CD19-targeted chimeric antigen receptors and uses thereof

Abstract

To provide a CD19-targeting chimeric antigen receptor, and to provide uses thereof.SOLUTION: Provided is a chimeric antigen receptor comprising a specific amino acid sequence. Also provided are a nucleic acid encoding the chimeric antigen receptor, a vector comprising the nucleic acid, an immune effector cell comprising the chimeric antigen receptor, the nucleic acid molecule and / or the vector, a method for preparing the immune effector cell, a composition comprising the immune effector cell, and use of the chimeric antigen receptor.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to the field of biomedicine. More specifically, the present disclosure relates to CD19-targeted chimeric antigen receptors and uses thereof. [Background technology]

[0002] Currently, clinical treatments for acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and B-cell lymphoma mainly involve chemotherapy, stem cell transplantation, and biological therapy. While these treatments can achieve some efficacy, relapsed or refractory leukemia remains a major challenge. As a novel therapeutic strategy, tumor cellular immunotherapy has become a hot topic in recent research. CD19 is widely expressed on the surface of almost all B-cell tumor cells, but is rarely expressed on other parenchymal cells or hematopoietic stem cells.

[0003] Tumor cells can produce immune escape by downregulating the expression of molecules involved in various pathways, such as T cell recognition and antigen response, or by reducing immunogenicity, thereby preventing the organism's immune system from effectively eliminating tumors.Research has shown that chimeric antigen receptor T cells (CAR-T cells) can recognize antigens on the surface of tumor cells and specifically kill the tumor cells, making them useful for tumor treatment. Summary of the Invention

[0004] The present disclosure provides a chimeric antigen receptor comprising the amino acid sequence set forth in SEQ ID NO: 1. The present disclosure further provides nucleic acids encoding the chimeric antigen receptor, vectors comprising the nucleic acids, immune effector cells comprising the chimeric antigen receptor, the nucleic acid molecule and / or the vector, methods for preparing the immune effector cells, compositions comprising the immune effector cells, and uses of the chimeric antigen receptor.

[0005] The chimeric antigen receptor of the present disclosure includes at least one of the following inventive advantages: (1) Stable expression on the surface of immune cells (e.g., T cells) at high levels; (2) Strong ability to kill CD19-positive target cells; (3) the ability to promote immune cell cytokine secretion, e.g., the ability to enhance the ability of T cells to secrete cytokines (INF-γ or IL-6) at least once, twice, or three times; (4) non-hemolytic and unlikely to induce hemolysis or red blood cell agglutination; (5) It is not vaso-irritating and does not cause local or systemic abnormalities after administration. (6) It is non-carcinogenic and non-carcinogenic in vivo or in vitro; (7) the ability to extend the survival time of cancer patients; (8) the ability to effectively ameliorate the severity of cancer (e.g., adult acute lymphoblastic leukemia, childhood acute lymphoblastic leukemia and / or non-Hodgkin's lymphoma); and (9) The ability to demonstrate a lower risk of inducing side effects (e.g., cytokine release syndrome or CAR-T cell-associated encephalopathy syndrome) and a higher safety profile.

[0006] One embodiment of the present disclosure provides a chimeric antigen receptor comprising the amino acid sequence set forth in SEQ ID NO:1.

[0007] Another aspect of the present disclosure provides an isolated nucleic acid molecule encoding a chimeric antigen receptor described herein.

[0008] Another aspect of the present disclosure further provides an isolated nucleic acid molecule encoding said chimeric antigen receptor comprising the nucleic acid sequence set forth in SEQ ID NO:2.

[0009] Another aspect of the present disclosure further provides a vector comprising a nucleic acid molecule described herein.

[0010] Another aspect of the present disclosure further provides immune effector cells comprising the chimeric antigen receptors, nucleic acid molecules and / or vectors described herein.

[0011] In certain embodiments, said immune effector cells are selected from the group consisting of T lymphocytes and natural killer cells.

[0012] In certain embodiments, the chimeric antigen receptor is expressed on the surface of an immune effector cell.

[0013] Another aspect of the present disclosure further provides a method of preparing immune effector cells comprising transducing immune effector cells with a vector described herein.

[0014] In certain embodiments, said immune effector cells are selected from the group consisting of T lymphocytes and natural killer cells.

[0015] Another aspect of the present disclosure further provides a composition comprising the immune effector cells described herein.

[0016] Another aspect of the present disclosure further provides the use of said chimeric antigen receptor, nucleic acid molecule, vector and / or immune effector cell in the manufacture of a medicament useful for the treatment of a disease or disorder associated with CD19 expression.

[0017] Another embodiment of the present disclosure further provides a method of treating a disease or disorder associated with CD19 expression, comprising the step of applying said chimeric antigen receptor, nucleic acid molecule, vector and / or immune effector cell.

[0018] Another aspect of the present disclosure further provides chimeric antigen receptors, nucleic acid molecules, vectors and / or immune effector cells for use in treating a disease or disorder associated with CD19 expression.

[0019] In certain embodiments, said disease or disorder associated with CD19 expression comprises a non-solid tumor.

[0020] In certain embodiments, the non-solid tumor comprises a leukemia and / or a lymphoma.

[0021] In certain embodiments, said disease or disorder associated with CD19 expression comprises acute lymphoblastic leukemia and / or B-cell lymphoma.

[0022] In certain embodiments, said acute lymphoblastic leukemia comprises acute lymphoblastic leukemia in adults and / or acute lymphoblastic leukemia in children.

[0023] In certain embodiments, the medicament for treating acute lymphoblastic leukemia is 0.25×10 8 ~0.5×10 8 The dose of CAR-positive T cells will be administered.

[0024] In certain embodiments, the B-cell lymphoma comprises non-Hodgkin's lymphoma.

[0025] In certain embodiments, the medicament for treating non-Hodgkin's lymphoma is 1×10 8 ~2×10 8 It will be administered at a dose of CAR-positive T cells.

[0026] Those skilled in the art will readily appreciate other aspects and advantages of the present disclosure from the following detailed description. The following detailed description shows and describes only exemplary embodiments of the present disclosure. As those skilled in the art will recognize, the contents of the present disclosure will enable those skilled in the art to make modifications to the specific embodiments disclosed herein without departing from the inventive spirit and scope of the present disclosure. Similarly, the drawings and examples in the description of the present disclosure are illustrative only, not restrictive. [Brief explanation of the drawings]

[0027] Particular features included in the present disclosure are set forth in the appended claims. The features and advantages of the disclosure contained herein can be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings, a brief description of which follows: [Figure 1] Figures 1A and 1B show the results of detecting CAR molecules expressed on the surface of CNCT19 cells. [Figure 2] Figure 2 shows the survival rate of tumor cells under different co-culture conditions. [Figure 3] Figure 3 shows the killing of target cells (CHO-CD19) by CNCT19 cells, monitored in real time by a real-time cell analysis (RTCA) dual-purpose (DP) system. [Figure 4] Figure 4A shows the variation in INF-γ concentration in the supernatant under different co-culture conditions, and Figure 4B shows the variation in IL-6 concentration in the supernatant under different co-culture conditions. [Figure 5] Figure 5A shows the observation results for each test tube before shaking for 3 hours, and Figure 5B shows the observation results for each test tube after shaking for 3 hours. [Figure 6] Figure 6A is a photomicrograph (HE staining, 10x objective) of the local injection site after administration of CAR-T cells. Figure 6B is a photomicrograph (HE staining, 10x objective) of the local injection site after administration of sodium chloride injection. [Figure 7] FIG. 7 shows soft agar colony formation in each group 3 weeks after cell inoculation. [Figure 8] FIG. 8 shows the survival curves of Nalm-6 xenograft tumor NCG mice treated with different cells. [Figure 9] FIG. 9 shows the tissue distribution after a single administration of CNCT19 cells. [Figure 10] FIG. 10 is a comparison of CNCT19 cell distribution in vivo in tumor-bearing and non-tumor-bearing animals. [Figure 11] FIG. 11 shows the variability of CNCT19 cells distributed in different tissues of animals after a single administration. DETAILED DESCRIPTION OF THE INVENTION

[0028] The following specific examples illustrate certain embodiments of the present disclosure. Those skilled in the art will readily appreciate other advantages and benefits of the present disclosure from the content disclosed herein.

[0029] The present disclosure will be further explained below.According to the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings that are generally understood by those skilled in the art.In addition, the relevant terms and experimental procedures used herein in protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology and immunology are all terms and routine procedures that are widely used in the corresponding fields.Here, in order to better understand the present disclosure, the definitions and explanations of relevant terms are provided below.

[0030] As used herein, the term "chimeric antigen receptor" (CAR) generally refers to a fusion protein comprising an extracellular domain capable of binding to an antigen and at least one intracellular domain. CAR is the core component of chimeric antigen receptor T cells (CAR-T), which may include a targeting moiety (e.g., a moiety that binds to a tumor-associated antigen (TAA)), a hinge region, a transmembrane region, and an intracellular domain. In the present disclosure, CAR may be combined with a T cell receptor activating intracellular domain based on the antigen specificity of an antibody. CAR-expressing T cells can specifically recognize and eliminate malignant cells that express the target antigen.

[0031] As used herein, the term "isolated" generally refers to something obtained by artificial means from a natural state. When a "isolated" substance or component occurs in nature, it means that the natural environment in which it exists has been changed, or that it has been isolated from its natural environment, or both. For example, when a non-isolated polynucleotide or polypeptide naturally occurs in a living animal, the same polynucleotide or polypeptide, isolated in high purity from this natural state, is called isolated. The term "isolated" does not exclude the possibility of mixing with artificial or synthetic substances, nor does it exclude the presence of other impurities that do not impair the activity of the substance.

[0032] As used herein, the term "immune effector cell" generally refers to a cell that is involved in an immune response, such as, for example, a cell that promotes an immune effector response. In the present disclosure, the immune effector cell may be selected from the group consisting of a T lymphocyte and a natural killer cell.

[0033] As used herein, the term "specifically bind and / or specifically recognize" generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody (or CAR structural fragment), which may determine the presence of the target in the presence of a heterogeneous cellular population of molecules (including biomolecules). For example, an antibody (or CAR structural fragment) that specifically binds to a target (which may be an epitope) is an antibody (or CAR structural fragment) that binds to the target in a simpler manner and / or for a longer period of time with higher affinity and avidity compared to binding to other targets.

[0034] As used herein, the term "isolated nucleic acid molecule" generally refers to nucleotides of any length, deoxyribonucleotides or ribonucleotides, in their isolated form, or analogs thereof that have been isolated from their natural environment or artificially synthesized.

[0035] As used herein, a "vector" generally refers to a tool for delivering nucleic acids into which a polynucleotide encoding a protein can be inserted, thereby enabling expression of the protein. A vector can be transformed, transduced, or transfected into a host cell so that the genetic material contained therein can be expressed in the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes (such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs)), phages such as λ phage or M13 phage, and animal viruses. Animal viruses that can serve as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papillomavirus (such as SV40). A vector may contain various elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. A vector may also contain an origin of replication. Vectors may contain components that aid in cell entry, such as, but not limited to, a viral particle, liposome, or protein coat.

[0036] As used herein, "composition" generally refers to a composition suitable for administration to a patient. For example, the composition of the present disclosure may include the immune effector cells described herein. Furthermore, the composition may also include one or more suitable formulations of (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. Acceptable components of the composition are non-toxic to recipients at any dosage and concentration used. Compositions of the present disclosure include, but are not limited to, liquids, and frozen or lyophilized compositions.

[0037] As used herein, "CD19" generally refers to cluster of differentiation (CD) 19 protein, which is a cluster of antigenic determinants that can be detected on leukemia progenitor cells. The amino acid and nucleic acid sequences of human and mouse CD19 are listed in public databases (such as GenBank, UniProt, and Swiss-Prot). For example, the amino acid sequence of human CD19 can be accessed under UniProt / Swiss-Prot accession number P15391, and the nucleotide sequence encoding human CD19 can be accessed under accession number NM_001178098. According to the present disclosure, "CD19" may also include proteins with mutations (e.g., point mutations, fragments, insertions, deletions, and splice variants of full-length wild-type CD19).

[0038] As used herein, the term "subject" generally refers to a human or non-human animal, including, but not limited to, a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

[0039] As used herein, the term "comprising" generally refers to the inclusion of the explicitly specified features and does not exclude other elements.

[0040] As used herein, the term "about" generally refers to a variation of 0.5% to 10% greater or less than the stated value, for example, a variation of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% greater or less than the stated value.

[0041] Chimeric antigen receptors, nucleic acids, vectors, immune effector cells, and compositions One embodiment of the present disclosure provides a chimeric antigen receptor comprising the amino acid sequence set forth in SEQ ID NO: 1. The present disclosure further provides a chimeric antigen receptor comprising an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

[0042] In certain embodiments, the chimeric antigen receptors described herein can specifically bind to and / or recognize a tumor antigen, for example, the chimeric antigen receptors described herein can specifically bind to and / or recognize the CD19 antigen.

[0043] In certain embodiments, the chimeric antigen receptors described herein can stimulate immune effector cells to secrete cytokines. The immune effector cells may be selected from the group consisting of T lymphocytes and natural killer cells. The cytokines may be selected from the group consisting of IFN-γ and IL-6. The immune effector cells may be mammalian immune effector cells. The T lymphocytes may be mammalian T lymphocytes, and the natural killer cells may be mammalian natural killer cells. The T lymphocytes may be human T lymphocytes, and the natural killer cells may be human natural killer cells.

[0044] In certain embodiments, the chimeric antigen receptors described herein are non-hemolytic and non-vasoirritating.

[0045] In certain embodiments, the chimeric antigen receptors described herein are non-oncogenic in vitro.

[0046] In certain embodiments, the chimeric antigen receptors described herein are non-oncogenic in vivo.

[0047] In certain embodiments, the chimeric antigen receptors described herein can effectively treat tumors. The tumors may be CD19-positive tumors. For example, the chimeric antigen receptors described herein can effectively prolong the survival of patients with CD19-positive tumors. For example, the chimeric antigen receptors described herein can effectively prolong the survival of patients with non-solid tumors. For example, the chimeric antigen receptors described herein can effectively prolong the survival of patients with lymphoma and / or leukemia. As another example, the chimeric antigen receptors described herein can effectively prolong the survival of adult patients with acute lymphoblastic leukemia. As another example, the chimeric antigen receptors described herein can effectively prolong the survival of pediatric patients with acute lymphoblastic leukemia. As another example, the chimeric antigen receptors described herein can effectively prolong the survival of patients with B-cell lymphoma (e.g., non-Hodgkin's lymphoma).

[0048] In certain embodiments, the chimeric antigen receptors described herein can effectively treat acute lymphoblastic leukemia in adults.

[0049] In certain embodiments, the chimeric antigen receptors described herein can effectively treat acute lymphoblastic leukemia in children.

[0050] In certain embodiments, the chimeric antigen receptors described herein can effectively treat non-Hodgkin's lymphoma.

[0051] Another aspect of the present disclosure provides an isolated nucleic acid molecule encoding a chimeric antigen receptor described herein.

[0052] Another aspect of the present disclosure provides an isolated nucleic acid molecule encoding a chimeric antigen receptor comprising the nucleic acid sequence set forth in SEQ ID NO:2.

[0053] Another aspect of the present disclosure provides an isolated nucleic acid molecule encoding a chimeric antigen receptor, the nucleic acid molecule comprising a nucleic acid sequence similar to the sequence set forth in SEQ ID NO:2 and encoding said chimeric antigen receptor.

[0054] In certain embodiments, a nucleic acid sequence similar to the sequence set forth in SEQ ID NO:2 refers to a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:2.

[0055] In certain embodiments, a nucleic acid sequence similar to the sequence set forth in SEQ ID NO:2 means that the nucleic acid molecule can encode a chimeric antigen receptor, but differs from the nucleic acid sequence set forth in SEQ ID NO:2 due to a base wobble (degeneracy) at position 3 of the nucleic acid codon.

[0056] The present disclosure includes gene and protein variants (e.g., variants of the amino acid sequence set forth in SEQ ID NO: 1 or variants of the nucleic acid sequence set forth in SEQ ID NO: 2 as described herein) that retain one or more biological activities. Such protein or polypeptide variants include proteins or polypeptides that have been or can be modified using recombinant DNA technology so that the protein or polypeptide has modified or additional properties, for example, the variant confers enhanced stability in plasma or increased activity to the protein. The variants may differ from the reference sequence, for example, from a naturally occurring polynucleotide, protein, or peptide. At the nucleotide sequence level, naturally occurring and non-naturally occurring variant genes typically have at least about 50%, more typically at least about 70%, and even more typically at least about 80% identity (90% or greater identity) to the reference gene. At the amino acid sequence level, naturally occurring and non-naturally occurring variant proteins typically have at least about 70%, more typically at least about 80%, and even more typically at least about 90% or more identity to the reference protein, while allowing for a higher percentage of non-identity in non-conserved regions (e.g., percent identity less than 70%, e.g., less than 60%, less than 50%, or even less than 40%). In other embodiments, the sequence has at least 60%, 70%, 75% or more identity (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity) with the reference sequence. Procedures for introducing nucleotide and amino acid modifications into polynucleotides, proteins, or polypeptides are known to those skilled in the art (see, e.g., Sambrook et al. (1989)).

[0057] As used herein, the terms "identity," "homology," and their grammatical variants generally mean that two or more entities are identical when their sequences are "aligned." Thus, for example, if two polypeptides have identical sequences, they have identical amino acid sequences, at least within a reference region or portion. If two polynucleotides have identical sequences, they have identical polynucleotide sequences, at least within a reference region or portion. The identity can be the identity of a defined zone (region or domain) of a sequence. The "zone" or "region" of identity refers to the identical portion of two or more reference entities. Thus, if two protein or nucleic acid sequences are identical in one or more sequence zones or regions, they share identity in that region. An "aligned" sequence refers to a larger number of polynucleotides or proteins (amino acids), often containing complementary or additional bases or amino acids (gaps) compared to the reference sequence. The degree of identity (homology) between two sequences can be determined using computer programs and mathematical algorithms. Such algorithms that calculate percent sequence identity (homology) typically calculate sequence gaps and mismatches in the regions or zones being compared. For example, for the BLAST (e.g., BLAST 2.0) search algorithm (see, e.g., Altschule et al., J. Mol. Biol. 215:403 (1990) (publicly available from NCBI)), exemplary search parameters include mismatches -2, gap openings 5, and gap extensions 2.

[0058] According to the present disclosure, a nucleic acid molecule, in isolated form, can be any length of nucleotides, deoxyribonucleotides or ribonucleotides, or analogs thereof isolated from their natural environment or artificially synthesized, so long as it is capable of encoding a chimeric antigen receptor as described herein.

[0059] Another aspect of the present disclosure provides a vector comprising a nucleic acid molecule described herein.

[0060] According to the present disclosure, the vector can be used to transform, transduce, or transfect host cells so that the genetic material elements carried therein can be expressed in the host cells. For example, the vector may include a plasmid, a phagemid, a cosmid, an artificial chromosome (e.g., yeast artificial chromosome (YAC)), a bacterial artificial chromosome (BAC), or a P1-derived artificial chromosome (PAC)), a phage such as λ phage or M13 phage, and an animal virus. Animal viruses that can serve as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papillomavirus vacuolar viruses (e.g., SV40). For another example, the vector may contain various expression control elements, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. Additionally, the vector may contain an origin of replication. Additionally, the vector may contain components that aid in its entry into cells, such as, but not limited to, a viral particle, a liposome, or a protein coat.

[0061] Another aspect of the present disclosure provides immune effector cells comprising the chimeric antigen receptors, nucleic acid molecules and / or vectors described herein.

[0062] According to the present disclosure, the immune effector cells may be selected from the group consisting of T lymphocytes and natural killer cells. In certain embodiments, the immune effector cells may be human immune effector cells. For example, the immune effector cells may be human T lymphocytes. As another example, the immune effector cells may be human natural killer cells.

[0063] In accordance with the present disclosure, the chimeric antigen receptors described herein are expressed on the surface of immune effector cells.

[0064] In certain embodiments, the immune effector cells described herein can effectively kill tumor cells. The tumor cells may be CD19-positive cells. For example, the immune effector cells described herein can significantly reduce the survival rate of CD19-positive human leukemia cell line Nalm-6 cells.

[0065] In certain embodiments, the immune effector cells described herein can effectively promote cytokine secretion when contacted with CD19-positive cells. The cytokine may be selected from the group consisting of IFN-γ and IL-6. For example, co-culturing the immune effector cells described herein with CD19-positive human leukemia cell line Nalm-6 cells significantly increases the secretion of IFN-γ and IL-6 cytokines.

[0066] In certain embodiments, the immune effector cells described herein are non-hemolytic and do not induce vasculitis. For example, in an in vitro hemolysis test, the immune effector cells described herein do not induce hemolysis or hemagglutination. In another example, the immune effector cells described herein do not induce vasculitis.

[0067] In certain embodiments, the immune effector cells described herein are non-tumorigenic in vitro.

[0068] In certain embodiments, the immune effector cells described herein are non-tumorigenic in vivo.

[0069] In certain embodiments, the immune effector cells described herein can effectively treat tumors. The tumors can be CD19-positive tumors. For example, the immune effector cells described herein can effectively prolong the survival of patients with CD19-positive tumors. As another example, the immune effector cells described herein can effectively prolong the survival of adult patients with acute lymphoblastic leukemia. As another example, the immune effector cells described herein can effectively prolong the survival of pediatric patients with acute lymphoblastic leukemia. As another example, the immune effector cells described herein can effectively prolong the survival of patients with B-cell lymphoma (e.g., non-Hodgkin's lymphoma).

[0070] In certain embodiments, the immune effector cells described herein can effectively treat acute lymphoblastic leukemia in adults.

[0071] In certain embodiments, the immune effector cells described herein can effectively treat acute lymphoblastic leukemia in children.

[0072] In certain embodiments, the immune effector cells described herein can effectively treat B-cell lymphoma (such as non-Hodgkin's lymphoma).

[0073] Another aspect of the present disclosure provides a composition comprising the immune effector cells described herein.

[0074] According to the present disclosure, the composition may contain one or more suitable formulations of (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. Acceptable components of the composition are non-toxic to recipients at any dosage and concentration used. Compositions of the present disclosure include, but are not limited to, liquids, and frozen or lyophilized compositions.

[0075] In certain embodiments, the composition may be for parenteral, transdermal, intraluminal, intraarterial, intrathecal, and / or intranasal administration, or by direct injection into tissue. For example, the composition may be administered to a patient or subject by infusion or injection. In other embodiments, the composition may be administered by a variety of methods, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration. In other embodiments, the composition may be administered without interruption. Uninterrupted (or continuous) administration may be achieved by a miniature pump system worn by the patient to meter the influx of therapeutic agent into the patient's body, as described in WO2015 / 036583.

[0076] According to the present disclosure, the dosing regimen of the composition may be a rapid infusion, multiple divided doses over time, or a variable dose depending on the severity and urgency of the treatment situation. In certain embodiments, the treatment regimen may be administered once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months. In certain embodiments, the dosing regimen includes intravenous administration and the dose is 0.1 x 10 8 ~3×10 8 CAR-positive T cells, for example, 0.15 x 10 8 ~2×10 8 CAR-positive T cells, 0.5 x 10 8 ~2×10 8 CAR-positive T cells, 1 x 10 8 ~2×10 8 CAR-positive T cells, 0.2 x 10 8 ~2×10 8 CAR-positive T cells, 0.2 x 10 8 ~1×10 8 CAR-positive T cells, 0.25 x 10 8 ~1×10 8 CAR-positive T cells, 0.25 x 10 8 ~0.5×10 8 CAR-positive T cells, or 0.5 x 10 8 CAR-positive T cells, or 2 x 10 8 The CAR-positive T cells may be

[0077] Dosages may vary depending on the indication. In certain embodiments, immune effector cells for the treatment of adult patients with relapsed and refractory acute lymphoblastic leukemia are administered in doses of 0.25 x 10 8 ~0.5×10 8 CAR-positive T cells, or 0.5 x 10 8 For example, 0.3 x 10 CAR-positive T cells may be administered at a dose of 0.3 x 10 8 ~0.5×10 8 , 0.4×10 8 ~0.5×10 8 , 0.25×10 8 ~0.4×10 8 , 0.3×10 8 ~0.4×10 8 , or 0.4 × 10 8 ~0.5×10 8 In certain embodiments, immune effector cells for the treatment of adult patients with relapsed and refractory acute lymphoblastic leukemia may be administered at a dose of 0.25 x 10 CAR-positive T cells. 8 , 0.26×10 8 , 0.27×10 8 , 0.28×10 8 , 0.29×10 8 , 0.3×10 8 , 0.31×10 8 , 0.32 × 10 8 , 0.33×10 8 , 0.34×10 8 , 0.35×10 8 , 0.36×10 8 , 0.37×10 8 , 0.38×10 8 , 0.39×10 8 , 0.4×10 8 , 0.41×10 8 , 0.42 × 10 8 , 0.43×10 8 , 0.44×10 8 , 0.45×10 8 , 0.46×10 8 , 0.47×10 8 , 0.48×10 8, 0.49×10 8 , or 0.5 × 10 8 The CAR-positive T cells may be administered at a dose of

[0078] In certain embodiments, immune effector cells for the treatment of pediatric patients with relapsed and refractory acute lymphoblastic leukemia are administered in amounts of 0.25 x 10 8 ~0.5×10 8 CAR-positive T cells, or 0.5 x 10 8 For example, 0.3 x 10 CAR-positive T cells may be administered at a dose of 0.3 x 10 8 ~0.5×10 8 , 0.4×10 8 ~0.5×10 8 , 0.25×10 8 ~0.4×10 8 , 0.3×10 8 ~0.4×10 8 , or 0.4 × 10 8 ~0.5×10 8 In some embodiments, immune effector cells for the treatment of pediatric patients with relapsed and refractory acute lymphoblastic leukemia may be administered at a dose of 0.25 x 10 CAR-positive T cells. 8 , 0.26×10 8 , 0.27×10 8 , 0.28×10 8 , 0.29×10 8 , 0.3×10 8 , 0.31×10 8 , 0.32 × 10 8 , 0.33×10 8 , 0.34×10 8 , 0.35×10 8 , 0.36×10 8 , 0.37×10 8 , 0.38×10 8 , 0.39×10 8 , 0.4×10 8 , 0.41×10 8 , 0.42 × 10 8 , 0.43×10 8 , 0.44×10 8 , 0.45×10 8 , 0.46×10 8, 0.47×10 8 , 0.48×10 8 , 0.49×10 8 , or 0.5 × 10 8 The CAR-positive T cells may be administered at a dose of

[0079] In another embodiment, the immune effector cells for the treatment of patients with relapsed and refractory non-Hodgkin's lymphoma are 1 x 10 8 ~2×10 8 CAR-positive T cells, or 2 x 10 8 For example, 1 x 10 CAR-positive T cells may be administered at a dose of 1 x 10 8 ~1.8×10 8 , 1×10 8 ~1.5×10 8 , 1×10 8 ~1.3×10 8 , 1.3 × 10 8 ~2×10 8 , 1.3 × 10 8 ~1.5×10 8 , 1.5×10 8 ~2×10 8 , 1.5×10 8 ~1.8×10 8 , or 1.8 × 10 8 ~2×10 8 In other embodiments, immune effector cells for the treatment of relapsed and refractory Hodgkin lymphoma patients may be administered at a dose of 1 x 10 CAR-positive T cells. 8 , 1.1×10 8 , 1.2 × 10 8 , 1.3 × 10 8 , 1.4×10 8 , 1.5×10 8 , 1.6×10 8 , 1.7×10 8 , 1.8×10 8 , 1.9×10 8 or 2.0 x 10 8 The CAR-positive T cells may be administered at a dose of

[0080] [Preparation method and use] Another aspect of the present disclosure further provides a method of preparing immune effector cells comprising transducing immune effector cells with a vector described herein.

[0081] In certain embodiments, said immune effector cells are selected from the group consisting of T lymphocytes and natural killer cells.

[0082] Another aspect of the present disclosure further provides use of a chimeric antigen receptor, a nucleic acid molecule, a vector, and / or an immune effector cell in the manufacture of a medicament useful for treating a disease or disorder associated with CD19 expression, the dosage of which can refer to the doses defined for immune effector cells as described above.

[0083] Another aspect of the present disclosure further provides a method of treating a disease or disorder associated with CD19 expression, comprising administering to a subject in need thereof a chimeric antigen receptor, nucleic acid molecule, vector, and / or immune effector cell described herein. According to the present disclosure, administration of the composition may be by various methods, for example, intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration.

[0084] In another embodiment, the chimeric antigen receptors, nucleic acid molecules, vectors and / or immune effector cells described herein are useful for treating a disease or disorder associated with CD19 expression.

[0085] According to the present disclosure, the medicament may comprise a T cell immunotherapeutic agent.

[0086] According to the present disclosure, the disease or disorder associated with CD19 expression may include non-solid tumors.

[0087] According to the present disclosure, the disease or disorder associated with CD19 expression may include leukemia and / or lymphoma.

[0088] In certain embodiments, said disease or disorder associated with CD19 expression may include acute lymphoblastic leukemia (ALL), such as adult acute lymphoblastic leukemia (ALL) and / or childhood acute lymphoblastic leukemia (ALL).

[0089] In another embodiment, the disease or disorder associated with CD19 expression may include adult chronic lymphocytic leukemia (CLL). In another embodiment, the disease or disorder associated with CD19 expression may include B-cell lymphoma. For example, B-cell lymphoma may include non-Hodgkin's lymphoma.

[0090] According to the present disclosure, the subject may include a human or a non-human animal, for example, but not limited to, a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

[0091] Without wishing to be bound by any theory, the following examples merely illustrate the chimeric antigen receptors, immune effector cells, preparation methods, and uses of the present disclosure and are not intended to limit the scope of the invention. The examples do not include detailed descriptions of traditional methods, such as how to construct vectors and plasmids, how to insert protein-encoding genes into such vectors and plasmids, or how to introduce plasmids into host cells. Such methods are well known to those skilled in the art and are described in many publications, including Sambrook, J., Fritsch, EF, and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.

[0092] All patents, applications, and references cited in this disclosure are incorporated herein by reference in their entirety to the same extent as if each were individually incorporated by reference. In the event that any material incorporated by reference contradicts or is inconsistent with the present specification, the present specification will control over such material.

[0093] [Example] [Example 1] Construction of lentiviral vector A fragment containing the CAR structure described in this disclosure (its amino acid sequence and nucleotide sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively) was artificially synthesized and constructed into a lentiviral vector (manufacturer: SBI Corporation, catalog number: CD500-CD800). The resulting expression vector was designated expression vector CNCT19 and transfected according to the instructions to obtain a lentivirus. The viral transfection titer was measured by flow cytometry to confirm that a functional lentiviral vector had been obtained.

[0094] [Example 2] Preparation of T cells infected with lentiviral vectors Infection experiments were performed according to conventional methods known to those skilled in the art. The infection steps are briefly described below.

[0095] 1. T cell selection Peripheral blood mononuclear cells (PBMCs) were isolated from the subjects' apheresis cells, and then T cells were selected from the PBMCs.

[0096] 2. T cell activation Culture the isolated T cells in complete lymphocyte culture medium (Xvivo15 medium + 5% FBS + 100 IU / mL IL-2 or Xvivo15 medium + 5% FBS + 20 ng / mL IL-21 + 10 ng / mL IL-7) at a concentration of (1–2) × 10 6 The cells were resuspended to a final concentration of 1000 cells / mL, and 5-10 μL of CD3 / CD28 stimulating magnetic beads were added. The mixture was mixed well and placed in a culture incubator at 37°C + 5% CO for at least 24 hours.

[0097] 3. Lentiviral Infection of T Cells The activated cultured T cells were removed and polybrene was added to a final concentration of 8 μg / mL and mixed thoroughly. The lentiviral vector was then slowly added at an MOI of 2. After thorough mixing, the mixture was placed in a centrifuge and centrifuged at 1500 rpm for 1.5 hours. The cells were then placed in a culture incubator at 37°C + 5% CO2 for at least 24 hours.

[0098] 4. Expansion of Infected T Cells Remove infected cells, monitor cell density, and adjust it to (0.5–1) × 10 6 cells / mL and used in subsequent examples. The resulting infected T cells were designated CNCT19 cells (i.e., immune effector cells as described herein).

[0099] [Example 3] Detection of CAR molecule expression on the surface of CNCT19 cells The experimental steps for this example are as follows: (1) Centrifuge the CNCT19 cell suspension at 300 g for 5 minutes, discard the supernatant, and add sheath fluid to obtain a viable cell density of (0.5–1) × 10. 7 The cells were resuspended to reach 100 cells / mL. (2) Two flow cytometry tubes were collected for each sample, labeled Tube 1 and Tube 2. Tube 1 was a blank control and did not require the addition of any antibody, while 10 μL of Alexa Fluor® 647-goat anti-mouse IgG F(ab')2 antibody (manufacturer: Jackson, catalog number: 115-605-072) diluted 10-fold was added to Tube 2. (3) 100 μL of cell suspension was added to each tube and incubated in the dark at room temperature for 15 to 20 minutes. (4) 2 mL of sheath washing fluid was added to each tube, and the tubes were centrifuged at 300 g for 5 minutes. (5) The supernatant was discarded, and 20 μL of FITC-CD3 (manufacturer: Tongsheng Shidai, catalog number: Z6410047-100T) was added to tube 2 and incubated in the dark at room temperature for 15 to 20 minutes. (6) 2 mL of sheath fluid for washing was added to each tube, followed by centrifugation at 300 g for 5 minutes. The supernatant was discarded, and another 2 mL of sheath fluid was added to each tube for washing, followed by centrifugation at 300 g for 5 minutes. (7) The supernatant was discarded, and 300 μL of sheath fluid was added to each tube to resuspend the cells, followed by detection by flow cytometry.

[0100] The results are shown in Figures 1A and 1B. As can be seen from the figures, Figure 1A represents the blank control tube, and its upper right CD3+CAR+ quadrant shows no cell population. Figure 1B represents the experimental detection tube. CART cells were labeled with IgG F(ab')2 and CD3 antibodies, and CD3 + In the CAR+ quadrant, a CAR expression rate of 68.6% was clearly detected by flow cytometry, demonstrating that the CAR molecules of the present disclosure are well expressed on the surface of CNCT19 cells.

[0101] [Example 4] Detection of the killing effect of CNCT19 cells on target cells in vitro The experimental steps for this example are as follows: 1) The CD19-positive human leukemia cell line Nalm-6 (purchased from Shanghai Enzyme Research Bioscience Co., Ltd., catalog number: CH179) and the CD19-negative human leukemia cell line KG-1a (purchased from Shanghai Enzyme Research Bioscience Co., Ltd., catalog number: CC-Y1305) were selected as tumor cells (i.e., target cells), respectively, and CD19CAR-T cells (i.e., CNCT19 cells obtained in Example 2) and non-transfected T cells (indicated as NTD) were selected as effector cells, respectively. 2) The target cells and effector cells were mixed at an effector / target ratio of 1:2 and 2:1, respectively, and seeded into a 24-well plate. The total number of cells co-cultured in each well was approximately 1 × 10 6 Each well was supplemented with 1 mL of culture medium, and the plate was placed in an incubator at 37°C with 5% CO2 for incubation. The incubation time was recorded. 3) After 24 hours of co-culture, the cell suspension in each well was collected, transferred to a 1.5 mL EP tube, and labeled. After centrifugation, the supernatant from each sample tube was aspirated into a new 1.5 mL EP tube and frozen at -20°C for subsequent cytokine detection (see Example 6 for details). 4) Depending on the type of tumor cell, the corresponding amount of antibody was added to each well of the mixed cells for labeling, and the procedure was performed according to the antibody's instructions. Nalm-6 cells were labeled with PE-CD10 antibody, and KG-1a cells were labeled with Percp-cy5.5-CD45 antibody. 5) Flow cytometry was used to detect changes in the proportion of different target tumor cells in each sample.

[0102] The results are shown in Figure 2. As can be seen, coculture of CD19-positive tumor cells Nalm-6 with CAR-T cells (i.e., CNCT19 cells) significantly reduced the survival rate of Nalm-6 compared with coculture with non-transfected T cells (i.e., NTD). However, there was no significant difference in the survival rate of CD19-negative tumor cells KG-1a after coculture with various effector cells. Specifically, at an effector / target ratio of 1:2, co-incubation of CNCT19 cells with Nalm-6 cells resulted in a target cell survival rate of 2.8 ± 1.3%, which was significantly lower than the target cell survival rate (12.1 ± 1.2% (P < 0.01)) resulting from co-incubation of non-transfected T cells with Nalm-6 cells. Similarly, at an effector / target ratio of 2:1, co-incubation of CNCT19 cells with Nalm-6 cells resulted in a target cell survival rate of 1.1 ± 0.1%, which was significantly lower than the target cell survival rate of 7.3 ± 1.2% (P < 0.01) resulting from co-incubation of untransfected T cells with Nalm-6 cells. Furthermore, the killing effect of CNCT19 cells against CD19-positive tumor cells increased with increasing effector / target ratio.

[0103] [Example 5] Real-time monitoring of the death function of CNCT19 cells The experimental steps for this example are as follows: (1) The target cells, CHO-CD19, were removed, the culture medium in the culture flask was aspirated and discarded, the culture flask was washed once with saline, 1 mL of EDTA-containing trypsin solution was added, and the cells were incubated in a 37°C incubator for 2-6 minutes before digestion was stopped. The CHO cells were purchased from Shanghai Enzyme Research Bioscience Co., Ltd. under catalog number CC-Y2110. The molecular sequence of the CD19 cells was obtained from NCBI. The CD19 molecular sequence was constructed in CHO cells using molecular biology techniques, and the target cell CHO-CD19 cell line was obtained by screening. (2) An appropriate amount of culture medium was added to a culture flask to prepare target cells from the cell suspension, and the cells were homogenized by pipetting. After counting the concentration of the cell suspension with a counting plate, the cell suspension was diluted to 1 × 10 required for the experiment. 5 The cell concentration was adjusted to cells / mL. (3) 50 μL of culture medium was added to the wells of the E-Plate 16 of the RTCA DP system. The E-Plate 16 was placed on the RTCA station. The RTCA system automatically scanned ("Scan Plate") to check whether the contact was good ("Connection OK" was displayed on the "Message" page). Baseline (background) detection was initiated to confirm that the selected wells were in normal contact. (4) Remove E-Plate 16 and add 100 μL of well-mixed target cell suspension to each well at a concentration of 1 × 10 4 Cells were added at 1000 x g / well. The E-Plate 16 was placed in a super clean bench at room temperature for 30 minutes, and then placed on the RTCA station in an incubator. After the system automatically scanned ("Scan Plate"), step 2 was initiated to dynamically detect the cell growth curve in real time. (5) The E-Plate 16 was removed, and the target cell suspension and CNCT19 cell suspension were added to some wells, and the target cell suspension and non-transfected T cell (i.e., NTD) suspension were added to other wells (as a control). The effector / target ratio (i.e., the ratio of effector cells to target cells, i.e., CNCT19 cells:target cells and NTD:target cells) was 1:1, and the volume of the target cell suspension was 50 μL. The E-Plate 16 detection plate was placed on the RTCA DP detection platform for 60 hours of real-time monitoring to observe the effect of CNCT19 cells on the target cells.

[0104] The results are shown in Figure 3. In the figure, line 1 represents the curve for the NTD control group, and line 2 represents the curve for the CNCT19-treated group. Comparing line 1 and line 2, it can be seen that the proliferation of tumor cells (i.e., target cells) was inhibited by CNCT19 cells over time. Specifically, the target cells after co-incubation with non-transfected T cells showed no significant change in proliferation tendency (line 1). On the other hand, the target cells after co-incubation with CNCT19 cells showed a significant tendency toward decreased proliferation and even began to show a decrease in their numbers (line 2). This indicates that CNCT19 has a strong killing ability against target cells.

[0105] [Example 6] Detection of secreted cytokines after co-culture of CNCT19 cells with target cells The experimental steps for this example are as follows: 1) The supernatant samples from the mixed cultures in each well of Example 4 (i.e., the samples obtained in step 3) were removed from the -20°C refrigerator and thawed at room temperature. 2) Each sample was processed using the LEGENDplex™ kit (manufacturer: Biolegend Co., Ltd., catalog number: 740013) according to the instructions. 3) The levels of different cytokines in each sample were detected by flow cytometry.

[0106] The results are shown in Figures 4A and 4B. Compared to non-transfected T cells (i.e., NTD), CAR-T cells (i.e., CNCT19 cells) and Nalm-6 cells (CD19 +Coculture with CNCT19 cells significantly increased the secretion of the cytokines IFN-γ and IL-6 by CAR-T cells. Specifically, after 24 hours of coculture with target cells at an effector / target ratio of 2:1, CNCT19 cells were stimulated by the target cells to secrete IFN-γ at a level of 6186.37 ± 861.13 pg / mL, which was significantly higher than the level of IFN-γ secreted by non-transfected T cells (2096.85 ± 228.16 pg / mL, P < 0.05). The level of IL-6 secreted by CNCT19 cells (32.22 ± 1.46 pg / mL) was significantly higher than that secreted by non-transfected T cells (12.23 ± 4.37 pg / mL, P < 0.05).

[0107] [Example 7] Detection of hemolysis and stimulation of CNCT19 cells 7.1. In vitro hemolysis test of CNCT19 cells The experimental steps for this example are as follows: 1) A total of seven glass test tubes were used, numbered 1 to 7, and 2.5 mL of 2% rabbit red blood cell suspension (collected from New Zealand rabbits and manufactured by the State Food and Drug Administration with quality approval number: 11400500032425) was added to each test tube. 2) Different doses (0.5–0.1 mL) of CNCT19 cells were added to 1 × 10 7 A concentration of 1000 cells / mL (based on the total number of T cells) was added to test tubes 1–5, which already contained different doses (2.0–2.4 mL) of sodium chloride injection. Meanwhile, 2.5 mL of sodium chloride injection (negative control) and 2.5 mL of sterile water for injection (positive control) were added to test tubes 6 and 7, respectively. 3) The total volume of each test tube was 5.0 mL. The test tubes were placed in an incubator at 37°C ± 0.5°C and incubated for 3 hours. After placing the test tubes in the incubator, they were observed for lysis or agglutination of red blood cells 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, and 3 hours later.

[0108] The results are shown in Figures 5A and 5B. Figure 5A shows the results of each test tube before shaking for 3 hours, and Figure 5B shows the results of each test tube after shaking for 3 hours. In the negative control tube (No. 6), the supernatant was clear and colorless, and the red blood cells settled to the bottom of the tube. After shaking, they were uniformly dispersed, indicating no hemolysis or agglutination. In the positive control tube (No. 7), the solution was clear and red, with no separated layer and no red blood cell residue at the bottom of the tube, indicating complete hemolysis. After 3 hours of observation of each test tube containing different doses of CNCT19 cells, the supernatant in each test tube was clear and colorless, and the red blood cells settled to the bottom of the tube. After shaking, they were uniformly dispersed, indicating no hemolysis or agglutination.

[0109] 7.2. Vascular irritation test by intravenous injection of CNCT19 cells The experimental steps for this example are as follows: 1) Six New Zealand rabbits (three males and three females) that passed quarantine inspection and had no abnormalities at the injection site were selected. Using the autologous control method, cryopreserved CAR-T cells (i.e., CNCT19 cells) and a negative control (sodium chloride injection) were intravenously injected into the right and left ears of each animal, respectively. 2) CAR-T cells were infused via the right marginal ear vein at a concentration of 1 × 10 7 cells / mL, 1 x 10 7 The dose was 1 mL / kg (based on the total number of T cells). Sodium chloride injection administered via the left marginal ear vein served as a negative control. The intravenous dose was 1 mL / kg in both the left and right marginal ear veins. 3) After intravenous administration, general observation, observation of the injection site, and pathological examination of the animals were performed.

[0110] The results are shown in Figures 6A and 6B. Figure 6A shows a micrograph (HE staining, 10x objective lens) of the injection site after CAR-T cell administration, and Figure 6B shows a micrograph (HE staining, 10x objective lens) of the injection site after sodium chloride injection. It can be seen that intravenous infusion of cryopreserved CAR-T cells did not result in any systemic or local symptoms or pathological abnormalities in the animals compared to the negative control side.

[0111] [Example 8] In vitro carcinogenicity experiment of CNCT19 cells The experimental steps for this example are as follows: 1) CAR-T cells (i.e., CNCT19 cells) from two donors were incubated in soft agar medium. The two donors were Donor 1 (healthy human donor T cells, lot number: TC20180613015) and Donor 2 (healthy human donor T cells, lot number: TC20180613016). The human embryonic lung fibroblast cell line MRC-5 was used as a negative control, and the human cervical cancer cell line HeLa was used as a positive control. 2) A 6-well culture plate was used for the experiment. The number of cells in each well was approximately 1 × 10 3 Each group had three parallel wells. The culture plates were placed in a CO2 incubator and observed for 3 weeks. 3) The culture plates were removed every week, and the presence or absence of clone formation was examined under a microscope and photographed. The experiment was stopped until clear clones were formed in the positive control group.

[0112] The results are shown in Figure 7. It can be seen that the positive control cells (Hela) formed clones in the culture medium, and the size of the clones significantly increased over time. On day 23 of observation, CAR-T cells derived from different donors and the negative control cells (MRC-5) did not show clonal expansion; they all died and were not characterized by immortalized growth in vitro.

[0113] [Example 9] In vivo tumorigenicity experiment of CNCT19 cells The experimental steps for this example are as follows: 1) BALB / c nude mice were subcutaneously inoculated with cryopreserved CAR-T cells (i.e., CNCT19 cells) and corresponding non-transfected T cells (i.e., NTD) from two donors: Donor 2 (healthy human donor T cells, lot number: TC20180613016) and Donor 3 (healthy human donor T cells, lot number: TC20180808019). 2) CAR-T cell inoculation was 1 × 10 7 The negative control group received 1 × 10 cells / mouse (based on the total number of T cells). 7 The positive control group was inoculated with 1 × 10 human embryonic lung fibroblast cell line MRC-5 cells per mouse. 6 The mice were inoculated with human cervical cancer cell line HeLa cells. 3) Continuous observation was carried out for 16 weeks to detect changes in body weight, the occurrence of nodules, and whether the nodules were induced to become tumor nodules. The results were compared between the negative and positive cell groups. 4) After observation, gross dissection was performed. Each organ was weighed and the organ index was calculated. The inoculation site and tissues or organs suspected of symptoms were subjected to histopathological examination.

[0114] The results demonstrate that CAR-T cells are not tumorigenic in vivo. In the negative control group (inoculated with MRC-5 cells), all liquid nodules disappeared by day 9 after inoculation. In the positive control group (inoculated with HeLa cells), subcutaneous nodules slowly increased in all patients. Pathological examination revealed that these nodules were due to tumor tissue proliferation, resulting in a 100% tumor formation rate. In summary, this experiment was valid. In the group inoculated with CAR-T cells (derived from two donors) and untransfected T cells, all liquid nodules disappeared by day 5 after inoculation, and no nodules reappeared before euthanasia on day 114. Pathological examination demonstrated no tumor formation at the inoculation site or metastatic sites.

[0115] [Example 10] Toxicity test of single intravenous injection of CNCT19 cells into Nalm-6 xenograft tumor-bearing NCG mice The experimental steps for this example are as follows: 1) Six to eight-week-old NCG mice (half of which were male) were used. Three days before administration, 2.5 × 10 Nalm-6 cell suspensions were injected into the tail vein. 6 cells / mL and at a dose of 10 mL / kg. 2) The screened animals were randomly divided into four gender-balanced groups (i.e., Groups 2 to 5) according to their body weight. Groups 2 to 5 were the vehicle control group, T cell control group, low-dose CAR-T cell group, and high-dose CAR-T cell group, with 40 animals (20 males and 20 females) in each group. Both the non-tumor-bearing control group (i.e., Group 1) and the vehicle control group (i.e., Group 2) received the vehicle control (i.e., saline containing 4% (W / V) human albumin). The T cell control group (i.e., Group 3) received 1 × 10 non-transfected T cells (i.e., NTD). 9 The low-dose CAR-T cell group (Group 4) received 1 × 10 cells / kg (based on the total number of T cells, same below). 8 cells / kg, and 1 × 10 9 cells / kg were inoculated. 3) The dose for each animal was 25 mL / kg, administered as a single intravenous injection at an administration rate of approximately 1 mL / min. 4) After administration, observation continued for four hours on the day of administration. During the study, general clinical observations were conducted once daily in the morning and once daily in the afternoon. Detailed clinical observations and measurements of body weight and food intake were conducted once a week. During the study, body temperature, clinical pathological indicators (blood cell counts and blood biochemistry indicators), and immunological indicators (T lymphocyte subsets, cytokines, and C-reactive protein) were detected. 5) In groups 1 to 5, 10 animals / sex / group were euthanized on day 2, and 5 animals / sex / group were euthanized on day 15. Their organs were weighed and gross anatomical observations were performed. The major organs of animals in groups 1 and 5 were subjected to histopathological examination.

[0116] The results of this study showed that the maximum tolerated dose of CAR-T cells (i.e., CNCT19 cells) for a single administration was 1 × 10 9 This indicates that the number of cells / kg was greater than that of the control group. During the study, no animals died or became moribund in either the low-dose or high-dose CAR-T cell groups. No abnormal reactions were observed in either the general condition or detailed clinical findings. No obvious abnormal changes were observed in body weight, food consumption, body temperature, C-reactive protein, or blood biochemical indices. Histopathological examination revealed no obvious abnormalities in the low-dose and high-dose CAR-T cell groups compared to the T-cell control group.

[0117] [Example 11] Treatment of animals with CNCT19 cells 11.1. Therapeutic effect of CNCT19 cells on Nalm-6 xenograft tumors in NCG mice The experimental steps for this example are as follows: 1) Six to eight-week-old female NCG mice were used. Three days before administration, 5 × 10 5 of Nalm-6 cells were injected into the tail vein, whereas 2.5 × 10 Nalm-6 cells 6 cells / mL in saline, and each mouse was injected with 200 μL of the cell resuspension. 2) Each experimental group was injected with the corresponding CAR-T cells (i.e., CNCT19 cells), non-transfected T cells (i.e., NTD), or cell storage solution (i.e., saline containing 4% (w / v) human albumin). A total of five groups were divided as follows: a CAR-T low-dose group (5 × 10 based on the total number of T cells); 6 cells / mouse injected with CNCT19 cells, and so on), CAR-T medium dose group (1 × 10 7 cells / mouse injected with CNCT19 cells), CAR-T high-dose group (2 × 10 7cells / mouse injected with CNCT19 cells), T cell control group (2 × 10 7 cells / mouse injected with non-transfected T cells), and a vehicle control group (injected with cell preservative solution at 200 μL / mouse). The injection volume per mouse was 200 μL. 3) Body weight was measured and general conditions were observed twice a week. Mice survival was recorded and survival curves were plotted.

[0118] The results are shown in Figure 8. As can be seen, CNCT19 cells significantly prolonged survival at all doses, and these effects were clearly dose-dependent. Specifically, the median survival time for the saline control group was 24 days, the median survival time for the NTD control group was 23 days, and the median survival time for the low-dose CNCT19 group was 40 days, while all experimental animals in the medium-dose and high-dose CNCT19 groups survived to the end of the observation period. Compared with the saline and NTD control groups, all CNCT19 dose groups were able to extend the survival time of leukemia animals by more than 16 days.

[0119] 11.2. Distribution of CNCT19 cells in animals The experimental steps for this example are as follows: 1) 6-8 week old NCG mice (half of which were male) were used. Nalm-6 xenograft tumor models were established according to the method described in Example 11.1. 2) 5 × 10 6 A single tail vein injection of CAR-T cells (i.e., CNCT19 cells) was performed at a dose of 100 cells / mouse (based on the total number of T cells). 3) Animals in the tumor-bearing group were euthanized at 24 hours (i.e., D2), 72 hours (i.e., D4), 168 hours (i.e., D8), 336 hours (i.e., D15), 504 hours (i.e., D22), and 672 hours (i.e., D29) after administration, as scheduled. Animals in the non-tumor-bearing group were euthanized at 24 hours (i.e., D2), 168 hours (i.e., D8), and 336 hours (i.e., D15) after administration, as scheduled. Animal whole blood (EDTA anticoagulation), brain, spinal cord (cervical segments), skeletal muscle, gonads (ovaries, testes, and epididymis), bladder, stomach, small intestine, mesenteric lymph nodes, bone marrow, liver, kidney, spleen, heart, lung, and other tissues or fluids were collected in this order. 4) The content of chimeric antigen receptor (i.e., CAR) in blood and various tissue samples was determined using a validated Q-PCR method.

[0120] From these results, 5 × 10 6 After administration to tumor-bearing and non-tumor-bearing mice via a single intravenous injection at a dose of 1000 cells / mouse, CNCT19 cells were primarily distributed in whole blood and tissues with large blood flows, such as the lungs, liver, heart, and spleen (see Figure 9). Among these, the heart and whole blood had the largest distribution, with an area under the curve (the number of copies of the nucleic acid molecule encoding the CAR in gDNA time, AUC) of approximately 150,000 h*copies / μg. Following lung and spinal cord tracking, the AUC was approximately 40,000–60,000 h*copies / μg. For the spleen, liver, and other tissues, the AUC was less than approximately 20,000 h*copies / μg.

[0121] These results also showed that the CNCT19 cell content in the tissues of tumor-bearing mice was slightly higher than that of non-tumor-bearing mice (see Figure 10). The CNCT19 cell concentrations in the whole blood, heart, and spinal cord of tumor-bearing mice were approximately 3-fold, 6-fold, and 5-fold higher than those of non-tumor-bearing mice 24 hours after administration.

[0122] Subsequently, the drug content in each tissue gradually decreased, and was essentially below the methodological detection limit 2 weeks after administration. As the disease progressed, CNCT19 cells in the mice proliferated in response to increased CD19 antigen stimulation (see Figure 11), and the concentrations of CNCT19 cells in the brain, lung, liver, whole blood, and spinal cord increased to more than 1200 copies / μg DNA, reaching 3500 copies / μg DNA.

[0123] [Example 12] Treatment of acute lymphoblastic leukemia with CNCT19 cells 12.1. Clinical Use of CNCT19 Cells The clinical application process is shown in Table 1, and specific experimental steps at several stages are described below.

[0124] [Table 1] TIFF0007734771000001.tif135170

[0125] 1. Lymph Node Dissection Preparation Lymph node dissection preparation is performed on day -5 before the CNCT19 cell suspension is re-injected as planned.

[0126] The pretreatment scheme is as follows: Fludarabine 30 mg / m 2 once daily for 2 to 4 consecutive days, and cyclophosphamide 500 mg / m 2 The two drugs in the conditioning chemotherapy should be administered on the same day.

[0127] 2. Re-injection of CNCT19 Cell Suspension (1) After successful cell preparation, the cells are cryopreserved at <-100°C, transported to the hospital under the same temperature conditions for use, and collected according to the experimental operating guide before infusion. (2) The method for cell reinfusion is as follows: Collect cells according to the experimental operation guide, and reinfusion of the cell suspension should be completed within 30 minutes of collection. The cell suspension is infused into the subject as a single infusion through a vein using a blood transfusion device. If there are two or more bags of cell suspension, they can be infused continuously without any time interval between the reinfusion of each bag. During cell reinfusion, the subject must be closely observed. If a serious adverse event occurs, the infusion should be discontinued and corresponding treatment should be administered according to the specific adverse event. If no serious adverse event occurs, follow-up observation can be performed according to the clinic workflow. (3) Subjects should be closely monitored for 24 hours after cell reinfusion. If serious adverse events occur, corresponding treatment should be administered according to the specific adverse event. If no serious adverse events occur, follow-up observation can be performed according to the clinic workflow. (4) After cell reinfusion, subjects will be required to remain hospitalized for observation for 14 days or for a period determined by the investigator after a comprehensive evaluation of the subject's condition.

[0128] 3. CNCT19 Cell Suspension Management To ensure strict management and use of the CNCT19 cell suspension, a strict system has been established for the management of the CNCT19 cell suspension by specially assigned personnel. Specific personnel are assigned to transport the research cell suspension to hospital departments, and a system for receiving and registering the research cell suspension has been established. After infusion, the cell suspension packaging is collected by pharmaceutical control personnel and stored / destroyed.

[0129] 4. Clinical efficacy and safety results of CNCT19 cell suspension in the treatment of relapsed or refractory acute lymphoblastic leukemia The study was conducted from September 2016 to October 2020. During the exploratory clinical trial and phase I clinical trial, 63 patients (23 adults and 40 pediatric patients) with relapsed or refractory acute lymphoblastic leukemia (ALL) were treated with CNCT. The exploratory dose administered to adult ALL patients was 0.25 × 10 8~0.5×10 8 The range of CAR-positive T cells was

[0130] (a) Clinical efficacy data are shown in Tables 2 and 3. Table 2 shows the recovery status of 63 cases, including adults and children. Table 3 shows the recovery status of 23 adult patients. Of the 63 patients with relapsed or refractory acute lymphoblastic leukemia, after injection and reinfusion of CNCT19 cell suspension, the majority of patients (93.7%) achieved complete remission, and 88.9% of patients were MRD-negative. These results indicate that injection and reinfusion of CNCT19 cell suspension can effectively treat adult and pediatric patients with relapsed or refractory acute lymphoblastic leukemia.

[0131] [Table 2] TIFF0007734771000002.tif49170

[0132] [Table 3] TIFF0007734771000003.tif52170 (Note) In Table 2 and Table 3, ORR represents the overall response rate; MRD (minimal residual disease) negative means that no tumor cells are detected (by the most sensitive method); CR stands for complete response; Cri indicates complete morphologic remission with incomplete recovery of blood counts.

[0133] b) Preliminary safety data are shown in Tables 4 and 5. Table 4 shows the safety results for 63 patients, including adult and pediatric patients, and Table 5 shows the safety results for 23 adult patients. Among the 63 ALL patients, the incidence of grade 3 or higher CRS and encephalopathy was 19% and 20.6%, respectively. Comparison between age groups revealed that the incidence of severe CRS in the adult group (39.1%) was higher than that in the pediatric group (7.5%), and the incidence of severe CRS in the adult group (17.4%) was slightly lower or similar to that in the pediatric group (22.6%). This is likely due to the fact that CNCT19 was administered by weight in the initial study, with adult patients receiving higher doses with larger weight portions. For example, the dose given to 46 patients in the initial study was 0.71 × 10 6 ~4.08×10 6 / kg, which is 0.16 × 10 8 ~2.36×10 8 As the initial dose exploration gradually uncovered the characteristics of the CNCT19 product, a safer dose range was selected for subsequent clinical trials, e.g., 0.2 × 10 8 ~1.1×10 8 of CAR-positive T cells (median: 0.5 × 10 8 Among 17 patients receiving the 500mg dose, the incidence of grade ≥3 CRS and CRES decreased to 5.9% (1 / 17).

[0134] Injection of CNCT19 cell suspension is unlikely to cause severe CRS or CRES side effects, and its overall safety is controllable, demonstrating that CNCT19 has a good safety profile. The good safety profile of CNCT19 improves product quality and reduces clinical risks.

[0135] [Table 4] TIFF0007734771000004.tif51170

[0136] [Table 5] TIFF0007734771000005.tif44170Note: In Tables 4 and 5, CRS stands for cytokine release syndrome, CRES stands for CAR-T cell-associated encephalopathy syndrome.

[0137] [Example 13] Treatment of relapsed or refractory non-Hodgkin's lymphoma using CNCT19 cells 13.1. Clinical Use of CNCT19 Cells The clinical application process is shown in Table 6, and specific experimental steps at several stages are described below.

[0138] [Table 6] TIFF0007734771000006.tif103170

[0139] 1. Lymph Node Dissection Preparation Lymph node dissection preparation will be performed on day -5 before the CNCT19 cell suspension is re-injected as planned.

[0140] The pretreatment scheme is as follows: Fludarabine 30 mg / m 2 once daily for 2 to 4 consecutive days, and cyclophosphamide 500 mg / m 2 Administer once daily for two consecutive days.

[0141] The use of the two drugs in the conditioning chemotherapy should begin on the same day.

[0142] 2. Re-injection of CNCT19 Cell Suspension (1) After successful cell preparation, the cells are cryopreserved at <-100°C, transported to the hospital under the same temperature conditions for use, and collected according to the experimental operating guide before infusion. (2) The method for cell reinfusion is as follows: Collect cells according to the experimental operation guide, and reinfusion of the cell suspension should be completed within 30 minutes of collection. The cell suspension is infused as a single infusion into the subject through a vein using a blood transfusion device. If there are two or more bags of cell suspension, they can be infused continuously without any time interval between the reinfusion of each bag. During cell reinfusion, the subject must be closely observed. If a serious adverse event occurs, the infusion should be discontinued and corresponding treatment should be administered according to the specific adverse event. If no serious adverse event occurs, follow-up observation can be performed according to the clinic workflow. (3) Subjects should be closely monitored for 24 hours after cell reinfusion. If serious adverse events occur, corresponding treatment will be administered according to the specific adverse event. If no serious adverse events occur, follow-up observation can be performed according to the clinic workflow. (4) After cell reinfusion, subjects will be required to remain hospitalized for observation for 14 days or for a period determined by the investigator after a comprehensive evaluation of the subject's condition.

[0143] 3. CNCT19 Cell Suspension Management To ensure strict management and use of the CNCT19 cell suspension, a strict system has been established for managing the CNCT19 cell suspension by specially assigned personnel. Specific personnel have been assigned to transport the research cell suspension to hospital departments, and specific personnel have been assigned to establish a receiving and registration system for the research cell suspension.

[0144] After injection, the cell suspension packaging is collected by drug control personnel and stored / destroyed.

[0145] 4. Clinical efficacy and safety results of CNCT19 cell suspension in the treatment of relapsed or refractory non-Hodgkin's lymphoma This study was conducted from September 2016 to October 2020. During the exploratory clinical trial and phase I clinical trial, 50 patients with relapsed or refractory non-Hodgkin's lymphoma (NHL) were administered a CNCT19 cell suspension. The exploratory dose for NHL (adults only) was 1 × 10 8 ~2×10 8 The range of CAR-positive T cells was

[0146] (a) Clinical efficacy data are shown in Table 7. Table 7 shows the recovery of 50 patients. Of the 50 patients with relapsed or refractory non-Hodgkin's lymphoma, approximately 80% of the patients were in complete or partial remission after injection and reinfusion of CNCT19 cell suspension. In this case, the complete remission rate was 54% (27 cases) and the partial remission rate was 24% (12 cases). This indicates that CNCT19 cell suspension can effectively treat patients with relapsed or refractory non-Hodgkin's lymphoma.

[0147] [Table 7] TIFF0007734771000007.tif56170Note: In Table 7, ORR represents the overall response rate; CR stands for complete response; PR stands for partial response.

[0148] b) Preliminary safety data are shown in Table 8. Table 8 shows the safety results for 50 patients. It can be seen that injection of CNCT19 cell suspension has a low probability of causing serious CRS or CRES side effects, with the probability of causing grade 3 or higher CRS being less than 10%, the probability of causing grade 3 or higher CRES being 0%, and the probability of causing grade 3 or higher CRES being 6%. Therefore, CNCT19 has a good safety profile.

[0149] [Table 8] TIFF0007734771000008.tif42170Note: In Table 8, CRS stands for cytokine release syndrome, CRES stands for CAR-T cell-associated encephalopathy syndrome.

[0150] The foregoing detailed description is provided by way of illustration and example, and is not intended to limit the scope of the appended claims. Various modifications to the embodiments recited in this disclosure will be apparent to those skilled in the art and are intended to fall within the scope of the appended claims and their equivalents.

Claims

1. 1. A composition suitable for administration to a patient comprising immune effector cells, comprising: The immune effector cells comprise one or more selected from the following (1), (2), and (3): The composition, wherein the composition is administered as an intravenous injection. (1) A chimeric antigen receptor comprising the amino acid sequence shown in SEQ ID NO: 1; (2) An isolated nucleic acid molecule encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO: 1; or (3) A vector comprising a nucleic acid molecule encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:

1.

2. 10. The composition of claim 1, wherein the composition further comprises one or more of a pharmaceutically effective carrier, stabilizer, excipient, diluent, solubilizer, surfactant, emulsifier and / or preservative.

3. The composition of claim 1 , wherein the composition is a cell suspension.

4. The composition of claim 1 , wherein the composition is a frozen or lyophilized composition.

5. The composition of claim 1 , wherein the administration is a single administration.

6. The composition is 0.25×10 ８ ~0.5 x 10 ８ The composition of claim 5, wherein the composition is administered at a dose ranging from 100 to 1000 CAR-positive T cells.

7. The composition is 1×10 ８ ~2 x 10 ８ The composition of claim 5, wherein the composition is administered at a dose ranging from 100 to 1000 CAR-positive T cells.

8. 2. The composition of claim 1, wherein the nucleic acid molecule encoding the chimeric antigen receptor comprises the nucleic acid sequence set forth in SEQ ID NO:

2.

9. The composition of claim 1 , wherein the vector is a lentiviral vector.

10. The composition of claim 1 , wherein the immune effector cells are selected from the group consisting of T lymphocytes and natural killer cells.

11. A composition according to any one of claims 1 to 10 for use in the treatment of a disease or disorder associated with CD19 expression.

12. The composition of claim 11 , wherein the disease or disorder associated with CD19 expression comprises a non-solid tumor.

13. The composition of claim 12 , wherein the non-solid tumor comprises a leukemia and / or a lymphoma.

14. The composition of claim 11 , wherein the disease or disorder associated with CD19 expression comprises acute lymphoblastic leukemia and / or B-cell lymphoma.

15. 15. The composition of claim 14, wherein the acute lymphoblastic leukemia comprises acute lymphoblastic leukemia in adults and / or acute lymphoblastic leukemia in children.

16. 15. The composition of claim 14, wherein the acute lymphoblastic leukemia comprises adult relapsed or refractory acute lymphoblastic leukemia and / or pediatric relapsed or refractory acute lymphoblastic leukemia.

17. 15. The composition of claim 14, wherein the B-cell lymphoma comprises non-Hodgkin's lymphoma.

18. 18. The composition of claim 17, wherein the non-Hodgkin's lymphoma comprises relapsed or refractory non-Hodgkin's lymphoma.

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