Therapeutic applications of chimeric antigen receptors targeting CD19

Abstract

Provides a chimeric antigen receptor targeting CD19 and its therapeutic and pharmaceutical uses in B-cell driven diseases such as cancer or autoimmune diseases, as well as methods for treating diseases such as cancer or autoimmune diseases.

Description

Therapeutic uses of chimeric antigen receptors targeting CD19

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority and related rights and interests of Chinese Patent Application No. 202311404971.8 filed on October 26, 2023 and Chinese Patent Application No. 202411406524.0 filed on October 9, 2024. The entire contents of the above Chinese patent applications are incorporated herein by reference. Technical Field

[0003] The present application relates generally to the fields of bioengineering and cell therapy, and more particularly to chimeric antigen receptors targeting CD19 and their therapeutic uses in tumors or autoimmune diseases. Background Art

[0004] B-cell malignancies are characterized by abnormal proliferation of B cells. Common B-cell malignancies include acute precursor B-lymphocytic leukemia (ALL), non-Hodgkin lymphoma (NHL), B-cell chronic lymphocytic leukemia (CLL), hairy cell leukemia, and acute common B-lymphocytic leukemia. In the search for new therapies for B-cell malignancies, CAR T therapy targeting the CD19 antigen on the surface of B-cell malignancies is a significant research advancement and has achieved significant clinical results.

[0005] Furthermore, B cell dysfunction may be highly correlated with the pathogenesis of autoimmune diseases. For example, systemic lupus erythematosus (SLE) is an autoimmune disease affecting multiple organs. Its primary pathogenesis lies in an autoimmune response caused by immune imbalance, with the most critical factor being the dysregulated activation of autoreactive B cells. CD19 is a characteristic surface antigen on B cells. Targeting CD19 and further exploring the function of B cells may provide new therapeutic strategies for autoimmune diseases.

[0006] There is a need to develop effective and safe chimeric antigen receptor-based cell products that specifically target CD19.

[0007] SUMMARY OF THE INVENTION

[0008] The present application provides immune cells comprising a chimeric antigen receptor targeting CD19, corresponding pharmaceutical compositions, and their use in treating B cell-driven related diseases, such as tumors or autoimmune diseases.

[0009] In a first aspect, the present application provides an immune effector cell, comprising a chimeric antigen receptor targeting CD19, wherein the chimeric antigen receptor comprises: an extracellular region comprising an antigen binding region that specifically binds to CD19, a transmembrane region connected to the extracellular region, and an intracellular domain connected to the transmembrane region, wherein the antigen binding region comprises a VH comprising HCDR1-3 and a VL comprising LCDR1-3, wherein:

[0010] (1) the HCDR1-3 have the sequences shown in SEQ ID NOs: 5-7, respectively, and the LCDR1-3 have the sequences shown in SEQ ID NOs: 8-10, respectively; or

[0011] (2) The HCDR1-3 each have at most 0-3 mutations compared with each corresponding CDR in (1) above, and / or the LCDR1-3 each have at most 0-3 mutations compared with each corresponding CDR in (1) above.

[0012] In a second aspect, the present application provides a pharmaceutical composition comprising the immune effector cells described in the first aspect and a pharmaceutically acceptable carrier.

[0013] In a third aspect, the present application provides the immune effector cell of the first aspect or the pharmaceutical composition of the second aspect, which is used to treat a disease, wherein the disease is a B cell-driven disease, preferably a CD19-positive B cell-driven disease.

[0014] In a fourth aspect, the present application provides the use of the immune effector cells described in the first aspect or the pharmaceutical composition described in the second aspect in the preparation of a drug for treating a disease, wherein the disease is a B cell-driven disease, preferably a CD19-positive B cell-driven disease.

[0015] In the fifth aspect, the present application provides a method for treating a disease, comprising administering an effective amount of the immune effector cells described in the first aspect or the pharmaceutical composition described in the second aspect to a subject in need; the disease is a B cell-driven disease, preferably a CD19-positive B cell-driven disease.

[0016] In some embodiments, the disease described in the third to fifth aspects is a tumor or cancer. In a preferred embodiment, the tumor or cancer is a CD19-related tumor or cancer. In a more preferred embodiment, the tumor or cancer is a B-cell malignancy, such as acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, multiple myeloma, plasmacytoma, monoclonal gammopathy of undetermined significance, Waldenstrom's macroglobulinemia (lymphoplasmacytic lymphoma), heavy chain disease, primary amyloidosis, post-transplant lymphoproliferative disorder, Hodgkin's lymphoma, MALT lymphoma, B-cell lymphoma, mantle cell lymphoma, (germinal center-like) diffuse large cell lymphoma, Burkitt's lymphoma, bi-lineage leukemia, biphenotypic leukemia, hairy cell leukemia, precursor B acute lymphoblastic leukemia / lymphoma, primary cutaneous follicle center lymphoma, follicular lymphoma, or marginal zone B-cell non-Hodgkin's lymphoma.

[0017] In some embodiments, the diseases described in the third to fifth aspects are autoimmune diseases, including systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), multiple sclerosis (MS), rheumatoid arthritis (RA), type 1 diabetes mellitus (T1DM), Sjögren's syndrome, myositis, vasculitis, scleroderma, etc. In some preferred embodiments, the disease is moderate to severe active systemic lupus erythematosus, refractory idiopathic myositis, refractory granulomatosis with polyangiitis, or systemic sclerosis.

[0018] After extensive research and testing, the inventors of this application have developed a new chimeric antigen receptor targeting CD19 and its applications, and provided a chimeric antigen receptor targeting CD19 and corresponding nucleic acid molecules, vectors, immune effector cells, preparation methods and products thereof, pharmaceutical compositions, therapeutic uses, pharmaceutical uses, and methods for treating tumors, cancers, or autoimmune diseases. The inventions of this application achieve at least one of the following beneficial effects: (1) specific killing of CD19-positive tumor cells and / or inhibitory effect on the growth of CD19-related tumors; (2) humanization of the chimeric antigen receptor targeting CD19 to reduce immunogenicity; (3) taking the exemplary effector cell NK cell as an example, it has the application prospect of "shelf-type" therapeutic products; (4) it is expected to have a good therapeutic effect in clinical practice on CD19-positive tumors, such as acute B-lymphocytic leukemia (B-ALL), non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), or autoimmune diseases, such as systemic lupus erythematosus, myositis, scleroderma, vasculitis, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG1 shows a schematic diagram of the structure of a chimeric antigen receptor targeting CD19.

[0020] Figure 2A shows the expansion of different CD19 CAR-NK cells, where UT stands for untreated.

[0021] Figure 2B shows the CAR cell positive rates of different CD19 CAR-NK cells during the expansion culture process.

[0022] Figures 3A and 3B show the killing effects of different CD19 CAR-NK cells on Raji cells, wherein Figure 3A shows the results at 0 h of recovery, and Figure 3B shows the results 24 h after recovery.

[0023] Figures 4A and 4B show the tumor imaging results of the Nalm6 xenograft model in animals after receiving different CD19 CAR-NK therapies, wherein Figure 4A shows the results from days 5 to 26, and Figure 4B shows the results from days 30 to 33.

[0024] Figure 4C shows the photon quantity results of Nalm6 xenograft model tumor imaging in animals after receiving different CD19 CAR-NK treatments (since death occurred in the negative control group starting from day 19, the photon quantity data were only counted up to day 19).

[0025] Figure 4D shows the body weight test results of Nalm6 xenograft model animals after receiving different CD19 CAR-NK treatments (since death occurred in the negative control group starting from day 19, the body weight data were only counted up to day 19).

[0026] Figure 4E shows the survival curves of Nalm6 xenograft model animals after receiving different CD19 CAR-NK therapies.

[0027] Figure 5A shows the expression of CD56 in peripheral blood samples obtained from Nalm6 xenograft model animals treated with different CD19 CAR-NK cells. + CAR19 + CD3 - Cell ratio.

[0028] Figure 5B shows the expression of CD56 in peripheral blood samples obtained from Nalm6 xenograft model animals treated with different CD19 CAR-NK cells. + CAR19 - CD3 - Cell ratio.

[0029] FIG6 shows a summary of the Nalm6 xenograft model efficacy testing for different CD19 CAR-NK treatment groups.

[0030] Figure 7A shows the statistical results of cell activity after CD19 CAR-NK cells short-term killed B cells from the peripheral blood of SLE patients in vitro.

[0031] Figure 7B shows the statistical results of cell activity after multiple rounds of killing of B cells from the peripheral blood of SLE patients by CD19 CAR-NK in vitro.

[0032] FIG8 shows a flowchart of the preliminary clinical study plan for CD19 CAR-NK for the treatment of B-cell malignancies.

[0033] FIG9 shows a flowchart of the preliminary clinical study plan for CD19 CAR-NK for the treatment of SLE.

[0034] Detailed Description of the Invention

[0035] Definitions and Explanations of Terms

[0036] Unless otherwise defined herein, scientific and technical terms related to the present application shall have the meanings understood by those of ordinary skill in the art.

[0037] Furthermore, unless otherwise indicated herein, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly indicated otherwise.

[0038] The terms "comprising," "including," and "having" are used interchangeably herein to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "comprising," "including," and "having" in this document also provides for "consisting of" solutions. For example, "a composition comprising A and B" should be understood to include the following technical solutions: a composition consisting of A and B, as well as a composition containing other components in addition to A and B, all fall within the scope of the aforementioned "a composition."

[0039] The term "and / or" as used herein includes the meanings of "and," "or," and "all or any other combination of elements linked by the associated term."

[0040] Although the numerical ranges and parameter approximations shown in the broad scope of this application, the numerical values ​​shown in the specific examples are recorded as accurately as possible. However, any numerical value is necessarily contained in a certain error, which is caused by the standard deviation present in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all subranges contained therein. For example, a range of "1 to 10" should be considered to include any and all subranges between a minimum of 1 and a maximum of 10 (including endpoints); that is, all subranges starting with a minimum of 1 or greater, such as 1 to 6.1, and subranges ending with a maximum of 10 or less, such as 5.5 to 10. In addition, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0041] As used herein, the term "CD19" is a 95kDa type I transmembrane glycoprotein with a single transmembrane domain, a cytoplasmic C-terminus, and an extracellular N-terminus, wherein the extracellular domain is composed of two C2-type Ig-like domains, and the highly conserved cytoplasmic domain is composed of 242 amino acids and 9 tyrosine residues near the C-terminus. CD19 plays an important role in establishing the threshold of intrinsic B cell signaling, which is achieved by regulating B cell receptor-dependent and -independent signaling. The CD19 antigen belongs to the immunoglobulin superfamily, which directly or indirectly recruits and binds to various downstream protein kinases, including the Src family (Lyn, Fyn), the Ras family, Abl, Btk, adapter molecules (Vav, Grb2), and PI3K protein kinases, by interacting with other B cell receptors or surface molecules. CD19, together with the complement receptor CD21, the tetraspanin CD81 (TAPA-1), and CD225, is the main signaling component of the multimolecular complex on the surface of mature B cells.

[0042] As used herein, "chimeric antigen receptor (CAR)" refers to an artificial immune effector cell surface receptor that is modified to express on immune effector cells and specifically binds to an antigen, and includes at least: (1) an extracellular antigen binding domain, such as a variable heavy chain or light chain of an antibody; (2) a transmembrane domain that anchors CAR into immune effector cells; and (3) an intracellular signaling domain. CAR can redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC restricted manner using the extracellular antigen binding domain. The extracellular domain of the chimeric antigen receptor can also include a signal peptide and / or a hinge region. The intracellular domain of the chimeric antigen receptor can also include a costimulatory domain.

[0043] As used herein, the term "signal peptide" in the context of chimeric antigen receptors refers to a segment of a protein or polypeptide that is used to guide the protein or polypeptide into the secretory pathway, translocate to the cell membrane and / or cell surface. A non-limiting example of a signal peptide is the CD8α signal peptide.

[0044] As used herein, the term "hinge region" in the context of a chimeric antigen receptor generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane region and the antigen binding region. Specifically, the hinge region is used to provide greater flexibility and accessibility to the antigen binding region. The hinge region can be derived in whole or in part from natural molecules, such as in whole or in part from the extracellular region of CD8, CD4 or CD28, or in whole or in part from an antibody constant region. Alternatively, the hinge region can be a synthetic sequence corresponding to a naturally occurring hinge sequence, or can be a fully synthetic hinge sequence.

[0045] As used herein, the term "transmembrane (TM) region" in the context of a chimeric antigen receptor refers to a polypeptide structure that enables a chimeric antigen receptor to be expressed on the surface of an immune cell (e.g., a lymphocyte, NK cell, or NKT cell) and guides the immune cell to respond to a target cell. The transmembrane domain may be natural or synthetic, or may be derived from any membrane-bound protein or transmembrane protein. When the chimeric antigen receptor binds to the target antigen, the transmembrane domain is capable of signal transduction. A non-limiting example of a hinge region is the CD8 transmembrane region.

[0046] As used herein, the term "intracellular signaling domain" in the context of a chimeric antigen receptor refers to a portion of a protein that transduces effector function signals and directs cells to perform designated functions. The intracellular signaling domain is responsible for primary signaling within the cell after the antigen binding domain binds to the antigen, leading to activation of immune cells and immune responses. In other words, the intracellular signaling domain is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is expressed. Exemplary intracellular signaling domains include CD3ζ.

[0047] As used herein, the term "costimulatory domain" in the context of a chimeric antigen receptor refers to the intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for the effective activation and function of T lymphocytes after binding to an antigen. A non-limiting example of a costimulatory domain is the CD28 costimulatory domain.

[0048] As used herein, the term "immune effector cell" or "effector cell" refers to a cell that participates in an immune response, such as a cell that promotes an immune effector response. Examples of immune effector cells include T cells, such as α / β T cells and γ / δ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.

[0049] The term "specific binding" herein refers to the ability of an antigen-binding molecule (e.g., an antibody or a ligand of an antigen) to specifically bind to an antigen and substantially the same antigen, typically with high affinity, but not to unrelated antigens with high affinity. Affinity is typically measured as an equilibrium dissociation constant (KD), where a lower KD indicates a higher affinity. For example, for antibodies, a high affinity typically refers to a specific affinity of about 10 -6 M or lower, about 10 -7 M or lower about 10 -8 M or less, about 1×10 -9 M or less, about 1×10 -10 M or less, 1×10 -11 M or lower or 1×10 -12 The KD is calculated as follows: KD = Kd / Ka, where Kd represents the off-rate and Ka represents the on-rate. The equilibrium dissociation constant, KD, can be measured using methods known in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis. For example, see the methods for obtaining KD values ​​described in the Examples herein.

[0050] The term "antibody" herein is used in the broadest sense to refer to a polypeptide or combination of polypeptides that contains sufficient sequence from the variable region of an immunoglobulin heavy chain and / or sufficient sequence from the variable region of an immunoglobulin light chain to specifically bind to an antigen. "Antibodies" herein encompass various forms and structures, as long as they exhibit the desired antigen-binding activity, including intact antibodies and antigen-binding fragments thereof.

[0051] The term "antibody" herein includes a typical "four-chain antibody," which is an immunoglobulin composed of two heavy chains (HC) and two light chains (LC). The heavy chain refers to a polypeptide chain that, from the N-terminus to the C-terminus, consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain. Furthermore, when the full-length antibody is of the IgE isotype, it optionally also includes a heavy chain constant region CH4 domain. The light chain is a polypeptide chain that, from the N-terminus to the C-terminus, consists of a light chain variable region (VL) and a light chain constant region (CL). Heavy chains are linked to each other and to each other through disulfide bonds, forming a "Y"-shaped structure. Due to the different amino acid composition and arrangement order of the constant regions of the heavy chains of immunoglobulins, their antigenicity also varies. Based on this, "immunoglobulins" as used herein can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE. Their corresponding heavy chains are μ, δ, γ, α, and ε, respectively. Igs within the same class are further divided into subclasses based on the amino acid composition of their hinge regions and the number and location of disulfide bonds in their heavy chains. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are classified as either kappa or lambda chains based on differences in their constant regions. Each of the five Ig classes can have either kappa or lambda chains.

[0052] "Antigen-binding fragment" and "antibody fragment" are used interchangeably herein and do not have the entire structure of an intact antibody, but only contain a portion or partial variant of an intact antibody that has the ability to bind to an antigen. "Antigen-binding fragment" or "antibody fragment" herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, scFv, and VHH.

[0053] The term "scFv" (single-chain variable fragment) herein refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers include GSTSGSGKPGSGEGSTKG and consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond can also exist between the VH and VL of the scFv to form a disulfide-linked Fv (dsFv).

[0054] As used herein, "antibodies" may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents, and vertebrates, such as camelids, llamas, ostriches, alpacas, sheep, rabbits, mice, rats, or cartilaginous fish (e.g., sharks).

[0055] The term "humanized antibody" means an antibody obtained by grafting CDR sequences derived from another mammalian species, such as a mouse germline, onto human framework sequences. In order to retain binding affinity, some residues of the backbone (called FR) segment may be modified. Humanized antibodies or fragments thereof according to the present application may be prepared by techniques known to those skilled in the art.

[0056] The term "variable region" herein refers to the region of an antibody heavy or light chain that is involved in binding the antibody to an antigen. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR," and "light chain variable region" is used interchangeably with "VL" and "LCVR." The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, each comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. The terms "complementarity determining region" and "CDR" are used interchangeably herein and generally refer to the hypervariable regions (HVRs) of the heavy chain variable region (VH) or light chain variable region (VL). These regions are also called complementarity determining regions because they form precise spatial complementarity with antigenic epitopes. The heavy chain variable region CDRs can be abbreviated as HCDRs, and the light chain variable region CDRs can be abbreviated as LCDRs. The terms "framework region" or "FR region" are used interchangeably and refer to the amino acid residues in the heavy chain variable region or light chain variable region of an antibody, excluding the CDRs. A typical antibody variable region is composed of four FR regions and three CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0057] For further description of CDRs, see Kabat et al., J. Biol. Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc et al., J. Mol. MP et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001). "CDRs" herein can be annotated and defined by methods well known in the art, including but not limited to the Kabat numbering system, using tool websites including but not limited to the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi).

[0058] The term "Kabat numbering system" herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0059] As used herein, the terms "percent (%) sequence identity" and "percent (%) sequence identity" are interchangeable and refer to the percentage of amino acid (or nucleotide) residues of a candidate sequence that are identical to the amino acid (or nucleotide) residues of a reference sequence after aligning sequences and introducing gaps (if necessary) to achieve maximum percent sequence identity (e.g., for optimal alignment, gaps can be introduced into one or both of the candidate and reference sequences, and for the purpose of comparison, non-homologous sequences can be ignored). For the purpose of determining percent sequence identity, alignment can be achieved in a variety of ways well known to those skilled in the art, such as using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAIi) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm that requires achieving maximum alignment over the full length of the compared sequences. For example, a reference sequence aligned for comparison with a candidate sequence can show that the candidate sequence exhibits from 50% to 100% sequence identity over the full length of the candidate sequence or a selected portion of the continuous amino acid (or nucleotide) residues of the candidate sequence. The length of the candidate sequence aligned for comparison purposes can be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence. When a position in the candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

[0060] As used herein, "mutation" includes insertion mutation, deletion mutation and substitution mutation. In some embodiments, the substitution mutation is preferably a substitution of a conservative amino acid.

[0061] As used herein, "conservative amino acids" generally refer to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). Exemplarily, the amino acids within each of the following groups are conservative amino acid residues of each other, and substitutions of amino acid residues within the group are substitutions of conservative amino acids:

[0062] Illustratively, the following six groups are examples of amino acids that are considered to be conservative substitutions for each other:

[0063] 1) Alanine (A), serine (S), threonine (T);

[0064] 2) Aspartic acid (D), glutamic acid (E);

[0065] 3) Asparagine (N), glutamine (Q);

[0066] 4) Arginine (R), Lysine (K), Histidine (H);

[0067] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0068] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0069] As used herein, "at most X mutations" means that the number of mutations can be selected from any natural number in the range of 0 to X.

[0070] As used herein, "at most 0-3 mutations" can mean 3 mutations, 2 mutations, 1 mutation, or 0 mutations. As used herein, "respectively" in "the HCDRs 1-3 each have at most 0-3 mutations" means that the number of mutations in each CDR in HCDRs 1-3 is independent of each other and can be independently selected from 0-3. Similarly, as used herein, "respectively" in "the LCDRs 1-3 each have at most 0-3 mutations" means that the number of mutations in each CDR in LCDRs 1-3 is independent of each other.

[0071] As used herein, a "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmids and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.

[0072] As used herein, the terms "subject," "object," and "patient" refer to an organism to which a nucleic acid molecule or vector or immune effector cell or product or pharmaceutical composition as described herein is administered, wherein "patient" also refers to an organism receiving treatment for a specific disease or condition as described herein (such as cancer, infectious disease, or autoimmune disease). Examples of patients include mammals such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the Bovidae family (such as domestic cattle, bison, buffalo, elk, and yak, etc.), cattle, sheep, horses, and bison, etc., who are receiving treatment for a disease or condition (such as a cell proliferative disorder, such as cancer or an infectious disease, or an autoimmune disease). Examples of subjects and objects include, in addition to patients, healthy mammals such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the Bovidae family (such as domestic cattle, bison, buffalo, elk, and yak, etc.), cattle, sheep, horses, and bison, etc.

[0073] As used herein, the term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition. As used herein, the term "tumor" or "neoplasm" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when used herein.

[0074] The term "B-cell malignancy" in this article refers to a malignant tumor characterized by abnormal proliferation of B cells. The more common ones include acute B-cell lymphocytic leukemia, non-Hodgkin's lymphoma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, acute common B-cell lymphocytic leukemia, etc.

[0075] In some cases, the term "acute lymphoblastic leukemia (ALL)" herein refers to a malignant clonal disease that occurs when the genes of lymphocyte progenitor cells change and cause uncontrolled cell proliferation. When lymphocyte progenitor cells undergo somatic mutations, uncontrolled proliferation occurs. This progressive clonal expansion leads to the replacement of normal hematopoietic cells in the bone marrow with early lymphoid precursor cells, further infiltration of various organs, and ultimately ALL. There are two types of ALL: acute B-lymphoblastic leukemia (B-ALL) (originating from B-cell progenitor cells) and acute T-lymphoblastic leukemia (T-ALL) (originating from T-cell progenitor cells), of which B-ALL is more common.

[0076] In some cases, the term "non-Hodgkin's Lymphoma (NHL)" in this article is a cancer that occurs in the lymphatic system. In NHL, white blood cells called lymphocytes grow abnormally and can form tumors throughout the body. NHL is the most common blood cancer in the world and in China. It is usually divided into two types: aggressive and indolent. Among them, diffuse large B-cell lymphoma (DLBCL) is the most common subtype of adult NHL. It is an aggressive and highly heterogeneous disease. Follicular lymphoma (FL) is the most common indolent B-cell NHL, accounting for about 1 / 4 of all NHLs. However, the FL 3B grade subgroup is rare.

[0077] In some cases, the term "chronic lymphocytic leukemia (CLL)" or small lymphocytic lymphoma (SLL) is used herein to refer to the same type of tumor arising in different locations. CLL tumor cells are mostly distributed in the blood and bone marrow, while SLL tumor cells are mainly distributed in the lymph nodes and spleen. CLL is a B-cell chronic lymphoproliferative disorder (B-CLPD) characterized by the accumulation of mature B lymphocytes in the peripheral blood, bone marrow, and lymphoid tissues, and resulting in corresponding clinical symptoms. It is the most common leukemia and primarily affects B cells.

[0078] The term "autoimmune disease" in this article refers to a condition caused by the body's abnormal immune response attacking normal cells, which can occur in almost any part of the body. Previous studies have shown that autoimmune diseases are highly correlated with cancer, and having autoimmune diseases increases the risk or probability of certain cancers. Autoimmune diseases cause inflammation through various mechanisms. Relatively speaking, the risk depends on whether autoimmune diseases increase chronic inflammation associated with cancer. Common autoimmune diseases associated with cancer include: celiac disease, inflammatory bowel disease (Crohn's disease and ulcerative colitis), multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus. The autoimmune diseases mentioned in this article also include myositis, vasculitis, scleroderma, etc. In this article, the terms "autoimmune disease" and "autoimmune-related diseases" can be used interchangeably.

[0079] In some cases, the term "systemic lupus erythematosus (SLE)" herein refers to a chronic autoimmune disease affecting multiple organs. Its main pathogenesis lies in an autoimmune reaction caused by immune imbalance, of which the most critical factor is the dysregulation of activation of autoreactive B cells. Studies have shown that senescent / autoimmune-related B cells have been found to be the main cells producing SLE autoantibodies. The B cells of SLE patients proliferate abnormally, producing a large number of autoantibodies against their own components, which bind to corresponding autoantigens in the body to form immune complexes, directly destroying cells, or causing acute and chronic inflammation and tissue necrosis under the action of complement effects, thereby leading to multi-system damage in the body. Current treatments for SLE mainly include plasma exchange, injection of immunoglobulins, and administration of drugs targeting B cells, such as injection of monoclonal antibodies targeting B cells.

[0080] In some cases, the term "myositis" herein refers to inflammatory myopathies, a heterogeneous group of diseases characterized by inflammatory cell infiltration of skeletal muscle and myofiber necrosis. These diseases primarily fall into two categories: infectious myopathies with clear etiologies, such as viral myositis, parasitic myositis, and tropical myositis; and idiopathic inflammatory myopathies (IIMs), which have unidentified etiologies but are associated with autoimmunity. These include polymyositis (PM), dermatomyositis (DM), and inclusion body myositis.

[0081] In some cases, the term "vasculitis" is used in this article to refer generally to systemic vasculitis, which are autoimmune diseases characterized by inflammation of blood vessels. Systemic vasculitis is a group of diseases that vary in organ involvement and clinical severity.

[0082] In some cases, the term "scleroderma" herein refers to a systemic autoimmune disease characterized by limited (limited systemic form) or diffuse (diffuse systemic form) thickening and hardening of the skin and fibrosis of the internal organs. Depending on the area of ​​skin involvement, scleroderma is classified as either localized scleroderma (limited to the skin symptoms without affecting the internal organs) or systemic sclerosis (SSc) (which affects the internal organs).

[0083] As used herein, the term "treatment" refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or lesions in the treated subject, such as the progression of a cell proliferative disorder (such as cancer or an infectious disease). Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, attenuation of the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Subjects in need of treatment include subjects already suffering from a disease or disease, as well as subjects susceptible to a disease or disease, or subjects intending to prevent a disease or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, and alleviating, their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0084] As used herein, the term "effective amount" refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or ameliorating a disease symptom or the progression of that disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of such a condition. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0085] The term "pharmaceutical composition" herein refers to a combination of at least one drug and optionally a pharmaceutically acceptable carrier or excipient that is combined together to achieve a specific purpose. In certain embodiments, the pharmaceutical composition includes a combination separated in time and / or space, as long as it can work together to achieve the purpose of the present application. For example, the ingredients contained in the pharmaceutical composition (such as CAR-NK cells according to the present application) can be administered to an individual as a whole, or separately. When the ingredients contained in the pharmaceutical composition are administered separately to an individual, the ingredients can be administered to the individual simultaneously or sequentially. The pharmaceutical composition according to the present application may include conventional components of cell culture to maintain the activity of CAR-NK cells. Pharmaceutically acceptable carriers may also include water, buffered aqueous solutions, isotonic saline solutions such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol or polyalkylene glycols such as polypropylene glycol, triglycerides, cell preservation fluids, colloidal dispersion systems, macromolecular complexes, nanocapsules, nanoparticles, microspheres, beads, etc. The pharmaceutical composition or pharmaceutical preparation according to the present application can be administered by any appropriate route, such as intravenous administration, intradermal, subcutaneous, intramuscular injection, etc. The composition according to the present application may contain a wetting agent, an emulsifier or a buffer substance as an additive.

[0086] The term "immune cell cryopreservative" herein refers to a cryopreservative obtained by resuspending immune cells in a cell freezing solution and freezing them. In some cases, the immune cell cryopreservative should be free of flocculent matter after thawing and should be a colorless or light yellow clear liquid.

[0087] The components in the term "cell preservation fluid" herein can effectively maintain cell morphology, prevent cell autolysis, ensure cell integrity, protein fixation, i.e., nucleic acid stabilization, and are used to preserve and transport cells taken from the human body. The main components include pH buffers, osmotic pressure maintainers, and nucleic acid stabilizers. In some cases, the cell preservation fluid herein can be a cell protection fluid or a cryoprotectant, which easily binds to water molecules in the solution, thereby lowering the freezing point and reducing the formation of ice crystals. It can also reduce the concentration of electrolytes in the unfrozen solution through its molar concentration, thereby protecting cells from solute damage and ice crystal damage in the cells, and the frozen cells still maintain their normal results and functions. In some cases, the cell preservation fluid described herein includes conventional compound electrolyte injections, human albumin injections, or freezing solutions in the art.

[0088] In some cases, the compound electrolyte injection described herein is a conventional compound electrolyte injection in the art, whose main components include: sodium chloride, sodium gluconate, sodium acetate (C2H3NaO2·3H2O), potassium chloride, magnesium chloride (MgCl2·6H2O). If necessary, the components can be optimized and adjusted; common products such as compound electrolyte injections of Luoxin Pharmaceutical, Sihuan Pharmaceutical, Huaren Pharmaceutical, Kelun Pharmaceutical, etc., or Compound electrolyte injection, Bolili A, etc.

[0089] In some cases, the human albumin injection described herein is a conventional human albumin injection in the art, comprising human albumin as a primary component and sodium octanoate and acetyltryptophan as excipients. In some cases, the human albumin concentration in the injection can range from 5% to 25%, for example, 5%, 10%, 20%, or 25%. Common products include human albumin injections from JetBelin, Octapharm, Griffiths, Takeda Pharmaceutical, Chengdu Rongsheng Pharmaceutical, Shenzhen Weiguang, and Guangdong Weilun.

[0090] In some cases, the freezing solution described herein is a conventional commercial freezing solution in the art, which should contain a cryoprotectant such as DMSO or glycerol. Common products include commercial freezing solutions from Sigma-Aldrich, Sartorius, Gibco, Ecosine, BioLife, etc. DETAILED DESCRIPTION

[0091] In the first aspect, the present application provides an immune effector cell, which comprises a chimeric antigen receptor targeting CD19, wherein the chimeric antigen receptor comprises an extracellular region comprising an antigen binding region that specifically binds to CD19, a transmembrane region connected to the extracellular region, and an intracellular domain connected to the transmembrane region, wherein the antigen binding region comprises a VH comprising HCDR1-3 and a VL comprising LCDR1-3. In some embodiments, the HCDR1-3 respectively have sequences as shown in SEQ ID NOs: 5-7, and the LCDR1-3 respectively have sequences as shown in SEQ ID NOs: 8-10. In some embodiments, the HCDR1-3 has at most 0-3 mutations (e.g., 0, 1, 2, 3 mutations) compared to the corresponding CDRs shown in SEQ ID NOs: 5-7. In some embodiments, the LCDR1-3 has at most 0-3 mutations (e.g., 0, 1, 2, 3 mutations) compared to the corresponding CDRs shown in SEQ ID NOs: 8-10.

[0092] In some embodiments, (1) the VH has the sequence set forth in SEQ ID NO: 20, and the VL has the sequence set forth in SEQ ID NO: 17; or (2) the VH has a sequence having at least 80% identity (e.g., 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%, at least 99%, or 100%) or at most 25 mutations (e.g., at most 20 mutations, at most 15 mutations, at most 10 mutations, at most 5 mutations, at most 4 mutations, at most 3 mutations, at most 2 mutations, or 1 mutation) to the corresponding VH in group (1), and the VL has a sequence having at least 80% identity (e.g., 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%, at least 99%, or 100%) ... ) group, or a sequence having at least 80% identity (e.g., 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%, at least 99%, or 100%) or at most 20 mutations (e.g., at most 15 mutations, at most 10 mutations, at most 5 mutations, at most 4 mutations, at most 3 mutations, at most 2 mutations, or 1 mutation) compared to the corresponding VL in the same group.

[0093] In some embodiments, the chimeric antigen receptor has a sequence as shown in SEQ ID NO: 23, or has a sequence similar to SEQ ID NO: NO:23 or a sequence having at least 80% identity (e.g., 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%, at least 99% or 100%) or at most 120 amino acid mutations (e.g., at most 110 mutations, at most 100 mutations, at most 90 mutations, at most 80 mutations, at most 70 mutations, at most 60 mutations, at most 50 mutations, at most 45 mutations, at most 40 mutations, at most 35 mutations, at most 30 mutations, at most 25 mutations, at most 20 mutations, at most 15 mutations, at most 10 mutations, at most 5 mutations, at most 4 mutations, at most 3 mutations, at most 2 mutations or 1 mutation) compared to NO:23.

[0094] In a second aspect, the present application provides a pharmaceutical composition comprising the immune effector cells described in the first aspect and a pharmaceutically acceptable carrier.

[0095] In some embodiments, the pharmaceutically acceptable carrier includes water, buffer, glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, polyalkylene glycols such as polypropylene glycol, triglycerides, cell preservation fluid, colloidal dispersion system, macromolecular complex, nanocapsule, nanoparticles, microspheres, beads.

[0096] In some embodiments, the pharmaceutical composition is a cryopreservative of immune cells.

[0097] In some embodiments, the pharmaceutically acceptable carrier is a cell preservation solution; in some preferred embodiments, the components of the cell preservation solution include compound electrolyte injection, human serum albumin injection and / or freezing solution.

[0098] In some preferred embodiments, the compound electrolyte injection is a conventional compound electrolyte injection in the art, and its main components include: sodium chloride, sodium gluconate, sodium acetate (C2H3NaO2·3H2O), potassium chloride, magnesium chloride (MgCl2·6H2O). If necessary, the components can be optimized and adjusted; common products such as compound electrolyte injections of Luoxin Pharmaceutical, Sihuan Pharmaceutical, Huaren Pharmaceutical, Kelun Pharmaceutical, etc., or Compound electrolyte injection, Bolili A, etc.

[0099] In some cases, the human albumin injection described herein is a conventional human albumin injection in the art, comprising human albumin as a primary component and sodium octanoate and acetyltryptophan as excipients. In some cases, the human albumin concentration in the injection can range from 5% to 25%, for example, 5%, 10%, 20%, or 25%. Common products include human albumin injections from JetBelin, Octapharm, Griffiths, Takeda Pharmaceutical, Chengdu Rongsheng Pharmaceutical, Shenzhen Weiguang, and Guangdong Weilun.

[0100] In some preferred embodiments, the freezing solution is a conventional commercial freezing solution in the art, which should contain a cryoprotectant such as DMSO or glycerol, such as commercial freezing solutions from Sigma-Aldrich, Sartorius, Gibco, Ecosine, BioLife, etc.

[0101] In some embodiments, the freezing density of immune effector cells in the immune cell cryopreservative is not less than 1×10 5 viable cells / ml; In some preferred embodiments, the freezing density of immune effector cells in the immune cell cryopreservative is not less than 1×10 6 viable cells / ml.

[0102] In some embodiments, the pharmaceutical composition is an injection;

[0103] In some preferred embodiments, the pharmaceutical composition is administered by intravenous drip.

[0104] In some embodiments, the CD3 + The proportion of cells does not exceed 5%;

[0105] In some preferred embodiments, the immune effector cells have CD3 + The proportion of cells does not exceed 3%;

[0106] In some more preferred embodiments, the immune effector cells have CD3 + The proportion of cells does not exceed 1%.

[0107] In a third aspect, the present application provides the immune effector cell described in the first aspect or the pharmaceutical composition described in the second aspect, which is used to prevent or treat a disease.

[0108] In a fourth aspect, the present application provides use of the immune effector cells described in the first aspect or the pharmaceutical composition described in the second aspect in the preparation of a drug for treating a disease.

[0109] In a fifth aspect, the present application provides a method for preventing or treating a disease, comprising administering an effective amount of the immune effector cells of the first aspect or the pharmaceutical composition of the second aspect to a subject in need thereof.

[0110] In some embodiments, the disease is a B cell driven disease.

[0111] In some preferred embodiments, the disease is a CD19-positive B cell-driven disease.

[0112] In some embodiments, the disease is a tumor or cancer.

[0113] In some embodiments, the tumor or cancer is a CD19-associated tumor or cancer. In some embodiments, the tumor or cancer is a B-cell malignancy. In some embodiments, the tumor or cancer is a relapsed or refractory CD19-positive B-cell malignancy. In some embodiments, the tumor or cancer is acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, multiple myeloma, plasmacytoma, monoclonal gammopathy of undetermined significance, Waldenstrom's macroglobulinemia (lymphoplasmacytic lymphoma), heavy chain disease, primary amyloidosis, post-transplant lymphoproliferative disorder, Hodgkin's lymphoma, MALT lymphoma, B-cell lymphoma, mantle cell lymphoma, (germinal center-like) diffuse large cell lymphoma, Burkitt's lymphoma, bi-lineage leukemia, biphenotypic leukemia, hairy cell leukemia, precursor B acute lymphoblastic leukemia / lymphoma, primary cutaneous follicle center lymphoma, follicular lymphoma, or marginal zone B-cell non-Hodgkin's lymphoma.

[0114] In some embodiments, a CD19-related tumor or cancer refers to tumor cells or cancer cells that express CD19 on their surface. In some embodiments, the tumor is a tumor that highly expresses CD19 (CD19+). In some embodiments, a tumor that highly expresses CD19 (CD19+) refers to at least 60% of tumor cells in a tumor cell population that express CD19. In some embodiments, a tumor that highly expresses CD19 (CD19+) refers to at least 70% of tumor cells in a tumor cell population that express CD19. In some embodiments, a tumor that highly expresses CD19 (CD19+) refers to at least 80% of tumor cells in a tumor cell population that express CD19. In some embodiments, a tumor that highly expresses CD19 (CD19+) refers to at least 90% of tumor cells in a tumor cell population that express CD19. In some embodiments, a tumor that highly expresses CD19 (CD19+) refers to at least 95% of tumor cells in a tumor cell population that express CD19. In some embodiments, a tumor that highly expresses CD19 (CD19+) refers to at least 98% of tumor cells in a tumor cell population that express CD19. In some embodiments, a tumor that highly expresses CD19 (CD19+) means that at least 99% of the tumor cells in a tumor cell population express CD19.

[0115] In some embodiments, the prevention or treatment comprises administering the immune cell of the first aspect or the pharmaceutical composition of the second aspect to a subject in need thereof.

[0116] In some embodiments, the subject has a tumor or cancer described herein.

[0117] In some embodiments, the subject has previously received at least 2 lines of therapy.

[0118] In some embodiments, the subject has at least one measurable lesion according to the Lugano 2014 Lymphoma Response Evaluation Criteria (Cheson 2014).

[0119] In some embodiments, the disease is an autoimmune disease.

[0120] In some embodiments, the autoimmune disease includes systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), multiple sclerosis (MS), rheumatoid arthritis (RA), type 1 diabetes mellitus (T1DM), Sjögren's syndrome, myositis, vasculitis, scleroderma, etc.

[0121] In some embodiments, the subject suffers from an autoimmune disease as described herein. In preferred embodiments, the autoimmune disease is systemic lupus erythematosus, myositis, vasculitis, or scleroderma. In more preferred embodiments, the autoimmune disease is moderately to severely active systemic lupus erythematosus, refractory idiopathic myositis, refractory granulomatosis with polyangiitis, or systemic sclerosis.

[0122] In some embodiments, the subject was diagnosed with SLE at least 24 weeks prior to screening.

[0123] In some embodiments, the European League of Societies of Rheumatology (EULAR) / American College of Rheumatology (ACR) 2019 SLE classification criteria are met at screening.

[0124] In some embodiments, the pharmaceutical composition is administered as a single dose or as multiple doses.

[0125] In some embodiments, the administration is once a week.

[0126] In some embodiments, the single administration is 1 administration.

[0127] In some embodiments, the multiple administrations are 2-6 administrations; in some preferred embodiments, the multiple administrations are 2, 3, 4, 5, or 6 administrations.

[0128] In some embodiments, the pharmaceutical composition is administered at a dose of 1×10 4cells / Kg~1×10 9 Cells / Kg administration.

[0129] In some preferred embodiments, the pharmaceutical composition is administered at a dose of 1×10 4 cells / Kg~1×10 5 cells / Kg administration; in some preferred embodiments, the pharmaceutical composition is administered at 1×10 5 cells / Kg~1×10 6 cells / Kg administration; in some preferred embodiments, the pharmaceutical composition is administered at 1×10 6 cells / Kg~1×10 7 cells / Kg administration; in some preferred embodiments, the pharmaceutical composition is administered at 1×10 7 cells / Kg~1×10 8 cells / Kg administration; in some preferred embodiments, the pharmaceutical composition is administered at 1×10 8 cells / Kg~1×10 9 Cells / Kg administration.

[0130] In some preferred embodiments, the pharmaceutical composition is administered at a dose of 1×10 6 cells / Kg~9×10 8 Cells / Kg, for example, 1×10 7 cells / Kg~9×10 8 Cells / Kg administration.

[0131] In some preferred embodiments, the pharmaceutical composition is administered at a dose of 1×10 6 cells / Kg~9×10 6 cells / Kg, 1×10 7 cells / Kg~9×10 7 cells / Kg, or 1×10 8 cells / Kg~9×10 8 Cells / Kg administration.

[0132] In some more preferred embodiments, the pharmaceutical composition is administered at a dose of 1×10 6 cells / Kg, 2×10 6 cells / Kg, 3×10 6 cells / Kg, 4×10 6 cells / Kg, 5×10 6 cells / Kg, 6×10 6 cells / Kg, 7×10 6 cells / Kg, 8×10 6 cells / Kg, 9×10 6cells / Kg, 1×10 7 cells / Kg, 2×10 7 cells / Kg, 3×10 7 cells / Kg, 4×10 7 cells / Kg, 5×10 7 cells / Kg, 6×10 7 cells / Kg, 7×10 7 cells / Kg, 8×10 7 cells / Kg, 9×10 7 cells / Kg, 1×10 8 cells / Kg, 2×10 8 cells / Kg, 3×10 8 cells / Kg, 4×10 8 cells / Kg, 5×10 8 cells / Kg, 6×10 8 cells / Kg, 7×10 8 cells / Kg, 8×10 8 cells / Kg, or 9×10 8 Cells / Kg administration.

[0133] It should be understood that the above detailed description is only for the purpose of enabling those skilled in the art to more clearly understand the content of the present application and is not intended to limit the present invention in any respect. Those skilled in the art can make various modifications and variations to the embodiments described.

[0134] Example

[0135] The present application will be further described below in conjunction with specific examples, and the advantages and features of the present application will become clearer as the description proceeds. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer were used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0136] The embodiments of the present application are merely exemplary and do not constitute any limitation on the scope of the present application. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present application may be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements shall fall within the scope of protection of the present application.

[0137] Materials and methods

[0138] SPR detection method

[0139] Anti-human CD19 antibodies were captured using a Protein A chip (GE Healthcare; 29-127-558). The sample and running buffer was HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) (GE Healthcare; BR-1006-69). The flow-through pool was set to 25°C. The sample block was set to 16°C. Both were pretreated with running buffer. In each cycle, the antibody to be tested was first captured using the Protein A chip, then a single concentration of CD19 antigen protein was injected, the binding and dissociation process of the antibody and antigen protein was recorded, and finally the chip was regenerated using Glycine pH 1.5 (GE Healthcare; BR-1003-54). Binding was measured by injecting different concentrations of recombinant human CD19-His in solution for 240 seconds with a flow rate of 30 μl / min, starting from 200 nM (see detailed results for the actual concentrations tested), diluted 1:1, for a total of 5 concentrations. The dissociation phase was monitored for up to 600 seconds and triggered by switching from the sample solution to the running buffer. The surface was regenerated by washing with a 10 mM glycine solution (pH 1.5) at a flow rate of 30 μl / min for 30 seconds. Bulk refractive index differences were corrected by subtracting the response obtained from the goat anti-human Fc surface. Blank injections (= double reference) were also subtracted. The apparent KD and other kinetic parameters were calculated using the Langmuir 1:1 model.

[0140] Virus packaging

[0141] The day before virus packaging, trypsinize 293T cells (purchased from ATCC) and seed them into 10 cm culture dishes at a density of 1E7 cells per dish. When transfecting cells, mix the packaging plasmid and the target plasmid and add them to α-MEM medium. In another centrifuge tube containing α-MEM medium, add HD transfection reagent (Promega, E2311). Add the diluted transfection reagent dropwise to the diluted plasmid, mix well, and let it stand at room temperature for 15 minutes. Finally, add the mixture of plasmid and transfection reagent to a 10cm culture dish, shake gently 10 times, mix well, and place it in the incubator. 3 days after cell transfection, harvest the virus and transfer 10ml of the virus-containing culture supernatant to a 50ml centrifuge tube. Incubate at 4°C, 1250rpm, for 5 minutes to remove dead 293T cells. Filter the virus-containing supernatant and concentrate it using Retro-X Concentrator (Clontech, 631455). Aliquot and store at -80°C until use.

[0142] NK cell purification and activation

[0143] NK cells are isolated from monocytes and the purification process is divided into two steps. The first step is to remove CD3 + Cells, second step enrichment of CD56 + Cells were washed, incubated with CD3 magnetic beads, and resuspended using Sepax, and then CD3 was removed using CliniMACS. + cells. Sepax was used to + CD3 after cell depletion - The cell product was washed, incubated with CD56 magnetic beads, and resuspended, and then CD56 was enriched using CliniMACS. + The enriched CD56 + The cells were washed and replaced with complete medium, K562 feeder cells were added, and the cells were transferred to a 37°C incubator for 5 days of activation.

[0144] Viral transduction

[0145] On day 1, add 500 μl / well of RetroNectin reagent to a 24-well plate and coat overnight at 4°C. On day 2, discard the upper layer of RetroNectin and wash with PBS. Then add the packaged retrovirus and centrifuge at 2000g at 4-8°C for 60 minutes. Discard the upper viral liquid. Add 5-day activated NK cells to a 24-well plate at 3E5 / well, centrifuge at 400g for 5 minutes at room temperature, and culture in a 37°C incubator (37°C, 5% CO2). On day 3, transfer the NK cells to a 6-well plate that has not been treated with tissue culture and continue to culture. On day 6, test for CAR expression to ensure that the CAR positivity rate of NK cells used for in vitro or in vivo killing is within the range of 60 to 80%.

[0146] Determination of tumor cell killing rate in vitro

[0147] Unless otherwise specified, the target cells referred to in the following examples were stably transfected with the luciferase gene and expressed luciferase. Fluorescence intensity was measured using a luciferase reporter assay, reflecting cell viability and NK cell cytotoxicity. The killing rate in the test wells was calculated as follows: Killing rate = (target cell well reading - test well reading) / target cell well reading × 100%.

[0148] Example 1 Preparation and Screening of Humanized Chimeric Antigen Receptors Targeting CD19

[0149] 1.1 Preparation of CD19 Antibody

[0150] Using human CD19 protein (NCBI: NP_001761.3) as the immunogen, hybridoma-positive clones were prepared using conventional antibody preparation and screening methods in the art. The heavy and light chain variable region sequences and CDRs (Kabat numbering system) of the preferred anti-CD19 antibody Mab01 were determined, along with the sequence information of the positive control FMC63, as shown in Tables 1 and 2. The SPR assay results, shown in Table 3, demonstrated that Mab01 had good affinity for human CD19 protein.

[0151] Table 1 Antibody sequence information

[0152] Table 2 Antibody CDRs

[0153] Table 3. Affinity of chimeric antibodies to human CD19 detected by SPR (Biacore)

[0154] 1.2 Antibody Humanization

[0155] The anti-CD19 antibody Mab01 obtained by the aforementioned screening was humanized, and the CDR amino acid residues of the resulting antibody were determined using the Kabat numbering system. The sequence information of the humanized VH and VL is shown in Table 4, and the resulting humanized antibody variable region combination is shown in Table 5. The binding ability of the chimeric antibody to the human CD19 protein was tested by SPR (Biacore), as shown in Tables 6 and 7. The resulting humanized antibody has strong binding ability to the human CD19 protein. Using the same method, the positive control FMC63 was humanized, and the humanized antibody hFMC63 was constructed and expressed. The antibody's binding ability to the human CD19 protein was tested by SPR (Biacore). The sequence information and binding ability of hFMC63 to the human CD19 protein are shown in Tables 4 and 7, respectively. The results show that hFMC63 has strong binding ability to human CD19.

[0156] Table 4. Sequence information of humanized antibodies Mab01 and FMC63

[0157] Table 5. Variable region combinations of Mab01 humanized antibody

[0158] Table 6. Affinity of Mab01 humanized antibody to human CD19 detected by SPR (Biacore)

[0159] Table 7. Affinity of FMC63 humanized antibody to human CD19 detected by SPR (Biacore)

[0160] Example 2 In vitro and in vivo validation of the killing activity of humanized chimeric antigen receptor targeting CD19

[0161] 2.1 Preparation of CD19 CAR-NK cells

[0162] According to the chimeric antigen structure diagram shown in Figure 1, the humanized antibodies Hab01-4, Hab01-11 and hFMC 63, FMC63 prepared in Example 1 were used to construct chimeric antigen receptors targeting CD19 (see Table 8 for specific sequence information), and the nucleic acid molecules of the numbered chimeric antigen receptors were cloned into conventional retroviral vectors in the art. The viral packaging, NK cell purification and activation, and viral transduction methods described in the aforementioned Materials and Methods section were used to prepare CD19 CAR NK cells, and the proliferation of NK cells and the CAR positivity rate after transfection were detected. The results are shown in Figures 2A-2B.

[0163] In vitro cytotoxicity of CD19 CAR-NK cells against Raji cells (Burkitt lymphoma cell line)

[0164] Before testing the target cell cytotoxicity, cryopreserved CD19 CAR-NK cells were revived and the same number of CD19 CAR-NK cells were mixed with target cells Raji at the time of resuscitation (0 h) and 24 h after resuscitation. The in vitro cytotoxicity of NK cells against target cells at different effector-target ratios (Raji: 1E6 cells / mL) was tested. The results are shown in Figures 3A-3B. FMC63-CAR, hFMC63-CAR, Hab01-4-CAR, and Hab01-11-CAR all had a strong cytotoxic effect on Raji cells just after resuscitation (Figure 3A) and 24 h after resuscitation (Figure 3B). In contrast, the cytotoxicity of CD19 CAR-NK cells was better after 24 h of resuscitation.

[0165] 2.3 In vivo efficacy evaluation of CD19 CAR-NK cells in a Nalm6 (human acute lymphoblastic leukemia (ALL) cell line) xenograft mouse model

[0166] A Nalm6 xenograft mouse model was constructed using conventional methods in the art to evaluate the tumor inhibitory effects of FMC63-CAR, hFMC63-CAR, Hab01-4-CAR, and Hab01-11-CAR.

[0167] Specifically, NOG mice were taken, weighed, and randomly divided into groups (see Table 9 for the grouping scheme), and injected with human acute lymphoblastic leukemia (ALL) cell line Nalm6 (carrying the luciferase gene) at a dose of 2.5E6 cells / mouse. Two days later, a single dose of CD19 CAR-NK cells (4E6 CD19 CAR-NK cells / mouse) was intravenously injected. The time point of CD19 CAR-NK cell injection was defined as day 0. Tumor development (fluorescence imaging), mouse weight, and survival status in mice in groups G1, G3, G4, G6, G8, and G10 were continuously observed. Peripheral blood was collected from mice in groups G2, G5, G7, G9, and G11 on days 0, 7, 16, and 21 for detection of CAR19. + CD56 + and CAR19 - CD56 + Peripheral blood was collected on days 0, 7, 14, and 28 for testing of routine blood tests and blood biochemical indicators. The results are shown in Figures 4A to 4E, 5A to 5B, and 6.

[0168] As shown in Figures 4A to 4E, tumor signals continued to increase in the negative control group (G1 and G3 groups) after tumor loading, and all four CD19 CAR-NK treatment groups (G4, G6, G8, and G10) showed significant anti-tumor efficacy and significantly prolonged mouse survival. Among the four CD19 CAR-NK treatment groups, the Hab01-4-CAR-NK treatment group (G8 group) had the best therapeutic effect. Although the G8 group, like the other treatment groups, experienced tumor metastasis to the head and face on day 26, dorsal and ventral imaging observations from days 30 to 33 revealed that the G8 group did not experience recurrence in other parts of the body, while the other treatment groups also experienced recurrence in other parts of the body besides the head and face.

[0169] At the same time, judging from the survival curve, the survival rate of mice in the G8 group was 80% (day 36), which was better than that of the other treatment groups. In terms of weight changes, the mice in the four CD19 CAR-NK treatment groups were in good condition before day 19 and did not experience treatment-related toxicity such as weight loss. On day 19, they began to lose weight, and fluorescent signals were detected at the mandible. The weight loss of mice was caused by tumor recurrence. As shown in Figures 5A and 5B, in the CD19 CAR + CD56 + In terms of proportion, G5 group>G9 group>G11 group>G7 group, in CD19 CAR - CD56 + In terms of proportion, G11 group < G9 group = G7 group < G5 group. Among the G5, G7, G9 and G11 groups using humanized antibodies, G9 group CAR +The relative number of NK cells in peripheral blood mononuclear cells was higher. For details of blood routine and biochemical indicators, see Figure 6. In the G9 group, alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) levels were lower than those in other CD19 CAR-NK treatment groups, indicating a safer overall outcome.

[0170] Table 8. Sequence information of chimeric antigen receptors targeting CD19

[0171] Table 9. Grouping of NOG mice

[0172] Example 3 Preparation of CD19 CAR-NK Cell Composition

[0173] The Hab01-4-CAR cells prepared in Example 2 were expanded. When the total number of cells per closed device exceeded 1.3E10, the cells were harvested, washed, and frozen on the same day. At the same time, samples were taken for cell counting, phenotype, mycoplasma, and cell function testing. If CD3 + If the cell count exceeds 1%, a CD3+ cell removal step is required before harvesting. Resuspend the harvested CD19 CAR-NK cell product in cell preservation solution (cell preservation solution components include: compound electrolyte injection, human serum albumin injection, and freezing solution) and aliquot and freeze. The cell freezing density should be no less than 1E6 viable cells / ml for subsequent use.

[0174] Example 4 Preliminary Clinical Exploration of CD19 CAR-NK Cell Combinations in Tumors

[0175] Two subjects were enrolled, one with B-cell NHL and the other with B-ALL. The regimen consisted of a screening phase with bridging therapy (as needed), pre-lymphocyte clearance, lymphocyte clearance, pre-infusion evaluation, multiple infusions, and a follow-up phase. The investigators determined whether to administer multiple doses according to the planned schedule based on the subject's condition.

[0176] The characteristics, distribution, and follow-up time of the study subjects are summarized in Tables 10 and 11, respectively. All patients received CD19 CAR-NK (hereinafter referred to as CD19 CAR-NK) cell transfusion therapy prepared in Example 3. A preliminary single-dose study was conducted, and the enrolled subjects included Subject 1 in the 1E7 cells / kg CD19 CAR-NK dose group and Subject 2 in the 2E7 cells / kg CD19 CAR-NK dose group.

[0177] As of the data cutoff date, subjects 1 and 2 completed the dose-limiting toxicity (DLT) observation period (28 days after the first CD19 CAR-NK infusion), and no protocol-specified related toxicity (DLT) events occurred during this period.

[0178] NHL subject 1 completed the first efficacy evaluation (28 days after the first CD19 CAR-NK infusion). During the primary follow-up phase, he received 1E7 cells / kg CD19 CAR-NK treatment after third-line treatment and achieved remission (CR (complete remission) / CMR (complete molecular biological remission)). ALL subject 2 completed the first bone marrow efficacy evaluation (14 days after the first CD19 CAR-NK infusion). During the primary follow-up phase, he received 2E7 cells / kg CD19 CAR-NK treatment after second-line treatment. The initial bone marrow biopsy results on D14 showed bone marrow remission (CRi), but extramedullary lesions were still not evaluated. On D28, combined with the imaging results of extramedullary lesions, complete remission (CR) was shown.

[0179] It can be seen that the CD19 CAR-NK combination has a certain clinical therapeutic effect on B-ALL and NHL.

[0180] Table 10 Summary of demographic and subject characteristics in preliminary exploratory clinical studies of tumors (all subjects who received CD19 CAR-NK transfusion)

[0181] Note: NR, not report; NA, not applicable

[0182] Table 11 List of individual efficacy evaluations in preliminary exploratory clinical studies of tumors (all subjects who received CD19 CAR-NK transfusion)

[0183] Example 5 B cell killing by CD19 CAR-NK cell combination

[0184] Systemic lupus erythematosus (SLE) is an autoimmune disease affecting multiple organs. Its primary pathogenesis lies in an autoimmune response caused by immune imbalance, with the most critical factor being the dysregulated activation of autoreactive B cells. CD19, a characteristic surface antigen on B cells, and anti-CD19 chimeric antigen receptor NK cells (CD19 CAR-NK) can specifically kill B cells, providing a new approach for the treatment of SLE.

[0185] To investigate the cytotoxicity of CD19 CAR-NK cells against B cells, an in vitro co-incubation system was established with CD19 CAR-NK cells and control mock-NK cells and B cells isolated from peripheral blood of systemic lupus erythematosus (SLE) patients. The specific cytotoxicity of NK cells against B cells from SLE patients was assessed. Specifically, CD19 CAR-NK cells and control mock-NK cells were co-incubated with SLE B cells for either 4-hour short-term killing or multiple rounds of long-term killing. Short-term killing was performed at effector-target ratios of 1:1 and 2:1 for 4 hours; multiple rounds of killing were performed at an effector-target ratio of 1:1 for 24 hours each round, for a total of two co-incubation rounds. At the endpoint of each co-incubation round, the NK cell-to-B cell ratio in the co-cultured cells was determined by flow cytometry and the residual cell count was determined by AO / PI cell counting. The cytotoxicity of CD19 CAR-NK cells against SLE B cells was calculated by combining flow cytometry and cell counting results.

[0186] The formula for calculating the killing efficiency is:

[0187] Note: The theoretical number of B cells refers to the number of cells co-incubated with the initial seeding.

[0188] The results of the in vitro 4-hour short-term killing experiment showed that, based on the total NK cell effective-to-target ratio, at an E:T ratio of 2:1, the CD19 CAR-NK cell killing efficiency against SLE-B cells was significantly better than that of the control NK cells (Mock-NK) (96.89% vs 15.72%). At a low effective-to-target ratio of 1:1, CD19 CAR-NK still had a significant killing effect (78.52%), which was significantly better than the control Mock-NK (12.92%) (see Figure 7A).

[0189] The results of multiple rounds of in vitro killing experiments showed that at the end of the first round of co-culture, the average killing efficiency of CD19 CAR-NK against SLE-B cells was 95.69%, significantly better than the control Mock-NK cells (39.43%); in the second round, the average killing efficiency of CD19 CAR-NK against SLE-B cells was 88.47%, still significantly better than the killing efficiency of Mock-NK cells (30.63%) (see Figure 7B).

[0190] The above results show that CD19 CAR-NK can effectively kill B cells from lupus erythematosus patients, and the killing activity is significantly improved compared with the control Mock-NK cells.

[0191] A similar method was used to study the killing effect of CD19 CAR-NK on B cells from patients with myositis, scleroderma, and vasculitis. The results showed that CD19 CAR-NK can effectively kill B cells from patients with myositis, scleroderma, and vasculitis, and the killing activity is significantly improved compared with the control Mock-NK cells.

[0192] Example 6 Preliminary clinical study of CD19 CAR-NK cell compositions for tumor treatment

[0193] The main purpose is to evaluate the safety and tolerability of CD19 CAR-NK in the treatment of relapsed / refractory non-Hodgkin's lymphoma subjects, determine the maximum tolerated dose (MTD) and / or recommended Phase II dose (RD), and preliminarily evaluate the efficacy of CD19 CAR-NK in the treatment of relapsed / refractory non-Hodgkin's lymphoma subjects.

[0194] The trial plans to enroll patients with large B-cell lymphoma and follicular lymphoma who have received at least two lines of treatment and have at least one measurable lesion according to the Lugano 2014 Lymphoma Response Evaluation Criteria (Cheson 2014).

[0195] The study is divided into two phases: dose escalation and dose expansion. In the dose escalation phase, a single-dose dose escalation study is first conducted, followed by a multiple-dose dose escalation study.

[0196] Single dose escalation

[0197] Three dose groups were pre-set for single-dose escalation, with dose levels ranging from low to high: (1) 1E9 CD19 CAR+NK cells, (2) 2E9 CD19 CAR+NK cells, and (3) 3E9 CD19 CAR+NK cells. Each dose group allowed a ±10% dose fluctuation. Subjects were planned to receive only one CD19 CAR-NK infusion. Before starting CD19 CAR-NK infusion, the investigators were required to assess whether the subject was suitable for CD19 CAR-NK administration. After the DLT observation period (28 days after CD19 CAR-NK infusion) of the first enrolled subject in each dose group, the data of the subject in this group was promptly cleaned up and the safety data was reviewed. The Safety Review Committee (SRC) discussed and decided whether to continue to enroll the remaining subjects in this group.

[0198] Multiple dose escalation

[0199] The selection of dose groups for multiple dose escalation, as well as the dosing frequency and dose per dose for each dose group, will be determined by the sponsor based on the recommendations of the SRC after the completion of the single dose escalation. Two dose levels are planned, from low to high: (1) a dose lower than the RD dose confirmed in the single dose escalation study, and (2) the RD dose confirmed in the single dose escalation study (each dose group allows for a ±10% dose fluctuation). Subjects will receive up to three CD19 CAR-NK treatments, once a week. Before each CD19 CAR-NK infusion is started for each subject in each dose group, the investigator must assess whether the subject is suitable for CD19 CAR-NK administration. After the DLT observation period (within 28 days after the first CD19 CAR-NK infusion) of the first enrolled subject in each dose group is completed, the data of the subject in this group will be cleaned up in a timely manner, and the safety data will be reviewed. The Safety Review Committee (SRC) will discuss and decide whether to enroll the remaining two subjects in this group. The sponsor may also consider adding dose groups to explore multiple dosing at higher doses. DLT events and DLT-inevaluable subjects were predefined, and the DLT observation period was 28 days after the first dose of CD19 CAR-NK (the first CD19 CAR-NK dose was considered day 0). For DLT-evaluable subjects, the SRC will determine the dose increase or decrease based on the "3+3" principle, combining safety, PK, and PD data (if applicable). The SRC can also make recommendations for exploring other dose levels and dosing frequencies based on the available data at the time.

[0200] Dose expansion phase

[0201] After the dose escalation phase is completed and the SRC determines the MTD and / or RD for single / multiple doses, a dose expansion study will be conducted based on the sponsor's decision to proceed with a single or multiple doses. In the dose expansion phase, at least approximately 10 subjects will be enrolled at the determined RD dose level (including up to 3 subjects who have previously received targeted CD19 CAR-T cell therapy) to further evaluate the safety and efficacy of CD19 CAR-NK. Whether to continue enrolling to the maximum sample size will be determined based on the efficacy, safety, PK, immunogenicity, and other data from approximately 10 subjects.

[0202] The study process consists of a screening period (days -34 to -6), a lymphoablative conditioning period (days -5 to -3), a CD19 CAR-NK treatment period (days 0 to +28), a safety follow-up period (days +28 to 3 months after the first CD19 CAR-NK dose), a primary follow-up period (from the end of the safety follow-up period to 2 years after the first CD19 CAR-NK dose), and a long-term follow-up period (from the completion of the primary follow-up period to 15 years after the first CD19 CAR-NK dose). Subjects were considered enrolled in the study once they began lymphoablative conditioning chemotherapy. The day of the first CD19 CAR-NK dose was defined as day 0. From the start of the CD19 CAR-NK treatment period, and at any time thereafter, if a subject experienced disease progression or initiated new anti-lymphoma therapy without disease progression, they should undergo an end-of-term visit, complete the relevant EOT assessments, and then proceed directly to survival follow-up. Patients withdrawing from study treatment for other reasons will enter the subsequent follow-up period after completing the EOT visit. If a patient voluntarily requests to withdraw from long-term follow-up, they will be withdrawn from the trial. Except for subjects who experience disease progression or those who receive new anti-lymphoma therapy without disease progression, all remaining subjects will undergo annual follow-up (±2 months) after the completion of the 2-year primary follow-up, provided they meet the long-term follow-up requirements, for a period of 15 years after infusion. PK testing will be performed at the central laboratory; laboratory testing will be performed at the study center; and efficacy assessment tests will be performed at the study center, with efficacy assessments being conducted by the investigator (see Figure 8).

[0203] The main study portion of this study will last two years after the last enrolled subject receives the first dose of CD19 CAR-NK therapy or until the last enrolled subject completes the clinical study, whichever occurs first. The overall study completion is defined as the completion of the 15-year long-term follow-up for the last enrolled subject.

[0204] Example 7 Preliminary Clinical Exploration of CD19 CAR-NK Cell Composition in Autoimmune Diseases

[0205] Three subjects were enrolled: one with moderately to severely active systemic lupus erythematosus (SLE), one with refractory granulomatosis with polyangiitis, and one with systemic sclerosis. The protocol consisted of a screening phase, a pre-lymphocyte clearance phase, a lymphocyte clearance phase, a pre-infusion evaluation phase, a CD19 CAR-NK cell therapy phase, and a follow-up phase.

[0206] The characteristics, distribution, and follow-up duration of the study subjects are summarized in Tables 12 and 13, respectively. All patients received CD19 CAR-NK cell transfusion therapy. Each subject received two CD19 CAR-NK cell infusions within 28 days, with a 7-day interval between the two infusions. The dose group for each subject is shown in Table 13.

[0207] As of the data cutoff date, all three subjects enrolled in this study completed the dose-limiting toxicity (DLT) observation period (28 days after the first CD19 CAR-NK infusion), and no protocol-specified DLT events occurred during this period.

[0208] Subjects with moderately to severely active SLE completed the first efficacy evaluation (28 days after the first CD19 CAR-NK cell infusion) and achieved SRI-4 (SRI: SLE Responder Index) at W4, W8, W12, W20, and W24 during the primary follow-up phase. Subjects with refractory granulomatosis with polyangiitis achieved a decrease in the Birmingham Vasculitis Activity Score (BVAS) from a baseline of 12 to 5 at W4, W8, and W12. Subjects with systemic sclerosis also achieved a decrease in the maximum mRSS (modified Rodnan Skin Scale) skin score from a baseline of 13 to 9 at W4 and W8. Safety follow-up is ongoing.

[0209] It can be seen that the CD19 CAR-NK combination has a certain clinical therapeutic effect on autoimmune diseases such as systemic lupus erythematosus, refractory granulomatosis with polyangiitis, and systemic sclerosis.

[0210] Table 12 Summary of demographic and subject characteristics in the autoimmune disease exploratory study (all subjects receiving CD19 CAR-NK transfusion)

[0211] Note: UK, unknown

[0212] Table 13 Individual efficacy evaluation list of autoimmune disease exploratory studies (all subjects who received CD19 CAR-NK transfusion)

[0213] Example 8 Preliminary clinical study of CD19 CAR-NK cell compositions for the treatment of autoimmune diseases

[0214] The main purpose is to evaluate the safety and tolerability of CD19 CAR-NK in the treatment of systemic lupus erythematosus (SLE), determine the maximum tolerated dose (MTD) and / or subsequent recommended dose (Recommended Phase I IDose, RD), and preliminarily evaluate the efficacy, pharmacokinetic (PK) characteristics, and immunogenicity of CD19 CAR-NK in the treatment of SLE subjects.

[0215] The study is planned to enroll subjects with moderate to severe refractory systemic lupus erythematosus (SLE) who had been diagnosed with SLE at least 24 weeks prior to screening and met the 2019 SLE classification criteria of the European League of Societies for Rheumatology (EULAR) / American College of Rheumatology (ACR) at the time of screening.

[0216] The study is divided into two phases: dose escalation and dose expansion. In the dose escalation phase, a single-dose dose escalation study is first conducted, and the MTD and / or subsequent recommended dose (RD) are determined based on the results of the single-dose dose escalation study, as well as whether multiple-dose dose escalation studies are needed. If a single dose is sufficient to meet the treatment needs, multiple-dose dose escalation can be skipped, and recommendations for the dose expansion phase are directly provided based on the single-dose MTD and / or RD dose.

[0217] Single dose escalation

[0218] The single-dose dose escalation phase was pre-configured into four dose groups, with dose levels ranging from low to high: (1) 0.5E9 CD19 CAR+NK cells, (2) 3E9 CD19 CAR+NK cells, (3) 6E9 CD19 CAR+NK cells, and (4) 9E9 CD19 CAR+NK cells. Each dose group was allowed to fluctuate ±10%. The single-dose dose escalation phase used an accelerated titration combined with a "3+3" design to guide dose escalation. In the single-dose dose escalation phase, subjects were planned to receive only one CD19 CAR-NK infusion. Before starting CD19 CAR-NK infusion, the investigator was required to assess whether the subject was suitable for CD19 CAR-NK administration.

[0219] Multiple dose escalation

[0220] The multiple dose escalation phase uses a "3+3" design to guide dose escalation. The selection of dose groups for multiple dose escalation and the dosing frequency for each dose group will be determined by the sponsor based on the SRC's recommendations after the completion of the single dose escalation. It is planned to set two dose levels, from low to high: (1) a dose lower than the RD dose confirmed in the single dose escalation study, and (2) the RD dose confirmed in the single dose escalation study (each dose group is allowed to fluctuate by ±10%). Subjects will receive a maximum of 2 CD19 CAR-NK treatments, once a week. Before each CD19 CAR-NK infusion is started for each subject in each dose group, the investigator needs to assess whether the subject is suitable for CD19 CAR-NK administration.

[0221] DLT events and DLT-inevaluable subjects are predefined, and the DLT observation period is 28 days after the last dose of CD19 CAR-NK. For DLT-evaluable subjects, the SRC will determine the dose increase or decrease based on safety, PK, and PD data (if applicable). The SRC may also make recommendations based on the available data at that time to explore other dose levels and dosing frequencies.

[0222] Dose expansion phase

[0223] After the dose escalation phase is completed and the SRC determines the MTD and / or RD for single / multiple doses, a dose expansion study will be conducted, either with a single dose or with multiple doses, based on the sponsor's decision. During the dose expansion phase, approximately 10 subjects will be enrolled at the determined RD dose level to further evaluate the safety and efficacy of the CD19 CAR-NK cell therapy. The decision to continue enrolling the patient to the maximum sample size will be made based on the efficacy, safety, PK, and immunogenicity data from these approximately 10 subjects.

[0224] The study process is divided into a screening period (days -34 to -6), a lympholytic conditioning period (days -5 to -3), a CD19 CAR-NK treatment period (days 0 to +28 days after the last F01 dose), a short-term follow-up period (days 8 to 96), and a long-term follow-up period (days 96 to 15 years). Once a subject begins receiving lympholytic conditioning chemotherapy, they are considered enrolled in the study. The day of the first CD19 CAR-NK administration is defined as day 0. Discontinuation of study treatment for any reason after the first CD19 CAR-NK infusion requires an end-of-term visit. If the subject does not withdraw from the study, they will enter the subsequent follow-up period after completing the end-of-term visit. If a subject experiences disease progression / relapse or prematurely initiates protocol-prohibited salvage therapy, survival follow-up and salvage treatment information will be collected directly after completing the end-of-term visit. If a patient voluntarily requests to withdraw from short-term follow-up, they will be withdrawn from the trial. After the 2-year short-term follow-up, all other subjects will be followed for up to 15 years, except for those who experience disease progression / recurrence and require protocol-prohibited salvage therapy. If a subject develops a second primary malignancy (SPM), lifelong follow-up is required. In this study, PK / PD testing will be performed at a central laboratory; laboratory tests will be performed at the study center; and tests related to efficacy assessment will be performed at the study center. Disease assessment will be assessed by the investigator (see Figure 9).

[0225] This study will collect and record any adverse events and serious adverse events (including AESI) that occur from the start of lymphoblastic conditioning chemotherapy until 12 weeks after the first CD19 CAR-NK product infusion or the start of protocol-prohibited salvage therapy (whichever occurs first). After 12 weeks after the first CD19 CAR-NK product infusion, only adverse events and serious adverse events (including AESI) assessed by the investigator as related to the study treatment will be collected. The duration of the main study portion of this study is 2 years after the last enrolled subject receives the first dose of CD19 CAR-NK treatment or the last subject in the group completes the clinical study, whichever occurs first. The overall end of the study is defined as the completion of the 15-year long-term follow-up for the last subject in the group.

[0226] The teachings of all patents, published applications, and references cited herein are incorporated by reference in their entirety.

[0227] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments as encompassed by the appended claims.

Claims

1. A pharmaceutical composition, wherein the pharmaceutical composition comprises an immune effector cell and a pharmaceutically acceptable carrier, wherein the immune effector cell comprises a chimeric antigen receptor targeting CD19, and the chimeric antigen receptor comprises: An extracellular region comprising an antigen binding region that specifically binds to CD19, a transmembrane region connected to the extracellular region, and an intracellular domain connected to the transmembrane region, wherein the antigen binding region comprises a VH comprising HCDR1-3 and a VL comprising LCDR1-3, wherein: (1) the HCDR1-3 have the sequences shown in SEQ ID NOs: 5-7, respectively, and the LCDR1-3 have the sequences shown in SEQ ID NOs: 8-10, respectively; or (2) The HCDR1-3 have 0-3 mutations compared with each corresponding CDR in group (1), and / or the LCDR1-3 have 0-3 mutations compared with each corresponding CDR in group (1).

2. The pharmaceutical composition according to claim 1, wherein (1) the VH has the sequence shown in SEQ ID NO: 20, and the VL has the sequence shown in SEQ ID NO: 17; or (2) The VH has a sequence that is at least 80% identical or has at most 25 mutations compared to the corresponding VH in group (1), and the VL has a sequence that is at least 80% identical or has at most 20 mutations compared to the corresponding VL in group (1).

3. The pharmaceutical composition according to claim 1 or 2, wherein the chimeric antigen receptor has a sequence as shown in SEQ ID NO: 23, or a sequence having at least 80% identity or at most 120 amino acid mutations compared to SEQ ID NO:

23.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition is a cryopreserved immune cell. The pharmaceutical composition according to claim 4 , wherein the pharmaceutically acceptable carrier is a cell preservation solution.

6. The pharmaceutical composition according to claim 4 or 5, wherein the freezing density of immune effector cells in the immune cell cryopreservation product is not less than 1×10 5 Preferably, the freezing density of immune effector cells in the immune cell cryopreserved material is not less than 1×10 6 Viable cells / ml.

7. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition is an injection.

8. The pharmaceutical composition according to any one of claims 1 to 6, wherein the CD3 + The proportion of cells does not exceed 5%. Preferably, the CD3 + More preferably, the proportion of CD3 + The proportion of cells does not exceed 1%.

9. A method for treating a disease, comprising administering the pharmaceutical composition of any one of claims 1 to 8 to a subject in need thereof, wherein the disease is a B cell-driven disease.

10. The method of claim 9, wherein the disease is a CD19-positive B cell-driven disease.

11. The method according to claim 9 or 10, wherein the disease is a tumor.

12. The method according to claim 11, wherein the tumor is a CD19-related tumor, preferably, the CD19-related tumor is a B-cell malignancy.

13. The method according to claim 12, wherein the CD19-related tumor is a CD19-positive B-cell malignancy; preferably, the CD19-positive B-cell malignancy is a relapsed or refractory CD19-positive B-cell malignancy.

14. The method according to claim 12 or 13, wherein the CD19-related tumor is selected from one or more of acute myeloid leukemia (ALL), chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), diffuse large cell lymphoma (DLBCL) and marginal zone B-cell non-Hodgkin's lymphoma (MZL).

15. The method according to any one of claims 11-14, wherein the subject has previously received at least 2 lines of treatment.

16. The method according to any one of claims 11-15, wherein the subject has at least one measurable lesion according to the Lugano 2014 Lymphoma Response Evaluation Criteria (Cheson 2014).

17. The method of claim 9 or 10, wherein the disease is an autoimmune disease.

18. The method of claim 17, wherein the autoimmune disease is selected from one or more of systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), multiple sclerosis (MS), rheumatoid arthritis (RA), type 1 diabetes mellitus (T1DM), Sjögren's syndrome, myositis, vasculitis and scleroderma.

19. A method for treating an autoimmune disease, comprising administering to a subject in need thereof an immune effector cell, wherein the immune effector cell comprises a chimeric antigen receptor targeting CD19, wherein the chimeric antigen receptor comprises: An extracellular region comprising an antigen binding region that specifically binds to CD19, a transmembrane region connected to the extracellular region, and an intracellular domain connected to the transmembrane region, wherein the antigen binding region comprises a VH comprising HCDR1-3 and a VL comprising LCDR1-3, wherein: (1) the HCDR1-3 have the sequences shown in SEQ ID NOs: 5-7, respectively, and the LCDR1-3 have the sequences shown in SEQ ID NOs: 8-10, respectively; or (2) The HCDR1-3 have 0-3 mutations compared with each corresponding CDR in group (1), and / or the LCDR1-3 have 0-3 mutations compared with each corresponding CDR in group (1).

20. The method according to claim 19, wherein: (1) the VH has the sequence shown in SEQ ID NO: 20, and the VL has the sequence shown in SEQ ID NO: 17; or (2) The VH has a sequence that is at least 80% identical or has at most 25 mutations compared to the corresponding VH in group (1), and the VL has a sequence that is at least 80% identical or has at most 20 mutations compared to the corresponding VL in group (1).

21. The method of claim 19 or 20, wherein the chimeric antigen receptor has a sequence as shown in SEQ ID NO: 23, or a sequence having at least 80% identity or at most 120 amino acid mutations compared to SEQ ID NO:

23.

22. The method according to any one of claims 19-21, wherein the autoimmune disease is selected from one or more of systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), multiple sclerosis (MS), rheumatoid arthritis (RA), type 1 diabetes mellitus (T1DM), Sjögren's syndrome, myositis, vasculitis, and scleroderma.

23. The method according to any one of claims 17-22, wherein the subject has been diagnosed with SLE at least 24 weeks prior to screening.

24. The method according to any one of claims 17-23, wherein the subject meets the European League of Societies for Rheumatology (EULAR) / American College of Rheumatology (ACR) 2019 SLE classification criteria when screened.

25. The method according to any one of claims 11 to 24, wherein the administration of the pharmaceutical composition is a single administration or multiple administrations; Optionally, the administration is once a week; and Optionally, the single administration is administration once; the multiple administrations are administrations 2-6 times; preferably, the multiple administrations are administrations 2, 3, 4, 5, 6 times.

26. The method according to any one of claims 11 to 25, wherein the pharmaceutical composition is administered at 1×10 4 Cells / Kg~1×10 9 Cells / Kg administration; Preferably, the pharmaceutical composition is administered at 1×10 6 Cells / Kg~9×10 8 Cells / Kg administration; Preferably, the pharmaceutical composition is administered at 1×10 6 Cells / Kg~9×10 6 Cells / Kg, 1×10 7 Cells / Kg~9×10 7 cells / Kg, or 1×10 8 Cells / Kg~9×10 8 Cells / Kg administration; More preferably, the pharmaceutical composition is administered at 5×10 6 Cells / Kg, 6×10 6 Cells / Kg, 7×10 6 Cells / Kg, 8×10 6 Cells / Kg, 9×10 6 Cells / Kg, 1×10 7 Cells / Kg, 2×10 7 Cells / Kg, 3×10 7 Cells / Kg, 4×10 7 Cells / Kg, 5×10 7 Cells / Kg, 6×10 7 Cells / Kg, 7×10 7 Cells / Kg, 8×10 7 Cells / Kg, 9×10 7 Cells / Kg, 1×10 8 Cells / Kg, 2×10 8 Cells / Kg, 3×10 8 Cells / Kg, 4×10 8 Cells / Kg, 5×10 8 Cells / Kg, 6×10 8 Cells / Kg, 7×10 8 Cells / Kg, 8×10 8 cells / Kg, or 9×10 8 Cells / Kg administration.

27. Use of a pharmaceutical composition or immune effector cells in the preparation of a drug for treating a disease, wherein the pharmaceutical composition is the pharmaceutical composition of any one of claims 1 to 8; the immune cells are the immune cells of any one of claims 1 to 8 or claims 19 to 21; The disease is a B cell driven disease; and The treatment comprises administering the pharmaceutical composition or the immune effector cell to a subject in need thereof.

28. A pharmaceutical composition or immune effector cell for treating a disease, wherein the pharmaceutical composition is the pharmaceutical composition of any one of claims 1 to 8; the immune cell is the immune cell of any one of claims 1 to 8 or claims 19 to 21; The disease is a B cell driven disease; and The treatment comprises administering the pharmaceutical composition or the immune effector cell to a subject in need thereof.

Images