and/or or TNF- ⁇ , etc.), the secretion of effector cells was increased by 0.1 times, 0.2 times, 0.3 times, 0.4 times, 0.5 times, 0.6 times, 0.7 times, 0.8 times, 0.9 times, 1 times, 2 times compared to the non-specific control effector cells , 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times or higher.
  • cytokines such as, but not limited to, IFN- ⁇ , IL-2, IL-6, IL-10, IL-17, and/or or TNF- ⁇ , etc.
  • Stronger direct oncolytic ability means that the number of direct killings of target tumor cells by the immune effector cells of the present invention is increased by 0.1 times, 0.2 times, 0.3 times, 0.4 times, 0.5 times, and 0.6 times compared to non-specific control effector cells. , 0.7 times, 0.8 times, 0.9 times, 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 Times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times or more.
  • the invention provides a method for preparing the immune effector cells of the invention, including introducing the nucleic acid, expression vector and/or virus of the invention into the cells, and/or causing the cells to express the chimeric antigen of the invention
  • the recipient for example, isolates T cells from human PBMC and transduces the isolated T cells with nucleic acids, expression vectors and/or viruses.
  • the invention provides methods of preventing or treating tumors (e.g., cancer) or providing anti-tumor immunity in a subject, comprising administering to said subject an effective amount of a chimeric gene expressing any of the foregoing.
  • Antigen receptor cells or any of the aforementioned T cells.
  • the patient has a tumor (e.g., a cancer) (e.g., elevated levels, e.g., nucleic acid or protein levels) of EGFRvIII, preferably the tumor is a glioma, more preferably the The tumor is glioblastoma.
  • a tumor e.g., a cancer
  • elevated levels e.g., nucleic acid or protein levels
  • the cells are administered in combination with one or more other therapies, such as therapeutic modalities and/or other therapeutic agents.
  • the other therapeutic agent is a chemotherapeutic agent, a cytokine, a cytotoxic agent, a therapeutic monoclonal antibody, a small molecule drug, an immunomodulatory agent (eg, an immunosuppressive agent), or any combination thereof.
  • the other therapeutic agent is a therapeutic monoclonal antibody, more preferably a PD-1 antibody.
  • the invention provides the use of the chimeric antigen receptor, nucleic acid, vector, virus or cell according to any of the preceding embodiments for the preparation of a medicament.
  • the medicine is used to prevent or treat tumors.
  • the tumor has (eg elevated levels, eg nucleic acid or protein levels) of EGFRvIII, preferably the tumor is a glioma, more preferably the tumor is a glioblastoma.
  • the invention provides the medicament, that is, the invention provides a pharmaceutical composition comprising the chimeric antigen receptor, nucleic acid, vector, virus or cell.
  • the drug is administered alone or in combination with one or more other therapies, such as therapeutic modalities and/or other therapeutic agents.
  • the other therapeutic agent is a chemotherapeutic agent, a cytokine, a cytotoxic agent, a therapeutic monoclonal antibody, a small molecule drug, an immunomodulatory agent (eg, an immunosuppressive agent), or any combination thereof.
  • the other therapeutic agent is a therapeutic monoclonal antibody, more preferably a PD-1 antibody.
  • FIG. 1 EGFRvIII CAR-T cells mediate excellent anti-tumor efficacy in vitro.
  • non-specific T cells non-transformed T cell non-specific CAR-T cells
  • EGFRvIII + CAR T cells were co-cultured with EGFRvIII + target cells.
  • Quantification of intracellular staining (ICS)-positive cells for CD107a+ revealed that EGFRvIII CAR-induced T cell activation resulted in significant CD107a expression.
  • C. Quantification of intracellular staining (ICS) positive cells for IFN- ⁇ + is shown. The effect of T cell transduction on the proportion of IFN- ⁇ + cells differed significantly between mock CAR cells and EGFRvIII+ CAR T cells.
  • D Cytokine production triggered by EGFRvIII-specific CAR.
  • EGFRvIII-specific CAR and non-specific T cells were co-cultured with target cells at an E:T ratio of 10:1. After 24 hours, IFN- ⁇ produced by T cells was measured by ELISA. .
  • EGFRvIII CAR is highly toxic to target cells (A represents U87-EGFRvIII cells, B represents U373-EGFRvIII cells). All results are expressed as the mean ⁇ SD of at least duplicates. (*p ⁇ 0.05, **p ⁇ 0.005, ***p ⁇ 0.005).
  • FIG. 1 EGFRvIII CAR-T cells exhibit EGFRvIII-specific tumor cell killing activity in vitro.
  • Using the xCELLigence System (Agilent) cell index values for tumor cells were continuously graphically output until the 50-hour time point during incubation of tumor cells with CAR T cells and incubation with tumor cells alone.
  • Real-time impedance analysis of mock- and EGFRvIII-CAR-T cells was performed according to the co-culture protocol.
  • FIG. 3 Intracranial delivery of EGFRvIII CAR T cells significantly extended mouse survival.
  • A U87-EGFRvIII cells (2 ⁇ 10 5 , left picture) and U373-EGFRvIII cells (2 ⁇ 10 5 , right picture) were orthotopically implanted into NOD-SCID mice. After implantation, intracranial effector cells ( 1 ⁇ 10 7 ) processing. Survival curves for each group were estimated using the Kaplan-Meier product limit estimation method. The main comparative analysis of the curves for each group was performed using the log-rank test.
  • FIG. 1 EGFRvIII CAR-T cells homing to the tumor site. Immunohistochemical analysis was performed to detect A. histomorphology, B. EGFRvIII protein, and C. T cells.
  • FIG. 5 A. Schematic representation of the EGFRvIII CAR molecule.
  • Figure 6.A Cell line stably transfected with green fluorescent protein and luciferase. Green fluorescent protein was performed using flow cytometry.
  • B Detection of EGFR and EGFRvIII expression by RT-PCR.
  • FIG. 7 Upregulation of T cell activation markers.
  • A. Mock T cells or B. EGFRvIII-CAR T cells were co-cultured with U87-EGFRvIII or U373-EGFRvIII.
  • Analysis of CD25 by flow cytometry via specific antibody-coupled APC showed that the expression ratio of CAR-T cells was significantly higher compared to control (mock) cells.
  • FIG. 8 Quantification of ICS-positive cells that are IFN- ⁇ + is shown.
  • A. The effect of T cell transduction on the proportion of IFN- ⁇ + cells was significantly different between mock CAR cells and EGFRvIII+CAR T cells. EGFRvIII+CAR T cells cultured alone (i.e., without target cells) or EGFRvIII+CAR T cells co-cultured with EGFRvIII-negative glioma cells were used as controls;
  • B Quantification of ICS-positive cells that are IFN- ⁇ + is negligible.
  • EGFRvIII+ cells induce significantly higher levels of various cytokines, especially IFN ⁇ and TNF, compared to EGFRvIII- cells.
  • IL17A did not reach the minimum detection sensitivity, so its changes were not included (not shown).
  • the U373 (tumor cells only) group served as a statistical negative control.
  • FIG. 10 Luciferase reporter assay.
  • EGFRvIII CAR T cells killed U87-EGFRvIII and U373-EGFRvIII cells, in contrast to EGFRvIII-negative cells.
  • the relative lysis rate (%) refers to the ratio of the higher lethality of EGFRvIII CAR to its basal lethality (Mock-CAR T cells).
  • T cells from the same donor were transduced with EGFRvIII CAR or Mock CAR and cultured overnight with EGFRvIII positive or negative tumor cells.
  • CAR-T cells were grouped using flow cytometry according to B.T cell phenotype or C.CD4 and CD8 gated groups.
  • D. Effector cells and target cells were co-cultured at an E:T ratio of 10:1. The phenotype of T cells was assessed by flow cytometry at day 1 (post-stimulation, top) and day 7 (bottom). We counted CD3 (left), CD4 (middle), and CD8 (right) positive T cell subsets respectively.
  • FIG. 12 Anti-tumor response produced by EGFRvIII CAR-T in vivo. Bioluminescent imaging of tumor growth over time, treated with intracranial effector cells after transplantation.
  • A. U373-EGFRvIII cells were implanted subcutaneously, n 5 mice.
  • B.U251 cells were implanted in situ, n 5 mice.
  • D. Use the Kaplan-Meier product limit estimation method to evaluate the survival curves of each group. The main comparative analysis of the curves of each group was performed using the log-rank test (subcutaneous model, top; in situ model, bottom).
  • the term “comprises” or “includes” means the inclusion of the stated element, integer or step, but not the exclusion of any other element, integer or step.
  • the term “comprises” or “includes” is used herein, it also encompasses combinations of the stated elements, integers, or steps unless otherwise indicated.
  • this is also intended to encompass elements/polypeptides consisting of that particular sequence.
  • EGFRvIII refers to the EGFRvIII (EGFR variant type-III) gene, or the protein expressed by the gene.
  • Enhanced expression of EGFR is frequently detected in a variety of cancers, including breast cancer, lung cancer, head and neck cancer, and glioblastoma.
  • Spontaneous rearrangements of the EGFR gene were first discovered in primary human glioblastomas, and in almost all cases, such alterations have been reported in EGFR-amplified tumors. These rearrangements result in three different types of mutants.
  • Type III is the most common of them and is known as type III EGF deletion mutant receptor. Compared with wild-type EGFR (epidermal growth factor receptor), the expression in its mRNA is Sub 2-7 are missing.
  • EGFRvIII is expressed in multiple tumor types, including glioblastoma multiforme (GBM), but is rarely observed in normal tissues. EGFRvIII is expressed in 24% to 67% of GBM cases and in surviving patients ⁇ 1 year later, EGFRvIII expression is an independent negative prognostic indicator.
  • anti-EGFRvIII antibody refers to an antibody that is capable of binding (human) EGFRvIII with sufficient affinity such that the antibody can For use as a therapeutic targeting (human) EGFRvIII.
  • the (human) EGFRvIII antibody binds (human) EGFRvIII with high affinity in vitro or in vivo.
  • antibody fragment includes a portion of an intact antibody.
  • the antibody fragment is an antigen-binding fragment.
  • Antigen-binding fragment refers to a molecule distinct from an intact antibody that contains a portion of an intact antibody and binds the antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; dAb (domain antibody); linear antibodies; single chain antibodies (such as scFv); single domain antibodies such as VHH ; Diavalent antibodies or fragments thereof; or camelid antibodies.
  • 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 and heavy chain variable regions are contiguous (for example via a synthetic linker such as a short flexible polypeptide linker), and can be expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • a scFv may have the VL and VH variable regions described in any order (eg, relative to the N-terminus and C-terminus of the polypeptide), the scFv may include VL-linker-VH or Can include VH-joint-VL.
  • anti-EGFRvIII antibody When reference is made herein to an “anti-EGFRvIII antibody,” “anti-EGFRvIII,” “EGFRvIII antibody,” or “antibody that binds EGFRvIII,” this may also refer to an antibody fragment that specifically binds EGFR, such as a scFv, unless otherwise stated.
  • CDR region is a region in an antibody variable domain that is highly variable in sequence and forms a structurally determined loop ("hypervariable loop") and/or contains antigen contact residues ("antigen contact points"). CDR is primarily responsible for binding to antigen epitopes.
  • the CDRs of the heavy and light chains are usually referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus.
  • the CDRs located in the variable domain of the heavy chain of an antibody are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located in the variable domain of the light chain of an antibody are referred to as LCDR1, LCDR2, and LCDR3.
  • 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 based on the three-dimensional structure of the antibody and the topology of the CDR loops (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)), Kabat based on the variability of antibody sequences (Kabat et al., Sequen ces of Proteins of Immunological Interest, 4th ed., U.S.
  • the residues of each CDR are as follows.
  • CDRs can also be determined based on having the same Kabat number position as a reference CDR sequence (eg, any of the exemplary CDRs of the invention).
  • residue positions in an antibody variable region refers 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)).
  • the heavy chain variable region CDRs of the antibodies of the invention are determined according to Kabat's rule
  • the light chain variable region CDRs of the antibodies of the invention are determined according to Kabat's rule.
  • the heavy chain variable region CDRs of an antibody of the invention are determined according to Kabat's rules; and the light chain variable region CDRs are determined according to Kabat's rules.
  • the heavy chain variable region CDR and/or light chain variable region CDR of the antibody of the invention are determined according to non-Kabat rules, such as Chothia, AbM, Contact, IMGT and North, etc. Commonly known rules.
  • the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the same antibody variable region defined under different assignment systems are different. Therefore, when referring to an antibody defined by a specific CDR sequence as defined in the invention, the scope of said antibody also encompasses antibodies whose variable region sequences comprise said specific CDR sequence but which differ due to the application of different protocols (e.g. Different assignment system rules or combinations) cause the claimed CDR boundaries to be different from the specific CDR boundaries defined in the present invention.
  • Antibodies with different specificities have different binding sites for different antigens
  • CDRs vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding.
  • the minimal overlapping region can be determined, thereby providing the "minimum binding unit" for antigen binding.
  • the smallest binding unit may be a subportion of a CDR.
  • the remainder of the CDR sequence can be determined from the structure of the antibody and protein folding. Therefore, variants of any CDR given herein are also contemplated by the present invention.
  • the amino acid residue of the minimal binding unit can remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia can be replaced by conserved amino acid residues.
  • a region “derived from human” or “derived from mouse” means that the region has the amino acid sequence or encoding nucleic acid sequence of the region in native human or native mouse, or is derived from the amino acid sequence or encoding nucleic acid sequence. sequence composition.
  • a region “derived from” human or mouse also encompasses a region that is substantially identical to, or at least 90%, 91% identical to, the amino acid sequence or encoding nucleic acid sequence of that region in native human or native mouse. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical, and still have the same or similar activity and/or function as that region.
  • the CD3 ⁇ chain when defining "the CD3 ⁇ chain is derived from humans", it means that the CD3 ⁇ chain has the amino acid sequence of the natural human CD3 ⁇ chain, or consists of the amino acid sequence; or it means that the amino acid sequence of the CD3 ⁇ chain is the same as that of the natural human CD3 ⁇ chain.
  • the amino acid sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity and still has the activity of the native human CD3 ⁇ chain and/or functionality.
  • a “humanized” antibody refers to an antibody that contains amino acid residues from a non-human CDR and amino acid residues from a human FR.
  • a humanized antibody will comprise substantially all of at least one, typically two, variable domains, wherein all or substantially all of the CDRs (e.g., CDRs) correspond to those of a non-human antibody, and all Or essentially all FRs correspond to those of human antibodies.
  • a humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has been humanized.
  • Human antibody or “fully human antibody” or “fully human antibody” are used interchangeably and refer to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody derived from a human or generated by human cells or derived from non-human sources, which utilize human antibody libraries or other human antibody coding sequences. This definition of human antibodies specifically excludes humanized antibodies containing non-human antigen-binding residues.
  • binding means that the binding is selective for the antigen and can be distinguished from undesired or non-specific interactions.
  • the ability of an antigen binding site to bind to a specific antigen can 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 method or surface plasmon resonance (SPR) or cell binding assay.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • MSD biofilm thin layer interferometry
  • SPR surface plasmon resonance
  • chimeric antigen receptor or alternatively “CAR” refers to a group of polypeptides that, when in an immune effector cell, provide said cell with specificity for a target cell (usually a cancer cell), and Has intracellular signal generation.
  • a CAR includes at least one extracellular binding domain, a transmembrane domain, and an intracellular signaling domain.
  • groups of polypeptides are adjacent to each other.
  • intracellular signaling domain refers to a functional portion of a protein that functions by transmitting information within a cell, serving to regulate the cell via a defined signaling pathway by producing a second messenger or acting as an effector in response to such a messenger. activity.
  • extracellular binding region refers to the part of the chimeric antigen receptor that recognizes the antigen.
  • the extracellular binding region is a scFv from a monoclonal antibody, or a dAb (domain antibody) or a VHH.
  • transmembrane region refers to the portion of the chimeric antigen receptor that spans the membrane.
  • the transmembrane region can be any protein structure that is thermodynamically stable in the membrane. This is usually an alpha helix containing several hydrophobic residues.
  • the transmembrane region of any transmembrane protein can be used to provide the transmembrane portion of the chimeric receptor, for example, the transmembrane region can be the transmembrane region from CD8 ⁇ or CD28, preferably comprising any one of SEQ ID NO: 15-17 An amino acid sequence or a sequence having at least 90%, 95%, 96%, 97%, 98% or 99% or more identity thereto.
  • CD3- ⁇ also known as CD247, is defined as a protein as provided in GenBan Acc. No. BAG36664.1 or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.); "CD3- ⁇ signaling domain” is defined as an amino acid residue from the cytoplasmic domain of CD3- ⁇ or a functional derivative thereof that is sufficient to functionally transmit the initiating signal required for T cell activation.
  • a "CD3- ⁇ signaling domain” comprises a sequence as provided in SEQ ID NO: 20 or is at least 90%, 95%, 96%, 97%, 98% or 99% or more identical thereto. sexual sequence.
  • 4-1BB refers to a member of the TNFR superfamily having the amino acid sequence as provided in GenBank Acc. No. AAA62478.2, or equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc. .
  • the "4-1BB costimulatory domain” is defined as amino acid residues 214-255 of GenBank Acc. No. AAA62478.2, or from a non-human species such as mouse, rodent, monkey, Equivalent residues of apes et al.
  • the "4-1BB costimulatory domain” is a sequence as provided in SEQ ID NO: 19 or equivalent residues from a non-human species such as mouse, rodent, monkey, ape, etc. or at least 90% identical thereto , 95%, 96%, 97%, 98% or 99% or more identical sequences.
  • CD28 refers to human leukocyte differentiation antigen 28. Its official name in NCBI GeneBank is CD28, and its ID number is 940. It has 3 isoforms (cDNA sequence/protein sequence), respectively NM_006139.3/NP_006130.1 , NM_001243077.1/NP_001230006.1, NM_001243078.1/NP_001230007.1.
  • CD28 costimulatory domain includes the sequence as provided in SEQ ID NO: 18 or is at least 90%, 95%, 96%, 97%, 98% or 99% or more identical thereto. sequence.
  • retrovirus refers to retroviruses, that is, RNA viruses, those viruses that are commonly used as vectors for genetic engineering/gene therapy.
  • retroviral vector used in the present invention is based on the Moloney murine leukemia virus (SFG) plasmid, also known as "vector pMSGV1".
  • FSG Moloney murine leukemia virus
  • retroviral vectors are available and known to those skilled in the art, for example, from the company Addgene.
  • therapeutic agent encompasses any substance that is effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, cytotoxic agents, therapeutic monoclonal antibodies, small molecule drugs or immunomodulatory agents (e.g. immunosuppressants).
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes cell death or destruction.
  • “Chemotherapeutic agents” include chemical compounds useful in treating cancer.
  • small molecule drugs refers to low molecular weight organic compounds capable of modulating biological processes.
  • Small molecules are defined as molecules with a molecular weight of less than 10 kD, usually less than 2 kD and preferably less than 1 kD.
  • Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimetics. As therapeutic agents, small molecules can be more permeable to cells, less susceptible to degradation, and less likely to elicit an immune response than larger molecules.
  • immunomodulator refers to natural or synthetic agents or drugs that inhibit or modulate immune responses.
  • the immune response can be a humoral response or a cellular response.
  • Immunomodulators include immunosuppressants.
  • an “immunosuppressant,” “immunosuppressive drug,” or “immunosuppressant” is a therapeutic agent used in immunosuppressive therapy to suppress or prevent the activity of the immune system.
  • an effective amount refers to an amount or dose of an antibody or fragment or conjugate or composition or combination of the invention that produces the desired effect in a patient in need of treatment or prophylaxis when administered to the patient in single or multiple doses.
  • a “therapeutically effective amount” means an amount effective to achieve the desired therapeutic result, at the required doses and for the required period of time.
  • a therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment, or conjugate or composition or combination thereof, are outweighed by the therapeutically beneficial effects.
  • a “therapeutically effective amount” preferably inhibits a measurable parameter (eg, 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 refers to an amount effective to achieve the desired prophylactic result at the required dosage and for the required period of time. Typically, since a prophylactic dose is used in a subject prior to or at an earlier stage of 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, domestic 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.
  • the sequences are aligned for optimal comparison purposes (e.g., a first and a second amino acid sequence or nucleic acid sequence may be aligned for optimal alignment). Introducing gaps in one or both may allow non-homologous sequences to be discarded for comparison purposes).
  • the length of the aligned reference sequences 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 reference sequence length.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. The molecules are identical when a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence.
  • Mathematical algorithms can be used to perform sequence comparison and calculation of percent identity between two sequences.
  • the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm is used which has been integrated into the GAP program of the GCG software package (available at http://www.gcg.com available), determine the distance between two amino acid sequences using the Blossum 62 matrix or the PAM250 matrix with gap weights 16, 14, 12, 10, 8, 6, or 4 and length weights 1, 2, 3, 4, 5, or 6 Percent identity.
  • the GAP program in the GCG software package (available at http://www.gcg.com) is used, using the NWSgapdna.CMP matrix and gap weights 40, 50, 60, 70 or 80 and A length weight of 1, 2, 3, 4, 5, or 6 determines the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set (and one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. You can also use the PAM120 weighted remainder table, gap length penalty 12, gap penalty 4), using the E. Meyers and W.
  • nucleic acid sequences and protein sequences described herein may further be used as "query sequences" to perform searches against public databases, for example to identify other family member sequences or related sequences.
  • anti-tumor effect refers to a biological effect that can be demonstrated by a variety of means, including but not limited to, for example, reduction in tumor volume, reduction in tumor cell number, reduction in tumor cell proliferation, or reduction in tumor cell survival.
  • tumor and cancer are used interchangeably herein to encompass both solid and liquid tumors.
  • cancer and “cancerous” refer to or describe a physiological disorder in mammals that is typically characterized by unregulated cell growth.
  • cancers suitable for treatment by the antibodies of the invention include gastric or pancreatic cancer, including metastatic forms of those cancers.
  • neoplastic refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • glioma is the abbreviation of glioma, also known as glioma.
  • Glioma is a tumor disease of the central nervous system, which refers to a tumor that occurs in the neuroectoderm and originates from the canceration of neuronal cells or mesenchymal cells. Glioma is the most common primary central nervous system tumor in the brain, accounting for about half of all primary intracranial tumors.
  • Glioblastoma is a relatively common type of glioma, also known as glioblastoma multiforme (GBM), which is the name of grade IV in the World Health Organization's grading of gliomas.
  • Grade IV glioma is the most malignant glioma. The peak age of onset of glioblastoma is generally 50-60 years old.
  • combination therapy refers to the administration of two or more therapeutic agents or treatment modalities (eg, radiation therapy or surgery) to treat a disease described herein.
  • administration involves co-administration of the therapeutic agents in a substantially simultaneous manner, for example, in a single capsule having a fixed ratio of the active ingredients.
  • administration involves co-administration of the individual active ingredients in multiple or separate containers (eg tablets, capsules, powders and liquids). Powders and/or liquids can be reconstituted or diluted to the desired dosage prior to administration.
  • administration includes administering each type of therapeutic agent at approximately the same time or at different times in a sequential manner. In either case, the treatment regimen will provide for the beneficial effects of the drug combination in treating the disorder or condition described herein.
  • treating means slowing, interrupting, retarding, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
  • prevention includes the inhibition of the onset or progression of a disease or condition or symptoms of a particular disease or condition.
  • subjects with a family history of cancer are candidates for a preventive regimen.
  • prevention refers to the administration of a drug before signs or symptoms of cancer occur, particularly in a subject at risk for cancer.
  • vector when 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 as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors.”
  • the term "marker” as used herein refers to a class of substances that are derived from in vivo or ex vivo samples and are relatively easy to detect in samples using existing or conventional experimental methods and tools in the field.
  • the presence level of the marker, and this presence level is related to a specific physiological or pathological state, so the presence level of the marker can be obtained to infer or assist in inferring this specific physiological state. or pathological condition.
  • the marker can be any substance present in the body, such as, but not limited to, nucleic acids, polysaccharides, proteins, inorganic or organic small molecules, or their polymers or hybrids (e.g., glycoproteins, phosphorylated proteins, methyl nucleic acid sequences, etc.), or their (e.g., protein) encoding genes.
  • the marker is IFN- ⁇ .
  • the specific physiological or pathological state that needs to be inferred refers to the ability to kill or inhibit the target tumor that the CAR-T cells of the present invention have shown or will show after entering the subject.
  • the sample is the in vitro culture supernatant of T cells transfected with CAR molecules, or the serum of a subject treated with CAR-T cells.
  • IFN- ⁇ refers to a subtype of the cytokine interferon, which is secreted by activated T cells and natural killer cells, especially type I helper T cells (Th1 cells), in the form of water-soluble Dimers exist.
  • the receptor of IFN- ⁇ is composed of two subunits. It is activated by IFN- ⁇ after binding to it and regulates the JAK-STAT pathway. Multiple studies have shown that IFN- ⁇ has antiviral, immunomodulatory and anti-tumor activities.
  • CD107a is a T cell surface marker. Cytotoxic granules in CTL and NK cells mainly exist in secretory lysosomes, and CD107a, also known as lysosome-associated membrane protein-1, is a protein that exists on the lysosomal membrane. When secretory lysosomes During the secretion process, lysosomes bind to the cell membrane, and CD107a is expressed on the cell membrane as the lysosome membrane binds to the cell membrane. Therefore, the CD107a molecule is a sensitive marker of CTL cell degranulation and is directly related to cytotoxic activity. Reflects the level of cytotoxic cell killing activity
  • the invention relates to an isolated chimeric antigen receptor (CAR) molecule comprising (e.g., sequentially linked) an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain (e.g., comprising a co- stimulation domain).
  • CAR chimeric antigen receptor
  • an extracellular binding region described herein that binds EGFRvIII binds wild-type EGFRvIII, such as human wild-type EGFRvIII.
  • the extracellular binding region that binds EGFRvIII can be any antigen-binding domain that binds EGFRvIII: 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 of camelid-derived Nanobodies (VHH), and in combination with those known in the art Alternative scaffolds function as antigen-binding domains. In some cases it may be advantageous for the antigen-binding domain to be derived from the same species as the CAR's ultimate use.
  • the antigen-binding domain of the CAR may comprise human or humanized residues for the antigen-binding domain of an antibody or antibody fragment.
  • the antigen binding domain comprises a human antibody or antibody fragment.
  • the antigen binding domain is a scFv.
  • the extracellular binding region that binds EGFRvIII includes one or more (eg, all 3) light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 described herein (LCDR2) and light chain complementarity determining region 3 (LCDR3) and one or more (e.g., all 3) of the heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2) and Heavy chain complementarity determining region 3 (HC CDR3).
  • LCDR1 light chain complementarity determining region 1
  • LCDR2 light chain complementarity determining region 2 described herein
  • LCDR3 light chain complementarity determining region 3
  • the extracellular binding region of the invention that binds EGFRvIII includes three complementarity determining regions (HCDR) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3.
  • HCDR complementarity determining regions
  • the extracellular binding region of the invention that binds EGFRvIII includes three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.
  • LCDRs complementarity determining regions
  • the extracellular binding region of the invention that binds EGFRvIII includes 3 complementarity determining regions (HCDR) from the heavy chain variable region and 3 complementarity determining regions (LCDR) from the light chain variable region.
  • HCDR complementarity determining regions
  • LCDR complementarity determining regions
  • the extracellular binding region of the present invention in conjunction with EGFRvIII comprises a variable region of heavy chain (VH). In some respects, the extracellular binding region of the present invention in conjunction with EGFRvIII comprises a variable region of light chain (VH). In some respects, the extracellular binding region of the present invention in conjunction with EGFRvIII comprises a variable region of heavy chain and a variable region of light chain (VH). In some embodiments, the variable region of heavy chain comprises 3 complementary determining regions (CDRs), HCDR1, HCDR2 and HCDR3 from the variable region of heavy chain. In some embodiments, the variable region of light chain comprises 3 complementary determining regions (CDRs), LCDR1, LCDR2 and LCDR3 from the variable region of light chain.
  • CDRs complementary determining regions
  • the heavy chain variable region of the invention that binds the extracellular binding region of EGFRvIII
  • amino acid (iii) Comprises one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably) compared to the amino acid sequence selected from SEQ ID NO:7
  • the amino acid sequence (amino acid substitution, more preferably amino acid conservative substitution) consists of the amino acid sequence.
  • the amino acid change does not occur in the CDR region.
  • the light chain variable region of the invention that binds the extracellular binding region of EGFRvIII
  • amino acid (iii) Comprises one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably) compared to the amino acid sequence selected from SEQ ID NO:8
  • the amino acid sequence (amino acid substitution, more preferably amino acid conservative substitution) consists of the amino acid sequence.
  • the amino acid change does not occur in the CDR region.
  • the three complementarity determining regions (HCDR) from the heavy chain variable region of the invention are selected from
  • the three HCDR regions comprise at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) sequence.
  • the three complementarity determining regions (LCDRs) from the light chain variable region of the invention, LCDR1, LCDR2 and LCDR3, are selected from
  • the three LCDR regions comprise at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) sequence.
  • the HCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO:1, or the HCDR1 contains one, two or three changes compared to the amino acid sequence of SEQ ID NO:1 (preferably Amino acid substitutions, preferably conservative substitutions) of the amino acid sequence.
  • HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:2, or HCDR2 comprises one, two or three changes compared to the amino acid sequence of SEQ ID NO:2 (preferably Amino acid substitutions, preferably conservative substitutions) of the amino acid sequence.
  • the HCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO:3, or the HCDR3 comprises one, two or three changes compared to the amino acid sequence of SEQ ID NO:3 (preferably Amino acid substitutions, preferably conservative substitutions) of the amino acid sequence.
  • LCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO:4, or LCDR1 contains one, two or three changes compared to the amino acid sequence of SEQ ID NO:4 (preferably Amino acid substitutions, preferably conservative substitutions) of the amino acid sequence.
  • LCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO:5, or LCDR2 comprises one, two or three changes compared to the amino acid sequence of SEQ ID NO:5 (preferably Amino acid substitutions, preferably conservative substitutions) of the amino acid sequence.
  • LCDR3 comprises the amino acid sequence of SEQ ID NO:6, or consists of the amino acid sequence, or LCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:6.
  • the extracellular binding region that binds EGFRvIII of the invention includes VH and VL, wherein the VH and VL respectively comprise or consist of the amino acid sequence shown below: SEQ ID NO:7 and 8.
  • the amino acid changes described herein include amino acid substitutions, insertions or deletions.
  • the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.
  • the amino acid changes described in the present invention occur in regions outside the CDRs (eg in the FR). More preferably, the amino acid changes described in the present invention occur in regions outside the heavy chain variable region and/or outside the light chain variable region.
  • substitutions are conservative substitutions.
  • a conservative substitution is when one amino acid is replaced by another amino acid within the same class, for example, an acidic amino acid is replaced by another acidic amino acid, a basic amino acid is replaced by another basic amino acid, or a neutral amino acid is replaced by another neutral amino acid Displacement. Exemplary substitutions are shown in the following table:
  • the substitution occurs in the CDR region of the extracellular binding domain of EGFRvIII.
  • the resulting variant will have modifications (eg, improvements) relative to the parent antibody with respect to certain biological properties (eg, increased affinity) and/or will have certain biological properties that are substantially retained from the parent.
  • modifications eg, improvements
  • exemplary substitution variants are affinity matured antibodies.
  • the extracellular binding region of EGFRvIII of the invention is an scFv fragment of an anti-EGFRvIII antibody.
  • the scFv fragment contains a linker.
  • the linker is a peptide linker.
  • the peptide linker may or may not consist primarily of 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 at least 1 (and preferably 2, 3, 4 , 5, 6, 7, 8, 9, 10) integers.
  • the linker comprises the amino acid sequence of (GSGGS)3 (SEQ ID NO: 11).
  • the linker comprises the amino acid sequence of (GSGGS)4 (SEQ ID NO: 12).
  • the scFv fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9 or 10 or is at least 80%, 85%, 90%, 91%, 92%, 93%, An amino acid sequence that is 94%, 95%, 96%, 97%, 98% or 99% identical or consists of said amino acid sequence.
  • 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 of the extracellular region , 2, 3, 4, 5, 6, 7, 8, 9, 10 to 15 amino acids) and/or one or more additional ones associated with the extracellular region of the protein from which the transmembrane protein is derived amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to 15 amino acids in the intracellular region).
  • the transmembrane region is a region related to one of the other regions of the CAR used, for example, in one embodiment, the transmembrane region can be derived from a signaling domain, a costimulatory domain, or a hinge structure The same protein from which the domain is derived. In another aspect, the transmembrane region is not derived from the same protein as any other region of the CAR. In some cases, the transmembrane region may be selected or modified by amino acid substitutions to avoid binding of such regions to transmembrane regions of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex. change.
  • the transmembrane region is capable of homodimerizing with another CAR on the cell surface of the CAR-expressing cell.
  • the amino acid sequence of the transmembrane region can be modified or substituted so as to minimize interaction with the binding domain of the native binding partner present in cells expressing the same CAR.
  • the transmembrane region can 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 conducting signals to the intracellular region whenever the CAR binds to the target.
  • the transmembrane region specifically used in the present invention may include at least the following transmembrane regions: for example, CD28, CD8 (eg, CD8 ⁇ , CD8 ⁇ ), CD4.
  • the transmembrane domain includes a transmembrane domain described herein, for example, having the sequence of any one of SEQ ID NO: 15-17, having the sequence of any one of SEQ ID NO: 15-17 An amino acid sequence with 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) to any of the amino acid sequences, or an amino acid sequence identical to SEQ ID NO: 15
  • the amino acid sequence of any one of -17 is a sequence with 90-99% identity.
  • the transmembrane region can be connected to an extracellular region of the CAR, such as the antigen-binding domain of the CAR, via a hinge (eg, a hinge from a human protein).
  • a hinge eg, a hinge from a human protein.
  • the hinge can be a human Ig (immunoglobulin) hinge (eg, IgG4 hinge, IgD hinge), GS linker (eg, GS linker described herein), KIR2DS2 hinge, or CD8a hinge.
  • the hinge region is a human IgG4 or CD8 hinge; in some embodiments, the hinge region sequence has at least 95% sequence identity with a human IgG4 or CD8 hinge; in one embodiment, the hinge region comprises ( For example, consisting of) the amino acid sequence of SEQ ID NO: 13 or 14.
  • the CAR molecule comprises an intracellular signaling region.
  • the intracellular signaling region comprises a co-stimulatory domain.
  • the co-stimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of: CD28 and/or 4-1BB (CD137) or a functional variant thereof.
  • the co-stimulatory domain comprises the following or consists of: a sequence of SEQ ID NO: 18 and/or a sequence of SEQ ID NO: 19.
  • the co-stimulatory domain comprises the following or consists of: an amino acid sequence having at least 1, 2 or 3 modifications (e.g., substitutions) but not 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 with the amino acid sequence of SEQ ID NO: 18 and/or SEQ ID NO: 19.
  • the intracellular signaling domain is generally responsible for the activation of at least one of the normal effector functions of the immune cell into which the CAR has been introduced.
  • effector function refers to a specialized function of a cell.
  • the effector function of T cells may be, for example, cytolytic activity or auxiliary activity, including secretion of cytokines.
  • intracellular signaling domain refers to the portion of a protein that transduces effector function signals and directs the cell to perform a specific function. Although the entire intracellular signaling domain can typically be used, in many cases it is not necessary to use the entire chain.
  • intracellular signaling domain is meant to include a truncated portion of the intracellular signaling domain sufficient to transduce an effector function signal.
  • the intracellular signaling region of a CAR for use in the present invention also includes the cytoplasmic sequences of the T cell receptor (TCR) and coreceptors that work together to initiate signal transduction following antigen receptor engagement, as well as any derivatives of these sequences or Variants and any recombinant sequences with the same functional capabilities. It is known that the signal generated by TCR alone is insufficient for complete activation of T cells, and secondary and/or costimulatory signals are also required.
  • TCR T cell receptor
  • T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to those that provide secondary or costimulatory signals (secondary cytoplasmic domains, such as costimulatory domains).
  • Primary cytoplasmic signaling domains regulate primary activation of the TCR complex in a stimulatory manner or in an inhibitory manner.
  • Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM-containing primary intracellular signaling regions specifically useful in the present invention include those listed below: CD3 ⁇ .
  • a CAR of the invention comprises an intracellular signaling region, such as the primary signaling domain of CD3- ⁇ .
  • the intracellular signaling region of the CAR may comprise the CD3- ⁇ signaling domain itself, or it may be combined with other intracellular signaling regions, such as costimulatory signaling domains, for use in the CAR of the invention.
  • the intracellular signaling region of the CAR may include a CD3 ⁇ chain portion and a costimulatory signaling domain.
  • the costimulatory signaling domain precedes the signaling domain of CD3 ⁇ .
  • the intracellular signal regions within the cytoplasmic portion of the CAR of the invention can be connected to each other in random or specified order.
  • short oligopeptides or polypeptide linkers for example, between 2 and 10 amino acids in length (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) can signal intracellularly. connections between areas.
  • glycine-serine doublets can be used as suitable linkers.
  • a single amino acid such as alanine, glycine can be used as a suitable linker.
  • the intracellular signaling domain is designed to contain two or more, such as 2, 3, 4, 5 or more costimulatory signaling domains. In one embodiment, two or more, eg, 2, 3, 4, 5 or more costimulatory signaling domains are separated by a linker molecule (eg, a linker as described herein). In one embodiment, the intracellular signaling region contains two costimulatory signaling domains. In certain embodiments, the linker molecule is a glycine residue. In certain embodiments, the linker is an alanine residue.
  • the intracellular signaling region is designed to include the signaling domain of CD3- ⁇ and the signaling domain of CD28. In one aspect, the intracellular signaling region is designed to include the signaling domain of CD3- ⁇ and the signaling domain of 4-1BB. In one aspect, the signaling domain of CD3- ⁇ comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% identical to the sequence set forth in SEQ ID NO: 20 , or consisting of an amino acid sequence that is 98% or 99% identical.
  • the CAR molecule may also comprise a signal peptide, for example, but not limited to, having a signal peptide as set forth in SEQ ID NO: 21 or at least 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98% thereof or a signal peptide with a sequence of 99% identity.
  • the CAR molecule of the present invention may comprise an extracellular binding region that binds to EGFRvIII, such as a scFv that binds to EGFRvIII, for example, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9 or 10 or having at least 80% or 85% similarity with said sequence.
  • a hinge region such as an IgG4 hinge A region, for example, comprising or consisting of the amino acid sequence of SEQ ID NO: 13, or, for example, a CD8 hinge region, for example, comprising or consisting of the amino acid sequence of SEQ ID NO: 14
  • a transmembrane region such as a CD28 transmembrane region,
  • it contains or consists of the amino acid sequence of SEQ ID NO:18, or for example, the CD8 transmembrane region, for example, it contains or consists of the amino acid sequence of SEQ ID NO:17, for example, the CD4 transmembrane region, for example, it contains SEQ ID NO:16
  • the amino acid sequence of or consisting of; the intracellular signal region, which includes the CD28 signaling domain for example, includes or consists of the amino acid sequence of SEQ ID NO:
  • the present invention provides the encoding nucleic acid of CAR, it comprises the nucleic acid sequence encoding one or more CAR of the present invention.
  • CAR encoding nucleic acid is provided in the form of DNA construct.
  • the CAR-encoding nucleic acid may contain a Kozak sequence at the 5' end to promote stability and translation efficiency.
  • the Kozak sequence comprises or consists of the nucleic acid sequence of GCCACCATGG (SEQ ID NO: 29).
  • the invention further provides vectors comprising a CAR transgene.
  • the CAR vector can be transduced directly into cells, such as T cells, such as T lymphocytes.
  • the vector is a cloning vector or an expression vector, including, for example, but not limited to, one or more plasmids (eg, expression plasmids, cloning vectors, minicircles, microvectors, double minichromosomes), retroviruses, and Lentiviral vector constructs.
  • the vector is capable of expressing the CAR molecule in mammalian T cells.
  • the mammalian T cells are human T cells, such as T lymphocytes.
  • the present invention also provides a vector in which the nucleic acid encoding the CAR of the present invention is inserted.
  • the vector is DNA, RNA, a plasmid, an adenoviral vector, a lentiviral vector or a retroviral vector.
  • the vector is a lentiviral vector.
  • the vector is a retrovirus.
  • the retrovirus is SFG.
  • the vector further comprises a promoter.
  • expression of a natural or synthetic nucleic acid encoding a CAR is generally achieved by operably linking a nucleic acid encoding a CAR polypeptide or a portion thereof to a promoter and incorporating the construct into an expression vector.
  • Vectors can be adapted for replication and integration into eukaryotes.
  • a typical cloning vector contains transcriptional and translational terminators, an initiation sequence, and a promoter for regulating expression of the desired nucleic acid sequence.
  • Expression constructs of the invention may also be used in 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 large number of types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Special purpose vectors include expression vectors, replication vectors, probe-generating vectors, and sequencing vectors.
  • the expression vector can be provided into the cell in the form of a viral vector.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY) and other virology and molecular biology handbooks.
  • Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers.
  • retroviral vectors are used.
  • the expression vector introduced into the cell may also contain a selectable marker gene or reporter gene or both to facilitate identification and selection from the cell population sought to be transfected or infected by the viral vector. expressing cells.
  • the reporter gene is GFP.
  • the reporter gene is a luciferase, such as firefly luciferase.
  • the vector can be readily introduced into a host cell, such as a mammalian, bacterial, yeast or insect cell, by any method in the art.
  • expression vectors can be transferred into host cells by physical, chemical, or biological means.
  • Physical methods used to introduce polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for generating cells including 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). One preferred method for introducing polynucleotides into host cells is lipofection.
  • the invention relates to cells comprising said vector or nucleic acid.
  • the cell is a human T cell, e.g., a T cell described herein.
  • the T cells are T lymphocytes.
  • the cells are autologous T cells.
  • the cells are allogeneic T cells.
  • the cell source eg, T cells or NK cells
  • T cells Prior to expansion and genetic modification, the cell source (eg, T cells or NK cells) is obtained from the subject.
  • subject is meant to include living organisms (eg, mammals) in which an immune response can 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, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. Many T cell lines available in the art can be used in certain aspects of the invention.
  • T cells can be obtained from a unit of blood collected from a subject using any of a variety of techniques known to those skilled in the art (such as Ficoll TM isolation).
  • the cells are obtained from the individual's circulating blood by apheresis.
  • T cells are separated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLLTM gradient or counterflow centrifuge elutriation.
  • Specific subpopulations of T cells such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells can be further separated by positive or negative selection techniques.
  • T cells are separated by culturing with anti-CD3/anti-CD28 (e.g., 3 ⁇ 28)-conjugated beads (e.g., M-450 CD3/CD28 T) for a period sufficient to positively select for the desired T cells.
  • the invention in another aspect, relates to a method of producing a cell comprising transducing a cell described herein (e.g., a T cell) with a vector containing a nucleic acid encoding a CAR molecule (e.g., a CAR molecule described herein).
  • a cell described herein e.g., a T cell
  • a vector containing a nucleic acid encoding a CAR molecule e.g., a CAR molecule described herein.
  • the vector is a lentiviral vector described herein.
  • One aspect of the invention provides a method of preventing or treating tumors (e.g., cancer) or providing anti-tumor immunity in a subject, comprising administering to said subject an effective amount of cells containing a CAR molecule, e.g., herein Cells expressing CAR molecules are described.
  • the cells are autologous T cells.
  • the cells are allogeneic T cells or NK cells.
  • the subject is human.
  • the T cells are T lymphocytes.
  • the tumor (eg, cancer) patient has (eg, elevated levels, eg, nucleic acid or protein levels) EGFRvIII.
  • the tumor such as cancer
  • the tumor includes solid and hematological tumors as well as metastatic lesions.
  • examples of solid tumors include malignant tumors. Cancer can be early, intermediate or late or metastatic.
  • the tumor treatment will benefit from inhibition of EGFRvIII at the nucleic acid or protein level. In some embodiments, the tumor treatment will benefit from the direct killing of tumor cells by the CAR-T cells of the invention. In some embodiments, the tumor treatment will benefit from the 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 regulation of the immune system in the body by the cytokines secreted by the CAR-T cells of the present invention, and the latter will cause growth inhibition or killing of tumor cells.
  • the anti-EGFRvIII antibodies of the invention are capable of inhibiting tumor cell proliferation, such as tumor cells expressing EGFRvIII, such as glioma cells, such as GBM cells.
  • the tumor is immune evasive.
  • the tumor is a cancer, such as a glioma, such as GBM.
  • the subject may be a mammal, eg, a primate, preferably a higher primate, eg, a human (eg, an individual suffering from or at risk of suffering from a disease described herein).
  • the subject suffers from or is at risk of suffering from a disease described herein (eg, cancer).
  • the subject receives or has received other treatments, such as chemotherapy treatment and/or radiation therapy.
  • the subject has previously received or is currently receiving immunotherapy.
  • 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 production or preparation of a medicament for use as described herein, e.g., for the prevention or treatment herein Related diseases or conditions mentioned.
  • the CAR molecule of the present invention or its encoding nucleic acid or a vector comprising the same or a cell comprising the same delays the onset of a disorder and/or symptoms associated with the disorder.
  • a CAR molecule of the invention or a nucleic acid encoding the same or a vector comprising the same or a cell comprising the same can also be administered in combination with one or more other therapies, such as therapeutic modalities and/or other therapeutic agents, for use herein.
  • therapies such as therapeutic modalities and/or other therapeutic agents, for use herein.
  • the described uses, for example, are used to prevent and/or treat the related diseases or conditions mentioned herein.
  • the treatment modality or therapeutic agent enhances the activity or adaptability of cells expressing the CAR molecule, or reduces the side effects associated with the administration of cells expressing the CAR molecule, or treats a disease associated with EGFRvIII.
  • treatment modalities include surgery; radiation therapy, localized or focused irradiation, and the like.
  • the therapeutic agent is selected from the group consisting of chemotherapeutic agents, cytokines, cytotoxic agents, vaccines, therapeutic monoclonal antibodies, small molecule drugs, or immunomodulatory agents.
  • chemotherapeutic agents include immunosuppressive or anti-inflammatory agents.
  • the immune effector cells stably expressing the EGFRvIII CAR molecule of the present invention have a killing effect on the human glioma cell lines U87-EGFRvIII and U373-EGFRvIII stably expressing EGFRvIII.
  • effector cells were significantly activated and secreted various cytokines related to immune function, especially INF- ⁇ .
  • tumor lysis can be assessed more visually.
  • EGFRvIII CAR showed similar cytolytic potential compared to other published reports.
  • the third-generation CAR-T cells we constructed showed stable cytolysis.
  • sequence SEQ ID NO number of the exemplary scFv of the present invention (all CDRs are defined using Kabat rules)
  • Glioma cell lines expressing EGFRvIII or wtEGFR were a generous donation from Webster K Cavenee at Memorial Sloan Kettering Cancer Center and maintained in DMEM supplemented with 1 ⁇ g/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 calf serum and 1% 10000IU/ml penicillin/10000 ⁇ g/ml streptomycin (Figure 6).
  • PG-13 and Phoenix ECO retrovirus-producing cell lines were cultured in DMEM supplemented with 10% FBS, 2mM glutamine, and 1mM sodium pyruvate (Lonza).
  • T cells were cultured in T cell culture medium, which consisted of X-VIVO-15 medium plus 5% antibody serum (SIGMA), 100 U/ml IL-2, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin. All cells were routinely tested for mycoplasma and the results were negative.
  • AF647-conjugated goat anti-mouse antibody Jackson ImmunoResearsh, 115-606-072
  • FITC-conjugated goat anti-mouse IgG H+L
  • GAM FITC-conjugated goat anti-mouse IgG
  • APC-conjugated CD107a BD Pharmingen TM , 560664
  • APC-R700 binds to CD3 (BD-Pharmingen TM , 565119);
  • BV421 binds to CD4 (BD Pharmingen TM , 562424);
  • PE-Cy7 binds to CD8 (BD-Pharmingen TM , 557746); PE binds to CD3 (555340, BD Biosciences);
  • FITC binds to CD3 (Invitrogen, 11-0036-42); PE-Cy7 binds to IFN- ⁇ (Biolegend, 502528);
  • V500 binds to CD
  • T cell activation rate was determined using APC combined with CD107a and protein transport inhibitor (BD Pharmingen TM , 554724).
  • IFN- ⁇ antibody was used to determine the expression of IFN- ⁇ in T cells according to the operating procedures of the intracellular fixation and permeabilization buffer set kit (Invitrogen, 88-8824). Flow cytometry was performed using BD-FacsCanto II Plus (BD Biosciences), and analysis was performed using FlowJo software (Tree star, Inc. Ashland, OR).
  • Freshly harvested PBMC are transduced with EGFRvIII-28BB ⁇ CAR and co-cultured with EGFRvIII-expressing cell lines or other cell lines for restimulation.
  • Cells were added to a U-shaped bottom 6-well microplate at a ratio of 10:1 (1 ⁇ 10 6 CAR-T: 1 ⁇ 10 5 target cells). After 24 hours, the supernatant was collected and incubated in liquid nitrogen. Quick freeze and then store at -80 °C until experimental use.
  • effector T cells (1x10 6 cells) and target cells in the ICCS (intracellular cytokine staining test) assay were separated in a 24-well microplate at 10:1E:T (effector cells: target cells) were co-cultured. Each well contains 1 ml of T cell culture medium and 2 ⁇ l of protein transport inhibitor (invitrogen, 00-4980-93). The cells were harvested after culturing the dishes at 37°C for 24 hours, and intracellular staining was performed to evaluate the expression of IFN- ⁇ .
  • ICCS intracellular cytokine staining test
  • EGFR and EGFRvIII fragments were amplified by PCR and assembled between constructs by restriction enzyme digestion using appropriately designed primers.
  • the final 196 base pair (bp) DNA fragment of the EGFR encoding product was used with specific primers at a concentration of 10 ⁇ M (forward, 5'-ATGCGACCCCGGGACGCGC-3', SEQ ID NO: 23; reverse, 5'-GCCCTTCGCACTTACTACTTGCGG-3 ', SEQ ID NO:24) was amplified, and the EGFRvIII fragment (150bp) was amplified with primers (forward, 5'-ATGCGCCTCCGGGACGGCC-3'SEQ ID NO:25; reverse, 5'-CTCATAGTCGGCCCCCAGG-3', SEQ ID NO: 26) Amplification, actin was amplified with primers (forward, 5'-ACCTGCCCATACAGG-3', SEQ ID NO:27; reverse, 5'-AGGGCCGGACTCGTCATACT-3', SEQ ID NO
  • Glioma cell lines (U251, U87, and U373) stably expressing EGFRvIII were constructed, and their expression levels were verified (see Figure 6B).
  • RTCA Real-time cell analysis
  • CI cell index
  • target cells were seeded onto the E plate at a density of 10,000 cells/well. After 30 min of incubation at room temperature, place the E-plate on the RTCA SP station in an incubator (5% CO2; 37°C) for continuous impedance recording. On day 1 after inoculation, the CI value was measured on another day of co-culture with 10x effector cells as an indicator of tumor cell density.
  • mice Female NOD/SCID mice aged 6 to 8 weeks were used in the experiments. All animals were housed in a specific pathogen-free environment and maintained in strict accordance with protocols and standard animal care requirements approved by Beijing Millennium Monument Hospital of Capital Medical University. Minimize the suffering of experimental animals.
  • mice received intracranial (i.c.) implantation of 2 ⁇ 10 ⁇ 5 EGFRvIII target cells or other glioma cell lines, using stereotaxic coordinates (2.5 mm posterior to bregma, 2.5mm laterally and 3.5mm deep into the parenchyma). Target cells were lightly resuspended in separate microcentrifuge tubes before each implantation. On the 6th day after intracranial tumor transplantation, 2 ⁇ 10 ⁇ 7 effector cells (EGFRvIII CAR or non-specific T cells) were injected intravenously.
  • EGFRvIII CAR effector cells
  • Protocol 2 Adapted feeding for 1 week, mice received intracranial (i.c.) implantation of 2 ⁇ 10 ⁇ 5 EGFRvIII target cells or other glioma cell lines, using stereotaxic coordinates (2.5 mm posterior to bregma, 2.5 lateral) mm, 3.5mm deep into the parenchyma). Mice recovered 3 days after the first surgery, and 1 ⁇ 10 ⁇ 7 effector cells (EGFRvIII CAR or non-specific T cells) were injected into the brain (0.3mm in front of bregma, 2mm to the left, and 3.5mm deep into the parenchyma) .
  • EGFRvIII CAR or non-specific T cells
  • Bioluminescence imaging was performed after tumor progression, and imaging data sets were acquired using the Xenogen IVIS imaging system (Xenogen) and in vivo imaging software (Xenogen). Mice in remission were then euthanized for further histological analysis. Briefly, mice were anesthetized using 1-2% isoflurane during an intraperitoneal injection of D-luciferin (150 mg/kg in 200 ⁇ l PBS;) 10 min before imaging. In high-sensitivity, cooled CCD cameras ( Bioluminescent imaging was performed every five minutes until peak emission values were obtained and a decrease in fluorescent signal was observed in all mice.
  • NOD/SCID mice received intracerebral or subcutaneous tumor implants and were euthanized before death. Brains were harvested and fixed in 4% frozen PFA at 4°C for 12 h before paraffin embedding. 5 ⁇ m 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, every 10 serial sections of the brain were stained with hematoxylin and eosin (H&E). Images were magnified 10x and 40x under an optical microscope.
  • H&E hematoxylin and eosin
  • the third-generation anti-EGFRvIII CAR ( Figure 5A) was generated by adding the CD28-4-1BB intracellular domain between the transmembrane region and the signaling domain molecule of CD3 ⁇ .
  • the CD28 transmembrane domain fragment was amplified by PCR and assembled between constructs by restriction enzyme digestion using appropriately designed primers.
  • the amino acid sequence is SEQ ID NO:15.
  • the EGFRvIII binding part comes from scFv, and its amino acid sequence is shown in SEQ ID NO:9.
  • the costimulatory domain and CD3 ⁇ signaling domain sequences derived from CD28.4-1BB are SEQ ID NO: 18, 19 and 20 respectively.
  • the ScFv and the transmembrane domain are connected through the hinge region (SEQ ID NO:13).
  • the vector retro-SFG-IgG4-CD28-4-1BB-CD3 ⁇ was obtained from the retroviral vector SFG (Addgene Company, also known as "vector pMSGV1"), and was digested with SmaI/MluI double enzyme, and then the aforementioned anti-EGFRvIII was cloned using cloning technology.
  • the nucleotide sequence of the scFv sequence was connected to the vector. After ligation for 1 hour at 37°C, the transformation was spread on an ampicillin-resistant LB plate.
  • the recombinant vector was identified by subcloning, plasmid extraction, enzyme digestion verification, and sequencing to construct the third Retroviral vector retro-SFG-EGFRvIII scFv-IgG4-CD28-4-1BB-CD3 ⁇ to generate CAR-T (shown in Figure 5A).
  • Retroviral concentrates were titer tested. Plate 0.5 ⁇ 10 Jurkat cells in each well of a 96-well sharp bottom plate at 200 ⁇ L/well. Dilute the retrovirus concentrate 100 times and add 400 ⁇ L, 40 ⁇ L, and 10 ⁇ L. Centrifuge at 32°C and 1200 ⁇ g for 90 minutes. After 4 hours, the cells were washed once with DPBS and spread on a 12-well plate. 48 hours after transduction, the cells were detected by flow cytometry. The results show that the retrovirus concentrate prepared in this example can infect Jurkat cells and express CAR after being diluted 100 times. Therefore, the retrovirus concentrate was used to generate CAR-T cells.
  • PBMC Peripheral blood mononuclear cells
  • Ficoll solution GE healthcare
  • anti-CD3/CD28 T cell activator Dynabeads Invitrogen was used to activate the cells at a bead-to-cell ratio of 1:1.
  • T cells were transduced with retroviral supernatants by centrifugation on retroconnexin (Takara)-coated plates to obtain EGFRvIII-28BB ⁇ CAR T cells. Verify transduction efficiency by flow cytometry or western blot after 9 days ( Figure 5B).
  • control T cells were transduced with a vector encoding a CD19-specific CAR gene.
  • 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).
  • FITC-labeled goat anti-mouse IgG (H+L) antibody Sigma.
  • We also used Western blot to assess the expression of CARs washed cells with PBS and lysed using RIPA buffer containing protease inhibitors, and separated the resulting proteins on a 15% SDS-PAGE gel (Bio-Rad). The separated proteins were subsequently transferred to PVDF membranes and detected using anti-human CD3- ⁇ monoclonal antibody (BD Pharmingen, clone 8D3). Bands were observed using the Odyssey Imaging System (LI-COR) with untransduced cells as a control.
  • LI-COR Odyssey Imaging System
  • PBMC Peripheral blood mononuclear cells
  • Example 4 Despite high levels of activation, EGFRvIII+CAR-expressing T cells exhibit similar levels of proliferation
  • Tn naive T cells defined by CD45RO, CCR7, CD27, and CD95 following priming with transduction and subsequent 7 days of in vitro EGFRvIII stimulation
  • Tn stem-like memory T cell
  • Tcm central memory T cell
  • Tem effector memory T cell
  • Tte effector T cell
  • Memory phenotype T cells were higher in EGFRvIII CAR than in non-specific CAR ( Figure 11B, 11D).
  • antigen-independent CAR-T signaling is characterized by the presence of multiple cell surface expressed co-inhibitory receptors (PD-1, Lag-3, and Tim-3).
  • PD-1, Lag-3, and Tim-3 cell surface expressed co-inhibitory receptors
  • antigen-activated CAR-T cells expressed exhaustion markers at the same level as T cells without EGFRvIII, that is, they did not exhibit exhaustion.
  • EGFRvIII CAR T cells expressed the same levels of exhaustion markers after EGFRvIII stimulation ( Figure 11E).
  • Example 5 Monitoring CAR-induced cytotoxic effects on EGFRvIII+ cells using real-time cell analysis (RTCA)
  • the xCELLigence system was used to measure the cellular impedance of target cells in real time, where the target cells were divided into groups co-cultured with EGFRvIII CARs and groups not co-cultured. Tumor cells were seeded in triplicate in 16-well E plates (e-plates). After tumor cells adhered for 24 hours, T cells were added at a ratio of 10 T cells per tumor cell. Electrical impedance was continuously recorded as an indicator of tumor cell density.
  • Figure 2A shows the measured tumor cell impedance according to the co-culture protocol used.
  • 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.
  • the CI maintained a steady increase and finally reached 2.42 ⁇ 0.03 and 1.52 ⁇ 0.02 (CI at the end of the experiment).
  • adding the same volume of culture medium but containing 10 times the number of effector cells (co-culture group) caused the CI to drop significantly to zero over time for both application regimens.
  • EGFRvIII-negative cells under effector cell treatment showed a lack of immune response, although the CI value decreased slightly, the detection value remained stable over longer co-culture times during the experiment.
  • Example 6 EGFRvIII CAR cells inhibit tumor growth and extend the survival of tumor-bearing mice in orthotopic xenograft models and subcutaneous models
  • mice bearing U87-EGFRvIII or U373-EGFRvIII cells showed a significant reduction in tumor growth after treatment with EGFRvIII CAR-T cells as measured by bioluminescence imaging compared to mice injected with Mock CAR-T cells (p ⁇ 0.01).
  • the reduction in tumor growth did not appear to be much different in mice bearing grade III and IV glioma cell lines or glioblastoma cell lines.
  • mice that received a single intratumoral injection of CAR-T cells did not significantly reduce tumor burden compared with the control group.
  • mice that received CAR-T cell treatment did not benefit from survival.
  • EGFRvIII CAR T cells can effectively target and kill EGFRvIII-expressing tumor cells in vivo.
  • Example 7 Assessment of EGFRvIII CAR cell migration and elongation after intracranial injection
  • Patent applications, patents, documents, books, etc. cited, mentioned and/or referred to in this article are hereby incorporated by reference in their entirety.
  • RTCA Real-Time Cell Analysis
  • Intratumoral IL-12 delivery empowers CAR-T cell immunotherapy in a pre-clinical model of glioblastoma.(2041-1723(Electronic)).

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Abstract

La présente invention concerne un nouveau récepteur antigénique chimérique se liant spécifiquement à l'EGFRvIII et son utilisation.
PCT/CN2022/120569 2022-09-19 2022-09-22 Récepteur antigénique chimérique anti-egfrviii et son utilisation WO2024060140A1 (fr)

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